U.S. patent application number 13/090682 was filed with the patent office on 2012-04-26 for phospholipases, nucleic acids encoding them and methods for making and using them.
This patent application is currently assigned to VERENIUM CORPORATION. Invention is credited to ADRIAN BADILLO, NELSON R. BARTON, ROBERT C. BROWN, MARK J. BURK, RODERICK JAMES FIELDING, SVETLANA GRAMATIKOVA, GEOFF HAZLEWOOD, CHARLES ISAAC, GISELLE JANSSEN, JOEL A. KREPS, DAVID LAM, JINCAI LI, DAN E. ROBERTSON, BLAKE G. STURGIS, XUQIU TAN, WILHELMUS P. VAN HOEK, AMIT VASAVADA.
Application Number | 20120100581 13/090682 |
Document ID | / |
Family ID | 46206116 |
Filed Date | 2012-04-26 |
United States Patent
Application |
20120100581 |
Kind Code |
A1 |
GRAMATIKOVA; SVETLANA ; et
al. |
April 26, 2012 |
PHOSPHOLIPASES, NUCLEIC ACIDS ENCODING THEM AND METHODS FOR MAKING
AND USING THEM
Abstract
The invention provides novel polypeptides having phospholipase
activity, including, e.g., phospholipase A, B, C and D activity,
patatin activity, phosphatidic acid phosphatases (PAP)) and/or
lipid acyl hydrolase (LAH) activity, nucleic acids encoding them
and antibodies that bind to them. Industrial methods, e.g., oil
degumming, and products comprising use of these phospholipases are
also provided.
Inventors: |
GRAMATIKOVA; SVETLANA; (SAN
DIEGO, CA) ; HAZLEWOOD; GEOFF; (NEWBURY, GB) ;
LAM; DAVID; (SAN MARCOS, CA) ; BARTON; NELSON R.;
(SAN DIEGO, CA) ; STURGIS; BLAKE G.; (CARLSBAD,
CA) ; ROBERTSON; DAN E.; (BELMONT, MA) ; LI;
JINCAI; (SAN DIEGO, CA) ; KREPS; JOEL A.;
(ENCINITAS, CA) ; FIELDING; RODERICK JAMES; (SAN
DIEGO, CA) ; BROWN; ROBERT C.; (SAN DIEGO, CA)
; VASAVADA; AMIT; (POWAY, CA) ; TAN; XUQIU;
(SAN DIEGO, CA) ; BADILLO; ADRIAN; (POWAY, CA)
; VAN HOEK; WILHELMUS P.; (CAMARILLO, CA) ;
JANSSEN; GISELLE; (DIXON, CA) ; ISAAC; CHARLES;
(CARLSBAD, CA) ; BURK; MARK J.; (SAN DIEGO,
CA) |
Assignee: |
VERENIUM CORPORATION
SAN DIEGO
CA
|
Family ID: |
46206116 |
Appl. No.: |
13/090682 |
Filed: |
April 20, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11625765 |
Jan 22, 2007 |
7943360 |
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13090682 |
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10592450 |
Jul 29, 2008 |
7977080 |
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PCT/US05/07908 |
Mar 8, 2005 |
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11625765 |
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10796907 |
Mar 8, 2004 |
7226771 |
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10592450 |
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10421654 |
Apr 21, 2003 |
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10796907 |
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60374313 |
Apr 19, 2002 |
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Current U.S.
Class: |
435/134 ;
435/198; 435/252.3; 435/252.31; 435/252.35; 435/254.11; 435/254.2;
435/254.21; 435/254.23; 435/262; 435/264; 435/280; 435/281;
435/320.1; 435/325; 435/348; 435/354; 435/358; 435/365; 435/366;
536/23.1; 536/23.2; 554/1 |
Current CPC
Class: |
C12N 15/8242 20130101;
C12P 7/6418 20130101; C12P 7/6445 20130101; C12N 9/20 20130101;
C12N 9/16 20130101; C12N 9/18 20130101; C12P 7/6481 20130101; C07K
2319/00 20130101; C12N 15/8247 20130101 |
Class at
Publication: |
435/134 ;
536/23.2; 536/23.1; 435/198; 435/262; 435/264; 435/280; 435/281;
554/1; 435/320.1; 435/252.3; 435/254.11; 435/254.2; 435/325;
435/348; 435/254.21; 435/254.23; 435/252.31; 435/252.35; 435/358;
435/365; 435/354; 435/366 |
International
Class: |
C12P 7/64 20060101
C12P007/64; C12N 9/20 20060101 C12N009/20; C12S 99/00 20100101
C12S099/00; C12S 3/00 20060101 C12S003/00; C12P 41/00 20060101
C12P041/00; C12N 5/10 20060101 C12N005/10; C07C 53/00 20060101
C07C053/00; C12N 15/63 20060101 C12N015/63; C12N 1/21 20060101
C12N001/21; C12N 1/15 20060101 C12N001/15; C12N 1/19 20060101
C12N001/19; C07H 21/00 20060101 C07H021/00; C12S 1/00 20060101
C12S001/00 |
Claims
1: An isolated, a synthetic, or a recombinant nucleic acid
comprising a nucleic acid sequence that is at least 85% identical
to SEQ ID NO:119, wherein the nucleic acid encodes a polypeptide
having a phospholipase C activity.
2: The nucleic acid of claim 1, wherein the nucleic acid sequence
is at least 90% identical to SEQ ID NO:119.
3: The nucleic acid of claim 1, wherein the nucleic acid sequence
is at least 95% identical to SEQ ID NO:119.
4: An isolated, synthetic or recombinant nucleic acid having a
sequence as set forth in SEQ ID NO:119.
5: The polypeptide of claim 1, wherein the polypeptide having a
Phospholipase C activity is an amino acid sequence as set forth in
SEQ ID NO:120.
6: The polypeptide of claim 1, wherein the polypeptide has
phospholipase C activity with a substrate selected from
phosphatidylinositol, phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, or phosphatidic
acid.
7: The polypeptide of claim 1, wherein the polypeptide has
phospholipase C activity at a temperature selected from about
37.degree. C. to about 95.degree. C.
8: The polypeptide of claim 7, wherein the polypeptide retains
phospholipase C activity within the range of about 55.degree. C. to
about 85.degree. C.
9: The polypeptide of claim 1, wherein the polypeptide lacks a
signal sequence.
10: The polypeptide of claim 1, wherein the polypeptide further
comprises a heterologous sequence.
11: The polypeptide of claim 10, wherein the heterologous sequence
comprises a signal sequence, a catalytic domain or active site, a
metal chelating peptide, or an N-terminal identification
peptide.
12: The polypeptide of claim 1, wherein the polypeptide has
phospholipase C activity with a specific activity of from about 100
to about 1000 units per milligram of protein at 37.degree. C.
13: The polypeptide of claim 1, wherein the polypeptide comprises
at least one glycosylation.
14: The polypeptide of claim 13, wherein the glycosylation is an
N-linked glycosylation or an O-linked glycosylation, or a
glycosylation upon expression in a P. pastoris or a S. pombe host
cell.
15: The polypeptide of claim 5, wherein the polypeptide comprises
at least one conservative amino acid substitution with respect to
SEQ ID NO:120.
16: A protein preparation comprising the polypeptide of claim 1,
wherein the protein preparation is a liquid, a solid, or a gel.
17: The polypeptide of claim 1, wherein the polypeptide is present
as a heterodimer with a second protein domain.
18: The polypeptide of claim 1, wherein the polypeptide
homodimerizes.
19: The polypeptide of claim 1, wherein the polypeptide is
immobilized on a cell, a metal, a resin, a polymer, a ceramic, a
glass, a microelectrode, a graphitic particle, a bead, a gel, a
plate, an array or a capillary tube.
20: A method for hydrolyzing, breaking up or disrupting a
phospholipid-comprising composition comprising: (i) (a) providing
the polypeptide of claim 1; (b) providing a composition comprising
a phospholipid; and (c) contacting the polypeptide of (a) with the
composition of (b) under conditions wherein the phospholipase
hydrolyzes, breaks up or disrupts the phospholipid-comprising
composition; or (ii) the method of (i), wherein the composition
comprises a phospholipid-comprising lipid bilayer or membrane, or
the composition comprises or is contained in a plant cell, a
bacterial cell, a yeast cell, an insect cell, or an animal
cell.
21: A method for liquefying or removing a phospholipid-comprising
composition comprising: (a) providing the polypeptide of claim 1;
(b) providing a composition comprising a phospholipid; and (c)
contacting the polypeptide of (a) with the composition of (b) under
conditions wherein the phospholipase removes or liquefies the
phospholipid-comprising composition.
22: A detergent composition comprising (a) the polypeptide of claim
1; (b) the detergent composition of (a), wherein the phospholipase
is a nonsurface-active phospholipase or a surface-active
phospholipase; or (c) the detergent composition of (a) or (b),
wherein the phospholipase is formulated in a non-aqueous liquid
composition, a cast solid, a granular form, a particulate form, a
compressed tablet, a gel form, a paste or a slurry form.
23: A method for washing an object comprising: (a) providing a
composition comprising the polypeptide of claim 1; (b) providing an
object; and (c) contacting the polypeptide of (a) and the object of
(b) under conditions wherein the composition can wash the
object.
24: A method for degumming an oil or a fat comprising: (i) (a)
providing the polypeptide of claim 1; (b) providing an composition
comprising an phospholipid-containing fat or oil; and (c)
contacting the polypeptide of (a) and the composition of (b) under
conditions wherein the polypeptide can catalyze the hydrolysis of a
phospholipid in the composition; (ii) the method of (i), wherein
the oil- or fat-comprising composition comprises a plant, an
animal, an algae or a fish oil or fat; (iii) the method of (ii),
wherein plant oil comprises a rice bran oil, a soybean oil, a
rapeseed oil, a corn oil, an oil from a palm kernel, a canola oil,
a sunflower oil, a sesame oil or a peanut oil; (iv) the method of
any or (i) to (iii), wherein the polypeptide hydrolyzes a
phosphatide from a hydratable and/or a non-hydratable phospholipid
in the oil-comprising composition; (v) the method of any or (i) to
(iii), wherein the polypeptide hydrolyzes a phosphatide at a
glyceryl phosphoester bond to generate a diglyceride and
water-soluble phosphate compound; (vi) the method of any or (i) to
(v), wherein the contacting comprises hydrolysis of a hydrated
phospholipid in an oil; (vii) the method of any or (i) to (vi),
wherein the hydrolysis conditions of (c) comprise a temperature of
about 20.degree. C. to 40.degree. C. at an alkaline pH, or the
alkaline conditions comprise a pH of about pH 8 to pH 10, or the
hydrolysis conditions of (c) comprise a reaction time of about 3 to
10 minutes, or the hydrolysis conditions of (c) comprise hydrolysis
of hydratable and non-hydratable phospholipids in oil at a
temperature of about 50.degree. C. to 60.degree. C., at a pH of
about pH 5 to pH 6.5 using a reaction time of about 30 to 60
minutes; (viii) the method of any or (i) to (vii), wherein the
polypeptide is bound to a filter and the phospholipid-containing
fat or oil is passed through the filter; or (ix) the method of any
or (i) to (viii), wherein the polypeptide is added to a solution
comprising the phospholipid-containing fat or oil and then the
solution is passed through a filter.
25: A method for converting a non-hydratable phospholipid to a
hydratable form comprising: (i) (a) providing the polypeptide of
claim 1; (b) providing an composition comprising a non-hydratable
phospholipid; and (c) contacting the polypeptide of (a) and the
composition of (b) under conditions wherein the polypeptide
converts the non-hydratable phospholipid to a hydratable form.
26: A method for caustic refining of a phospholipid-containing
composition comprising: (i) (a) providing the polypeptide of claim
1; (b) providing an composition comprising a phospholipid; and (c)
contacting the polypeptide of (a) with the composition of (b)
before, during or after the caustic refining; (ii) the method of
(i), wherein the polypeptide having a phospholipase activity is
added before caustic refining and the composition comprising the
phospholipid comprises a plant and the polypeptide is expressed
transgenic ally in the plant, the polypeptide having a
phospholipase activity added during crushing of a seed or other
plant part, or, the polypeptide having a phospholipase activity
added following crushing or prior to refining; (iii) the method of
any of (i) to (ii), wherein the polypeptide having a phospholipase
activity is added during caustic refining and varying levels of
acid and caustic are added depending on levels of phosphorous and
levels of free fatty acids; or (iv) the method of any of (i) to
(iii), wherein the polypeptide having a phospholipase activity is
added after caustic refining: in an intense mixer or retention
mixer prior to separation; following a heating step; in a
centrifuge; in a soapstock; in a washwater; or, during bleaching or
deodorizing steps.
27: A method for purification of a phytosterol or a triterpene
comprising: (i) (a) providing the polypeptide of claim 1; (b)
providing an composition comprising a phytosterol or a triterpene;
and (c) contacting the polypeptide of (a) with the composition of
(b) under conditions wherein the polypeptide can catalyze the
hydrolysis of a phospholipid in the composition; (ii) the method of
(i), wherein the phytosterol or a triterpene comprises a plant
sterol, or the plant sterol is derived from a vegetable oil, or the
vegetable oil comprises a coconut oil, canola oil, cocoa butter
oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil,
peanut oil, oil derived from a rice bran, safflower oil, sesame
oil, soybean oil or a sunflower oil; (iii) the method of any of (i)
to (ii), further comprising use of nonpolar solvents to
quantitatively extract free phytosterols and phytosteryl fatty-acid
esters; or (iv) the method of any of (i) to (iii), wherein the
phytosterol or a triterpene comprises a .beta.-sitosterol, a
campesterol, a stigmasterol, a stigmastanol, a .beta.-sitostanol, a
sitostanol, a desmosterol, a chalinasterol, a poriferasterol, a
clionasterol or a brassicasterol.
28: A method for refining an oil comprising: (i) (a) providing the
polypeptide of claim 1; (b) providing a composition comprising an
oil comprising a phospholipid; and (c) contacting the polypeptide
of (a) with the composition of (b) under conditions wherein the
polypeptide can catalyze the hydrolysis of a phospholipid in the
composition; (ii) the method of (i), wherein the polypeptide having
a phospholipase activity is in a water solution that is added to
the composition; (iii) the method of any of (i) to (ii), wherein
the water level is between about 0.5 to 5%; (iv) the method of any
of (i) to (iii), wherein the process time is less than about 2
hours, or the process time is less than about 60 minutes, or the
process time is less than about 30 minutes, less than about 15
minutes, or less than about 5 minutes; (v) the method of any of (i)
to (iv), wherein the hydrolysis conditions comprise a temperature
of between about 25.degree. C. to 70.degree. C., or the hydrolysis
conditions comprise use of caustics, or the hydrolysis conditions
comprise a pH of between about pH 3 and pH 10, or the hydrolysis
conditions comprise addition of emulsifiers and/or mixing after the
contacting of (i)(c); (vi) the method of any of (i) to (v),
comprising addition of an emulsion-breaker and/or heat to promote
separation of an aqueous phase; (vii) the method of any of (i) to
(vi), comprising degumming before the contacting to collect
lecithin by centrifugation and then adding a PLC, a PLC and/or a
PLA to remove non-hydratable phospholipids; (viii) the method of
any of (i) to (vii), comprising water degumming of crude oil to
less than 10 ppm for edible oils and subsequent physical refining
to less than about 50 ppm for biodiesel oils; or (ix) the method of
any of (i) to (viii), comprising addition of acid to promote
hydration of non-hydratable phospholipids.
29: A method for treating an oil or a fat comprising: (a) providing
the polypeptide of claim 1; (b) providing an composition comprising
an phospholipid-containing fat or oil; and (c) contacting the
polypeptide of (a) and the composition of (b) under conditions
wherein the polypeptide can catalyze the hydrolysis of a
phospholipid in the composition.
30: A composition comprising the polypeptide of claim 1.
31: A treated oil or fat made by the method of claim 1.
32: An expression cassette, a vector, or a cloning vehicle
comprising the nucleic acid sequence of claim 1.
33: A transformed cell comprising the nucleic acid sequence of
claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. ("USSN") 11/625,765, filed Jan. 22, 2007, currently
pending; which is a divisional of U.S. Ser. No. 10/592,450, filed
Jul. 29, 2008, currently pending; which is a National Stage Filing
under 35 U.S.C. .sctn.371 of international patent application
number PCT/US2005/07908, filed Mar. 8, 2005; which claims priority
to U.S. Ser. No. 10/796,907, filed Mar. 8, 2004, now U.S. Pat. No.
7,226,771; which is a continuation-in-part of U.S. Ser. No.
10/421,654, filed Apr. 21, 2003, now abandon; which is a
non-provisional filing of U.S. Ser. No. 60/374,313, filed Apr. 19,
2002, now expired, all of which are incorporated by reference into
the specification of this application in there entirety and for all
purposes.
REFERENCE TO SEQUENCE LISTING
[0002] This application includes a sequence listing that is
identical to a sequence filed Jan. 22, 2007, in computer readable
form with application Ser. No. 11/625,765. A request for transfer
of a computer readable form under 37 .sctn.C.F.R. 1.821(e) from
application Ser. No. 11/625,765, to this application is being
concurrently filed herewith.
FIELD OF THE INVENTION
[0003] This invention relates generally to phospholipase enzymes,
polynucleotides encoding the enzymes, methods of making and using
these polynucleotides and polypeptides. In particular, the
invention provides novel polypeptides having phospholipase
activity, nucleic acids encoding them and antibodies that bind to
them. Industrial methods and products comprising use of these
phospholipases are also provided.
BACKGROUND
[0004] Phospholipases are enzymes that hydrolyze the ester bonds of
phospholipids. Corresponding to their importance in the metabolism
of phospholipids, these enzymes are widespread among prokaryotes
and eukaryotes. The phospholipases affect the metabolism,
construction and reorganization of biological membranes and are
involved in signal cascades. Several types of phospholipases are
known which differ in their specificity according to the position
of the bond attacked in the phospholipid molecule. Phospholipase A1
(PLA1) removes the 1-position fatty acid to produce free fatty acid
and 1-lyso-2-acylphospholipid. Phospholipase A2 (PLA2) removes the
2-position fatty acid to produce free fatty acid and
1-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes can be intra- or
extra-cellular, membrane-bound or soluble. Intracellular PLA2 is
found in almost every mammalian cell. Phospholipase C (PLC) removes
the phosphate moiety to produce 1,2 diacylglycerol and phosphate
ester. Phospholipase D (PLD) produces 1,2-diacylglycerophosphate
and base group. PLC and PLD are important in cell function and
signaling. PLD had been the dominant phospholipase in biocatalysis
(see, e.g., Godfrey, T. and West S. (1996) Industrial enzymology,
299-300, Stockton Press, New York). Patatins are another type of
phospholipase, thought to work as a PLA (see for example,
Hirschberg H J, et al., (2001), Eur J Biochem 268
(19):5037-44).
[0005] Common oilseeds, such as soybeans, rapeseed, sunflower
seeds, rice bran oil, sesame and peanuts are used as sources of
oils and feedstock. In the oil extraction process, the seeds are
mechanically and thermally treated. The oil is separated and
divided from the meal by a solvent. Using distillation, the solvent
is then separated from the oil and recovered. The oil is "degummed"
and refined. The solvent content in the meal can be evaporated by
thermal treatment in a "desolventizer toaster," followed by meal
drying and cooling. After a solvent had been separated by
distillation, the produced raw oil is processed into edible oil,
using special degumming procedures and physical refining. It can
also be utilized as feedstock for the production of fatty acids and
methyl ester. The meal can be used for animal rations.
[0006] Degumming is the first step in vegetable oil refining and it
is designed to remove contaminating phosphatides that are extracted
with the oil but interfere with the subsequent oil processing.
These phosphatides are soluble in the vegetable oil only in an
anhydrous form and can be precipitated and removed if they are
simply hydrated. Hydration is usually accomplished by mixing a
small proportion of water continuously with substantially dry oil.
Typically, the amount of water is 75% of the phosphatides content,
which is typically 1 to 1.5%. The temperature is not highly
critical, although separation of the hydrated gums is better when
the viscosity of the oil is reduced at 50.degree. C. to 80.degree.
C.
[0007] Many methods for oil degumming are currently used. The
process of oil degumming can be enzymatically assisted by using
phospholipase enzymes. Phospholipases A1 and A2 have been used for
oil degumming in various commercial processes, e.g., "ENZYMAX.TM.
degumming" (Lurgi Life Science Technologies GmbH, Germany).
Phospholipase C (PLC) also has been considered for oil degumming
because the phosphate moiety generated by its action on
phospholipids is very water soluble and easy to remove and the
diglyceride would stay with the oil and reduce losses; see e.g.,
Godfrey, T. and West S. (1996) Industrial Enzymology, pp. 299-300,
Stockton Press, New York; Dahlke (1998) "An enzymatic process for
the physical refining of seed oils," Chem. Eng. Technol.
21:278-281; Clausen (2001) "Enzymatic oil degumming by a novel
microbial phospholipase," Eur. J. Lipid Sci. Technol.
103:333-340.
[0008] High phosphatide oils such as soy, canola and sunflower are
processed differently than other oils such as palm. Unlike the
steam or "physical refining" process for low phosphatide oils,
these high phosphorus oils require special chemical and mechanical
treatments to remove the phosphorus-containing phospholipids. These
oils are typically refined chemically in a process that entails
neutralizing the free fatty acids to form soap and an insoluble gum
fraction. The neutralization process is highly effective in
removing free fatty acids and phospholipids but this process also
results in significant yield losses and sacrifices in quality. In
some cases, the high phosphatide crude oil is degummed in a step
preceding caustic neutralization. This is the case for soy oil
utilized for lecithin wherein the oil is first water or acid
degummed.
[0009] Phytosterols (plant sterols) are members of the "triterpene"
family of natural products, which includes more than 100 different
phytosterols and more than 4000 other types of triterpenes. In
general, phytosterols are thought to stabilize plant membranes,
with an increase in the sterol/phospholipid ration leading to
membrane rigidification. Chemically, phytosterols closely resemble
cholesterol in structure and are thought to regulate membrane
fluidity in plant membranes, as does cholesterol in animal
membranes. The major phytosterols are .beta.-sitosterol,
campesterol and stigmasterol. Others include stigmastanol
(.beta.-sitostanol), sitostanol, desmosterol,
dihydrobrassicasterol, chalinasterol, poriferasterol, clionasterol
and brassicasterol.
[0010] Plant sterols are important agricultural products for health
and nutritional industries. They are useful emulsifiers for
cosmetic manufacturers and supply the majority of steroidal
intermediates and precursors for the production of hormone
pharmaceuticals. The saturated analogs of phytosterols and their
esters have been suggested as effective cholesterol-lowering agents
with cardiologic health benefits. Plant sterols reduce serum
cholesterol levels by inhibiting cholesterol absorption in the
intestinal lumen and have immunomodulating properties at extremely
low concentrations, including enhanced cellular response of T
lymphocytes and cytotoxic ability of natural killer cells against a
cancer cell line. In addition, their therapeutic effect has been
demonstrated in clinical studies for treatment of pulmonary
tuberculosis, rheumatoid arthritis, management of HIV-infested
patients and inhibition of immune stress in marathon runners.
[0011] Plant sterol esters, also referred to as phytosterol esters,
were approved as GRAS (Generally Recognized As Safe) by the US Food
and Drug Administration (FDA) for use in margarines and spreads in
1999. In September 2000, the FDA also issued an interim rule that
allows health-claims labeling of foods containing phytosterol
ester. Consequently enrichment of foods with phytosterol esters is
highly desired for consumer acceptance.
[0012] Soybean oil is widely used and is an important foodstuff,
accounting for .about.30% of the oil production from seeds and
fruits. Soybeans contain only 20% oil, and the extraction is
usually done by using a solvent such as hexane on a commercial
scale. The recognized quality of its oil and the nutritive value of
the meal protein make soya bean a primary oilseed. Before
extraction, soybeans must be cleaned, cracked and flaked as
efficient solvent extraction of oil requires that every oil cell is
broken to improve the mass transfer. Cell walls mostly composed of
cellulose, associated with hemicelluloses, pectic substances and
lignin), could also be broken by means of enzymes, to achieve a
significant improvement in extraction yields and rates.
[0013] Diacylglycerol (DAG) oil is an edible oil containing 80% or
greater amount of DAG than natural fatty acids. It has been shown
in humans that postprandial elevation of triglyceride in
chylomicrons is markedly smaller after ingestion of a DAG oil
emulsion compared to a TAG oil with a similar fatty acid
composition. In studies using Japanese men and American men and
women, long-term DAG oil consumption promoted weight loss and body
fat reduction. One study showed that substitution of DAG oil for
ordinary cooking oil reduces the incidence of obesity and other
risk factors.
SUMMARY OF THE INVENTION
[0014] The invention provides isolated or recombinant nucleic acids
comprising a nucleic acid sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to an exemplary nucleic acid of the
invention, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID
NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ
ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,
SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID
NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109,
SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID
NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127,
SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID
NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,
SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID
NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ ID NO:161, SEQ ID NO:163,
SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 or SEQ
ID NO:173, over a region of at least about 10, 15, 20, 25, 30, 35,
40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,
1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300,
2350, 2400, 2450, 2500, or more residues, and in one aspect the
nucleic acid encodes at least one polypeptide having a
phospholipase (PL) activity, e.g., a phospholipase A, C or D
activity, or any combination of phospholipase activity, for
example, a PL A, PL C and/or PL D activity--as a multifunctional
activity. In one aspect, the sequence identities are determined by
analysis with a sequence comparison algorithm or by a visual
inspection.
[0015] The invention provides isolated or recombinant nucleic acids
comprising a nucleic acid sequence having at least 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99%, or more, or complete (100%) sequence identity to
SEQ ID NO:1 over a region of at least about 10, 15, 20, 25, 30, 35,
40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850 more consecutive residues, and in one
aspect the nucleic acid encodes at least one polypeptide having a
phospholipase (PL) activity, e.g., a phospholipase A, C or D
activity, or any combination of phospholipase activity, for
example, a PL A, PL C and/or PL D activity--as a multifunctional
activity. In one aspect, the sequence identities are determined by
analysis with a sequence comparison algorithm or by a visual
inspection.
[0016] The invention provides isolated or recombinant nucleic acids
comprising a nucleic acid sequence having at least 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity to SEQ ID NO:3 over a region of at least about 10, 15, 20,
25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850 or more residues, and in one
aspect the nucleic acid encodes at least one polypeptide having a
phospholipase (PL) activity, e.g., a phospholipase A, C or D
activity, or any combination of phospholipase activity, for
example, a PL A, PL C and/or PL D activity--as a multifunctional
activity. In one aspect, the sequence identities are determined by
analysis with a sequence comparison algorithm or by a visual
inspection.
[0017] The invention provides isolated or recombinant nucleic acids
comprising a nucleic acid sequence having at least 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity to SEQ ID NO:5 over a region of at least about 10, 15, 20,
25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850 or more residues, and in one
aspect the nucleic acid encodes at least one polypeptide having a
phospholipase (PL) activity, e.g., a phospholipase A, C or D
activity, or any combination of phospholipase activity, for
example, a PL A, PL C and/or PL D activity--as a multifunctional
activity. In one aspect, the sequence identities are determined by
analysis with a sequence comparison algorithm or by a visual
inspection.
[0018] The invention provides isolated or recombinant nucleic acids
comprising a nucleic acid sequence having at least 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)
sequence identity to SEQ ID NO:7 over a region of at least about
10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300,
350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more
residues, and in one aspect the nucleic acid encodes at least one
polypeptide having a phospholipase (PL) activity, e.g., a
phospholipase A, C or D activity, or any combination of
phospholipase activity, for example, a PL A, PL C and/or PL D
activity--as a multifunctional activity. In one aspect, the
sequence identities are determined by analysis with a sequence
comparison algorithm or by a visual inspection.
[0019] In alternative aspects, the isolated or recombinant nucleic
acid encodes a polypeptide comprising a sequence as set forth in
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ
ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,
SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID
NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ
ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66,
SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ
ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,
SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112,
SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID
NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,
SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID
NO:140; SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID
NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158,
SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID
NO:168, SEQ ID NO:170, SEQ ID NO:172, or SEQ ID NO:174. In one
aspect these polypeptides have a phospholipase, e.g., a
phospholipase A, B, C or D activity, or any combination of
phospholipase activity, for example, a PL A, PL C and/or PL D
activity--as a multifunctional activity.
[0020] In one aspect, the sequence comparison algorithm is a BLAST
algorithm, such as a BLAST version 2.2.2 algorithm. In one aspect,
the filtering setting is set to blastall-p blastp-d "nr pataa"-F F
and all other options are set to default.
[0021] In one aspect, the phospholipase activity comprises
catalyzing hydrolysis of a glycerolphosphate ester linkage (i.e.,
cleavage of glycerolphosphate ester linkages). The phospholipase
activity can comprise catalyzing hydrolysis of an ester linkage in
a phospholipid in a vegetable oil. The vegetable oil phospholipid
can comprise an oilseed phospholipid. The phospholipase activity
can comprise a phospholipase C (PLC) activity; a phospholipase A
(PLA) activity, such as a phospholipase A1 or phospholipase A2
activity; a phospholipase D (PLD) activity, such as a phospholipase
D1 or a phospholipase D2 activity; a phospholipase B (PLB)
activity, e.g., a phospholipase and a lysophospholipase (LPL)
activity or a phospholipase and a lysophospholipase-transacylase
(LPTA) activity or a phospholipase and a lysophospholipase (LPL)
activity and lysophospholipase-transacylase (LPTA) activity; or
patatin activity, or a combination thereof. The phospholipase
activity can comprise hydrolysis of a glycoprotein, e.g., as a
glycoprotein found in a potato tuber. The phospholipase activity
can comprise a patatin enzymatic activity. The phospholipase
activity can comprise a lipid acyl hydrolase (LAH) activity. In one
aspect, a phospholipase of the invention can have multifunctional
activity, e.g., a combination of one or more of the enzyme
activities described herein, for example, a phospholipase of the
invention can have PLC and PLA activity; PLB and PLA activity; PLC
and PLD activity; PLC and PLB activity; PLB and patatin activity;
PLC and patatin activity; PLD and PLA; PLD, PLA, PLB and PLC
activity; or PLD, PLA, PLB, PLC and patatin activity; or, a
phospholipase and a lysophospholipase (LPL) activity or a
phospholipase and a lysophospholipase-transacylase (LPTA) activity
or a phospholipase and a lysophospholipase (LPL) activity and
lysophospholipase-transacylase (LPTA) activity, or any combination
thereof.
[0022] For example, in one aspect, a polypeptide of the invention
is enzymatically active, but lacks a lipase activity, e.g., lacks
any enzymatic activity that affects a neutral oil (triglyceride)
fraction. It may be desirable to use such a polypeptide in a
particular process, e.g., in a degumming process where it is
important that the neutral oil fraction not be harmed (diminished,
e.g., hydrolyzed). Thus, in one aspect, the invention provides a
degumming process comprising use of a polypeptide of the invention
having a phospholipase activity, but not a lipase activity.
[0023] In one aspect, the isolated or recombinant nucleic acid
encodes a polypeptide having a phospholipase activity which is
thermostable. The polypeptide can retain a phospholipase activity
under conditions comprising a temperature range of between about
20.degree. C. to about 30.degree. C., between about 25.degree. C.
to about 40.degree. C., between about 37.degree. C. to about
95.degree. C.; between about 55.degree. C. to about 85.degree. C.,
between about 70.degree. C. to about 95.degree. C., or, between
about 90.degree. C. to about 95.degree. C. In another aspect, the
isolated or recombinant nucleic acid encodes a polypeptide having a
phospholipase activity which is thermotolerant. The polypeptide can
retain a phospholipase activity after exposure to a temperature in
the range from greater than 37.degree. C. to about 95.degree. C. or
anywhere in the range from greater than 55.degree. C. to about
85.degree. C. In one aspect, the polypeptide retains a
phospholipase activity after exposure to a temperature in the range
from greater than 90.degree. C. to about 95.degree. C. at pH
4.5.
[0024] The polypeptide can retain a phospholipase activity under
conditions comprising about pH 8, pH 7.5, pH 7, pH 6.5, pH 6.0, pH
5.5, pH 5, or pH 4.5. The polypeptide can retain a phospholipase
activity under conditions comprising a temperature range of between
about 40.degree. C. to about 70.degree. C.
[0025] In one aspect, the isolated or recombinant nucleic acid
comprises a sequence that hybridizes under stringent conditions to
a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ
ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71,
SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID
NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ
ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99,
SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID
NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,
SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID
NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135,
SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID
NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,
SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID
NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171
or SEQ ID NO:173, wherein the nucleic acid encodes a polypeptide
having a phospholipase activity. The nucleic acid can at least
about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,
350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or residues
in length or the full length of the gene or transcript, with or
without a signal sequence, as described herein. The stringent
conditions can be highly stringent, moderately stringent or of low
stringency, as described herein. The stringent conditions can
include a wash step, e.g., a wash step comprising a wash in
0.2.times.SSC at a temperature of about 65.degree. C. for about 15
minutes.
[0026] The invention provides a nucleic acid probe for identifying
a nucleic acid encoding a polypeptide with a phospholipase, e.g., a
phospholipase, activity, wherein the probe comprises at least 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, or more, consecutive
bases of a sequence of the invention, e.g., a sequence as set forth
in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, and the
probe identifies the nucleic acid by binding or hybridization. The
probe can comprise an oligonucleotide comprising at least about 10
to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60
to 100 consecutive bases of a sequence as set forth in SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7.
[0027] The invention provides a nucleic acid probe for identifying
a nucleic acid encoding a polypeptide with a phospholipase, e.g., a
phospholipase activity, wherein the probe comprises a nucleic acid
of the invention, e.g., a nucleic acid having at least 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5
and/or SEQ ID NO:7, or a subsequence thereof, over a region of at
least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more
consecutive residues; and, in one aspect, the sequence identities
are determined by analysis with a sequence comparison algorithm or
by visual inspection.
[0028] The invention provides an amplification primer sequence pair
for amplifying a nucleic acid encoding a polypeptide having a
phospholipase activity, wherein the primer pair is capable of
amplifying a nucleic acid comprising a sequence of the invention,
or fragments or subsequences thereof. One or each member of the
amplification primer sequence pair can comprise an oligonucleotide
comprising at least about 10 to 50 consecutive bases of the
sequence, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, or 25 or more consecutive bases of the sequence.
[0029] The invention provides amplification primer pairs, wherein
the primer pair comprises a first member having a sequence as set
forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 or more residues of a nucleic acid of the
invention, and a second member having a sequence as set forth by
about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, or 25 or more residues of the complementary strand of
the first member.
[0030] The invention provides phospholipases generated by
amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. The invention provides
methods of making a phospholipase by amplification, e.g.,
polymerase chain reaction (PCR), using an amplification primer pair
of the invention. In one aspect, the amplification primer pair
amplifies a nucleic acid from a library, e.g., a gene library, such
as an environmental library.
[0031] The invention provides methods of amplifying a nucleic acid
encoding a polypeptide having a phospholipase activity comprising
amplification of a template nucleic acid with an amplification
primer sequence pair capable of amplifying a nucleic acid sequence
of the invention, or fragments or subsequences thereof. The
amplification primer pair can be an amplification primer pair of
the invention.
[0032] The invention provides expression cassettes comprising a
nucleic acid of the invention or a subsequence thereof. In one
aspect, the expression cassette can comprise the nucleic acid that
is operably linked to a promoter. The promoter can be a viral,
bacterial, mammalian or plant promoter. In one aspect, the plant
promoter can be a potato, rice, corn, wheat, tobacco or barley
promoter. The promoter can be a constitutive promoter. The
constitutive promoter can comprise CaMV35S. In another aspect, the
promoter can be an inducible promoter. In one aspect, the promoter
can be a tissue-specific promoter or an environmentally regulated
or a developmentally regulated promoter. Thus, the promoter can be,
e.g., a seed-specific, a leaf-specific, a root-specific, a
stem-specific or an abscission-induced promoter. In one aspect, the
expression cassette can further comprise a plant or plant virus
expression vector.
[0033] The invention provides cloning vehicles comprising an
expression cassette (e.g., a vector) of the invention or a nucleic
acid of the invention. The cloning vehicle can be a viral vector, a
plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage
or an artificial chromosome. The viral vector can comprise an
adenovirus vector, a retroviral vector or an adeno-associated viral
vector. The cloning vehicle can comprise a bacterial artificial
chromosome (BAC), a plasmid, a bacteriophage P1-derived vector
(PAC), a yeast artificial chromosome (YAC), or a mammalian
artificial chromosome (MAC).
[0034] The invention provides transformed cell comprising a nucleic
acid of the invention or an expression cassette (e.g., a vector) of
the invention, or a cloning vehicle of the invention. In one
aspect, the transformed cell can be a bacterial cell, a mammalian
cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
In one aspect, the plant cell can be a potato, wheat, rice, corn,
tobacco or barley cell.
[0035] The invention provides transgenic non-human animals
comprising a nucleic acid of the invention or an expression
cassette (e.g., a vector) of the invention. In one aspect, the
animal is a mouse, a rat, a cow, a sheep or another mammal.
[0036] The invention provides transgenic plants comprising a
nucleic acid of the invention or an expression cassette (e.g., a
vector) of the invention. The transgenic plant can be a corn plant,
a potato plant, a tomato plant, a wheat plant, an oilseed plant, a
rapeseed plant, a soybean plant, a rice plant, a barley plant or a
tobacco plant. The invention provides transgenic seeds comprising a
nucleic acid of the invention or an expression cassette (e.g., a
vector) of the invention. The transgenic seed can be a corn seed, a
wheat kernel, an oilseed, a rapeseed (a canola plant), a soybean
seed, a palm kernel, a sunflower seed, a sesame seed, a peanut,
rice or a tobacco plant seed.
[0037] The invention provides an antisense oligonucleotide
comprising a nucleic acid sequence complementary to or capable of
hybridizing under stringent conditions to a nucleic acid of the
invention. The invention provides methods of inhibiting the
translation of a phospholipase message in a cell comprising
administering to the cell or expressing in the cell an antisense
oligonucleotide comprising a nucleic acid sequence complementary to
or capable of hybridizing under stringent conditions to a nucleic
acid of the invention.
[0038] The invention provides an antisense oligonucleotide
comprising a nucleic acid sequence complementary to or capable of
hybridizing under stringent conditions to a nucleic acid of the
invention. The invention provides methods of inhibiting the
translation of a phospholipase message in a cell comprising
administering to the cell or expressing in the cell an antisense
oligonucleotide comprising a nucleic acid sequence complementary to
or capable of hybridizing under stringent conditions to a nucleic
acid of the invention. The antisense oligonucleotide can be between
about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80,
about 60 to 100, about 70 to 110, or about 80 to 120 bases in
length.
[0039] The invention provides methods of inhibiting the translation
of a phospholipase, e.g., a phospholipase, message in a cell
comprising administering to the cell or expressing in the cell an
antisense oligonucleotide comprising a nucleic acid sequence
complementary to or capable of hybridizing under stringent
conditions to a nucleic acid of the invention. The invention
provides double-stranded inhibitory RNA (RNAi) molecules comprising
a subsequence of a sequence of the invention. In one aspect, the
RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more
duplex nucleotides in length. The invention provides methods of
inhibiting the expression of a phospholipase, e.g., a
phospholipase, in a cell comprising administering to the cell or
expressing in the cell a double-stranded inhibitory RNA (iRNA),
wherein the RNA comprises a subsequence of a sequence of the
invention.
[0040] The invention provides an isolated or recombinant
polypeptide comprising an amino acid sequence having at least about
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more,
or complete (100%) sequence identity to an exemplary polypeptide or
peptide of the invention (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ
ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90,
SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID
NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108
SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID
NO:118, SEQ ID NO:120 or SEQ ID NO:122, SEQ ID NO:124, SEQ ID
NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134,
SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID
NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID
NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162,
SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID
NO:172, or SEQ ID NO:174) over a region of at least about 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125,
150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 450, 500, 550 or
600 or more residues, or over the full length of the polypeptide;
and, in one aspect, the sequence identities are determined by
analysis with a sequence comparison algorithm or by a visual
inspection.
[0041] In one aspect, the invention provides an isolated or
recombinant polypeptide comprising an amino acid sequence having at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)
sequence identity to SEQ ID NO:2. In one aspect, the invention
provides an isolated or recombinant polypeptide comprising an amino
acid sequence having at least about 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ
ID NO:4. In one aspect, the invention provides an isolated or
recombinant polypeptide comprising an amino acid sequence having at
least about 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or
complete (100%) sequence identity to SEQ ID NO:6. In one aspect,
the invention provides an isolated or recombinant polypeptide
comprising an amino acid sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to SEQ ID NO:8.
[0042] The invention provides isolated or recombinant polypeptides
encoded by a nucleic acid of the invention. In alternative aspects,
the polypeptide can have a sequence as set forth in SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40,
SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ
ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,
SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID
NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ
ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96,
SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114,
SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID
NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132,
SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQ ID
NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160,
SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID
NO:170, SEQ ID NO:172, or SEQ ID NO:174. The polypeptide can have a
phospholipase activity, e.g., a phospholipase A, B, C or D
activity, or any combination of phospholipase activity, for
example, a PL A, PL C and/or PL D activity--as a multifunctional
activity. For example, in one aspect, a polypeptide of the
invention is enzymatically active, but lacks a lipase activity,
e.g., lacks any enzymatic activity that affects a neutral oil
(triglyceride) fraction. In one aspect, the invention provides a
degumming process comprising use of a polypeptide of the invention
having a phospholipase activity, but not a lipase activity, such
that in the degumming process any neutral oil fraction is not
harmed (diminished, altered, degraded, e.g., hydrolyzed).
[0043] The invention provides isolated or recombinant polypeptides
comprising a polypeptide of the invention lacking a signal
sequence. In one aspect, the polypeptide lacking a signal sequence
has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to
residues 30 to 287 of SEQ ID NO:2, an amino acid sequence having at
least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence
identity to residues 25 to 283 of SEQ ID NO:4, an amino acid
sequence having at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or more sequence identity to residues 26 to 280 of SEQ ID
NO:6, or, an amino acid sequence having at least 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity
to residues 40 to 330 of SEQ ID NO:8. The sequence identities can
be determined by analysis with a sequence comparison algorithm or
by visual inspection.
[0044] Another aspect of the invention provides an isolated or
recombinant polypeptide or peptide including at least 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100
or more consecutive bases of a polypeptide or peptide sequence of
the invention, sequences substantially identical thereto, and the
sequences complementary thereto. The peptide can be, e.g., an
immunogenic fragment, a motif (e.g., a binding site) or an active
site.
[0045] In one aspect, the isolated or recombinant polypeptide of
the invention (with or without a signal sequence) has a
phospholipase activity. In one aspect, the phospholipase activity
comprises catalyzing hydrolysis of a glycerolphosphate ester
linkage (i.e., cleavage of glycerolphosphate ester linkages). The
phospholipase activity can comprise catalyzing hydrolysis of an
ester linkage in a phospholipid in a vegetable oil. The vegetable
oil phospholipid can comprise an oilseed phospholipid. The
phospholipase activity can comprise a phospholipase C (PLC)
activity; a phospholipase A (PLA) activity, such as a phospholipase
A1 or phospholipase A2 activity; a phospholipase D (PLD) activity,
such as a phospholipase D1 or a phospholipase D2 activity; a
phospholipase B (PLB) activity, e.g., a phospholipase and a
lysophospholipase (LPL) activity or a phospholipase and a
lysophospholipase-transacylase (LPTA) activity or a phospholipase
and a lysophospholipase (LPL) activity and
lysophospholipase-transacylase (LPTA) activity; or patatin
activity, or a combination thereof. For example, in one aspect a
phospholipase comprises a combination of one or more of the enzyme
activities described herein, for example, an phospholipase can have
PLC and PLA activity; PLB and PLA activity; PLC and PLD activity;
PLC and PLB activity; PLB and patatin activity; PLC and patatin
activity; PLD and PLA; PLD, PLA, PLB and PLC activity; or PLD, PLA,
PLB, PLC and patatin activity; or, a phospholipase and a
lysophospholipase (LPL) activity or a phospholipase and a
lysophospholipase-transacylase (LPTA) activity or a phospholipase
and a lysophospholipase (LPL) activity and
lysophospholipase-transacylase (LPTA) activity, or any combination
thereof.
[0046] The phospholipase activity can comprise hydrolysis of a
glycoprotein, e.g., as a glycoprotein found in a potato tuber. The
phospholipase activity can comprise a patatin enzymatic activity.
The phospholipase activity can comprise a lipid acyl hydrolase
(LAH) activity.
[0047] In one aspect, the phospholipase activity is thermostable.
The polypeptide can retain a phospholipase activity under
conditions comprising a temperature range of between about 20 to
about 30.degree. C., between about 25.degree. C. to about
40.degree. C., between about 37.degree. C. to about 95.degree. C.,
between about 55.degree. C. to about 85.degree. C., between about
70.degree. C. to about 95.degree. C., or between about 90.degree.
C. to about 95.degree. C. In another aspect, the phospholipase
activity can be thermotolerant. The polypeptide can retain a
phospholipase activity after exposure to a temperature in the range
from greater than 37.degree. C. to about 95.degree. C., or in the
range from greater than 55.degree. C. to about 85.degree. C. In one
aspect, the polypeptide can retain a phospholipase activity after
exposure to a temperature in the range from greater than 90.degree.
C. to about 95.degree. C. at pH 4.5.
[0048] In one aspect, the polypeptide can retain a phospholipase
activity under conditions comprising about pH 6.5, pH 6, pH 5.5, pH
5, pH 4.5 or pH 4 or less (more acidic). In one aspect, the
polypeptide can retain a phospholipase activity under conditions
comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10,
pH 10.5 or pH 11 or more (more basic).
[0049] In one aspect, the isolated or recombinant polypeptide can
comprise the polypeptide of the invention that lacks a signal
sequence. In one aspect, the isolated or recombinant polypeptide
can comprise the polypeptide of the invention comprising a
heterologous signal sequence, such as a heterologous phospholipase
or non-phospholipase signal sequence.
[0050] The invention provides isolated or recombinant peptides
comprising an amino acid sequence having at least 95%, 96%, 97%,
98%, 99%, or more sequence identity to residues 1 to 29 of SEQ ID
NO:2, at least 95%, 96%, 97%, 98%, 99%, or more sequence identity
to residues 1 to 24 of SEQ ID NO:4, at least 95%, 96%, 97%, 98%,
99%, or more sequence identity to residues 1 to 25 of SEQ ID NO:6,
or at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to
residues 1 to 39 of SEQ ID NO:8, and to other signal sequences as
set forth in the SEQ ID listing, wherein the sequence identities
are determined by analysis with a sequence comparison algorithm or
by visual inspection. These peptides can act as signal sequences on
its endogenous phospholipase, on another phospholipase, or a
heterologous protein (a non-phospholipase enzyme or other protein).
In one aspect, the invention provides chimeric proteins comprising
a first domain comprising a signal sequence of the invention and at
least a second domain. The protein can be a fusion protein. The
second domain can comprise an enzyme. The enzyme can be a
phospholipase.
[0051] The invention provides chimeric polypeptides comprising at
least a first domain comprising signal peptide (SP) of the
invention or a catalytic domain (CD), or active site, of a
phospholipase of the invention and at least a second domain
comprising a heterologous polypeptide or peptide, wherein the
heterologous polypeptide or peptide is not naturally associated
with the signal peptide (SP) or catalytic domain (CD). In one
aspect, the heterologous polypeptide or peptide is not a
phospholipase. The heterologous polypeptide or peptide can be amino
terminal to, carboxy terminal to or on both ends of the signal
peptide (SP) or catalytic domain (CD).
[0052] The invention provides isolated or recombinant nucleic acids
encoding a chimeric polypeptide, wherein the chimeric polypeptide
comprises at least a first domain comprising signal peptide (SP) or
a catalytic domain (CD), or active site, of a polypeptide of the
invention, and at least a second domain comprising a heterologous
polypeptide or peptide, wherein the heterologous polypeptide or
peptide is not naturally associated with the signal peptide (SP) or
catalytic domain (CD).
[0053] In one aspect, the phospholipase activity comprises a
specific activity at about 37.degree. C. in the range from about 10
units per milligram to about 100 units per milligram of protein. In
another aspect, the phospholipase activity comprises a specific
activity from about 100 units per milligram to about 1000 units per
milligram, from about 500 units per milligram to about 750 units
per milligram of protein. Alternatively, the phospholipase activity
comprises a specific activity at 37.degree. C. in the range from
about 100 to about 500 units per milligram of protein. In one
aspect, the phospholipase activity comprises a specific activity at
37.degree. C. in the range from about 500 to about 1200 units per
milligram of protein. In another aspect, the phospholipase activity
comprises a specific activity at 37.degree. C. in the range from
about 750 to about 1000 units per milligram of protein. In another
aspect, the thermotolerance comprises retention of at least half of
the specific activity of the phospholipase at 37.degree. C. after
being heated to the elevated temperature. Alternatively, the
thermotolerance can comprise retention of specific activity at
37.degree. C. in the range from about 500 to about 1200 units per
milligram of protein after being heated to the elevated
temperature.
[0054] The invention provides an isolated or recombinant
polypeptide of the invention, wherein the polypeptide comprises at
least one glycosylation site. In one aspect, glycosylation can be
an N-linked glycosylation. In one aspect, the polypeptide can be
glycosylated after being expressed in a P. pastoris or a S.
pombe.
[0055] The invention provides phospholipase enzymes, and the
nucleic acids that encode them, having a sequence of any of the
exemplary phospholipases of the invention with one or more or all
of the glycosylation sites altered, as described above. Thus, the
invention provides methods of making variant phospholipase coding
sequences having increased expression in a host cell, where the
method comprises modifying a phospholipase coding sequence of the
invention such that one, several or all N-linked glycosylation site
coding motifs are modified to a non-glycosylated motif. The
invention also provides phospholipase coding sequence made by this
process, and the enzymes they encode.
[0056] The invention provides methods for making a variant
phospholipase coding sequence encoding a phospholipase having
increased resistance to a protease comprising modifying an amino
acid equivalent to position 131 of SEQ ID NO:2 to one, several or
all of the following residues: Lysine (K); Serine (S); Glycine (G);
Arginine (R); Glutamine (Q); Alanine (A); Isoleucine (I); Histidine
(H); Phenylalanine (F); Threonine (T); Methionine (M) Leucine (L),
including variants to SEQ ID NO:2 (and the nucleic acid that encode
them) having these exemplary modifications. The invention also
provides isolated, synthetic or recombinant phospholipases encoded
by a sequence made by this method.
[0057] The invention provides methods for making a variant
phospholipase coding sequence encoding a phospholipase having
decreased resistance to a protease comprising modifying an amino
acid equivalent to position 131 of SEQ ID NO:2 to one, several or
all of the following residues: Tryptophan (W); Glutamate (E);
Tyrosine (Y), including variants to SEQ ID NO:2 (and the nucleic
acid that encode them) having these exemplary modifications. The
invention also provides isolated, synthetic or recombinant
phospholipases encoded by a sequence made by this method.
[0058] The invention provides protein preparations comprising a
polypeptide of the invention, wherein the protein preparation
comprises a liquid, a solid or a gel.
[0059] The invention provides heterodimers comprising a polypeptide
of the invention and a second protein or domain. The second member
of the heterodimer can be a different phospholipase, a different
enzyme or another protein. In one aspect, the second domain can be
a polypeptide and the heterodimer can be a fusion protein. In one
aspect, the second domain can be an epitope or a tag. In one
aspect, the invention provides homodimers comprising a polypeptide
of the invention.
[0060] The invention provides immobilized polypeptides having a
phospholipase activity, wherein the polypeptide comprises a
polypeptide of the invention, a polypeptide encoded by a nucleic
acid of the invention, or a polypeptide comprising a polypeptide of
the invention and a second domain (e.g., a fusion protein). In one
aspect, a polypeptide of the invention is immobilized on a cell, a
vesicle, a liposome, a film, a membrane, a metal, a resin, a
polymer, a ceramic, a glass, a microelectrode, a graphitic
particle, a bead, a gel, a plate, crystals, a tablet, a pill, a
capsule, a powder, an agglomerate, a surface, a porous structure,
an array or a capillary tube. In one aspect, a polypeptide of the
invention is immobilized on materials such as grains, husks, bark,
skin, hair, enamel, bone, shell and materials deriving from them,
or animal feed materials, or a combination thereof.
[0061] Polypeptides of the invention (e.g., phospholipases) can be
also present alone or as mixture of phospholipases or
phospholipases and other hydrolytic enzymes such as cellulases,
xylanases, proteases, lipases, amylases, or redox enzymes such as
laccases, peroxidases, catalases, oxidases, or reductases. They can
be formulated in a solid form such as a powder, lyophilized
preparations, granules, tablets, bars, crystals, capsules, pills,
pellets, or in a liquid form such as an aqueous solution, an
aerosol, a gel, a paste, a slurry, an aqueous/oil emulsion, a
cream, a capsule, vesicular, or micellar suspension. In one aspect,
these formulations of the invention can comprise any or a
combination of the following ingredients: polyols such as
polyethylene glycols, polyvinylalcohols, glycerol, sugars such as
sucrose, sorbitol, trehalose, glucose, fructose, maltose, gelling
agents such as guar gums, carageenans, alginates, dextrans,
cellulosic derivatives, pectins, salts such as sodium chloride,
sodium sulfate, ammonium sulfate, calcium chloride, magnesium
chloride, zinc chloride, zinc sulfate, salts of fatty acids and
their derivatives, metal chelators such as EDTA, EGTA, sodium
citrate, antimicrobial agents such as fatty acids, derivatives
thereof, parabens, sorbates, benzoates, additionally compounds to
block the impact of proteases such as bulk proteins such as BSA,
wheat hydrolysates, borate compounds, emulsifiers such as non-ionic
and ionic detergents may used alone or in combination,
phytosterols, vitamins, amino acids, reducing agents, such as
cysteine or antioxidant compounds such as ascorbic acid may be
included as well dispersants.
[0062] In one aspect, cross-linking and protein modification such
as pegylation, fatty acid modification and glycosylation are used
to improve the stability of a polypeptide of the invention (e.g.,
enzyme stability). In one aspect, the polyols and/or sugars
comprise from about 5% to about 60%, or more, of the formulation,
from about 10% to about 50% of the formulation, from about 20% to
about 40% of the formulation, or from about 5% to about 20% of the
formulation. In another aspect, the gelling agents comprise from
about 0.5% to about 10% of the formulation, from about 1% to about
8% of the formulation, from about 2% to about 5% of the
formulation, or from about 0.5% to about 3% of the formulation. In
another aspect, the salts such as sodium chloride, sodium sulfate,
ammonium sulfate, calcium chloride and/or magnesium chloride
comprise from about 1% to about 30% of the formulation, from about
2% to about 20% of the formulation, from about 5% to about 15% of
the formulation, or from about 1% to about 10% of the formulation.
In another aspect, zinc chloride is present in the formulation at
concentrations comprising from about 0.1 mM to about 20 mM, from
about 0.5 mM to about 10 mM, from about 1 mM to about 5 mM, or from
about 0.1 mM to about 5 mM). In yet another aspect, zinc sulfate is
present in the formulation at concentrations comprising from about
0.1 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about
1 mM to about 5 mM, or from about 0.1 mM to about 5 mM). In another
aspect, salts of fatty acids and/or their derivatives comprise from
about 5% to about 40% of the formulation, from about 10% to about
30% of the formulation, from about 15% to about 25% of the
formulation, or from about 5% to about 20% of the formulation. In
another aspect, metal chelators such as EDTA, EGTA, and/or sodium
citrate are present in the formulation at concentrations comprising
from 0.1 mM to about 10 mM), from about 0.5 mM to about 8 mM, from
about 1 mM to about 5 mM, or from about 0.1 mM to about 1 mM. In
another aspect, antimicrobials such as parabens, sorbates, and/or
benzoates comprise from about 0.01% to about 10% of the
formulation, from about 0.05% to about 5% of the formulation, from
about 0.1% to about 1% of the formulation, or from about 0.05% to
about 0.5% of the formulation. In yet another aspect, bulk proteins
such as BSA and/or wheat hydrolysates comprise from about 1% to
about 20% of the formulation, from about 5% to about 15% of the
formulation, from about 2.5% to about 7.5% of the formulation, or
from about 1% to about 5% of the formulation. In another aspect,
emulsifiers such as non-ionic and/or ionic detergents are present
in the formulation at concentrations comprising from about 1.times.
critical micelle concentration (CMC) to about 10.times.CMC, from
about 2.5.times.CMC to about 7.5.times.CMC, from about 1.times.CMC
to about 5.times.CMC, or from about 3.times.CMC to about
6.times.CMC. In another aspect, vitamins, amino acids, reducing
agents and/or antioxidant compounds comprise from about 0.1% to
about 5% of the formulation, from about 0.5% to about 4% of the
formulation, from about 1% to about 2.5% of the formulation, or
from about 0.1% to about 1% of the formulation.
[0063] The invention provides arrays comprising an immobilized
polypeptide, wherein the polypeptide is a phospholipase of the
invention or is a polypeptide encoded by a nucleic acid of the
invention. The invention provides arrays comprising an immobilized
nucleic acid of the invention. The invention provides an array
comprising an immobilized antibody of the invention.
[0064] The invention provides isolated or recombinant antibodies
that specifically bind to a polypeptide of the invention or to a
polypeptide encoded by a nucleic acid of the invention. The
antibody can be a monoclonal or a polyclonal antibody. The
invention provides hybridomas comprising an antibody of the
invention.
[0065] The invention provides methods of isolating or identifying a
polypeptide with a phospholipase activity comprising the steps of:
(a) providing an antibody of the invention; (b) providing a sample
comprising polypeptides; and, (c) contacting the sample of step (b)
with the antibody of step (a) under conditions wherein the antibody
can specifically bind to the polypeptide, thereby isolating or
identifying a phospholipase. The invention provides methods of
making an anti-phospholipase antibody comprising administering to a
non-human animal a nucleic acid of the invention, or a polypeptide
of the invention, in an amount sufficient to generate a humoral
immune response, thereby making an anti-phospholipase antibody.
[0066] The invention provides methods of producing a recombinant
polypeptide comprising the steps of: (a) providing a nucleic acid
of the invention operably linked to a promoter; and, (b) expressing
the nucleic acid of step (a) under conditions that allow expression
of the polypeptide, thereby producing a recombinant polypeptide.
The nucleic acid can comprise a sequence having at least 85%
sequence identity to SEQ ID NO:1 over a region of at least about
100 residues, having at least 80% sequence identity to SEQ ID NO:3
over a region of at least about 100 residues, having at least 80%
sequence identity to SEQ ID NO:5 over a region of at least about
100 residues, or having at least 70% sequence identity to SEQ ID
NO:7 over a region of at least about 100 residues, wherein the
sequence identities are determined by analysis with a sequence
comparison algorithm or by visual inspection. The nucleic acid can
comprise a nucleic acid that hybridizes under stringent conditions
to a nucleic acid as set forth in SEQ ID NO:1, or a subsequence
thereof; a sequence as set forth in SEQ ID NO:3, or a subsequence
thereof; a sequence as set forth in SEQ ID NO:5, or a subsequence
thereof; or, a sequence as set forth in SEQ ID NO:7, or a
subsequence thereof. The method can further comprise transforming a
host cell with the nucleic acid of step (a) followed by expressing
the nucleic acid of step (a), thereby producing a recombinant
polypeptide in a transformed cell. The method can further comprise
inserting into a host non-human animal the nucleic acid of step (a)
followed by expressing the nucleic acid of step (a), thereby
producing a recombinant polypeptide in the host non-human
animal.
[0067] The invention provides methods for identifying a polypeptide
having a phospholipase activity comprising the following steps: (a)
providing a polypeptide of the invention or a polypeptide encoded
by a nucleic acid of the invention, or a fragment or variant
thereof, (b) providing a phospholipase substrate; and, (c)
contacting the polypeptide or a fragment or variant thereof of step
(a) with the substrate of step (b) and detecting an increase in the
amount of substrate or a decrease in the amount of reaction
product, wherein a decrease in the amount of the substrate or an
increase in the amount of the reaction product detects a
polypeptide having a phospholipase activity. In alternative
aspects, the nucleic acid comprises a sequence having at least 85%
sequence identity to SEQ ID NO:1 over a region of at least about
100 residues, having at least 80% sequence identity to SEQ ID NO:3
over a region of at least about 100 residues, having at least 80%
sequence identity to SEQ ID NO:5 over a region of at least about
100 residues, or having at least 70% sequence identity to SEQ ID
NO:7 over a region of at least about 100 residues, wherein the
sequence identities are determined by analysis with a sequence
comparison algorithm or by visual inspection. In alternative
aspects the nucleic acid hybridizes under stringent conditions a
sequence as set forth in SEQ ID NO:1, or a subsequence thereof; a
sequence as set forth in SEQ ID NO:3, or a subsequence thereof; a
sequence as set forth in SEQ ID NO:5, or a subsequence thereof; or,
a sequence as set forth in SEQ ID NO:7, or a subsequence
thereof.
[0068] The invention provides methods for identifying a
phospholipase substrate comprising the following steps: (a)
providing a polypeptide of the invention or a polypeptide encoded
by a nucleic acid of the invention; (b) providing a test substrate;
and, (c) contacting the polypeptide of step (a) with the test
substrate of step (b) and detecting an increase in the amount of
substrate or a decrease in the amount of reaction product, wherein
a decrease in the amount of the substrate or an increase in the
amount of the reaction product identifies the test substrate as a
phospholipase substrate. In alternative aspects, the nucleic acid
can have at least 85% sequence identity to SEQ ID NO:1 over a
region of at least about 100 residues, at least 80% sequence
identity to SEQ ID NO:3 over a region of at least about 100
residues, at least 80% sequence identity to SEQ ID NO:5 over a
region of at least about 100 residues, or, at least 70% sequence
identity to SEQ ID NO:7 over a region of at least about 100
residues, wherein the sequence identities are determined by
analysis with a sequence comparison algorithm or by visual
inspection. In alternative aspects, the nucleic acid hybridizes
under stringent conditions to a sequence as set forth in SEQ ID
NO:1, or a subsequence thereof; a sequence as set forth in SEQ ID
NO:3, or a subsequence thereof; a sequence as set forth in SEQ ID
NO:5, or a subsequence thereof; or, a sequence as set forth in SEQ
ID NO:7, or a subsequence thereof.
[0069] The invention provides methods of determining whether a
compound specifically binds to a phospholipase comprising the
following steps: (a) expressing a nucleic acid or a vector
comprising the nucleic acid under conditions permissive for
translation of the nucleic acid to a polypeptide, wherein the
nucleic acid and vector comprise a nucleic acid or vector of the
invention; or, providing a polypeptide of the invention (b)
contacting the polypeptide with the test compound; and, (c)
determining whether the test compound specifically binds to the
polypeptide, thereby determining that the compound specifically
binds to the phospholipase. In alternative aspects, the nucleic
acid sequence has at least 85% sequence identity to SEQ ID NO:1
over a region of at least about 100 residues, at least 80% sequence
identity to SEQ ID NO:3 over a region of at least about 100
residues, least 80% sequence identity to SEQ ID NO:5 over a region
of at least about 100 residues, or, at least 70% sequence identity
to SEQ ID NO:7 over a region of at least about 100 residues,
wherein the sequence identities are determined by analysis with a
sequence comparison algorithm or by visual inspection. In
alternative aspects, the nucleic acid hybridizes under stringent
conditions to a sequence as set forth in SEQ ID NO:1, or a
subsequence thereof; a sequence as set forth in SEQ ID NO:3, or a
subsequence thereof; a sequence as set forth in SEQ ID NO:5, or a
subsequence thereof; or, a sequence as set forth in SEQ ID NO:7, or
a subsequence thereof.
[0070] The invention provides methods for identifying a modulator
of a phospholipase activity comprising the following steps: (a)
providing a polypeptide of the invention or a polypeptide encoded
by a nucleic acid of the invention; (b) providing a test compound;
(c) contacting the polypeptide of step (a) with the test compound
of step (b); and, measuring an activity of the phospholipase,
wherein a change in the phospholipase activity measured in the
presence of the test compound compared to the activity in the
absence of the test compound provides a determination that the test
compound modulates the phospholipase activity. In alternative
aspects, the nucleic acid can have at least 85% sequence identity
to SEQ ID NO:1 over a region of at least about 100 residues, at
least 80% sequence identity to SEQ ID NO:3 over a region of at
least about 100 residues, at least 80% sequence identity to SEQ ID
NO:5 over a region of at least about 100 residues, or, at least 70%
sequence identity to SEQ ID NO:7 over a region of at least about
100 residues, wherein the sequence identities are determined by
analysis with a sequence comparison algorithm or by visual
inspection. In alternative aspects, the nucleic acid can hybridize
under stringent conditions to a nucleic acid sequence selected from
the group consisting of a sequence as set forth in SEQ ID NO:1, or
a subsequence thereof; a sequence as set forth in SEQ ID NO:3, or a
subsequence thereof; a sequence as set forth in SEQ ID NO:5, or a
subsequence thereof; and, a sequence as set forth in SEQ ID NO:7,
or a subsequence thereof.
[0071] In one aspect, the phospholipase activity is measured by
providing a phospholipase substrate and detecting an increase in
the amount of the substrate or a decrease in the amount of a
reaction product. The decrease in the amount of the substrate or
the increase in the amount of the reaction product with the test
compound as compared to the amount of substrate or reaction product
without the test compound identifies the test compound as an
activator of phospholipase activity. The increase in the amount of
the substrate or the decrease in the amount of the reaction product
with the test compound as compared to the amount of substrate or
reaction product without the test compound identifies the test
compound as an inhibitor of phospholipase activity.
[0072] The invention provides computer systems comprising a
processor and a data storage device wherein said data storage
device has stored thereon a polypeptide sequence of the invention
or a nucleic acid sequence of the invention.
[0073] In one aspect, the computer system can further comprise a
sequence comparison algorithm and a data storage device having at
least one reference sequence stored thereon. The sequence
comparison algorithm can comprise a computer program that indicates
polymorphisms. The computer system can further comprising an
identifier that identifies one or more features in said
sequence.
[0074] The invention provides computer readable mediums having
stored thereon a sequence comprising a polypeptide sequence of the
invention or a nucleic acid sequence of the invention.
[0075] The invention provides methods for identifying a feature in
a sequence comprising the steps of: (a) reading the sequence using
a computer program which identifies one or more features in a
sequence, wherein the sequence comprises a polypeptide sequence of
the invention or a nucleic acid sequence of the invention; and, (b)
identifying one or more features in the sequence with the computer
program.
[0076] The invention provides methods for comparing a first
sequence to a second sequence comprising the steps of: (a) reading
the first sequence and the second sequence through use of a
computer program which compares sequences, wherein the first
sequence comprises a polypeptide sequence of the invention or a
nucleic acid sequence of the invention; and, (b) determining
differences between the first sequence and the second sequence with
the computer program. In one aspect, the step of determining
differences between the first sequence and the second sequence
further comprises the step of identifying polymorphisms. In one
aspect, the method further comprises an identifier (and use of the
identifier) that identifies one or more features in a sequence. In
one aspect, the method comprises reading the first sequence using a
computer program and identifying one or more features in the
sequence.
[0077] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide with a phospholipase activity
from an environmental sample comprising the steps of: (a) providing
an amplification primer sequence pair for amplifying a nucleic acid
encoding a polypeptide with a phospholipase activity, wherein the
primer pair is capable of amplifying a nucleic acid of the
invention (e.g., SEQ ID NO:1, or a subsequence thereof; SEQ ID
NO:3, or a subsequence thereof; SEQ ID NO:5, or a subsequence
thereof; or SEQ ID NO:7, or a subsequence thereof, etc.); (b)
isolating a nucleic acid from the environmental sample or treating
the environmental sample such that nucleic acid in the sample is
accessible for hybridization to the amplification primer pair; and,
(c) combining the nucleic acid of step (b) with the amplification
primer pair of step (a) and amplifying nucleic acid from the
environmental sample, thereby isolating or recovering a nucleic
acid encoding a polypeptide with a phospholipase activity from an
environmental sample. In one aspect, each member of the
amplification primer sequence pair comprises an oligonucleotide
comprising at least about 10 to 50 consecutive bases of a nucleic
acid sequence of the invention. In one aspect, the amplification
primer sequence pair is an amplification pair of the invention.
[0078] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide with a phospholipase activity
from an environmental sample comprising the steps of: (a) providing
a polynucleotide probe comprising a nucleic acid sequence of the
invention, or a subsequence thereof; (b) isolating a nucleic acid
from the environmental sample or treating the environmental sample
such that nucleic acid in the sample is accessible for
hybridization to a polynucleotide probe of step (a); (c) combining
the isolated nucleic acid or the treated environmental sample of
step (b) with the polynucleotide probe of step (a); and, (d)
isolating a nucleic acid that specifically hybridizes with the
polynucleotide probe of step (a), thereby isolating or recovering a
nucleic acid encoding a polypeptide with a phospholipase activity
from the environmental sample. In alternative aspects, the
environmental sample comprises a water sample, a liquid sample, a
soil sample, an air sample or a biological sample. In alternative
aspects, the biological sample is derived from a bacterial cell, a
protozoan cell, an insect cell, a yeast cell, a plant cell, a
fungal cell, an algal (algae) cell, a lichen, or a mammalian
cell.
[0079] The invention provides methods of generating a variant of a
nucleic acid encoding a phospholipase comprising the steps of: (a)
providing a template nucleic acid comprising a nucleic acid of the
invention; (b) modifying, deleting or adding one or more
nucleotides in the template sequence, or a combination thereof, to
generate a variant of the template nucleic acid.
[0080] In one aspect, the method further comprises expressing the
variant nucleic acid to generate a variant phospholipase
polypeptide. In alternative aspects, the modifications, additions
or deletions are introduced by error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.), synthetic ligation reassembly (SLR)
and/or a combination thereof. In alternative aspects, the
modifications, additions or deletions are introduced by a method
selected from the group consisting of recombination, recursive
sequence recombination, phosphothioate-modified DNA mutagenesis,
uracil-containing template mutagenesis, gapped duplex mutagenesis,
point mismatch repair mutagenesis, repair-deficient host strain
mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion
mutagenesis, restriction-selection mutagenesis,
restriction-purification mutagenesis, artificial gene synthesis,
ensemble mutagenesis, chimeric nucleic acid multimer creation
and/or a combination thereof.
[0081] In one aspect, the method is iteratively repeated until a
phospholipase having an altered or different activity or an altered
or different stability from that of a phospholipase encoded by the
template nucleic acid is produced. In one aspect, the altered or
different activity is a phospholipase activity under an acidic
condition, wherein the phospholipase encoded by the template
nucleic acid is not active under the acidic condition. In one
aspect, the altered or different activity is a phospholipase
activity under a high temperature, wherein the phospholipase
encoded by the template nucleic acid is not active under the high
temperature. In one aspect, the method is iteratively repeated
until a phospholipase coding sequence having an altered codon usage
from that of the template nucleic acid is produced. The method can
be iteratively repeated until a phospholipase gene having higher or
lower level of message expression or stability from that of the
template nucleic acid is produced.
[0082] The invention provides methods for modifying codons in a
nucleic acid encoding a phospholipase to increase its expression in
a host cell, the method comprising (a) providing a nucleic acid of
the invention encoding a phospholipase; and, (b) identifying a
non-preferred or a less preferred codon in the nucleic acid of step
(a) and replacing it with a preferred or neutrally used codon
encoding the same amino acid as the replaced codon, wherein a
preferred codon is a codon over-represented in coding sequences in
genes in the host cell and a non-preferred or less preferred codon
is a codon under-represented in coding sequences in genes in the
host cell, thereby modifying the nucleic acid to increase its
expression in a host cell.
[0083] The invention provides methods for modifying codons in a
nucleic acid encoding a phospholipase, the method comprising (a)
providing a nucleic acid of the invention encoding a phospholipase;
and, (b) identifying a codon in the nucleic acid of step (a) and
replacing it with a different codon encoding the same amino acid as
the replaced codon, thereby modifying codons in a nucleic acid
encoding a phospholipase.
[0084] The invention provides methods for modifying codons in a
nucleic acid encoding a phospholipase to increase its expression in
a host cell, the method comprising (a) providing a nucleic acid of
the invention encoding a phospholipase; and, (b) identifying a
non-preferred or a less preferred codon in the nucleic acid of step
(a) and replacing it with a preferred or neutrally used codon
encoding the same amino acid as the replaced codon, wherein a
preferred codon is a codon over-represented in coding sequences in
genes in the host cell and a non-preferred or less preferred codon
is a codon under-represented in coding sequences in genes in the
host cell, thereby modifying the nucleic acid to increase its
expression in a host cell.
[0085] The invention provides methods for modifying a codon in a
nucleic acid encoding a phospholipase to decrease its expression in
a host cell, the method comprising (a) providing a nucleic acid of
the invention encoding a phospholipase; and, (b) identifying at
least one preferred codon in the nucleic acid of step (a) and
replacing it with a non-preferred or less preferred codon encoding
the same amino acid as the replaced codon, wherein a preferred
codon is a codon over-represented in coding sequences in genes in a
host cell and a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell,
thereby modifying the nucleic acid to decrease its expression in a
host cell. In alternative aspects, the host cell is a bacterial
cell, a fungal cell, an insect cell, a yeast cell, a plant cell, an
algal (algae) cell, a lichen, or a mammalian cell.
[0086] The invention provides methods for producing a library of
nucleic acids encoding a plurality of modified phospholipase active
sites or substrate binding sites, wherein the modified active sites
or substrate binding sites are derived from a first nucleic acid
comprising a sequence encoding a first active site or a first
substrate binding site the method comprising: (a) providing a first
nucleic acid encoding a first active site or first substrate
binding site, wherein the first nucleic acid sequence comprises a
nucleic acid of the invention; (b) providing a set of mutagenic
oligonucleotides that encode naturally-occurring amino acid
variants at a plurality of targeted codons in the first nucleic
acid; and, (c) using the set of mutagenic oligonucleotides to
generate a set of active site-encoding or substrate binding
site-encoding variant nucleic acids encoding a range of amino acid
variations at each amino acid codon that was mutagenized, thereby
producing a library of nucleic acids encoding a plurality of
modified phospholipase active sites or substrate binding sites. In
alternative aspects, the method comprises mutagenizing the first
nucleic acid of step (a) by a method comprising an optimized
directed evolution system, Gene Site Saturation Mutagenesis.TM.
(GSSM.TM.), and synthetic ligation reassembly (SLR). The method can
further comprise mutagenizing the first nucleic acid of step (a) or
variants by a method comprising error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.), synthetic ligation reassembly (SLR) and
a combination thereof. The method can further comprise mutagenizing
the first nucleic acid of step (a) or variants by a method
comprising recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation and a combination
thereof.
[0087] The invention provides methods for making a small molecule
comprising the steps of: (a) providing a plurality of biosynthetic
enzymes capable of synthesizing or modifying a small molecule,
wherein one of the enzymes comprises a phospholipase enzyme encoded
by a nucleic acid of the invention; (b) providing a substrate for
at least one of the enzymes of step (a); and, (c) reacting the
substrate of step (b) with the enzymes under conditions that
facilitate a plurality of biocatalytic reactions to generate a
small molecule by a series of biocatalytic reactions.
[0088] The invention provides methods for modifying a small
molecule comprising the steps: (a) providing a phospholipase enzyme
encoded by a nucleic acid of the invention; (b) providing a small
molecule; and, (c) reacting the enzyme of step (a) with the small
molecule of step (b) under conditions that facilitate an enzymatic
reaction catalyzed by the phospholipase enzyme, thereby modifying a
small molecule by a phospholipase enzymatic reaction. In one
aspect, the method comprises providing a plurality of small
molecule substrates for the enzyme of step (a), thereby generating
a library of modified small molecules produced by at least one
enzymatic reaction catalyzed by the phospholipase enzyme. In one
aspect, the method further comprises a plurality of additional
enzymes under conditions that facilitate a plurality of
biocatalytic reactions by the enzymes to form a library of modified
small molecules produced by the plurality of enzymatic reactions.
In one aspect, the method further comprises the step of testing the
library to determine if a particular modified small molecule that
exhibits a desired activity is present within the library. The step
of testing the library can further comprises the steps of
systematically eliminating all but one of the biocatalytic
reactions used to produce a portion of the plurality of the
modified small molecules within the library by testing the portion
of the modified small molecule for the presence or absence of the
particular modified small molecule with a desired activity, and
identifying at least one specific biocatalytic reaction that
produces the particular modified small molecule of desired
activity.
[0089] The invention provides methods for determining a functional
fragment of a phospholipase enzyme comprising the steps of: (a)
providing a phospholipase enzyme comprising an amino acid sequence
of the invention; and, (b) deleting a plurality of amino acid
residues from the sequence of step (a) and testing the remaining
subsequence for a phospholipase activity, thereby determining a
functional fragment of a phospholipase enzyme. In one aspect, the
phospholipase activity is measured by providing a phospholipase
substrate and detecting an increase in the amount of the substrate
or a decrease in the amount of a reaction product. In one aspect, a
decrease in the amount of an enzyme substrate or an increase in the
amount of the reaction product with the test compound as compared
to the amount of substrate or reaction product without the test
compound identifies the test compound as an activator of
phospholipase activity.
[0090] The invention provides methods for cleaving a
glycerolphosphate ester linkage comprising the following steps: (a)
providing a polypeptide having a phospholipase activity, wherein
the polypeptide comprises an amino acid sequence of the invention,
or the polypeptide is encoded by a nucleic acid of the invention;
(b) providing a composition comprising a glycerolphosphate ester
linkage; and, (c) contacting the polypeptide of step (a) with the
composition of step (b) under conditions wherein the polypeptide
cleaves the glycerolphosphate ester linkage. In one aspect, the
conditions comprise between about pH 5 to about 8.5, or, between
about pH 4.5 (or more acidic, i.e., pH<4.5) to about 9.0 (or
more alkaline (i.e., pH>9). In one aspect, the conditions
comprise a temperature of between about 40.degree. C. and about
70.degree. C. In one aspect, the composition comprises a vegetable
oil. In one aspect, the composition comprises an oilseed
phospholipid. In one aspect, the cleavage reaction can generate a
water extractable phosphorylated base and a diglyceride.
[0091] The invention provides methods hydrolyzing, breaking up or
disrupting a phospholipid-comprising composition comprising
providing at least one polypeptide of the invention having a
phospholipase activity, or a polypeptide having a phospholipase
activity encoded by at least one nucleic acid of the invention;
providing a composition comprising a phospholipid; and contacting
the polypeptide with the composition under conditions wherein the
phospholipase hydrolyzes, breaks up or disrupts the
phospholipid-comprising composition. In one aspect, the method
comprises use of high shear mixing of the composition, followed by
no or low shear mixing with the at least one polypeptide of the
invention having a phospholipase activity to allow adequate
"contacting" of the phospholipid substrate with the phospholipase.
The at least one polypeptide having a phospholipase activity can
also be present in the high shear mixing step. The process can be
practiced at any scale, e.g., at a scale comprising about 1 gram
(g) to about 500, 1000, 2000, 2500, 5000 g, or more, or any amount
in this range.
[0092] The invention provides methods for oil degumming comprising
the following steps: (a) providing at least one polypeptide having
a phospholipase activity, wherein the polypeptide comprises an
amino acid sequence of the invention, or the polypeptide is encoded
by a nucleic acid of the invention; (b) providing a composition
comprising a vegetable oil; and, (c) contacting the polypeptide of
step (a) and the vegetable oil of step (b) under conditions wherein
the polypeptide can cleave ester linkages in the vegetable oil,
thereby degumming the oil. In one aspect, the vegetable oil
comprises oilseed. The vegetable oil can comprise rice bran oils,
palm oil, rapeseed oil, corn oil, soybean oil, canola oil, sesame
oil, peanut oil or sunflower oil. In one aspect, the method further
comprises addition of a phospholipase of the invention, another
phospholipase or a combination thereof. In one aspect, more than
one polypeptide having a phospholipase activity is added to the
process, wherein at least one polypeptide is an enzyme of the
invention. In one aspect, the enzymes are added in a specific
order, e.g., PLCs with differing specificities in are added in a
specific order, for example, an enzyme with PC and PE activity is
added first (or two enzymes are added together, one with PC and the
other with PE activity), then an enzyme with PI PLC activity is
added, or any combination thereof.
[0093] In one aspect of the oil degumming process, the
oil-comprising composition comprises a plant, an animal, an algae
or a fish oil or fat. The plant oil can comprise a rice bran oil, a
soybean oil, a rapeseed oil, a corn oil, an oil from a palm kernel,
a canola oil, a sunflower oil, a sesame oil or a peanut oil. The
polypeptide can hydrolyze a phosphatide from a hydratable and/or a
non-hydratable phospholipid in the oil-comprising composition. In
one aspect, the polypeptide hydrolyzes a phosphatide at a glyceryl
phosphoester bond to generate a diglyceride and water-soluble
phosphate compound. In one aspect, the polypeptide has a
phospholipase C activity. In one aspect, the polypeptide is a
phospholipase D and a phosphatase enzyme is also added.
[0094] In one aspect of the oil degumming process, the contacting
comprises hydrolysis of a hydrated phospholipid in an oil. The
hydrolysis conditions can comprise alkaline conditions, e.g., in
one aspect, the conditions comprise a temperature of about
20.degree. C. to 40.degree. C. at the alkaline pH. The alkaline
conditions can comprise a pH of about pH 8 to pH 10, or more. The
hydrolysis conditions can be made alkaline at any time in the
process, e.g., in one aspect, a phospholipase, such as a PLC, is
added before the conditions are made alkaline (e.g., a "caustic
neutralization" of an acid-comprising oil, such as phosphatidic
acid).
[0095] In one aspect of the oil degumming process, the base causes
the isomerization of 1,2-DAG, produced by PLC, into 1,3-DAG which
provides a nutritional health benefit over 1,2-DAG, e.g., the
1,3-DAG is burned as energy instead of being stored as fat (as is
1,2-DAG). Thus, the invention provides a caustic oil refining
process wherein a phospholipase, e.g., an enzyme of the invention,
including a PLC, is added "at the front end", i.e., before adding
any acid and caustic, e.g., as illustrated in the exemplary process
of FIG. 13. One of the consequences of adding the PLC at the front
end of a caustic refining process of the invention (see further
discussion, below), and adding the acid and caustic subsequently,
is the generation of an elevated level of 1,3-DAG (not 1,2-DAG).
This may be a consequence of acid or base-catalyzed acyl migration.
Nutritionally, 1,3-DAG is better than 1,2-DAG. Thus, the invention
comprises an oil degumming process using a PLC of the invention,
whereby the final degummed oil product contains not less than about
0.5%, 1.0%, 2.0%, 3.0%, 4.0% or 5.0% 1,3-DAG.
[0096] In one aspect of the oil degumming process, the hydrolysis
conditions can comprise a reaction time of about 3 to 10 or more
minutes. The hydrolysis conditions can comprise hydrolysis of
hydratable and non-hydratable phospholipids in oil at a temperature
of between about 50.degree. C. to 60.degree. C., at a pH of between
about pH 5 to pH 6.5, or between about pH 5 to pH 7.5, or between
about pH 5 to pH 8.0, using a reaction time of about 30 to 60
minutes.
[0097] In one aspect of the oil degumming process, the polypeptide
is bound to a filter and the phospholipid-containing fat or oil is
passed through the filter. The polypeptide can be added to a
solution comprising the phospholipid-containing fat or oil and then
the solution is passed through a filter.
[0098] In one aspect the oil degumming method further comprises
physical removal of gum produced by the degumming process by
addition of a hardening substance, e.g., a talc or equivalent. In
one aspect, this increases oil gain.
[0099] The invention also provides methods for converting a
non-hydratable phospholipid to a hydratable form comprising the
following steps: (a) providing a polypeptide having a phospholipase
activity, wherein the polypeptide comprises an amino acid sequence
of the invention, or the polypeptide is encoded by a nucleic acid
of the invention; (b) providing a composition comprising a
non-hydratable phospholipid; and, (c) contacting the polypeptide of
step (a) and the non-hydratable phospholipid of step (b) under
conditions wherein the polypeptide can cleave ester linkages in the
non-hydratable phospholipid, thereby converting a non-hydratable
phospholipid to a hydratable form.
[0100] The invention provides methods for degumming an oil
comprising the following steps: (a) providing a composition
comprising a polypeptide of the invention having a phospholipase
activity or a polypeptide encoded by a nucleic acid of the
invention; (b) providing an composition comprising a fat or an oil
comprising a phospholipid; and (c) contacting the polypeptide of
step (a) and the composition of step (b) under conditions wherein
the polypeptide can degum the phospholipid-comprising composition
(under conditions wherein the polypeptide of the invention can
catalyze the hydrolysis of a phospholipid). In one aspect the
oil-comprising composition comprises a plant, an animal, an algae
or a fish oil. The plant oil can comprise a rice bran oil, a
soybean oil, a rapeseed oil, a corn oil, an oil from a palm kernel,
a canola oil, a sunflower oil, a sesame oil or a peanut oil. The
polypeptide can hydrolyze a phosphatide from a hydratable and/or a
non-hydratable phospholipid in the oil-comprising composition. The
polypeptide can hydrolyze a phosphatide at a glyceryl phosphoester
bond to generate a diglyceride and water-soluble phosphate
compound. The polypeptide can have a phospholipase C, B, A or D
activity. In one aspect, a phospholipase D activity and a
phosphatase enzyme are added. The contacting can comprise
hydrolysis of a hydrated phospholipid in an oil. The hydrolysis
conditions of can comprise a temperature of about 20.degree. C. to
40.degree. C. at an alkaline pH. The alkaline conditions can
comprise a pH of about pH 8 to pH 10. The hydrolysis conditions can
comprise a reaction time of about 3 to 10 minutes. The hydrolysis
conditions can comprise hydrolysis of hydratable and non-hydratable
phospholipids in oil at a temperature of about 50.degree. C. to
60.degree. C., at a pH of about pH 5 to pH 6.5 using a reaction
time of about 30 to 60 minutes. The polypeptide can be bound to a
filter and the phospholipid-containing fat or oil is passed through
the filter. The polypeptide can be added to a solution comprising
the phospholipid-containing fat or oil and then the solution is
passed through a filter.
[0101] The invention provides methods for converting a
non-hydratable phospholipid to a hydratable form comprising the
following steps: (a) providing a composition comprising a
polypeptide having a phospholipase activity of the invention, or a
polypeptide encoded by a nucleic acid of the invention; (b)
providing an composition comprising a non-hydratable phospholipid;
and (c) contacting the polypeptide of step (a) and the composition
of step (b) under conditions wherein the polypeptide converts the
non-hydratable phospholipid to a hydratable form. The polypeptide
can have a phospholipase C activity. The polypeptide can have a
phospholipase D activity and a phosphatase enzyme is also
added.
[0102] The invention provides methods for caustic refining of a
phospholipid-containing composition comprising the following steps:
(a) providing a composition comprising a phospholipase, which can
be a polypeptide of the invention having a phospholipase activity,
or a polypeptide encoded by a nucleic acid of the invention; (b)
providing an composition comprising a phospholipid; and (c)
contacting the polypeptide of step (a) with the composition of step
(b) before, during or after the caustic refining. The polypeptide
can have a phospholipase activity, e.g., PLC, PLB, PLD and/or PLA
activity. The polypeptide can be added before caustic refining,
i.e., at the "front end" of the process, before adding acid or
caustic, as illustrated in FIG. 13.
[0103] The polypeptide (which can be an enzyme, e.g., a PLC, of the
invention) can be added during caustic refining and varying levels
of acid and caustic can be added depending on levels of phosphorus
and levels of free fatty acids. The polypeptide (which can be an
enzyme of the invention) can be added before caustic refining, or,
after caustic refining: in an intense mixer or retention mixer
prior to separation; following a heating step; in a centrifuge; in
a soapstock; in a washwater; and/or, during bleaching or
deodorizing steps. The method can comprise use of concentrated
solutions of caustic, e.g., more concentrated than the industrial
standard of 11%, to decrease mass of gum. In alternative aspects,
the concentrated solution of caustic is between about 12% and 50%
concentrated, e.g., about 20%, 30%, 40%, 50% or 60%, or more,
concentrated.
[0104] The composition comprising the phospholipid can comprise a
plant. The polypeptide can be expressed transgenically in the
plant. The polypeptide having a phospholipase activity can be added
during crushing of a seed or other plant part, or, the polypeptide
having a phospholipase activity is added following crushing or
prior to refining.
[0105] Also provided is a caustic refining process for hydrolyzing
phospholipids in oil (e.g., plant oil) using a polypeptide of the
invention to generate diacylglycerol (DAG) and water-soluble
phosphate ester. In one aspect, the enzyme of the invention must
operate in a caustic refining process, including, optionally low
water and/or in a temperature range of about 55.degree. C. to about
70.degree. C. Use of a caustic refining process with low water in
this temperature range will maximize yield by increasing DAG and
reducing entrained oil. In one aspect, the enzyme used in this
caustic refining process of the invention has both very good
activity on phosphatidylcholine (PC) and phosphatidylethanolamine
(PE), is active between a pH of about pH 6 to pH 9, is active up to
75.degree. C., and is active in low water in oil, e.g., about 2% to
5% water, e.g., the enzyme encoded by the sequence of SEQ ID NO:2,
encoded e.g., by SEQ ID NO:1.
[0106] In another aspect of the invention's caustic refining
process for hydrolyzing phospholipids in oils, two enzymes are
used: a PI-specific PLC (hydrolyzes PI), and a PC-PLC that
hydrolyzes PC, PE and PA. This embodiment generates oil suitable
for chemical or physical refining and maximizes yield increase from
DAG and less entrained oil.
[0107] The invention provides methods for purification of a
phytosterol or a triterpene comprising the following steps: (a)
providing a composition comprising a polypeptide of the invention
having a phospholipase activity, or a polypeptide encoded by a
nucleic acid of the invention; (b) providing an composition
comprising a phytosterol or a triterpene; and (c) contacting the
polypeptide of step (a) with the composition of step (b) under
conditions wherein the polypeptide can catalyze the hydrolysis of a
phospholipid in the composition. The polypeptide can have a
phospholipase C activity. The phytosterol or a triterpene can
comprise a plant sterol. The plant sterol can be derived from a
vegetable oil. The vegetable oil can comprise a rice bran oil, a
coconut oil, canola oil, cocoa butter oil, corn oil, cottonseed
oil, linseed oil, olive oil, palm oil, peanut oil, oil derived from
a rice bran, safflower oil, sesame oil, soybean oil or a sunflower
oil. The method can comprise use of nonpolar solvents to
quantitatively extract free phytosterols and phytosteryl fatty-acid
esters. The phytosterol or a triterpene can comprise a
.beta.-sitosterol, a campesterol, a stigmasterol, a stigmastanol, a
.beta.-sitostanol, a sitostanol, a desmosterol, a chalinasterol, a
poriferasterol, a clionasterol or a brassicasterol.
[0108] The invention provides methods for refining a crude oil
comprising the following steps: (a) providing a composition
comprising a polypeptide of the invention having a phospholipase
activity, or a polypeptide encoded by a nucleic acid of the
invention; (b) providing a composition comprising an oil comprising
a phospholipid; and (c) contacting the polypeptide of step (a) with
the composition of step (b) under conditions wherein the
polypeptide can catalyze the hydrolysis of a phospholipid in the
composition. The polypeptide can have a phospholipase C activity.
The polypeptide can have a phospholipase activity is in a water
solution that is added to the composition. The water level can be
between about 0.5 to 5%. The process time can be less than about 2
hours, less than about 60 minutes, less than about 30 minutes, less
than 15 minutes, or less than 5 minutes. The hydrolysis conditions
can comprise a temperature of between about 25.degree.
C.-70.degree. C. The hydrolysis conditions can comprise use of
caustics. Concentrated solutions of caustic, e.g., more
concentrated than the industrial standard of 11%, to decrease mass
of gum can be used. In alternative aspects, the concentrated
solution of caustic is between about 12% and 50% concentrated,
e.g., about 20%, 30%, 40%, 50%, or 60% or more concentrated.
[0109] The hydrolysis conditions can comprise a pH of between about
pH 3 and pH 10, between about pH 4 and pH 9, or between about pH 5
and pH 8. The hydrolysis conditions can comprise addition of
emulsifiers and/or mixing after the contacting of step (c). The
methods can comprise addition of an emulsion-breaker and/or heat or
cooling (e.g. to between about 4.degree. C. to about -20.degree.
C., or less) to promote separation of an aqueous phase. The methods
can comprise degumming before the contacting step to collect
lecithin by centrifugation and then adding a PLC, a PLC and/or a
PLA to remove non-hydratable phospholipids. The methods can
comprise water degumming of crude oil to less than 10 ppm
phosphorus for edible oils and subsequent physical refining to less
than about 50 ppm phosphorus for biodiesel oils. The methods can
comprise addition of acid to promote hydration of non-hydratable
phospholipids. In one aspect, addition of acid promotes lowering of
the calcium and magnesium metal content.
[0110] The invention provides a method for ameliorating or
preventing lipopolysaccharide (LPS)-mediated toxicity comprising
administering to a patient a pharmaceutical composition comprising
a polypeptide of the invention. The invention provides a method for
detoxifying an endotoxin comprising contacting the endotoxin with a
polypeptide of the invention. The invention provides a method for
deacylating a 2' or a 3' fatty acid chain from a lipid A comprising
contacting the lipid A with a polypeptide of the invention.
[0111] The invention provides a method for refining a lubricant
comprising the following steps: (a) providing a composition
comprising an enzyme of the invention; (b) providing a lubricant;
and (c) treating the lubricant with an enzyme under conditions
wherein the enzyme can selective hydrolyze oils in the lubricant,
thereby refining it. The lubricant can be a hydraulic oil.
[0112] The invention provides a method of treating a fabric
comprising the following steps: (a) providing a composition
comprising an enzyme of the invention, (b) providing a fabric; and
(c) treating the fabric with the enzyme. The treatment of the
fabric can comprise improvement of the hand and drape of the final
fabric, dyeing, obtaining flame retardancy, obtaining water
repellency, obtaining optical brightness, or obtaining resin
finishing. The fabric can comprise cotton, viscose, rayon, lyocell,
flax, linen, ramie, all blends thereof, or blends thereof with
polyesters, wool, polyamides acrylics or polyacrylics. The
invention provides a fabric, yarn or fiber comprising an enzyme of
the invention. The enzyme can be adsorbed, absorbed or immobilized
on the surface of the fabric, yarn or fiber.
[0113] The invention provides methods for expressing phospholipase
C comprising providing a Pichia strain with a Mut.sup.+ phenotype;
inserting a heterologous phospholipase C-encoding nucleic acid in
the Pichia strain; and, culturing the Pichia strain under
conditions whereby the phospholipase C is expressed. The method can
further comprise supplementing the culture conditions with zinc.
The invention also provides cell systems, isolated cells and cell
lines for expressing phospholipase C comprising a Mut.sup.+
phenotype Pichia strain comprising a heterologous phospholipase
C-encoding nucleic acid operably linked to a promoter operable in
the Pichia strain.
[0114] The invention provides zeocin-resistant yeast cell systems
(e.g., yeast cells, cell lines, individual cells) for expressing a
heterologous protein comprising the steps of providing a Pichia sp.
(e.g., P. pastoris) cell comprising a heterologous nucleic acid
capable of expressing a heterologous protein; culturing the cell
under conditions comprising zeocin at an initial concentration;
selecting cells resistant to the initial concentration of zeocin,
and reculturing under conditions comprising a higher concentration
of zeocin; and selecting the cells cultured in step (c) resistant
to the higher concentration of zeocin. In one aspect, the
heterologous protein is an enzyme, or optionally, a phospholipase,
or optionally a phospholipase C (PLC), e.g., any enzyme of the
invention.
[0115] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0116] All publications, patents, patent applications, GenBank
sequences and ATCC deposits, cited herein are hereby expressly
incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0117] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0118] FIG. 1 is a block diagram of a computer system, as described
in detail, below.
[0119] FIG. 2 is a flow diagram illustrating one aspect of a
process 200 for comparing a new nucleotide or protein sequence with
a database of sequences in order to determine the homology levels
between the new sequence and the sequences in the database, as
described in detail, below.
[0120] FIG. 3 is a flow diagram illustrating one embodiment of a
process in a computer for determining whether two sequences are
homologous, as described in detail, below.
[0121] FIG. 4 is a flow diagram illustrating one aspect of an
identifier process for detecting the presence of a feature in a
sequence, as described in detail, below.
[0122] FIGS. 5A, 5B and 5C schematically illustrate a model
two-phase system for simulation of PLC-mediated degumming, as
described in detail in Example 2, below.
[0123] FIG. 6 schematically illustrates an exemplary vegetable oil
refining process using the phospholipases of the invention.
[0124] FIG. 7 schematically illustrates an exemplary degumming
process of the invention for physically refined oils, as discussed
in detail, below.
[0125] FIG. 8 schematically illustrates phosphatide hydrolysis with
a phospholipase C of the invention, as discussed in detail,
below.
[0126] FIG. 9 schematically illustrates an exemplary caustic
refining process of the invention, and illustrates an alternative
embodiment comprising application of a phospholipase C of the
invention as a "Caustic Refining Aid" (Long Mix Caustic Refining),
as discussed in detail, below.
[0127] FIG. 10 schematically illustrates application of a
phospholipase C of the invention as a degumming aid, as discussed
in detail, below.
[0128] FIG. 11 is a chart describing selected characteristics of
exemplary nucleic acids and polypeptides of the invention, as
described in further detail, below.
[0129] FIG. 12 schematically illustrates data from a two enzyme
system of the invention, as described in Example 3, below.
[0130] FIG. 13 schematically illustrates an exemplary caustic
refining process of the invention, and illustrates an alternative
embodiment comprising application of a phospholipase C of the
invention as a "Caustic Refining Aid" (Long Mix Caustic Refining),
as discussed in detail, below.
[0131] FIG. 14 illustrates another variation of methods of the
invention where two centrifugation steps are used in the process,
as discussed in detail, below.
[0132] FIG. 15 illustrates another variation of methods of the
invention where three centrifugation steps are used in the process,
as discussed in detail, below.
[0133] FIG. 16 illustrates another exemplary variation of this
process using acid treatment and having a centrifugation step
before a degumming step, as discussed in detail, below.
[0134] FIG. 17 illustrates the results of the in vitro digestion
experiments wherein the phospholipase C variants of the invention,
as discussed in detail in Example 4, below.
[0135] FIG. 18 illustrates the results of a batch fermentor culture
using an exemplary enzyme of the invention, as discussed in detail
in Example 5, below.
[0136] FIG. 19 illustrates the results of Oxygen Uptake Rate
("OUR") comparisons of cultures of P. pastoris MutS strains of the
invention, as discussed in detail in Example 5, below.
[0137] FIG. 20 illustrates a methanol consumption profile
comparison in P. pastoris MutS strains of the invention, as
discussed in detail in Example 5, below.
[0138] FIG. 21 illustrates an "OUR" profile of a culture of a
recombinant form of the exemplary PLC enzyme of the invention SEQ
ID NO:2, as discussed in detail in Example 5, below.
[0139] FIG. 22 illustrates results from an SDS-PAGE showing the
quality of PLC protein produced in a culture, and a corresponding
OUR profile, of a culture of a recombinant form of the exemplary
PLC enzyme of the invention SEQ ID NO:2, as discussed in detail in
Example 5, below.
[0140] FIG. 23 illustrates results from an SDS-PAGE showing the
quantity of active PLC located intracellularly in a culture of a
recombinant form of the exemplary PLC enzyme of the invention SEQ
ID NO:2, as discussed in detail in Example 5, below.
[0141] FIG. 24 illustrates a visualization of the morphological
changes in yeast cells associated with active PLC--a recombinant
form of the exemplary PLC enzyme of the invention SEQ ID NO:2, as
discussed in detail in Example 5, below.
[0142] FIG. 25 graphically summarizes data showing the status of a
PLC production performance at 95 h TFT (total fermentation time) in
Pichia using an exemplary PLC enzyme of the invention SEQ ID NO:2,
as discussed in detail in Example 5, below.
[0143] FIG. 26 is a table summary of data from expression screening
of exemplary zeocin-adapted cell colonies of the invention, as
discussed in detail in Example 5, below.
[0144] FIG. 27 illustrates data showing that PLC protein levels
were higher in cultures comprising exemplary zeocin-adapted cell
colonies of the invention, as discussed in detail in Example 5,
below.
[0145] FIG. 28 illustrates data showing a growth comparison of
zeo-adapted colonies of the invention vs control, as discussed in
detail in Example 5, below.
[0146] FIG. 29 illustrates the results of a heating experiment
demonstrating the thermostability of the exemplary enzyme of the
invention SEQ ID NO:2, with the conditions indicated in the figure,
as discussed in detail in Example 6, below.
[0147] FIG. 30 illustrates NMR data summarizing the heating
experiment demonstrating the thermostability of the exemplary
enzyme of the invention SEQ ID NO:2, as discussed in detail in
Example 6, below.
[0148] FIGS. 31, 32 and 33 illustrate data demonstrating the
thermal stability of SEQ ID NO:2 using p-NPPC, at the conditions
shown in the figure, as discussed in detail in Example 6,
below.
[0149] FIG. 34 illustrates data demonstrating the thermal stability
of SEQ ID NO:2 using DSC analysis, as discussed in detail in
Example 6, below.
[0150] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION OF THE INVENTION
[0151] The present invention provides phospholipases, e.g.,
polypeptides having phospholipase A, B, C, D, patatin, phosphatidic
acid phosphatases (PAP) and/or lipid acyl hydrolase (LAH) or
equivalent activity, polynucleotides encoding them and methods for
making and using them. The invention provides enzymes that
efficiently cleave glycerolphosphate ester linkage in oils, such as
vegetable oils, e.g., oilseed phospholipids, to generate a water
extractable phosphorylated base and a diglyceride. In one aspect,
the phospholipases of the invention have a lipid acyl hydrolase
(LAH) activity. In alternative aspects, the phospholipases of the
invention can cleave glycerolphosphate ester linkages in
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic
acid, and/or sphingomyelin, or a combination thereof. For example,
in one aspect a phospholipase of the invention is specific for one
or more specific substrates, e.g., an enzyme of the invention can
have a specificity of action for PE and PC; PE an PI; PE and PS; PS
and PE; PS and PI; PI and PE; PS, PI and PC; PE, PI and PC; or, PE,
PS, PI and PC.
[0152] A phospholipase of the invention (e.g., polypeptides having
phospholipase A, B, C, D, patatin, phosphatidic acid phosphatases
(PAP) and/or lipid acyl hydrolase (LAH) or equivalent activity) can
be used for enzymatic degumming of vegetable oils because the
phosphate moiety is soluble in water and easy to remove. The
diglyceride product will remain in the oil and therefore will
reduce losses. The PLCs of the invention can be used in addition to
or in place of PLA1s and PLA2s in commercial oil degumming, such as
in the ENZYMAX.RTM. process, where phospholipids are hydrolyzed by
PLA1 and PLA2.
[0153] In one aspect, the phospholipases of the invention are
active at a high and/or at a low temperature, or, over a wide range
of temperature, e.g., they can be active in the temperatures
ranging between 20.degree. C. to 90.degree. C., between 30.degree.
C. to 80.degree. C., or between 40.degree. C. to 70.degree. C. The
invention also provides phospholipases of the invention have
activity at alkaline pHs or at acidic pHs, e.g., low water acidity.
In alternative aspects, the phospholipases of the invention can
have activity in acidic pHs as low as pH 6.5, pH 6.0, pH 5.5, pH
5.0, pH 4.5, pH 4.0 and pH 3.5 or more acidic (i.e., <pH 3.5).
In alternative aspects, the phospholipases of the invention can
have activity in alkaline pHs as high as pH 7.5, pH 8.0, pH 8.5, pH
9.0, pH 9.5, pH 10 or more alkaline (i.e., >pH 10). In one
aspect, the phospholipases of the invention are active in the
temperature range of between about 40.degree. C. to about
70.degree. C., 75.degree. C., or 80.degree. C., or more, under
conditions of low water activity (low water content).
[0154] The invention also provides methods for further modifying
the exemplary phospholipases of the invention to generate enzymes
with desirable properties. For example, phospholipases generated by
the methods of the invention can have altered substrate
specificities, substrate binding specificities, substrate cleavage
patterns, thermal stability, pH/activity profile, pH/stability
profile (such as increased stability at low, e.g. pH<6 or
pH<5, or high, e.g. pH>9, pH values), stability towards
oxidation, Ca.sup.2+ dependency, specific activity and the like.
The invention provides for altering any property of interest. For
instance, the alteration may result in a variant which, as compared
to a parent phospholipase, has altered pH and temperature activity
profile.
[0155] In one aspect, the phospholipases of the invention are used
in various vegetable oil processing steps, such as in vegetable oil
extraction, particularly, in the removal of "phospholipid gums" in
a process called "oil degumming," as described herein. The
invention provides compositions (e.g., comprising enzymes of the
invention) and processes for the production of vegetable oils from
various sources, such as oil from rice bran, soybeans, rapeseed,
peanut, sesame, sunflower and corn. The phospholipase enzymes of
the invention can be used in place of PLA, e.g., phospholipase A2,
in any vegetable oil processing step.
DEFINITIONS
[0156] The term "phospholipase" encompasses enzymes having any
phospholipase activity, for example, cleaving a glycerolphosphate
ester linkage (catalyzing hydrolysis of a glycerolphosphate ester
linkage), e.g., in an oil, such as a vegetable oil. The
phospholipase activity of the invention can generate a water
extractable phosphorylated base and a diglyceride. The
phospholipase activity of the invention also includes hydrolysis of
glycerolphosphate ester linkages at high temperatures, low
temperatures, alkaline pHs and at acidic pHs. The term "a
phospholipase activity" also includes cleaving a glycerolphosphate
ester to generate a water extractable phosphorylated base and a
diglyceride. The term "a phospholipase activity" also includes
cutting ester bonds of glycerin and phosphoric acid in
phospholipids. The term "a phospholipase activity" also includes
other activities, such as the ability to bind to and hydrolyze a
substrate, such as an oil, e.g. a vegetable oil, substrate also
including plant and animal phosphatidylcholines,
phosphatidyl-ethanolamines, phosphatidylserines and sphingomyelins.
The phospholipase activity can comprise a phospholipase C (PLC)
activity; a phospholipase A (PLA) activity, such as a phospholipase
A1 or phospholipase A2 activity; a phospholipase B (PLB) activity,
such as a phospholipase B1 or phospholipase B2 activity, including
lysophospholipase (LPL) activity and/or
lysophospholipase-transacylase (LPTA) activity; a phospholipase D
(PLD) activity, such as a phospholipase D1 or a phospholipase D2
activity; and/or a patatin activity or any combination thereof. The
phospholipase activity can comprise hydrolysis of a glycoprotein,
e.g., as a glycoprotein found in a potato tuber or any plant of the
genus Solanum, e.g., Solanum tuberosum. The phospholipase activity
can comprise a patatin enzymatic activity, such as a patatin
esterase activity (see, e.g., Jimenez (2002) Biotechnol. Prog.
18:635-640). The phospholipase activity can comprise a lipid acyl
hydrolase (LAH) activity. The phospholipase activity can comprise
being specific for one or more specific substrates, e.g., an enzyme
of the invention can have a specificity of action for PE and PC; PE
an PI; PE and PS; PS and PE; PS and PI; PI and PE; PS, PI and PC;
PE, PI and PC; or, PE, PS, PI and PC, or any combination
thereof.
[0157] In one aspect, a phospholipase of the invention can have
multifunctional activity, e.g., a combination of one or more of the
enzyme activities described herein. For example, in one aspect, a
polypeptide of the invention is enzymatically active, but lacks a
lipase activity or lacks any enzymatic activity that affects a
neutral oil (triglyceride) fraction. It may be desirable to use
such a polypeptide in a particular process, e.g., in a degumming
process where it is important that the neutral oil fraction not be
harmed (diminished, degraded, e.g., hydrolyzed). Thus, in one
aspect, the invention provides a degumming process comprising use
of a polypeptide of the invention having a phospholipase activity,
but not a lipase activity.
[0158] In one aspect, PLC phospholipases of the invention utilize
(e.g., catalyze hydrolysis of) a variety of phospholipid substrates
including phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylserine (PS), phosphatidylinositol (PI), and/or
phosphatidic acid or a combination thereof. In addition, these
enzymes can have varying degrees of activity on the
lysophospholipid forms of these phospholipids. In various aspects,
PLC enzymes of the invention may show a preference for
phosphatidylcholine and phosphatidylethanolamine as substrates.
[0159] In one aspect, phosphatidylinositol PLC phospholipases of
the invention utilize a variety of phospholipid substrates
including phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, and phosphatidic acid, or
a combination thereof. In addition, these enzymes can have varying
degrees of activity on the lysophospholipid forms of these
phospholipids. In various aspects, phosphatidylinositol PLC enzymes
of the invention may show a preference for phosphatidylinositol as
a substrate.
[0160] In one aspect, patatin enzymes of the invention utilize a
variety of phospholipid substrates including phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
and phosphatidic acid, or a combination thereof. In addition, these
enzymes can have varying degrees of activity on the
lysophospholipid forms of these phospholipids. In various aspects,
patatins of the invention are based on a conservation of amino acid
sequence similarity. In various aspects, these enzymes display a
diverse set of biochemical properties and may perform reactions
characteristic of PLA1, PLA2, PLC, or PLD enzyme classes.
[0161] In one aspect, PLD phospholipases of the invention utilize a
variety of phospholipid substrates including phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
and phosphatidic acid, or a combination thereof. In addition, these
enzymes can have varying degrees of activity on the
lysophospholipid forms of these phospholipids. In one aspect, these
enzymes are useful for carrying out transesterification reactions
to produce structured phospholipids.
[0162] The term "antibody" includes a peptide or polypeptide
derived from, modeled after or substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof,
capable of specifically binding an antigen or epitope, see, e.g.
Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven
Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273;
Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term
antibody includes antigen-binding portions, i.e., "antigen binding
sites," (e.g., fragments, subsequences, complementarity determining
regions (CDRs)) that retain capacity to bind antigen, including (i)
a Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Single chain
antibodies are also included by reference in the term
"antibody."
[0163] The terms "array" or "microarray" or "biochip" or "chip" as
used herein is a plurality of target elements, each target element
comprising a defined amount of one or more polypeptides (including
antibodies) or nucleic acids immobilized onto a defined area of a
substrate surface, as discussed in further detail, below.
[0164] As used herein, the terms "computer," "computer program" and
"processor" are used in their broadest general contexts and
incorporate all such devices, as described in detail, below.
[0165] A "coding sequence of" or a "sequence encodes" a particular
polypeptide or protein, is a nucleic acid sequence which is
transcribed and translated into a polypeptide or protein when
placed under the control of appropriate regulatory sequences.
[0166] The term "expression cassette" as used herein refers to a
nucleotide sequence which is capable of affecting expression of a
structural gene (i.e., a protein coding sequence, such as a
phospholipase of the invention) in a host compatible with such
sequences. Expression cassettes include at least a promoter
operably linked with the polypeptide coding sequence; and,
optionally, with other sequences, e.g., transcription termination
signals. Additional factors necessary or helpful in effecting
expression may also be used, e.g., enhancers. "Operably linked" as
used herein refers to linkage of a promoter upstream from a DNA
sequence such that the promoter mediates transcription of the DNA
sequence. Thus, expression cassettes also include plasmids,
expression vectors, recombinant viruses, any form of recombinant
"naked DNA" vector, and the like. A "vector" comprises a nucleic
acid which can infect, transfect, transiently or permanently
transduce a cell. It will be recognized that a vector can be a
naked nucleic acid, or a nucleic acid complexed with protein or
lipid. The vector optionally comprises viral or bacterial nucleic
acids and/or proteins, and/or membranes (e.g., a cell membrane, a
viral lipid envelope, etc.). Vectors include, but are not limited
to replicons (e.g., RNA replicons, bacteriophages) to which
fragments of DNA may be attached and become replicated. Vectors
thus include, but are not limited to RNA, autonomous
self-replicating circular or linear DNA or RNA (e.g., plasmids,
viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and
includes both the expression and non-expression plasmids. Where a
recombinant microorganism or cell culture is described as hosting
an "expression vector" this includes both extra-chromosomal
circular and linear DNA and DNA that has been incorporated into the
host chromosome(s). Where a vector is being maintained by a host
cell, the vector may either be stably replicated by the cells
during mitosis as an autonomous structure, or is incorporated
within the host's genome.
[0167] "Plasmids" are designated by a lower case "p" preceded
and/or followed by capital letters and/or numbers. The starting
plasmids herein are either commercially available, publicly
available on an unrestricted basis, or can be constructed from
available plasmids in accord with published procedures. In
addition, equivalent plasmids to those described herein are known
in the art and will be apparent to the ordinarily skilled
artisan.
[0168] The term "gene" means the segment of DNA involved in
producing a polypeptide chain, including, inter alia, regions
preceding and following the coding region, such as leader and
trailer, promoters and enhancers, as well as, where applicable,
intervening sequences (introns) between individual coding segments
(exons).
[0169] The phrases "nucleic acid" or "nucleic acid sequence" as
used herein refer to an oligonucleotide, nucleotide,
polynucleotide, or to a fragment of any of these, to DNA or RNA
(e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which
may be single-stranded or double-stranded and may represent a sense
or antisense strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like material, natural or synthetic in origin,
including, e.g., iRNA, ribonucleoproteins (e.g., double stranded
iRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e.,
oligonucleotides, containing known analogues of natural
nucleotides. The term also encompasses nucleic-acid-like structures
with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl.
Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry
36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev
6:153-156.
[0170] "Amino acid" or "amino acid sequence" as used herein refer
to an oligopeptide, peptide, polypeptide, or protein sequence, or
to a fragment, portion, or subunit of any of these, and to
naturally occurring or synthetic molecules.
[0171] The terms "polypeptide" and "protein" as used herein, refer
to amino acids joined to each other by peptide bonds or modified
peptide bonds, i.e., peptide isosteres, and may contain modified
amino acids other than the 20 gene-encoded amino acids. The term
"polypeptide" also includes peptides and polypeptide fragments,
motifs and the like. The term also includes glycosylated
polypeptides. The peptides and polypeptides of the invention also
include all "mimetic" and "peptidomimetic" forms, as described in
further detail, below.
[0172] As used herein, the term "isolated" means that the material
is removed from its original environment (e.g., the natural
environment if it is naturally occurring). For example, a naturally
occurring polynucleotide or polypeptide present in a living animal
is not isolated, but the same polynucleotide or polypeptide,
separated from some or all of the coexisting materials in the
natural system, is isolated. Such polynucleotides could be part of
a vector and/or such polynucleotides or polypeptides could be part
of a composition, and still be isolated in that such vector or
composition is not part of its natural environment. As used herein,
an isolated material or composition can also be a "purified"
composition, i.e., it does not require absolute purity; rather, it
is intended as a relative definition. Individual nucleic acids
obtained from a library can be conventionally purified to
electrophoretic homogeneity. In alternative aspects, the invention
provides nucleic acids which have been purified from genomic DNA or
from other sequences in a library or other environment by at least
one, two, three, four, five or more orders of magnitude.
[0173] As used herein, the term "recombinant" means that the
nucleic acid is adjacent to a "backbone" nucleic acid to which it
is not adjacent in its natural environment. In one aspect, nucleic
acids represent 5% or more of the number of nucleic acid inserts in
a population of nucleic acid "backbone molecules." "Backbone
molecules" according to the invention include nucleic acids such as
expression vectors, self-replicating nucleic acids, viruses,
integrating nucleic acids, and other vectors or nucleic acids used
to maintain or manipulate a nucleic acid insert of interest. In one
aspect, the enriched nucleic acids represent 15%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or more of the number of nucleic acid
inserts in the population of recombinant backbone molecules.
"Recombinant" polypeptides or proteins refer to polypeptides or
proteins produced by recombinant DNA techniques; e.g., produced
from cells transformed by an exogenous DNA construct encoding the
desired polypeptide or protein. "Synthetic" polypeptides or protein
are those prepared by chemical synthesis, as described in further
detail, below.
[0174] A promoter sequence is "operably linked to" a coding
sequence when RNA polymerase which initiates transcription at the
promoter will transcribe the coding sequence into mRNA, as
discussed further, below.
[0175] "Oligonucleotide" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0176] The phrase "substantially identical" in the context of two
nucleic acids or polypeptides, refers to two or more sequences that
have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%
nucleotide or amino acid residue (sequence) identity, when compared
and aligned for maximum correspondence, as measured using one any
known sequence comparison algorithm, as discussed in detail below,
or by visual inspection. In alternative aspects, the invention
provides nucleic acid and polypeptide sequences having substantial
identity to an exemplary sequence of the invention, e.g., SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, etc., over a region of at least
about 100 residues, 150 residues, 200 residues, 300 residues, 400
residues, or a region ranging from between about 50 residues to the
full length of the nucleic acid or polypeptide. Nucleic acid
sequences of the invention can be substantially identical over the
entire length of a polypeptide coding region.
[0177] Additionally a "substantially identical" amino acid sequence
is a sequence that differs from a reference sequence by one or more
conservative or non-conservative amino acid substitutions,
deletions, or insertions, particularly when such a substitution
occurs at a site that is not the active site of the molecule, and
provided that the polypeptide essentially retains its functional
properties. A conservative amino acid substitution, for example,
substitutes one amino acid for another of the same class (e.g.,
substitution of one hydrophobic amino acid, such as isoleucine,
valine, leucine, or methionine, for another, or substitution of one
polar amino acid for another, such as substitution of arginine for
lysine, glutamic acid for aspartic acid or glutamine for
asparagine). One or more amino acids can be deleted, for example,
from a phospholipase polypeptide, resulting in modification of the
structure of the polypeptide, without significantly altering its
biological activity. For example, amino- or carboxyl-terminal amino
acids that are not required for phospholipase biological activity
can be removed. Modified polypeptide sequences of the invention can
be assayed for phospholipase biological activity by any number of
methods, including contacting the modified polypeptide sequence
with a phospholipase substrate and determining whether the modified
polypeptide decreases the amount of specific substrate in the assay
or increases the bioproducts of the enzymatic reaction of a
functional phospholipase with the substrate, as discussed further,
below.
[0178] "Hybridization" refers to the process by which a nucleic
acid strand joins with a complementary strand through base pairing.
Hybridization reactions can be sensitive and selective so that a
particular sequence of interest can be identified even in samples
in which it is present at low concentrations. Suitably stringent
conditions can be defined by, for example, the concentrations of
salt or formamide in the prehybridization and hybridization
solutions, or by the hybridization temperature, and are well known
in the art. For example, stringency can be increased by reducing
the concentration of salt, increasing the concentration of
formamide, or raising the hybridization temperature, altering the
time of hybridization, as described in detail, below. In
alternative aspects, nucleic acids of the invention are defined by
their ability to hybridize under various stringency conditions
(e.g., high, medium, and low), as set forth herein.
[0179] The term "variant" refers to polynucleotides or polypeptides
of the invention modified at one or more base pairs, codons,
introns, exons, or amino acid residues (respectively) yet still
retain the biological activity of a phospholipase of the invention.
Variants can be produced by any number of means included methods
such as, for example, error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, GSSM.TM. and any
combination thereof. Techniques for producing variant
phospholipases having activity at a pH or temperature, for example,
that is different from a wild-type phospholipase, are included
herein.
[0180] The term "saturation mutagenesis", Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.) or "GSSM.TM." includes a method that
uses degenerate oligonucleotide primers to introduce point
mutations into a polynucleotide, as described in detail, below.
[0181] The term "optimized directed evolution system" or "optimized
directed evolution" includes a method for reassembling fragments of
related nucleic acid sequences, e.g., related genes, and explained
in detail, below.
[0182] The term "synthetic ligation reassembly" or "SLR" includes a
method of ligating oligonucleotide fragments in a non-stochastic
fashion, and explained in detail, below.
Generating and Manipulating Nucleic Acids
[0183] The invention provides isolated and recombinant nucleic
acids (e.g., the exemplary SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ
ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71,
SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID
NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ
ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99,
SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID
NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,
SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID
NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135,
SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID
NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,
SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID
NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171
or SEQ ID NO:173), including expression cassettes such as
expression vectors, encoding the polypeptides and phospholipases of
the invention. The invention also includes methods for discovering
new phospholipase sequences using the nucleic acids of the
invention. Also provided are methods for modifying the nucleic
acids of the invention by, e.g., synthetic ligation reassembly,
optimized directed evolution system and/or saturation
mutagenesis.
[0184] The nucleic acids of the invention can be made, isolated
and/or manipulated by, e.g., cloning and expression of cDNA
libraries, amplification of message or genomic DNA by PCR, and the
like. In practicing the methods of the invention, homologous genes
can be modified by manipulating a template nucleic acid, as
described herein. The invention can be practiced in conjunction
with any method or protocol or device known in the art, which are
well described in the scientific and patent literature.
[0185] General Techniques
[0186] The nucleic acids used to practice this invention, whether
RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors,
viruses or hybrids thereof, may be isolated from a variety of
sources, genetically engineered, amplified, and/or
expressed/generated recombinantly. Recombinant polypeptides
generated from these nucleic acids can be individually isolated or
cloned and tested for a desired activity. Any recombinant
expression system can be used, including bacterial, mammalian,
yeast, insect or plant cell expression systems.
[0187] Alternatively, these nucleic acids can be synthesized in
vitro by well-known chemical synthesis techniques, as described in,
e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997)
Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.
Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang
(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;
Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
[0188] Techniques for the manipulation of nucleic acids, such as,
e.g., subcloning, labeling probes (e.g., random-primer labeling
using Klenow polymerase, nick translation, amplification),
sequencing, hybridization and the like are well described in the
scientific and patent literature, see, e.g., Sambrook, ed.,
MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold
Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);
LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:
HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic
Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
[0189] Another useful means of obtaining and manipulating nucleic
acids used to practice the methods of the invention is to clone
from genomic samples, and, if desired, screen and re-clone inserts
isolated or amplified from, e.g., genomic clones or cDNA clones.
Sources of nucleic acid used in the methods of the invention
include genomic or cDNA libraries contained in, e.g., mammalian
artificial chromosomes (MACS), see, e.g., U.S. Pat. Nos. 5,721,118;
6,025,155; human artificial chromosomes, see, e.g., Rosenfeld
(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);
bacterial artificial chromosomes (BAC); P1 artificial chromosomes,
see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors
(PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids,
recombinant viruses, phages or plasmids.
[0190] In one aspect, a nucleic acid encoding a polypeptide of the
invention is assembled in appropriate phase with a leader sequence
capable of directing secretion of the translated polypeptide or
fragment thereof.
[0191] The invention provides fusion proteins and nucleic acids
encoding them. A polypeptide of the invention can be fused to a
heterologous peptide or polypeptide, such as N-terminal
identification peptides which impart desired characteristics, such
as increased stability or simplified purification. Peptides and
polypeptides of the invention can also be synthesized and expressed
as fusion proteins with one or more additional domains linked
thereto for, e.g., producing a more immunogenic peptide, to more
readily isolate a recombinantly synthesized peptide, to identify
and isolate antibodies and antibody-expressing B cells, and the
like. Detection and purification facilitating domains include,
e.g., metal chelating peptides such as polyhistidine tracts and
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle
Wash.). The inclusion of a cleavable linker sequences such as
Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a
purification domain and the motif-comprising peptide or polypeptide
to facilitate purification. For example, an expression vector can
include an epitope-encoding nucleic acid sequence linked to six
histidine residues followed by a thioredoxin and an enterokinase
cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797;
Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine
residues facilitate detection and purification while the
enterokinase cleavage site provides a means for purifying the
epitope from the remainder of the fusion protein. Technology
pertaining to vectors encoding fusion proteins and application of
fusion proteins are well described in the scientific and patent
literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
[0192] Transcriptional and Translational Control Sequences
[0193] The invention provides nucleic acid (e.g., DNA) sequences of
the invention operatively linked to expression (e.g.,
transcriptional or translational) control sequence(s), e.g.,
promoters or enhancers, to direct or modulate RNA
synthesis/expression. The expression control sequence can be in an
expression vector. Exemplary bacterial promoters include lacI,
lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary eukaryotic
promoters include CMV immediate early, HSV thymidine kinase, early
and late SV40, LTRs from retrovirus, and mouse metallothionein
I.
[0194] Promoters suitable for expressing a polypeptide in bacteria
include the E. coli lac or trp promoters, the lad promoter, the
lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter,
the lambda PR promoter, the lambda PL promoter, promoters from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), and the acid phosphatase promoter. Eukaryotic
promoters include the CMV immediate early promoter, the HSV
thymidine kinase promoter, heat shock promoters, the early and late
SV40 promoter, LTRs from retroviruses, and the mouse
metallothionein-I promoter. Other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their
viruses may also be used.
[0195] Expression Vectors and Cloning Vehicles
[0196] The invention provides expression vectors and cloning
vehicles comprising nucleic acids of the invention, e.g., sequences
encoding the phospholipases of the invention. Expression vectors
and cloning vehicles of the invention can comprise viral particles,
baculovirus, phage, plasmids, phagemids, cosmids, fosmids,
bacterial artificial chromosomes, viral DNA (e.g., vaccinia,
adenovirus, foul pox virus, pseudorabies and derivatives of SV40),
P1-based artificial chromosomes, yeast plasmids, yeast artificial
chromosomes, and any other vectors specific for specific hosts of
interest (such as Bacillus, Aspergillus and yeast). Vectors of the
invention can include chromosomal, non-chromosomal and synthetic
DNA sequences. Large numbers of suitable vectors are known to those
of skill in the art, and are commercially available. Exemplary
vectors are include: bacterial: pQE vectors (Qiagen), pBluescript
plasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,
pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5
(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any
other plasmid or other vector may be used so long as they are
replicable and viable in the host. Low copy number or high copy
number vectors may be employed with the present invention.
[0197] The expression vector may comprise a promoter, a
ribosome-binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression. Mammalian expression vectors
can comprise an origin of replication, any necessary ribosome
binding sites, a polyadenylation site, splice donor and acceptor
sites, transcriptional termination sequences, and 5' flanking
non-transcribed sequences. In some aspects, DNA sequences derived
from the SV40 splice and polyadenylation sites may be used to
provide the required non-transcribed genetic elements.
[0198] In one aspect, the expression vectors contain one or more
selectable marker genes to permit selection of host cells
containing the vector. Such selectable markers include genes
encoding dihydrofolate reductase or genes conferring neomycin
resistance for eukaryotic cell culture, genes conferring
tetracycline or ampicillin resistance in E. coli, and the S.
cerevisiae TRP1 gene. Promoter regions can be selected from any
desired gene using chloramphenicol transferase (CAT) vectors or
other vectors with selectable markers.
[0199] Vectors for expressing the polypeptide or fragment thereof
in eukaryotic cells may also contain enhancers to increase
expression levels. Enhancers are cis-acting elements of DNA,
usually from about 10 to about 300 bp in length that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin bp 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and the adenovirus
enhancers.
[0200] A DNA sequence may be inserted into a vector by a variety of
procedures. In general, the DNA sequence is ligated to the desired
position in the vector following digestion of the insert and the
vector with appropriate restriction endonucleases. Alternatively,
blunt ends in both the insert and the vector may be ligated. A
variety of cloning techniques are known in the art, e.g., as
described in Ausubel and Sambrook. Such procedures and others are
deemed to be within the scope of those skilled in the art.
[0201] The vector may be in the form of a plasmid, a viral
particle, or a phage. Other vectors include chromosomal,
non-chromosomal and synthetic DNA sequences, derivatives of SV40;
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A
variety of cloning and expression vectors for use with prokaryotic
and eukaryotic hosts are described by, e.g., Sambrook.
[0202] Particular bacterial vectors which may be used include the
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia
Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison,
Wis., USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174 pBluescript
II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a,
pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8 and pCM7.
Particular eukaryotic vectors include pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any
other vector may be used as long as it is replicable and viable in
the host cell.
[0203] Host Cells and Transformed Cells
[0204] The invention also provides a transformed cell comprising a
nucleic acid sequence of the invention, e.g., a sequence encoding a
phospholipase of the invention, a vector of the invention. The host
cell may be any of the host cells familiar to those skilled in the
art, including prokaryotic cells, eukaryotic cells, such as
bacterial cells, fungal cells, yeast cells, mammalian cells, insect
cells, or plant cells. Enzymes of the invention can be expressed in
any host cell, e.g., any bacterial cell, any yeast cell, e.g.,
Pichia pastoris, Saccharomyces cerevisiae or Schizosaccharomyces
pombe. Exemplary bacterial cells include E. coli, Lactococcus
lactis, Streptomyces, Bacillus subtilis, Bacillus cereus,
Salmonella typhimurium or any species within the genera Bacillus,
Streptomyces and Staphylococcus. Exemplary insect cells include
Drosophila S2 and Spodoptera Sf9. Exemplary animal cells include
CHO, COS or Bowes melanoma or any mouse or human cell line. The
selection of an appropriate host is within the abilities of those
skilled in the art.
[0205] The vector may be introduced into the host cells using any
of a variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene
transfer. Particular methods include calcium phosphate
transfection, DEAE-Dextran mediated transfection, lipofection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0206] Where appropriate, the engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the invention. Following transformation of a suitable host
strain and growth of the host strain to an appropriate cell
density, the selected promoter may be induced by appropriate means
(e.g., temperature shift or chemical induction) and the cells may
be cultured for an additional period to allow them to produce the
desired polypeptide or fragment thereof.
[0207] Cells can be harvested by centrifugation, disrupted by
physical or chemical means, and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0208] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts and
other cell lines capable of expressing proteins from a compatible
vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.
[0209] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Depending upon the host employed in a recombinant
production procedure, the polypeptides produced by host cells
containing the vector may be glycosylated or may be
non-glycosylated. Polypeptides of the invention may or may not also
include an initial methionine amino acid residue.
[0210] Cell-free translation systems can also be employed to
produce a polypeptide of the invention. Cell-free translation
systems can use mRNAs transcribed from a DNA construct comprising a
promoter operably linked to a nucleic acid encoding the polypeptide
or fragment thereof. In some aspects, the DNA construct may be
linearized prior to conducting an in vitro transcription reaction.
The transcribed mRNA is then incubated with an appropriate
cell-free translation extract, such as a rabbit reticulocyte
extract, to produce the desired polypeptide or fragment
thereof.
[0211] The expression vectors can contain one or more selectable
marker genes to provide a phenotypic trait for selection of
transformed host cells such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
[0212] An exemplary phospholipase C enzyme (having a sequence as
set forth in SEQ ID NO:2) has been over-expressed in active form in
a variety of host systems including gram negative bacteria, such as
E. coli, gram positive bacteria, such as any Bacillus sp. (e.g.,
Bacillus subtilis, Bacillus cereus), yeast host cells (including,
e.g., Pichia pastoris, Saccharomyces sp., such as S. cerevisiae and
S. pombe) and Lactococcus lactis, or mammalian, fungi, plant or
insect cells. The active enzyme is expressed from a variety of
constructs in each host system. These nucleic acid expression
constructs can comprise nucleotides encoding the full-length open
reading frame (composed of the signal sequence, the pro-sequence,
and the mature protein coding sequence) or they can comprise a
subset of these genetic elements either alone or in combination
with heterologous genetic elements that serve as the signal
sequence and/or the pro-sequence for the mature open reading frame.
Each of these systems can serve as a commercial production host for
the expression of PLC for use in the previously described enzymatic
oil degumming processes.
Amplification of Nucleic Acids
[0213] In practicing the invention, nucleic acids encoding the
polypeptides of the invention, or modified nucleic acids, can be
reproduced by, e.g., amplification. The invention provides
amplification primer sequence pairs for amplifying nucleic acids
encoding polypeptides with a phospholipase activity. In one aspect,
the primer pairs are capable of amplifying nucleic acid sequences
of the invention, e.g., including the exemplary SEQ ID NO:1, or a
subsequence thereof; a sequence as set forth in SEQ ID NO:3, or a
subsequence thereof; a sequence as set forth in SEQ ID NO:5, or a
subsequence thereof; and, a sequence as set forth in SEQ ID NO:7,
or a subsequence thereof, etc. One of skill in the art can design
amplification primer sequence pairs for any part of or the full
length of these sequences.
[0214] The invention provides an amplification primer sequence pair
for amplifying a nucleic acid encoding a polypeptide having a
phospholipase activity, wherein the primer pair is capable of
amplifying a nucleic acid comprising a sequence of the invention,
or fragments or subsequences thereof. One or each member of the
amplification primer sequence pair can comprise an oligonucleotide
comprising at least about 10 to 50 consecutive bases of the
sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 consecutive bases of the sequence.
[0215] The invention provides amplification primer pairs, wherein
the primer pair comprises a first member having a sequence as set
forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 residues of a nucleic acid of the
invention, and a second member having a sequence as set forth by
about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, or 25 residues of the complementary strand of the first
member. The invention provides phospholipases generated by
amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. The invention provides
methods of making a phospholipase by amplification, e.g.,
polymerase chain reaction (PCR), using an amplification primer pair
of the invention. In one aspect, the amplification primer pair
amplifies a nucleic acid from a library, e.g., a gene library, such
as an environmental library.
[0216] Amplification reactions can also be used to quantify the
amount of nucleic acid in a sample (such as the amount of message
in a cell sample), label the nucleic acid (e.g., to apply it to an
array or a blot), detect the nucleic acid, or quantify the amount
of a specific nucleic acid in a sample. In one aspect of the
invention, message isolated from a cell or a cDNA library are
amplified. The skilled artisan can select and design suitable
oligonucleotide amplification primers. Amplification methods are
also well known in the art, and include, e.g., polymerase chain
reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND
APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR
STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase
chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;
Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);
transcription amplification (see, e.g., Kwoh (1989) Proc. Natl.
Acad. Sci. USA 86:1173); and, self-sustained sequence replication
(see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q
Beta replicase amplification (see, e.g., Smith (1997) J. Clin.
Microbiol. 35:1477-1491), automated Q-beta replicase amplification
assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and
other RNA polymerase mediated techniques (e.g., NASBA, Cangene,
Mississauga, Ontario); see also Berger (1987) Methods Enzymol.
152:307-316; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and
4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
Determining the Degree of Sequence Identity
[0217] The invention provides isolated and recombinant nucleic
acids comprising sequences having at least about 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)
sequence identity to an exemplary nucleic acid of the invention
(e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27,
SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID
NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ
ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,
SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID
NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ
ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83,
SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID
NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ
ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID
NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,
SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID
NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137,
SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID
NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,
SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID
NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 or SEQ ID
NO:173, and nucleic acids encoding SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ
ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90,
SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID
NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108
SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID
NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126,
SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID
NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144;
NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154,
SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID
NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,
or SEQ ID NO:174) over a region of at least about 50, 75, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,
1400, 1450, 1500, 1550 or more, residues. The invention provides
polypeptides comprising sequences having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to an exemplary polypeptide of the
invention. The extent of sequence identity (homology) may be
determined using any computer program and associated parameters,
including those described herein, such as BLAST 2.2.2. or FASTA
version 3.0t78, with the default parameters. In alternative
embodiments, the sequence identify can be over a region of at least
about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400
consecutive residues, or the full length of the nucleic acid or
polypeptide. The extent of sequence identity (homology) may be
determined using any computer program and associated parameters,
including those described herein, such as BLAST 2.2.2. or FASTA
version 3.0t78, with the default parameters.
[0218] FIG. 11 is a chart describing selected characteristics of
exemplary nucleic acids and polypeptides of the invention,
including sequence identity comparison of the exemplary sequences
to public databases. All sequences described in FIG. 11 have been
subject to a BLAST search (as described in detail, below) against
two sets of databases. The first database set is available through
NCBI (National Center for Biotechnology Information). All results
from searches against these databases are found in the columns
entitled "NR Description", "NR Accession Code", "NR Evalue" or "NR
Organism". "NR" refers to the Non-Redundant nucleotide database
maintained by NCBI. This database is a composite of GenBank,
GenBank updates, and EMBL updates. The entries in the column "NR
Description" refer to the definition line in any given NCBI record,
which includes a description of the sequence, such as the source
organism, gene name/protein name, or some description of the
function of the sequence. The entries in the column "NR Accession
Code" refer to the unique identifier given to a sequence record.
The entries in the column "NR Evalue" refer to the Expect value
(Evalue), which represents the probability that an alignment score
as good as the one found between the query sequence (the sequences
of the invention) and a database sequence would be found in the
same number of comparisons between random sequences as was done in
the present BLAST search. The entries in the column "NR Organism"
refer to the source organism of the sequence identified as the
closest BLAST hit. The second set of databases is collectively
known as the Geneseq.TM. database, which is available through
Thomson Derwent (Philadelphia, Pa.). All results from searches
against this database are found in the columns entitled "Geneseq
Protein Description", "Geneseq Protein Accession Code", "Geneseq
Protein Evalue", "Geneseq DNA Description", "Geneseq DNA Accession
Code" or "Geneseq DNA Evalue". The information found in these
columns is comparable to the information found in the NR columns
described above, except that it was derived from BLAST searches
against the Geneseq database instead of the NCBI databases. In
addition, this table includes the column "Predicted EC No.". An EC
number is the number assigned to a type of enzyme according to a
scheme of standardized enzyme nomenclature developed by the Enzyme
Commission of the Nomenclature Committee of the International Union
of Biochemistry and Molecular Biology (IUBMB). The results in the
"Predicted EC No." column are determined by a BLAST search against
the Kegg (Kyoto Encyclopedia of Genes and Genomes) database. If the
top BLAST match has an Evalue equal to or less than e.sup.-6, the
EC number assigned to the top match is entered into the table. The
EC number of the top hit is used as a guide to what the EC number
of the sequence of the invention might be. The columns "Query DNA
Length" and "Query Protein Length" refer to the number of
nucleotides or the number amino acids, respectively, in the
sequence of the invention that was searched or queried against
either the NCBI or Geneseq databases. The columns "Geneseq or NR
DNA Length" and "Geneseq or NR Protein Length" refer to the number
of nucleotides or the number amino acids, respectively, in the
sequence of the top match from the BLAST search. The results
provided in these columns are from the search that returned the
lower Evalue, either from the NCBI databases or the Geneseq
database. The columns "Geneseq or NR % ID Protein" and "Geneseq or
NR % ID DNA" refer to the percent sequence identity between the
sequence of the invention and the sequence of the top BLAST match.
The results provided in these columns are from the search that
returned the lower Evalue, either from the NCBI databases or the
Geneseq database.
[0219] Homologous sequences also include RNA sequences in which
uridines replace the thymines in the nucleic acid sequences. The
homologous sequences may be obtained using any of the procedures
described herein or may result from the correction of a sequencing
error. It will be appreciated that the nucleic acid sequences as
set forth herein can be represented in the traditional single
character format (see, e.g., Stryer, Lubert. Biochemistry, 3rd Ed.,
W. H Freeman & Co., New York) or in any other format which
records the identity of the nucleotides in a sequence.
[0220] Various sequence comparison programs identified herein are
used in this aspect of the invention. Protein and/or nucleic acid
sequence identities (homologies) may be evaluated using any of the
variety of sequence comparison algorithms and programs known in the
art. Such algorithms and programs include, but are not limited to,
TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman,
Proc. Natl. Acad. Sci. USA 85 (8):2444-2448, 1988; Altschul et al.,
J. Mol. Biol. 215 (3):403-410, 1990; Thompson et al., Nucleic Acids
Res. 22 (2):4673-4680, 1994; Higgins et al., Methods Enzymol.
266:383-402, 1996; Altschul et al., J. Mol. Biol. 215 (3):403-410,
1990; Altschul et al., Nature Genetics 3:266-272, 1993).
[0221] Homology or identity can be measured using sequence analysis
software (e.g., Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). Such software matches
similar sequences by assigning degrees of homology to various
deletions, substitutions and other modifications. The terms
"homology" and "identity" in the context of two or more nucleic
acids or polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same when compared
and aligned for maximum correspondence over a comparison window or
designated region as measured using any number of sequence
comparison algorithms or by manual alignment and visual inspection.
For sequence comparison, one sequence can act as a reference
sequence (an exemplary sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, etc.) to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
entered into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. Default program parameters can be used, or alternative
parameters can be designated. The sequence comparison algorithm
then calculates the percent sequence identities for the test
sequences relative to the reference sequence, based on the program
parameters.
[0222] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous residues. For
example, in alternative aspects of the invention, contiguous
residues ranging anywhere from 20 to the full length of an
exemplary sequence of the invention, e.g., SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, etc., are compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned. If the reference sequence has the requisite
sequence identity to an exemplary sequence of the invention, e.g.,
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
sequence identity to a sequence of the invention, e.g., SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, etc., that sequence is within the
scope of the invention. In alternative embodiments, subsequences
ranging from about 20 to 600, about 50 to 200, and about 100 to 150
are compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequence for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by
the search for similarity method of person & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection. Other algorithms for determining
homology or identity include, for example, in addition to a BLAST
program (Basic Local Alignment Search Tool at the National Center
for Biological Information), ALIGN, AMAS (Analysis of Multiply
Aligned Sequences), AMPS (Protein Multiple Sequence Alignment),
ASSET (Aligned Segment Statistical Evaluation Tool), BANDS,
BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node),
BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points,
BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS,
Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced
Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC,
FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global
Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive
Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local
Content Program), MACAW (Multiple Alignment Construction &
Analysis Workbench), MAP (Multiple Alignment Program), MBLKP,
MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA
(Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such
alignment programs can also be used to screen genome databases to
identify polynucleotide sequences having substantially identical
sequences. A number of genome databases are available, for example,
a substantial portion of the human genome is available as part of
the Human Genome Sequencing Project (Gibbs, 1995). Several genomes
have been sequenced, e.g., M. genitalium (Fraser et al., 1995), M.
jannaschii (Butt et al., 1996), H. influenzae (Fleischmann et al.,
1995), E. coli (Blattner et al., 1997), and yeast (S. cerevisiae)
(Mewes et al., 1997), and D. melanogaster (Adams et al., 2000).
Significant progress has also been made in sequencing the genomes
of model organism, such as mouse, C. elegans, and Arabadopsis sp.
Databases containing genomic information annotated with some
functional information are maintained by different organization,
and are accessible via the internet.
[0223] BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to
practice the invention. They are described, e.g., in Altschul
(1977) Nuc. Acids Res. 25:3389-3402; Altschul (1990) J. Mol. Biol.
215:403-410. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul (1990) supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands. The BLAST algorithm also performs a
statistical analysis of the similarity between two sequences (see,
e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA
90:5873). One measure of similarity provided by BLAST algorithm is
the smallest sum probability (P(N)), which provides an indication
of the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a references sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001. In one
aspect, protein and nucleic acid sequence homologies are evaluated
using the Basic Local Alignment Search Tool ("BLAST"). For example,
five specific BLAST programs can be used to perform the following
task: (1) BLASTP and BLAST3 compare an amino acid query sequence
against a protein sequence database; (2) BLASTN compares a
nucleotide query sequence against a nucleotide sequence database;
(3) BLASTX compares the six-frame conceptual translation products
of a query nucleotide sequence (both strands) against a protein
sequence database; (4) TBLASTN compares a query protein sequence
against a nucleotide sequence database translated in all six
reading frames (both strands); and, (5) TBLASTX compares the
six-frame translations of a nucleotide query sequence against the
six-frame translations of a nucleotide sequence database. The BLAST
programs identify homologous sequences by identifying similar
segments, which are referred to herein as "high-scoring segment
pairs," between a query amino or nucleic acid sequence and a test
sequence which is preferably obtained from a protein or nucleic
acid sequence database. High-scoring segment pairs are preferably
identified (i.e., aligned) by means of a scoring matrix, many of
which are known in the art. Preferably, the scoring matrix used is
the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;
Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably,
the PAM or PAM250 matrices may also be used (see, e.g., Schwartz
and Dayhoff, eds., 1978, Matrices for Detecting Distance
Relationships: Atlas of Protein Sequence and Structure, Washington:
National Biomedical Research Foundation).
[0224] In one aspect of the invention, to determine if a nucleic
acid has the requisite sequence identity to be within the scope of
the invention, the NCBI BLAST 2.2.2 programs is used. default
options to blastp. There are about 38 setting options in the BLAST
2.2.2 program. In this exemplary aspect of the invention, all
default values are used except for the default filtering setting
(i.e., all parameters set to default except filtering which is set
to OFF); in its place a "-F F" setting is used, which disables
filtering. Use of default filtering often results in
Karlin-Altschul violations due to short length of sequence.
[0225] The default values used in this exemplary aspect of the
invention, and to determine the values in FIG. 11, as discussed
above, include: [0226] "Filter for low complexity: ON [0227] Word
Size: 3 [0228] Matrix: Blosum62 [0229] Gap Costs: Existence:11
[0230] Extension:1" Other default settings are: filter for low
complexity OFF, word size of 3 for protein, BLOSUM62 matrix, gap
existence penalty of -11 and a gap extension penalty of -1.
[0231] An exemplary NCBI BLAST 2.2.2 program setting is set forth
in Example 1, below. Note that the "-W" option defaults to 0. This
means that, if not set, the word size defaults to 3 for proteins
and 11 for nucleotides.
Computer Systems and Computer Program Products
[0232] To determine and identify sequence identities, structural
homologies, motifs and the like in silico, a polypeptide or nucleic
acid sequence of the invention can be stored, recorded, and
manipulated on any medium which can be read and accessed by a
computer. Accordingly, the invention provides computers, computer
systems, computer readable mediums, computer programs products and
the like recorded or stored thereon the nucleic acid and
polypeptide sequences of the invention, e.g., an exemplary sequence
of the invention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, etc.
As used herein, the words "recorded" and "stored" refer to a
process for storing information on a computer medium. A skilled
artisan can readily adopt any known methods for recording
information on a computer readable medium to generate manufactures
comprising one or more of the nucleic acid and/or polypeptide
sequences of the invention.
[0233] Another aspect of the invention is a computer readable
medium having recorded thereon at least one nucleic acid and/or
polypeptide sequence of the invention. Computer readable media
include magnetically readable media, optically readable media,
electronically readable media, magnetic/optical media, flash
memories. For example, the computer readable media may be a hard
disk, a floppy disk, a magnetic tape, a flash memory, CD-ROM,
Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read
Only Memory (ROM), or any type of media known to those skilled in
the art.
[0234] Aspects of the invention include systems (e.g., internet
based systems), particularly computer systems, which store and
manipulate the sequences and sequence information described herein.
One example of a computer system 100 is illustrated in block
diagram form in FIG. 1. As used herein, "a computer system" refers
to the hardware components, software components, and data storage
components used to analyze a nucleotide or polypeptide sequence of
the invention. The computer system 100 can include a processor for
processing, accessing and manipulating the sequence data. The
processor 105 can be any well-known type of central processing
unit, such as, for example, the Pentium III from Intel Corporation,
or similar processor from Sun, Motorola, Compaq, AMD or
International Business Machines. The computer system 100 is a
general purpose system that comprises the processor 105 and one or
more internal data storage components 110 for storing data, and one
or more data retrieving devices for retrieving the data stored on
the data storage components. A skilled artisan can readily
appreciate that any one of the currently available computer systems
are suitable.
[0235] In one aspect, the computer system 100 includes a processor
105 connected to a bus which is connected to a main memory 115
(preferably implemented as RAM) and one or more internal data
storage devices 110, such as a hard drive and/or other computer
readable media having data recorded thereon. The computer system
100 can further include one or more data retrieving device 118 for
reading the data stored on the internal data storage devices
110.
[0236] The data retrieving device 118 may represent, for example, a
floppy disk drive, a compact disk drive, a magnetic tape drive, or
a modem capable of connection to a remote data storage system
(e.g., via the internet) etc. In some embodiments, the internal
data storage device 110 is a removable computer readable medium
such as a floppy disk, a compact disk, a magnetic tape, etc.
containing control logic and/or data recorded thereon. The computer
system 100 may advantageously include or be programmed by
appropriate software for reading the control logic and/or the data
from the data storage component once inserted in the data
retrieving device.
[0237] The computer system 100 includes a display 120 which is used
to display output to a computer user. It should also be noted that
the computer system 100 can be linked to other computer systems
125a-c in a network or wide area network to provide centralized
access to the computer system 100. Software for accessing and
processing the nucleotide or amino acid sequences of the invention
can reside in main memory 115 during execution.
[0238] In some aspects, the computer system 100 may further
comprise a sequence comparison algorithm for comparing a nucleic
acid sequence of the invention. The algorithm and sequence(s) can
be stored on a computer readable medium. A "sequence comparison
algorithm" refers to one or more programs which are implemented
(locally or remotely) on the computer system 100 to compare a
nucleotide sequence with other nucleotide sequences and/or
compounds stored within a data storage means. For example, the
sequence comparison algorithm may compare the nucleotide sequences
of an exemplary sequence, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, etc. stored on a computer readable medium to reference
sequences stored on a computer readable medium to identify
homologies or structural motifs.
[0239] The parameters used with the above algorithms may be adapted
depending on the sequence length and degree of homology studied. In
some aspects, the parameters may be the default parameters used by
the algorithms in the absence of instructions from the user. FIG. 2
is a flow diagram illustrating one aspect of a process 200 for
comparing a new nucleotide or protein sequence with a database of
sequences in order to determine the homology levels between the new
sequence and the sequences in the database. The database of
sequences can be a private database stored within the computer
system 100, or a public database such as GENBANK that is available
through the Internet. The process 200 begins at a start state 201
and then moves to a state 202 wherein the new sequence to be
compared is stored to a memory in a computer system 100. As
discussed above, the memory could be any type of memory, including
RAM or an internal storage device.
[0240] The process 200 then moves to a state 204 wherein a database
of sequences is opened for analysis and comparison. The process 200
then moves to a state 206 wherein the first sequence stored in the
database is read into a memory on the computer. A comparison is
then performed at a state 210 to determine if the first sequence is
the same as the second sequence. It is important to note that this
step is not limited to performing an exact comparison between the
new sequence and the first sequence in the database. Well-known
methods are known to those of skill in the art for comparing two
nucleotide or protein sequences, even if they are not identical.
For example, gaps can be introduced into one sequence in order to
raise the homology level between the two tested sequences. The
parameters that control whether gaps or other features are
introduced into a sequence during comparison are normally entered
by the user of the computer system.
[0241] Once a comparison of the two sequences has been performed at
the state 210, a determination is made at a decision state 210
whether the two sequences are the same. Of course, the term "same"
is not limited to sequences that are absolutely identical.
Sequences that are within the homology parameters entered by the
user will be marked as "same" in the process 200. If a
determination is made that the two sequences are the same, the
process 200 moves to a state 214 wherein the name of the sequence
from the database is displayed to the user. This state notifies the
user that the sequence with the displayed name fulfills the
homology constraints that were entered. Once the name of the stored
sequence is displayed to the user, the process 200 moves to a
decision state 218 wherein a determination is made whether more
sequences exist in the database. If no more sequences exist in the
database, then the process 200 terminates at an end state 220.
However, if more sequences do exist in the database, then the
process 200 moves to a state 224 wherein a pointer is moved to the
next sequence in the database so that it can be compared to the new
sequence. In this manner, the new sequence is aligned and compared
with every sequence in the database.
[0242] It should be noted that if a determination had been made at
the decision state 212 that the sequences were not homologous, then
the process 200 would move immediately to the decision state 218 in
order to determine if any other sequences were available in the
database for comparison. Accordingly, one aspect of the invention
is a computer system comprising a processor, a data storage device
having stored thereon a nucleic acid sequence of the invention and
a sequence comparer for conducting the comparison. The sequence
comparer may indicate a homology level between the sequences
compared or identify structural motifs, or it may identify
structural motifs in sequences which are compared to these nucleic
acid codes and polypeptide codes.
[0243] FIG. 3 is a flow diagram illustrating one embodiment of a
process 250 in a computer for determining whether two sequences are
homologous. The process 250 begins at a start state 252 and then
moves to a state 254 wherein a first sequence to be compared is
stored to a memory. The second sequence to be compared is then
stored to a memory at a state 256. The process 250 then moves to a
state 260 wherein the first character in the first sequence is read
and then to a state 262 wherein the first character of the second
sequence is read. It should be understood that if the sequence is a
nucleotide sequence, then the character would normally be either A,
T, C, G or U. If the sequence is a protein sequence, then it can be
a single letter amino acid code so that the first and sequence
sequences can be easily compared. A determination is then made at a
decision state 264 whether the two characters are the same. If they
are the same, then the process 250 moves to a state 268 wherein the
next characters in the first and second sequences are read. A
determination is then made whether the next characters are the
same. If they are, then the process 250 continues this loop until
two characters are not the same. If a determination is made that
the next two characters are not the same, the process 250 moves to
a decision state 274 to determine whether there are any more
characters either sequence to read. If there are not any more
characters to read, then the process 250 moves to a state 276
wherein the level of homology between the first and second
sequences is displayed to the user. The level of homology is
determined by calculating the proportion of characters between the
sequences that were the same out of the total number of sequences
in the first sequence. Thus, if every character in a first 100
nucleotide sequence aligned with a every character in a second
sequence, the homology level would be 100%.
[0244] Alternatively, the computer program can compare a reference
sequence to a sequence of the invention to determine whether the
sequences differ at one or more positions. The program can record
the length and identity of inserted, deleted or substituted
nucleotides or amino acid residues with respect to the sequence of
either the reference or the invention. The computer program may be
a program which determines whether a reference sequence contains a
single nucleotide polymorphism (SNP) with respect to a sequence of
the invention, or, whether a sequence of the invention comprises a
SNP of a known sequence. Thus, in some aspects, the computer
program is a program which identifies SNPs. The method may be
implemented by the computer systems described above and the method
illustrated in FIG. 3. The method can be performed by reading a
sequence of the invention and the reference sequences through the
use of the computer program and identifying differences with the
computer program.
[0245] In other aspects the computer based system comprises an
identifier for identifying features within a nucleic acid or
polypeptide of the invention. An "identifier" refers to one or more
programs which identifies certain features within a nucleic acid
sequence. For example, an identifier may comprise a program which
identifies an open reading frame (ORF) in a nucleic acid sequence.
FIG. 4 is a flow diagram illustrating one aspect of an identifier
process 300 for detecting the presence of a feature in a sequence.
The process 300 begins at a start state 302 and then moves to a
state 304 wherein a first sequence that is to be checked for
features is stored to a memory 115 in the computer system 100. The
process 300 then moves to a state 306 wherein a database of
sequence features is opened. Such a database would include a list
of each feature's attributes along with the name of the feature.
For example, a feature name could be "Initiation Codon" and the
attribute would be "ATG". Another example would be the feature name
"TAATAA Box" and the feature attribute would be "TAATAA". An
example of such a database is produced by the University of
Wisconsin Genetics Computer Group. Alternatively, the features may
be structural polypeptide motifs such as alpha helices, beta
sheets, or functional polypeptide motifs such as enzymatic active
sites, helix-turn-helix motifs or other motifs known to those
skilled in the art. Once the database of features is opened at the
state 306, the process 300 moves to a state 308 wherein the first
feature is read from the database. A comparison of the attribute of
the first feature with the first sequence is then made at a state
310. A determination is then made at a decision state 316 whether
the attribute of the feature was found in the first sequence. If
the attribute was found, then the process 300 moves to a state 318
wherein the name of the found feature is displayed to the user. The
process 300 then moves to a decision state 320 wherein a
determination is made whether move features exist in the database.
If no more features do exist, then the process 300 terminates at an
end state 324. However, if more features do exist in the database,
then the process 300 reads the next sequence feature at a state 326
and loops back to the state 310 wherein the attribute of the next
feature is compared against the first sequence. If the feature
attribute is not found in the first sequence at the decision state
316, the process 300 moves directly to the decision state 320 in
order to determine if any more features exist in the database.
Thus, in one aspect, the invention provides a computer program that
identifies open reading frames (ORFs).
[0246] A polypeptide or nucleic acid sequence of the invention may
be stored and manipulated in a variety of data processor programs
in a variety of formats. For example, a sequence can be stored as
text in a word processing file, such as MicrosoftWORD or
WORDPERFECT or as an ASCII file in a variety of database programs
familiar to those of skill in the art, such as DB2, SYBASE, or
ORACLE. In addition, many computer programs and databases may be
used as sequence comparison algorithms, identifiers, or sources of
reference nucleotide sequences or polypeptide sequences to be
compared to a nucleic acid sequence of the invention. The programs
and databases used to practice the invention include, but are not
limited to: MacPattern (EMBL), DiscoveryBase (Molecular
Applications Group), GeneMine (Molecular Applications Group), Look
(Molecular Applications Group), MacLook (Molecular Applications
Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al,
J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc.
Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp.
App. Biosci. 6:237-245, 1990), Catalyst (Molecular Simulations
Inc.), Catalyst/SHAPE (Molecular Simulations Inc.),
Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (Molecular
Simulations Inc.), Insight II, (Molecular Simulations Inc.),
Discover (Molecular Simulations Inc.), CHARMm (Molecular
Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,
(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations
Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular
Simulations Inc.), ISIS (Molecular Simulations Inc.),
Quanta/Protein Design (Molecular Simulations Inc.), WebLab
(Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular
Simulations Inc.), Gene Explorer (Molecular Simulations Inc.),
SeqFold (Molecular Simulations Inc.), the MDL Available Chemicals
Directory database, the MDL Drug Data Report data base, the
Comprehensive Medicinal Chemistry database, Derwent's World Drug
Index database, the BioByteMasterFile database, the Genbank
database, and the Genseqn database. Many other programs and data
bases would be apparent to one of skill in the art given the
present disclosure.
[0247] Motifs which may be detected using the above programs
include sequences encoding leucine zippers, helix-turn-helix
motifs, glycosylation sites, ubiquitination sites, alpha helices,
and beta sheets, signal sequences encoding signal peptides which
direct the secretion of the encoded proteins, sequences implicated
in transcription regulation such as homeoboxes, acidic stretches,
enzymatic active sites, substrate binding sites, and enzymatic
cleavage sites.
Hybridization of Nucleic Acids
[0248] The invention provides isolated or recombinant nucleic acids
that hybridize under stringent conditions to an exemplary sequence
of the invention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ
ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ
ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,
SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID
NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ
ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77,
SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID
NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ
ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID
NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113,
SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID
NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131,
SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID
NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,
SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID
NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167,
SEQ ID NO:169, SEQ ID NO:171 or SEQ ID NO:173, or a nucleic acid
that encodes a polypeptide comprising a sequence as set forth in
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ
ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,
SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID
NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ
ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66,
SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ
ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,
SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112,
SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID
NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,
SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID
NO:140; SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID
NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158,
SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID
NO:168, SEQ ID NO:170, SEQ ID NO:172, or SEQ ID NO:174. The
stringent conditions can be highly stringent conditions, medium
stringent conditions, low stringent conditions, including the high
and reduced stringency conditions described herein. In alternative
embodiments, nucleic acids of the invention as defined by their
ability to hybridize under stringent conditions can be between
about five residues and the full length of the molecule, e.g., an
exemplary nucleic acid of the invention. For example, they can be
at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,
90, 100, 150, 200, 250, 300, 350, 400 or more residues in length.
Nucleic acids shorter than full length are also included. These
nucleic acids are useful as, e.g., hybridization probes, labeling
probes, PCR oligonucleotide probes, iRNA (single or double
stranded), antisense or sequences encoding antibody binding
peptides (epitopes), motifs, active sites, binding domains,
regulatory domains and the like.
[0249] In one aspect, nucleic acids of the invention are defined by
their ability to hybridize under high stringency comprises
conditions of about 50% formamide at about 37.degree. C. to
42.degree. C. In one aspect, nucleic acids of the invention are
defined by their ability to hybridize under reduced stringency
comprising conditions in about 35% to 25% formamide at about
30.degree. C. to 35.degree. C. Alternatively, nucleic acids of the
invention are defined by their ability to hybridize under high
stringency comprising conditions at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic
acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and
denatured salmon sperm DNA). In one aspect, nucleic acids of the
invention are defined by their ability to hybridize under reduced
stringency conditions comprising 35% formamide at a reduced
temperature of 35.degree. C.
[0250] Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate" conditions above 25% formamide and
"low" conditions below 25% formamide. A specific example of
"moderate" hybridization conditions is when the above hybridization
is conducted at 30% formamide. A specific example of "low
stringency" hybridization conditions is when the above
hybridization is conducted at 10% formamide.
[0251] The temperature range corresponding to a particular level of
stringency can be further narrowed by calculating the purine to
pyrimidine ratio of the nucleic acid of interest and adjusting the
temperature accordingly. Nucleic acids of the invention are also
defined by their ability to hybridize under high, medium, and low
stringency conditions as set forth in Ausubel and Sambrook.
Variations on the above ranges and conditions can be used to
practice the invention and are well known in the art. Hybridization
conditions are discussed further, below.
Oligonucleotides Probes and Methods for Using them
[0252] The invention also provides nucleic acid probes for
identifying nucleic acids encoding a polypeptide having a
phospholipase activity. In one aspect, the probe comprises at least
10 consecutive bases of a sequence as set forth in SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ
ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ
ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,
SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID
NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ
ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77,
SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID
NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ
ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID
NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113,
SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID
NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131,
SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID
NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,
SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID
NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167,
SEQ ID NO:169, SEQ ID NO:171 or SEQ ID NO:173. Alternatively, a
probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,
50, 55, 60, 65, 70, 75, 80, 90, 100, or 150, or more, or about 10
to 50, about 20 to 60 about 30 to 70, consecutive bases of a
sequence as set forth in a sequence of the invention. The probes
identify a nucleic acid by binding or hybridization. The probes can
be used in arrays of the invention, see discussion below,
including, e.g., capillary arrays. The probes of the invention can
also be used to isolate other nucleic acids or polypeptides.
[0253] The probes of the invention can be used to determine whether
a biological sample, such as a soil sample, contains an organism
having a nucleic acid sequence of the invention or an organism from
which the nucleic acid was obtained. In such procedures, a
biological sample potentially harboring the organism from which the
nucleic acid was isolated is obtained and nucleic acids are
obtained from the sample. The nucleic acids are contacted with the
probe under conditions which permit the probe to specifically
hybridize to any complementary sequences present in the sample.
Where necessary, conditions which permit the probe to specifically
hybridize to complementary sequences may be determined by placing
the probe in contact with complementary sequences from samples
known to contain the complementary sequence, as well as control
sequences which do not contain the complementary sequence.
Hybridization conditions, such as the salt concentration of the
hybridization buffer, the formamide concentration of the
hybridization buffer, or the hybridization temperature, may be
varied to identify conditions which allow the probe to hybridize
specifically to complementary nucleic acids (see discussion on
specific hybridization conditions).
[0254] If the sample contains the organism from which the nucleic
acid was isolated, specific hybridization of the probe is then
detected. Hybridization may be detected by labeling the probe with
a detectable agent such as a radioactive isotope, a fluorescent dye
or an enzyme capable of catalyzing the formation of a detectable
product. Many methods for using the labeled probes to detect the
presence of complementary nucleic acids in a sample are familiar to
those skilled in the art. These include Southern Blots, Northern
Blots, colony hybridization procedures, and dot blots. Protocols
for each of these procedures are provided in Ausubel and
Sambrook.
[0255] Alternatively, more than one probe (at least one of which is
capable of specifically hybridizing to any complementary sequences
which are present in the nucleic acid sample), may be used in an
amplification reaction to determine whether the sample contains an
organism containing a nucleic acid sequence of the invention (e.g.,
an organism from which the nucleic acid was isolated). In one
aspect, the probes comprise oligonucleotides. In one aspect, the
amplification reaction may comprise a PCR reaction. PCR protocols
are described in Ausubel and Sambrook (see discussion on
amplification reactions). In such procedures, the nucleic acids in
the sample are contacted with the probes, the amplification
reaction is performed, and any resulting amplification product is
detected. The amplification product may be detected by performing
gel electrophoresis on the reaction products and staining the gel
with an intercalator such as ethidium bromide. Alternatively, one
or more of the probes may be labeled with a radioactive isotope and
the presence of a radioactive amplification product may be detected
by autoradiography after gel electrophoresis.
[0256] Probes derived from sequences near the 3' or 5' ends of a
nucleic acid sequence of the invention can also be used in
chromosome walking procedures to identify clones containing
additional, e.g., genomic sequences. Such methods allow the
isolation of genes which encode additional proteins of interest
from the host organism.
[0257] In one aspect, nucleic acid sequences of the invention are
used as probes to identify and isolate related nucleic acids. In
some aspects, the so-identified related nucleic acids may be cDNAs
or genomic DNAs from organisms other than the one from which the
nucleic acid of the invention was first isolated. In such
procedures, a nucleic acid sample is contacted with the probe under
conditions which permit the probe to specifically hybridize to
related sequences. Hybridization of the probe to nucleic acids from
the related organism is then detected using any of the methods
described above.
[0258] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter. Hybridization may be carried out under conditions of low
stringency, moderate stringency or high stringency. As an example
of nucleic acid hybridization, a polymer membrane containing
immobilized denatured nucleic acids is first prehybridized for 30
minutes at 45.degree. C. in a solution consisting of 0.9 M NaCl, 50
mM NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 10.times.Denhardt's,
and 0.5 mg/ml polyriboadenylic acid. Approximately 2.times.107 cpm
(specific activity 4-9.times.108 cpm/ug) of .sup.32P end-labeled
oligonucleotide probe are then added to the solution. After 12-16
hours of incubation, the membrane is washed for 30 minutes at room
temperature (RT) in 1.times.SET (150 mM NaCl, 20 mM Tris
hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed
by a 30 minute wash in fresh 1.times.SET at Tm-10.degree. C. for
the oligonucleotide probe. The membrane is then exposed to
auto-radiographic film for detection of hybridization signals.
[0259] By varying the stringency of the hybridization conditions
used to identify nucleic acids, such as cDNAs or genomic DNAs,
which hybridize to the detectable probe, nucleic acids having
different levels of homology to the probe can be identified and
isolated. Stringency may be varied by conducting the hybridization
at varying temperatures below the melting temperatures of the
probes. The melting temperature, Tm, is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly complementary probe. Very stringent
conditions are selected to be equal to or about 5.degree. C. lower
than the Tm for a particular probe. The melting temperature of the
probe may be calculated using the following exemplary formulas. For
probes between 14 and 70 nucleotides in length the melting
temperature (Tm) is calculated using the formula: Tm=81.5+16.6(log
[Na+])+0.41(fraction G+C)-(600/N) where N is the length of the
probe. If the hybridization is carried out in a solution containing
formamide, the melting temperature may be calculated using the
equation: Tm=81.5+16.6(log [Na+])+0.41(fraction G+C)-(0.63%
formamide)-(600/N) where N is the length of the probe.
Prehybridization may be carried out in 6.times.SSC,
5.times.Denhardt's reagent, 0.5% SDS, 100 .mu.g denatured
fragmented salmon sperm DNA or 6.times.SSC, 5.times.Denhardt's
reagent, 0.5% SDS, 100 .mu.g denatured fragmented salmon sperm DNA,
50% formamide. Formulas for SSC and Denhardt's and other solutions
are listed, e.g., in Sambrook.
[0260] Hybridization is conducted by adding the detectable probe to
the prehybridization solutions listed above. Where the probe
comprises double stranded DNA, it is denatured before addition to
the hybridization solution. The filter is contacted with the
hybridization solution for a sufficient period of time to allow the
probe to hybridize to cDNAs or genomic DNAs containing sequences
complementary thereto or homologous thereto. For probes over 200
nucleotides in length, the hybridization may be carried out at
15-25.degree. C. below the Tm. For shorter probes, such as
oligonucleotide probes, the hybridization may be conducted at
5-10.degree. C. below the Tm. In one aspect, hybridizations in
6.times.SSC are conducted at approximately 68.degree. C. In one
aspect, hybridizations in 50% formamide containing solutions are
conducted at approximately 42.degree. C. All of the foregoing
hybridizations would be considered to be under conditions of high
stringency.
[0261] Following hybridization, the filter is washed to remove any
non-specifically bound detectable probe. The stringency used to
wash the filters can also be varied depending on the nature of the
nucleic acids being hybridized, the length of the nucleic acids
being hybridized, the degree of complementarity, the nucleotide
sequence composition (e.g., GC v. AT content), and the nucleic acid
type (e.g., RNA v. DNA). Examples of progressively higher
stringency condition washes are as follows: 2.times.SSC, 0.1% SDS
at room temperature for 15 minutes (low stringency); 0.1.times.SSC,
0.5% SDS at room temperature for 30 minutes to 1 hour (moderate
stringency); 0.1.times.SSC, 0.5% SDS for 15 to 30 minutes at
between the hybridization temperature and 68.degree. C. (high
stringency); and 0.15M NaCl for 15 minutes at 72.degree. C. (very
high stringency). A final low stringency wash can be conducted in
0.1.times.SSC at room temperature. The examples above are merely
illustrative of one set of conditions that can be used to practice
the invention, e.g., to wash filters. One of skill in the art would
know that there are numerous recipes for different stringency
washes, all of which can be used to practice the invention.
[0262] Nucleic acids which have hybridized to the probe can be
identified by autoradiography or other conventional techniques. The
above procedure may be modified to identify nucleic acids having
decreasing levels of homology to the probe sequence. For example,
to obtain nucleic acids of decreasing homology to the detectable
probe, less stringent conditions may be used. For example, the
hybridization temperature may be decreased in increments of
5.degree. C. from 68.degree. C. to 42.degree. C. in a hybridization
buffer having a Na+ concentration of approximately 1M. Following
hybridization, the filter may be washed with 2.times.SSC, 0.5% SDS
at the temperature of hybridization. These conditions are
considered to be "moderate" conditions above 50.degree. C. and
"low" conditions below 50.degree. C. An example of "moderate"
hybridization conditions is when the above hybridization is
conducted at 55.degree. C. An example of "low stringency"
hybridization conditions is when the above hybridization is
conducted at 45.degree. C.
[0263] Alternatively, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. C. In this case, the concentration of formamide in
the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to
the probe. Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate" conditions above 25% formamide and
"low" conditions below 25% formamide. A specific example of
"moderate" hybridization conditions is when the above hybridization
is conducted at 30% formamide. A specific example of "low
stringency" hybridization conditions is when the above
hybridization is conducted at 10% formamide.
[0264] These probes and methods of the invention can be used to
isolate nucleic acids having a sequence with at least about 99%, at
least 98%, at least 97%, at least 96%, at least 95%, at least 90%,
at least 85%, at least 80%, at least 75%, at least 70%, at least
65%, at least 60%, at least 55%, or at least 50% homology to a
nucleic acid sequence of the invention comprising at least about
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350,
400, or 500 consecutive bases thereof, and the sequences
complementary thereto. Homology may be measured using an alignment
algorithm, as discussed herein. For example, the homologous
polynucleotides may have a coding sequence which is a naturally
occurring allelic variant of one of the coding sequences described
herein. Such allelic variants may have a substitution, deletion or
addition of one or more nucleotides when compared to nucleic acids
of the invention.
[0265] Additionally, the probes and methods of the invention may be
used to isolate nucleic acids which encode polypeptides having at
least about 99%, at least 95%, at least 90%, at least 85%, at least
80%, at least 75%, at least 70%, at least 65%, at least 60%, at
least 55%, or at least 50% sequence identity (homology) to a
polypeptide of the invention comprising at least 5, 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof as
determined using a sequence alignment algorithm (e.g., such as the
FASTA version 3.0t78 algorithm with the default parameters, or a
BLAST 2.2.2 program with exemplary settings as set forth
herein).
Inhibiting Expression of Phospholipases
[0266] The invention further provides for nucleic acids
complementary to (e.g., antisense sequences to) the nucleic acids
of the invention, e.g., phospholipase-encoding nucleic acids.
Antisense sequences are capable of inhibiting the transport,
splicing or transcription of phospholipase-encoding genes. The
inhibition can be effected through the targeting of genomic DNA or
messenger RNA. The transcription or function of targeted nucleic
acid can be inhibited, for example, by hybridization and/or
cleavage. One particularly useful set of inhibitors provided by the
present invention includes oligonucleotides which are able to
either bind phospholipase gene or message, in either case
preventing or inhibiting the production or function of
phospholipase enzyme. The association can be though sequence
specific hybridization. Another useful class of inhibitors includes
oligonucleotides which cause inactivation or cleavage of
phospholipase message. The oligonucleotide can have enzyme activity
which causes such cleavage, such as ribozymes. The oligonucleotide
can be chemically modified or conjugated to an enzyme or
composition capable of cleaving the complementary nucleic acid. One
may screen a pool of many different such oligonucleotides for those
with the desired activity.
[0267] Inhibition of phospholipase expression can have a variety of
industrial applications. For example, inhibition of phospholipase
expression can slow or prevent spoilage. Spoilage can occur when
lipids or polypeptides, e.g., structural lipids or polypeptides,
are enzymatically degraded. This can lead to the deterioration, or
rot, of fruits and vegetables. In one aspect, use of compositions
of the invention that inhibit the expression and/or activity of
phospholipase, e.g., antibodies, antisense oligonucleotides,
ribozymes and RNAi, are used to slow or prevent spoilage. Thus, in
one aspect, the invention provides methods and compositions
comprising application onto a plant or plant product (e.g., a
fruit, seed, root, leaf, etc.) antibodies, antisense
oligonucleotides, ribozymes and RNAi of the invention to slow or
prevent spoilage. These compositions also can be expressed by the
plant (e.g., a transgenic plant) or another organism (e.g., a
bacterium or other microorganism transformed with a phospholipase
gene of the invention).
[0268] The compositions of the invention for the inhibition of
phospholipase expression (e.g., antisense, iRNA, ribozymes,
antibodies) can be used as pharmaceutical compositions.
[0269] Antisense Oligonucleotides
[0270] The invention provides antisense oligonucleotides capable of
binding phospholipase message which can inhibit phospholipase
activity by targeting mRNA. Strategies for designing antisense
oligonucleotides are well described in the scientific and patent
literature, and the skilled artisan can design such phospholipase
oligonucleotides using the novel reagents of the invention. For
example, gene walking/RNA mapping protocols to screen for effective
antisense oligonucleotides are well known in the art, see, e.g., Ho
(2000) Methods Enzymol. 314:168-183, describing an RNA mapping
assay, which is based on standard molecular techniques to provide
an easy and reliable method for potent antisense sequence
selection. See also Smith (2000) Eur. J. Pharm. Sci.
11:191-198.
[0271] Naturally occurring nucleic acids are used as antisense
oligonucleotides. The antisense oligonucleotides can be of any
length; for example, in alternative aspects, the antisense
oligonucleotides are between about 5 to 100, about 10 to 80, about
15 to 60, about 18 to 40. The optimal length can be determined by
routine screening. The antisense oligonucleotides can be present at
any concentration. The optimal concentration can be determined by
routine screening. A wide variety of synthetic, non-naturally
occurring nucleotide and nucleic acid analogues are known which can
address this potential problem. For example, peptide nucleic acids
(PNAs) containing non-ionic backbones, such as
N-(2-aminoethyl)glycine units can be used. Antisense
oligonucleotides having phosphorothioate linkages can also be used,
as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl
Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana
Press, Totowa, N.J., 1996). Antisense oligonucleotides having
synthetic DNA backbone analogues provided by the invention can also
include phosphoro-dithioate, methylphosphonate, phosphoramidate,
alkyl phosphotriester, sulfamate, 3'-thioacetal,
methylene(methylimino), 3'-N-carbamate, and morpholino carbamate
nucleic acids, as described above.
[0272] Combinatorial chemistry methodology can be used to create
vast numbers of oligonucleotides that can be rapidly screened for
specific oligonucleotides that have appropriate binding affinities
and specificities toward any target, such as the sense and
antisense phospholipase sequences of the invention (see, e.g., Gold
(1995) J. of Biol. Chem. 270:13581-13584).
[0273] Inhibitory Ribozymes
[0274] The invention provides for with ribozymes capable of binding
phospholipase message which can inhibit phospholipase enzyme
activity by targeting mRNA. Strategies for designing ribozymes and
selecting the phospholipase-specific antisense sequence for
targeting are well described in the scientific and patent
literature, and the skilled artisan can design such ribozymes using
the novel reagents of the invention. Ribozymes act by binding to a
target RNA through the target RNA binding portion of a ribozyme
which is held in close proximity to an enzymatic portion of the RNA
that cleaves the target RNA. Thus, the ribozyme recognizes and
binds a target RNA through complementary base-pairing, and once
bound to the correct site, acts enzymatically to cleave and
inactivate the target RNA. Cleavage of a target RNA in such a
manner will destroy its ability to direct synthesis of an encoded
protein if the cleavage occurs in the coding sequence. After a
ribozyme has bound and cleaved its RNA target, it is typically
released from that RNA and so can bind and cleave new targets
repeatedly.
[0275] In some circumstances, the enzymatic nature of a ribozyme
can be advantageous over other technologies, such as antisense
technology (where a nucleic acid molecule simply binds to a nucleic
acid target to block its transcription, translation or association
with another molecule) as the effective concentration of ribozyme
necessary to effect a therapeutic treatment can be lower than that
of an antisense oligonucleotide. This potential advantage reflects
the ability of the ribozyme to act enzymatically. Thus, a single
ribozyme molecule is able to cleave many molecules of target RNA.
In addition, a ribozyme is typically a highly specific inhibitor,
with the specificity of inhibition depending not only on the base
pairing mechanism of binding, but also on the mechanism by which
the molecule inhibits the expression of the RNA to which it binds.
That is, the inhibition is caused by cleavage of the RNA target and
so specificity is defined as the ratio of the rate of cleavage of
the targeted RNA over the rate of cleavage of non-targeted RNA.
This cleavage mechanism is dependent upon factors additional to
those involved in base pairing. Thus, the specificity of action of
a ribozyme can be greater than that of antisense oligonucleotide
binding the same RNA site.
[0276] The enzymatic ribozyme RNA molecule can be formed in a
hammerhead motif, but may also be formed in the motif of a hairpin,
hepatitis delta virus, group I intron or RNaseP-like RNA (in
association with an RNA guide sequence). Examples of such
hammerhead motifs are described by Rossi (1992) Aids Research and
Human Retroviruses 8:183; hairpin motifs by Hampel (1989)
Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the
hepatitis delta virus motif by Perrotta (1992) Biochemistry 31:16;
the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and the
group I intron by Cech U.S. Pat. No. 4,987,071. The recitation of
these specific motifs is not intended to be limiting; those skilled
in the art will recognize that an enzymatic RNA molecule of this
invention has a specific substrate binding site complementary to
one or more of the target gene RNA regions, and has nucleotide
sequence within or surrounding that substrate binding site which
imparts an RNA cleaving activity to the molecule.
[0277] RNA Interference (RNAi)
[0278] In one aspect, the invention provides an RNA inhibitory
molecule, a so-called "RNAi" molecule, comprising a phospholipase
sequence of the invention. The RNAi molecule comprises a
double-stranded RNA (dsRNA) molecule. The RNAi can inhibit
expression of a phospholipase gene. In one aspect, the RNAi is
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex
nucleotides in length. While the invention is not limited by any
particular mechanism of action, the RNAi can enter a cell and cause
the degradation of a single-stranded RNA (ssRNA) of similar or
identical sequences, including endogenous mRNAs. When a cell is
exposed to double-stranded RNA (dsRNA), mRNA from the homologous
gene is selectively degraded by a process called RNA interference
(RNAi). A possible basic mechanism behind RNAi is the breaking of a
double-stranded RNA (dsRNA) matching a specific gene sequence into
short pieces called short interfering RNA, which trigger the
degradation of mRNA that matches its sequence. In one aspect, the
RNAi's of the invention are used in gene-silencing therapeutics,
see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one
aspect, the invention provides methods to selectively degrade RNA
using the RNAi's of the invention. The process may be practiced in
vitro, ex vivo or in vivo. In one aspect, the RNAi molecules of the
invention can be used to generate a loss-of-function mutation in a
cell, an organ or an animal. Methods for making and using RNAi
molecules for selectively degrade RNA are well known in the art,
see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109;
6,489,127.
Modification of Nucleic Acids
[0279] The invention provides methods of generating variants of the
nucleic acids of the invention, e.g., those encoding a
phospholipase enzyme. In alternative embodiment, the invention
provides methods for modifying an enzyme of the invention, e.g., by
mutation of its coding sequence by random or stochastic methods,
or, non-stochastic, or "directed evolution," such as Gene Site
Saturation Mutagenesis.TM. (GSSM.TM.), to alter the enzymes pH
range of activity or range of optimal activity, temperature range
of activity or range of optimal activity, specificity, activity
(kinetics); the enzyme's use of glycosylation, phosphorylation or
metals (e.g., Ca, Mg, Zn, Fe, Na), e.g., to impact pH/temperature
stability. The invention provides methods for modifying an enzyme
of the invention, e.g., by mutation of its coding sequence, e.g.,
by GSSM.TM., to increase its resistance to protease activity. The
invention provides methods for modifying an enzyme of the
invention, e.g., by mutation of its coding sequence, e.g., by
GSSM.TM., to modify the enzyme's use of metal chelators specific
for Ca, Mg, Na that would not chelate Zn. The invention provides
methods for modifying an enzyme of the invention, e.g., by mutation
of its coding sequence, e.g., by GSSM.TM., that would have a
desired combination of activities, e.g., PI, PA and PC/PE specific
PLCs.
[0280] These methods can be repeated or used in various
combinations to generate phospholipase enzymes having an altered or
different activity or an altered or different stability from that
of a phospholipase encoded by the template nucleic acid. These
methods also can be repeated or used in various combinations, e.g.,
to generate variations in gene/message expression, message
translation or message stability. In another aspect, the genetic
composition of a cell is altered by, e.g., modification of a
homologous gene ex vivo, followed by its reinsertion into the
cell.
[0281] A nucleic acid of the invention can be altered by any means.
For example, random or stochastic methods, or, non-stochastic, or
"directed evolution," methods.
[0282] Methods for random mutation of genes are well known in the
art, see, e.g., U.S. Pat. No. 5,830,696. For example, mutagens can
be used to randomly mutate a gene. Mutagens include, e.g.,
ultraviolet light or gamma irradiation, or a chemical mutagen,
e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or
in combination, to induce DNA breaks amenable to repair by
recombination. Other chemical mutagens include, for example, sodium
bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
Other mutagens are analogues of nucleotide precursors, e.g.,
nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. These
agents can be added to a PCR reaction in place of the nucleotide
precursor thereby mutating the sequence. Intercalating agents such
as proflavine, acriflavine, quinacrine and the like can also be
used.
[0283] Any technique in molecular biology can be used, e.g., random
PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA
89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,
e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively,
nucleic acids, e.g., genes, can be reassembled after random, or
"stochastic," fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242;
6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238;
5,605,793. In alternative aspects, modifications, additions or
deletions are introduced by error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.), synthetic ligation reassembly (SLR),
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation, and/or a combination of
these and other methods.
[0284] The following publications describe a variety of recursive
recombination procedures and/or methods which can be incorporated
into the methods of the invention: Stemmer (1999) "Molecular
breeding of viruses for targeting and other clinical properties"
Tumor Targeting 4:1-4; Ness (1999) Nature Biotechnology 17:893-896;
Chang (1999) "Evolution of a cytokine using DNA family shuffling"
Nature Biotechnology 17:793-797; Minshull (1999) "Protein evolution
by molecular breeding" Current Opinion in Chemical Biology
3:284-290; Christians (1999) "Directed evolution of thymidine
kinase for AZT phosphorylation using DNA family shuffling" Nature
Biotechnology 17:259-264; Crameri (1998) "DNA shuffling of a family
of genes from diverse species accelerates directed evolution"
Nature 391:288-291; Crameri (1997) "Molecular evolution of an
arsenate detoxification pathway by DNA shuffling," Nature
Biotechnology 15:436-438; Zhang (1997) "Directed evolution of an
effective fucosidase from a galactosidase by DNA shuffling and
screening" Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten et al.
(1997) "Applications of DNA Shuffling to Pharmaceuticals and
Vaccines" Current Opinion in Biotechnology 8:724-733; Crameri et
al. (1996) "Construction and evolution of antibody-phage libraries
by DNA shuffling" Nature Medicine 2:100-103; Crameri et al. (1996)
"Improved green fluorescent protein by molecular evolution using
DNA shuffling" Nature Biotechnology 14:315-319; Gates et al. (1996)
"Affinity selective isolation of ligands from peptide libraries
through display on a lac repressor `headpiece dimer`" Journal of
Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and
Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH
Publishers, New York. pp. 447-457; Crameri and Stemmer (1995)
"Combinatorial multiple cassette mutagenesis creates all the
permutations of mutant and wildtype cassettes" BioTechniques
18:194-195; Stemmer et al. (1995) "Single-step assembly of a gene
and entire plasmid form large numbers of oligodeoxyribonucleotides"
Gene, 164:49-53; Stemmer (1995) "The Evolution of Molecular
Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence
Space" Bio/Technology 13:549-553; Stemmer (1994) "Rapid evolution
of a protein in vitro by DNA shuffling" Nature 370:389-391; and
Stemmer (1994) "DNA shuffling by random fragmentation and
reassembly: In vitro recombination for molecular evolution." Proc.
Natl. Acad. Sci. USA 91:10747-10751.
[0285] Mutational methods of generating diversity include, for
example, site-directed mutagenesis (Ling et al. (1997) "Approaches
to DNA mutagenesis: an overview" Anal Biochem. 254 (2): 157-178;
Dale et al. (1996) "Oligonucleotide-directed random mutagenesis
using the phosphorothioate method" Methods Mol. Biol. 57:369-374;
Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19:423-462;
Botstein & Shortle (1985) "Strategies and applications of in
vitro mutagenesis" Science 229:1193-1201; Carter (1986)
"Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987)
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic
Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.
eds., Springer Verlag, Berlin)); mutagenesis using uracil
containing templates (Kunkel (1985) "Rapid and efficient
site-specific mutagenesis without phenotypic selection" Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and
efficient site-specific mutagenesis without phenotypic selection"
Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant
Trp repressors with new DNA-binding specificities" Science
242:240-245); oligonucleotide-directed mutagenesis (Methods in
Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350
(1987); Zoller & Smith (1982) "Oligonucleotide-directed
mutagenesis using M13-derived vectors: an efficient and general
procedure for the production of point mutations in any DNA
fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith
(1983) "Oligonucleotide-directed mutagenesis of DNA fragments
cloned into M13 vectors" Methods in Enzymol. 100:468-500; and
Zoller & Smith (1987) "Oligonucleotide-directed mutagenesis: a
simple method using two oligonucleotide primers and a
single-stranded DNA template" Methods in Enzymol. 154:329-350);
phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985)
"The use of phosphorothioate-modified DNA in restriction enzyme
reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764;
Taylor et al. (1985) "The rapid generation of
oligonucleotide-directed mutations at high frequency using
phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787
(1985); Nakamaye (1986) "Inhibition of restriction endonuclease Nci
I cleavage by phosphorothioate groups and its application to
oligonucleotide-directed mutagenesis" Nucl. Acids Res. 14:
9679-9698; Sayers et al. (1988) "Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl.
Acids Res. 16:791-802; and Sayers et al. (1988) "Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide"
Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA
(Kramer et al. (1984) "The gapped duplex DNA approach to
oligonucleotide-directed mutation construction" Nucl. Acids Res.
12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol.
"Oligonucleotide-directed construction of mutations via gapped
duplex DNA" 154:350-367; Kramer et al. (1988) "Improved enzymatic
in vitro reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations" Nucl. Acids
Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed
construction of mutations: a gapped duplex DNA procedure without
enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).
[0286] Additional protocols used in the methods of the invention
include point mismatch repair (Kramer (1984) "Point Mismatch
Repair" Cell 38:879-887), mutagenesis using repair-deficient host
strains (Carter et al. (1985) "Improved oligonucleotide
site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13:
4431-4443; and Carter (1987) "Improved oligonucleotide-directed
mutagenesis using M13 vectors" Methods in Enzymol. 154: 382-403),
deletion mutagenesis (Eghtedarzadeh (1986) "Use of oligonucleotides
to generate large deletions" Nucl. Acids Res. 14: 5115),
restriction-selection and restriction-selection and
restriction-purification (Wells et al. (1986) "Importance of
hydrogen-bond formation in stabilizing the transition state of
subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis
by total gene synthesis (Nambiar et al. (1984) "Total synthesis and
cloning of a gene coding for the ribonuclease S protein" Science
223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and
expression of a gene for the a-subunit of bovine rod outer segment
guanine nucleotide-binding protein (transducin)" Nucl. Acids Res.
14: 6361-6372; Wells et al. (1985) "Cassette mutagenesis: an
efficient method for generation of multiple mutations at defined
sites" Gene 34:315-323; and Grundstrom et al. (1985)
"Oligonucleotide-directed mutagenesis by microscale `shot-gun` gene
synthesis" Nucl. Acids Res. 13: 3305-3316), double-strand break
repair (Mandecki (1986); Arnold (1993) "Protein engineering for
unusual environments" Current Opinion in Biotechnology 4:450-455.
"Oligonucleotide-directed double-strand break repair in plasmids of
Escherichia coli: a method for site-specific mutagenesis" Proc.
Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many of
the above methods can be found in Methods in Enzymology Volume 154,
which also describes useful controls for trouble-shooting problems
with various mutagenesis methods.
[0287] See also U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997),
"Methods for In Vitro Recombination;" U.S. Pat. No. 5,811,238 to
Stemmer et al. (Sep. 22, 1998) "Methods for Generating
Polynucleotides having Desired Characteristics by Iterative
Selection and Recombination;" U.S. Pat. No. 5,830,721 to Stemmer et
al. (Nov. 3, 1998), "DNA Mutagenesis by Random Fragmentation and
Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10,
1998) "End-Complementary Polymerase Reaction;" U.S. Pat. No.
5,837,458 to Minshull, et al. (Nov. 17, 1998), "Methods and
Compositions for Cellular and Metabolic Engineering;" WO 95/22625,
Stemmer and Crameri, "Mutagenesis by Random Fragmentation and
Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End
Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer
and Crameri "Methods for Generating Polynucleotides having Desired
Characteristics by Iterative Selection and Recombination;" WO
97/35966 by Minshull and Stemmer, "Methods and Compositions for
Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al.
"Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et
al. "Antigen Library Immunization;" WO 99/41369 by Punnonen et al.
"Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et
al. "Optimization of Immunomodulatory Properties of Genetic
Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by
Random Fragmentation and Reassembly;" EP 0932670 by Stemmer
"Evolving Cellular DNA Uptake by Recursive Sequence Recombination;"
WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and
Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al.,
"Human Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al.
"Evolution of Whole Cells and Organisms by Recursive Sequence
Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and
Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer
et al., "Methods for Optimization of Gene Therapy by Recursive
Sequence Shuffling and Selection," WO 00/00632, "Methods for
Generating Highly Diverse Libraries," WO 00/09679, "Methods for
Obtaining in Vitro Recombined Polynucleotide Sequence Banks and
Resulting Sequences," WO 98/42832 by Arnold et al., "Recombination
of Polynucleotide Sequences Using Random or Defined Primers," WO
99/29902 by Arnold et al., "Method for Creating Polynucleotide and
Polypeptide Sequences," WO 98/41653 by Vind, "An in vitro Method
for Construction of a DNA Library," WO 98/41622 by Borchert et al.,
"Method for Constructing a Library Using DNA Shuffling," and WO
98/42727 by Pati and Zarling, "Sequence Alterations using
Homologous Recombination."
[0288] Certain U.S. applications provide additional details
regarding various diversity generating methods, including
"SHUFFLING OF CODON ALTERED GENES" by Patten et al. filed Sep. 28,
1999, (U.S. Ser. No. 09/407,800); "EVOLUTION OF WHOLE CELLS AND
ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION" by del Cardayre et
al., filed Jul. 15, 1998 (U.S. Ser. No. 09/166,188), and Jul. 15,
1999 (U.S. Ser. No. 09/354,922); "OLIGONUCLEOTIDE MEDIATED NUCLEIC
ACID RECOMBINATION" by Crameri et al., filed Sep. 28, 1999 (U.S.
Ser. No. 09/408,392), and "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID
RECOMBINATION" by Crameri et al., filed Jan. 18, 2000
(PCT/US00/01203); "USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESIS
FOR SYNTHETIC SHUFFLING" by Welch et al., filed Sep. 28, 1999 (U.S.
Ser. No. 09/408,393); "METHODS FOR MAKING CHARACTER STRINGS,
POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS"
by Selifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and,
e.g. "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES &
POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al.,
filed Jul. 18, 2000 (U.S. Ser. No. 09/618,579); "METHODS OF
POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" by
Selifonov and Stemmer, filed Jan. 18, 2000 (PCT/US00/01138); and
"SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATED RECOMBINATION AND
NUCLEIC ACID FRAGMENT ISOLATION" by Affholter, filed Sep. 6, 2000
(U.S. Ser. No. 09/656,549).
[0289] Non-stochastic, or "directed evolution," methods include,
e.g., saturation mutagenesis (e.g., GSSM.TM.), synthetic ligation
reassembly (SLR), or a combination thereof are used to modify the
nucleic acids of the invention to generate phospholipases with new
or altered properties (e.g., activity under highly acidic or
alkaline conditions, high temperatures, and the like). Polypeptides
encoded by the modified nucleic acids can be screened for an
activity before testing for a phospholipase or other activity. Any
testing modality or protocol can be used, e.g., using a capillary
array platform. See, e.g., U.S. Pat. Nos. 6,280,926; 5,939,250.
[0290] Saturation Mutagenesis, or, GSSM.TM.
[0291] In one aspect of the invention, non-stochastic gene
modification, a "directed evolution process," is used to generate
phospholipases with new or altered properties. Variations of this
method have been termed "gene site mutagenesis," "site-saturation
mutagenesis," "Gene Site Saturation Mutagenesis.TM." or simply
"GSSM.TM.." It can be used in combination with other mutagenization
processes. See, e.g., U.S. Pat. Nos. 6,171,820; 6,238,884. In one
aspect, GSSM.TM. comprises providing a template polynucleotide and
a plurality of oligonucleotides, wherein each oligonucleotide
comprises a sequence homologous to the template polynucleotide,
thereby targeting a specific sequence of the template
polynucleotide, and a sequence that is a variant of the homologous
gene; generating progeny polynucleotides comprising non-stochastic
sequence variations by replicating the template polynucleotide with
the oligonucleotides, thereby generating polynucleotides comprising
homologous gene sequence variations.
[0292] In one aspect, codon primers containing a degenerate N,N,G/T
sequence are used to introduce point mutations into a
polynucleotide, so as to generate a set of progeny polypeptides in
which a full range of single amino acid substitutions is
represented at each amino acid position, e.g., an amino acid
residue in an enzyme active site or ligand binding site targeted to
be modified. These oligonucleotides can comprise a contiguous first
homologous sequence, a degenerate N,N,G/T sequence, and,
optionally, a second homologous sequence. The downstream progeny
translational products from the use of such oligonucleotides
include all possible amino acid changes at each amino acid site
along the polypeptide, because the degeneracy of the N,N,G/T
sequence includes codons for all 20 amino acids. In one aspect, one
such degenerate oligonucleotide (comprised of, e.g., one degenerate
N,N,G/T cassette) is used for subjecting each original codon in a
parental polynucleotide template to a full range of codon
substitutions. In another aspect, at least two degenerate cassettes
are used--either in the same oligonucleotide or not, for subjecting
at least two original codons in a parental polynucleotide template
to a full range of codon substitutions. For example, more than one
N,N,G/T sequence can be contained in one oligonucleotide to
introduce amino acid mutations at more than one site. This
plurality of N,N,G/T sequences can be directly contiguous, or
separated by one or more additional nucleotide sequence(s). In
another aspect, oligonucleotides serviceable for introducing
additions and deletions can be used either alone or in combination
with the codons containing an N,N,G/T sequence, to introduce any
combination or permutation of amino acid additions, deletions,
and/or substitutions.
[0293] In one aspect, simultaneous mutagenesis of two or more
contiguous amino acid positions is done using an oligonucleotide
that contains contiguous N,N,G/T triplets, i.e. a degenerate
(N,N,G/T)n sequence. In another aspect, degenerate cassettes having
less degeneracy than the N,N,G/T sequence are used. For example, it
may be desirable in some instances to use (e.g. in an
oligonucleotide) a degenerate triplet sequence comprised of only
one N, where said N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two positions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.
[0294] In one aspect, use of degenerate triplets (e.g., N,N,G/T
triplets) allows for systematic and easy generation of a full range
of possible natural amino acids (for a total of 20 amino acids)
into each and every amino acid position in a polypeptide (in
alternative aspects, the methods also include generation of less
than all possible substitutions per amino acid residue, or codon,
position). For example, for a 100 amino acid polypeptide, 2000
distinct species (i.e. 20 possible amino acids per
position.times.100 amino acid positions) can be generated. Through
the use of an oligonucleotide or set of oligonucleotides containing
a degenerate N,N,G/T triplet, 32 individual sequences can code for
all 20 possible natural amino acids. Thus, in a reaction vessel in
which a parental polynucleotide sequence is subjected to saturation
mutagenesis using at least one such oligonucleotide, there are
generated 32 distinct progeny polynucleotides encoding 20 distinct
polypeptides. In contrast, the use of a non-degenerate
oligonucleotide in site-directed mutagenesis leads to only one
progeny polypeptide product per reaction vessel. Nondegenerate
oligonucleotides can optionally be used in combination with
degenerate primers disclosed; for example, nondegenerate
oligonucleotides can be used to generate specific point mutations
in a working polynucleotide. This provides one means to generate
specific silent point mutations, point mutations leading to
corresponding amino acid changes, and point mutations that cause
the generation of stop codons and the corresponding expression of
polypeptide fragments.
[0295] In one aspect, each saturation mutagenesis reaction vessel
contains polynucleotides encoding at least 20 progeny polypeptide
(e.g., phospholipase) molecules such that all 20 natural amino
acids are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide (other aspects use less than all 20 natural
combinations). The 32-fold degenerate progeny polypeptides
generated from each saturation mutagenesis reaction vessel can be
subjected to clonal amplification (e.g. cloned into a suitable
host, e.g., E. coli host, using, e.g., an expression vector) and
subjected to expression screening. When an individual progeny
polypeptide is identified by screening to display a favorable
change in property (when compared to the parental polypeptide, such
as increased phospholipase activity under alkaline or acidic
conditions), it can be sequenced to identify the correspondingly
favorable amino acid substitution contained therein.
[0296] In one aspect, upon mutagenizing each and every amino acid
position in a parental polypeptide using saturation mutagenesis as
disclosed herein, favorable amino acid changes may be identified at
more than one amino acid position. One or more new progeny
molecules can be generated that contain a combination of all or
part of these favorable amino acid substitutions. For example, if 2
specific favorable amino acid changes are identified in each of 3
amino acid positions in a polypeptide, the permutations include 3
possibilities at each position (no change from the original amino
acid, and each of two favorable changes) and 3 positions. Thus,
there are 3.times.3.times.3 or 27 total possibilities, including 7
that were previously examined--6 single point mutations (i.e. 2 at
each of three positions) and no change at any position.
[0297] In another aspect, site-saturation mutagenesis can be used
together with another stochastic or non-stochastic means to vary
sequence, e.g., synthetic ligation reassembly (see below),
shuffling, chimerization, recombination and other mutagenizing
processes and mutagenizing agents. This invention provides for the
use of any mutagenizing process(es), including saturation
mutagenesis, in an iterative manner.
[0298] Synthetic Ligation Reassembly (SLR)
[0299] The invention provides a non-stochastic gene modification
system termed "synthetic ligation reassembly," or simply "SLR," a
"directed evolution process," to generate phospholipases with new
or altered properties. SLR is a method of ligating oligonucleotide
fragments together non-stochastically. This method differs from
stochastic oligonucleotide shuffling in that the nucleic acid
building blocks are not shuffled, concatenated or chimerized
randomly, but rather are assembled non-stochastically. See, e.g.,
U.S. patent application Ser. No. (USSN) 09/332,835 entitled
"Synthetic Ligation Reassembly in Directed Evolution" and filed on
Jun. 14, 1999 ("U.S. Ser. No. 09/332,835"). In one aspect, SLR
comprises the following steps: (a) providing a template
polynucleotide, wherein the template polynucleotide comprises
sequence encoding a homologous gene; (b) providing a plurality of
building block polynucleotides, wherein the building block
polynucleotides are designed to cross-over reassemble with the
template polynucleotide at a predetermined sequence, and a building
block polynucleotide comprises a sequence that is a variant of the
homologous gene and a sequence homologous to the template
polynucleotide flanking the variant sequence; (c) combining a
building block polynucleotide with a template polynucleotide such
that the building block polynucleotide cross-over reassembles with
the template polynucleotide to generate polynucleotides comprising
homologous gene sequence variations.
[0300] SLR does not depend on the presence of high levels of
homology between polynucleotides to be rearranged. Thus, this
method can be used to non-stochastically generate libraries (or
sets) of progeny molecules comprised of over 10.sup.100 different
chimeras. SLR can be used to generate libraries comprised of over
10.sup.1000 different progeny chimeras. Thus, aspects of the
present invention include non-stochastic methods of producing a set
of finalized chimeric nucleic acid molecule shaving an overall
assembly order that is chosen by design. This method includes the
steps of generating by design a plurality of specific nucleic acid
building blocks having serviceable mutually compatible ligatable
ends, and assembling these nucleic acid building blocks, such that
a designed overall assembly order is achieved.
[0301] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus the overall
assembly order in which the nucleic acid building blocks can be
coupled is specified by the design of the ligatable ends. If more
than one assembly step is to be used, then the overall assembly
order in which the nucleic acid building blocks can be coupled is
also specified by the sequential order of the assembly step(s). In
one aspect, the annealed building pieces are treated with an
enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent
bonding of the building pieces.
[0302] In one aspect, the design of the oligonucleotide building
blocks is obtained by analyzing a set of progenitor nucleic acid
sequence templates that serve as a basis for producing a progeny
set of finalized chimeric polynucleotides. These parental
oligonucleotide templates thus serve as a source of sequence
information that aids in the design of the nucleic acid building
blocks that are to be mutagenized, e.g., chimerized or
shuffled.
[0303] In one aspect of this method, the sequences of a plurality
of parental nucleic acid templates are aligned in order to select
one or more demarcation points. The demarcation points can be
located at an area of homology, and are comprised of one or more
nucleotides. These demarcation points are preferably shared by at
least two of the progenitor templates. The demarcation points can
thereby be used to delineate the boundaries of oligonucleotide
building blocks to be generated in order to rearrange the parental
polynucleotides. The demarcation points identified and selected in
the progenitor molecules serve as potential chimerization points in
the assembly of the final chimeric progeny molecules. A demarcation
point can be an area of homology (comprised of at least one
homologous nucleotide base) shared by at least two parental
polynucleotide sequences. Alternatively, a demarcation point can be
an area of homology that is shared by at least half of the parental
polynucleotide sequences, or, it can be an area of homology that is
shared by at least two thirds of the parental polynucleotide
sequences. Even more preferably a serviceable demarcation points is
an area of homology that is shared by at least three fourths of the
parental polynucleotide sequences, or, it can be shared by at
almost all of the parental polynucleotide sequences. In one aspect,
a demarcation point is an area of homology that is shared by all of
the parental polynucleotide sequences.
[0304] In one aspect, a ligation reassembly process is performed
exhaustively in order to generate an exhaustive library of progeny
chimeric polynucleotides. In other words, all possible ordered
combinations of the nucleic acid building blocks are represented in
the set of finalized chimeric nucleic acid molecules. At the same
time, in another embodiment, the assembly order (i.e. the order of
assembly of each building block in the 5' to 3 sequence of each
finalized chimeric nucleic acid) in each combination is by design
(or non-stochastic) as described above. Because of the
non-stochastic nature of this invention, the possibility of
unwanted side products is greatly reduced.
[0305] In another aspect, the ligation reassembly method is
performed systematically. For example, the method is performed in
order to generate a systematically compart-mentalized library of
progeny molecules, with compartments that can be screened
systematically, e.g. one by one. In other words this invention
provides that, through the selective and judicious use of specific
nucleic acid building blocks, coupled with the selective and
judicious use of sequentially stepped assembly reactions, a design
can be achieved where specific sets of progeny products are made in
each of several reaction vessels. This allows a systematic
examination and screening procedure to be performed. Thus, these
methods allow a potentially very large number of progeny molecules
to be examined systematically in smaller groups. Because of its
ability to perform chimerizations in a manner that is highly
flexible yet exhaustive and systematic as well, particularly when
there is a low level of homology among the progenitor molecules,
these methods provide for the generation of a library (or set)
comprised of a large number of progeny molecules. Because of the
non-stochastic nature of the instant ligation reassembly invention,
the progeny molecules generated preferably comprise a library of
finalized chimeric nucleic acid molecules having an overall
assembly order that is chosen by design. The saturation mutagenesis
and optimized directed evolution methods also can be used to
generate different progeny molecular species. It is appreciated
that the invention provides freedom of choice and control regarding
the selection of demarcation points, the size and number of the
nucleic acid building blocks, and the size and design of the
couplings. It is appreciated, furthermore, that the requirement for
intermolecular homology is highly relaxed for the operability of
this invention. In fact, demarcation points can even be chosen in
areas of little or no intermolecular homology. For example, because
of codon wobble, i.e. the degeneracy of codons, nucleotide
substitutions can be introduced into nucleic acid building blocks
without altering the amino acid originally encoded in the
corresponding progenitor template. Alternatively, a codon can be
altered such that the coding for an originally amino acid is
altered. This invention provides that such substitutions can be
introduced into the nucleic acid building block in order to
increase the incidence of intermolecularly homologous demarcation
points and thus to allow an increased number of couplings to be
achieved among the building blocks, which in turn allows a greater
number of progeny chimeric molecules to be generated.
[0306] In another aspect, the synthetic nature of the step in which
the building blocks are generated allows the design and
introduction of nucleotides (e.g., one or more nucleotides, which
may be, for example, codons or introns or regulatory sequences)
that can later be optionally removed in an in vitro process (e.g.
by mutagenesis) or in an in vivo process (e.g. by utilizing the
gene splicing ability of a host organism). It is appreciated that
in many instances the introduction of these nucleotides may also be
desirable for many other reasons in addition to the potential
benefit of creating a serviceable demarcation point.
[0307] In one aspect, a nucleic acid building block is used to
introduce an intron. Thus, functional introns are introduced into a
man-made gene manufactured according to the methods described
herein. The artificially introduced intron(s) can be functional in
a host cells for gene splicing much in the way that
naturally-occurring introns serve functionally in gene
splicing.
[0308] Optimized Directed Evolution System
[0309] The invention provides a non-stochastic gene modification
system termed "optimized directed evolution system" to generate
phospholipases with new or altered properties. Optimized directed
evolution is directed to the use of repeated cycles of reductive
reassortment, recombination and selection that allow for the
directed molecular evolution of nucleic acids through
recombination. Optimized directed evolution allows generation of a
large population of evolved chimeric sequences, wherein the
generated population is significantly enriched for sequences that
have a predetermined number of crossover events.
[0310] A crossover event is a point in a chimeric sequence where a
shift in sequence occurs from one parental variant to another
parental variant. Such a point is normally at the juncture of where
oligonucleotides from two parents are ligated together to form a
single sequence. This method allows calculation of the correct
concentrations of oligonucleotide sequences so that the final
chimeric population of sequences is enriched for the chosen number
of crossover events. This provides more control over choosing
chimeric variants having a predetermined number of crossover
events.
[0311] In addition, this method provides a convenient means for
exploring a tremendous amount of the possible protein variant space
in comparison to other systems. Previously, if one generated, for
example, 10.sup.13 chimeric molecules during a reaction, it would
be extremely difficult to test such a high number of chimeric
variants for a particular activity. Moreover, a significant portion
of the progeny population would have a very high number of
crossover events which resulted in proteins that were less likely
to have increased levels of a particular activity. By using these
methods, the population of chimerics molecules can be enriched for
those variants that have a particular number of crossover events.
Thus, although one can still generate 10.sup.13 chimeric molecules
during a reaction, each of the molecules chosen for further
analysis most likely has, for example, only three crossover events.
Because the resulting progeny population can be skewed to have a
predetermined number of crossover events, the boundaries on the
functional variety between the chimeric molecules is reduced. This
provides a more manageable number of variables when calculating
which oligonucleotide from the original parental polynucleotides
might be responsible for affecting a particular trait.
[0312] One method for creating a chimeric progeny polynucleotide
sequence is to create oligonucleotides corresponding to fragments
or portions of each parental sequence. Each oligonucleotide
preferably includes a unique region of overlap so that mixing the
oligonucleotides together results in a new variant that has each
oligonucleotide fragment assembled in the correct order. Additional
information can also be found in U.S. Ser. No. 09/332,835. The
number of oligonucleotides generated for each parental variant
bears a relationship to the total number of resulting crossovers in
the chimeric molecule that is ultimately created. For example,
three parental nucleotide sequence variants might be provided to
undergo a ligation reaction in order to find a chimeric variant
having, for example, greater activity at high temperature. As one
example, a set of 50 oligonucleotide sequences can be generated
corresponding to each portions of each parental variant.
Accordingly, during the ligation reassembly process there could be
up to 50 crossover events within each of the chimeric sequences.
The probability that each of the generated chimeric polynucleotides
will contain oligonucleotides from each parental variant in
alternating order is very low. If each oligonucleotide fragment is
present in the ligation reaction in the same molar quantity it is
likely that in some positions oligonucleotides from the same
parental polynucleotide will ligate next to one another and thus
not result in a crossover event. If the concentration of each
oligonucleotide from each parent is kept constant during any
ligation step in this example, there is a 1/3 chance (assuming 3
parents) that an oligonucleotide from the same parental variant
will ligate within the chimeric sequence and produce no
crossover.
[0313] Accordingly, a probability density function (PDF) can be
determined to predict the population of crossover events that are
likely to occur during each step in a ligation reaction given a set
number of parental variants, a number of oligonucleotides
corresponding to each variant, and the concentrations of each
variant during each step in the ligation reaction. The statistics
and mathematics behind determining the PDF is described below. By
utilizing these methods, one can calculate such a probability
density function, and thus enrich the chimeric progeny population
for a predetermined number of crossover events resulting from a
particular ligation reaction. Moreover, a target number of
crossover events can be predetermined, and the system then
programmed to calculate the starting quantities of each parental
oligonucleotide during each step in the ligation reaction to result
in a probability density function that centers on the predetermined
number of crossover events. These methods are directed to the use
of repeated cycles of reductive reassortment, recombination and
selection that allow for the directed molecular evolution of a
nucleic acid encoding an polypeptide through recombination. This
system allows generation of a large population of evolved chimeric
sequences, wherein the generated population is significantly
enriched for sequences that have a predetermined number of
crossover events. A crossover event is a point in a chimeric
sequence where a shift in sequence occurs from one parental variant
to another parental variant. Such a point is normally at the
juncture of where oligonucleotides from two parents are ligated
together to form a single sequence. The method allows calculation
of the correct concentrations of oligonucleotide sequences so that
the final chimeric population of sequences is enriched for the
chosen number of crossover events. This provides more control over
choosing chimeric variants having a predetermined number of
crossover events.
[0314] In addition, these methods provide a convenient means for
exploring a tremendous amount of the possible protein variant space
in comparison to other systems. By using the methods described
herein, the population of chimerics molecules can be enriched for
those variants that have a particular number of crossover events.
Thus, although one can still generate 10.sup.13 chimeric molecules
during a reaction, each of the molecules chosen for further
analysis most likely has, for example, only three crossover events.
Because the resulting progeny population can be skewed to have a
predetermined number of crossover events, the boundaries on the
functional variety between the chimeric molecules is reduced. This
provides a more manageable number of variables when calculating
which oligonucleotide from the original parental polynucleotides
might be responsible for affecting a particular trait.
[0315] In one aspect, the method creates a chimeric progeny
polynucleotide sequence by creating oligonucleotides corresponding
to fragments or portions of each parental sequence. Each
oligonucleotide preferably includes a unique region of overlap so
that mixing the oligonucleotides together results in a new variant
that has each oligonucleotide fragment assembled in the correct
order. See also U.S. Ser. No. 09/332,835.
[0316] The number of oligonucleotides generated for each parental
variant bears a relationship to the total number of resulting
crossovers in the chimeric molecule that is ultimately created. For
example, three parental nucleotide sequence variants might be
provided to undergo a ligation reaction in order to find a chimeric
variant having, for example, greater activity at high temperature.
As one example, a set of 50 oligonucleotide sequences can be
generated corresponding to each portions of each parental variant.
Accordingly, during the ligation reassembly process there could be
up to 50 crossover events within each of the chimeric sequences.
The probability that each of the generated chimeric polynucleotides
will contain oligonucleotides from each parental variant in
alternating order is very low. If each oligonucleotide fragment is
present in the ligation reaction in the same molar quantity it is
likely that in some positions oligonucleotides from the same
parental polynucleotide will ligate next to one another and thus
not result in a crossover event. If the concentration of each
oligonucleotide from each parent is kept constant during any
ligation step in this example, there is a 1/3 chance (assuming 3
parents) that a oligonucleotide from the same parental variant will
ligate within the chimeric sequence and produce no crossover.
[0317] Accordingly, a probability density function (PDF) can be
determined to predict the population of crossover events that are
likely to occur during each step in a ligation reaction given a set
number of parental variants, a number of oligonucleotides
corresponding to each variant, and the concentrations of each
variant during each step in the ligation reaction. The statistics
and mathematics behind determining the PDF is described below. One
can calculate such a probability density function, and thus enrich
the chimeric progeny population for a predetermined number of
crossover events resulting from a particular ligation reaction.
Moreover, a target number of crossover events can be predetermined,
and the system then programmed to calculate the starting quantities
of each parental oligonucleotide during each step in the ligation
reaction to result in a probability density function that centers
on the predetermined number of crossover events.
[0318] Determining Crossover Events
[0319] Embodiments of the invention include a system and software
that receive a desired crossover probability density function
(PDF), the number of parent genes to be reassembled, and the number
of fragments in the reassembly as inputs. The output of this
program is a "fragment PDF" that can be used to determine a recipe
for producing reassembled genes, and the estimated crossover PDF of
those genes. The processing described herein is preferably
performed in MATLAB.RTM. (The Mathworks, Natick, Mass.) a
programming language and development environment for technical
computing.
[0320] Iterative Processes
[0321] In practicing the invention, these processes can be
iteratively repeated. For example a nucleic acid (or, the nucleic
acid) responsible for an altered phospholipase phenotype is
identified, re-isolated, again modified, re-tested for activity.
This process can be iteratively repeated until a desired phenotype
is engineered. For example, an entire biochemical anabolic or
catabolic pathway can be engineered into a cell, including
phospholipase activity.
[0322] Similarly, if it is determined that a particular
oligonucleotide has no affect at all on the desired trait (e.g., a
new phospholipase phenotype), it can be removed as a variable by
synthesizing larger parental oligonucleotides that include the
sequence to be removed. Since incorporating the sequence within a
larger sequence prevents any crossover events, there will no longer
be any variation of this sequence in the progeny polynucleotides.
This iterative practice of determining which oligonucleotides are
most related to the desired trait, and which are unrelated, allows
more efficient exploration all of the possible protein variants
that might be provide a particular trait or activity.
[0323] In Vivo Shuffling
[0324] In vivo shuffling of molecules is use in methods of the
invention that provide variants of polypeptides of the invention,
e.g., antibodies, phospholipase enzymes, and the like. In vivo
shuffling can be performed utilizing the natural property of cells
to recombine multimers. While recombination in vivo has provided
the major natural route to molecular diversity, genetic
recombination remains a relatively complex process that involves 1)
the recognition of homologies; 2) strand cleavage, strand invasion,
and metabolic steps leading to the production of recombinant
chiasma; and finally 3) the resolution of chiasma into discrete
recombined molecules. The formation of the chiasma requires the
recognition of homologous sequences.
[0325] In one aspect, the invention provides a method for producing
a hybrid polynucleotide from at least a first polynucleotide and a
second polynucleotide. The invention can be used to produce a
hybrid polynucleotide by introducing at least a first
polynucleotide and a second polynucleotide which share at least one
region of partial sequence homology into a suitable host cell. The
regions of partial sequence homology promote processes which result
in sequence reorganization producing a hybrid polynucleotide. The
term "hybrid polynucleotide", as used herein, is any nucleotide
sequence which results from the method of the present invention and
contains sequence from at least two original polynucleotide
sequences. Such hybrid polynucleotides can result from
intermolecular recombination events which promote sequence
integration between DNA molecules. In addition, such hybrid
polynucleotides can result from intramolecular reductive
reassortment processes which utilize repeated sequences to alter a
nucleotide sequence within a DNA molecule.
[0326] Producing Sequence Variants
[0327] The invention also provides methods of making sequence
variants of the nucleic acid and phospholipase sequences of the
invention or isolating phospholipase enzyme, e.g., phospholipase,
sequence variants using the nucleic acids and polypeptides of the
invention. In one aspect, the invention provides for variants of a
phospholipase gene of the invention, which can be altered by any
means, including, e.g., random or stochastic methods, or,
non-stochastic, or "directed evolution," methods, as described
above.
[0328] The isolated variants may be naturally occurring. Variant
can also be created in vitro. Variants may be created using genetic
engineering techniques such as site directed mutagenesis, random
chemical mutagenesis, Exonuclease III deletion procedures, and
standard cloning techniques. Alternatively, such variants,
fragments, analogs, or derivatives may be created using chemical
synthesis or modification procedures. Other methods of making
variants are also familiar to those skilled in the art. These
include procedures in which nucleic acid sequences obtained from
natural isolates are modified to generate nucleic acids which
encode polypeptides having characteristics which enhance their
value in industrial or laboratory applications. In such procedures,
a large number of variant sequences having one or more nucleotide
differences with respect to the sequence obtained from the natural
isolate are generated and characterized. These nucleotide
differences can result in amino acid changes with respect to the
polypeptides encoded by the nucleic acids from the natural
isolates.
[0329] For example, variants may be created using error prone PCR.
In error prone PCR, PCR is performed under conditions where the
copying fidelity of the DNA polymerase is low, such that a high
rate of point mutations is obtained along the entire length of the
PCR product. Error prone PCR is described, e.g., in Leung, D. W.,
et al., Technique, 1:11-15, 1989) and Caldwell, R. C. & Joyce
G. F., PCR Methods Applic., 2:28-33, 1992. Briefly, in such
procedures, nucleic acids to be mutagenized are mixed with PCR
primers, reaction buffer, MgCl2, MnCl2, Taq polymerase and an
appropriate concentration of dNTPs for achieving a high rate of
point mutation along the entire length of the PCR product. For
example, the reaction may be performed using 20 fmoles of nucleic
acid to be mutagenized, 30 pmole of each PCR primer, a reaction
buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01%
gelatin, 7 mM MgCl2, 0.5 mM MnCl2, 5 units of Taq polymerase, 0.2
mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR may be
performed for 30 cycles of 94.degree. C. for 1 min, 45.degree. C.
for 1 min, and 72.degree. C. for 1 min. However, it will be
appreciated that these parameters may be varied as appropriate. The
mutagenized nucleic acids are cloned into an appropriate vector and
the activities of the polypeptides encoded by the mutagenized
nucleic acids is evaluated.
[0330] Variants may also be created using oligonucleotide directed
mutagenesis to generate site-specific mutations in any cloned DNA
of interest. Oligonucleotide mutagenesis is described, e.g., in
Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such
procedures a plurality of double stranded oligonucleotides bearing
one or more mutations to be introduced into the cloned DNA are
synthesized and inserted into the cloned DNA to be mutagenized.
Clones containing the mutagenized DNA are recovered and the
activities of the polypeptides they encode are assessed.
[0331] Another method for generating variants is assembly PCR.
Assembly PCR involves the assembly of a PCR product from a mixture
of small DNA fragments. A large number of different PCR reactions
occur in parallel in the same vial, with the products of one
reaction priming the products of another reaction. Assembly PCR is
described in, e.g., U.S. Pat. No. 5,965,408.
[0332] Still another method of generating variants is sexual PCR
mutagenesis. In sexual PCR mutagenesis, forced homologous
recombination occurs between DNA molecules of different but highly
related DNA sequence in vitro, as a result of random fragmentation
of the DNA molecule based on sequence homology, followed by
fixation of the crossover by primer extension in a PCR reaction.
Sexual PCR mutagenesis is described, e.g., in Stemmer (1994) Proc.
Natl. Acad. Sci. USA 91:10747-10751. Briefly, in such procedures a
plurality of nucleic acids to be recombined are digested with DNase
to generate fragments having an average size of 50-200 nucleotides.
Fragments of the desired average size are purified and resuspended
in a PCR mixture. PCR is conducted under conditions which
facilitate recombination between the nucleic acid fragments. For
example, PCR may be performed by resuspending the purified
fragments at a concentration of 10-30 ng/.mu.l in a solution of 0.2
mM of each dNTP, 2.2 mM MgCl.sub.2, 50 mM KCL, 10 mM Tris HCl, pH
9.0, and 0.1% Triton X-100. 2.5 units of Taq polymerase per 100:1
of reaction mixture is added and PCR is performed using the
following regime: 94.degree. C. for 60 seconds, 94.degree. C. for
30 seconds, 50-55.degree. C. for 30 seconds, 72.degree. C. for 30
seconds (30-45 times) and 72.degree. C. for 5 minutes. However, it
will be appreciated that these parameters may be varied as
appropriate. In some aspects, oligonucleotides may be included in
the PCR reactions. In other aspects, the Klenow fragment of DNA
polymerase I may be used in a first set of PCR reactions and Taq
polymerase may be used in a subsequent set of PCR reactions.
Recombinant sequences are isolated and the activities of the
polypeptides they encode are assessed.
[0333] Variants may also be created by in vivo mutagenesis. In some
embodiments, random mutations in a sequence of interest are
generated by propagating the sequence of interest in a bacterial
strain, such as an E. coli strain, which carries mutations in one
or more of the DNA repair pathways. Such "mutator" strains have a
higher random mutation rate than that of a wild-type parent.
Propagating the DNA in one of these strains will eventually
generate random mutations within the DNA. Mutator strains suitable
for use for in vivo mutagenesis are described, e.g., in PCT
Publication No. WO 91/16427.
[0334] Variants may also be generated using cassette mutagenesis.
In cassette mutagenesis a small region of a double stranded DNA
molecule is replaced with a synthetic oligonucleotide "cassette"
that differs from the native sequence. The oligonucleotide often
contains completely and/or partially randomized native
sequence.
[0335] Recursive ensemble mutagenesis may also be used to generate
variants. Recursive ensemble mutagenesis is an algorithm for
protein engineering (protein mutagenesis) developed to produce
diverse populations of phenotypically related mutants whose members
differ in amino acid sequence. This method uses a feedback
mechanism to control successive rounds of combinatorial cassette
mutagenesis. Recursive ensemble mutagenesis is described, e.g., in
Arkin (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.
[0336] In some embodiments, variants are created using exponential
ensemble mutagenesis. Exponential ensemble mutagenesis is a process
for generating combinatorial libraries with a high percentage of
unique and functional mutants, wherein small groups of residues are
randomized in parallel to identify, at each altered position, amino
acids which lead to functional proteins. Exponential ensemble
mutagenesis is described, e.g., in Delegrave (1993) Biotechnology
Res. 11:1548-1552. Random and site-directed mutagenesis are
described, e.g., in Arnold (1993) Current Opinion in Biotechnology
4:450-455.
[0337] In some embodiments, the variants are created using
shuffling procedures wherein portions of a plurality of nucleic
acids which encode distinct polypeptides are fused together to
create chimeric nucleic acid sequences which encode chimeric
polypeptides as described in, e.g., U.S. Pat. Nos. 5,965,408;
5,939,250.
[0338] The invention also provides variants of polypeptides of the
invention comprising sequences in which one or more of the amino
acid residues (e.g., of an exemplary polypeptide of the invention)
are substituted with a conserved or non-conserved amino acid
residue (e.g., a conserved amino acid residue) and such substituted
amino acid residue may or may not be one encoded by the genetic
code. Conservative substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Thus, polypeptides of the invention include those
with conservative substitutions of sequences of the invention,
including but not limited to the following replacements:
replacements of an aliphatic amino acid such as Alanine, Valine,
Leucine and Isoleucine with another aliphatic amino acid;
replacement of a Serine with a Threonine or vice versa; replacement
of an acidic residue such as Aspartic acid and Glutamic acid with
another acidic residue; replacement of a residue bearing an amide
group, such as Asparagine and Glutamine, with another residue
bearing an amide group; exchange of a basic residue such as Lysine
and Arginine with another basic residue; and replacement of an
aromatic residue such as Phenylalanine, Tyrosine with another
aromatic residue. Other variants are those in which one or more of
the amino acid residues of the polypeptides of the invention
includes a substituent group.
[0339] Other variants within the scope of the invention are those
in which the polypeptide is associated with another compound, such
as a compound to increase the half-life of the polypeptide, for
example, polyethylene glycol.
[0340] Additional variants within the scope of the invention are
those in which additional amino acids are fused to the polypeptide,
such as a leader sequence, a secretory sequence, a proprotein
sequence or a sequence which facilitates purification, enrichment,
or stabilization of the polypeptide.
[0341] In some aspects, the variants, fragments, derivatives and
analogs of the polypeptides of the invention retain the same
biological function or activity as the exemplary polypeptides,
e.g., a phospholipase activity, as described herein. In other
aspects, the variant, fragment, derivative, or analog includes a
proprotein, such that the variant, fragment, derivative, or analog
can be activated by cleavage of the proprotein portion to produce
an active polypeptide.
Optimizing Codons to Achieve High Levels of Protein Expression in
Host Cells
[0342] The invention provides methods for modifying
phospholipase-encoding nucleic acids to modify codon usage. In one
aspect, the invention provides methods for modifying codons in a
nucleic acid encoding a phospholipase to increase or decrease its
expression in a host cell. The invention also provides nucleic
acids encoding a phospholipase modified to increase its expression
in a host cell, phospholipase enzymes so modified, and methods of
making the modified phospholipase enzymes. The method comprises
identifying a "non-preferred" or a "less preferred" codon in
phospholipase-encoding nucleic acid and replacing one or more of
these non-preferred or less preferred codons with a "preferred
codon" encoding the same amino acid as the replaced codon and at
least one non-preferred or less preferred codon in the nucleic acid
has been replaced by a preferred codon encoding the same amino
acid. A preferred codon is a codon over-represented in coding
sequences in genes in the host cell and a non-preferred or less
preferred codon is a codon under-represented in coding sequences in
genes in the host cell.
[0343] Host cells for expressing the nucleic acids, expression
cassettes and vectors of the invention include bacteria, yeast,
fungi, plant cells, insect cells and mammalian cells. Thus, the
invention provides methods for optimizing codon usage in all of
these cells, codon-altered nucleic acids and polypeptides made by
the codon-altered nucleic acids. Exemplary host cells include gram
negative bacteria, such as Escherichia coli; gram positive
bacteria, such as any Bacillus (e.g., B. cereus) or Streptomyces,
Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris,
Bacillus subtilis. Exemplary host cells also include eukaryotic
organisms, e.g., various yeast, such as Saccharomyces sp.,
including Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha,
Aspergillus niger, and mammalian cells and cell lines and insect
cells and cell lines. Thus, the invention also includes nucleic
acids and polypeptides optimized for expression in these organisms
and species.
[0344] For example, the codons of a nucleic acid encoding a
phospholipase isolated from a bacterial cell are modified such that
the nucleic acid is optimally expressed in a bacterial cell
different from the bacteria from which the phospholipase was
derived, a yeast, a fungi, a plant cell, an insect cell or a
mammalian cell. Methods for optimizing codons are well known in the
art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.
Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;
Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001)
Infect. Immun 69:7250-7253, describing optimizing codons in mouse
systems; Outchkourov (2002) Protein Expr. Purif. 24:18-24,
describing optimizing codons in yeast; Feng (2000) Biochemistry
39:15399-15409, describing optimizing codons in E. coli; Humphreys
(2000) Protein Expr. Purif. 20:252-264, describing optimizing codon
usage that affects secretion in E. coli.
Transgenic Non-Human Animals
[0345] The invention provides transgenic non-human animals
comprising a nucleic acid, a polypeptide, an expression cassette or
vector or a transfected or transformed cell of the invention. The
transgenic non-human animals can be, e.g., goats, rabbits, sheep,
pigs, cows, rats and mice, comprising the nucleic acids of the
invention. These animals can be used, e.g., as in vivo models to
study phospholipase activity, or, as models to screen for
modulators of phospholipase activity in vivo. The coding sequences
for the polypeptides to be expressed in the transgenic non-human
animals can be designed to be constitutive, or, under the control
of tissue-specific, developmental-specific or inducible
transcriptional regulatory factors. Transgenic non-human animals
can be designed and generated using any method known in the art;
see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952;
6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070;
5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742; 5,087,571,
describing making and using transformed cells and eggs and
transgenic mice, rats, rabbits, sheep, pigs and cows. See also,
e.g., Pollock (1999) J. Immunol. Methods 231:147-157, describing
the production of recombinant proteins in the milk of transgenic
dairy animals; Baguisi (1999) Nat. Biotechnol. 17:456-461,
demonstrating the production of transgenic goats. U.S. Pat. No.
6,211,428, describes making and using transgenic non-human mammals
which express in their brains a nucleic acid construct comprising a
DNA sequence. U.S. Pat. No. 5,387,742, describes injecting cloned
recombinant or synthetic DNA sequences into fertilized mouse eggs,
implanting the injected eggs in pseudo-pregnant females, and
growing to term transgenic mice whose cells express proteins
related to the pathology of Alzheimer's disease. U.S. Pat. No.
6,187,992, describes making and using a transgenic mouse whose
genome comprises a disruption of the gene encoding amyloid
precursor protein (APP).
[0346] "Knockout animals" can also be used to practice the methods
of the invention. For example, in one aspect, the transgenic or
modified animals of the invention comprise a "knockout animal,"
e.g., a "knockout mouse," engineered not to express or to be unable
to express a phospholipase.
Transgenic Plants and Seeds
[0347] The invention provides transgenic plants and seeds
comprising a nucleic acid, a polypeptide (e.g., a phospholipase),
an expression cassette or vector or a transfected or transformed
cell of the invention. The invention also provides plant products,
e.g., oils, seeds, leaves, extracts and the like, comprising a
nucleic acid and/or a polypeptide (e.g., a phospholipase) of the
invention. The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). The invention also provides methods
of making and using these transgenic plants and seeds. The
transgenic plant or plant cell expressing a polypeptide of the
invention may be constructed in accordance with any method known in
the art. See, for example, U.S. Pat. No. 6,309,872.
[0348] Nucleic acids and expression constructs of the invention can
be introduced into a plant cell by any means. For example, nucleic
acids or expression constructs can be introduced into the genome of
a desired plant host, or, the nucleic acids or expression
constructs can be episomes. Introduction into the genome of a
desired plant can be such that the host's phospholipase production
is regulated by endogenous transcriptional or translational control
elements. The invention also provides "knockout plants" where
insertion of gene sequence by, e.g., homologous recombination, has
disrupted the expression of the endogenous gene. Means to generate
"knockout" plants are well-known in the art, see, e.g., Strepp
(1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao (1995) Plant J
7:359-365. See discussion on transgenic plants, below.
[0349] The nucleic acids of the invention can be used to confer
desired traits on essentially any plant, e.g., on oil-seed
containing plants, such as rice, soybeans, rapeseed, sunflower
seeds, sesame and peanuts. Nucleic acids of the invention can be
used to manipulate metabolic pathways of a plant in order to
optimize or alter host's expression of phospholipase. The can
change phospholipase activity in a plant. Alternatively, a
phospholipase of the invention can be used in production of a
transgenic plant to produce a compound not naturally produced by
that plant. This can lower production costs or create a novel
product.
[0350] In one aspect, the first step in production of a transgenic
plant involves making an expression construct for expression in a
plant cell. These techniques are well known in the art. They can
include selecting and cloning a promoter, a coding sequence for
facilitating efficient binding of ribosomes to mRNA and selecting
the appropriate gene terminator sequences. One exemplary
constitutive promoter is CaMV35S, from the cauliflower mosaic
virus, which generally results in a high degree of expression in
plants. Other promoters are more specific and respond to cues in
the plant's internal or external environment. An exemplary
light-inducible promoter is the promoter from the cab gene,
encoding the major chlorophyll a/b binding protein.
[0351] In one aspect, the nucleic acid is modified to achieve
greater expression in a plant cell. For example, a sequence of the
invention is likely to have a higher percentage of A-T nucleotide
pairs compared to that seen in a plant, some of which prefer G-C
nucleotide pairs. Therefore, A-T nucleotides in the coding sequence
can be substituted with G-C nucleotides without significantly
changing the amino acid sequence to enhance production of the gene
product in plant cells.
[0352] Selectable marker gene can be added to the gene construct in
order to identify plant cells or tissues that have successfully
integrated the transgene. This may be necessary because achieving
incorporation and expression of genes in plant cells is a rare
event, occurring in just a few percent of the targeted tissues or
cells. Selectable marker genes encode proteins that provide
resistance to agents that are normally toxic to plants, such as
antibiotics or herbicides. Only plant cells that have integrated
the selectable marker gene will survive when grown on a medium
containing the appropriate antibiotic or herbicide. As for other
inserted genes, marker genes also require promoter and termination
sequences for proper function.
[0353] In one aspect, making transgenic plants or seeds comprises
incorporating sequences of the invention and, optionally, marker
genes into a target expression construct (e.g., a plasmid), along
with positioning of the promoter and the terminator sequences. This
can involve transferring the modified gene into the plant through a
suitable method. For example, a construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation and microinjection of plant cell
protoplasts, or the constructs can be introduced directly to plant
tissue using ballistic methods, such as DNA particle bombardment.
For example, see, e.g., Christou (1997) Plant Mol. Biol.
35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987)
Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,
discussing use of particle bombardment to introduce transgenes into
wheat; and Adam (1997) supra, for use of particle bombardment to
introduce YACs into plant cells. For example, Rinehart (1997)
supra, used particle bombardment to generate transgenic cotton
plants. Apparatus for accelerating particles is described U.S. Pat.
No. 5,015,580; and, the commercially available BioRad (Biolistics)
PDS-2000 particle acceleration instrument; see also, John, U.S.
Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describing
particle-mediated transformation of gymnosperms.
[0354] In one aspect, protoplasts can be immobilized and injected
with nucleic acids, e.g., an expression construct. Although plant
regeneration from protoplasts is not easy with cereals, plant
regeneration is possible in legumes using somatic embryogenesis
from protoplast derived callus. Organized tissues can be
transformed with naked DNA using gene gun technique, where DNA is
coated on tungsten microprojectiles, shot 1/100th the size of
cells, which carry the DNA deep into cells and organelles.
Transformed tissue is then induced to regenerate, usually by
somatic embryogenesis. This technique has been successful in
several cereal species including maize and rice.
[0355] Nucleic acids, e.g., expression constructs, can also be
introduced in to plant cells using recombinant viruses. Plant cells
can be transformed using viral vectors, such as, e.g., tobacco
mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol.
33:989-999), see Porta (1996) "Use of viral replicons for the
expression of genes in plants," Mol. Biotechnol. 5:209-221.
[0356] Alternatively, nucleic acids, e.g., an expression construct,
can be combined with suitable T-DNA flanking regions and introduced
into a conventional Agrobacterium tumefaciens host vector. The
virulence functions of the Agrobacterium tumefaciens host will
direct the insertion of the construct and adjacent marker into the
plant cell DNA when the cell is infected by the bacteria.
Agrobacterium tumefaciens-mediated transformation techniques,
including disarming and use of binary vectors, are well described
in the scientific literature. See, e.g., Horsch (1984) Science
233:496-498; Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803
(1983); Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag,
Berlin 1995). The DNA in an A. tumefaciens cell is contained in the
bacterial chromosome as well as in another structure known as a Ti
(tumor-inducing) plasmid. The Ti plasmid contains a stretch of DNA
termed T-DNA (.about.20 kb long) that is transferred to the plant
cell in the infection process and a series of vir (virulence) genes
that direct the infection process. A. tumefaciens can only infect a
plant through wounds: when a plant root or stem is wounded it gives
off certain chemical signals, in response to which, the vir genes
of A. tumefaciens become activated and direct a series of events
necessary for the transfer of the T-DNA from the Ti plasmid to the
plant's chromosome. The T-DNA then enters the plant cell through
the wound. One speculation is that the T-DNA waits until the plant
DNA is being replicated or transcribed, then inserts itself into
the exposed plant DNA. In order to use A. tumefaciens as a
transgene vector, the tumor-inducing section of T-DNA have to be
removed, while retaining the T-DNA border regions and the vir
genes. The transgene is then inserted between the T-DNA border
regions, where it is transferred to the plant cell and becomes
integrated into the plant's chromosomes.
[0357] The invention provides for the transformation of
monocotyledonous plants using the nucleic acids of the invention,
including important cereals, see Hiei (1997) Plant Mol. Biol.
35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley
(1983) Proc. Natl. Acad. Sci. USA 80:4803; Thykjaer (1997) supra;
Park (1996) Plant Mol. Biol. 32:1135-1148, discussing T-DNA
integration into genomic DNA. See also D'Halluin, U.S. Pat. No.
5,712,135, describing a process for the stable integration of a DNA
comprising a gene that is functional in a cell of a cereal, or
other monocotyledonous plant.
[0358] In one aspect, the third step can involve selection and
regeneration of whole plants capable of transmitting the
incorporated target gene to the next generation. Such regeneration
techniques rely on manipulation of certain phytohormones in a
tissue culture growth medium, typically relying on a biocide and/or
herbicide marker that has been introduced together with the desired
nucleotide sequences. Plant regeneration from cultured protoplasts
is described in Evans et al., Protoplasts Isolation and Culture,
Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing
Company, New York, 1983; and Binding, Regeneration of Plants, Plant
Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration
can also be obtained from plant callus, explants, organs, or parts
thereof. Such regeneration techniques are described generally in
Klee (1987) Ann Rev. of Plant Phys. 38:467-486. To obtain whole
plants from transgenic tissues such as immature embryos, they can
be grown under controlled environmental conditions in a series of
media containing nutrients and hormones, a process known as tissue
culture. Once whole plants are generated and produce seed,
evaluation of the progeny begins.
[0359] After the expression cassette is stably incorporated in
transgenic plants, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed. Since transgenic
expression of the nucleic acids of the invention leads to
phenotypic changes, plants comprising the recombinant nucleic acids
of the invention can be sexually crossed with a second plant to
obtain a final product. Thus, the seed of the invention can be
derived from a cross between two transgenic plants of the
invention, or a cross between a plant of the invention and another
plant. The desired effects (e.g., expression of the polypeptides of
the invention to produce a plant in which flowering behavior is
altered) can be enhanced when both parental plants express the
polypeptides (e.g., a phospholipase) of the invention. The desired
effects can be passed to future plant generations by standard
propagation means.
[0360] The nucleic acids and polypeptides of the invention are
expressed in or inserted in any plant or seed. Transgenic plants of
the invention can be dicotyledonous or monocotyledonous. Examples
of monocot transgenic plants of the invention are grasses, such as
meadow grass (blue grass, Poa), forage grass such as festuca,
lolium, temperate grass, such as Agrostis, and cereals, e.g.,
wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples
of dicot transgenic plants of the invention are tobacco, legumes,
such as lupins, potato, sugar beet, pea, bean and soybean, and
cruciferous plants (family Brassicaceae), such as cauliflower, rape
seed, and the closely related model organism Arabidopsis thaliana.
Thus, the transgenic plants and seeds of the invention include a
broad range of plants, including, but not limited to, species from
the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica,
Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis,
Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,
Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum,
Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago,
Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus,
Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,
Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella,
Triticum, Vicia, Vitis, Vigna, and Zea.
[0361] In alternative embodiments, the nucleic acids of the
invention are expressed in plants (e.g., as transgenic plants),
such as oil-seed containing plants, e.g., rice, soybeans, rapeseed,
sunflower seeds, sesame and peanuts. The nucleic acids of the
invention can be expressed in plants which contain fiber cells,
including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra),
desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp,
roselle, jute, sisal abaca and flax. In alternative embodiments,
the transgenic plants of the invention can be members of the genus
Gossypium, including members of any Gossypium species, such as G.
arboreum; G. herbaceum, G. barbadense, and G. hirsutum.
[0362] The invention also provides for transgenic plants to be used
for producing large amounts of the polypeptides (e.g., a
phospholipase or antibody) of the invention. For example, see
Palmgren (1997) Trends Genet. 13:348; Chong (1997) Transgenic Res.
6:289-296 (producing human milk protein beta-casein in transgenic
potato plants using an auxin-inducible, bidirectional mannopine
synthase (mas1',2') promoter with Agrobacterium
tumefaciens-mediated leaf disc transformation methods).
[0363] Using known procedures, one of skill can screen for plants
of the invention by detecting the increase or decrease of transgene
mRNA or protein in transgenic plants. Means for detecting and
quantitation of mRNAs or proteins are well known in the art.
Polypeptides and Peptides
[0364] The invention provides isolated or recombinant polypeptides
having a sequence identity (e.g., at least 50%, 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity) to an exemplary sequence of the invention, e.g., SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID
NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ
ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86,
SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104,
SEQ ID NO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQ ID
NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,
SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID
NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140;
SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150,
SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID
NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168,
SEQ ID NO:170, SEQ ID NO:172, or SEQ ID NO:174. As discussed above,
the identity can be over the full length of the polypeptide, or,
the identity can be over a subsequence thereof, e.g., a region of
at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700 or more residues. Polypeptides of
the invention can also be shorter than the full length of exemplary
polypeptides (e.g., SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID
NO:8, etc.). In alternative embodiment, the invention provides
polypeptides (peptides, fragments) ranging in size between about 5
and the full length of a polypeptide, e.g., an enzyme, such as a
phospholipase, e.g., phospholipase; exemplary sizes being of about
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 100, 125, 150, 175, 200, 250, 300, 350, 400 or more residues,
e.g., contiguous residues of the exemplary phospholipases of SEQ ID
NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8, etc. Peptides of the
invention can be useful as, e.g., labeling probes, antigens,
toleragens, motifs, phospholipase active sites, binding domains,
regulatory domains, and the like.
[0365] In one aspect, the polypeptide has a phospholipase activity,
e.g., cleavage of a glycerolphosphate ester linkage, the ability to
hydrolyze phosphate ester bonds, including patatin, lipid acyl
hydrolase (LAH), phospholipase A, B, C and/or phospholipase D
activity, or any combination thereof.
[0366] In alternative aspects, exemplary polypeptides of the
invention have a phospholipase activity, Signal Sequence Location,
and an initial source, as set forth in the following Table 1,
below. To aid in reading the table, for example, in the first row,
where SEQ ID NO: 143, 144, means the polypeptide having a sequence
as set forth in SEQ ID NO:144, and encoded by, e.g., SEQ ID NO:143,
having a PLA-specific PLA activity, initially isolated from an
unknown source; another example in the SEQ ID NO:167, 168 row where
167, 168 means the polypeptide having a sequence as set forth in
SEQ ID NO:168, and encoded by, e.g., SEQ ID NO:167, having a
phosphatidic acid phosphatase activity, a signal sequence at
residues 1 to 30 ("AA1-30" means amino acid residues 1 to 30,
etc.), i.e., MARSWKWRPLLSSFLLVSLAPFSTSVPCFK, and initially isolated
from an unknown source. The invention also provides peptides
comprising signal sequences, and chimeric polypeptides, where the
peptides or chimerics comprise signal sequences as set forth in
Table 1, and as described below.
TABLE-US-00001 TABLE 1 Signal Seq. Location (AA = Amino SEQ ID NO:
Enzyme type Acid) Signal (AA) Source 143, 144 PA-specific PLA
Unknown 25, 26 Patatin Unknown 77, 78 Patatin Unknown 35, 36
Patatin Unknown 125, 126 Patatin Unknown 135, 136 Patatin Unknown
99, 100 Patatin Unknown 65, 66 Patatin Unknown 87, 88 Patatin
Unknown 86, 87 Patatin Unknown 45, 46 Patatin Unknown 59, 60
Patatin Unknown 13, 14 Patatin Unknown 71, 72 Patatin Unknown 55,
56 Patatin Unknown 33, 34 Patatin Unknown 91, 92 Patatin Unknown
103, 104 Patatin Unknown 11, 12 Patatin Unknown 17, 18 Patatin
Unknown 95, 96 Patatin Unknown 43, 44 Patatin Unknown 27, 28
Patatin Unknown 131, 132 Patatin Unknown 127, 128 Patatin Unknown
133, 134 Patatin Unknown 137, 138 Patatin Unknown 165, 166 Patatin
Unknown 167, 168 Phosphatidic acid AA1-30
MARSWKWRPLLSSFLLVSLAPFSTSVPCFK Unknown phosphatases 169, 170
Phosphatidic acid Unknown phosphatases 171, 172 Phosphatidic acid
Unknown phosphatases 173, 174 Phosphatidic acid Unknown
phosphatases 111, 112 Phosphatidyl- AA1-16 MGAGAILLTGAPTASA
Bacteria inositol PLC 107, 108 Phosphatidyl- AA1-23
MSNKKFILKLFICSTILSTFVFA Unknown inositol PLC 109, 110 Phosphatidyl-
AA1-23 MSNKKFILKLFICSTILSTFVFA Unknown inositol PLC 113, 114
phosphatidyl- AA1-23 MSNKKFILKLFICSTILSTFVFA Unknown inositol PLC
117, 118 Phosphatidyl- AA1-23 MNNKKFILKLFICSMVLSAFVFA Unknown
inositol PLC 119, 120 phosphatidyl- AA1-23 MNNKKFILKLFICSMVLSAFVFA
Unknown inositol PLC 115, 116 Phosphatidyl- AA1-23
MNNKKFILKLFICSMVLSAFVFA Unknown inositol PLC 121, 122 Phosphatidyl-
AA1-23 MRNKKFILKLLICSTVLSTFVFA Unknown inositol PLC 141, 142
Phospholipase Unknown 155, 156 Phospholipase AA1-36
MRTTTTNWRQIVKSLKLFLMGLCLFISASF Unknown ASSAYA 159, 160
Phospholipase Unknown 145, 146 PLA Unknown 147, 148 PLA Unknown
149, 150 PLA Unknown 151, 152 PLA Unknown 153, 154 PLA Unknown 157,
158 PLA Unknown 163, 164 PLA Unknown 101, 102 PLC AA1-39
LSLVASLRRAPGAALALALAAATLAVTAQG Bacteria ATAAPAAAAA 1, 2 PLC AA1-24
MKKKVLALAAMVALAAPVQSVVFAQ Unknown 3, 4 PLC AA1-24
MKRKILAIASVIALTAPIQSVAFAH Unknown 5, 6 PLC AA1-24
MKRKILAIASVIALTAPIQSVAFAH Unknown 97, 98 PLC AA1-25
MKRKLCTWALVTAIASSTAVIPTAAE Unknown 7, 8 PLC AA1-29
MITLIKKCLLVLTMTLLLGVFVPLQPSHAT Unknown 31, 32 PLC AA1-20
MKKKLCTWALVTAISSGVVAI Unknown 81, 82 PLC AA1-25
MKKKLCTMALVTAISSGVVTIPTEAQ Unknown 93, 94 PLC AA1-29
MITLIKKCLLVLTMTLLSGVFVPLQPSYAT Unknown 89, 90 PLC AA1-25
MKKKLCTLAFVTAISSIAITIPTEAQ Unknown 123, 124 PLC AA1-24
MKKKVLALAAMVALAAPVQSVVFA Unknown 129, 130 PLC AA1-27
MKKKICTLALVSAITSGVVTIPTVASA Unknown 139, 140 PLC AA1-20
MKIKPLTFSFGLAVTSSVQA Unknown 105, 106 PLC AA1-30
MNRCRNSLNLQLRAVTVAALVVVASSAALAW Unknown 9, 10 PLC AA1-20
MKLLRVFVCVFALLSAHSKAD Unknown 47, 48 PLD Unknown 15, 16 PLD Unknown
41, 42 PLD Unknown 23, 24 PLD Unknown 51, 52 PLD Unknown 53, 54 PLD
Unknown 19, 20 PLD AA1-19 MKKTTLVLALLMPFGAASAQ Unknown 75, 76 PLD
Unknown 57, 58 PLD Unknown 63, 64 PLD AA1-18 MKNTLILAGCILAAPAVAD
Unknown 79, 80 PLD AA1-23 MRNFSKGLTSILLSIATSTSAMAF Unknown 37, 38
PLD AA1-23 MRNFSKGLTSILLSIATSTSAMAF Unknown 61, 62 PLD AA1-21
MTLKLSLLIASLSAVSPAVLAN Unknown 67, 68 PLD No Unknown 83, 84 PLD
AA1-21 MKKIVIYSFVAGVMTSGGVFAA Unknown 49, 50 PLD AA1-23
MNFWSFLLSITLPMGVGVAHAQPD Unknown 39, 40 PLD Unknown 73, 74 PLD
Unknown 29, 30 PLD Unknown 21, 22 PLD AA1-28
MQQHKLRNFNKGLTGVVLSVLTSTSAMAF Unknown 71, 72 PLD Unknown 161, 162
PLD AA1-24 MNRKLLSLCLGATSCIALSLPVHA Unknown
[0367] In one aspect, the invention provides polypeptides having
sequences as set forth in SEQ ID NO:107, SEQ ID NO:109, SEQ ID
NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,
SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID
NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137,
SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID
NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,
SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID
NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 and/or SEQ ID
NO:173, and subsequences thereof, e.g., their active sites
("catalytic domains") having a phospholipase activity, e.g., a
phospholipase C (PLC) activity. In one aspect, the polypeptide has
a phospholipase activity but lacks neutral oil (triglyceride)
hydrolysis activity. For example, in one aspect, the polypeptide
has a phospholipase activity but lacks any activity that affects a
neutral oil (triglyceride) fraction. In one aspect, the invention
provides a degumming process comprising use of a polypeptide of the
invention having a phospholipase activity, but not a lipase
activity.
[0368] Polypeptides and peptides of the invention can be isolated
from natural sources, be synthetic, or be recombinantly generated
polypeptides. Peptides and proteins can be recombinantly expressed
in vitro or in vivo. The peptides and polypeptides of the invention
can be made and isolated using any method known in the art.
Polypeptide and peptides of the invention can also be synthesized,
whole or in part, using chemical methods well known in the art. See
e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn
(1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K.,
Therapeutic Peptides and Proteins, Formulation, Processing and
Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa.
For example, peptide synthesis can be performed using various
solid-phase techniques (see e.g., Roberge (1995) Science 269:202;
Merrifield (1997) Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 431A Peptide
Synthesizer (Perkin Elmer) in accordance with the instructions
provided by the manufacturer.
[0369] The peptides and polypeptides of the invention can also be
glycosylated. The glycosylation can be added post-translationally
either chemically or by cellular biosynthetic mechanisms, wherein
the later incorporates the use of known glycosylation motifs, which
can be native to the sequence or can be added as a peptide or added
in the nucleic acid coding sequence. The glycosylation can be
O-linked or N-linked.
[0370] The peptides and polypeptides of the invention, as defined
above, include all "mimetic" and "peptidomimetic" forms. The terms
"mimetic" and "peptidomimetic" refer to a synthetic chemical
compound which has substantially the same structural and/or
functional characteristics of the polypeptides of the invention.
The mimetic can be either entirely composed of synthetic,
non-natural analogues of amino acids, or, is a chimeric molecule of
partly natural peptide amino acids and partly non-natural analogs
of amino acids. The mimetic can also incorporate any amount of
natural amino acid conservative substitutions as long as such
substitutions also do not substantially alter the mimetic's
structure and/or activity. As with polypeptides of the invention
which are conservative variants, routine experimentation will
determine whether a mimetic is within the scope of the invention,
i.e., that its structure and/or function is not substantially
altered. Thus, in one aspect, a mimetic composition is within the
scope of the invention if it has a phospholipase activity.
[0371] Polypeptide mimetic compositions of the invention can
contain any combination of non-natural structural components. In
alternative aspect, mimetic compositions of the invention include
one or all of the following three structural groups: a) residue
linkage groups other than the natural amide bond ("peptide bond")
linkages; b) non-natural residues in place of naturally occurring
amino acid residues; or c) residues which induce secondary
structural mimicry, i.e., to induce or stabilize a secondary
structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix
conformation, and the like. For example, a polypeptide of the
invention can be characterized as a mimetic when all or some of its
residues are joined by chemical means other than natural peptide
bonds. Individual peptidomimetic residues can be joined by peptide
bonds, other chemical bonds or coupling means, such as, e.g.,
glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or
N,N'-di-isopropylcarbodiimide (DIC). Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH2- for
--C(.dbd.O)--NH--), aminomethylene (CH2-NH), ethylene, olefin
(CH.dbd.CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-),
thiazole, retroamide, thioamide, or ester (see, e.g., Spatola
(1983) in Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications,"
Marcell Dekker, NY).
[0372] A polypeptide of the invention can also be characterized as
a mimetic by containing all or some non-natural residues in place
of naturally occurring amino acid residues. Non-natural residues
are well described in the scientific and patent literature; a few
exemplary non-natural compositions useful as mimetics of natural
amino acid residues and guidelines are described below. Mimetics of
aromatic amino acids can be generated by replacing by, e.g., D- or
L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;
D- or L-1, -2,3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine;
D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or
L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;
D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or
L-p-biphenyl-phenylalanine; K- or
L-p-methoxy-biphenylphenylalanine; D- or
L-2-indole-(alkyl)alanines; and, D- or L-alkylainines, where alkyl
can be substituted or unsubstituted methyl, ethyl, propyl, hexyl,
butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a
non-acidic amino acids. Aromatic rings of a non-natural amino acid
include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl,
naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
[0373] Mimetics of acidic amino acids can be generated by
substitution by, e.g., non-carboxylate amino acids while
maintaining a negative charge; (phosphono)alanine; sulfated
threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can
also be selectively modified by reaction with carbodiimides
(R'--N--C--N--R') such as, e.g.,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide Aspartyl or
glutamyl can also be converted to asparaginyl and glutaminyl
residues by reaction with ammonium ions. Mimetics of basic amino
acids can be generated by substitution with, e.g., (in addition to
lysine and arginine) the amino acids ornithine, citrulline, or
(guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where
alkyl is defined above. Nitrile derivative (e.g., containing the
CN-moiety in place of COOH) can be substituted for asparagine or
glutamine. Asparaginyl and glutaminyl residues can be deaminated to
the corresponding aspartyl or glutamyl residues. Arginine residue
mimetics can be generated by reacting arginyl with, e.g., one or
more conventional reagents, including, e.g., phenylglyoxal,
2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, preferably
under alkaline conditions. Tyrosine residue mimetics can be
generated by reacting tyrosyl with, e.g., aromatic diazonium
compounds or tetranitromethane. N-acetylimidizol and
tetranitromethane can be used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Cysteine residue mimetics can be
generated by reacting cysteinyl residues with, e.g.,
alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide
and corresponding amines; to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteine residue mimetics can also
be generated by reacting cysteinyl residues with, e.g.,
bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic
acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl
disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate;
2-chloromercuri-4 nitrophenol; or,
chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be
generated (and amino terminal residues can be altered) by reacting
lysinyl with, e.g., succinic or other carboxylic acid anhydrides.
Lysine and other alpha-amino-containing residue mimetics can also
be generated by reaction with imidoesters, such as methyl
picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,
trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione,
and transamidase-catalyzed reactions with glyoxylate. Mimetics of
methionine can be generated by reaction with, e.g., methionine
sulfoxide. Mimetics of proline include, e.g., pipecolic acid,
thiazolidine carboxylic acid, 3- or 4-hydroxy proline,
dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline.
Histidine residue mimetics can be generated by reacting histidyl
with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
Other mimetics include, e.g., those generated by hydroxylation of
proline and lysine; phosphorylation of the hydroxyl groups of seryl
or threonyl residues; methylation of the alpha-amino groups of
lysine, arginine and histidine; acetylation of the N-terminal
amine; methylation of main chain amide residues or substitution
with N-methyl amino acids; or amidation of C-terminal carboxyl
groups.
[0374] A residue, e.g., an amino acid, of a polypeptide of the
invention can also be replaced by an amino acid (or peptidomimetic
residue) of the opposite chirality. Thus, any amino acid naturally
occurring in the L-configuration (which can also be referred to as
the R or S, depending upon the structure of the chemical entity)
can be replaced with the amino acid of the same chemical structural
type or a peptidomimetic, but of the opposite chirality, referred
to as the D-amino acid, but also can be referred to as the R- or
S-form.
[0375] The invention also provides methods for modifying the
polypeptides of the invention by either natural processes, such as
post-translational processing (e.g., phosphorylation, acylation,
etc), or by chemical modification techniques, and the resulting
modified polypeptides. Modifications can occur anywhere in the
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also a given polypeptide may have many types of
modifications. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of a phosphatidylinositol,
cross-linking cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cysteine, formation
of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI anchor formation, hydroxylation, iodination, methylation,
myristolyation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation, and transfer-RNA mediated addition of amino acids to
protein such as arginylation. See, e.g., Creighton, T. E.,
Proteins--Structure and Molecular Properties 2nd Ed., W.H. Freeman
and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York, pp. 1-12 (1983).
[0376] Solid-phase chemical peptide synthesis methods can also be
used to synthesize the polypeptide or fragments of the invention.
Such method have been known in the art since the early 1960's
(Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See
also Stewart, J. M. and Young, J. D., Solid Phase Peptide
Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., pp.
11-12)) and have recently been employed in commercially available
laboratory peptide design and synthesis kits (Cambridge Research
Biochemicals). Such commercially available laboratory kits have
generally utilized the teachings of H. M. Geysen et al, Proc. Natl.
Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing
peptides upon the tips of a multitude of "rods" or "pins" all of
which are connected to a single plate. When such a system is
utilized, a plate of rods or pins is inverted and inserted into a
second plate of corresponding wells or reservoirs, which contain
solutions for attaching or anchoring an appropriate amino acid to
the pin's or rods tips. By repeating such a process step, i.e.,
inverting and inserting the rod's and pin's tips into appropriate
solutions, amino acids are built into desired peptides. In
addition, a number of available FMOC peptide synthesis systems are
available. For example, assembly of a polypeptide or fragment can
be carried out on a solid support using an Applied Biosystems, Inc.
Model 431A.TM. automated peptide synthesizer. Such equipment
provides ready access to the peptides of the invention, either by
direct synthesis or by synthesis of a series of fragments that can
be coupled using other known techniques.
Phospholipase Enzymes
[0377] The invention provides novel phospholipases, nucleic acids
encoding them, antibodies that bind them, peptides representing the
enzyme's antigenic sites (epitopes) and active sites, regulatory
and binding domains, and methods for making and using them. In one
aspect, polypeptides of the invention have a phospholipase
activity, or any combination of phospholipase activities, as
described herein (e.g., cleavage of a glycerolphosphate ester
linkage, lacking lipase activity, etc.). In alternative aspects,
the phospholipases of the invention have activities that have been
modified from those of the exemplary phospholipases described
herein.
[0378] The invention includes phospholipases with and without
signal sequences and the signal sequences themselves. The invention
includes fragments or subsequences of enzymes of the invention,
e.g., peptides or polypeptides comprising or consisting of
catalytic domains ("active sites"), binding sites, regulatory
domains, epitopes, signal sequences, prepro domains, and the like.
The invention also includes immobilized phospholipases,
anti-phospholipase antibodies and fragments thereof. The invention
includes heterocomplexes, e.g., fusion proteins, heterodimers,
etc., comprising the phospholipases of the invention. Determining
peptides representing the enzyme's antigenic sites (epitopes),
active sites, binding sites, signal sequences, and the like can be
done by routine screening protocols.
[0379] These enzymes and processes of the invention can be used to
achieve a more complete degumming of high phosphorus oils, in
particular, rice, soybean, corn, canola, and sunflower oils. For
example, in one aspect, upon cleavage by PI-PLC,
phosphatidylinositol is converted to diacylglycerol and
phosphoinositol. The diacylglycerol partitions to the aqueous phase
(improving oil yield) and the phosphoinositol partitions to the
aqueous phase where it is removed as a component of the heavy phase
during centrifugation. An enzyme of the invention, e.g., a PI-PLC
of the invention, can be incorporated into either a chemical or
physical oil refining process.
[0380] In alternative aspects, enzymes of the invention have
phosphatidylinositol-specific phospholipase C (PI-PLC) activity,
phosphatidylcholine-specific phospholipase C activity, phosphatidic
acid phosphatase activity, phospholipase A activity and/or
patatin-related phospholipase activity. These enzymes can be used
alone or in combination each other or with other enzymes of the
invention, or other enzymes. In one aspect, the invention provides
methods wherein these enzymes (including
phosphatidylinositol-specific phospholipase C (PIPLC),
phosphatidylcholine-specific phospholipase C, and/or phospholipase
D (in conjunction with a phosphatase), phosphatidic acid
phosphatase, phospholipase A, patatin-related phospholipases of the
invention) are used alone or in combination in the degumming of
oils, e.g., vegetable oils, e.g., high phosphorus oils, such as
soybean, corn, canola, rice bran and sunflower oils. These enzymes
and processes of the invention can be used to achieve a more
complete degumming of high phosphorus oils, in particular, soybean,
corn, canola, rice bran and sunflower oils. Upon cleavage by
PI-PLC, phosphatidylinositol is converted to diacylglycerol and
phosphoinositol. The diacylglycerol partitions to the aqueous phase
(improving oil yield) and the phosphoinositol partitions to the
aqueous phase where it is removed as a component of the heavy phase
during centrifugation. An enzyme of the invention, e.g., a PI-PLC
of the invention, can be incorporated into either a chemical or
physical oil refining process.
[0381] In one aspect, the invention provides compositions, e.g.,
solutions, comprising sodium citrate at neutral pH to hydrate
non-hydratables. For example, the invention provides sodium citrate
solutions in a pH range of between about 4 to 9, or, 5 to 8, or, 6
to 7, that can be used to hydrate non-hydratable phospholipids
(including enzymes of the invention) in high phosphorus oils. In
one aspect, the hydration of non-hydratable phospholipids is by
chelating the calcium and magnesium associated with the
phospholipids, thereby allowing the formerly insoluble phospholipid
salts to more readily partition in the aqueous phase. In one
aspect, once phospholipids move to the water/oil interface or into
the aqueous phase, a phospholipase of the invention (e.g., a
phospholipase-specific phosphohydrolase of the invention), or
another phospholipase, will convert the phospholipid to
diacylglycerol and a phosphate-ester. In one aspect, calcium and
magnesium metal content are lowered upon addition of acid and
caustic (see discussion on caustic processes).
[0382] The enzymes of the invention are highly selective catalysts.
As with other enzymes, they catalyze reactions with exquisite
stereo-, regio-, and chemo-selectivities that are unparalleled in
conventional synthetic chemistry. Moreover, the enzymes of the
invention are remarkably versatile. They can be tailored to
function in organic solvents, operate at extreme pHs (for example,
high pHs and low pHs) extreme temperatures (for example, high
temperatures and low temperatures), extreme salinity levels (for
example, high salinity and low salinity), and catalyze reactions
with compounds that are structurally unrelated to their natural,
physiological substrates. Enzymes of the invention can be designed
to be reactive toward a wide range of natural and unnatural
substrates, thus enabling the modification of virtually any organic
lead compound. Enzymes of the invention can also be designed to be
highly enantio- and regio-selective. The high degree of functional
group specificity exhibited by these enzymes enables one to keep
track of each reaction in a synthetic sequence leading to a new
active compound. Enzymes of the invention can also be designed to
catalyze many diverse reactions unrelated to their native
physiological function in nature.
[0383] The present invention exploits the unique catalytic
properties of enzymes. Whereas the use of biocatalysts (i.e.,
purified or crude enzymes, non-living or living cells) in chemical
transformations normally requires the identification of a
particular biocatalyst that reacts with a specific starting
compound. The present invention uses selected biocatalysts, i.e.,
the enzymes of the invention, and reaction conditions that are
specific for functional groups that are present in many starting
compounds. Each biocatalyst is specific for one functional group,
or several related functional groups, and can react with many
starting compounds containing this functional group. The
biocatalytic reactions produce a population of derivatives from a
single starting compound. These derivatives can be subjected to
another round of biocatalytic reactions to produce a second
population of derivative compounds. Thousands of variations of the
original compound can be produced with each iteration of
biocatalytic derivatization.
[0384] Enzymes react at specific sites of a starting compound
without affecting the rest of the molecule, a process that is very
difficult to achieve using traditional chemical methods. This high
degree of biocatalytic specificity provides the means to identify a
single active enzyme within a library. The library is characterized
by the series of biocatalytic reactions used to produce it, a
so-called "biosynthetic history". Screening the library for
biological activities and tracing the biosynthetic history
identifies the specific reaction sequence producing the active
compound. The reaction sequence is repeated and the structure of
the synthesized compound determined. This mode of identification,
unlike other synthesis and screening approaches, does not require
immobilization technologies, and compounds can be synthesized and
tested free in solution using virtually any type of screening
assay. It is important to note, that the high degree of specificity
of enzyme reactions on functional groups allows for the "tracking"
of specific enzymatic reactions that make up the biocatalytically
produced library.
[0385] The invention also provides methods of discovering new
phospholipases using the nucleic acids, polypeptides and antibodies
of the invention. In one aspect, lambda phage libraries are
screened for expression-based discovery of phospholipases. Use of
lambda phage libraries in screening allows detection of toxic
clones; improved access to substrate; reduced need for engineering
a host, by-passing the potential for any bias resulting from mass
excision of the library; and, faster growth at low clone densities.
Screening of lambda phage libraries can be in liquid phase or in
solid phase. Screening in liquid phase gives greater flexibility in
assay conditions; additional substrate flexibility; higher
sensitivity for weak clones; and ease of automation over solid
phase screening.
[0386] Many of the procedural steps are performed using robotic
automation enabling the execution of many thousands of biocatalytic
reactions and screening assays per day as well as ensuring a high
level of accuracy and reproducibility (see discussion of arrays,
below). As a result, a library of derivative compounds can be
produced in a matter of weeks. For further teachings on
modification of molecules, including small molecules, see
PCT/US94/09174.
Phospholipase Signal Sequences
[0387] The invention provides phospholipase signal sequences (e.g.,
signal peptides (SPs)), e.g., peptides comprising signal sequences
and/or chimeric polypeptides, where the peptides or chimerics have
a signal sequence as set forth in Table 1, or as set forth, below.
The invention provides nucleic acids encoding these signal
sequences (SPs, e.g., a peptide having a sequence
comprising/consisting of amino terminal residues of a polypeptide
of the invention). In one aspect, the invention provides a signal
sequence comprising a peptide comprising/consisting of a sequence
as set forth in residues 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to
24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31,
1 to 32 or 1 to 33 of a polypeptide of the invention, e.g., SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID
NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ
ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86,
SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104,
SEQ ID NO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQ ID
NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,
SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID
NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140;
SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150,
SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID
NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168,
SEQ ID NO:170, SEQ ID NO:172, or SEQ ID NO:174. Any of these
peptides can be part of a chimeric protein, e.g., a recombinant
protein. A signal sequence peptide can be matched with another
enzyme of the invention (e.g., a phospholipase of the invention
from which is was not derived), or, with another phospholipase, or
with any polypeptide, as discussed further, below.
[0388] Exemplary signal sequences are set forth in Table 1 and the
SEQ ID listing, e.g., residues 1 to 24 of SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6; residues 1 to 29 of SEQ ID NO:8; residues 1 to 20 of
SEQ ID NO:10; residues 1 to 19 of SEQ ID NO:20; residues 1 to 28 of
SEQ ID NO:22; residues 1 to 20 of SEQ ID NO:32; residues 1 to 23 of
SEQ ID NO:38; see Table 1 and the SEQ ID listing for other
exemplary signal sequences of the invention.
[0389] In some aspects phospholipases of the invention do not have
signal sequences. In one aspect, the invention provides the
phospholipases of the invention lacking all or part of a signal
sequence. In one aspect, the invention provides a nucleic acid
sequence encoding a signal sequence from one phospholipase operably
linked to a nucleic acid sequence of a different phospholipase or,
optionally, a signal sequence from a non-phospholipase protein may
be desired.
[0390] Phospholipase Prepro Domains, Binding Domains and Catalytic
Domains
[0391] In addition to signal sequences (e.g., signal peptides
(SPs)), as discussed above, the invention provides prepro domains,
binding domains (e.g., substrate binding domain) and catalytic
domains (CDs). The SP domains, binding domains, prepro domains
and/or CDs of the invention can be isolated or recombinant peptides
or can be part of a fusion protein, e.g., as a heterologous domain
in a chimeric protein. The invention provides nucleic acids
encoding these catalytic domains (CDs) (e.g., "active sites"),
prepro domains, binding domains and signal sequences (SPs, e.g., a
peptide having a sequence comprising/consisting of amino terminal
residues of a polypeptide of the invention).
[0392] The phospholipase signal sequences (SPs), binding domains,
catalytic domains (CDs) and/or prepro sequences of the invention
can be isolated peptides, or, sequences joined to another
phospholipase or a non-phospholipase polypeptide, e.g., as a fusion
(chimeric) protein. In one aspect, polypeptides comprising
phospholipase signal sequences SPs and/or prepro of the invention
comprise sequences heterologous to phospholipases of the invention
(e.g., a fusion protein comprising an SP and/or prepro of the
invention and sequences from another phospholipase or a
non-phospholipase protein). In one aspect, the invention provides
phospholipases of the invention with heterologous CDs, SPs and/or
prepro sequences, e.g., sequences with a yeast signal sequence. A
phospholipase of the invention can comprise a heterologous CD, SP
and/or prepro in a vector, e.g., a pPIC series vector (Invitrogen,
Carlsbad, Calif.).
[0393] In one aspect, SPs, CDs, and/or prepro sequences of the
invention are identified following identification of novel
phospholipase polypeptides. The pathways by which proteins are
sorted and transported to their proper cellular location are often
referred to as protein targeting pathways. One of the most
important elements in all of these targeting systems is a short
amino acid sequence at the amino terminus of a newly synthesized
polypeptide called the signal sequence. This signal sequence
directs a protein to its appropriate location in the cell and is
removed during transport or when the protein reaches its final
destination. Most lysosomal, membrane, or secreted proteins have an
amino-terminal signal sequence that marks them for translocation
into the lumen of the endoplasmic reticulum. The signal sequences
can vary in length from 13 to 45 or more amino acid residues.
Various methods of recognition of signal sequences are known to
those of skill in the art. For example, in one aspect, novel
hydrolase signal peptides are identified by a method referred to as
SignalP. SignalP uses a combined neural network which recognizes
both signal peptides and their cleavage sites. (Nielsen, et al.,
"Identification of prokaryotic and eukaryotic signal peptides and
prediction of their cleavage sites." Protein Engineering, vol. 10,
no. 1, p. 1-6 (1997).
[0394] In some aspects, a phospholipase of the invention may not
have SPs and/or prepro sequences, and/or catalytic domains (CDs).
In one aspect, the invention provides phospholipases lacking all or
part of an SP, a CD and/or a prepro domain. In one aspect, the
invention provides a nucleic acid sequence encoding a signal
sequence (SP), a CD and/or prepro from one phospholipase operably
linked to a nucleic acid sequence of a different phospholipase or,
optionally, a signal sequence (SPs), a CD and/or prepro domain from
a non-phospholipase protein may be desired.
[0395] The invention also provides isolated or recombinant
polypeptides comprising signal sequences (SPs), prepro domain
and/or catalytic domains (CDs) of the invention and heterologous
sequences. The heterologous sequences are sequences not naturally
associated (e.g., to a phospholipase) with an SP, prepro domain
and/or CD. The sequence to which the SP, prepro domain and/or CD
are not naturally associated can be on the SP's, prepro domain
and/or CD's amino terminal end, carboxy terminal end, and/or on
both ends of the SP and/or CD. In one aspect, the invention
provides an isolated or recombinant polypeptide comprising (or
consisting of) a polypeptide comprising a signal sequence (SP),
prepro domain and/or catalytic domain (CD) of the invention with
the proviso that it is not associated with any sequence to which it
is naturally associated (e.g., phospholipase sequence). Similarly
in one aspect, the invention provides isolated or recombinant
nucleic acids encoding these polypeptides. Thus, in one aspect, the
isolated or recombinant nucleic acid of the invention comprises
coding sequence for a signal sequence (SP), prepro domain and/or
catalytic domain (CD) of the invention and a heterologous sequence
(i.e., a sequence not naturally associated with the a signal
sequence (SP), prepro domain and/or catalytic domain (CD) of the
invention). The heterologous sequence can be on the 3' terminal
end, 5' terminal end, and/or on both ends of the SP, prepro domain
and/or CD coding sequence.
[0396] The polypeptides of the invention include phospholipases in
an active or inactive form. For example, the polypeptides of the
invention include proproteins before "maturation" or processing of
prepro sequences, e.g., by a proprotein-processing enzyme, such as
a proprotein convertase to generate an "active" mature protein. The
polypeptides of the invention include phospholipases inactive for
other reasons, e.g., before "activation" by a post-translational
processing event, e.g., an endo- or exo-peptidase or proteinase
action, a phosphorylation event, an amidation, a glycosylation, a
de-glycosylation, a sulfation, a dimerization event, and/or the
like. Methods for identifying "prepro" domain sequences, CDs,
binding domains and signal sequences are routine and well known in
the art, see, e.g., Van de Ven (1993) Crit. Rev. Oncog.
4(2):115-136; yeast two-hybrid screenings for identifying
protein-protein interactions, described e.g., by Miller (2004)
Methods Mol. Biol. 261:247-62; Heyninck (2004) Methods Mol. Biol.
282:223-41, U.S. Pat. Nos. 6,617,122; 6,190,874. For example, to
identify a prepro sequence, the protein is purified from the
extracellular space and the N-terminal protein sequence is
determined and compared to the unprocessed form.
[0397] The polypeptides of the invention can be formulated as a
protein preparation into any liquid, solid, semi-solid or gel form.
For example, a protein preparation of the invention can comprise a
formulation comprising a non-aqueous liquid composition, a cast
solid, a powder, a lyophilized powder, a granular form, a
particulate form, a compressed tablet, a pellet, a pill, a gel
form, a hydrogel, a paste, an aerosol, a spray, a lotion or a
slurry formulation.
[0398] The polypeptides of the invention include all active forms,
including active subsequences, e.g., catalytic domains (CDs) or
active sites, of an enzyme of the invention. In one aspect, the
invention provides catalytic domains or active sites as set forth
below. In one aspect, the invention provides a peptide or
polypeptide comprising or consisting of an active site domain as
predicted through use of a database such as Pfam (which is a large
collection of multiple sequence alignments and hidden Markov models
covering many common protein families, The Pfam protein families
database, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L.
Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall,
and E. L. L. Sonnhammer, Nucleic Acids Research, 30 (1):276-280,
2002) or equivalent.
[0399] The invention provides fusion of N-terminal or C-terminal
subsequences of enzymes of the invention (e.g., signal sequences,
prepro sequences) with other polypeptides, active proteins or
protein fragments. The production of an enzyme of the invention
(e.g., a phospholipase C enzyme) may also be accomplished by
expressing the enzyme as an inactive fusion protein that is later
activated by a proteolytic cleavage event (using either an
endogenous or exogenous protease activity, e.g. trypsin) that
results in the separation of the fusion protein partner and the
mature enzyme, e.g., phospholipase C enzyme. In one aspect, the
fusion protein of the invention is expressed from a hybrid
nucleotide construct that encodes a single open reading frame
containing the following elements: the nucleotide sequence for the
fusion protein, a linker sequence (defined as a nucleotide sequence
that encodes a flexible amino acid sequence that joins two less
flexible protein domains), protease cleavage recognition site, and
the mature enzyme (e.g., any enzyme of the invention, e.g., a
phospholipase) sequence. In alternative aspects, the fusion protein
can comprise a pectate lyase sequence, a xylanase sequence, a
phosphatidic acid phosphatase sequence, or another sequence, e.g.,
a sequence that has previously been shown to be over-expressed in a
host system of interest.
[0400] Any host system can be used (see discussion, above), for
example, any bacteria, e.g., a gram positive bacteria, such as
Bacillus, or a gram negative bacteria, such as E. coli, or any
yeast, e.g., Pichia pastoris. The arrangement of the nucleotide
sequences in the chimeric nucleotide construction can be determined
based on the protein expression levels achieved with each fusion
construct. Proceeding from the 5' end of the nucleotide construct
to the 3' prime end of the construct, in one aspect, the nucleotide
sequences is assembled as follows: Signal sequence/fusion
protein/linker sequence/protease cleavage recognition site/mature
enzyme (e.g., any enzyme of the invention, e.g., a phospholipase)
or Signal sequence/pro sequence/mature enzyme/linker
sequence/fusion protein. The expression of enzyme (e.g., any enzyme
of the invention, e.g., a phospholipase) as an inactive fusion
protein may improve the overall expression of the enzyme's
sequence, may reduce any potential toxicity associated with the
overproduction of active enzyme and/or may increase the shelf life
of enzyme prior to use because enzyme would be inactive until the
fusion protein e.g. pectate lyase is separated from the enzyme,
e.g., phospholipase protein.
[0401] In various aspects, the invention provides specific
formulations for the activation of phospholipase of the invention
expressed as a fusion protein. In one aspect, the activation of the
phospholipase activity initially expressed as an inactive fusion
protein is accomplished using a proteolytic activity or potentially
a proteolytic activity in combination with an amino-terminal or
carboxyl-terminal peptidase. This activation event may be
accomplished in a variety of ways and at variety of points in the
manufacturing/storage process prior to application in oil
degumming. Exemplary processes of the invention include: Cleavage
by an endogenous activity expressed by the manufacturing host upon
secretion of the fusion construct into the fermentation media;
Cleavage by an endogenous protease activity that is activated or
comes in contact with intracellularly expressed fusion construct
upon rupture of the host cells; Passage of the crude or purified
fusion construct over a column of immobilized protease activity to
accomplish cleavage and enzyme (e.g., phospholipase of the
invention, e.g., a phospholipase C) activation prior to enzyme
formulation; Treatment of the crude or purified fusion construct
with a soluble source of proteolytic activity; Activation of a
phospholipase (e.g., a phospholipase of the invention, e.g., a
phospholipase C) at the oil refinery using either a soluble or
insoluble source of proteolytic activity immediately prior to use
in the process; and/or, Activation of the phospholipase (e.g., a
phospholipase of the invention, e.g., a phospholipase C) activity
by continuously circulating the fusion construct formulation
through a column of immobilized protease activity at reduced
temperature (for example, any between about 4.degree. C. and
20.degree. C.). This activation event may be accomplished prior to
delivery to the site of use or it may occur on-site at the oil
refinery.
[0402] Glycosylation
[0403] The peptides and polypeptides of the invention (e.g.,
hydrolases, antibodies) can also be glycosylated, for example, in
one aspect, comprising at least one glycosylation site, e.g., an
N-linked or O-linked glycosylation. In one aspect, the polypeptide
can be glycosylated after being expressed in a P. pastoris or a S.
pombe. The glycosylation can be added post-translationally either
chemically or by cellular biosynthetic mechanisms, wherein the
later incorporates the use of known glycosylation motifs, which can
be native to the sequence or can be added as a peptide or added in
the nucleic acid coding sequence.
[0404] In one aspect, the invention provides a polypeptide
comprising an N-linked glycosylated SEQ ID NO:2, as described,
e.g., in the following table:
TABLE-US-00002 Site Amino acid position of number Glycosylation
site Length glycosylation site 1 Match: NNS Length: 3 Start: 27
Stop: 29 2 Match: NTT Length: 3 Start: 65 Stop: 67 3 Match: NET
Length: 3 Start: 72 Stop: 74 4 Match: NST Length: 3 Start: 100
Stop: 102 5 Match: NFT Length: 3 Start: 168 Stop: 170 6 Match: NLS
Length: 3 Start: 171 Stop: 173 7 Match: NDT Length: 3 Start: 229
Stop: 231
[0405] The full-length SEQ ID NO:2 (which in one aspect is encoded
by SEQ ID NO:1) open reading frame encodes seven (7) potential
asparagine-linked (N-linked) glycosylation sites. The expression of
the wild-type SEQ ID NO:2 open reading frame in a glycosylating
host (e.g. Pichia pastoris, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, or a mammalian cell) results in the
production of a glycosylated SEQ ID NO:2 phospholipase enzyme that
is essentially inactive due to the presence of N-linked
glycosylation. Enzymatic deglycosylation of the wild-type,
glycosylated SEQ ID NO:2 with PNGase F or Endoglycosidase H results
in the activation of the SEQ ID NO:2 activity. In addition,
modification of one or more of the N-linked glycosylation sites
through mutagenesis (so that the site is no longer recognized as an
N-linked glycosylation site and glycosylation no longer occurs at
that site) results in the production of SEQ ID NO:2 with varying
degrees of increased activity.
[0406] Mutagenesis of the nucleotide codon encoding the asparagine
in SEQ ID NO:2 glycosylation sites 4,5, and/or 6 (e.g. converting
the asparagine to an aspartic acid) results in the production of an
enzyme with increased PLC activity compared to the wild-type open
reading frame expressed in the same host (the triple mutant
expressed in Pichia pastoris possesses a specific activity and a
functional activity that is essentially identical to that of the
wild-type sequence expressed in a non-glycosylating host like E.
coli. It is also possible to abolish the N-linked glycosylation
site by mutagenesis of the serine or threonine residue in the
N-linked glycosylation consensus sequence (NXS/T), for example by
converting these nucleotide codons to produce valine or isoleucine
at these positions instead of serine or threonine. The use of this
strategy to remove N-linked glycosylation sites also results in the
production of active SEQ ID NO:2 phospholipase in glycosylating
host expression systems.
Assays for Phospholipase Activity
[0407] The invention provides isolated, synthetic or recombinant
polypeptides (e.g., enzymes, antibodies) having a phospholipase
activity, or any combination of phospholipase activities, and
nucleic acids encoding them. Any of the many phospholipase activity
assays known in the art can be used to determine if a polypeptide
has a phospholipase activity and is within the scope of the
invention. Routine protocols for determining phospholipase A, B, D
and C, patatin and lipid acyl hydrolase activities, or lipase
activity, are well known in the art.
[0408] Exemplary activity assays include turbidity assays,
methylumbelliferyl phosphocholine (fluorescent) assays, Amplex red
(fluorescent) phospholipase assays, thin layer chromatography
assays (TLC), cytolytic assays and p-nitrophenylphosphorylcholine
assays. Using these assays polypeptides, peptides or antibodies can
be quickly screened for a phospholipase activity.
[0409] The phospholipase activity can comprise a lipid acyl
hydrolase (LAH) activity. See, e.g., Jimenez (2001) Lipids
36:1169-1174, describing an octaethylene glycol monododecyl
ether-based mixed micellar assay for determining the lipid acyl
hydrolase activity of a patatin. Pinsirodom (2000) J. Agric. Food
Chem. 48:155-160, describes an exemplary lipid acyl hydrolase (LAH)
patatin activity.
[0410] Turbidity assays to determine phospholipase activity are
described, e.g., in Kauffmann (2001) "Conversion of Bacillus
thermocatenulatus lipase into an efficient phospholipase with
increased activity towards long-chain fatty acyl substrates by
directed evolution and rational design," Protein Engineering
14:919-928; Ibrahim (1995) "Evidence implicating phospholipase as a
virulence factor of Candida albicans," Infect. Immun
63:1993-1998.
[0411] Methylumbelliferyl (fluorescent) phosphocholine assays to
determine phospholipase activity are described, e.g., in Goode
(1997) "Evidence for cell surface and internal phospholipase
activity in ascidian eggs," Develop. Growth Differ. 39:655-660;
Diaz (1999) "Direct fluorescence-based lipase activity assay,"
BioTechniques 27:696-700.
[0412] Amplex Red (fluorescent) Phospholipase Assays to determine
phospholipase activity are available as kits, e.g., the detection
of phosphatidylcholine-specific phospholipase using an Amplex Red
phosphatidylcholine-specific phospholipase assay kit from Molecular
Probes Inc. (Eugene, Oreg.), according to manufacturer's
instructions. Fluorescence is measured in a fluorescence microplate
reader using excitation at 560.+-.10 nm and fluorescence detection
at 590.+-.10 nm. The assay is sensitive at very low enzyme
concentrations.
[0413] Thin layer chromatography assays (TLC) to determine
phospholipase activity are described, e.g., in Reynolds (1991)
Methods in Enzymol. 197:3-13; Taguchi (1975) "Phospholipase from
Clostridium novyi type A.I," Biochim Biophys. Acta 409:75-85. Thin
layer chromatography (TLC) is a widely used technique for detection
of phospholipase activity. Various modifications of this method
have been used to extract the phospholipids from the aqueous assay
mixtures. In some PLC assays the hydrolysis is stopped by addition
of chloroform/methanol (2:1) to the reaction mixture. The unreacted
starting material and the diacylglycerol are extracted into the
organic phase and may be fractionated by TLC, while the head group
product remains in the aqueous phase. For more precise measurement
of the phospholipid digestion, radiolabeled substrates can be used
(see, e.g., Reynolds (1991) Methods in Enzymol. 197:3-13). The
ratios of products and reactants can be used to calculate the
actual number of moles of substrate hydrolyzed per unit time. If
all the components are extracted equally, any losses in the
extraction will affect all components equally. Separation of
phospholipid digestion products can be achieved by silica gel TLC
with chloroform/methanol/water (65:25:4) used as a solvent system
(see, e.g., Taguchi (1975) Biochim Biophys. Acta 409:75-85).
[0414] p-Nitrophenylphosphorylcholine assays to determine
phospholipase activity are described, e.g., in Korbsrisate (1999)
J. Clin. Microbiol. 37:3742-3745; Berka (1981) Infect. Immun.
34:1071-1074. This assay is based on enzymatic hydrolysis of the
substrate analog p-nitrophenylphosphorylcholine to liberate a
yellow chromogenic compound p-nitrophenol, detectable at 405 nm.
This substrate is convenient for high-throughput screening.
[0415] A cytolytic assay can detect phospholipases with cytolytic
activity based on lysis of erythrocytes. Toxic phospholipases can
interact with eukaryotic cell membranes and hydrolyze
phosphatidylcholine and sphingomyelin, leading to cell lysis. See,
e.g., Titball (1993) Microbiol. Rev. 57:347-366.
Hybrid (Chimeric) Phospholipases and Peptide Libraries
[0416] In one aspect, the invention provides hybrid phospholipases
and fusion proteins, including peptide libraries, comprising
sequences of the invention. The peptide libraries of the invention
can be used to isolate peptide modulators (e.g., activators or
inhibitors) of targets, such as phospholipase substrates,
receptors, enzymes. The peptide libraries of the invention can be
used to identify formal binding partners of targets, such as
ligands, e.g., cytokines, hormones and the like. In one aspect, the
invention provides chimeric proteins comprising a signal sequence
(SP) and/or catalytic domain (CD) of the invention and a
heterologous sequence (see above).
[0417] The invention also provides methods for generating
"improved" and hybrid phospholipases using the nucleic acids and
polypeptides of the invention. For example, the invention provides
methods for generating enzymes that have activity, e.g.,
phospholipase activity (such as, e.g., phospholipase A, B, C or D
activity, patatin esterase activity, cleavage of a
glycerolphosphate ester linkage, cleavage of an ester linkage in a
phospholipid in a vegetable oil) at extreme alkaline pHs and/or
acidic pHs, high and low temperatures, osmotic conditions and the
like. The invention provides methods for generating hybrid enzymes
(e.g., hybrid phospholipases).
[0418] In one aspect, the methods of the invention produce new
hybrid polypeptides by utilizing cellular processes that integrate
the sequence of a first polynucleotide such that resulting hybrid
polynucleotides encode polypeptides demonstrating activities
derived from the first biologically active polypeptides. For
example, the first polynucleotides can be an exemplary nucleic acid
sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
etc.) encoding an exemplary phospholipase of the invention (e.g.,
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, etc.). The
first nucleic acid can encode an enzyme from one organism that
functions effectively under a particular environmental condition,
e.g. high salinity. It can be "integrated" with an enzyme encoded
by a second polynucleotide from a different organism that functions
effectively under a different environmental condition, such as
extremely high temperatures. For example, when the two nucleic
acids can produce a hybrid molecule by e.g., recombination and/or
reductive reassortment. A hybrid polynucleotide containing
sequences from the first and second original polynucleotides may
encode an enzyme that exhibits characteristics of both enzymes
encoded by the original polynucleotides. Thus, the enzyme encoded
by the hybrid polynucleotide may function effectively under
environmental conditions shared by each of the enzymes encoded by
the first and second polynucleotides, e.g., high salinity and
extreme temperatures.
[0419] Alternatively, a hybrid polypeptide resulting from this
method of the invention may exhibit specialized enzyme activity not
displayed in the original enzymes. For example, following
recombination and/or reductive reassortment of polynucleotides
encoding phospholipase activities, the resulting hybrid polypeptide
encoded by a hybrid polynucleotide can be screened for specialized
activities obtained from each of the original enzymes, i.e. the
type of bond on which the phospholipase acts and the temperature at
which the phospholipase functions. Thus, for example, the
phospholipase may be screened to ascertain those chemical
functionalities which distinguish the hybrid phospholipase from the
original phospholipases, such as: (a) amide (peptide bonds), i.e.,
phospholipases; (b) ester bonds, i.e., phospholipases and lipases;
(c) acetals, i.e., glycosidases and, for example, the temperature,
pH or salt concentration at which the hybrid polypeptide
functions.
[0420] Sources of the polynucleotides to be "integrated" with
nucleic acids of the invention may be isolated from individual
organisms ("isolates"), collections of organisms that have been
grown in defined media ("enrichment cultures"), or, uncultivated
organisms ("environmental samples"). The use of a
culture-independent approach to derive polynucleotides encoding
novel bioactivities from environmental samples is most preferable
since it allows one to access untapped resources of biodiversity.
"Environmental libraries" are generated from environmental samples
and represent the collective genomes of naturally occurring
organisms archived in cloning vectors that can be propagated in
suitable prokaryotic hosts. Because the cloned DNA is initially
extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. Additionally, a normalization of the environmental
DNA present in these samples could allow more equal representation
of the DNA from all of the species present in the original sample.
This can dramatically increase the efficiency of finding
interesting genes from minor constituents of the sample that may be
under-represented by several orders of magnitude compared to the
dominant species.
[0421] For example, gene libraries generated from one or more
uncultivated microorganisms are screened for an activity of
interest. Potential pathways encoding bioactive molecules of
interest are first captured in prokaryotic cells in the form of
gene expression libraries. Polynucleotides encoding activities of
interest are isolated from such libraries and introduced into a
host cell. The host cell is grown under conditions that promote
recombination and/or reductive reassortment creating potentially
active biomolecules with novel or enhanced activities.
[0422] The microorganisms from which hybrid polynucleotides may be
prepared include prokaryotic microorganisms, such as Eubacteria and
Archaebacteria, and lower eukaryotic microorganisms such as fungi,
some algae and protozoa. Polynucleotides may be isolated from
environmental samples. Nucleic acid may be recovered without
culturing of an organism or recovered from one or more cultured
organisms. In one aspect, such microorganisms may be extremophiles,
such as hyperthermophiles, psychrophiles, psychrotrophs,
halophiles, barophiles and acidophiles. In one aspect,
polynucleotides encoding phospholipase enzymes isolated from
extremophilic microorganisms are used to make hybrid enzymes. Such
enzymes may function at temperatures above 100.degree. C. in, e.g.,
terrestrial hot springs and deep sea thermal vents, at temperatures
below 0.degree. C. in, e.g., arctic waters, in the saturated salt
environment of, e.g., the Dead Sea, at pH values around 0 in, e.g.,
coal deposits and geothermal sulfur-rich springs, or at pH values
greater than 11 in, e.g., sewage sludge. For example,
phospholipases cloned and expressed from extremophilic organisms
can show high activity throughout a wide range of temperatures and
pHs.
[0423] Polynucleotides selected and isolated as described herein,
including at least one nucleic acid of the invention, are
introduced into a suitable host cell. A suitable host cell is any
cell that is capable of promoting recombination and/or reductive
reassortment. The selected polynucleotides can be in a vector that
includes appropriate control sequences. The host cell can be a
higher eukaryotic cell, such as a mammalian cell, or a lower
eukaryotic cell, such as a yeast cell, or preferably, the host cell
can be a prokaryotic cell, such as a bacterial cell. Introduction
of the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis et al., 1986).
[0424] Exemplary appropriate hosts include bacterial cells, such as
E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such
as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9;
animal cells such as CHO, COS or Bowes melanoma; adenoviruses; and
plant cells (see also, discussion above). The selection of an
appropriate host for recombination and/or reductive reassortment or
just for expression of recombinant protein is deemed to be within
the scope of those skilled in the art from the teachings herein.
Mammalian cell culture systems that can be employed for
recombination and/or reductive reassortment or just for expression
of recombinant protein include, e.g., the COS-7 lines of monkey
kidney fibroblasts, described in "SV40-transformed simian cells
support the replication of early SV40 mutants" (Gluzman, 1981), the
C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression
vectors can comprise an origin of replication, a suitable promoter
and enhancer, and necessary ribosome binding sites, polyadenylation
site, splice donor and acceptor sites, transcriptional termination
sequences, and 5' flanking non-transcribed sequences. DNA sequences
derived from the SV40 splice, and polyadenylation sites may be used
to provide the required non-transcribed genetic elements.
[0425] Host cells containing the polynucleotides of interest (for
recombination and/or reductive reassortment or just for expression
of recombinant protein) can be cultured in conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants or amplifying genes. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan. The clones which are identified as
having the specified enzyme activity may then be sequenced to
identify the polynucleotide sequence encoding an enzyme having the
enhanced activity.
[0426] In another aspect, the nucleic acids and methods of the
present invention can be used to generate novel polynucleotides for
biochemical pathways, e.g., pathways from one or more operons or
gene clusters or portions thereof. For example, bacteria and many
eukaryotes have a coordinated mechanism for regulating genes whose
products are involved in related processes. The genes are
clustered, in structures referred to as "gene clusters," on a
single chromosome and are transcribed together under the control of
a single regulatory sequence, including a single promoter which
initiates transcription of the entire cluster. Thus, a gene cluster
is a group of adjacent genes that are either identical or related,
usually as to their function.
[0427] Gene cluster DNA can be isolated from different organisms
and ligated into vectors, particularly vectors containing
expression regulatory sequences which can control and regulate the
production of a detectable protein or protein-related array
activity from the ligated gene clusters. Use of vectors which have
an exceptionally large capacity for exogenous DNA introduction are
particularly appropriate for use with such gene clusters and are
described by way of example herein to include the f-factor (or
fertility factor) of E. coli. This f-factor of E. coli is a plasmid
which affects high-frequency transfer of itself during conjugation
and is ideal to achieve and stably propagate large DNA fragments,
such as gene clusters from mixed microbial samples. "Fosmids,"
cosmids or bacterial artificial chromosome (BAC) vectors can be
used as cloning vectors. These are derived from E. coli f-factor
which is able to stably integrate large segments of genomic DNA.
When integrated with DNA from a mixed uncultured environmental
sample, this makes it possible to achieve large genomic fragments
in the form of a stable "environmental DNA library." Cosmid vectors
were originally designed to clone and propagate large segments of
genomic DNA. Cloning into cosmid vectors is described in detail in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor Laboratory Press (1989). Once ligated into an
appropriate vector, two or more vectors containing different
polyketide synthase gene clusters can be introduced into a suitable
host cell. Regions of partial sequence homology shared by the gene
clusters will promote processes which result in sequence
reorganization resulting in a hybrid gene cluster. The novel hybrid
gene cluster can then be screened for enhanced activities not found
in the original gene clusters.
[0428] Thus, in one aspect, the invention relates to a method for
producing a biologically active hybrid polypeptide using a nucleic
acid of the invention and screening the polypeptide for an activity
(e.g., enhanced activity) by:
[0429] (1) introducing at least a first polynucleotide (e.g., a
nucleic acid of the invention) in operable linkage and a second
polynucleotide in operable linkage, said at least first
polynucleotide and second polynucleotide sharing at least one
region of partial sequence homology, into a suitable host cell;
[0430] (2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage;
[0431] (3) expressing a hybrid polypeptide encoded by the hybrid
polynucleotide;
[0432] (4) screening the hybrid polypeptide under conditions which
promote identification of the desired biological activity (e.g.,
enhanced phospholipase activity); and
[0433] (5) isolating the a polynucleotide encoding the hybrid
polypeptide.
[0434] Methods for screening for various enzyme activities are
known to those of skill in the art and are discussed throughout the
present specification. Such methods may be employed when isolating
the polypeptides and polynucleotides of the invention.
[0435] In vivo reassortment can be focused on "inter-molecular"
processes collectively referred to as "recombination." In bacteria
it is generally viewed as a "RecA-dependent" phenomenon. The
invention can rely on recombination processes of a host cell to
recombine and re-assort sequences, or the cells' ability to mediate
reductive processes to decrease the complexity of quasi-repeated
sequences in the cell by deletion. This process of "reductive
reassortment" occurs by an "intra-molecular", RecA-independent
process. Thus, in one aspect of the invention, using the nucleic
acids of the invention novel polynucleotides are generated by the
process of reductive reassortment. The method involves the
generation of constructs containing consecutive sequences (original
encoding sequences), their insertion into an appropriate vector,
and their subsequent introduction into an appropriate host cell.
The reassortment of the individual molecular identities occurs by
combinatorial processes between the consecutive sequences in the
construct possessing regions of homology, or between quasi-repeated
units. The reassortment process recombines and/or reduces the
complexity and extent of the repeated sequences, and results in the
production of novel molecular species.
[0436] Various treatments may be applied to enhance the rate of
reassortment. These could include treatment with ultra-violet
light, or DNA damaging chemicals, and/or the use of host cell lines
displaying enhanced levels of "genetic instability". Thus the
reassortment process may involve homologous recombination or the
natural property of quasi-repeated sequences to direct their own
evolution.
[0437] Repeated or "quasi-repeated" sequences play a role in
genetic instability. "Quasi-repeats" are repeats that are not
restricted to their original unit structure. Quasi-repeated units
can be presented as an array of sequences in a construct;
consecutive units of similar sequences. Once ligated, the junctions
between the consecutive sequences become essentially invisible and
the quasi-repetitive nature of the resulting construct is now
continuous at the molecular level. The deletion process the cell
performs to reduce the complexity of the resulting construct
operates between the quasi-repeated sequences. The quasi-repeated
units provide a practically limitless repertoire of templates upon
which slippage events can occur. The constructs containing the
quasi-repeats thus effectively provide sufficient molecular
elasticity that deletion (and potentially insertion) events can
occur virtually anywhere within the quasi-repetitive units. When
the quasi-repeated sequences are all ligated in the same
orientation, for instance head to tail or vice versa, the cell
cannot distinguish individual units. Consequently, the reductive
process can occur throughout the sequences. In contrast, when for
example, the units are presented head to head, rather than head to
tail, the inversion delineates the endpoints of the adjacent unit
so that deletion formation will favor the loss of discrete units.
Thus, in one aspect of the invention, the sequences to be
reassorted are in the same orientation. Random orientation of
quasi-repeated sequences will result in the loss of reassortment
efficiency, while consistent orientation of the sequences will
offer the highest efficiency. However, while having fewer of the
contiguous sequences in the same orientation decreases the
efficiency, it may still provide sufficient elasticity for the
effective recovery of novel molecules. Constructs can be made with
the quasi-repeated sequences in the same orientation to allow
higher efficiency.
[0438] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following: a)
Primers that include a poly-A head and poly-T tail which when made
single-stranded would provide orientation can be utilized. This is
accomplished by having the first few bases of the primers made from
RNA and hence easily removed RNase H. b) Primers that include
unique restriction cleavage sites can be utilized. Multiple sites,
a battery of unique sequences, and repeated synthesis and ligation
steps would be required. c) The inner few bases of the primer could
be thiolated and an exonuclease used to produce properly tailed
molecules.
[0439] The recovery of the re-assorted sequences relies on the
identification of cloning vectors with a reduced repetitive index
(RI). The re-assorted encoding sequences can then be recovered by
amplification. The products are re-cloned and expressed. The
recovery of cloning vectors with reduced RI can be affected by: 1)
The use of vectors only stably maintained when the construct is
reduced in complexity. 2) The physical recovery of shortened
vectors by physical procedures. In this case, the cloning vector
would be recovered using standard plasmid isolation procedures and
size fractionated on either an agarose gel, or column with a low
molecular weight cut off utilizing standard procedures. 3) The
recovery of vectors containing interrupted genes which can be
selected when insert size decreases. 4) The use of direct selection
techniques with an expression vector and the appropriate
selection.
[0440] Encoding sequences (for example, genes) from related
organisms may demonstrate a high degree of homology and encode
quite diverse protein products. These types of sequences are
particularly useful in the present invention as quasi-repeats.
However, this process is not limited to such nearly identical
repeats.
[0441] The following is an exemplary method of the invention.
Encoding nucleic acid sequences (quasi-repeats) are derived from
three (3) species, including a nucleic acid of the invention. Each
sequence encodes a protein with a distinct set of properties,
including an enzyme of the invention. Each of the sequences differs
by a single or a few base pairs at a unique position in the
sequence. The quasi-repeated sequences are separately or
collectively amplified and ligated into random assemblies such that
all possible permutations and combinations are available in the
population of ligated molecules. The number of quasi-repeat units
can be controlled by the assembly conditions. The average number of
quasi-repeated units in a construct is defined as the repetitive
index (RI). Once formed, the constructs may, or may not be size
fractionated on an agarose gel according to published protocols,
inserted into a cloning vector, and transfected into an appropriate
host cell. The cells are then propagated and "reductive
reassortment" is effected. The rate of the reductive reassortment
process may be stimulated by the introduction of DNA damage if
desired. Whether the reduction in R1 is mediated by deletion
formation between repeated sequences by an "intra-molecular"
mechanism, or mediated by recombination-like events through
"inter-molecular" mechanisms is immaterial. The end result is a
reassortment of the molecules into all possible combinations. In
one aspect, the method comprises the additional step of screening
the library members of the shuffled pool to identify individual
shuffled library members having the ability to bind or otherwise
interact, or catalyze a particular reaction (e.g., such as
catalytic domain of an enzyme) with a predetermined macromolecule,
such as for example a proteinaceous receptor, an oligosaccharide,
virion, or other predetermined compound or structure. The
polypeptides, e.g., phospholipases, that are identified from such
libraries can be used for various purposes, e.g., the industrial
processes described herein and/or can be subjected to one or more
additional cycles of shuffling and/or selection.
[0442] In another aspect, it is envisioned that prior to or during
recombination or reassortment, polynucleotides generated by the
method of the invention can be subjected to agents or processes
which promote the introduction of mutations into the original
polynucleotides. The introduction of such mutations would increase
the diversity of resulting hybrid polynucleotides and polypeptides
encoded therefrom. The agents or processes which promote
mutagenesis can include, but are not limited to: (+)-CC-1065, or a
synthetic analog such as (+)-CC-1065-(N3-Adenine (See Sun and
Hurley, (1992); an N-acetylated or deacetylated
4'-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA
synthesis (See, for example, van de Poll et al. (1992)); or a
N-acetylated or deacetylated 4-aminobiphenyl adduct capable of
inhibiting DNA synthesis (See also, van de Poll et al. (1992), pp.
751-758); trivalent chromium, a trivalent chromium salt, a
polycyclic aromatic hydrocarbon (PAH) DNA adduct capable of
inhibiting DNA replication, such as 7-bromomethyl-benz[a]anthracene
("BMA"), tris(2,3-dibromopropyl)phosphate ("Tris-BP"),
1,2-dibromo-3-chloropropane ("DBCP"), 2-bromoacrolein (2BA),
benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide ("BPDE"), a
platinum(II) halogen salt,
N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline
("N-hydroxy-IQ"), and
N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine
("N-hydroxy-PhIP"). Especially preferred means for slowing or
halting PCR amplification consist of UV light (+)-CC-1065 and
(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA
adducts or polynucleotides comprising the DNA adducts from the
polynucleotides or polynucleotides pool, which can be released or
removed by a process including heating the solution comprising the
polynucleotides prior to further processing.
Screening Methodologies and "On-Line" Monitoring Devices
[0443] In practicing the methods of the invention, a variety of
apparatus and methodologies can be used to in conjunction with the
polypeptides and nucleic acids of the invention, e.g., to screen
polypeptides for phospholipase activity, to screen compounds as
potential modulators of activity (e.g., potentiation or inhibition
of enzyme activity), for antibodies that bind to a polypeptide of
the invention, for nucleic acids that hybridize to a nucleic acid
of the invention, and the like.
[0444] Immobilized Enzyme Solid Supports
[0445] The phospholipase enzymes, fragments thereof and nucleic
acids that encode the enzymes and fragments can be affixed to a
solid support. This is often economical and efficient in the use of
the phospholipases in industrial processes. For example, a
consortium or cocktail of phospholipase enzymes (or active
fragments thereof), which are used in a specific chemical reaction,
can be attached to a solid support and dunked into a process vat.
The enzymatic reaction can occur. Then, the solid support can be
taken out of the vat, along with the enzymes affixed thereto, for
repeated use. In one embodiment of the invention, an isolated
nucleic acid of the invention is affixed to a solid support. In
another embodiment of the invention, the solid support is selected
from the group of a gel, a resin, a polymer, a ceramic, a glass, a
microelectrode and any combination thereof.
[0446] For example, solid supports useful in this invention include
gels. Some examples of gels include Sepharose, gelatin,
glutaraldehyde, chitosan-treated glutaraldehyde,
albumin-glutaraldehyde, chitosan-Xanthan, toyopearl gel (polymer
gel), alginate, alginate-polylysine, carrageenan, agarose, glyoxyl
agarose, magnetic agarose, dextran-agarose, poly(Carbamoyl
Sulfonate) hydrogel, BSA-PEG hydrogel, phosphorylated polyvinyl
alcohol (PVA), monoaminoethyl-N-aminoethyl (MANA), amino, or any
combination thereof.
[0447] Another solid support useful in the present invention are
resins or polymers. Some examples of resins or polymers include
cellulose, acrylamide, nylon, rayon, polyester, anion-exchange
resin, AMBERLITE.TM. XAD-7, AMBERLITE.TM. XAD-8, AMBERLITE.TM.
IRA-94, AMBERLITE.TM. IRC-50, polyvinyl, polyacrylic,
polymethacrylate, or any combination thereof.
[0448] Another type of solid support useful in the present
invention is ceramic. Some examples include non-porous ceramic,
porous ceramic, SiO.sub.2, Al.sub.2O.sub.3. Another type of solid
support useful in the present invention is glass. Some examples
include non-porous glass, porous glass, aminopropyl glass or any
combination thereof. Another type of solid support that can be used
is a microelectrode. An example is a polyethyleneimine-coated
magnetite. Graphitic particles can be used as a solid support.
[0449] Other exemplary solid supports used to practice the
invention comprise diatomaceous earth products and silicates. Some
examples include CELITE.RTM. KENITE.RTM., DIACTIV.RTM.,
PRIMISIL.RTM., DIAFIL.RTM. diatomites and MICRO-CEL.RTM.,
CALFLO.RTM., SILASORB.TM., and CELKATE.RTM. synthetic calcium and
magnesium silicates. Another example of a solid support is a cell,
such as a red blood cell.
[0450] Methods of Immobilization
[0451] There are many methods that would be known to one of skill
in the art for immobilizing enzymes or fragments thereof, or
nucleic acids, onto a solid support. Some examples of such methods
include, e.g., electrostatic droplet generation, electrochemical
means, via adsorption, via covalent binding, via cross-linking, via
a chemical reaction or process, via encapsulation, via entrapment,
via calcium alginate, or via poly(2-hydroxyethyl methacrylate).
Like methods are described in Methods in Enzymology, Immobilized
Enzymes and Cells, Part C. 1987. Academic Press. Edited by S. P.
Colowick and N. O. Kaplan. Volume 136; and Immobilization of
Enzymes and Cells. 1997. Humana Press. Edited by G. F. Bickerstaff.
Series: Methods in Biotechnology, Edited by J. M. Walker.
[0452] Capillary Arrays
[0453] Capillary arrays, such as the GIGAMATRIX.TM., Diversa
Corporation, San Diego, Calif., can be used to in the methods of
the invention. Nucleic acids or polypeptides of the invention can
be immobilized to or applied to an array, including capillary
arrays. Arrays can be used to screen for or monitor libraries of
compositions (e.g., small molecules, antibodies, nucleic acids,
etc.) for their ability to bind to or modulate the activity of a
nucleic acid or a polypeptide of the invention. Capillary arrays
provide another system for holding and screening samples. For
example, a sample screening apparatus can include a plurality of
capillaries formed into an array of adjacent capillaries, wherein
each capillary comprises at least one wall defining a lumen for
retaining a sample. The apparatus can further include interstitial
material disposed between adjacent capillaries in the array, and
one or more reference indicia formed within of the interstitial
material. A capillary for screening a sample, wherein the capillary
is adapted for being bound in an array of capillaries, can include
a first wall defining a lumen for retaining the sample, and a
second wall formed of a filtering material, for filtering
excitation energy provided to the lumen to excite the sample.
[0454] A polypeptide or nucleic acid, e.g., a ligand, can be
introduced into a first component into at least a portion of a
capillary of a capillary array. Each capillary of the capillary
array can comprise at least one wall defining a lumen for retaining
the first component. An air bubble can be introduced into the
capillary behind the first component. A second component can be
introduced into the capillary, wherein the second component is
separated from the first component by the air bubble. A sample of
interest can be introduced as a first liquid labeled with a
detectable particle into a capillary of a capillary array, wherein
each capillary of the capillary array comprises at least one wall
defining a lumen for retaining the first liquid and the detectable
particle, and wherein the at least one wall is coated with a
binding material for binding the detectable particle to the at
least one wall. The method can further include removing the first
liquid from the capillary tube, wherein the bound detectable
particle is maintained within the capillary, and introducing a
second liquid into the capillary tube.
[0455] The capillary array can include a plurality of individual
capillaries comprising at least one outer wall defining a lumen.
The outer wall of the capillary can be one or more walls fused
together. Similarly, the wall can define a lumen that is
cylindrical, square, hexagonal or any other geometric shape so long
as the walls form a lumen for retention of a liquid or sample. The
capillaries of the capillary array can be held together in close
proximity to form a planar structure. The capillaries can be bound
together, by being fused (e.g., where the capillaries are made of
glass), glued, bonded, or clamped side-by-side. The capillary array
can be formed of any number of individual capillaries, for example,
a range from 100 to 4,000,000 capillaries. A capillary array can
form a microtiter plate having about 100,000 or more individual
capillaries bound together.
[0456] Arrays, or "BioChips"
[0457] Nucleic acids or polypeptides of the invention can be
immobilized to or applied to an array. Arrays can be used to screen
for or monitor libraries of compositions (e.g., small molecules,
antibodies, nucleic acids, etc.) for their ability to bind to or
modulate the activity of a nucleic acid or a polypeptide of the
invention. For example, in one aspect of the invention, a monitored
parameter is transcript expression of a phospholipase gene. One or
more, or, all the transcripts of a cell can be measured by
hybridization of a sample comprising transcripts of the cell, or,
nucleic acids representative of or complementary to transcripts of
a cell, by hybridization to immobilized nucleic acids on an array,
or "biochip." By using an "array" of nucleic acids on a microchip,
some or all of the transcripts of a cell can be simultaneously
quantified. Alternatively, arrays comprising genomic nucleic acid
can also be used to determine the genotype of a newly engineered
strain made by the methods of the invention. "Polypeptide arrays"
can also be used to simultaneously quantify a plurality of
proteins.
[0458] The present invention can be practiced with any known
"array," also referred to as a "microarray" or "nucleic acid array"
or "polypeptide array" or "antibody array" or "biochip," or
variation thereof. Arrays are generically a plurality of "spots" or
"target elements," each target element comprising a defined amount
of one or more biological molecules, e.g., oligonucleotides,
immobilized onto a defined area of a substrate surface for specific
binding to a sample molecule, e.g., mRNA transcripts.
[0459] In practicing the methods of the invention, any known array
and/or method of making and using arrays can be incorporated in
whole or in part, or variations thereof, as described, for example,
in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606;
6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452;
5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752;
5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752;
5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313;
WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol.
8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern
(1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes,
Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics
Supp. 21:25-32. See also published U.S. patent applications Nos.
20010018642; 20010019827; 20010016322; 20010014449; 20010014448;
20010012537; 20010008765.
Antibodies and Antibody-Based Screening Methods
[0460] The invention provides isolated or recombinant antibodies
that specifically bind to a phospholipase of the invention. These
antibodies can be used to isolate, identify or quantify the
phospholipases of the invention or related polypeptides. These
antibodies can be used to inhibit the activity of an enzyme of the
invention. These antibodies can be used to isolated polypeptides
related to those of the invention, e.g., related phospholipase
enzymes.
The antibodies can be used in immunoprecipitation, staining (e.g.,
FACS), immunoaffinity columns, and the like. If desired, nucleic
acid sequences encoding for specific antigens can be generated by
immunization followed by isolation of polypeptide or nucleic acid,
amplification or cloning and immobilization of polypeptide onto an
array of the invention. Alternatively, the methods of the invention
can be used to modify the structure of an antibody produced by a
cell to be modified, e.g., an antibody's affinity can be increased
or decreased. Furthermore, the ability to make or modify antibodies
can be a phenotype engineered into a cell by the methods of the
invention.
[0461] Methods of immunization, producing and isolating antibodies
(polyclonal and monoclonal) are known to those of skill in the art
and described in the scientific and patent literature, see, e.g.,
Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991);
Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical
Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New
York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988)
ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications,
New York. Antibodies also can be generated in vitro, e.g., using
recombinant antibody binding site expressing phage display
libraries, in addition to the traditional in vivo methods using
animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70;
Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
[0462] The polypeptides can be used to generate antibodies which
bind specifically to the polypeptides of the invention. The
resulting antibodies may be used in immunoaffinity chromatography
procedures to isolate or purify the polypeptide or to determine
whether the polypeptide is present in a biological sample. In such
procedures, a protein preparation, such as an extract, or a
biological sample is contacted with an antibody capable of
specifically binding to one of the polypeptides of the
invention.
[0463] In immunoaffinity procedures, the antibody is attached to a
solid support, such as a bead or other column matrix. The protein
preparation is placed in contact with the antibody under conditions
in which the antibody specifically binds to one of the polypeptides
of the invention. After a wash to remove non-specifically bound
proteins, the specifically bound polypeptides are eluted.
[0464] The ability of proteins in a biological sample to bind to
the antibody may be determined using any of a variety of procedures
familiar to those skilled in the art. For example, binding may be
determined by labeling the antibody with a detectable label such as
a fluorescent agent, an enzymatic label, or a radioisotope.
Alternatively, binding of the antibody to the sample may be
detected using a secondary antibody having such a detectable label
thereon. Particular assays include ELISA assays, sandwich assays,
radioimmunoassays, and Western Blots.
[0465] Polyclonal antibodies generated against the polypeptides of
the invention can be obtained by direct injection of the
polypeptides into an animal or by administering the polypeptides to
an animal, for example, a nonhuman. The antibody so obtained will
then bind the polypeptide itself. In this manner, even a sequence
encoding only a fragment of the polypeptide can be used to generate
antibodies which may bind to the whole native polypeptide. Such
antibodies can then be used to isolate the polypeptide from cells
expressing that polypeptide.
[0466] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique, the trioma
technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0467] Techniques described for the production of single chain
antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to
produce single chain antibodies to the polypeptides of the
invention. Alternatively, transgenic mice may be used to express
humanized antibodies to these polypeptides or fragments
thereof.
[0468] Antibodies generated against the polypeptides of the
invention may be used in screening for similar polypeptides from
other organisms and samples. In such techniques, polypeptides from
the organism are contacted with the antibody and those polypeptides
which specifically bind the antibody are detected. Any of the
procedures described above may be used to detect antibody
binding.
Kits
[0469] The invention provides kits comprising the compositions,
e.g., nucleic acids, expression cassettes, vectors, cells,
polypeptides (e.g., a kit having at least one phospholipase of the
invention) and/or antibodies (e.g., a kit having at least one
antibody of the invention. The kits also can contain instructional
material teaching the methodologies and industrial uses of the
invention, as described herein.
Industrial and Medical Uses of the Enzymes of the Invention
[0470] The invention provides many industrial uses and medical
applications using polypeptides of the invention, e.g., a
phospholipase and other enzymes of the invention, e.g.,
phospholipases A, B, C and D, patatins, including converting a
non-hydratable phospholipid to a hydratable form, oil degumming,
processing of oils from plants, fish, algae and the like, to name
just a few applications. In any of these alternative industrial
uses and medical applications, an enzymes can be added in a
specific order, e.g., phospholipases with differing specificities
are added in a specific order, for example, an enzyme with PC- and
PE-hydrolyzing activity is added first (or two enzymes are added,
one with PC-hydrolyzing activity and the other with PE-hydrolyzing
activity), then an enzyme with PI-hydrolyzing activity (e.g., PLC
activity) is added, or any combination thereof.
[0471] Any or all of the methods of the invention can be used on a
"process scale", e.g., an oil processes or refining on a scale from
about 15,000; 25,000; 50,000; 75,000; or 100,000 lbs of refined
oil/day up to about 1, 2, 3, 4, 5 or 6 or more million lbs refined
oil/day.
[0472] Methods of using phospholipase enzymes in industrial
applications are well known in the art. For example, the
phospholipases and methods of the invention can be used for the
processing of fats and oils as described, e.g., in JP Patent
Application Publication H6-306386, describing converting
phospholipids present in the oils and fats into water-soluble
substances containing phosphoric acid groups.
[0473] Phospholipases of the invention can be used to process plant
oils and phospholipids such as those derived from or isolated from
rice bran, soy, canola, palm, cottonseed, corn, palm kernel,
coconut, peanut, sesame, sunflower. Phospholipases of the invention
can be used to process essential oils, e.g., those from fruit seed
oils, e.g., grapeseed, apricot, borage, etc. Phospholipases of the
invention can be used to process oils and phospholipids in
different forms, including crude forms, degummed, gums, wash water,
clay, silica, soapstock, and the like. The phospholipids of the
invention can be used to process high phosphorus oils, fish oils,
animal oils, plant oils, algae oils and the like. In any aspect of
the invention, any time a phospholipase C can be used, an
alternative comprises use of a phospholipase D of the invention and
a phosphatase (e.g., using a PLD/phosphatase combination to improve
yield in a high phosphorus oil, such as a soy bean oil).
[0474] Phospholipases of the invention can be used to process and
make edible oils, biodiesel oils, liposomes for pharmaceuticals and
cosmetics, structured phospholipids and structured lipids.
Phospholipases of the invention can be used in oil extraction.
Phospholipases of the invention can be used to process and make
various soaps.
[0475] Processing Edible Oils: Generation of 1,3-diacylglycerol
(1,3 DAG)
[0476] The invention provides processes using enzyme(s) of the
invention to make 1,3-diacylglycerol (1,3 DAG). In one aspect, a
phospholipase C or phospholipase D plus a phosphatase generates
1,2-diacylglycerol; this improves oil yield during edible oil
refining. When used in a process that includes a caustic
neutralization step, for example as a caustic refining aid, as much
as 70% of the 1,2-diacylglyceride (1,2-DAG) undergoes acyl
migration and is converted to 1,3-DAG. 1,3-DAG possesses increased
health benefits and therefore the use of PLC as a caustic refining
aid produces an oil with increased nutritional value.
[0477] The invention provides processes using enzyme(s) of the
invention to make and process edible oils, including generation of
edible oils with increased amounts of 1,3-DAG. Diacylglycerols are
naturally occurring compounds found in many edible oils. In one
aspect of a method of the invention, e.g., the oil degumming
process, a base (caustic) causes the isomerization of 1,2-DAG,
produced by PLC, into 1,3-DAG which provides a nutritional health
benefit over 1,2-DAG, e.g., the 1,3-DAG is burned as energy instead
of being stored as fat (as is 1,2-DAG). By adding the PLC at the
front end of caustic refining process (and the acid and caustic
subsequently), the methods of the invention generate an elevated
level of 1,3-DAG (decreasing 1,2-DAG). Nutritionally, 1,3-DAG is
better for you than 1,2-DAG. In alternative aspects, the invention
comprises an oil degumming process using a PLC of the invention,
whereby the final degummed oil product contains not less than 0.5%,
1.0%, 2.0% or 3.0% or more 1,3-DAG.
[0478] Thus, the invention provides a process for making (through
interesterification) a refined oil (e.g., a diacylglycerol oil),
including edible oils, containing increased levels of
1,3-diacylglycerol (1,3-DAG), e.g., as illustrated in Example 13,
where a phospholipase, such as an enzyme of the invention, is
"front-loaded" or added before addition of acid or caustic. The
generation by enzymatic hydrolysis of a DAG from a triglyceride
generates by interesterification 1,3 DAG from 1,2 DAG. The 1,3
DAG-comprising edible oil shows different metabolic effects
compared to conventional edible oils. Differences in metabolic
pathways between 1,3 DAG and either 1,2 DAG or triglycerides allow
a greater portion of fatty acids from 1,3 diacylglycerol to be
burned as energy rather than being stored as fat. Clinical studies
have shown that regular consumption of DAG oil as part of a
sensible diet can help individuals to manage their body weight and
body fat. In addition, metabolism of 1,3 DAG reduces circulating
postmeal triglycerides in the bloodstream. Since obesity and
elevated blood lipids are associated as risk factors for chronic
diseases including cardiovascular disease and Type II diabetes,
these lifestyle-related health conditions may be impacted in a
beneficial manner with regular consumption of DAG oils.
[0479] Consumption of DAG-comprising oil can take place through a
variety of means. Thus, in one aspect, the invention provides a
process using an enzyme of the invention for making a food, e.g., a
baked good, having increased levels of 1,3-DAG diacylglycerol and
baked goods comprising diacylglycerol oils. In one aspect, the
baked goods are cookies, cakes and similar baked goods.
[0480] In alternative embodiments, combination of enzymes that can
be used in the methods of the invention, including the processing
of edible oils, include (where one, several or all of the enzymes
in the combination comprise an enzyme of the instant invention):
[0481] PLC+PI-PLC+PLA (PLA added after completion of PLC
reactions); [0482] PLD+phosphatase+PI-PLC followed by PLA; or,
[0483] PLC or (PLC+PI-PLC)+PLA specific for phosphatidic acid (all
enzymes added together or sequentially).
[0484] Oil Degumming and Vegetable Oil Processing
[0485] The enzymes of the invention (e.g., polypeptides of the
invention having lipase, phospholipase, esterase and/or glycosidase
or equivalent activity) can be used in various vegetable oil
processing steps, such as in vegetable oil extraction,
particularly, in the removal of "phospholipid gums" in a process
called "oil degumming".
[0486] These processes of the invention can be used on a "process
scale", e.g., on a scale from about 15,000; 25,000; 50,000; 75,000;
or 100,000 lbs of refined oil/day up to about 1, 2, 3, 4, 5 or 6 or
more million lbs refined oil/day.
[0487] In one aspect, the invention provides oil degumming
processes comprising use of a phospholipase of the invention, e.g.,
a PLC of the invention. In one aspect, the process further
comprises addition of another phospholipase (which can also be a
phospholipase of the invention), e.g., another PLC, a PLA, a PLB, a
PLB or a patatin of the invention, or an enzyme (which can also be
an enzyme of the invention) having a lysophospholipase-transacylase
(LPTA) activity or lysophospholipase (LPL) activity and
lysophospholipase-transacylase (LPTA), or a combination thereof,
and/or a patatin-like phospholipase (which can also be an enzyme of
the invention). In one aspect, all enzymes are added together, or,
alternatively, the enzymes are added in a specific order, e.g., PLC
addition is followed by PLA and/or patatin addition; or, an enzyme
or enzymes of the invention having PC and PE activity added first,
then PI PLC added second.
[0488] In one aspect, this process provides a yield improvement as
a result of the phospholipase (e.g., PLC of the invention)
treatment. In one aspect, this process provides an additional
decrease of the phosphorus content of the oil as a result of the
phospholipase (e.g., PLA of the invention) treatment.
[0489] In one aspect, the invention provides processes comprising
use of a phospholipase of the invention, e.g., a PLC of the
invention, to reduce gum mass and increase neutral oil
(triglyceride) gain through reduced oil entrapment. In one aspect,
the invention provides processes comprising use of a phospholipase
of the invention, e.g., a PLC of the invention, for increasing
neutral oils and diacylglycerol (DAG) production to contribute to
the oil phase. In alternative aspects, processes of the invention
(e.g., degumming processes) may comprise one or more other enzymes
such as a protease, an amylase, a lipase, a cutinase, another
phospholipase (including, e.g., an enzyme of the invention), a
carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase,
a galactanase, a xylanase, an oxidase, e.g., a lactase, and/or a
peroxidase, or polypeptides with equivalent activity, or a
combination thereof.
[0490] The phospholipases of the invention can be used in various
vegetable oil processing steps, such as in vegetable oil
extraction, particularly, in the removal of "phospholipid gums" in
a process called "oil degumming," as described above. The invention
provides methods for processing vegetable oils from various
sources, such as rice bran, soybeans, rapeseed, peanuts and other
nuts, sesame, sunflower, palm and corn. The methods can used in
conjunction with processes based on extraction with as hexane, with
subsequent refining of the crude extracts to edible oils, including
use of the methods and enzymes of the invention. The first step in
the refining sequence is the so-called "degumming" process, which
serves to separate phosphatides by the addition of water. The
material precipitated by degumming is separated and further
processed to mixtures of lecithins. The commercial lecithins, such
as soybean lecithin and sunflower lecithin, are semi-solid or very
viscous materials. They consist of a mixture of polar lipids,
mainly phospholipids, and oil, mainly triglycerides.
[0491] The phospholipases of the invention can be used in any
"degumming" procedure, including water degumming, ALCON oil
degumming (e.g., for soybeans), safinco degumming, "super
degumming," UF degumming, TOP degumming, uni-degumming, dry
degumming and ENZYMAX.TM. degumming See, e.g., U.S. Pat. Nos.
6,355,693; 6,162,623; 6,103,505; 6,001,640; 5,558,781; 5,264,367.
Various "degumming" procedures incorporated by the methods of the
invention are described in Bockisch, M. (1998) In Fats and Oils
Handbook, The extraction of Vegetable Oils (Chapter 5), 345-445,
AOCS Press, Champaign, Ill. The phospholipases of the invention can
be used in the industrial application of enzymatic degumming of
triglyceride oils as described, e.g., in EP 513 709.
[0492] In one aspect, phospholipases of the invention are used to
treat vegetable oils, e.g., crude oils, such as rice bran, soy,
canola, flower and the like. In one aspect, this improves the
efficiency of the degumming process. In one aspect, the invention
provides methods for enzymatic degumming under conditions of low
water, e.g., in the range of between about 0.1% to 20% water, or,
0.5% to 10% water. In one aspect, this results in the improved
separation of a heavy phase from the oil phase during
centrifugation. The improved separation of these phases can result
in more efficient removal of phospholipids from the oil, including
both hydratable and nonhydratable oils. In one aspect, this can
produce a gum fraction that contains less entrained neutral oil
(triglycerides), thereby improving the overall yield of oil during
the degumming process.
[0493] In one aspect, phospholipases of the invention, e.g., a
polypeptide having PLC activity, are used to treat oils (e.g.,
vegetable oils, including crude oils, such as rice bran, soy,
canola, flower and the like), e.g., in degumming processes, to
reduce gum mass and increase neutral oil gain through reduced oil
entrapment. In one aspect, phospholipases of the invention e.g., a
polypeptide having PLC activity, are used for diacylglycerol (DAG)
production and to contribute to the oil phase.
[0494] The phospholipases of the invention can be used in the
industrial application of enzymatic degumming as described, e.g.,
in CA 1102795, which describes a method of isolating polar lipids
from cereal lipids by the addition of at least 50% by weight of
water. This method is a modified degumming in the sense that it
utilizes the principle of adding water to a crude oil mixture.
[0495] In one aspect, the invention provides enzymatic processes
comprising use of phospholipases of the invention (e.g., a PLC)
comprising hydrolysis of hydrated phospholipids in oil at a
temperature of about 20.degree. C. to 40.degree. C., at an alkaline
pH, e.g., a pH of about pH 8 to pH 10, using a reaction time of
about 3 to 10 minutes. This can result in less than 10 ppm final
oil phosphorus levels. The invention also provides enzymatic
processes comprising use of phospholipases of the invention (e.g.,
a PLC) comprising hydrolysis of hydratable and non-hydratable
phospholipids in oil at a temperature of about 50.degree. C. to
60.degree. C., at a pH slightly below neutral, e.g., of about pH 5
to pH 6.5, using a reaction time of about 30 to 60 minutes. This
can result in less than 10 ppm final oil phosphorus levels.
[0496] In one aspect, the invention provides enzymatic processes
that utilize a phospholipase C enzyme to hydrolyze a glyceryl
phosphoester bond and thereby enable the return of the
diacylglyceride portion of phospholipids back to the oil, e.g., a
vegetable, fish or algae oil (a "phospholipase C (PLC) caustic
refining aid"); and, reduce the phospholipid content in a degumming
step to levels low enough for high phosphorus oils to be physically
refined (a "phospholipase C (PLC) degumming aid"). The two
approaches can generate different values and have different target
applications.
[0497] In various exemplary processes of the invention, a number of
distinct steps compose the degumming process preceding the core
bleaching and deodorization refining processes. These steps include
heating, mixing, holding, separating and drying. Following the
heating step, water and often acid are added and mixed to allow the
insoluble phospholipid "gum" to agglomerate into particles which
may be separated. While water separates many of the phosphatides in
degumming, portions of the phospholipids are non-hydratable
phosphatides (NHPs) present as calcium or magnesium salts.
Degumming processes address these NHPs by the addition of acid.
Following the hydration of phospholipids, the oil is mixed, held
and separated by centrifugation. Finally, the oil is dried and
stored, shipped or refined, as illustrated, e.g., in FIG. 6. The
resulting gums are either processed further for lecithin products
or added back into the meal.
[0498] In various exemplary processes of the invention phosphorus
levels are reduced low enough for physical refining. The separation
process can result in potentially higher yield losses than caustic
refining. Additionally, degumming processes may generate waste
products that may not be sold as commercial lecithin, see, e.g.,
FIG. 7 for an exemplary degumming process for physically refined
oils. Therefore, these processes have not achieved a significant
share of the market and caustic refining processes continue to
dominate the industry for rice bran, soy, canola and sunflower.
Note however, that a phospholipase C enzyme employed in a special
degumming process would decrease gum formation and return the
diglyceride portion of the phospholipid back to the oil.
[0499] In one aspect, the invention provides methods using a PLC of
the invention in the gum fraction. In one aspect of this method,
oil is added to the crude oil to create an emulsion that results in
the movement of the phosphatidylcholine, phosphatidyl-ethanolamine
and phosphatidylinositol into the aqueous phase (water degumming).
Following centrifugation, these phospholipids are major components
of the aqueous gum fraction. The phospholipids in the gum fraction
can be treated with phospholipase C or phospholipase D plus
phosphatase (or other combinations, noted below) to generate
diacylglycerol (DAG) and a phosphate ester. At this point, the DAG
can be extracted from the other gum components and treated with a
lipase under conditions suitable for the transesterification of the
DAG to produce a desired triacylglycerol (structured lipid).
[0500] In another aspect, the majority of the 1,2-DAG can be
converted to 1,3-DAG by increasing the pH of the gum following the
PLC reaction, for example, by adding caustic. The 1,3-DAG can then
be extracted from the gum and reacted with a lipase under the
appropriate conditions to transesterify the 1,3-DAG at the sn2
position to create the desired structured triacylglycerol.
[0501] In alternative aspects, the fatty acids used in the
transesterification reaction could come from a variety of sources
including the free fatty acids found in the crude oil.
[0502] In one aspect, the phospholipids from water degumming are
used in combination with a PLC of the invention to create
structured lipids. The water-degummed oil can be exposed to a PLC
and/or PLD (either or both can be enzymes of the invention) plus
phosphatase or one of these combinations followed by PLA (can be an
enzyme of the invention) to reduce the phosphorus to levels
suitable for caustic or physical refining.
[0503] In alternative embodiments, combination of enzymes that can
be used in the methods of the invention, including these degumming
processes, include (where one, several or all of the enzymes in the
combination comprise an enzyme of the instant invention): [0504]
PLC+PI-PLC+PLA (PLA added after completion of PLC reactions);
[0505] PLD+phosphatase+PI-PLC followed by PLA; or, [0506] PLC or
(PLC+PI-PLC)+PLA specific for phosphatidic acid (all enzymes added
together or sequentially).
[0507] Caustic Refining
[0508] The invention provides processes using phospholipases
(including enzymes of the invention) in caustic refining, where the
enzymes are used as caustic refining aids. In alternative aspects,
a PLC or PLD and/or a phosphatase are used in the processes as a
drop-in, either before, during, or after a caustic neutralization
refining process (either continuous or batch refining). The amount
of enzyme added may vary according to the process. The water level
used in the process can be low, e.g., about 0.5 to 5%.
Alternatively, caustic is be added to the process multiple times.
In addition, the process may be performed at different temperatures
(25.degree. C. to 70.degree. C.), with different acids orcaustics,
and at varying pH (4-12). Concentrated solutions of caustic, e.g.,
more concentrated than the industrial standard of 11%, to decrease
mass of gum can be used. In alternative aspects, the concentrated
solution of caustic is between about 12% and 50% concentrated,
e.g., about 20%, 30%, 40%, 50%, or 60% or more concentrated.
[0509] In one aspect, a phospholipase C enzyme of the invention
hydrolyzes a phosphatide at a glyceryl phosphoester bond to
generate a diglyceride and water-soluble phosphate compound. The
hydrolyzed phosphatide moves to the aqueous phase, leaving the
diglyceride in the oil phase, as illustrated in FIG. 8. One
objective of the PLC "Caustic Refining Aid" is to convert the
phospholipid gums formed during neutralization into a
diacylglyceride that will migrate back into the oil phase. In
contrast, one objective of the "PLC Degumming Aid" is to reduce the
phospholipids in crude oil to a phosphorus equivalent of less than
10 parts per million (ppm).
[0510] Acids that may be used in a caustic refining process
include, but are not limited to, phosphoric, citric, ascorbic,
sulfuric, fumaric, maleic, hydrochloric and/or acetic acids. Acids
are used to hydrate non-hydratable phospholipids. Caustics that may
be used include, but are not limited to, KOH-- and NaOH. Caustics
are used to neutralize free fatty acids. Alternatively,
phospholipases, or more particularly a PLC or a PLD and a
phosphatase, are used for purification of phytosterols from the
gum/soapstock.
[0511] An alternate embodiment of the invention to add the
phospholipase before caustic refining is to express the
phospholipase in a plant. In another embodiment, the phospholipase
is added during crushing of the plant, seeds or other plant part.
Alternatively, the phospholipase is added following crushing, but
prior to refining (i.e. in holding vessels). In addition,
phospholipase is added as a refining pre-treatment, either with or
without acid.
[0512] Another embodiment of the invention, already described, is
to add the phospholipase during a caustic refining process. In this
process, the levels of acid and caustic are varied depending on the
level of phosphorus and the level of free fatty acids. In addition,
broad temperature and pH ranges are used in the process, dependent
upon the type of enzyme used.
[0513] In another embodiment of the invention, the phospholipase is
added after caustic refining (FIG. 9). In one instance, the
phospholipase is added in an intense mixer or in a retention mixer,
prior to separation. Alternatively, the phospholipase is added
following the heat step. In another embodiment, the phospholipase
is added in the centrifugation step. In an additional embodiment,
the phospholipase is added to the soapstock. Alternatively, the
phospholipase is added to the washwater. In another instance, the
phospholipase is added during the bleaching and/or deodorizing
steps.
[0514] In one aspect, a phospholipase, e.g., a phospholipase C,
enzyme of the invention will hydrolyze the phosphatide from both
hydratable and non-hydratable phospholipids in neutralized crude
and degummed oils before bleaching and deodorizing. Exemplary
"caustic refining" processes of the invention are illustrated in
FIG. 9 and FIG. 13. FIG. 9 includes exemplary times, temperature
and pHs for static mixer (30 to 60 min, 50 to 60.degree. C., pH 5
to 6.5) and retention mixer (3 to 10 min, 20 to 40.degree. C.). The
target enzyme can be applied as a drop-in product in the existing
caustic neutralization process, as illustrated in FIG. 9. In this
aspect, the enzyme will not be required to withstand extreme pH
levels if it is added after the addition of caustic. As illustrated
in FIG. 13 (an enzyme "front loading" exemplary process), any
phospholipase, including, e.g., a phospholipase of the invention,
such as a PLC, PLB, PLA and/or PLC, can be used in a crude oil
degumming process, as described, e.g., in Bailey's Industrial Oil
& Fat Products v.4 (ed. Y. H. Hui). FIG. 14 and FIG. 15
illustrate variations of methods of the invention where two or
three centrifugation steps, respectively, are used in the process,
which can be used to process any oil, e.g., a vegetable oil such as
crude soy oil, as shown in the figure. The exemplary method of FIG.
15 has a centrifugation step before caustic refining (in addition
to a centrifugation step after caustic refining and before the
water wash, and, after the water wash), while the exemplary method
of FIG. 14 does not have a centrifugation step before caustic
refining. FIG. 16 illustrates another exemplary variation of this
process using acid treatment and having a centrifugation step
before a degumming step; this exemplary process can be used to
process any oil, e.g., a vegetable oil such as crude soy oil, as
shown in the figure.
[0515] In one aspect, a phospholipase of the invention enables
phosphorus to be removed to the low levels acceptable in physical
refining. In one aspect, a PLC of the invention will hydrolyze the
phosphatide from both hydratable and non-hydratable phospholipids
in crude oils before bleaching and deodorizing. The target enzyme
can be applied as a drop-in product in an existing degumming
operation, see, e.g., FIG. 10. Given sub-optimal mixing in
commercial equipment, it is likely that acid will be required to
bring the non-hydratable phospholipids in contact with the enzyme
at the oil/water interface. Therefore, in one aspect, an
acid-stable PLC of the invention is used.
[0516] In one aspect, a PLC Degumming Aid process of the invention
can eliminate losses in one, or all three, areas noted in Table 2.
Losses associated in a PLC process can be estimated to be about
0.8% versus 5.2% on a mass basis due to removal of the
phosphatide.
TABLE-US-00003 TABLE 2 Losses Addressed by PLC Products Caustic
Refining Degumming Aid Aid 1) Oil lost in gum formation & 2.1%
X X separation 2) Saponified oil in caustic addition 3.1% X 3) Oil
trapped in clay in bleaching* <1.0% X X Total Yield Loss ~5.2%
~2.1% ~5.2%
[0517] Additional potential benefits of this process of the
invention include the following: [0518] Reduced adsorbents--less
adsorbents required with lower (<5 ppm) phosphorus [0519] Lower
chemical usage--less chemical and processing costs associated with
hydration of non-hydratable phospholipids [0520] Lower waste
generation--less water required to remove phosphorus from oil
[0521] Oils processed (e.g., "degummed") by the methods of the
invention include plant oilseeds, e.g., soybean oil, rapeseed oil,
rice bran oil and sunflower oil. In one aspect, the "PLC Caustic
Refining Aid" of the invention can save 1.2% over existing caustic
refining processes. The refining aid application addresses soy oil
that has been degummed for lecithin and these are also excluded
from the value/load calculations.
[0522] Performance targets of the processes of the invention can
vary according to the applications and more specifically to the
point of enzyme addition, see Table 3.
TABLE-US-00004 TABLE 3 Performance Targets by Application Caustic
Refining Aid Degumming Aid Incoming Oil Phosphorus <200 ppm*
600-1,400 ppm Levels Final Oil Phosphorus Levels <10
ppm.sup..dagger. <10 ppm Hydratable & Non-hydratable Yes Yes
gums Residence Time 3-10 minutes 30 minutes.sup..dagger-dbl. Liquid
Formulation Yes Yes Target pH
8-10.sup..dagger-dbl..dagger-dbl..dagger-dbl.
5.0-5.5.sup..dagger-dbl..dagger-dbl. Target Temperature
20-40.degree. C. ~50-60.degree. C. Water Content <5% 1-1.25%
Enzyme Formulation Purity No lipase/protease.sup.1 No
lipase/protease Other Key Requirements Removal of Fe Removal of Fe
*Water degummed oil .sup..dagger.Target levels achieved in upstream
caustic neutralization step but must be maintained
.sup..dagger-dbl.1-2 hours existing
.sup..dagger-dbl..dagger-dbl.Acid degumming will require an enzyme
that is stable in much more acidic conditions: pH at 2.3 for citric
acid at 5%. (~Roehm USPN 6,001,640).
.sup..dagger-dbl..dagger-dbl..dagger-dbl.The pH of neutralized oil
is NOT neutral. Testing at POS indicates that the pH will be in the
alkaline range from 6.5-10 (Dec. 9, 2002). Typical pH range needs
to be determined.
[0523] Other processes that can be used with a phospholipase of the
invention, e.g., a phospholipase A.sub.1 can convert non-hydratable
native phospholipids to a hydratable form. In one aspect, the
enzyme is sensitive to heat. This may be desirable, since heating
the oil can destroy the enzyme. However, the degumming reaction
must be adjusted to pH 4-5 and 60.degree. C. to accommodate this
enzyme. At 300 Units/kg oil saturation dosage, this exemplary
process is successful at taking previously water-degummed oil
phosphorus content down to .ltoreq.10 ppm P. Advantages can be
decreased H.sub.2O content and resultant savings in usage, handling
and waste. Table 4 lists exemplary applications for industrial uses
for enzymes of the invention:
TABLE-US-00005 TABLE 4 Exemplary Application Caustic Refining
Degumming Aid Aid Soy oil w/ lecithin production X Chemical refined
soy oil, Sunflower oil, X X Canola oil Low phosphatide oils (e.g.
palm) X
[0524] In addition to these various "degumming" processes, the
phospholipases of the invention can be used in any vegetable oil
processing step. For example, phospholipase enzymes of the
invention can be used in place of PLA, e.g., phospholipase A2, in
any vegetable oil processing step. Oils that are "processed" or
"degummed" in the methods of the invention include soybean oils,
rapeseed oils, corn oils, oil from palm kernels, canola oils,
sunflower oils, sesame oils, peanut oils, rice bran oil and the
like. The main products from this process include
triglycerides.
[0525] In one exemplary process, when the enzyme is added to and
reacted with a crude oil, the amount of phospholipase employed is
about 10-10,000 units, or, alternatively, about, 100-2,000 units,
per 1 kg of crude oil. The enzyme treatment is conducted for 5 min
to 10 hours at a temperature of 30.degree. C. to 90.degree. C., or,
alternatively, about, 40.degree. C. to 70.degree. C. The conditions
may vary depending on the optimum temperature of the enzyme. The
amount of water added to dissolve the enzyme is 5-1,000 wt. parts
per 100 wt. parts of crude oil, or, alternatively, about, 10 to 200
wt. parts per 100 wt. parts of crude oil.
[0526] Upon completion of such enzyme treatment, the enzyme liquid
is separated with an appropriate means such as a centrifugal
separator and the processed oil is obtained. Phosphorus-containing
compounds produced by enzyme decomposition of gummy substances in
such a process are practically all transferred into the aqueous
phase and removed from the oil phase. Upon completion of the enzyme
treatment, if necessary, the processed oil can be additionally
washed with water or organic or inorganic acid such as, e.g.,
acetic acid, citric acid, phosphoric acid, succinic acid, and
equivalent acids, or with salt solutions.
[0527] In one exemplary process for ultra-filtration degumming, the
enzyme is bound to a filter or the enzyme is added to an oil prior
to filtration or the enzyme is used to periodically clean
filters.
[0528] In one exemplary process for a phospholipase-mediated
physical refining aid, water and enzyme are added to crude oil
(e.g., crude vegetable oil). In one aspect, a PLC or a PLD of the
invention and a phosphatase are used in the process. In
phospholipase-mediated physical refining, the water level can be
low, i.e. 0.5-5% and the process time should be short (less than 2
hours, or, less than 60 minutes, or, less than 30 minutes, or, less
than 15 minutes, or, less than 5 minutes). The process can be run
at different temperatures (25.degree. C. to 70.degree. C.), using
different acids and/or caustics, at different pHs (e.g., 3-10).
[0529] In alternate aspects, water degumming is performed first to
collect lecithin by centrifugation and then PLC or PLC and PLA of
the invention is added to remove non-hydratable phospholipids (the
process should be performed under low water concentration). In
another aspect, water degumming of crude oil to less than 10 ppm
(edible oils) and subsequent physical refining (less than 50 ppm
for biodiesel) is performed. In one aspect, an emulsifier is added
and/or the crude oil is subjected to an intense mixer to promote
mixing. Alternatively, an emulsion-breaker is added and/or the
crude oil is heated to promote separation of the aqueous phase. In
another aspect, an acid is added to promote hydration of
non-hydratable phospholipids. Additionally, phospholipases can be
used to mediate purification of phytosterols from the
gum/soapstock.
[0530] In one aspect, the invention provides compositions and
methods (which can comprise use of phospholipases of the invention)
for oil degumming comprising using varying amounts of acid and base
without making soapstock. Using this aspect of the invention for
oil degumming, acid (including phosphoric and/or citric) can be
used to hydrate non-hydratable phospholipids in high phosphorus
oils (including soybean, canola, and sunflower). Once the
phospholipids are hydrated, the pH of the aqueous phase can be
raised using caustic addition: the amount of caustic added can
create a favorable pH for enzyme activity but will not result in
the formation of a significant soapstock fraction in the oil.
Because a soapstock is not formed, the free fatty acids in the oil
can be removed downstream, following the degumming step, during
bleaching and deodorization.
[0531] Enzymes of the invention are used to improve oil extraction
and oil degumming (e.g., vegetable oils). In one aspect, a PLC of
the invention and at least one plant cell wall degrader (e.g., a
cellulase, a hemicellulase or the like, to soften walls and
increase yield at extraction) is used in a process of the
invention. In this exemplary approach to using enzymes of the
invention to improve oil extraction and oil degumming, a
phospholipase C of the invention as well as other hydrolases (e.g.,
a cellulase, a hemicellulase, an esterase, a protease and/or a
phosphatase) are used during the crushing steps associated with oil
production (including but not limited to soybean, canola,
sunflower, rice bran oil). By using enzymes prior to or in place of
solvent extraction, it is possible to increase oil yield and reduce
the amount of hydratable and non-hydratable phospholipids in the
crude oil. The reduction in non-hydratable phospholipids may result
from conversion of potentially non-hydratable phospholipids to
diacylglycerol and corresponding phosphate-ester prior to
complexation with calcium or magnesium. The overall reduction of
phospholipids in the crude oil will result in improved yields
during refining with the potential for eliminating the requirement
for a separate degumming step prior to bleaching and
deodorization.
[0532] In one aspect, the invention provides processes using a
phospholipase of the invention (e.g., a phospholipase-specific
phosphohydrolase of the invention), or another phospholipase, in a
modified "organic refining process," which can comprise addition of
the enzyme (e.g., a PLC) in a citric acid holding tank.
[0533] The enzymes of the invention can be used in any oil
processing method, e.g., degumming or equivalent processes. For
example, the enzymes of the invention can be used in processes as
described in U.S. Pat. Nos. 5,558,781; 5,264,367; 6,001,640. The
process described in U.S. Pat. No. 5,558,781 uses either
phospholipase A1, A2 or B, essentially breaking down lecithin in
the oil that behaves as an emulsifier.
[0534] The enzymes and methods of the invention can be used in
processes for the reduction of phosphorus-containing components in
edible oils comprising a high amount of non-hydratable phosphorus
by using of a phospholipase of the invention, e.g., a polypeptide
having a phospholipase A and/or B activity, as described, e.g., in
EP Patent Number: EP 0869167. In one aspect, the edible oil is a
crude oil, a so-called "non-degummed oil." In one aspect, the
method treat a non-degummed oil, including pressed oils or
extracted oils, or a mixture thereof, from, e.g., rapeseed,
soybean, sesame, peanut, corn, rice bran or sunflower. The
phosphatide content in a crude oil can vary from 0.5 to 3% w/w
corresponding to a phosphorus content in the range of 200 to 1200
ppm, or, in the range of 250 to 1200 ppm. Apart from the
phosphatides, the crude oil can also contains small concentrations
of carbohydrates, sugar compounds and metal/phosphatide acid
complexes of Ca, Mg and Fe. In one aspect, the process comprises
treatment of a phospholipid or lysophospholipid with the
phospholipase of the invention so as to hydrolyze fatty acyl
groups. In one aspect, the phospholipid or lysophospholipid
comprises lecithin or lysolecithin. In one aspect of the process
the edible oil has a phosphorus content from between about 50 to
250 ppm, and the process comprises treating the oil with a
phospholipase of the invention so as to hydrolyze a major part of
the phospholipid and separating an aqueous phase containing the
hydrolyzed phospholipid from the oil. In one aspect, prior to the
enzymatic degumming process the oil is water-degummed. In one
aspect, the methods provide for the production of an animal feed
comprising mixing the phospholipase of the invention with feed
substances and at least one phospholipid.
[0535] The enzymes and methods of the invention can be used in
processes of oil degumming as described, e.g., in WO 98/18912. The
phospholipases of the invention can be used to reduce the content
of phospholipid in an edible oil. The process can comprise treating
the oil with a phospholipase of the invention to hydrolyze a major
part of the phospholipid and separating an aqueous phase containing
the hydrolyzed phospholipid from the oil. This process is
applicable to the purification of any edible oil, which contains a
phospholipid, e.g. vegetable oils, such as soybean oil, rice bran
oil, rapeseed oil and sunflower oil, fish oils, algae and animal
oils and the like. Prior to the enzymatic treatment, the vegetable
oil is preferably pretreated to remove slime (mucilage), e.g. by
wet refining. The oil can contain between about 50 to 250 ppm, or
between 50 to about 1500 ppm, or more, of phosphorus, as
phospholipid at the start of the treatment with phospholipase, and
the process of the invention can reduce this value to below between
about 5 to 10 ppm.
[0536] The enzymes of the invention can be used in processes as
described in JP Application No.: H5-132283, filed Apr. 25, 1993,
which comprises a process for the purification of oils and fats
comprising a step of converting phospholipids present in the oils
and fats into water-soluble substances containing phosphoric acid
groups and removing them as water-soluble substances. An enzyme
action is used for the conversion into water-soluble substances. An
enzyme having a phospholipase C activity is preferably used as the
enzyme.
[0537] The enzymes of the invention can be used in processes as
described as the "Organic Refining Process," (ORP) (IPH, Omaha,
Nebr.) which is a method of refining seed oils. ORP may have
advantages over traditional chemical refining, including improved
refined oil yield, value added co-products, reduced capital costs
and lower environmental costs.
[0538] The enzymes of the invention can be used in processes for
the treatment of an oil or fat, animal or vegetal, raw,
semi-processed or refined, comprising adding to such oil or fat at
least one enzyme of the invention that allows hydrolyzing and/or
depolymerizing the non-glyceridic compounds contained in the oil,
as described, e.g., in EP Application number: 82870032.8. Exemplary
methods of the invention for hydrolysis and/or depolymerization of
non-glyceridic compounds in oils are: [0539] 1) The addition and
mixture in oils and fats of an enzyme of the invention or enzyme
complexes previously dissolved in a small quantity of appropriate
solvent (for example water). A certain number of solvents are
possible, but a non-toxic and suitable solvent for the enzyme is
chosen. This addition may be done in processes with successive
loads, as well as in continuous processes. The quantity of
enzyme(s) necessary to be added to oils and fats, according to this
process, may range, depending on the enzymes and the products to be
processed, from between about 5 to 400 ppm, or between about 20 to
400 ppm; e.g., 0.005 kg to 0.4 kg of enzyme for 1000 kg of oil or
fat, and preferably from 5 to 100 ppm, i.e., from 0.005 to 0.1 kg
of enzyme for 1000 kg of oil, these values being understood to be
for concentrated enzymes, i.e., without diluent or solvent. [0540]
2) Passage of the oil or fat through a fixed or insoluble filtering
bed of enzyme(s) of the invention on solid or semi-solid supports,
preferably presenting a porous or fibrous structure. In this
technique, the enzymes are trapped in the micro-cavities of the
porous or fibrous structure of the supports. These consist, for
example, of resins or synthetic polymers, cellulose carbonates,
gels such as agarose, filaments of polymers or copolymers with
porous structure, trapping small droplets of enzyme in solution in
their cavities. Concerning the enzyme concentration, it is possible
to go up to the saturation of the supports. [0541] 3) Dispersion of
the oils and fats in the form of fine droplets, in a diluted
enzymatic solution, in alternative aspects containing between about
0.05 to 4%, or containing between about 0.2 to 4%, in volume of an
enzyme of the invention. This technique is described, e.g., in
Belgian patent No. 595,219. A cylindrical column with a height of
several meters, with conical lid, is filled with a diluted
enzymatic solution. For this purpose, a solvent that is non-toxic
and non-miscible in the oil or fat to be processed, preferably
water, is chosen. The bottom of the column is equipped with a
distribution system in which the oil or fat is continuously
injected in an extremely divided form (approximately 10,000 flux
per m.sup.2). Thus an infinite number of droplets of oil or fat are
formed, which slowly rise in the solution of enzymes and meet at
the surface, to be evacuated continuously at the top of the conical
lid of the reactor.
[0542] Palm oil can be pre-treated before treatment with an enzyme
of the invention. For example, about 30 kg of raw palm oil is
heated to +50.degree. C. 1% solutions were prepared in distilled
water with cellulases and pectinases. 600 g of each of these was
added to aqueous solutions of the oil under strong agitation for a
few minutes. The oil is then kept at +50.degree. C. under moderate
agitation, for a total reaction time of two hours. Then,
temperature is raised to +90.degree. C. to deactivate the enzymes
and prepare the mixture for filtration and further processing. The
oil is dried under vacuum and filtered with a filtering aid.
[0543] The enzymes of the invention can be used in processes as
described in EP patent EP 0 513 709 B2. For example, the invention
provides a process for the reduction of the content process for the
reduction of the content of phosphorus-containing components in
animal and vegetable oils by enzymatic decomposition using a
phospholipase of the invention. In alternative aspects,
predemucilaginated animal and vegetable oil with a phosphorus
content of between about of 50 to 1500 ppm, or, between about 50 to
250 ppm, is agitated with an organic carboxylic acid and the pH
value of the resulting mixture set to between about pH 4 to pH 6,
an enzyme solution which contains phospholipase A.sub.1, A.sub.2,
or B of the invention is added to the mixture in a mixing vessel
under turbulent stirring and with the formation of fine droplets,
where an emulsion with 0.5 to 5% by weight relative to the oil is
formed, said emulsion being conducted through at least one
subsequent reaction vessel under turbulent motion during a reaction
time of 0.1 to 10 hours at temperatures in the range of 20 to
80.degree. C. and where the treated oil, after separation of the
aqueous solution, has a phosphorus content under 5 ppm.
[0544] The organic refining process is applicable to both crude and
degummed oil. The process uses inline addition of an organic acid
under controlled process conditions, in conjunction with
conventional centrifugal separation. The water separated naturally
from the vegetable oil phospholipids ("VOP") is recycled and
reused. The total water usage can be substantially reduced as a
result of the Organic Refining Process.
[0545] The phospholipases and methods of the invention can also be
used in the enzymatic treatment of edible oils, as described, e.g.,
in U.S. Pat. No. 6,162,623. In this exemplary methods, the
invention provides an amphiphilic enzyme. It can be immobilized,
e.g., by preparing an emulsion containing a continuous hydrophobic
phase and a dispersed aqueous phase containing the enzyme and a
carrier for the enzyme and removing water from the dispersed phase
until this phase turns into solid enzyme coated particles. The
enzyme can be a lipase. The immobilized lipase can be used for
reactions catalyzed by lipase such as interesterification of mono-,
di- or triglycerides, de-acidification of a triglyceride oil, or
removal of phospholipids from a triglyceride oil when the lipase is
a phospholipase. The aqueous phase may contain a fermentation
liquid, an edible triglyceride oil may be the hydrophobic phase,
and carriers include sugars, starch, dextran, water soluble
cellulose derivatives and fermentation residues. This exemplary
method can be used to process triglycerides, diglycerides,
monoglycerides, glycerol, phospholipids, glycolipids or fatty
acids, which may be in the hydrophobic phase. In one aspect, the
process for the removal of phospholipids from triglyceride oil
comprising mixing a triglyceride oil containing phospholipids with
a preparation containing a phospholipase of the invention;
hydrolyzing the phospholipids to lysophospholipid; separating the
hydrolyzed phospholipids from the oil, wherein the phospholipase is
an immobilized phospholipase.
[0546] The phospholipases and methods of the invention can also be
used in the enzymatic treatment of edible oils, as described, e.g.,
in U.S. Pat. No. 6,127,137. This exemplary method hydrolyzes both
fatty acyl groups in intact phospholipid. The phospholipase of the
invention used in this exemplary method has no lipase activity and
is active at very low pH. These properties make it very suitable
for use in oil degumming, as enzymatic and alkaline hydrolysis
(saponification) of the oil can both be suppressed. In one aspect,
the invention provides a process for hydrolyzing fatty acyl groups
in a phospholipid or lysophospholipid comprising treating the
phospholipid or lysophospholipid with the phospholipase that
hydrolyzes both fatty acyl groups in a phospholipid and is
essentially free of lipase activity. In one aspect, the
phospholipase of the invention has a temperature optimum at about
50.degree. C., measured at pH 3 to pH 4 for 10 minutes, and a pH
optimum of about pH 3, measured at 40.degree. C. for about 10
minutes. In one aspect, the phospholipid or lysophospholipid
comprises lecithin or lysolecithin. In one aspect, after
hydrolyzing a major part of the phospholipid, an aqueous phase
containing the hydrolyzed phospholipid is separated from the oil.
In one aspect, the invention provides a process for removing
phospholipid from an edible oil, comprising treating the oil at pH
1.5 to 3 with a dispersion of an aqueous solution of the
phospholipase of the invention, and separating an aqueous phase
containing the hydrolyzed phospholipid from the oil. In one aspect,
the oil is treated to remove mucilage prior to the treatment with
the phospholipase. In one aspect, the oil prior to the treatment
with the phospholipase contains the phospholipid in an amount
corresponding to 50 to 250 ppm of phosphorus. In one aspect, the
treatment with phospholipase is done at 30.degree. C. to 45.degree.
C. for 1 to 12 hours at a phospholipase dosage of 0.1 to 10 mg/l in
the presence of 0.5 to 5% of water.
[0547] The phospholipases and methods of the invention can also be
used in the enzymatic treatment of edible oils, as described, e.g.,
in U.S. Pat. No. 6,025,171. In this exemplary methods, enzymes of
the invention are immobilized by preparing an emulsion containing a
continuous hydrophobic phase, such as a triglyceride oil, and a
dispersed aqueous phase containing an amphiphilic enzyme, such as
lipase or a phospholipase of the invention, and carrier material
that is partly dissolved and partly undissolved in the aqueous
phase, and removing water from the aqueous phase until the phase
turns into solid enzyme coated carrier particles. The undissolved
part of the carrier material may be a material that is insoluble in
water and oil, or a water soluble material in undissolved form
because the aqueous phase is already saturated with the water
soluble material. The aqueous phase may be formed with a crude
lipase fermentation liquid containing fermentation residues and
biomass that can serve as carrier materials. Immobilized lipase is
useful for ester re-arrangement and de-acidification in oils. After
a reaction, the immobilized enzyme can be regenerated for a
subsequent reaction by adding water to obtain partial dissolution
of the carrier, and with the resultant enzyme and
carrier-containing aqueous phase dispersed in a hydrophobic phase
evaporating water to again form enzyme coated carrier
particles.
[0548] The phospholipases and methods of the invention can also be
used in the enzymatic treatment of edible oils, as described, e.g.,
in U.S. Pat. No. 6,143,545. This exemplary method is used for
reducing the content of phosphorus containing components in an
edible oil comprising a high amount of non-hydratable phosphorus
content using a phospholipase of the invention. In one aspect, the
method is used to reduce the content of phosphorus containing
components in an edible oil having a non-hydratable phosphorus
content of at least 50 ppm measured by pre-treating the edible oil,
at 60.degree. C., by addition of a solution comprising citric acid
monohydrate in water (added water vs. oil equals 4.8% w/w; (citric
acid) in water phase=106 mM, in water/oil emulsion=4.6 mM) for 30
minutes; transferring 10 ml of the pre-treated water in oil
emulsion to a tube; heating the emulsion in a boiling water bath
for 30 minutes; centrifuging at 5000 rpm for 10 minutes,
transferring about 8 ml of the upper (oil) phase to a new tube and
leaving it to settle for 24 hours; and drawing 2 g from the upper
clear phase for measurement of the non-hydratable phosphorus
content (ppm) in the edible oil. The method also can comprise
contacting an oil at a pH from about pH 5 to 8 with an aqueous
solution of a phospholipase A or B of the invention (e.g., PLA1,
PLA2, or a PLB), which solution is emulsified in the oil until the
phosphorus content of the oil is reduced to less than 11 ppm, and
then separating the aqueous phase from the treated oil.
[0549] The phospholipases and methods of the invention can also be
used in the enzymatic treatment of edible oils, as described, e.g.,
in U.S. Pat. No. 5,532,163. The invention provides processes for
the refining of oil and fat by which phospholipids in the oil and
fat to be treated can be decomposed and removed efficiently. In one
aspect, the invention provides a process for the refining of oil
and fat which comprises reacting, in an emulsion, the oil and fat
with an enzyme of the invention, e.g., an enzyme having an activity
to decompose glycerol-fatty acid ester bonds in
glycerophospholipids (e.g., a PLA2 of the invention); and another
process in which the enzyme-treated oil and fat is washed with
water or an acidic aqueous solution. In one aspect, the acidic
aqueous solution to be used in the washing step is a solution of at
least one acid, e.g., citric acid, acetic acid, phosphoric acid and
salts thereof. In one aspect, the emulsified condition is formed
using 30 weight parts or more of water per 100 weight parts of the
oil and fat. Since oil and fat can be purified without employing
the conventional alkali refining step, generation of washing waste
water and industrial waste can be reduced. In addition, the
recovery yield of oil is improved because loss of neutral oil and
fat due to their inclusion in these wastes does not occur in the
inventive process. In one aspect, the invention provides a process
for refining oil and fat containing about 100 to 10,000 ppm of
phospholipids which comprises: reacting, in an emulsified
condition, said oil and fat with an enzyme of the invention having
activity to decompose glycerol-fatty acid ester bonds in
glycerophospholipids. In one aspect, the invention provides
processes for refining oil and fat containing about 100 to 10,000
ppm of phospholipids which comprises reacting, in an emulsified
condition, oil and fat with an enzyme of the invention having
activity to decompose glycerol-fatty acid ester bonds in
glycerophospholipids; and subsequently washing the treated oil and
fat with a washing water.
[0550] The phospholipases and methods of the invention can also be
used in the enzymatic treatment of edible oils, as described, e.g.,
in U.S. Pat. No. 5,264,367. The content of phosphorus-containing
components and the iron content of an edible vegetable or animal
oil, such as an oil, e.g., soybean oil, which has been wet-refined
to remove mucilage, are reduced by enzymatic decomposition by
contacting the oil with an aqueous solution of an enzyme of the
invention, e.g., a phospholipase A1, A2, or B, and then separating
the aqueous phase from the treated oil. In one aspect, the
invention provides an enzymatic method for decreasing the content
of phosphorus- and iron-containing components in oils, which have
been refined to remove mucilage. An oil, which has been refined to
remove mucilage, can be treated with an enzyme of the invention,
e.g., phospholipase C, A1, A2, or B. Phosphorus contents below 5
ppm and iron contents below 1 ppm can be achieved. The low iron
content can be advantageous for the stability of the oil.
[0551] The phospholipases and methods of the invention can also be
used for preparing transesterified oils, as described, e.g., in
U.S. Pat. No. 5,288,619. The invention provides methods for
enzymatic transesterification for preparing a margarine oil having
both low trans-acid and low intermediate chain fatty acid content.
The method includes the steps of providing a transesterification
reaction mixture containing a stearic acid source material and an
edible liquid vegetable oil, transesterifying the stearic acid
source material and the vegetable oil using a 1-, 3-positionally
specific lipase, and then finally hydrogenating the fatty acid
mixture to provide a recycle stearic acid source material for a
recyclic reaction with the vegetable oil. The invention also
provides a counter-current method for preparing a transesterified
oil. The method includes the steps of providing a
transesterification reaction zone containing a 1-, 3-positionally
specific lipase, introducing a vegetable oil into the
transesterification zone, introducing a stearic acid source
material, conducting a supercritical gas or subcritical liquefied
gas counter-current fluid, carrying out a transesterification
reaction of the triglyceride stream with the stearic acid or
stearic acid monoester stream in the reaction zone, withdrawing a
transesterified triglyceride margarine oil stream, withdrawing a
counter-current fluid phase, hydrogenating the transesterified
stearic acid or stearic acid monoester to provide a hydrogenated
recycle stearic acid source material, and introducing the
hydrogenated recycle stearic acid source material into the reaction
zone.
[0552] In one aspect, the highly unsaturated phospholipid compound
may be converted into a triglyceride by appropriate use of a
phospholipase C of the invention to remove the phosphate group in
the sn-3 position, followed by 1,3 lipase acyl ester synthesis. The
2-substituted phospholipid may be used as a functional food
ingredient directly, or may be subsequently selectively hydrolyzed
in reactor 160 using an immobilized phospholipase C of the
invention to produce a 1-diglyceride, followed by enzymatic
esterification as described herein to produce a triglyceride
product having a 2-substituted polyunsaturated fatty acid
component.
[0553] The phospholipases and methods of the invention can also be
used in a vegetable oil enzymatic degumming process as described,
e.g., in U.S. Pat. No. 6,001,640. This method of the invention
comprises a degumming step in the production of edible oils.
Vegetable oils from which hydratable phosphatides have been
eliminated by a previous aqueous degumming process are freed from
non-hydratable phosphatides by enzymatic treatment using a
phospholipase of the invention. The process can be gentle,
economical and environment-friendly. Phospholipases that only
hydrolyze lysolecithin, but not lecithin, are used in this
degumming process.
[0554] In one aspect, to allow the enzyme of the invention to act,
both phases, the oil phase and the aqueous phase that contain the
enzyme, must be intimately mixed. It may not be sufficient to
merely stir them. Good dispersion of the enzyme in the oil is aided
if it is dissolved in a small amount of water, e.g., 0.5-5 weight-%
(relative to the oil), and emulsified in the oil in this form, to
form droplets of less than 10 micrometers in diameter (weight
average). The droplets can be smaller than 1 micrometer. Turbulent
stirring can be done with radial velocities above 100 cm/sec. The
oil also can be circulated in the reactor using an external rotary
pump. The aqueous phase containing the enzyme can also be finely
dispersed by means of ultrasound action. A dispersion apparatus can
be used.
[0555] The enzymatic reaction probably takes place at the border
surface between the oil phase and the aqueous phase. It is the goal
of all these measures for mixing to create the greatest possible
surface for the aqueous phase which contains the enzyme. The
addition of surfactants increases the microdispersion of the
aqueous phase. In some cases, therefore, surfactants with HLB
values above 9, such as Na-dodecyl sulfate, are added to the enzyme
solution, as described, e.g., in EP-A 0 513 709. A similar
effective method for improving emulsification is the addition of
lysolecithin. The amounts added can lie in the range of 0.001% to
1%, with reference to the oil. The temperature during enzyme
treatment is not critical. Temperatures between 20.degree. C. and
80.degree. C. can be used, but the latter can only be applied for a
short time. In this aspect, a phospholipase of the invention having
a good temperature and/or low pH tolerance is used. Application
temperatures of between 30.degree. C. and 50.degree. C. are
optimal. The treatment period depends on the temperature and can be
kept shorter with an increasing temperature. Times of 0.1 to 10
hours, or, 1 to 5 hours are generally sufficient. The reaction
takes place in a degumming reactor, which can be divided into
stages, as described, e.g., in DE-A 43 39 556. Therefore continuous
operation is possible, along with batch operation. The reaction can
be carried out in different temperature stages. For example,
incubation can take place for 3 hours at 40.degree. C., then for 1
hour at 60.degree. C. If the reaction proceeds in stages, this also
opens up the possibility of adjusting different pH values in the
individual stages. For example, in the first stage the pH of the
solution can be adjusted to 7, for example, and in a second stage
to 2.5, by adding citric acid. In at least one stage, however, the
pH of the enzyme solution must be below 4, or, below 3. If the pH
was subsequently adjusted below this level, a deterioration of
effect may be found. Therefore the citric acid can be added to the
enzyme solution before the latter is mixed into the oil.
[0556] After completion of the enzyme treatment, the enzyme
solution, together with the decomposition products of the NHP
contained in it, can be separated from the oil phase, in batches or
continuously, e.g., by means of centrifugation. Since the enzymes
are characterized by a high level of stability and the amount of
the decomposition products contained in the solution is slight
(they may precipitate as sludge) the same aqueous enzyme phase can
be used several times. There is also the possibility of freeing the
enzyme of the sludge, see, e.g., DE-A 43 39 556, so that an enzyme
solution which is essentially free of sludge can be used again. In
one aspect of this degumming process, oils which contain less than
15 ppm phosphorus are obtained. One goal is phosphorus contents of
less than 10 ppm; or, less than 5 ppm. With phosphorus contents
below 10 ppm, further processing of the oil according to the
process of distillative de-acidification is easily possible. A
number of other ions, such as magnesium, calcium, zinc, as well as
iron, can be removed from the oil, e.g., below 0.1 ppm. Thus, this
product possesses ideal prerequisites for good oxidation resistance
during further processing and storage.
[0557] The phospholipases and methods of the invention also can
also be used for reducing the amount of phosphorus-containing
components in vegetable and animal oils as described, e.g., in EP
patent EP 0513709. In this method, the content of
phosphorus-containing components, especially phosphatides, such as
lecithin, and the iron content in vegetable and animal oils, which
have previously been deslimed, e.g. soya oil, are reduced by
enzymatic breakdown using a phospholipase A1, A2 or B of the
invention.
[0558] The phospholipases and methods of the invention can also be
used for refining fat or oils as described, e.g., in JP 06306386.
The invention provides processes for refining a fat or oil
comprising a step of converting a phospholipid in a fat or an oil
into a water-soluble phosphoric-group-containing substance and
removing this substance. The action of an enzyme of the invention
(e.g., a PLC) is utilized to convert the phospholipid into the
substance. Thus, it is possible to refine a fat or oil without
carrying out an alkali refining step from which industrial wastes
containing alkaline waste water and a large amount of oil are
produced. Improvement of yields can be accomplished because the
loss of neutral fat or oil from escape with the wastes can be
reduced to zero. In one aspect, gummy substances are converted into
water-soluble substances and removed as water-soluble substances by
adding an enzyme of the invention having a phospholipase C activity
in the stage of degumming the crude oil and conducting enzymatic
treatment. In one aspect, the phospholipase C of the invention has
an activity that cuts ester bonds of glycerin and phosphoric acid
in phospholipids. If necessary, the method can comprise washing the
enzyme-treated oil with water or an acidic aqueous solution. In one
aspect, the enzyme of the invention is added to and reacted with
the crude oil. The amount of phospholipase C employed can be 10 to
10,000 units, or, about 100 to 2,000 units, per 1 kg of crude
oil.
[0559] The phospholipases and methods of the invention can also be
used for water-degumming processes as described, e.g., in Dijkstra,
Albert J., et al., Oleagineux, Corps Gras, Lipides (1998), 5 (5),
367-370. In this exemplary method, the water-degumming process is
used for the production of lecithin and for dry degumming processes
using a degumming acid and bleaching earth. This method may be
economically feasible only for oils with a low phosphatide content,
e.g., palm oil, lauric oils, etc. For seed oils having a high
NHP-content, the acid refining process is used, whereby this
process is carried out at the oil mill to allow gum disposal via
the meal. In one aspect, this acid refined oil is a possible
"polishing" operation to be carried out prior to physical
refining.
[0560] The phospholipases and methods of the invention can also be
used for degumming processes as described, e.g., in Dijkstra, et
al., Res. Dev. Dep., N.V. Vandemoortele Coord. Cent., Izegem, Belg.
JAOCS, J. Am. Oil Chem. Soc. (1989), 66:1002-1009. In this
exemplary method, the total degumming process involves dispersing
an acid such as H.sub.3PO.sub.4 or citric acid into soybean oil,
allowing a contact time, and then mixing a base such as caustic
soda or Na silicate into the acid-in-oil emulsion. This keeps the
degree of neutralization low enough to avoid forming soaps, because
that would lead to increased oil loss. Subsequently, the oil passed
to a centrifugal separator where most of the gums are removed from
the oil stream to yield a gum phase with minimal oil content. The
oil stream is then passed to a second centrifugal separator to
remove all remaining gums to yield a dilute gum phase, which is
recycled. Washing and drying or in-line alkali refining complete
the process. After the adoption of the total degumming process, in
comparison with the classical alkali refining process, an overall
yield improvement of about 0.5% is realized. The totally degummed
oil can be subsequently alkali refined, bleached and deodorized, or
bleached and physically refined.
[0561] The phospholipases and methods of the invention can also be
used for the removal of nonhydratable phospholipids from a plant
oil, e.g., soybean oil, as described, e.g., in Hvolby, et al.,
Sojakagefabr., Copenhagen, Den., J. Amer. Oil Chem. Soc. (1971)
48:503-509. In this exemplary method, water-degummed oil is mixed
at different fixed pH values with buffer solutions with and without
Ca.sup.++, Mg/Ca-binding reagents, and surfactants. The
nonhydratable phospholipids can be removed in a nonconverted state
as a component of micelles or of mixed emulsifiers. Furthermore,
the nonhydratable phospholipids are removable by conversion into
dissociated forms, e.g., by removal of Mg and Ca from the
phosphatidates, which can be accomplished by acidulation or by
treatment with Mg/Ca-complexing or Mg/Ca-precipitating reagents.
Removal or chemical conversion of the nonhydratable phospholipids
can result in reduced emulsion formation and in improved separation
of the deacidified oil from the emulsion layer and the
soapstock.
[0562] The phospholipases and methods of the invention can also be
used for the degumming of vegetable oils as described, e.g.,
Buchold, et al., Frankfurt/Main, Germany. Fett Wissenschaft
Technologie (1993), 95 (8), 300-304. In this exemplary process of
the invention for the degumming of edible vegetable oils, aqueous
suspensions of an enzyme of the invention, e.g., phospholipase A2,
is used to hydrolyze the fatty acid bound at the sn2 position of
the phospholipid, resulting in 1-acyl-lysophospholipids which are
insoluble in oil and thus more amenable to physical separation.
Even the addition of small amounts corresponding to about 700
lecitase units/kg oil results in a residual P concentration of less
than 10 ppm, so that chemical refining is replaceable by physical
refining, eliminating the necessity for neutralization, soapstock
splitting, and wastewater treatment.
[0563] The phospholipases and methods of the invention can also be
used for the degumming of vegetable oils as described, e.g., by
EnzyMax, Dahlke, Klaus, Dept. G-PDO, Lurgi O1-Gas, Chemie, GmbH,
Frankfurt, Germany. Oleagineux, Corps Gras, Lipides (1997), 4 (1),
55-57. This exemplary process is a degumming process for the
physical refining of almost any kind of oil. By an
enzymatic-catalyzed hydrolysis, phosphatides are converted to
water-soluble lysophosphatides which are separated from the oil by
centrifugation. The residual phosphorus content in the
enzymatically degummed oil can be as low as 2 ppm P.
[0564] The phospholipases and methods of the invention can also be
used for the degumming of vegetable oils as described, e.g., by
Cleenewerck, et al., N. V. Vamo Mills, Izegem, Belg. Fett
Wissenschaft Technologie (1992), 94:317-22; and, Clausen, Kim;
Nielsen, Munk. Novozymes A/S, Den. Dansk Kemi (2002) 83 (2):24-27.
The phospholipases and methods of the invention can incorporate the
pre-refining of vegetable oils with acids as described, e.g., by
Nilsson-Johansson, et al., Fats Oils Div., Alfa-Laval Food Eng. AB,
Tumba, Swed. Fett Wissenschaft Technologie (1988), 90 (11), 447-51;
and, Munch, Ernst W. Cereol Deutschland GmbH, Mannheim, Germany.
Editor(s): Wilson, Richard F. Proceedings of the World Conference
on Oilseed Processing Utilization, Cancun, MX, Nov. 12-17, (2001),
Meeting Date 2000, 17-20.
[0565] The phospholipases and methods of the invention can also be
used for the degumming of vegetable oils as described, e.g., by
Jerzewska, et al., Inst. Przemyslu Miesnego i Tluszczowego, Warsaw,
Pol., Tluszcze Jadalne (2001), 36 (3/4), 97-110. In this process of
the invention, enzymatic degumming of hydrated low-erucic acid
rapeseed oil is by use of a phospholipase A2 of the invention. The
enzyme can catalyze the hydrolysis of fatty acid ester linkages to
the central carbon atom of the glycerol moiety in phospholipids. It
can hydrolyze non-hydratable phospholipids to their corresponding
hydratable lyso-compounds. With a nonpurified enzyme preparation,
better results can be achieved with the addition of 2% preparation
for 4 hours (87% P removal).
[0566] In another exemplary process of the invention for oil
degumming (or an oil degumming process using an enzyme of the
invention), an acidic polymer, e.g., an alginate or pectin, is
added. In this oil degumming process of the invention, an acidic
polymer (e.g. alginic acid or pectin or a more soluble salt form)
is added to the crude oil with a low amount of water (e.g., in a
range of between about 0.5 to 5%). In this aspect, the acidic
polymers can reduce and/or disrupt phospholipid-metal complexes by
binding calcium and/or magnesium in the crude oil, thereby
improving the solubility of nonhydratable phospholipids. In
alternative aspects, these phospholipids will move to the oil/water
interface or enter the aqueous phase and either be converted to
diacylglycerol and the corresponding side chain or the intact
phospholipid will be removed by subsequent centrifugation as a
component of the heavy phase. The presence of the acidic polymer in
the aqueous phase can also increase the density of the aqueous
phase and result in an improved separation of the heavy phase from
the oil (light) phase.
[0567] One exemplary process of the invention for oil degumming (or
an oil degumming process using an enzyme of the invention) alters
the deodorization procedure to get a diacylglycerol (DAG) fraction.
In alternative aspect, if necessary or desired, following
enzyme-assisted degumming, the deodorization conditions
(temperature, pressure, configuration of the distillation
apparatus) can be modified with the goal of improving the
separation of the free fatty acids (FFA) from the
diacylglycerol/triacylglycerol fraction or further modified to
separate the diacylglycerol from the triacylglycerol fraction. As a
result of these modifications, using this method of the invention,
it is possible to obtain food grade FFA and diacylglycerol if an
enzyme of the invention (e.g., a phosphatase, or, a PLC or a
combination of PLC and phosphatases) are used to degum edible oil
in a physical refining process.
[0568] In various aspects, practicing the methods of the invention
as described herein (or using the enzymes of the invention), have
advantages such as: decrease or eliminate solvent and solvent
recovery; lower capital costs; decrease downstream refining costs,
decrease chemical usage, equipment, process time, energy (heat) and
water usage/wastewater generation; produce higher quality oil;
expeller pressed oil may be used without refining in some cooking
and sauteing applications (this pressed oil may have superior
stability, color and odor characteristics and high tocopherol
content); produce higher quality meal; produce a lower fat content
in meal (currently, meal coming out of mechanical press causes
digestion problems in ruminants); produce improved nutritional
attributes--reduced levels of glucosinolates, tannins, sinapine,
phytic acid (as described, e.g., in Technology and Solvents for
Extracting Oilseeds and Nonpetroleum Oils, AOCS 1997).
[0569] In one aspect, the invention provides methods for refining
vegetable oils (e.g., soybean oil, corn oil, cottonseed oil, palm
oil, peanut oil, rapeseed oil, safflower oil, sunflower seed oil,
sesame seed oil, rice bran oil, coconut oil or canola oil) and
their byproducts, and processes for deodorizing lecithin, for
example, as described in U.S. Pat. No. 6,172,248, or 6,172,247,
wherein the methods comprise use of at least one enzyme of the
invention, e.g., a phospholipase C of the invention. Thus, the
invention provides lecithin and vegetable oils comprising at least
one enzyme of the invention. In an exemplary organic acid refining
process, vegetable oil is combined with a dilute aqueous organic
acid solution and subjected to high shear to finely disperse the
acid solution in the oil. The resulting acid-and-oil mixture is
mixed at low shear for a time sufficient to sequester contaminants
into a hydrated impurities phase, producing a purified vegetable
oil phase. In this exemplary process, a mixer or recycle system
(e.g., recycle water tank) and/or a phosphatide or lecithin storage
tank can be used, e.g., as described in U.S. Pat. No. 4,240,972,
4,049,686, 6,172,247 or 6,172,248. These processes can be conducted
as a batch or continuous process. Crude or degummed vegetable oil
can be supplied from a storage tank (e.g., through a pump) and can
be heated. The vegetable oil to be purified can be either crude or
"degummed" oil.
[0570] In one aspect, phosphatidylinositol-PLC (PI-PLC) enzymes of
the invention are used for vegetable oil degumming PI-PLC enzymes
of the invention can be used alone or in combination with other
enzymes (for instance PLC, PLD, phosphatase enzymes of the
invention) to improve oil yield during the degumming of vegetable
oils (including soybean, canola, and sunflower). The PI-PLC may
preferentially convert phosphatidylinositol to 1,2-diacylglycerol
(DAG) and phosphoinositol but it may also demonstrate activity on
other phospholipids including phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid,
or a combination thereof. The improvement in yield will be realized
as an increase in the amount of DAG in the enzyme-treated vegetable
oil and an increase in neutral oil, due to a decrease in the amount
of oil entrained in the smaller gum fraction that results from
enzyme treatment of the vegetable oil.
[0571] Enzymatic Processing of Oilseeds
[0572] The invention provides compositions (e.g., enzymes) and
methods for enzymatic processing of oilseeds, including soybean,
canola, coconut, avocado and olive paste. In one aspect, these
processes of the invention can increase the oil yield and to
improve the nutritional quality of the obtained meals. In some
aspects, enzymatic processing of oilseeds using the enzymes and
methods of the invention will provide economical and environmental
benefits, as well as alternative technologies for oil extraction
and processing food for human and animal consumption. In
alternative aspects, the processes of the invention comprise use of
phospholipases of the invention, other phospholipases, proteases,
phosphatases, phytases, xylanases, amylases (e.g.,
.alpha.-amylases), glucanases (e.g., .beta.-glucanases),
polygalacturonases, galactolipases, cellulases, hemicellulases,
pectinases and other plant cell wall degrading enzymes, as well as
mixed enzyme preparations and cell lysates.
[0573] In alternative aspects, the processes of the invention can
be practiced in conjunction with other processes, e.g., enzymatic
treatments, e.g., with carbohydrases, including cellulase,
hemicellulase and other side degrading activities, or, chemical
processes, e.g., hexane extraction of soybean oil. The enzymatic
treatment can increase the oil extractability by 8-10% when the
enzymatic treatment is carried out prior to the solvent
extraction.
[0574] In alternative aspects, the processes of the invention can
be practiced with aqueous extraction processes. The aqueous
extraction methods can be environmentally cleaner alternative
technologies for oil extraction. Low extraction yields of aqueous
process can be overcome by using enzymes that hydrolyze the
structural polysaccharides forming the cell wall of oilseeds, or
that hydrolyze the proteins which form the cell and lipid body
membranes, e.g., utilizing digestions comprising cellulase,
hemicellulase, and/or protopectinase for extraction of oil from
soybean cells. In one aspect, methods are practiced with an enzyme
of the invention as described by Kasai (2003) J. Agric. Food Chem.
51:6217-6222, who reported that the most effective enzyme to digest
the cell wall was cellulase.
[0575] In one aspect, proteases are used in combination with the
methods of the invention. The combined effect of operational
variables and enzyme activity of protease and cellulase on oil and
protein extraction yields combined with other process parameters,
such as enzyme concentration, time of hydrolysis, particle size and
solid-to-liquid ratio has been evaluated. In one aspect, methods
are practiced with an enzyme of the invention as described by
Rosenthal (2001) Enzyme and Microb. Tech. 28:499-509, who reported
that use of protease can result in significantly higher yields of
oil and protein over the control when heat treated flour is
used.
[0576] In one aspect, complete protein, pectin, and hemicellulose
extraction are used in combination with the methods of the
invention. The plant cell consists of a series of polysaccharides
often associated with or replaced by proteins or phenolic
compounds. Most of these carbohydrates are only partially digested
or poorly utilized by the digestive enzymes. The disruption of
these structures through processing or degrading enzymes can
improve their nutrient availability. In one aspect, methods are
practiced with an enzyme of the invention as described by Ouhida
(2002) J. Agric. Food Chem. 50:1933-1938, who reported that a
significant degradation of the soybean cell wall cellulose (up to
20%) has been achieved after complete protein, pectin, and
hemicellulose extraction.
[0577] In one aspect, the methods of the invention further comprise
incorporation of various enzymatic treatments in the treatment of
seeds, e.g., canola seeds, these treatments comprising use of
proteases, cellulases, and hemicellulases (in various combinations
with each other and with one or more enzymes of the invention). For
example, the methods can comprise enzymatic treatments of canola
seeds at 20 to 40 moisture during the incubation with enzymes prior
to a conventional process; as described, e.g., by Sosulski (1990)
Proc. Can. Inst. Food Sci. Technol. 3:656. The methods of the
invention can further comprise incorporation of proteases,
.alpha.-amylases, polygalacturonases (in various combinations with
each other and with one or more enzymes of the invention) to
hydrolyze cellular material in coconut meal and release the coconut
oil, which can be recovered by centrifugation, as described, e.g.,
by McGlone (1986) J. of Food Sci. 51:695-697. The methods of the
invention can further comprise incorporation of pectinases,
.alpha.-amylases, proteases, cellulases in different combinations
(with each other and with one or more enzymes of the invention) to
result in significant yield improvement (.about.70% in the best
case) during enzymatic extraction of avocado oil, as described,
e.g., by Buenrostro (1986) Biotech. Letters 8 (7):505-506. In
processes of the invention for olive oil extraction, olive paste is
treated with cellulase, hemicellulase, poligalacturonase,
pectin-methyltransferase, protease and their combinations (with
each other and with one or more enzymes of the invention), as
described, e.g., by Montedoro (1976) Acta Vitamin. Enzymol.
(Milano) 30:13.
[0578] Purification of Phytosterols from Vegetable Oils
[0579] The invention provides methods for purification of
phytosterols and triterpenes, or plant sterols, from vegetable
oils. Phytosterols that can be purified using phospholipases and
methods of the invention include .beta.-sitosterol, campesterol,
stigmasterol, stigmastanol, .beta.-sitostanol, sitostanol,
desmosterol, chalinasterol, poriferasterol, clionasterol and
brassicasterol. Plant sterols are important agricultural products
for health and nutritional industries. Thus, phospholipases and
methods of the invention are used to make emulsifiers for cosmetic
manufacturers and steroidal intermediates and precursors for the
production of hormone pharmaceuticals. Phospholipases and methods
of the invention are used to make (e.g., purify) analogs of
phytosterols and their esters for use as cholesterol-lowering
agents with cardiologic health benefits. Phospholipases and methods
of the invention are used to purify plant sterols to reduce serum
cholesterol levels by inhibiting cholesterol absorption in the
intestinal lumen. Phospholipases and methods of the invention are
used to purify plant sterols that have immunomodulating properties
at extremely low concentrations, including enhanced cellular
response of T lymphocytes and cytotoxic ability of natural killer
cells against a cancer cell line. Phospholipases and methods of the
invention are used to purify plant sterols for the treatment of
pulmonary tuberculosis, rheumatoid arthritis, management of
HIV-infested patients and inhibition of immune stress, e.g., in
marathon runners.
[0580] Phospholipases and methods of the invention are used to
purify sterol components present in the sterol fractions of
commodity vegetable oils (e.g., coconut, canola, cocoa butter,
corn, cottonseed, linseed, olive, palm, peanut, rice bran,
safflower, sesame, soybean, sunflower oils), such as sitosterol
(40.2-92.3%), campesterol (2.6-38.6%), stigmasterol (0-31%) and
5-avenasterol (1.5-29%).
[0581] Methods of the invention can incorporate isolation of
plant-derived sterols in oil seeds by solvent extraction with
chloroform-methanol, hexane, methylene chloride, or acetone,
followed by saponification and chromatographic purification for
obtaining enriched total sterols. Alternatively, the plant samples
can be extracted by supercritical fluid extraction with
supercritical carbon dioxide to obtain total lipid extracts from
which sterols can be enriched and isolated. For subsequent
characterization and quantification of sterol compounds, the crude
isolate can be purified and separated by a wide variety of
chromatographic techniques including column chromatography (CC),
gas chromatography, thin-layer chromatography (TLC), normal phase
high-performance liquid chromatography (HPLC), reversed-phase HPLC
and capillary electrochromatography. Of all chromatographic
isolation and separation techniques, CC and TLC procedures employ
the most accessible, affordable and suitable for sample clean up,
purification, qualitative assays and preliminary estimates of the
sterols in test samples.
[0582] Phytosterols are lost in the vegetable oils lost as
byproducts during edible oil refining processes. Phospholipases and
methods of the invention use phytosterols isolated from such
byproducts to make phytosterol-enriched products isolated from such
byproducts. Phytosterol isolation and purification methods of the
invention can incorporate oil processing industry byproducts and
can comprise operations such as molecular distillation,
liquid-liquid extraction and crystallization.
[0583] Methods of the invention can incorporate processes for the
extraction of lipids to extract phytosterols. For example, methods
of the invention can use nonpolar solvents as hexane (commonly used
to extract most types of vegetable oils) quantitatively to extract
free phytosterols and phytosteryl fatty-acid esters. Steryl
glycosides and fatty-acylated steryl glycosides are only partially
extracted with hexane, and increasing polarity of the solvent gave
higher percentage of extraction. One procedure that can be used is
the Bligh and Dyer chloroform-methanol method for extraction of all
sterol lipid classes, including phospholipids. One exemplary method
to both qualitatively separate and quantitatively analyze
phytosterol lipid classes comprises injection of the lipid extract
into HPLC system.
[0584] Phospholipases and methods of the invention can be used to
remove sterols from fats and oils, as described, e.g., in U.S. Pat.
No. 6,303,803. This is a method for reducing sterol content of
sterol-containing fats and oils. It is an efficient and cost
effective process based on the affinity of cholesterol and other
sterols for amphipathic molecules that form hydrophobic, fluid
bilayers, such as phospholipid bilayers. Aggregates of
phospholipids are contacted with, for example, a sterol-containing
fat or oil in an aqueous environment and then mixed. The molecular
structure of this aggregated phospholipid mixture has a high
affinity for cholesterol and other sterols, and can selectively
remove such molecules from fats and oils. The aqueous separation
mixture is mixed for a time sufficient to selectively reduce the
sterol content of the fat/oil product through partitioning of the
sterol into the portion of phospholipid aggregates. The
sterol-reduced fat or oil is separated from the aqueous separation
mixture. Alternatively, the correspondingly sterol-enriched
fraction also may be isolated from the aqueous separation mixture.
These steps can be performed at ambient temperatures, costs
involved in heating are minimized, as is the possibility of thermal
degradation of the product. Additionally, a minimal amount of
equipment is required, and since all required materials are food
grade, the methods require no special precautions regarding
handling, waste disposal, or contamination of the final
product(s).
[0585] Phospholipases and methods of the invention can be used to
remove sterols from fats and oils, as described, e.g., in U.S. Pat.
No. 5,880,300. Phospholipid aggregates are contacted with, for
example, a sterol-containing fat or oil in an aqueous environment
and then mixed. Following adequate mixing, the sterol-reduced fat
or oil is separated from the aqueous separation mixture.
Alternatively, the correspondingly sterol-enriched phospholipid
also may be isolated from the aqueous separation mixture. Plant
(e.g., vegetable) oils contain plant sterols (phytosterols) that
also may be removed using the methods of the present invention.
This method is applicable to a fat/oil product at any stage of a
commercial processing cycle. For example, the process of the
invention may be applied to refined, bleached and deodorized oils
("RBD oils"), or to any stage of processing prior to attainment of
RBD status. Although RBD oil may have an altered density compared
to pre-RBD oil, the processes of the are readily adapted to either
RBD or pre-RBD oils, or to various other fat/oil products, by
variation of phospholipid content, phospholipid composition,
phospholipid:water ratios, temperature, pressure, mixing
conditions, and separation conditions as described below.
[0586] Alternatively, the enzymes and methods of the invention can
be used to isolate phytosterols or other sterols at intermediate
steps in oil processing. For example, it is known that phytosterols
are lost during deodorization of plant oils. A sterol-containing
distillate fraction from, for example, an intermediate stage of
processing can be subjected to the sterol-extraction procedures
described above. This provides a sterol-enriched lecithin or other
phospholipid material that can be further processed in order to
recover the extracted sterols.
[0587] Detergent Compositions
[0588] The invention provides detergent compositions comprising one
or more phospholipase of the invention, and methods of making and
using these compositions. The invention incorporates all methods of
making and using detergent compositions, see, e.g., U.S. Pat. Nos.
6,413,928; 6,399,561; 6,365,561; 6,380,147. The detergent
compositions can be a one and two part aqueous composition, a
non-aqueous liquid composition, a cast solid, a granular form, a
particulate form, a compressed tablet, a gel and/or a paste and a
slurry form. The invention also provides methods capable of a rapid
removal of gross food soils, films of food residue and other minor
food compositions using these detergent compositions.
Phospholipases of the invention can facilitate the removal of
stains by means of catalytic hydrolysis of phospholipids.
Phospholipases of the invention can be used in dishwashing
detergents in textile laundering detergents.
[0589] The actual active enzyme content depends upon the method of
manufacture of a detergent composition and is not critical,
assuming the detergent solution has the desired enzymatic activity.
In one aspect, the amount of phospholipase present in the final
solution ranges from about 0.001 mg to 0.5 mg per gram of the
detergent composition. The particular enzyme chosen for use in the
process and products of this invention depends upon the conditions
of final utility, including the physical product form, use pH, use
temperature, and soil types to be degraded or altered. The enzyme
can be chosen to provide optimum activity and stability for any
given set of utility conditions. In one aspect, the polypeptides of
the present invention are active in the pH ranges of from about 4
to about 12 and in the temperature range of from about 20.degree.
C. to about 95.degree. C. The detergents of the invention can
comprise cationic, semi-polar nonionic or zwitterionic surfactants;
or, mixtures thereof.
[0590] Phospholipases of the present invention can be formulated
into powdered and liquid detergents having pH between 4.0 and 12.0
at levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by
weight. These detergent compositions can also include other enzymes
such as known proteases, cellulases, lipases or endoglycosidases,
as well as builders and stabilizers. The addition of phospholipases
of the invention to conventional cleaning compositions does not
create any special use limitation. In other words, any temperature
and pH suitable for the detergent is also suitable for the present
compositions as long as the pH is within the above range, and the
temperature is below the described enzyme's denaturing temperature.
In addition, the polypeptides of the invention can be used in a
cleaning composition without detergents, again either alone or in
combination with builders and stabilizers.
[0591] The present invention provides cleaning or disinfecting
compositions including detergent and/or disinfecting compositions
for cleaning and/or disinfecting hard surfaces, detergent
compositions for cleaning and/or disinfecting fabrics, dishwashing
compositions, oral cleaning compositions, denture cleaning
compositions, and/or contact lens cleaning solutions.
[0592] In one aspect, the invention provides a method for washing
an object comprising contacting the object with a phospholipase of
the invention under conditions sufficient for washing. A
phospholipase of the invention may be included as a detergent
additive. The detergent composition of the invention may, for
example, be formulated as a hand or machine laundry detergent
composition comprising a phospholipase of the invention. A laundry
additive suitable for pre-treatment of stained fabrics can comprise
a phospholipase of the invention. A fabric softener composition can
comprise a phospholipase of the invention. Alternatively, a
phospholipase of the invention can be formulated as a detergent
composition for use in general household hard surface cleaning
operations. In alternative aspects, detergent additives and
detergent compositions of the invention may comprise one or more
other enzymes such as a protease, a lipase, a cutinase, another
phospholipase, a carbohydrase, a cellulase, a pectinase, a
mannanase, an arabinase, a galactanase, a xylanase, an oxidase,
e.g., a lactase, and/or a peroxidase. The properties of the
enzyme(s) of the invention are chosen to be compatible with the
selected detergent (i.e. pH-optimum, compatibility with other
enzymatic and non-enzymatic ingredients, etc.) and the enzyme(s) is
present in effective amounts. In one aspect, phospholipase enzymes
of the invention are used to remove malodorous materials from
fabrics. Various detergent compositions and methods for making them
that can be used in practicing the invention are described in,
e.g., U.S. Pat. Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038;
6,309,871; 6,204,232; 6,197,070; 5,856,164.
Waste Treatment
[0593] The phospholipases of the invention can be used in waste
treatment. In one aspect, the invention provides a solid waste
digestion process using phospholipases of the invention. The
methods can comprise reducing the mass and volume of substantially
untreated solid waste. Solid waste can be treated with an enzymatic
digestive process in the presence of an enzymatic solution
(including phospholipases of the invention) at a controlled
temperature. The solid waste can be converted into a liquefied
waste and any residual solid waste. The resulting liquefied waste
can be separated from said any residual solidified waste. See e.g.,
U.S. Pat. No. 5,709,796.
[0594] Detoxification
[0595] The phospholipases (e.g., PLCs, patatins of the invention)
can be used in detoxification processes, e.g., for the
detoxification of endotoxins, e.g., compositions comprising
lipopolysaccharides (LPS), and, the invention provides
detoxification processes using at least one enzyme of the
invention, e.g., a patatin having a sequence as set forth in SEQ ID
NO:12 (encoded by SEQ ID NO:11), SEQ ID NO:14 (encoded by SEQ ID
NO:13), SEQ ID NO:18 (encoded by SEQ ID NO:17), SEQ ID NO:26
(encoded by SEQ ID NO:25), SEQ ID NO:28 (encoded by SEQ ID NO:27),
SEQ ID NO:34 (encoded by SEQ ID NO:33), SEQ ID NO:36 (encoded by
SEQ ID NO:35), SEQ ID NO:44 (encoded by SEQ ID NO:43), SEQ ID NO:46
(encoded by SEQ ID NO:45), SEQ ID NO:56 (encoded by SEQ ID NO:55),
SEQ ID NO:60 (encoded by SEQ ID NO:59), SEQ ID NO:66 (encoded by
SEQ ID NO:65), SEQ ID NO:72 (encoded by SEQ ID NO:71), SEQ ID NO:78
(encoded by SEQ ID NO:77), SEQ ID NO:87 (encoded by SEQ ID NO:86),
SEQ ID NO:88 (encoded by SEQ ID NO:87), SEQ ID NO:92 (encoded by
SEQ ID NO:91), SEQ ID NO:96 (encoded by SEQ ID NO:95), SEQ ID
NO:100 (encoded by SEQ ID NO:99), SEQ ID NO:104 (encoded by SEQ ID
NO:103), SEQ ID NO:126 (encoded by SEQ ID NO:125), SEQ ID NO:128
(encoded by SEQ ID NO:127), SEQ ID NO:132 (encoded by SEQ ID
NO:131), SEQ ID NO:134 (encoded by SEQ ID NO:133), SEQ ID NO:136
(encoded by SEQ ID NO:135), or SEQ ID NO:138 (encoded by SEQ ID
NO:137). In one aspect, a phospholipase of the invention is used to
detoxify a lipopolysaccharide (LPS). In one aspect, this
detoxification is by deacylation of 2' and/or 3' fatty acid chains
from lipid A. In one aspect, a phospholipase (e.g., a PLC, a
patatin) of the invention is used to hydrolyze a 2'-lauroyl and/or
a 3'-myristoyl chain from a lipid, e.g., a lipid A (e.g., from a
bacterial endotoxin). In one aspect, the process of the invention
is used to destroy an endotoxin, e.g., a toxin from a gram negative
bacteria, as from E. coli. In one aspect, a phospholipase (e.g., a
PLC, a patatin) of the invention is used to ameliorate the effects
of toxin poisoning (e.g., from an on-going gram negative
infection), or, to prophylactically to prevent the effects of
endotoxin during an infection (e.g., an infection in an animal or a
human). Accordingly, the invention provides a pharmaceutical
composition comprising a phospholipase (e.g., a PLC, a patatin) of
the invention, and method using a hydrolase of the invention, for
the amelioration or prevention of lipopolysaccharide (LPS) toxic
effects, e.g., during sepsis.
Processing Foods
[0596] The phospholipases of the invention can be used to process
foods, e.g., to change their stability, shelf-life, flavor,
texture, improve on their nutritional status, and the like. For
example, in one aspect, phospholipases of the invention are used to
generate acidic phospholipids for controlling bitter taste in
foods.
[0597] In one aspect, the invention provides cheese-making
processes using phospholipases of the invention (and, thus, the
invention also provides cheeses comprising phospholipases of the
invention). In one aspect, the enzymes of the invention (e.g.,
phospholipase A, lysophospholipase or a combination thereof) are
used to process cheeses for flavor enhancement, to increase yield
and/or for "stabilizing" cheeses, e.g., by reducing the tendency
for "oil-off," or, in one aspect, the enzymes of the invention are
used to produce cheese from cheese milk. These processes of the
invention can incorporate any method or protocol, e.g., as
described, e.g., in U.S. Pat. Nos. 6,551,635, and 6,399,121, WO
03/070013, WO 00/054601. For example, in one aspect, the
phospholipases of the invention are used to stabilize fat emulsion
in milk or milk-comprising compositions, e.g. cream, and are used
to stabilize milk compositions, e.g. for the manufacturing of
creams or cream liquors. In one aspect, the invention provides a
process for enhancing the favor of a cheese using at least one
enzyme of the invention, the process comprising incubating a
protein, a fat and a protease and a lipase in an aqueous medium
under conditions that produce an enhanced cheese flavor (e.g.,
reduced bitterness), e.g., as described in WO 99/66805. In one
aspect, phospholipases of the invention are used to enhance flavor
in a cheese (e.g., a curd) by mixing with water, a protease, and a
lipase (of the invention) at an elevated temperature, e.g., between
about 75.degree. C. to 95.degree. C., as described, e.g., in U.S.
Pat. No. 4,752,483. In one aspect, phospholipases of the invention
are used to accelerate cheese aging by adding an enzyme of the
invention (e.g., a lipase or a phospholipase) to a cheese (e.g., a
cheese milk) before adding a coagulant to the milk, or, adding an
enzyme of the invention to a curd with salt before pressing, e.g.,
as described, e.g., in U.S. Pat. No. 4,707,364. In one aspect, a
lipase of the invention is used degrade a triglyceride in milk fat
to liberate free fatty acids, resulting in flavor enhancement. A
protease also can be used in any of these processes of the
invention, see, e.g., Brindisi (2001) J. of Food Sci. 66:1100-1107.
In another aspect, a combination of esterases, lipases,
phospholipases and/or proteases can be used in these or any process
of the invention.
[0598] In one aspect, a phospholipase of the invention is used to
reduce the content of phosphorus components in a food, e.g., an
oil, such as a vegetable oil having a high non-hydratable
phosphorus content, e.g., as described in WO 98/26057.
[0599] Other Uses for the Phospholipases of the Invention
[0600] The phospholipases of the invention can also be used to
study the phosphoinositide (PI) signaling system; in the diagnosis,
prognosis and development of treatments for bipolar disorders (see,
e.g., Pandey (2002) Neuropsychopharmacology 26:216-228); as
antioxidants; as modified phospholipids; as foaming and gelation
agents; to generate angiogenic lipids for vascularizing tissues; to
identify phospholipase, e.g., PLA, PLB, PLC, PLD and/or patatin
modulators (agonists or antagonists), e.g., inhibitors for use as
anti-neoplastics, anti-inflammatory and as analgesic agents. They
can be used to generate acidic phospholipids for controlling the
bitter taste in food and pharmaceuticals. They can be used in fat
purification. They can be used to identify peptides inhibitors for
the treatment of viral, inflammatory, allergic and cardiovascular
diseases. They can be used to make vaccines. They can be used to
make polyunsaturated fatty acid glycerides and
phosphatidylglycerols.
[0601] The phospholipases of the invention, for example PLA and PLC
enzymes, are used to generate immunotoxins and various therapeutics
for anti-cancer treatments.
[0602] The phospholipases of the invention can be used in
conjunction with other enzymes for decoloring (i.e. chlorophyll
removal) and in detergents (see above), e.g., in conjunction with
other enzymes (e.g., lipases, proteases, esterases, phosphatases).
For example, in any instance where a PLC is used, a PLD and a
phosphatase may be used in combination, to produce the same result
as a PLC alone.
[0603] The following table summaries several exemplary processes
and formulations of the invention:
TABLE-US-00006 Exemplary Process of the invention Purpose Chemical
usage in PLC oil degumming No use of acid Chemical elimination No
use of caustic Chemical elimination Range of acid and caustic use
(no excess Chemical reduction/degumming process to excess)
alternative embodiment Other types of acid and caustic Degumming
process alternative embodiments Impact of water in PLC oil
degumming Use of silica gel Replacement of water wash step Use of
water drying agent Elimination of water in final product Impact of
lower water during caustic Elimination of water in final product
treatment Minimal water content (<5%) Elimination of water in
final product Maximal water content (>5%) Process alternative
Humidity profiles on PLC degumming Degumming process alternative
embodiment Oil dependence on water content for PLC Degumming
process alternative degumming embodiment In situ removal of free
fatty acids, FFAs Addition of FFA chelating agent Degumming process
alternative embodiment; improves conditions in oil from spoilt
beans Impact of mixing regimen on PLC oil degumming PLC degumming
with minimal mixing Protection of enzyme from mixing induced
denaturation, energy savings PLC degumming with initial shear
Degumming process alternative mixing, followed by paddle mixing
embodiment Order of addition of chemicals Order of addition:
enzyme-water followed Allow the PLC to work before exposure to by
acid then caustic acid and or caustic, causing potential pH or
metal chelation PLC inactivation PLC oil degumming process
alternative embodiments for temperature and time Enzyme treatment
step (time): <60 min, Degumming process alternative preferably
<30 min embodiment Enzyme treatment step (temperature):
50-70.degree. C., Degumming process alternative possibly
<50.degree. C. (e.g. RT) embodiment Benefits from PLC oil
degumming Producing soapstock with minimized PL Degumming process
alternative content and enriched in water soluble embodiment
phosphate esters Reduced neutral oil in gum through use of
Degumming process alternative PLC embodiment Process of generating
increase of DAG in Degumming process alternative vegetable oils
(for ex, 1,3-DAG) embodiment Benefits of using increased DAG
Exemplary Product benefit vegetable oils with other oils for health
benefits Investigate degumming process that Degumming process
alternative leaves no PLC activity in oil embodiment/regulatory
improvement Investigate degumming process that Degumming process
alternative leaves no detectable PLC protein in oil
embodiment/regulatory improvement Use of an enzyme to produce DAG
from Exemplary Product benefit lecithin gum mass Use of PLC with
specialty oils (PA, PI Exemplary Product benefit enriched) Use of
PA/PI specific enzymes (e.g. Degumming process alternative
596ES2/PI specific) embodiment Use of PA/PI specific enzymes (e.g.
Degumming process alternative 596ES2/PI specific) + PC/PE specific
embodiment enzymes; impact of order of addition Batch or continuous
process Degumming process alternative embodiment Use of resuspended
PLC treated gum for Degumming process alternative further oil
degumming operations embodiment Mass balance for DAG, FFA, P,
metals, Degumming process alternative neutral oil in gum embodiment
Miscellaneous Addition of PLC to flaked oil seed kernels Process
alternative embodiment before extrusion Small scale degumming assay
Degumming process alternative embodiment Use of other enzymes to
reduce gum mass Degumming process alternative (e.g., PYROLASE .TM.
enzyme, embodiment chlorophyllase, peroxidase, lipase, laccase,
mannanase, protease, lactase, amylase, etc. or combinations
thereof) Use of compound to better facilitate Degumming process
alternative oil/gum separation embodiment Harden gum from PLC
treated oil Degumming process alternative embodiment
Glycosylated/deglycosylated variants of Degumming process
alternative phospholipase embodiment
TABLE-US-00007 Exemplary Formulations of the invention Purpose
Exemplary Liquid formulation for stability Use of compounds to
increase the stability Stabilization of enzyme for maximum of PLC
at different pH and temp. ranges DAG production, possibly for
altering (polyols, salts, metals . . .) substrate specificity or
directing product formation towards the 1,3-DAG type Use of a
hydrophobic delivery system for Stabilization of enzyme for maximum
PLC (liposomes, hydrated enzyme in DAG production, possibly for
altering refined oil droplets) substrate specificity or directing
product formation towards the 1,3-DAG type Solid formulation for
stability Use of different PLC, phospholipase Stabilization of the
enzyme(s) and ease of carrier systems (immobilization resins,
separation of the enzyme from the oil or porous matrices, gels,
granules, powders, gum phase after degumming; recyclability
tablets, vesicles/micelles, encapsulates, of the enzyme
preparation; physical structured liquids, etc) to stabilize
separation of the enzyme phase during oil phospholipase and
co-enzymes processing; attack of PI/PA by PLC Use of degumming
waste materials (gum Cost reduction of formulation ingredient,
components, seed hulls) for PLC formulation better miscibility of
enzyme with oil, thermostabilization of enzyme Exemplary
Formulation and processes for activity boost Use of chemical or
enzyme to help Faster reaction time/degumming disperse the enzyme
better in oil (e.g. process/reduction of chemical usage
effervescent matrix, etc)
TABLE-US-00008 Exemplary Process of the invention Purpose Re-use of
gums/enzyme for further Recyclability of enzyme degumming reactions
Use of formulations that enhance the Faster reaction time/degumming
segregation or enzyme capture of PLs for process/reduction of
chemical usage hydrolysis Use of multiple formulations to
Versatility of process; different enzymes accommodate PLCs with
different PL may require different formulations or may
specificities be added at different stages in the process Use of
multiple formulations to prevent Protection of PLC activities in a
multi- inactivation of one PLC by a component enzyme format
embodiment in the prep of another PLC with a different substrate
specificity Use of multiple formulations to prevent Protection of
PLC activity in a multi-enzyme inactivation of one PLC by a
component format embodiment in the prep of another enzyme
(hydrolase, oxidase) Use of intermittent caustic additions as in
Protection of enzyme from mixing time released caustic addition
formulation induced denaturation, energy savings
Inactivating and Modulating Activity of Enzymes by
Glycosylation
[0604] This invention provides methods comprising use of
recombinant technology to make and expressing enzymes or other
proteins with biological activity, e.g., noxious or toxic enzymes,
(wherein the enzymes or other proteins are not normally
glycosylated) in an inactive or less active, but re-activatable,
form. The method comprises adding one or more glycosylation sites
(e.g., N-linked or O-linked glycosylation) into the enzymes or
other proteins with biological activity (e.g., an enzyme of the
present invention) by engineering a coding sequence incorporating
the new glycosylation site(s); expressing the variant coding
sequences in eukaryotic cells or an equivalent engineered or in
vitro system capable of post-translational glycosylation. For
example, the 3 amino acid sequence NXS/T is the site for
glycosylation in eukaryotic cells, prokaryotic cells do not do
this. Thus, the invention comprises adding at least one 3 amino
acid sequence NXS/T to the protein such that its activity is
decreased or inactivated because of post-translational
glycosylation.
[0605] The glycosylation can result in 2 molecules of N-acetyl
glucosamine (NGlucNac) being added to the N residue. Subsequent
additions can be organism specific. In most species mannose (Mann)
sugars are then added onto the NGlucNac, with the number Mann
residues ranging from 10 to 100. Sialic acid can also be added in
some species. In Pichia after the NGlucNac is added, 10 to 25 Mann
residues can be added.
[0606] These methods comprise using any deglycosylating enzyme or
set of enzymes, many of which can have been identified and/or are
commercially available. For example, the endoglycosidase H enzyme
cleaves at the last NGlucNac leaving one NClucNac still attached to
the N residue. The PNGaseF enzyme cleaves off all of the sugars and
converts the amino side chain of the N residue into a hydroxyl
group resulting in the N amino acid becoming an aspartate (D) amino
acid in the enzyme. Thus, the methods comprise using
endoglycosidase H and/or PNGaseF or equivalent enzymes in vivo or
in vitro to re-activate partially or completely the engineered
"temporarily inactivated" proteins.
[0607] The method comprises targeting the enzymes or other
polypeptides to the host secretory pathway so that the enzymes will
be glycosylated. The new glycosylation sites are designed such that
glycosylation inactivates the enzyme or modifies its activity,
e.g., decreases it activity or other otherwise modifies activity,
such as blocks a substrate binding site. Because the enzyme is
inactive or less active, noxious or toxic enzymes could be
expressed at higher levels since the negative effects of their
activity are no longer a limitation to how much of the protein can
accumulate in the host cells. The inactive, glycosylated enzyme can
be re-activated (partially or completely) by removing the sugars,
e.g., using commercially available deglycosylating enzymes, for
example, by removing the sugars in vitro, or removing the sugars in
vivo using whole cell engineering approaches.
[0608] In one aspect, a eukaryotic glycosylation target site such
as NXS/T is added to any protein, for example, an enzyme of the
invention. This enables one skilled in the art to add glycosylation
sites to a protein of interest with the expectation of converting
that protein into one that is temporarily inactive when that
protein is glycosylated by expressing that protein in a eukaryotic
host cell and targeting the protein to the host cell's secretory
pathway.
[0609] Thus, the invention provides methods for the production of
enzymes that normally are too noxious or toxic to be tolerated in
large amounts by a host cell. The effect can temporary as it is
possible to regenerate the active enzyme (by deglycosylation, e.g.,
by post-translational modification/deglycosylation) for future work
requiring an active enzyme.
[0610] In one aspect, the invention provides methods for making and
expressing a protein having a biological activity whose activity is
temporarily inactivated by glycosylation comprising: (a) providing
a nucleic acid encoding a protein having a biological activity,
wherein the protein is not naturally glycosylated; (b) inserting at
least one glycosylation motif coding sequence into the
protein-encoding nucleic acid, wherein the glycosylated form of the
protein is inactive; (c) inserting a targeting sequence into the
protein such that it is directed to a host cell's secretory
pathway, wherein the host cell is capable of recognizing the
glycosylation motif and glycosylating the protein; and (d)
expressing the modified nucleic acid in the host cell. In one
aspect, the method further comprises deglycosylating the expressed
the protein, thereby re-activating the activity of the protein,
e.g., an enzyme, such as an enzyme of the invention. In one aspect,
the host cell is a eukaryotic cell. In one aspect, the inactivated
expressed recombinant protein is re-activated in vitro by
deglycosylation, either chemical or enzymatic.
[0611] Determining the placement of one or more glycosylation
motifs to temporarily inactivate a protein involves only routine
methods of making variant protein-encoding nucleic acids, e.g., by
GSSM.TM., and routine screening protocols, e.g., activity or
binding assays.
[0612] An enzyme whose activity was detrimental to the host cell
was rendered inactive because of glycosylation. Because it was
inactive it could accumulate in much higher levels in the
eukaryotic host cells. Because it was no longer active it could no
longer able to exert its negative effects. The inactivation of the
toxic enzyme was temporary because deglycosylating the enzyme using
EndoH or PNGase F resulted in a complete restoration of normal
activity to the enzyme. A large amount of the glycosylated,
inactive enzyme accumulated in the medium suggesting that it was
tolerated well by the host as the inactive form.
[0613] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
EXAMPLES
Example 1
Blast Program Used for Sequence Identify Profiling
[0614] This example describes an exemplary sequence identity
program to determine if a nucleic acid is within the scope of the
invention. An NCBI BLAST 2.2.2 program is used, default options to
blastp. All default values were used except for the default
filtering setting (i.e., all parameters set to default except
filtering which is set to OFF); in its place a "-F F" setting is
used, which disables filtering. Use of default filtering often
results in Karlin-Altschul violations due to short length of
sequence. The default values used in this example: [0615] "Filter
for low complexity: ON [0616] Word Size: 3 [0617] Matrix: Blosum62
[0618] Gap Costs: Existence:11 [0619] Extension:1"
[0620] Other default settings were: filter for low complexity OFF,
word size of 3 for protein, BLOSUM62 matrix, gap existence penalty
of -11 and a gap extension penalty of -1. The "-W" option was set
to default to 0. This means that, if not set, the word size
defaults to 3 for proteins and 11 for nucleotides. The settings
read:
TABLE-US-00009 <<README.bls.txt>> >
----------------------------------------------------------------------
----- > blastall arguments: > > -p Program Name [String]
> -d Database [String] > default = nr > -i Query File
[File In] > default = stdin > -e Expectation value (E) [Real]
> default = 10.0 > -m alignment view options: > 0 =
pairwise, > 1 = query-anchored showing identities, > 2 =
query-anchored no identities, > 3 = flat query-anchored, show
identities, > 4 = flat query-anchored, no identities, > 5 =
query-anchored no identities and blunt ends, > 6 = flat
query-anchored, no identities and blunt ends, > 7 = XML Blast
output, > 8 = tabular, > 9 tabular with comment lines
[Integer] > default = 0 > -o BLAST report Output File [File
Out] Optional > default = stdout > -F Filter query sequence
(DUST with blastn, SEG with others) [String] > default = T >
-G Cost to open a gap (zero invokes default behavior) [Integer]
> default = 0 > -E Cost to extend a gap (zero invokes default
behavior) [Integer] > default = 0 > -X X dropoff value for
gapped alignment (in bits) (zero invokes default > behavior)
[Integer] > default = 0 > -I Show GI's in deflines [T/F] >
default = F > -q Penalty for a nucleotide mismatch (blastn only)
[Integer] > default = -3 > -r Reward for a nucleotide match
(blastn only) [Integer] > default = 1 > -v Number of database
sequences to show one-line descriptions for (V) > [Integer] >
default = 500 > -b Number of database sequence to show
alignments for (B) [Integer] > default = 250 > -f Threshold
for extending hits, default if zero [Integer] > default = 0 >
-g Perform gapped alignment (not available with tblastx) [T/F] >
default = T > -Q Query Genetic code to use [Integer] >
default = 1 > -D DB Genetic code (for tblast[nx] only) [Integer]
> default = 1 > -a Number of processors to use [Integer] >
default = 1 > -O SeqAlign file [File Out] Optional > -J
Believe the query defline [T/F] > default = F > -M Matrix
[String] > default = BLOSUM62 > -W Word size, default if zero
[Integer] > default = 0 > -z Effective length of the database
(use zero for the real size) > [String] > default = 0 > -K
Number of best hits from a region to keep (off by default, if used
a > value of 100 is recommended) [Integer] > default = 0 >
-P 0 for multiple hits 1-pass, 1 for single hit 1-pass, 2 for
2-pass > [Integer] > default = 0 > -Y Effective length of
the search space (use zero for the real size) > [Real] >
default = 0 > -S Query strands to search against database (for
blast[nx], and > tblastx). 3 is both, 1 is top, 2 is bottom
[Integer] > default = 3 > -T Produce HTML output [T/F] >
default = F > -l Restrict search of database to list of GI's
[String] Optional > -U Use lower case filtering of FASTA
sequence [T/F] Optional > default = F > -y Dropoff (X) for
blast extensions in bits (0.0 invokes default > behavior) [Real]
> default = 0.0 > -Z X dropoff value for final gapped
alignment (in bits) [Integer] > default = 0 > -R PSI-TBLASTN
checkpoint file [File In] Optional > -n MegaBlast search [T/F]
> default = F > -L Location on query sequence [String]
Optional > -A Multiple Hits window size (zero for single hit
algorithm) [Integer] > default = 40
Example 2
Simulation of PLC Mediated Degumming
[0621] This example describes the simulation of phospholipase C
(PLC)-mediated degumming.
[0622] Due to its poor solubility in water phosphatidylcholine (PC)
was originally dissolved in ethanol (100 mg/ml). For initial
testing, a stock solution of PC in 50 mM
3-morpholinopropanesulpholic acid or 60 mM citric acid/NaOH at pH 6
was prepared. The PC stock solution (10 .mu.l, 1 .mu.g/.mu.l) was
added to 500 .mu.l of refined soybean oil (2% water) in an
Eppendorf tube. To generate an emulsion the content of the tube was
mixed for 3 min by vortexing (see FIG. 5A). The oil and the water
phase were separated by centrifugation for 1 min at 13,000 rpm
(FIG. 5B). The reaction tubes were pre-incubated at the desired
temperature (37.degree. C., 50.degree. C., or 60.degree. C.) and 3
.mu.l of PLC from Bacillus cereus (0.9 U/.mu.l) were added to the
water phase (FIG. 5C). The disappearance of PC was analyzed by TLC
using chloroform/methanol/water (65:25:4) as a solvent system (see,
e.g., Taguchi (1975) supra) and was visualized after exposure to
I.sub.2 vapor.
[0623] FIG. 5 schematically illustrates a model two-phase system
for simulation of PLC-mediated degumming FIG. 5A: Generation of
emulsion by mixing crude oil with 2% water to hydrate the
contaminating phosphatides (P). FIG. 5B: The oil and water phases
are separated after centrifugation and PLC is added to the water
phase, which contains the precipitated phosphatides ("gums"). The
PLC hydrolysis takes place in the water phase. FIG. 5C: The time
course of the reaction is monitored by withdrawing aliquots from
the water phase and analyzing them by TLC.
Example 3
Expression of Phospholipases
[0624] This example describes the construction of a commercial
production strain of the invention that can express multiple
phospholipases (including enzymes of the invention). In order to
produce a multi-enzyme formulation suitable for use in the
degumming of food-grade vegetable oils (including soybean, canola,
and sunflower), a recombinant expression strain can be generated
that expresses two different phospholipase sequences in the same
expression host. For example, this strain may be constructed to
contain one or more copies of a PLC gene and one or more copies of
a phosphatidylinositol-PLC gene. These genes may exist on one
plasmid, multiple plasmids, or the genes may be inserted into the
genome of the expression host by homologous recombination. When the
genes are introduced by homologous recombination, the genes may be
introduced into a single site in the host genome as a DNA
expression cassette that contains one or more copies of both genes.
Alternatively, one or more copies of each gene may be introduced
into distinct sites in the host chromosome. The expression of these
two gene sequences could be driven by one type of promoter or each
gene sequence may be driven by an independent promoter. Depending
on the number of copies of each gene and the type of promoter, the
final strain will express varying ratios of each active enzyme
type. The expression strains can be constructed using any Bacillus
(e.g., B. cereus) or Streptomyces, E. coli, S. pombe, P. pastoris,
or other gram-negative, gram-positive, or yeast expression
systems.
[0625] In one aspect, the invention provides a two-enzyme system
for degumming of soybean oil, wherein at least one enzyme is an
enzyme of the invention. PLC plus PI-PLC produces more DAG than
either enzyme alone. However both enzymes produce more DAG than a
no enzyme control sample. In one aspect, reaction conditions
comprise 1 milliliter soybean oil, .about.0.4% initial moisture in
the oil before any additions, 50.degree. C., 0.2% Citric acid
neutralized with 2.75M NaOH, 10U PLC, 15 .mu.L PI-PLC (0.45 mg
total protein), 1 hour total reaction time. FIG. 12 illustrates a
table summarizing data from this two-enzyme degumming system of the
invention.
[0626] In another aspect, a PI-PLC enzyme of the invention can be
used under the same conditions described for PLC. These include
chemical refining of vegetable oils and water degumming of
vegetable oils.
Example 4
Phospholipases with Improved Expression and Altered Protease
Resistance
[0627] The invention provides method for selectioning Phospholipase
C variants (mutants) having improved expression in a glycosylating
host and altered resistance to secreted proteases.
Improved Expression in a Glycosylating Host.
[0628] Potential asparagines-linked glycosylation sites with the
amino acid consensus sequence, asparagine-any amino acid-serine or
threonine (NXS/T in the one letter amino acid code), were knocked
out using mutagenesis methods to change the asparagines or the
serine or the threonine in the glycosylation recognition motif to a
different amino acid so the sequence no longer encodes a potential
glycosylation site. The elimination of the glycosylation sites was
effected as indicated below: amino acid positions amino acid 63,
amino acid 131, and amino acid 134, of the phospholipase C enzyme
of the invention having an amino acid sequence as set forth in SEQ
ID NO:2, encoded, e.g., by SEQ ID NO:1. This elimination of the
glycosylation sites improved expression of this variant, active
phospholipase C enzyme (PLC, SEQ ID NO:2) when the protein was
heterologously expressed in the yeast Pichia pastoris. This
strategy of reducing or eliminating potential glycosylation sites
in the PLC enzyme can improve the expression of active PLC in any
glycosylating host. Thus, the invention provides phospholipase
enzymes (and the nucleic acids that encode them) having a sequence
of any of the exemplary phospholipases of the invention with one or
more or all of the glycosylation sites altered, as described above.
Thus, the invention provides methods of making variant
phospholipase coding sequences having increased expression in a
host cell, where the method comprises modifying a phospholipase
coding sequence of the invention such that one, several or all
N-linked glycosylation site coding motifs are modified to a
non-glycosylated motif. The invention also provides phospholipase
coding sequence made by this process, and the enzymes they
encode.
Altered Resistance to Protease
[0629] The invention provides methods for making a variant
phospholipase coding sequence encoding a phospholipase having
increased resistance to a protease comprising modifying an amino
acid equivalent to position 131 of SEQ ID NO:2 to one, several or
all of the following residues: Lysine (K); Serine (S); Glycine (G);
Arginine (R); Glutamine (Q); Alanine (A); Isoleucine (I); Histidine
(H); Phenylalanine (F); Threonine (T); Methionine (M) Leucine (L),
including variants to SEQ ID NO:2 (and the nucleic acid that encode
them) having these exemplary modifications. The invention also
provides isolated, synthetic or recombinant phospholipases encoded
by a sequence made by this method. The invention also provides
methods for making a variant phospholipase coding sequence encoding
a phospholipase having decreased resistance to a protease
comprising modifying an amino acid equivalent to position 131 of
SEQ ID NO:2 to one, several or all of the following residues:
Tryptophan (W); Glutamate (E); Tyrosine (Y), including variants to
SEQ ID NO:2 (and the nucleic acid that encode them) having these
exemplary modifications. The invention also provides isolated,
synthetic or recombinant phospholipases encoded by a sequence made
by this method.
[0630] Supernatant containing a mixture of native secreted Pichia
pastoris proteases is mixed and incubated with wild type and mutant
PLC enzyme preparations. Reactions are quenched and degradation
visualized by SDS-PAGE versus the no protease negative control.
Degradation may also determined by measurement of residual PLC
activity. Novelty was derived from the observation that certain
mutations to knock-out glycosylation significantly change the
susceptibility of the expressed phospholipase to degradation during
fermentation. An advantage to the method is direct selection of
mutants with increased or decreased resistance to the proteases
secreted by the host organism during production.
[0631] This process of the invention can employ site directed
mutagenesis (e.g., GSSM.TM.) to change the amino acid sequence of a
phospholipase C enzyme of the invention, e.g., as shown below--a
subsequence of SEQ ID NO:2 encoded by SEQ ID NO:1. Each of the
amino acids highlighted in red (below) were changed from asparagine
(N in single letter code) to Aspartate (D), serine (S), or another
amino acid as described below. These amino acids are designated as
amino acid 63, amino acid 131, and amino acid 134 of the sequence
below where tryptophan (W) is designated amino acid 1. These
mutations were made to increase the expression of active
phospholipase C protein by reducing glycosylation of the expressed
protein in the Pichia pastoris expression system. These same
mutations can increase expression of any active phospholipase C of
the invention in any other expression system that glycosylates
asparagines (N-linked glycosylation) according to the NXS/T system
where N is asparagine, X is any amino acid, and S/T is serine or
threonine. Thus, the invention also provides a process for changing
the susceptibility of the expressed phospholipase C by changing the
amino acid in position 131.
[0632] Amino Acids 39-286 of SEQ ID NO:2:
NOTE: To count the positions changed, count the first amino acid
(W) as position 1.
TABLE-US-00010 WSAEDKHNEGINSHLWIVNRAIDIMSRNTTIVNPNETALLNEWRADLENG
##STR00001## ##STR00002##
TIKNNYIVSDSNGYWNWKGANPEDWIEGAAVAAKQDYPGVVNDTTKDWFV
KAAVSQEYADKWRAEVTPVTGKRLMEAQRVTAGYIHLWFDTYVNR-
[0633] The expressed phospholipase C variants were incubated in the
presence of P. pastoris proteases as described below and the
following results were obtained.
[0634] The following amino acids at amino acid position 131 of SEQ
ID NO:2 increased the resistance of the expressed phospholipase C
to degradation by P. pastoris proteases: Lysine (K); Serine (S);
Glycine (G); Arginine (R); Glutamine (Q); Alanine (A); Isoleucine
(I); Histidine (H); Phenylalanine (F); Threonine (T); Methionine
(M) Leucine (L). The following amino acids at amino acid position
131 of SEQ ID NO:2 decreased the resistance of the expressed
phospholipase C to degradation by P. pastoris proteases: Tryptophan
(W); Glutamate (E); Tyrosine (Y). Thus, the invention provides
variant phospholipases having any one of, or several or all of
these modifications, depending on whether it was desired to
increase or decrease the resistance of the expressed phospholipase
C to degradation by protease. The invention provides variant
phospholipases having any one of, or several or all of these
modifications in positions equivalent to position 131 of SEQ ID
NO:2. Which residue is equivalent to position 131 of SEQ ID NO:2,
and whether any particular amino acid residue modification can
increase or decrease the resistance of the enzyme to degradation by
a protease, can be routinely and predictably ascertained by
protocols well known in the art, e.g., the exemplary assay used to
evaluate protease susceptibility of the (SEQ ID NO:2, encoded by
SEQ ID NO:1) phospholipase C described below:
[0635] Buffers: [0636] 1.0 M MES, pH 6.2 [0637] 0.7 M sodium
acetate ("NaAc"), pH 5.2
[0638] Challenge: [0639] Use separate 1.5 mL microfuge tubes [0640]
To 25 .mu.L PLC enzyme sample add 5 .mu.L NaAc or 7 .mu.L MES
buffer and mix [0641] Add 25 .mu.L protease-containing Pichia
pastoris supernatant and mix [0642] Add 2 .mu.L 5% sodium azide and
mix [0643] Place tubes in floating rack in prewarmed beaker of
water in a humidified incubator [0644] Controls include
PLC+buffer+dH.sub.2O and Pichia SN+buffer+dH.sub.2O [0645] Incubate
from 0-24 hours, sampling multiple timepoints if desired
[0646] Detection: [0647] Visualize on SDS-PAGE by mixing samples
1:2 with sample buffer containing 5 mM EDTA, heat 100.degree. C., 4
minutes, cool, centrifuge, mix, load 5 .mu.L sample per lane,
Coomassie stain. [0648] Samples and timepoints may also be taken
directly to standard PLC activity assay.
[0649] Results: SDS-PAGE gels were run and the results are
illustrated in FIG. 17; which shows the results of the in vitro
digestion experiments wherein the phospholipase C variants were
incubated in crude protease extracts for up to 22 hr at 37.degree.
C. Each PLC mutant is named according to the amino acid found in
the "X" position of the sequence "DXD" (Aspartate at amino acid
position 63-any amino acid at amino acid position 131-Aspartate at
amino acid position 134). The gels show the stability or
sensitivity of the expressed PLC mutant protein following
incubation with crude protease. A stable mutant shows a PLC band of
similar staining intensity in the "-" (control no protease
reaction) and the "+" (reaction contains protease). A mutant more
sensitive to protease will show a reduction in PLC protein band
staining intensity in the "=" lane compared to the "-" lane.
Example 5
Process for Stable High Level Expression PLC
[0650] The invention provides a fermentation process for stable,
high level expression and high specific activity of phospholipase
enzymes, e.g., PLC, in yeast cultures, e.g., Pichia pastoris
cultures. The enzymes produced by this method can be used, e.g., in
vegetable oil refinement, such as soybean, canola, sunflower or
other oils.
[0651] The invention provides a production process comprising
characteristics that enable production of active phospholipase,
e.g., PLC, in a yeast cell culture, e.g., Pichia pastoris, as
fed-batch cultures at a g/l scale. Heterologous expression of
active PLC protein in microbial cultures had occasionally been
described in the literature only at the mg/l scale. The process of
the present invention is based, inter alia, on the finding that
expression of PLC protein in Pichia cultures impairs the MeOH
uptake capacity, but no other studied physiological growth
characteristics. In contrast to conventional heterologous protein
expression in Pichia cultures, high co-feed rates (glucose/or
glycerol) are required. In addition to improving enzyme production
characteristics, higher co-feeding also eliminates the expression
of general protease activity which is correlated with PLC
degradation. In addition, the poor MeOH utilization characteristics
can be overcome, thereby improving the production characteristics
further, by producing PLC in Pichia strains with a Mut.sup.+
phenotype without compromising scalability challenges normally
associated with a Mut.sup.+ phenotype (and are therefore, not used
on industrial scale). Thus, this process of the invention improves
the production of active PLC by >50-fold (from 100 U/ml using
conventional methods to >5000 U/ml whole broth; >5 g/l
protein) compared to conditions that are normally applied in
industrial scale Pichia systems. In addition, because PLC is a
metallo-enzyme requiring binding of zinc for proper folding and
activity, in one aspect the invention comprises a zinc
supplementation. This zinc supplementation strategy for the
cultures of the invention renders the PLC activity nearly
completely stable (<5% loss in activity) as a whole broth, e.g.,
at 4.degree. C. for >5 days. This significantly aides the
recovery process since 1) production of unstable protein activity
continues to worsen during the recovery process, and 2) it allows
for more processing flexibility, especially at large-scale.
Tryptophanyl Aminopeptidase Microplate Assay
[0652] The invention provides a Tryptophanyl Aminopeptidase
Microplate Assay, which was developed for determination of relative
tryptophanyl aminopeptidase activities in Pichia fermentation
timepoint samples. The throughput capacity of this assay is
sufficient for sampling of multiple timepoints from numerous
fermentations.
[0653] Materials and Methods
[0654] Buffer: [0655] 15 mM NaPO.sub.4, 2 mM MnCl.sub.2, pH 7.5,
aq.
[0656] Substrate: [0657] HTrp-AMC (Bachem, I1670)
[0658] Substrate Solution: [0659] Dissolve Substrate to 10 mM in
methanol [0660] Add 100 .mu.L 10 mM substrate to 6 mL of buffer
[0661] Samples: [0662] Pichia fermentation timepoints [0663]
Centrifuge to remove cells.
[0664] Microplate Preparation: [0665] Aliquot 90 .mu.l substrate
solution per well of black 96-well for each sample replicate,
blanks and references [0666] Place microplate on fluorescent
microplate reader stage (e.g. SpectraMax, Molecular Dynamics)
[0667] Sample Addition and Reaction Kinetics: [0668] Set-up
fluorescent microplate reader: [0669] Ex. 350 nm/Em. 460 nm; auto
cutoff (455 nm); PMT medium; 3 reads per well; autocalibrate "on"
[0670] RT [0671] 0-30 minute timecourse; read every 30 seconds
[0672] Initialize the instrument plate mix function to mix for 5
seconds before first read [0673] Aliquot samples in a 96-well
format and use a multichannel pipet to transfer samples at 10 .mu.L
per well [0674] With lid removed, replace microplate in microplate
reader [0675] Begin reading
[0676] Depending on the inherent activity of unknown samples, it
may be desirable to vary sample dilution, assay duration and
kinetic sampling, all variables that can be determined by routine
screening.
[0677] The substrate has been shown to be very stable under these
conditions and a negative control blank should show no increase in
absorbance over time.
[0678] Bodipy BSA Protease Microplate Assay
[0679] The invention provides a Bodipy BSA Protease Microplate
Assay to aid in the determination of general protease activity in
Pichia fermentation timepoint samples. The throughput capacity of
this assay is sufficient for sampling of multiple timepoints from
numerous fermentations.
[0680] Materials and Methods
[0681] Substrate: [0682] DQ BSA green (Molecular Probes,
D12050)
[0683] Substrate Solution: [0684] Dissolve contents of one vial of
substrate (1 mg) in 1 mL water containing 0.1% sodium azide
[0685] Samples: [0686] Pichia fermentation timepoints [0687]
Centrifuge to remove cells.
[0688] Positive Control: [0689] 0.2 mg/mL subtilisin (Sigma, P5380)
in 50 mM NaPO.sub.4, pH 7.5 [0690] Serially dilute in water
[0691] Microplate Preparation: [0692] Aliquot 90 .mu.l substrate
solution per well of black 96-well for each sample replicate,
blanks and references
[0693] Sample Addition and Reaction: [0694] Aliquot samples in a
96-well format and use a multichannel pipet to transfer samples at
10 .mu.L per well [0695] Replace microplate cover, wrap with foil
and place in humidified incubator at 37.degree. C. and allow to
incubate 3-4 hours or overnight
[0696] Fluorescence Measurement: [0697] Set-up fluorescent
microplate reader (SpectraMax): [0698] Ex. 495 nm/Em. 525 nm; auto
cutoff (515 nm); PMT low; 3 reads per well; autocalibrate "on"
[0699] RT
[0700] Bodipy BSA was selected as a general protease substrate.
Lack of hydrolysis of bodipy BSA does not indicate the absence of
protease(s) but it has been shown to correlate to hydrolysis of PLC
enzyme and loss of PLC activity. It has been demonstrated that BSA
may be substituted with bodipy ovalbumin or casein.
[0701] In one aspect, it is useful to characterize protease
activity across a fermentation timecourse since the activity may be
temporal and transient.
[0702] The substrate has been shown to be very stable under these
conditions and a negative control blank should show no increase in
absorbance over time
[0703] PLC Activity Measurement in Whole Culture Broth or
Supernatant:
[0704] The invention provides a PLC activity measurement assay in
whole culture broth or supernatant; this is a modification of a
method described, e.g., by Edward A. Dennis (1973) Kinetic
dependence of phospholipase A2 activity on the detergent Triton
X-100. J. Lipid Res. 14:152-159, USP 24/NF 19, Pancrealipase-Assay
for lipase activity. Page 1256-1257. The PLC activity measurement
assay of the invention comprises:
Solutions:
[0705] 100 mM Zinc Sulfate Solution [0706] 100 mM Calcium Chloride
Solution [0707] Substrate Solution (20 mM Phosphatidyl Choline, 40
mM Triton X-100, 5 mM Calcium Chloride) [0708] Dilution Buffer
(0.1% Triton X-100, 1 mM Zinc Sulfate, 1% Gum Arabic)
Assay Procedure:
[0708] [0709] Prepare dilutions of the samples to be assayed using
the dilution buffer (1.0% Gum Arabic, 1.0% Triton X-100, 1 mM zinc
sulfate). Prepare dilutions immediately before assay, using
ice-cold buffer, and store in an ice bath until used.
[0710] Transfer 20 mL of the substrate solution into a jacketed
glass vessel of about 50 mL capacity, the outer chamber of which is
connected to a thermostatically controlled water bath. Cover the
mixture, and stir continuously with a mechanical stirring device.
With mixture maintained at a temperature of 37.+-.0.1.degree. C.
pre-titrate the substrate with 0.01 N KOH VS, from a microburet
inserted through an opening in the cover, to adjust the pH to 7.3.
Add 50 .mu.L of enzyme dilution, and then continue automatically to
add the 0.01 N KOH VS for 6 minutes to maintain the pH at 7.
[0711] In addition, standard PAGE gel electrophoresis, Western and
Northern blot analysis on fermenter cultures as well as standard
analysis techniques for on-line/off-line fermentation parameters
(biomass levels, gas analysis etc.)
[0712] Generating the Mut.sup.+ Phenotype Pichia Strains
[0713] The invention provides cells, cell systems and methods for
expressing phospholipase C comprising using a Pichia strain with a
Mut.sup.+ phenotype. The method comprises inserting a heterologous
PLC-encoding nucleic acid in the Pichia strain. The cell is then
cultured under conditions whereby the PLC is expressed. The method
can further comprise supplementing the culture conditions with
zinc.
[0714] In one aspect, these methods, cells and cell systems use SEQ
ID NO:2, which is a zinc-requiring metalloenzyme. In one aspect, it
is used at 3 moles/mole. It has a MW of approximately 28 kDa and a
pI of approximately 5.2, and has a broad substrate tolerance:
PC>PE>PS>>PI. The unprocessed enzyme has a signal
sequence of 24 amino acids, a prosequence of 13 amino acids, and a
"mature" enzyme of 245 amino acid residues.
[0715] In one aspect, the Mut.sup.+ Pichia strains of the invention
have two copies of alcohol oxidase (AOX) genes, AOX1 and AOX2,
affected during transformation ("Mut" stands for "Methanol
Utilization"), as follows: [0716] Mut.sup.+ [0717] Single crossover
event, AOX1 and AOX2 genes intact [0718] Growth and expression on
methanol alone. Co-feeding possible [0719] Mut.sup.S [0720] Double
crossover event disrupts the AOX1 gene [0721] Growth and expression
improved with co-feeding [0722] Mut.sup.- [0723] Recombination
event disrupts AOX1 and 2 genes [0724] Cannot metabolize methanol,
requires co-feeding In summary:
Mut.sup.-<Mut.sup.S.sub.plc<Mut.sup.S/Mut.sup.+.sub.plc<Mut.sup.-
+
[0725] There are fermentation differences between Mut.sup.+ and
Mut.sup.S, including: [0726] Optimal Induction Concentration of
Methanol [0727] Oxygen Consumption Rate [0728] Mut.sup.+ grows
faster than Mut.sup.S on Methanol due to faster uptake capacity
[0729] Ease of Transition Period after Induction [0730] Mut.sup.+
not used for expression at large scale [0731] Aeration/cooling
capacity, MeOH sensitivity
[0732] The methanol utilization pathway in Pichia pastoris is well
known in the art. Alcohol oxidase (AOX) catalyzes the conversion of
methanol to formaldehyde; thus, if the AOX is overexpressed,
results in a "pickled" yeast cell.
[0733] An exemplary fermentation protocol for Pichia pastoris used
in one aspect of the invention comprises: [0734] Seed Culture
(flask or tank) [0735] Batch fermentation in rich medium to enhance
biomass [0736] Fed-Batch Fermentor Culture [0737] Batch Phase
(Glycerol) [0738] Biomass growth as initial carbon source is
consumed. [0739] Glucose or Glycerol Feeding Phase [0740] Addition
of feed triggered by D.O. content or linear/exponential feeding
[0741] Growth to sufficient biomass for induction and expression
(absence of Ethanol, C-limited) [0742] Methanol Induction [0743]
Addition of feed regulated (D.O. %, MeOH sensor, RQ) or preset
feeding profiles [0744] Co-feeding with glucose or glycerol
dependent on phenotype and expression parameters [0745] Mut.sup.+
Induction at 1-3 g/L MeOH [0746] Mut.sup.S Induction at 4-7 g/L
MeOH
[0747] FIG. 18 illustrates the results of a batch fermentor
culture, as discussed above, using only glycerol. Protease activity
is from an endogenous protease in Pichia. The batch fermentation
can be rich medium to enhance biomass. As noted in FIG. 18, the
progressive increase in protease activity beginning at about 69
hours corresponds to a progressive decrease in PLC activity. A
higher co-feed rate of glycerol (glyc) improves active PLC
expression and decreases (eliminates) protease production, as the
following data summary table illustrates:
TABLE-US-00011 C-source PLC MeOH Co-feed rate before/after
Induction activity consumed Bodipy Final (ml/min) induction OD
(U/ml sup) (L) protease OD 0.5 Glyc/Glyc 250-300 100 1 Yes 450 1.5
Glyc/Glyc 1100 1.7 No 680 2 Glyc/Glyc 1550 1.3 No 860 2.5 Glyc/Glyc
1550 1.4 No 900 3 Glyc/Glyc 1715 1.4 No 820
[0748] These studies were done in 30-L BB fermenters with DSD-PLC.
The OUR, or Vol. Oxygen Uptake Rate ("OUR"), as an `overall culture
health` indicator or `Biomarker` for good expression, was measured.
FIG. 19 illustrates the results of such a study, an OUR profile
comparison of cultures of P. pastoris MutS 30 L cultures producing
DSD-PLC, using 1700 U/ml, 1100 U/ml and 100 U/ml PLC, 30.degree.
C., glycerol co-feed, as discussed above.
[0749] FIG. 20 illustrates a methanol consumption profile
comparison in P. pastoris MutS 30 L cultures producing DSD-PLC, pH
6.2 (1100 U/ml and 100 U/ml PLC), or a heterologous protein, with a
glycerol co-feed, as discussed above. This was a demand-driven MeOH
feeding, and the residual MeOH level was controlled at 4 g/l.
[0750] In addition, Mut.sup.+ phenotype improves active PLC
expression and enhances MeOH uptake, as this data table
summarizes:
TABLE-US-00012 Co-feed PLC MeOH rate Induction activity consumed
Bodipy Final Mut (ml/min) OD (U/ml sup) (L) protease OD S 0.5
250-300 100 1 Yes 450 1.5 1100 1.7 No 680 2 1550 1.3 No 860 2.5
1550 1.4 No 900 3 1715 1.4 No 820 + 0.5 250-300 1001 5.6 yes 871
0.5 1200 7 No 908 1 1786 5.9 No 988 1 2010 6.8 No 930 1 1768 7.9 No
700 1.5 2669 10 No 701 1.5 2693 7.1 No 818 1.5 2597 8.1 No 804 2
2154 8.3 No 752
[0751] PLC does not seem to affect physiological growth
characteristics of this Mut.sup.+ phenotype strain--which expresses
recombinant PLC SEQ ID NO:2, in a 6.times. copy number, the data
illustrated in FIG. 21, an OUR profile as set forth in the figure
description. This is a supply-driven MeOH feeding with no residual
glucose or MeOH in Mut.sup.+ cultures.
[0752] Additionally, the quality of PLC protein produced is
unpredictably variable, e.g., <<or >>50% of total PLC
protein is active, as illustrated by the representation of the
results from SDS-PAGE, in FIG. 22. The OUR profile (discussed
above) graphic summary of data is inserted into the upper section
of the SDS-PAGE illustration. The control is designated JG=0.5
.mu.l 1.6 mg ml-1. There was no correlation with protease or
aminopeptidase activity. A significant quantity of active PLC was
located intracellularly, as illustrated in FIG. 23 (also showing
the study's protocol), where >700 U/ml PLC was detected
intracellularly (in FIG. 23, PLC (SEQ ID NO:2)+an alpha signal
peptide (from Saccharomyces)+glycosylation). Morphological changes
were correlated with active PLC concentration, as illustrated in
FIG. 24. Magnitude of the morphological change was strain and
C-source dependent.
[0753] Increased Zn did not boost expression in a Pichia strain
having 2.times. copy number Mut+ SEQ ID NO:2 with DSD mutation, as
summarizes in the data chart, below (excess over 1.times. supplied
via co-feed) (first, upper row is empty vector control). Increased
Zn did improve storage stability as whole broth (similar activity
level after >100 h at 4.degree. C.) and overall robustness of
process.
TABLE-US-00013 MeOH Base 70% (v/v) PLC Zn (L) (L) Glycerol (L)
OD600 (U/ml) 1X 7.1 2.3 9.6 765 0 (2.2 mM) 0.2X 7.4 2.1 8.6 731 392
1X 7.1 2.8 9.0 776 2700 4X 6.1 2.2 10 780 2448 12X 6.4 2.3 9.8 776
2498
[0754] FIG. 25 graphically summarizes data showing the status of a
PLC production performance at 95 h TFT (total fermentation time) in
Pichia. The five bars on the right side of the graph show results
from the "Zeo strain", or Zeocin adaptation of the PLC producing
Pichia pastoris strain. This strain is an antibiotic-resistant
markerless strain expressing as a heterologous gene a PLC of the
invention (SEQ ID NO:2) in a Pichia pastoris strain. It has been
demonstrated that by adapting the strain with zeocin, an
antibiotic, one can obtain a new stable strain with greatly
improved expression level for the protein of interest.
[0755] The original antibiotic-resistant markerless strain, strain
#1 (containing SEQ ID NO:2), was grown in a series of dilution
steps, each time with an increasing concentration of zeocin, which
is an antibiotic. On each step, a portion of the culture from
previous step was diluted to an optical density at 600 nm (OD600)
of 1.0 with fresh medium and an increasing amount of zeocin was
added to the new culture for another 24 hours of growth. At the
final stage, a zeocin concentration of 200 ug/ml was used and the
final culture was streaked to a MD/YPD plate to allow individual
colonies to grow. It was found that the colonies from the final
stage culture show high tolerance to zeocin, while the parent
strain exhibits very little tolerance. One of the colonies, strain
#2 (containing SEQ ID NO:2), showed dramatic improvement (about 70%
higher) in PLC expression compared to the original PLC strain,
strain #1. It was also demonstrated that strain #2 is stable both
in zeocin tolerance and PLC expression after a 40-generation
passage, indicating that the new strain acquired the "permanent"
trait of high PLC expression and zeocin tolerance.
[0756] A high level of PLC activity was achieved using the "Zeo
strain" (Zeocin Pichia adaptation) of the invention: 4100 u/ml
achieved in mini-tanks. This result comes from the Pichia strain
comprising 6.times.DSD SEQ ID NO:2. Briefly, this SEQ ID
NO:2-expressing strain was "adapted" by growing it in a series of
steps, each with increasing concentration of zeocin. Apparently,
this adaptation process forced some changes (in molecular or
genetic level) to the strain/construct and resulted in significant
improvement of PLC activity level. Exemplary results are: [0757]
Tank 1, 2, and 4 (each representing different colonies) all
out-performed the original pre-adapted SEQ ID NO:2-expressing
strain, with tank 1 & 4 both got to 4100 u/ml and tank 2 got to
3500 u/ml. [0758] Tank 1 & 4 got to over 3000 u/ml as early as
in 75 hrs, representing a much faster activity accumulation
comparing to the original pre-adapted SEQ ID NO:2-expressing strain
(which is normally well below 2000 u/ml at the time).
[0759] Details of the Experimental Design and Result are:
[0760] Rationale of Zeocin Adaptation:
[0761] Earlier stage of work on PLC expression was done in the
Pichia pPICZa vector, which contains the zeocin-resistance marker.
Zeocin was thus used for transformation selection. Later on, we
switched to the AMR-less version construct to develop commercial
product candidates. While doing mini-tank fermentations, we
observed a significant drop of PLC activity level obtained using
the AMR-less constructs: supernatant activity reached 4000 u/ml in
pPICZa-DSD constructs, whereas only ca. 2000 u/ml was obtained in
the 2.times.DSD. Significant physiological differences, e.g., lower
methanol consumption rates and a lot more cell lysis, were also
observed with the AMR-less constructs, especially when testing
higher copy number (5.times., 6.times.) constructs using the same
fermentation protocols.
[0762] With one of the apparent differences between the pPIZa
construct and the AMR-less construct being the use of zeocin in
transformation, the question was raised on what the cells might
have gone through with zeocin selection. The invention provides
growing the AMR-less construct in the presence of zeocin--the cells
then go through some changes beneficial to PLC expression.
[0763] Zeocin Adaptation Experiment on 2.times.DSD:
[0764] The experiment was first used with the 2.times.DSD (as it
was the transfer molecule at the time). The study started with a
zeocin concentration of 1 ug/ml ("zeo 1") and grew the culture for
.about.24 hrs. From there, step increase of zeocin concentration to
zeo 5, zeo 10, zeo 15, zeo 20, zeo 40, zeo 60, zeo 80, zeo 100 and
finally to zeo 200 were carried out (zeo 100 is normally used for
transformation selection). Each step fresh medium was used and
previous stage culture was used to inoculate the next stage culture
with OD of 1.0 and grown for .about.24 hrs. Cultures of each stage
were also streaked to YPD plates for preservation and to obtain
individual colonies.
[0765] Mini-Tank Fermentation Results of Zeo-Adapted Colonies:
[0766] To test the effects of zeocin adaptation, a dozen of
colonies from zeo 200 and zeo 100 cultures (that were streaked to
YPD plates) was picked and screened with mini-tanks. The results
are summarized in slide 6. We were able to find several colonies
that significantly outperformed the original construct (Pichia
strain comprising SEQ ID NO:2). Among them, colony #5 from zeo 200
culture showed about 50% improvement on PLC activity level.
Observations on the screening: [0767] There were no apparent
differences on growth profiles between the zeo-adapted cultures and
the original SEQ ID NO:2-expressing strain. [0768] Although
stability of the adapted cultures was not extensively tested, they
were re-streaked several times on YPD and/or MD plates without the
presence of zeocin. All fermentation was also done without the
presence of zeocin. [0769] There were apparent colony to colony
variations, both on growth and on PLC expression. [0770] Some
technical problems with the fermentation might be partly
responsible for the variations.
[0771] Zeocin Adaptation Experiment on 6.times.DSD:
[0772] Encouraged by the results from the zeo-adapted 2.times.DSD,
we then carried the same experiment on the 6.times.DSD (which at
the time was determined as being superior to the 2.times.DSD). We
started with zeocin concentration of 5 ug/ml ("zeo 5") and grew the
culture for .about.24 hrs. From there, step increase of zeocin
concentration to zeo 15, zeo 30, zeo 50, zeo 100 and finally to zeo
200 were carried out. Same as with the 2.times.DSD, each step fresh
medium was used and previous stage culture was used to inoculate
the next stage culture with OD of 1.0 and grown for .about.24 hrs.
Cultures of each stage were also streaked to YPD plates for
preservation and to obtain individual colonies.
[0773] Mini-Tank Results of Zeo-Adapted 6.times.DSD Colonies:
[0774] Six colonies from the zeo 200 culture (that was streaked to
MD plate) were picked and tested together with the original SEQ ID
NO:2-expressing strain in the mini-tanks. Key observations are as
below: [0775] All three colonies (tank 1, 2, and 4) out-performed
the original SEQ ID NO:2-expressing strain, with tank 1 & 4
both got to 4100 u/ml and tank 2 got to 3500 u/ml. [0776] Tank 1
& 4 got to over 3000 u/ml as early as in 75 hrs, representing a
much faster activity accumulation comparing to the SEQ ID
NO:2-expressing strain (which is normally well below 2000 u/ml at
the time). [0777] PLC protein level also seems to be higher in
tanks 1, 2, & 4 comparing to the 3000 u/ml run in 10-L tank
(see slide 4). It is thus not clear whether apparent specific
activity is higher in tanks 1, 2, & 4., i.e., whether the PLC
being produced is different than from the original SEQ ID
NO:2-expressing strain. [0778] The control, tank 7 & 8, did not
get to 3000 u/ml this time. It's not clear whether tank 1, 2, &
4 might be able to reach even higher level. Note that the percent
increase (35%, 4100 u/ml vs 3000 u/ml) is smaller than the 2.times.
adapted culture. [0779] A summary of expression screening from the
6.times.DSD zeocin-adapted colonies is found in FIG. 26. The
highest activity level seen with the original strain was
.about.3000 u/ml (mini-tank & 10-L); the level achieved with
zeocin-adapted 6.times.DSD was 4100 u/ml (.about.35% increase).
FIG. 27 illustrates data showing that PLC protein level was higher
in tanks 1, 2, & 4 comparing to the 3000 u/ml run in 10-L tank
(and tank conditions), as discussed above (the gel loading was at
1.0 ul of 5.times. diluted broth, 0.2 ul of whole broth). FIG. 28
shows the growth comparison of zeo-adapted colonies vs control. The
Zeocin-adapted 6.times.DSD colonies have similar growth profile
compared to the original SEQ ID NO:2-expressing strain
(6.times.DSD).
[0780] The Qp of secreted protein in C-limited aerobic yeast
cultures is generally 0.5-2.5 mg/gh-1 at .mu.=0.10 h-1. Based on
protein content of 400 mg/g DW, `metabolic burden` is <10% of
overall protein production rate. PLC mRNA level remains high
throughout fermentation and does not correlate with expression.
Based on 5 g/l (150 g) PLC protein, less than 0.1 mol C/h of total
5 mol C/h (.about.2% of total C consumed) goes to PLC carbon and
.about.25% goes to biomass. PLC activity does not seem to impact
general growth physiological characteristics under these production
conditions (except MeOH utilization capacity is affected).
[0781] In summary, the invention provides zeocin-resistant yeast
cell systems, such as yeast cells, cell lines and/or individual
cells, for expressing a heterologous protein (e.g., an enzyme, such
as a PLC) made by a process comprising the steps of providing a
Pichia sp. (e.g., P. pastoris) cell comprising a heterologous
nucleic acid (e.g., a vector comprising an enzyme coding sequence;
an ORF operably linked to a promoter) capable of expressing a
heterologous protein; culturing the cell(s) under conditions
comprising zeocin at an initial concentration (a concentration low
enough that some cells survive, but, high enough to select for
antibiotic resistant cells); selecting cells resistant to the
initial concentration of zeocin, and reculturing under conditions
comprising a higher concentration of zeocin; and selecting the
cells resistant to the higher concentration of zeocin. The
invention also provides yeast cells, cell lines and/or individual
cells made by this process. Routine screening can determine which
initial concentration of antibiotic to use, how many rounds of
selection are needed, or desired, and how quickly to increase
concentrations of antibiotic between selection rounds.
Example 6
Thermostable PLC
[0782] The invention provide thermostable phospholipase enzymes.
The thermostability for the exemplary enzyme having a sequence as
set forth in SEQ ID NO:2 was demonstrated. Thermostability of
comparable phospholipids of the invention was demonstrated using
SEQ ID NO:2. The activity of SEQ ID NO:2 was tested in two
different systems: aqueous and in oil. In the aqueous system, a
surrogate substrate (p-nppc) was used to measure activity; the
enzyme began to loose activity at 86 C. However in the oil assays,
the enzyme showed good activity in hydrolyzing PC and PE substrates
present in soy oil at 85 C. Tm of the same enzyme was checked and
found that it was 86 C @ 15 mg/mL, and not reversible.
[0783] FIG. 29 illustrates the results of an 85.degree. C. heating
experiment with 10 U of SEQ ID NO:2, with the conditions indicated
in the figure. FIG. 30 illustrates NMR data summarizing this
heating experiment. FIGS. 31, 32 and 33 illustrate data summarizing
the thermal stability of SEQ ID NO:2 using p-NPPC, at the
conditions shown in the figure. FIG. 34 illustrates data from DSC
analysis showing the thermostability of SEQ ID NO:2, with the
enzyme at a concentration of 15 mg/mL and the Tm at 86.degree. C.
Sequence CWU 1
1
1401849DNAUnknownObtained from an environmental sample. 1atgaaaaaga
aagtattagc actagcagct atggttgctt tagctgcgcc agttcaaagt 60gtagtatttg
cacaaacaaa taatagtgaa agtcctgcac cgattttaag atggtcagct
120gaggataagc ataatgaggg gattaactct catttgtgga ttgtaaatcg
tgcaattgac 180atcatgtctc gtaatacaac gattgtgaat ccgaatgaaa
ctgcattatt aaatgagtgg 240cgtgctgatt tagaaaatgg tatttattct
gctgattacg agaatcctta ttatgataat 300agtacatatg cttctcactt
ttatgatccg gatactggaa caacatatat tccttttgcg 360aaacatgcaa
aagaaacagg cgcaaaatat tttaaccttg ctggtcaagc ataccaaaat
420caagatatgc agcaagcatt cttctactta ggattatcgc ttcattattt
aggagatgtg 480aatcagccaa tgcatgcagc aaactttacg aatctttctt
atccaatggg tttccattct 540aaatacgaaa attttgttga tacaataaaa
aataactata ttgtttcaga tagcaatgga 600tattggaatt ggaaaggagc
aaacccagaa gattggattg aaggagcagc ggtagcagct 660aaacaagatt
atcctggcgt tgtgaacgat acgacaaaag attggtttgt aaaagcagcc
720gtatctcaag aatatgcaga taaatggcgt gcggaagtaa caccggtgac
aggaaagcgt 780ttaatggaag cgcagcgcgt tacagctggt tatattcatt
tgtggtttga tacgtatgta 840aatcgctaa 8492282PRTUnknownObtained from
an environmental sample. 2Met Lys Lys Lys Val Leu Ala Leu Ala Ala
Met Val Ala Leu Ala Ala1 5 10 15Pro Val Gln Ser Val Val Phe Ala Gln
Thr Asn Asn Ser Glu Ser Pro 20 25 30Ala Pro Ile Leu Arg Trp Ser Ala
Glu Asp Lys His Asn Glu Gly Ile 35 40 45Asn Ser His Leu Trp Ile Val
Asn Arg Ala Ile Asp Ile Met Ser Arg 50 55 60Asn Thr Thr Ile Val Asn
Pro Asn Glu Thr Ala Leu Leu Asn Glu Trp65 70 75 80Arg Ala Asp Leu
Glu Asn Gly Ile Tyr Ser Ala Asp Tyr Glu Asn Pro 85 90 95Tyr Tyr Asp
Asn Ser Thr Tyr Ala Ser His Phe Tyr Asp Pro Asp Thr 100 105 110Gly
Thr Thr Tyr Ile Pro Phe Ala Lys His Ala Lys Glu Thr Gly Ala 115 120
125Lys Tyr Phe Asn Leu Ala Gly Gln Ala Tyr Gln Asn Gln Asp Met Gln
130 135 140Gln Ala Phe Phe Tyr Leu Gly Leu Ser Leu His Tyr Leu Gly
Asp Val145 150 155 160Asn Gln Pro Met His Ala Ala Asn Phe Thr Asn
Leu Ser Tyr Pro Met 165 170 175Gly Phe His Ser Lys Tyr Glu Asn Phe
Val Asp Thr Ile Lys Asn Asn 180 185 190Tyr Ile Val Ser Asp Ser Asn
Gly Tyr Trp Asn Trp Lys Gly Ala Asn 195 200 205Pro Glu Asp Trp Ile
Glu Gly Ala Ala Val Ala Ala Lys Gln Asp Tyr 210 215 220Pro Gly Val
Val Asn Asp Thr Thr Lys Asp Trp Phe Val Lys Ala Ala225 230 235
240Val Ser Gln Glu Tyr Ala Asp Lys Trp Arg Ala Glu Val Thr Pro Val
245 250 255Thr Gly Lys Arg Leu Met Glu Ala Gln Arg Val Thr Ala Gly
Tyr Ile 260 265 270His Leu Trp Phe Asp Thr Tyr Val Asn Arg 275
2803852DNAUnknownObtained from an environmental sample. 3atgaaaagaa
aaattttagc tatagcttcc gtaattgctt taacagctcc tatccaaagt 60gtggcgtttg
cgcatgaaaa tggtcaccaa gatccaccaa ttgctctaaa gtggtcagca
120gaatctatac ataatgaagg agtaagttct catttatgga ttgtaaacag
agccattgat 180attatgtccc aaaatacgac tgttgtgaag caaaatgaga
cagctctatt aaatgaatgg 240cgtacggatc tagagaaagg catttactct
gcggattatg aaaacccata ctatgataat 300tccacattcg cttcacactt
ctatgatcct gattcaggaa aaacgtatat tccatttgct 360aaacaagcaa
agcaaacagg agcgaaatat tttaaattag ctggtgaagc ttatcaaaat
420aaagatctga aaaacgcatt cttttattta ggattatcac ttcactattt
aggggatgtc 480aaccaaccaa tgcatgcagc aaactttact aatatttcgc
atccatttgg cttccactca 540aaatatgaaa atttcgttga tacagtgaaa
gacaattata gagtaacgga tggaaatggc 600tattggaatt ggcaaagtgc
aaatccagaa gagtgggttc atgcatcagc atcagcagca 660aaagctgatt
ttccatcaat tgttaatgat aagacgaaaa attggttcct aaaagcagct
720gtatcacaag actctgctga taaatggcgt gcagaagtaa caccgataac
aggaaaacgt 780ttaatggaag cgcagcgtgt tacagctgga tatatccatt
tatggtttga tacgtacgtg 840aataacaaat aa 8524283PRTUnknownObtained
from an environmental sample. 4Met Lys Arg Lys Ile Leu Ala Ile Ala
Ser Val Ile Ala Leu Thr Ala1 5 10 15Pro Ile Gln Ser Val Ala Phe Ala
His Glu Asn Gly His Gln Asp Pro 20 25 30Pro Ile Ala Leu Lys Trp Ser
Ala Glu Ser Ile His Asn Glu Gly Val 35 40 45Ser Ser His Leu Trp Ile
Val Asn Arg Ala Ile Asp Ile Met Ser Gln 50 55 60Asn Thr Thr Val Val
Lys Gln Asn Glu Thr Ala Leu Leu Asn Glu Trp65 70 75 80Arg Thr Asp
Leu Glu Lys Gly Ile Tyr Ser Ala Asp Tyr Glu Asn Pro 85 90 95Tyr Tyr
Asp Asn Ser Thr Phe Ala Ser His Phe Tyr Asp Pro Asp Ser 100 105
110Gly Lys Thr Tyr Ile Pro Phe Ala Lys Gln Ala Lys Gln Thr Gly Ala
115 120 125Lys Tyr Phe Lys Leu Ala Gly Glu Ala Tyr Gln Asn Lys Asp
Leu Lys 130 135 140Asn Ala Phe Phe Tyr Leu Gly Leu Ser Leu His Tyr
Leu Gly Asp Val145 150 155 160Asn Gln Pro Met His Ala Ala Asn Phe
Thr Asn Ile Ser His Pro Phe 165 170 175Gly Phe His Ser Lys Tyr Glu
Asn Phe Val Asp Thr Val Lys Asp Asn 180 185 190Tyr Arg Val Thr Asp
Gly Asn Gly Tyr Trp Asn Trp Gln Ser Ala Asn 195 200 205Pro Glu Glu
Trp Val His Ala Ser Ala Ser Ala Ala Lys Ala Asp Phe 210 215 220Pro
Ser Ile Val Asn Asp Lys Thr Lys Asn Trp Phe Leu Lys Ala Ala225 230
235 240Val Ser Gln Asp Ser Ala Asp Lys Trp Arg Ala Glu Val Thr Pro
Ile 245 250 255Thr Gly Lys Arg Leu Met Glu Ala Gln Arg Val Thr Ala
Gly Tyr Ile 260 265 270His Leu Trp Phe Asp Thr Tyr Val Asn Asn Lys
275 2805843DNAUnknownObtained from an environmental sample.
5atgaaaagaa aaattttagc tatagcttct gtaattgctt taacagctcc tattcaaagt
60gtggcgtttg cgcatgaatc tgatgggcct attgctttaa gatggtcagc ggaatctgta
120cataatgaag gagtaagttc tcatttatgg attgtaaaca gagcaattga
tattatgtcc 180caaaatacga ctgtggtgaa gcaaaatgag acagctctat
taaatgaatg gcgtacgaat 240ttggaggaag gtatttattc tgcagattat
aaaaacccat actatgataa ttccacattc 300gcttcacact tctatgatcc
tgattcagaa aaaacgtata ttccatttgc taaacaagca 360aagcaaacgg
gagcaaagta ttttaaatta gctggtgaag cttatcaaaa taaagatctg
420aaaaatgcat tcttttattt aggattatca cttcattatt taggggatgt
caatcaacca 480atgcatgcag caaactttac taacatttcg catccatttg
gcttccactc aaaatatgaa 540aacttcgttg atacagtgaa agacaattat
agagtaacag atggagatgg ctattggaat 600tggaaaagtg caaatccaga
agagtgggtt catgcatcag catcagcagc aaaagctgat 660ttcccatcaa
ttgttaatga taatacgaaa agttggttcc taaaagcagc ggtatcacaa
720gactctgctg acaaatggcg tgctgaagta acaccggtaa caggaaaacg
tttaatggaa 780gcacagcgta ttacagctgg atatattcat ttatggtttg
atacgtacgt gaataacaaa 840taa 8436280PRTUnknownObtained from an
environmental sample. 6Met Lys Arg Lys Ile Leu Ala Ile Ala Ser Val
Ile Ala Leu Thr Ala1 5 10 15Pro Ile Gln Ser Val Ala Phe Ala His Glu
Ser Asp Gly Pro Ile Ala 20 25 30Leu Arg Trp Ser Ala Glu Ser Val His
Asn Glu Gly Val Ser Ser His 35 40 45Leu Trp Ile Val Asn Arg Ala Ile
Asp Ile Met Ser Gln Asn Thr Thr 50 55 60Val Val Lys Gln Asn Glu Thr
Ala Leu Leu Asn Glu Trp Arg Thr Asn65 70 75 80Leu Glu Glu Gly Ile
Tyr Ser Ala Asp Tyr Lys Asn Pro Tyr Tyr Asp 85 90 95Asn Ser Thr Phe
Ala Ser His Phe Tyr Asp Pro Asp Ser Glu Lys Thr 100 105 110Tyr Ile
Pro Phe Ala Lys Gln Ala Lys Gln Thr Gly Ala Lys Tyr Phe 115 120
125Lys Leu Ala Gly Glu Ala Tyr Gln Asn Lys Asp Leu Lys Asn Ala Phe
130 135 140Phe Tyr Leu Gly Leu Ser Leu His Tyr Leu Gly Asp Val Asn
Gln Pro145 150 155 160Met His Ala Ala Asn Phe Thr Asn Ile Ser His
Pro Phe Gly Phe His 165 170 175Ser Lys Tyr Glu Asn Phe Val Asp Thr
Val Lys Asp Asn Tyr Arg Val 180 185 190Thr Asp Gly Asp Gly Tyr Trp
Asn Trp Lys Ser Ala Asn Pro Glu Glu 195 200 205Trp Val His Ala Ser
Ala Ser Ala Ala Lys Ala Asp Phe Pro Ser Ile 210 215 220Val Asn Asp
Asn Thr Lys Ser Trp Phe Leu Lys Ala Ala Val Ser Gln225 230 235
240Asp Ser Ala Asp Lys Trp Arg Ala Glu Val Thr Pro Val Thr Gly Lys
245 250 255Arg Leu Met Glu Ala Gln Arg Ile Thr Ala Gly Tyr Ile His
Leu Trp 260 265 270Phe Asp Thr Tyr Val Asn Asn Lys 275
2807963DNAUnknownObtained from an environmental sample. 7gtgattactt
tgataaaaaa atgtttatta gtattgacga tgactctatt gttaggggtt 60ttcgtaccgc
tgcagccatc acatgctact gaaaattatc caaatgattt taaactgttg
120caacataatg tatttttatt gcctgaatca gtttcttatt ggggtcagga
cgaacgtgca 180gattatatga gtaatgcaga ttacttcaag ggacatgatg
ctctgctctt aaatgagctt 240tttgacaatg gaaattcgaa catgctgcta
atgaacttat ccacggaata tccatatcaa 300acgccagtgc ttggccgttc
gatgagtgga tgggatgaaa ctagaggaag ctattctaat 360tttgtacccg
aagatggcgg tgtagcaatt atcagtaaat ggccaatcgt ggagaaaata
420cagcatgttt acgcgaatgg ttgcggtgca gactattatg caaataaagg
atttgtttat 480gcaaaagtac aaaaagggga taaattctat catcttatca
gcactcatgc tcaagccgaa 540gatactgggt gtgatcaggg tgaaggagca
gaaattcgtc attcacagtt tcaagaaatc 600aacgacttta ttaaaaataa
aaacattccg aaagatgaag tggtatttat tggtggtgac 660tttaatgtga
tgaagagtga cacaacagag tacaatagca tgttatcaac attaaatgtc
720aatgcgccta ccgaatattt agggcatagc tctacttggg acccagaaac
gaacagcatt 780acaggttaca attaccctga ttatgcgcca cagcatttag
attatatttt tgtggaaaaa 840gatcataaac aaccaagttc atgggtaaat
gaaacgatta ctccgaagtc tccaacttgg 900aaggcaatct atgagtataa
tgattattcc gatcactatc ctgttaaagc atacgtaaaa 960taa
9638320PRTUnknownObtained from an environmental sample. 8Met Ile
Thr Leu Ile Lys Lys Cys Leu Leu Val Leu Thr Met Thr Leu1 5 10 15Leu
Leu Gly Val Phe Val Pro Leu Gln Pro Ser His Ala Thr Glu Asn 20 25
30Tyr Pro Asn Asp Phe Lys Leu Leu Gln His Asn Val Phe Leu Leu Pro
35 40 45Glu Ser Val Ser Tyr Trp Gly Gln Asp Glu Arg Ala Asp Tyr Met
Ser 50 55 60Asn Ala Asp Tyr Phe Lys Gly His Asp Ala Leu Leu Leu Asn
Glu Leu65 70 75 80Phe Asp Asn Gly Asn Ser Asn Met Leu Leu Met Asn
Leu Ser Thr Glu 85 90 95Tyr Pro Tyr Gln Thr Pro Val Leu Gly Arg Ser
Met Ser Gly Trp Asp 100 105 110Glu Thr Arg Gly Ser Tyr Ser Asn Phe
Val Pro Glu Asp Gly Gly Val 115 120 125Ala Ile Ile Ser Lys Trp Pro
Ile Val Glu Lys Ile Gln His Val Tyr 130 135 140Ala Asn Gly Cys Gly
Ala Asp Tyr Tyr Ala Asn Lys Gly Phe Val Tyr145 150 155 160Ala Lys
Val Gln Lys Gly Asp Lys Phe Tyr His Leu Ile Ser Thr His 165 170
175Ala Gln Ala Glu Asp Thr Gly Cys Asp Gln Gly Glu Gly Ala Glu Ile
180 185 190Arg His Ser Gln Phe Gln Glu Ile Asn Asp Phe Ile Lys Asn
Lys Asn 195 200 205Ile Pro Lys Asp Glu Val Val Phe Ile Gly Gly Asp
Phe Asn Val Met 210 215 220Lys Ser Asp Thr Thr Glu Tyr Asn Ser Met
Leu Ser Thr Leu Asn Val225 230 235 240Asn Ala Pro Thr Glu Tyr Leu
Gly His Ser Ser Thr Trp Asp Pro Glu 245 250 255Thr Asn Ser Ile Thr
Gly Tyr Asn Tyr Pro Asp Tyr Ala Pro Gln His 260 265 270Leu Asp Tyr
Ile Phe Val Glu Lys Asp His Lys Gln Pro Ser Ser Trp 275 280 285Val
Asn Glu Thr Ile Thr Pro Lys Ser Pro Thr Trp Lys Ala Ile Tyr 290 295
300Glu Tyr Asn Asp Tyr Ser Asp His Tyr Pro Val Lys Ala Tyr Val
Lys305 310 315 3209999DNAUnknownObtained from an environmental
sample. 9atgaaattac tgcgtgtctt tgtgtgcgtt tttgctttac tcagcgcaca
cagcaaagcc 60gatacactta aagtaatggc ttataatatt atgcaactaa acgtacaaga
ttgggatcaa 120gcaaatcgtg cacagcgctt gccaaacgtc atatctcaat
taagtgacag tcctgatgtc 180attcttatca gcgaagcgtt tagcagccaa
tcagaatctg cgttagcgca acttgctcaa 240ctttaccctt atcaaactcc
caatgttggc gaagactgta gtggcgctgg ctggcaaagc 300ttaacgggta
actgctcgaa tagccccttt gtgatccgcg gtggagtggt gattttatct
360aagtacccca tcattacgca aaaagcccat gtgtttaata acagcctgac
tgatagttgg 420gattatttag caaacaaagg tttcgcttat gttgaaatag
aaaaacatgg caaacgttac 480caccttattg gcacgcattt acaagcaacg
catgatggcg acacagaagc tgagcatatt 540gtgagaatgg gtcaattaca
agagatacaa gatttcattc aaagcgagca aattcacact 600tctgagccgg
tcattatcgg cggtgatatg aacgtagagt ggagcaagca atctgaaatt
660acagatatgc tcgaagtggt tcgcagccgt ctaattttca acacacctga
agttggctct 720ttctctgcaa aacacaactg gtttaccaaa gctaacgcct
actatttcga ctacagctta 780gagtataacg acacgctcga ttatgtactt
tggcatgcag accataagca acccaccaat 840accccagaaa tgttagtacg
ttacccaaaa gcagagcgtg acttttactg gcgttactta 900cgcggaaatt
ggaacttacc ttctggccgt tattatcatg atggatacta taacgaactg
960tctgatcact acccagtgca agttaacttt gaattttaa
99910332PRTUnknownObtained from an environmental sample. 10Met Lys
Leu Leu Arg Val Phe Val Cys Val Phe Ala Leu Leu Ser Ala1 5 10 15His
Ser Lys Ala Asp Thr Leu Lys Val Met Ala Tyr Asn Ile Met Gln 20 25
30Leu Asn Val Gln Asp Trp Asp Gln Ala Asn Arg Ala Gln Arg Leu Pro
35 40 45Asn Val Ile Ser Gln Leu Ser Asp Ser Pro Asp Val Ile Leu Ile
Ser 50 55 60Glu Ala Phe Ser Ser Gln Ser Glu Ser Ala Leu Ala Gln Leu
Ala Gln65 70 75 80Leu Tyr Pro Tyr Gln Thr Pro Asn Val Gly Glu Asp
Cys Ser Gly Ala 85 90 95Gly Trp Gln Ser Leu Thr Gly Asn Cys Ser Asn
Ser Pro Phe Val Ile 100 105 110Arg Gly Gly Val Val Ile Leu Ser Lys
Tyr Pro Ile Ile Thr Gln Lys 115 120 125Ala His Val Phe Asn Asn Ser
Leu Thr Asp Ser Trp Asp Tyr Leu Ala 130 135 140Asn Lys Gly Phe Ala
Tyr Val Glu Ile Glu Lys His Gly Lys Arg Tyr145 150 155 160His Leu
Ile Gly Thr His Leu Gln Ala Thr His Asp Gly Asp Thr Glu 165 170
175Ala Glu His Ile Val Arg Met Gly Gln Leu Gln Glu Ile Gln Asp Phe
180 185 190Ile Gln Ser Glu Gln Ile His Thr Ser Glu Pro Val Ile Ile
Gly Gly 195 200 205Asp Met Asn Val Glu Trp Ser Lys Gln Ser Glu Ile
Thr Asp Met Leu 210 215 220Glu Val Val Arg Ser Arg Leu Ile Phe Asn
Thr Pro Glu Val Gly Ser225 230 235 240Phe Ser Ala Lys His Asn Trp
Phe Thr Lys Ala Asn Ala Tyr Tyr Phe 245 250 255Asp Tyr Ser Leu Glu
Tyr Asn Asp Thr Leu Asp Tyr Val Leu Trp His 260 265 270Ala Asp His
Lys Gln Pro Thr Asn Thr Pro Glu Met Leu Val Arg Tyr 275 280 285Pro
Lys Ala Glu Arg Asp Phe Tyr Trp Arg Tyr Leu Arg Gly Asn Trp 290 295
300Asn Leu Pro Ser Gly Arg Tyr Tyr His Asp Gly Tyr Tyr Asn Glu
Leu305 310 315 320Ser Asp His Tyr Pro Val Gln Val Asn Phe Glu Phe
325 330111041DNAUnknownObtained from an environmental sample.
11atggcttcac aattcaggaa tctggttttt gaaggaggcg gtgtaaaggg aatcgcctat
60atcggcgcca tgcaggtgct ggagcagcgc ggacatttgg agcacgttgt gagggtggga
120ggaacaagtg caggggctat taacgctctc attttttcgc tgggctttac
cattaaagag 180cagcaggata ttctcaattc caccaacttc agggagttta
tggacagctc tttcggattt 240gtgcgaaact tcagaaggct ctggagtgaa
ttcgggtgga accgcggtga tgtgttttcg 300gagtgggcag gagagctggt
gaaagagaaa ctcggcaaga agaacgccac cttcggcgat 360ctgaaaaaag
cgaagcgccc cgatctctac gttatcggaa ccaacctctc caccgggttt
420tccgagactt tttcgcatga acgccacgcc aacatgccgc tggtggatgc
ggtgcggatc 480agcatgtcga tcccgctctt ttttgcggca cgcagacttg
gcaaacgaag cgatgtgtat 540gtggatggag gtgttatgct caactacccg
gtaaagctgt tcgacaggga gaaatacatc 600gatttggaga aggagaaaga
ggcagcccgc tacgtggagt actacaatca agagaatgcc 660cggtttctgc
ttgagcggcc cggccgaagc ccgtacgttt acaaccggca gaccctaggc
720ctgcggctcg actcgcagga agagatcggc ctgttccgtt acgatgagcc
gctgaagggc 780aaacagatca accgcttccc cgaatatgcc aaagccctga
tcggtgcact gatgcaggtg 840caggagaaca tccacctgaa aagcgacgac
tggcagcgaa cgctctacat caacacgctg 900gatgtgggta ccacagattt
cgacattaat gacgagaaga aaaaagtgct ggtgaatgag 960ggaatcaagg
gagcggaaac ctacttccgc tggtttgagg atcccgaagc taaaccggtg
1020aacaaggtgg atttggtctg a 104112346PRTUnknownObtained from an
environmental sample. 12Met Ala Ser Gln Phe Arg Asn Leu Val Phe Glu
Gly Gly Gly Val Lys1 5 10 15Gly Ile Ala Tyr Ile Gly Ala Met Gln Val
Leu Glu Gln Arg Gly His 20 25 30Leu Glu His Val Val Arg Val Gly Gly
Thr Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ser Leu Gly Phe
Thr Ile Lys Glu Gln Gln Asp Ile 50 55 60Leu Asn Ser Thr Asn Phe Arg
Glu Phe Met Asp Ser Ser Phe Gly Phe65 70 75 80Val Arg Asn Phe Arg
Arg Leu Trp Ser Glu Phe Gly Trp Asn Arg Gly 85 90 95Asp Val Phe Ser
Glu Trp Ala Gly Glu Leu Val Lys Glu Lys Leu Gly 100 105 110Lys Lys
Asn Ala Thr Phe Gly Asp Leu Lys Lys Ala Lys Arg Pro Asp 115 120
125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ser Glu Thr Phe
130 135 140Ser His Glu Arg His Ala Asn Met Pro Leu Val Asp Ala Val
Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala Ala Arg
Arg Leu Gly Lys Arg 165 170 175Ser Asp Val Tyr Val Asp Gly Gly Val
Met Leu Asn Tyr Pro Val Lys 180 185 190Leu Phe Asp Arg Glu Lys Tyr
Ile Asp Leu Glu Lys Glu Lys Glu Ala 195 200 205Ala Arg Tyr Val Glu
Tyr Tyr Asn Gln Glu Asn Ala Arg Phe Leu Leu 210 215 220Glu Arg Pro
Gly Arg Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230 235
240Leu Arg Leu Asp Ser Gln Glu Glu Ile Gly Leu Phe Arg Tyr Asp Glu
245 250 255Pro Leu Lys Gly Lys Gln Ile Asn Arg Phe Pro Glu Tyr Ala
Lys Ala 260 265 270Leu Ile Gly Ala Leu Met Gln Val Gln Glu Asn Ile
His Leu Lys Ser 275 280 285Asp Asp Trp Gln Arg Thr Leu Tyr Ile Asn
Thr Leu Asp Val Gly Thr 290 295 300Thr Asp Phe Asp Ile Asn Asp Glu
Lys Lys Lys Val Leu Val Asn Glu305 310 315 320Gly Ile Lys Gly Ala
Glu Thr Tyr Phe Arg Trp Phe Glu Asp Pro Glu 325 330 335Ala Lys Pro
Val Asn Lys Val Asp Leu Val 340 345131038DNAUnknownObtained from an
environmental sample. 13atgacaacac aatttagaaa cttgatattt gaaggcggcg
gtgtaaaagg tgttgcttac 60attggcgcca tgcagattct cgaaaatcgt ggcgtgttgc
aagatattca cagagtcgga 120gggtgcagtg cgggtgcgat caacgcgctg
atttttgcgc tgggttacac ggtccgtgag 180caaaaagaga tcttacaagc
cacggatttt aaccagttta tggataactc ttggggtgtt 240attcgtgata
ttcgcaggct tgctcgagac tttggctggc acaagggtga cttctttaat
300agctggatag gtgatttgat tcatcgtcgt ttggggaatc gccgagcgac
gttcaaagat 360ctgcaaaagg ccaagcttcc tgatctttat gtcatcggta
ctaatctgtc tacagggtat 420gcagaggttt tttcagccga aagacacccc
gatatggagc tagcgacagc ggtgcgtatc 480tccatgtcga taccgctgtt
ctttgcggcc gtgcgtcacg gtgaacgaca agatgtgtat 540gtcgatgggg
gtgttcaact taactatccg attaaactgt ttgatcggga gcgttacatt
600gatctggtca aagatcccgg tgccgttcgg cgaacgggtt attacaacaa
agaaaacgct 660cgctttcagc ttgagcggcc gggccatagc ccctatgttt
acaatcgcca gaccttgggt 720ttgcgactgg atagtcgaga ggagataggg
ctctttcgtt atgacgaacc cctcaagggc 780aaacccatta agtccttcac
tgactacgct cgacaacttt tcggtgcgtt gatgaatgca 840caggaaaaca
ttcatctaca tggcgatgat tgggcgcgca cggtctatat cgatacattg
900gatgtgggta cgacggattt caatctttct gatgcaacca agcaagcact
gattgagcaa 960ggaattaacg gcaccgaaaa ttatttcgac tggtttgata
atccgttaga gaagcctgtg 1020aatagagtgg agtcatag
103814345PRTUnknownObtained from an environmental sample. 14Met Thr
Thr Gln Phe Arg Asn Leu Ile Phe Glu Gly Gly Gly Val Lys1 5 10 15Gly
Val Ala Tyr Ile Gly Ala Met Gln Ile Leu Glu Asn Arg Gly Val 20 25
30Leu Gln Asp Ile His Arg Val Gly Gly Cys Ser Ala Gly Ala Ile Asn
35 40 45Ala Leu Ile Phe Ala Leu Gly Tyr Thr Val Arg Glu Gln Lys Glu
Ile 50 55 60Leu Gln Ala Thr Asp Phe Asn Gln Phe Met Asp Asn Ser Trp
Gly Val65 70 75 80Ile Arg Asp Ile Arg Arg Leu Ala Arg Asp Phe Gly
Trp His Lys Gly 85 90 95Asp Phe Phe Asn Ser Trp Ile Gly Asp Leu Ile
His Arg Arg Leu Gly 100 105 110Asn Arg Arg Ala Thr Phe Lys Asp Leu
Gln Lys Ala Lys Leu Pro Asp 115 120 125Leu Tyr Val Ile Gly Thr Asn
Leu Ser Thr Gly Tyr Ala Glu Val Phe 130 135 140Ser Ala Glu Arg His
Pro Asp Met Glu Leu Ala Thr Ala Val Arg Ile145 150 155 160Ser Met
Ser Ile Pro Leu Phe Phe Ala Ala Val Arg His Gly Glu Arg 165 170
175Gln Asp Val Tyr Val Asp Gly Gly Val Gln Leu Asn Tyr Pro Ile Lys
180 185 190Leu Phe Asp Arg Glu Arg Tyr Ile Asp Leu Val Lys Asp Pro
Gly Ala 195 200 205Val Arg Arg Thr Gly Tyr Tyr Asn Lys Glu Asn Ala
Arg Phe Gln Leu 210 215 220Glu Arg Pro Gly His Ser Pro Tyr Val Tyr
Asn Arg Gln Thr Leu Gly225 230 235 240Leu Arg Leu Asp Ser Arg Glu
Glu Ile Gly Leu Phe Arg Tyr Asp Glu 245 250 255Pro Leu Lys Gly Lys
Pro Ile Lys Ser Phe Thr Asp Tyr Ala Arg Gln 260 265 270Leu Phe Gly
Ala Leu Met Asn Ala Gln Glu Asn Ile His Leu His Gly 275 280 285Asp
Asp Trp Ala Arg Thr Val Tyr Ile Asp Thr Leu Asp Val Gly Thr 290 295
300Thr Asp Phe Asn Leu Ser Asp Ala Thr Lys Gln Ala Leu Ile Glu
Gln305 310 315 320Gly Ile Asn Gly Thr Glu Asn Tyr Phe Asp Trp Phe
Asp Asn Pro Leu 325 330 335Glu Lys Pro Val Asn Arg Val Glu Ser 340
345151344DNAUnknownObtained from an environmental sample.
15atgctggtca tcattcatgg ctggagcgat gaggcgggct cgttcaagac cctggccaga
60cgtttggcca aggcgccacc cgagggcctc gggacgcagg tcacggaaat ccatctgggt
120gattatgtgt ccctggatga ccaggtgacg ttcaatgatc tggtcgatgc
catggccaga 180gcctggagcg atcgtggtct gcccacggcc ccgcgcagcg
tcgatgccgt cgtgcacagc 240accggcggcc tggtgatccg cgactggctc
acgcagctgt acacgccgga aacagccccc 300attcgtcgcc tgctgatgct
cgctccggcc aatttcggct cgccgctggc acacaccgga 360cgcagcatga
tcggccgggt caccaagggc tggaagggca cgcggctctt tgaaacgggc
420aagcacattc tcaaagggct cgaactggcc agcccctacg cctgggcgct
ggccgaacgc 480gatctgttca gcgatcagaa ctattatggc gccgggcgca
tcctgtgcac tgtcctggtg 540ggcaacgccg gttatcgcgg catcagcgcc
gtcgccaacc ggcccggcac ggacggcacc 600gtgcgcgtca gcagcgccaa
tctccaagcg gccaggatgc tgctcgattt cagcgccagt 660ccacaggctg
agccggaatt caccctgcac gacagcaccg cggaaattgc cttcggcatc
720gccgacgagg aagaccacag caccatcgcc gccaaggatc gcggcccgcg
caaggcagtc 780acctgggaac tgattctcaa agccctgcag atcgaggatg
caagctttgc tcaatggtgc 840cggcagatgc aggagcattc cgcggccgtg
acggaaacgg cggaaaagcg ccgcaatgtt 900cactacaaca gcttccagaa
taccgtcgtg cgcgtggtgg acaaccacgg tgccgccgtg 960caggattatc
tcatcgagtt ttacatgaat gatgatcgca aactccgcga tcagcgcctc
1020acccagcgcc tgcaggagca ggtgattacc aacgtgcacg gctacggtga
cgacaagtcc 1080tatcgcagca tgctgatcaa ctgcacggag ctctatgcgc
tgatgtccag accgcaggat 1140cgcctgaaca tcagcatcac cgcctatccg
gatctctcca agggactggt ggggtatcgc 1200acctacacgg acgaggatat
cggttccctc tctctggatg cagcgcagat ccgaaagctc 1260tttaagccgc
accgtaccct gttgatgaca ctgtgcctgc aacgctatca gaaagatgat
1320gtgttccgat tcagggatgt ttga 134416447PRTUnknownObtained from an
environmental sample. 16Met Leu Val Ile Ile His Gly Trp Ser Asp Glu
Ala Gly Ser Phe Lys1 5 10 15Thr Leu Ala Arg Arg Leu Ala Lys Ala Pro
Pro Glu Gly Leu Gly Thr 20 25 30Gln Val Thr Glu Ile His Leu Gly Asp
Tyr Val Ser Leu Asp Asp Gln 35 40 45Val Thr Phe Asn Asp Leu Val Asp
Ala Met Ala Arg Ala Trp Ser Asp 50 55 60Arg Gly Leu Pro Thr Ala Pro
Arg Ser Val Asp Ala Val Val His Ser65 70 75 80Thr Gly Gly Leu Val
Ile Arg Asp Trp Leu Thr Gln Leu Tyr Thr Pro 85 90 95Glu Thr Ala Pro
Ile Arg Arg Leu Leu Met Leu Ala Pro Ala Asn Phe 100 105 110Gly Ser
Pro Leu Ala His Thr Gly Arg Ser Met Ile Gly Arg Val Thr 115 120
125Lys Gly Trp Lys Gly Thr Arg Leu Phe Glu Thr Gly Lys His Ile Leu
130 135 140Lys Gly Leu Glu Leu Ala Ser Pro Tyr Ala Trp Ala Leu Ala
Glu Arg145 150 155 160Asp Leu Phe Ser Asp Gln Asn Tyr Tyr Gly Ala
Gly Arg Ile Leu Cys 165 170 175Thr Val Leu Val Gly Asn Ala Gly Tyr
Arg Gly Ile Ser Ala Val Ala 180 185 190Asn Arg Pro Gly Thr Asp Gly
Thr Val Arg Val Ser Ser Ala Asn Leu 195 200 205Gln Ala Ala Arg Met
Leu Leu Asp Phe Ser Ala Ser Pro Gln Ala Glu 210 215 220Pro Glu Phe
Thr Leu His Asp Ser Thr Ala Glu Ile Ala Phe Gly Ile225 230 235
240Ala Asp Glu Glu Asp His Ser Thr Ile Ala Ala Lys Asp Arg Gly Pro
245 250 255Arg Lys Ala Val Thr Trp Glu Leu Ile Leu Lys Ala Leu Gln
Ile Glu 260 265 270Asp Ala Ser Phe Ala Gln Trp Cys Arg Gln Met Gln
Glu His Ser Ala 275 280 285Ala Val Thr Glu Thr Ala Glu Lys Arg Arg
Asn Val His Tyr Asn Ser 290 295 300Phe Gln Asn Thr Val Val Arg Val
Val Asp Asn His Gly Ala Ala Val305 310 315 320Gln Asp Tyr Leu Ile
Glu Phe Tyr Met Asn Asp Asp Arg Lys Leu Arg 325 330 335Asp Gln Arg
Leu Thr Gln Arg Leu Gln Glu Gln Val Ile Thr Asn Val 340 345 350His
Gly Tyr Gly Asp Asp Lys Ser Tyr Arg Ser Met Leu Ile Asn Cys 355 360
365Thr Glu Leu Tyr Ala Leu Met Ser Arg Pro Gln Asp Arg Leu Asn Ile
370 375 380Ser Ile Thr Ala Tyr Pro Asp Leu Ser Lys Gly Leu Val Gly
Tyr Arg385 390 395 400Thr Tyr Thr Asp Glu Asp Ile Gly Ser Leu Ser
Leu Asp Ala Ala Gln 405 410 415Ile Arg Lys Leu Phe Lys Pro His Arg
Thr Leu Leu Met Thr Leu Cys 420 425 430Leu Gln Arg Tyr Gln Lys Asp
Asp Val Phe Arg Phe Arg Asp Val 435 440 445171137DNAUnknownObtained
from an environmental sample. 17atgaaaaaaa gccttcaaca acatcttgcc
gctgacggca gcccaaagaa tattctttct 60ctcgacgggg gaggaatcag aggggctttg
acccttggtt ttctcaaaaa aatagaaagc 120atcctgcagg aaaaacatgg
gaaggactat ctcctttgcg atcactttga tttgatcggt 180ggaacttcca
caggctccat cattgcagca gcattggcta taggcatgac agtggaggaa
240atcactaaaa tgtatatgga tctgggcgga aaaattttcg gcaagaaaag
gagtttctgg 300agaccctggg aaactgcgaa atacttgaaa gcaggatatg
accacaaagc tcttgaaaag 360agtctgaaag atgctttcca ggattttctt
ttaggaagtg accaaattag aacaggtctt 420tgtatagtag ccaaaagagc
agataccaat agtatatggc cattgattaa ccaccccaaa 480ggaaaattct
atgattcaga acaaggcaaa aacaaaaata tccccttatg gcaggcagta
540agggcgagta ccgctgctcc aacctatttc gctccacaat taatagatgt
gggtgatggt 600caaaaggctg cttttgtgga cggaggggta agcatggcca
ataaccccgc attaaccctg 660ttaaaagtgg ctacacttaa aggttttcct
tttcattggc caatgggaga agacaaactg 720accatagttt cagtaggcac
cggatatagt gttttccaaa gacaaaaggg tgaaatcacc 780aaagcttcct
tattaacttg ggccaaaaac gtcccggaaa tgttgatgca ggatgcttct
840tggcagaatc agaccatact tcagtggatt tctaaatccc ccactgcaca
ttccatagat 900atggaaatgg aagaccttag agatgacttt ctaggcggaa
gaccactcat caaatacctc 960aggtacaact tccccttgac agtaaatgat
ctcaatggat tgaagcttgg gaaaagcttt 1020acccaaaaag aggtcgaaga
tttggtggaa atgagcaatg cacataaccg agaggagttg 1080tataggattg
gggagaaggc ggctgaaggg tcggtaaaaa aagaacattt tgaataa
113718378PRTUnknownObtained from an environmental sample. 18Met Lys
Lys Ser Leu Gln Gln His Leu Ala Ala Asp Gly Ser Pro Lys1 5 10 15Asn
Ile Leu Ser Leu Asp Gly Gly Gly Ile Arg Gly Ala Leu Thr Leu 20 25
30Gly Phe Leu Lys Lys Ile Glu Ser Ile Leu Gln Glu Lys His Gly Lys
35 40 45Asp Tyr Leu Leu Cys Asp His Phe Asp Leu Ile Gly Gly Thr Ser
Thr 50 55 60Gly Ser Ile Ile Ala Ala Ala Leu Ala Ile Gly Met Thr Val
Glu Glu65 70 75 80Ile Thr Lys Met Tyr Met Asp Leu Gly Gly Lys Ile
Phe Gly Lys Lys 85 90 95Arg Ser Phe Trp Arg Pro Trp Glu Thr Ala Lys
Tyr Leu Lys Ala Gly 100 105 110Tyr Asp His Lys Ala Leu Glu Lys Ser
Leu Lys Asp Ala Phe Gln Asp 115 120 125Phe Leu Leu Gly Ser Asp Gln
Ile Arg Thr Gly Leu Cys Ile Val Ala 130 135 140Lys Arg Ala Asp Thr
Asn Ser Ile Trp Pro Leu Ile Asn His Pro Lys145 150 155 160Gly Lys
Phe Tyr Asp Ser Glu Gln Gly Lys Asn Lys Asn Ile Pro Leu 165 170
175Trp Gln Ala Val Arg Ala Ser Thr Ala Ala Pro Thr Tyr Phe Ala Pro
180 185 190Gln Leu Ile Asp Val Gly Asp Gly Gln Lys Ala Ala Phe Val
Asp Gly 195 200 205Gly Val Ser Met Ala Asn Asn Pro Ala Leu Thr Leu
Leu Lys Val Ala 210 215 220Thr Leu Lys Gly Phe Pro Phe His Trp Pro
Met Gly Glu Asp Lys Leu225 230 235 240Thr Ile Val Ser Val Gly Thr
Gly Tyr Ser Val Phe Gln Arg Gln Lys 245 250 255Gly Glu Ile Thr Lys
Ala Ser Leu Leu Thr Trp Ala Lys Asn Val Pro 260 265 270Glu Met Leu
Met Gln Asp Ala Ser Trp Gln Asn Gln Thr Ile Leu Gln 275 280 285Trp
Ile Ser Lys Ser Pro Thr Ala His Ser Ile Asp Met Glu Met Glu 290 295
300Asp Leu Arg Asp Asp Phe Leu Gly Gly Arg Pro Leu Ile Lys Tyr
Leu305 310 315 320Arg Tyr Asn Phe Pro Leu Thr Val Asn Asp Leu Asn
Gly Leu Lys Leu 325 330 335Gly Lys Ser Phe Thr Gln Lys Glu Val Glu
Asp Leu Val Glu Met Ser 340 345 350Asn Ala His Asn Arg Glu Glu Leu
Tyr Arg Ile Gly Glu Lys Ala Ala 355 360 365Glu Gly Ser Val Lys Lys
Glu His Phe Glu 370 375191248DNAUnknownObtained from an
environmental sample. 19atgaaaaaga caacgttagt tttggctcta ttgatgccat
ttggtgccgc ctccgcacaa 60gacaatagta tgactccaga agcaatcaca tcagctcaag
tcgcacaaac acaatcagcc 120tccacctata cctacgttag gtgttggtat
cgaacagacg caagccatga ttcaccagca 180accgactggg agtgggctag
aaaggaaaac ggagactatt acaccattga cggttactgg 240tggtcatcga
tctcctttaa aaatatgttc tatagcgaga ctcctcaaca agagatcaag
300cagcgttgtg tagacacctt ggatgttcag cacgacaaag ccgacatcac
ctactttgcc 360gctgacaacc gcttctctta caaccattct atctggacta
acgatcacgg ctttcaagcg 420aaccaaatca accgaatagt cgcttttggc
gatagtcttt cagacacggg caacctattt 480aatgggtcac aatggatttt
ccctaaccct aattcttggt tcttgggtca cttctctaac 540ggcttcgttt
ggactgaata cttggctaac gctaagggcg ttccactcta taactgggct
600gtgggtggcg cagcaggaac caaccaatat gtcgctctaa ctggtgtcta
tgatcaggtc 660acttcgtacc tgacttacat gaagatggcg aaaaattatc
gcccagagaa cacactattc 720acattagagt ttggattgaa tgactttatg
aattacggac gtgaagtagc tgatgtaaaa 780gctgacttta gtagcgcact
gattcgcctc accgacgctg gcgcaaaaaa cattctgttg 840ttcaccctac
cagatgcgac caaagcccct cagtttaagt actcaacggc ccaagaaatc
900gagacagttc gtggcaagat tctggcgttc aaccagttca tcaaagaaca
agcagagtac 960tatcaaagca aaggtgacaa cgtgatccta tttgatgcgc
acgctctatt ctctagcatc 1020accagcgacc cacaaaaaca cgggttcaga
aacgcaaaag atgcctgcct agatattaat 1080cgtagtgcat ctcaagacta
cctatacagc catagtctga ccaacgactg tgcaacctat 1140ggttctgata
gctatgtatt ttggggcgta acacacccaa ccacagcaac tcataaatac
1200atcgcaacgc atatactgat gaattcaatg tcgaccttcg acttttaa
124820415PRTUnknownObtained from an environmental sample. 20Met Lys
Lys Thr Thr Leu Val Leu Ala Leu Leu Met Pro Phe Gly Ala1 5
10 15Ala Ser Ala Gln Asp Asn Ser Met Thr Pro Glu Ala Ile Thr Ser
Ala 20 25 30Gln Val Ala Gln Thr Gln Ser Ala Ser Thr Tyr Thr Tyr Val
Arg Cys 35 40 45Trp Tyr Arg Thr Asp Ala Ser His Asp Ser Pro Ala Thr
Asp Trp Glu 50 55 60Trp Ala Arg Lys Glu Asn Gly Asp Tyr Tyr Thr Ile
Asp Gly Tyr Trp65 70 75 80Trp Ser Ser Ile Ser Phe Lys Asn Met Phe
Tyr Ser Glu Thr Pro Gln 85 90 95Gln Glu Ile Lys Gln Arg Cys Val Asp
Thr Leu Asp Val Gln His Asp 100 105 110Lys Ala Asp Ile Thr Tyr Phe
Ala Ala Asp Asn Arg Phe Ser Tyr Asn 115 120 125His Ser Ile Trp Thr
Asn Asp His Gly Phe Gln Ala Asn Gln Ile Asn 130 135 140Arg Ile Val
Ala Phe Gly Asp Ser Leu Ser Asp Thr Gly Asn Leu Phe145 150 155
160Asn Gly Ser Gln Trp Ile Phe Pro Asn Pro Asn Ser Trp Phe Leu Gly
165 170 175His Phe Ser Asn Gly Phe Val Trp Thr Glu Tyr Leu Ala Asn
Ala Lys 180 185 190Gly Val Pro Leu Tyr Asn Trp Ala Val Gly Gly Ala
Ala Gly Thr Asn 195 200 205Gln Tyr Val Ala Leu Thr Gly Val Tyr Asp
Gln Val Thr Ser Tyr Leu 210 215 220Thr Tyr Met Lys Met Ala Lys Asn
Tyr Arg Pro Glu Asn Thr Leu Phe225 230 235 240Thr Leu Glu Phe Gly
Leu Asn Asp Phe Met Asn Tyr Gly Arg Glu Val 245 250 255Ala Asp Val
Lys Ala Asp Phe Ser Ser Ala Leu Ile Arg Leu Thr Asp 260 265 270Ala
Gly Ala Lys Asn Ile Leu Leu Phe Thr Leu Pro Asp Ala Thr Lys 275 280
285Ala Pro Gln Phe Lys Tyr Ser Thr Ala Gln Glu Ile Glu Thr Val Arg
290 295 300Gly Lys Ile Leu Ala Phe Asn Gln Phe Ile Lys Glu Gln Ala
Glu Tyr305 310 315 320Tyr Gln Ser Lys Gly Asp Asn Val Ile Leu Phe
Asp Ala His Ala Leu 325 330 335Phe Ser Ser Ile Thr Ser Asp Pro Gln
Lys His Gly Phe Arg Asn Ala 340 345 350Lys Asp Ala Cys Leu Asp Ile
Asn Arg Ser Ala Ser Gln Asp Tyr Leu 355 360 365Tyr Ser His Ser Leu
Thr Asn Asp Cys Ala Thr Tyr Gly Ser Asp Ser 370 375 380Tyr Val Phe
Trp Gly Val Thr His Pro Thr Thr Ala Thr His Lys Tyr385 390 395
400Ile Ala Thr His Ile Leu Met Asn Ser Met Ser Thr Phe Asp Phe 405
410 415211716DNAUnknownObtained from an environmental sample.
21atgcagcagc ataaattgag gaatttcaac aagggattga ccggcgtcgt attgagcgta
60ttgacctcta ccagcgccat ggcttttaca caaatcggtg gcggcggcgc gattccgatg
120ggccatgaat ggctcacgcg cagatccgca ctggaattat taaatgcaga
ccatatcgtc 180tccaacgacc cgctcgaccc acgcttgggc tggagccagg
gcttggccaa aaatttggat 240ctctccaatg cattgaacga agtgcagcgc
atccagagcg ttaccaagac caacgcactt 300tatgaaccac gctatgatga
cgtgttttct gcgattgtcg gcgaacgctg ggtggacacg 360gccggtttca
acgttgcgaa ggctaccgtc ggtaaaatcg attgtttcag cgcggtcgcg
420caagaacctg ccgatgttca gcaagaccat ttcatgcgtc gttacgatga
cgtgggcgga 480caaggtggcg ttaacgccgc acgccgcggg caacaacgtt
tcatcaccca tttcatcaac 540gccgcgatgg ccgaagaaaa aagcataaaa
gcgtgggacg gcggtggata ctccacgctg 600gaaaaagtca gccacaatta
tttcttgttt ggtcgcgctg tgcatttgtt ccaggattct 660ttcagcccgg
aacacaccgt gcgtctgccg caagacaact acgaaaaagt acgtcaggta
720aaagcctatc tgtgttccga aggcgcagag caacatacgc ataacgcgca
ggatgcgatc 780agcttcacca gcggcgacgt tatctggaag aaaaacaccc
gtctggatgc cggctggagc 840acctacaaac ccagcaatat gaaacccgtt
gccttggtgg cgatggaagc ctcgaaggac 900ttgtgggccg ccttcattcg
caccatggcc gcaccgcgca gcgagcgtcg cgccattgct 960cagcaagagg
cacaaacgct ggtaaacaac tggttgtcgt tcgacgaaca ggaaatgctg
1020agctggtacg acgaagaaac tcatcgcgat cacacttacg tgctcgaacc
cggccagaac 1080ggccccggta tttccatgtt cgattgcatg gtgggtctgg
gcgtgacgtc tggcagccag 1140gctgcgcgtg tggccgaact ggatcaacaa
cgtcgccagt gcttgttcaa cgtcaaggcc 1200accaccggtt acagcgatct
gaacgatccg cacatggata tcccgtataa ctggcaatgg 1260acgtcgacca
cgcagtggaa agtgccaagc gcgagctgga cgattccgca gttgccggcc
1320gacgcaggca agaaagtgac gatcaaaaac gccatcaacg gcaatccgct
ggtagcgccg 1380gctggcgtca aacacaacag cgatatttat tccgcgccgg
gtgaagccat cgaattcatt 1440ttcgtcggtg actacaacaa tgagtcttat
ctgcgctcga aaaaagatgc ggatttgttc 1500ttgagctaca gtgcggtatc
cggcaagggc ttgctgtaca acacaccgaa tcaggcaggt 1560tatcgcgtga
aaccggcggg cgtgctgtgg acgatcgaga acacctactg gaatgatttc
1620ctgtggttca acagttcgaa caaccgcatc tacgtaagcg gcacgggcga
tgccaacaag 1680ttacattcac agtggatcat tgacggtctg aaataa
171622571PRTUnknownObtained from an environmental sample. 22Met Gln
Gln His Lys Leu Arg Asn Phe Asn Lys Gly Leu Thr Gly Val1 5 10 15Val
Leu Ser Val Leu Thr Ser Thr Ser Ala Met Ala Phe Thr Gln Ile 20 25
30Gly Gly Gly Gly Ala Ile Pro Met Gly His Glu Trp Leu Thr Arg Arg
35 40 45Ser Ala Leu Glu Leu Leu Asn Ala Asp His Ile Val Ser Asn Asp
Pro 50 55 60Leu Asp Pro Arg Leu Gly Trp Ser Gln Gly Leu Ala Lys Asn
Leu Asp65 70 75 80Leu Ser Asn Ala Leu Asn Glu Val Gln Arg Ile Gln
Ser Val Thr Lys 85 90 95Thr Asn Ala Leu Tyr Glu Pro Arg Tyr Asp Asp
Val Phe Ser Ala Ile 100 105 110Val Gly Glu Arg Trp Val Asp Thr Ala
Gly Phe Asn Val Ala Lys Ala 115 120 125Thr Val Gly Lys Ile Asp Cys
Phe Ser Ala Val Ala Gln Glu Pro Ala 130 135 140Asp Val Gln Gln Asp
His Phe Met Arg Arg Tyr Asp Asp Val Gly Gly145 150 155 160Gln Gly
Gly Val Asn Ala Ala Arg Arg Gly Gln Gln Arg Phe Ile Thr 165 170
175His Phe Ile Asn Ala Ala Met Ala Glu Glu Lys Ser Ile Lys Ala Trp
180 185 190Asp Gly Gly Gly Tyr Ser Thr Leu Glu Lys Val Ser His Asn
Tyr Phe 195 200 205Leu Phe Gly Arg Ala Val His Leu Phe Gln Asp Ser
Phe Ser Pro Glu 210 215 220His Thr Val Arg Leu Pro Gln Asp Asn Tyr
Glu Lys Val Arg Gln Val225 230 235 240Lys Ala Tyr Leu Cys Ser Glu
Gly Ala Glu Gln His Thr His Asn Ala 245 250 255Gln Asp Ala Ile Ser
Phe Thr Ser Gly Asp Val Ile Trp Lys Lys Asn 260 265 270Thr Arg Leu
Asp Ala Gly Trp Ser Thr Tyr Lys Pro Ser Asn Met Lys 275 280 285Pro
Val Ala Leu Val Ala Met Glu Ala Ser Lys Asp Leu Trp Ala Ala 290 295
300Phe Ile Arg Thr Met Ala Ala Pro Arg Ser Glu Arg Arg Ala Ile
Ala305 310 315 320Gln Gln Glu Ala Gln Thr Leu Val Asn Asn Trp Leu
Ser Phe Asp Glu 325 330 335Gln Glu Met Leu Ser Trp Tyr Asp Glu Glu
Thr His Arg Asp His Thr 340 345 350Tyr Val Leu Glu Pro Gly Gln Asn
Gly Pro Gly Ile Ser Met Phe Asp 355 360 365Cys Met Val Gly Leu Gly
Val Thr Ser Gly Ser Gln Ala Ala Arg Val 370 375 380Ala Glu Leu Asp
Gln Gln Arg Arg Gln Cys Leu Phe Asn Val Lys Ala385 390 395 400Thr
Thr Gly Tyr Ser Asp Leu Asn Asp Pro His Met Asp Ile Pro Tyr 405 410
415Asn Trp Gln Trp Thr Ser Thr Thr Gln Trp Lys Val Pro Ser Ala Ser
420 425 430Trp Thr Ile Pro Gln Leu Pro Ala Asp Ala Gly Lys Lys Val
Thr Ile 435 440 445Lys Asn Ala Ile Asn Gly Asn Pro Leu Val Ala Pro
Ala Gly Val Lys 450 455 460His Asn Ser Asp Ile Tyr Ser Ala Pro Gly
Glu Ala Ile Glu Phe Ile465 470 475 480Phe Val Gly Asp Tyr Asn Asn
Glu Ser Tyr Leu Arg Ser Lys Lys Asp 485 490 495Ala Asp Leu Phe Leu
Ser Tyr Ser Ala Val Ser Gly Lys Gly Leu Leu 500 505 510Tyr Asn Thr
Pro Asn Gln Ala Gly Tyr Arg Val Lys Pro Ala Gly Val 515 520 525Leu
Trp Thr Ile Glu Asn Thr Tyr Trp Asn Asp Phe Leu Trp Phe Asn 530 535
540Ser Ser Asn Asn Arg Ile Tyr Val Ser Gly Thr Gly Asp Ala Asn
Lys545 550 555 560Leu His Ser Gln Trp Ile Ile Asp Gly Leu Lys 565
570231473DNAUnknownObtained from an environmental sample.
23atgacgatcc gctcgaccga ctacgcgctg ctcgcgcagg agagctacca cgacagccag
60gtcgatgctg acgtcaagct cgatggcatc tcctacaagg tattcgccac cacggacgac
120cccctcaccg gcttccaggc caccgcttac cagcgccagg atacgggcga
ggtggtcatc 180gcctaccgcg gcacggaatt cgaccgcgaa cccgtgcgcg
atggcggcgt cgacgcaggc 240atggtgttgc ttggcgtcaa cgcccagtca
cctgcatccg aggtattcac ccgcgaagtg 300atcgaaaagg cgaagcacga
agccgagctc aacgatcgcg agccgaagat caccgtcacc 360gggcattccc
tcggcggcac cctcgccgaa atcaatgccg cgaaatacgg cctccacggc
420gaaaccttca atgcctacgg tgcggccagc ctcaagggca tccccgaggg
cggcgacacg 480gtgatcgacc atgtccgcgc cggcgatctc gtcagcgccg
ccagcccgca ctacgggcag 540gtgcgtgtgt acgcagctca gcaggatatc
gataccctgc aacatgccgg ctaccgcgac 600gacagtggca tcttcagcct
gcgcaacccc atcaaggcca cggatttcga cgcccacgcg 660atcgataact
tcgtgcccaa cagcaagctg cttggccaat cgatcatcgc tcctgagaac
720gaagcccgtt acgaagccca caagggcatg atcgatcgct atcgcgatga
cgtggccgat 780atccggaaag gcatctccgc tccctgggaa atccccaagg
ccgtcggcga gctgaaggac 840aagctcgaac acgaagcctt cgagctggcc
ggcaagggca tcctcgccgt cgagcacggt 900gtagccgagg tcgttcacga
ggcgaaggaa gggttcgatc atctcaagga aggcttgcac 960cacgtcaggg
aagagatcag cgagggcatc cacgccgtgg aagagaaggc ttccagcgca
1020tggcacaccc tcacccaccc gaaggaatgg ttcgagcacg acaaacctca
agtgaatctc 1080gaccatcccc agcatccaga caacgccttg ttcaagcagg
cgcagggcgc ggtacacgcc 1140ctcgatgcca cgcaaggccg cacgccagat
aggacgagcg accagatcgc aggttctctg 1200gtggtcgcgg cgcgacgcga
tggtctcgag cgggtggacc gcgccgtgct cagcgatgac 1260actagccggc
tctacggcgt gcagggtgcg acggattcgc ccttgaagca gttcaccgag
1320gtgaacacga cagtggcggc gcaaacgtca ctgcagcaaa gcagccaggc
atggcagcag 1380caagcagaga tcgcgcgaca gaaccaggca accagccagg
ctcagcgcat ggaaccgcag 1440gtgcccccgc aggcaccggc acatggcatg taa
147324490PRTUnknownObtained from an environmental sample. 24Met Thr
Ile Arg Ser Thr Asp Tyr Ala Leu Leu Ala Gln Glu Ser Tyr1 5 10 15His
Asp Ser Gln Val Asp Ala Asp Val Lys Leu Asp Gly Ile Ser Tyr 20 25
30Lys Val Phe Ala Thr Thr Asp Asp Pro Leu Thr Gly Phe Gln Ala Thr
35 40 45Ala Tyr Gln Arg Gln Asp Thr Gly Glu Val Val Ile Ala Tyr Arg
Gly 50 55 60Thr Glu Phe Asp Arg Glu Pro Val Arg Asp Gly Gly Val Asp
Ala Gly65 70 75 80Met Val Leu Leu Gly Val Asn Ala Gln Ser Pro Ala
Ser Glu Val Phe 85 90 95Thr Arg Glu Val Ile Glu Lys Ala Lys His Glu
Ala Glu Leu Asn Asp 100 105 110Arg Glu Pro Lys Ile Thr Val Thr Gly
His Ser Leu Gly Gly Thr Leu 115 120 125Ala Glu Ile Asn Ala Ala Lys
Tyr Gly Leu His Gly Glu Thr Phe Asn 130 135 140Ala Tyr Gly Ala Ala
Ser Leu Lys Gly Ile Pro Glu Gly Gly Asp Thr145 150 155 160Val Ile
Asp His Val Arg Ala Gly Asp Leu Val Ser Ala Ala Ser Pro 165 170
175His Tyr Gly Gln Val Arg Val Tyr Ala Ala Gln Gln Asp Ile Asp Thr
180 185 190Leu Gln His Ala Gly Tyr Arg Asp Asp Ser Gly Ile Phe Ser
Leu Arg 195 200 205Asn Pro Ile Lys Ala Thr Asp Phe Asp Ala His Ala
Ile Asp Asn Phe 210 215 220Val Pro Asn Ser Lys Leu Leu Gly Gln Ser
Ile Ile Ala Pro Glu Asn225 230 235 240Glu Ala Arg Tyr Glu Ala His
Lys Gly Met Ile Asp Arg Tyr Arg Asp 245 250 255Asp Val Ala Asp Ile
Arg Lys Gly Ile Ser Ala Pro Trp Glu Ile Pro 260 265 270Lys Ala Val
Gly Glu Leu Lys Asp Lys Leu Glu His Glu Ala Phe Glu 275 280 285Leu
Ala Gly Lys Gly Ile Leu Ala Val Glu His Gly Val Ala Glu Val 290 295
300Val His Glu Ala Lys Glu Gly Phe Asp His Leu Lys Glu Gly Leu
His305 310 315 320His Val Arg Glu Glu Ile Ser Glu Gly Ile His Ala
Val Glu Glu Lys 325 330 335Ala Ser Ser Ala Trp His Thr Leu Thr His
Pro Lys Glu Trp Phe Glu 340 345 350His Asp Lys Pro Gln Val Asn Leu
Asp His Pro Gln His Pro Asp Asn 355 360 365Ala Leu Phe Lys Gln Ala
Gln Gly Ala Val His Ala Leu Asp Ala Thr 370 375 380Gln Gly Arg Thr
Pro Asp Arg Thr Ser Asp Gln Ile Ala Gly Ser Leu385 390 395 400Val
Val Ala Ala Arg Arg Asp Gly Leu Glu Arg Val Asp Arg Ala Val 405 410
415Leu Ser Asp Asp Thr Ser Arg Leu Tyr Gly Val Gln Gly Ala Thr Asp
420 425 430Ser Pro Leu Lys Gln Phe Thr Glu Val Asn Thr Thr Val Ala
Ala Gln 435 440 445Thr Ser Leu Gln Gln Ser Ser Gln Ala Trp Gln Gln
Gln Ala Glu Ile 450 455 460Ala Arg Gln Asn Gln Ala Thr Ser Gln Ala
Gln Arg Met Glu Pro Gln465 470 475 480Val Pro Pro Gln Ala Pro Ala
His Gly Met 485 490251098DNAUnknownObtained from an environmental
sample. 25atgtgcgcca aagttaaagt agtcaaaata aagacaaaca caggcagccc
aaacaaatac 60cacttcaaga acctcgtctt cgaaggcggc ggcgtgaaag gcattgccta
tgtgggagcc 120cttaccaagc tcgacgagga aggcatcctt caaaacatta
agcgcgtggc cggcacctca 180gcaggagcaa tggtggccgt cctcgtcgga
ttgggcttca ccgctaagga gataagcgac 240atcctgtggg acatcaaatt
ccagaacttt ttagacaact catggggcgt gatacgcaac 300accaatcgtc
tgctgacgga atacggctgg tataagggcg agtttttccg cgacctcatg
360gctgattaca tcaaaagaaa gacagacgat ggcgagatta ctttcgggga
gttggaggcc 420atgagaaaag agggcaagcc cttcttggaa atccatctgg
ttggctccga cctcacgaca 480gggtattcca gagtgttcaa ctccaaaaac
accccaaatg tgaaagtcgc cgatgccgcc 540cgcatctcca tgtcgatacc
gctgtttttc tccgctgtga gaggcgtgca aggcgacgac 600cacctctatg
tggacggtgg gcttttggac aactacgcca tcaagatttt cgaccagtcg
660aaactcgttt cagacaaaaa caacaaaagg aagaccgagt attacaacag
gctcaaccag 720caagtgaacg cgaaagcaac gaaaagcaag acggaatctg
tagagtatgt ctacaacaag 780gagactttgg gcttccgctt ggatgccaaa
gaggacatca acctcttcct caaccacgat 840gatgcccctc aaaaagaaat
caagagtttc ttctcttaca ccaaagcttt ggtttccacg 900ctcatcgatt
tccagaacaa tgtacacctg cacagcgacg actggcagcg tacggtctac
960atcgacacac tcggtgtcag ctccattgac ttcggtctgt caaacacaac
gaaacaagct 1020cttgtcgatt cgggctacaa ctacaccaca gcctacctcg
actggtacaa caacgacgag 1080gataaagcca acaagtaa
109826365PRTUnknownObtained from an environmental sample. 26Met Cys
Ala Lys Val Lys Val Val Lys Ile Lys Thr Asn Thr Gly Ser1 5 10 15Pro
Asn Lys Tyr His Phe Lys Asn Leu Val Phe Glu Gly Gly Gly Val 20 25
30Lys Gly Ile Ala Tyr Val Gly Ala Leu Thr Lys Leu Asp Glu Glu Gly
35 40 45Ile Leu Gln Asn Ile Lys Arg Val Ala Gly Thr Ser Ala Gly Ala
Met 50 55 60Val Ala Val Leu Val Gly Leu Gly Phe Thr Ala Lys Glu Ile
Ser Asp65 70 75 80Ile Leu Trp Asp Ile Lys Phe Gln Asn Phe Leu Asp
Asn Ser Trp Gly 85 90 95Val Ile Arg Asn Thr Asn Arg Leu Leu Thr Glu
Tyr Gly Trp Tyr Lys 100 105 110Gly Glu Phe Phe Arg Asp Leu Met Ala
Asp Tyr Ile Lys Arg Lys Thr 115 120 125Asp Asp Gly Glu Ile Thr Phe
Gly Glu Leu Glu Ala Met Arg Lys Glu 130 135 140Gly Lys Pro Phe Leu
Glu Ile His Leu Val Gly Ser Asp Leu Thr Thr145 150 155 160Gly Tyr
Ser Arg Val Phe Asn Ser Lys Asn Thr Pro Asn Val Lys Val 165 170
175Ala Asp Ala Ala Arg Ile Ser Met Ser Ile Pro Leu Phe Phe Ser Ala
180 185 190Val Arg Gly Val Gln Gly Asp Asp His Leu Tyr Val Asp Gly
Gly Leu 195 200 205Leu Asp Asn Tyr Ala Ile Lys Ile Phe Asp Gln Ser
Lys Leu Val Ser 210 215 220Asp Lys Asn Asn Lys Arg Lys Thr Glu Tyr
Tyr Asn Arg Leu Asn Gln225 230 235
240Gln Val Asn Ala Lys Ala Thr Lys Ser Lys Thr Glu Ser Val Glu Tyr
245 250 255Val Tyr Asn Lys Glu Thr Leu Gly Phe Arg Leu Asp Ala Lys
Glu Asp 260 265 270Ile Asn Leu Phe Leu Asn His Asp Asp Ala Pro Gln
Lys Glu Ile Lys 275 280 285Ser Phe Phe Ser Tyr Thr Lys Ala Leu Val
Ser Thr Leu Ile Asp Phe 290 295 300Gln Asn Asn Val His Leu His Ser
Asp Asp Trp Gln Arg Thr Val Tyr305 310 315 320Ile Asp Thr Leu Gly
Val Ser Ser Ile Asp Phe Gly Leu Ser Asn Thr 325 330 335Thr Lys Gln
Ala Leu Val Asp Ser Gly Tyr Asn Tyr Thr Thr Ala Tyr 340 345 350Leu
Asp Trp Tyr Asn Asn Asp Glu Asp Lys Ala Asn Lys 355 360
365271287DNAUnknownObtained from an environmental sample.
27gtgtcgatta ccgtttaccg gaagccctcc ggcgggtttg gagcgatagt tcctcaagcg
60aaaattgaga accttgtttt cgagggcggc ggaccaaagg gcctggtcta tgtcggcgcg
120gtcgaggttc tcggcgaaag gggactgctg gaagggatcg caaatgtcgg
cggcgcttca 180gcaggcgcca tgaccgctct agccgtcggt ctgggactga
gccccaggga aattcgcgcg 240gtcgtcttta accagaacat tgcggacctc
accgatatcg agaagaccgt cgagccgtcc 300tccgggatta caggcatgtt
caagagcgtg ttcaagaagg gttggcaggc ggtgcgcaac 360gtaaccggca
cctctgacga gcgcgggcgc gggctctatc gcggcgagaa gttgcgagcc
420tggatcagag acctgattgc acagcgagtc gaggcggggc gctccgaggt
cctgagccga 480gccgacgccg atggacggaa cttctatgag aaagccgccg
caaagaaggg cgccctgaca 540tttgccgagc ttgatcgggt ggcgcaaatg
gcgccgggcc tgcggcttcg ccgcctggcc 600ttcaccggaa ccaacttcac
gtcgaagaag ctcgaagtgt tcagtctgca cgagaccccg 660gacatgccga
tcgacgtcgc ggtacgcatc tccgcatcgt tgccatggtt tttcaaatcc
720gtgaaatgga acggctccga atacatagat ggcggctgcc tgtcgaactt
cccaatgccg 780atattcgacg tcgatcccta tcgtggcgac gcatcgtcga
aaatccggct cggcatcttc 840ggccagaacc tcgcgacgct cggcttcaag
gtcgacagcg aggaggagat ccgcgacatt 900ctctggcgta gccccgagag
cacgagcgac ggctttttcc aaggcatcct gtcaagcgtg 960aaagcttctg
cagaacactg ggtcgtcggc atcgacgtcg aaggcgccac ccgcgcgtcg
1020aacgtggccg ttcacggcaa gtatgctcag cgaacgatcc agataccgga
cctcggatat 1080agcacgttca agttcgatct ttcggacgct gacaaggagc
gcatggccga ggccggcgca 1140aaggccacgc gggaatggct ggcgctgtac
ttcgacgacg ccggaataga ggtcgaattt 1200tctgatccga acgaattgcg
cggccagttg tccgacgccg cattcgcaga cctcgaggat 1260tcgtttcgag
ccttgatcgc ggcctag 128728428PRTUnknownObtained from an
environmental sample. 28Met Ser Ile Thr Val Tyr Arg Lys Pro Ser Gly
Gly Phe Gly Ala Ile1 5 10 15Val Pro Gln Ala Lys Ile Glu Asn Leu Val
Phe Glu Gly Gly Gly Pro 20 25 30Lys Gly Leu Val Tyr Val Gly Ala Val
Glu Val Leu Gly Glu Arg Gly 35 40 45Leu Leu Glu Gly Ile Ala Asn Val
Gly Gly Ala Ser Ala Gly Ala Met 50 55 60Thr Ala Leu Ala Val Gly Leu
Gly Leu Ser Pro Arg Glu Ile Arg Ala65 70 75 80Val Val Phe Asn Gln
Asn Ile Ala Asp Leu Thr Asp Ile Glu Lys Thr 85 90 95Val Glu Pro Ser
Ser Gly Ile Thr Gly Met Phe Lys Ser Val Phe Lys 100 105 110Lys Gly
Trp Gln Ala Val Arg Asn Val Thr Gly Thr Ser Asp Glu Arg 115 120
125Gly Arg Gly Leu Tyr Arg Gly Glu Lys Leu Arg Ala Trp Ile Arg Asp
130 135 140Leu Ile Ala Gln Arg Val Glu Ala Gly Arg Ser Glu Val Leu
Ser Arg145 150 155 160Ala Asp Ala Asp Gly Arg Asn Phe Tyr Glu Lys
Ala Ala Ala Lys Lys 165 170 175Gly Ala Leu Thr Phe Ala Glu Leu Asp
Arg Val Ala Gln Met Ala Pro 180 185 190Gly Leu Arg Leu Arg Arg Leu
Ala Phe Thr Gly Thr Asn Phe Thr Ser 195 200 205Lys Lys Leu Glu Val
Phe Ser Leu His Glu Thr Pro Asp Met Pro Ile 210 215 220Asp Val Ala
Val Arg Ile Ser Ala Ser Leu Pro Trp Phe Phe Lys Ser225 230 235
240Val Lys Trp Asn Gly Ser Glu Tyr Ile Asp Gly Gly Cys Leu Ser Asn
245 250 255Phe Pro Met Pro Ile Phe Asp Val Asp Pro Tyr Arg Gly Asp
Ala Ser 260 265 270Ser Lys Ile Arg Leu Gly Ile Phe Gly Gln Asn Leu
Ala Thr Leu Gly 275 280 285Phe Lys Val Asp Ser Glu Glu Glu Ile Arg
Asp Ile Leu Trp Arg Ser 290 295 300Pro Glu Ser Thr Ser Asp Gly Phe
Phe Gln Gly Ile Leu Ser Ser Val305 310 315 320Lys Ala Ser Ala Glu
His Trp Val Val Gly Ile Asp Val Glu Gly Ala 325 330 335Thr Arg Ala
Ser Asn Val Ala Val His Gly Lys Tyr Ala Gln Arg Thr 340 345 350Ile
Gln Ile Pro Asp Leu Gly Tyr Ser Thr Phe Lys Phe Asp Leu Ser 355 360
365Asp Ala Asp Lys Glu Arg Met Ala Glu Ala Gly Ala Lys Ala Thr Arg
370 375 380Glu Trp Leu Ala Leu Tyr Phe Asp Asp Ala Gly Ile Glu Val
Glu Phe385 390 395 400Ser Asp Pro Asn Glu Leu Arg Gly Gln Leu Ser
Asp Ala Ala Phe Ala 405 410 415Asp Leu Glu Asp Ser Phe Arg Ala Leu
Ile Ala Ala 420 42529753DNAUnknownObtained from an environmental
sample. 29atgggaaacg gtgcagcagt tggttcgaat gataatggta gagaagaaag
tgtttacgta 60ctttctgtga tcgcctgtaa tgtttattat ttacaaaagt gtgaaggtgg
ggcatcgcgt 120gatagcgtga ttagagaaat caatagccaa actcaacctt
taggatatga gattgtagca 180gattctattc gtgatggtca tattggctct
tttgcctgta agatggctgt ctttagaaat 240aatggaaacg gcaattgtgt
tttagcaatc aaagggactg atatgaataa tatcaatgac 300ttggtgaatg
acctaaccat gatattagga ggtattggtt ctgttgctgc aatccaacca
360acgattaaca tggcacaaga actcatcgac caatatggag tgaatttgat
tacaggtcac 420tcccttggag gctacatgac tgagatcatc gccaccaatc
gtggacttcc aggtattgca 480ttttgcgcac caggttcaaa tggtcccatt
gtaaaattag gtggacaaga gacacctggc 540tttcacaatg tgaactttga
acatgatcca gcaggtaacg ttatgacggg ggtttatact 600catgtccaat
ggagtattta tgtaggatgt gatggtatga ctcatggtat tgaaaatatg
660gtgaattatt ttaaagataa aagagattta accaatcgca atattcaagg
aagaagtgaa 720agtcataata cgggttatta ttacccaaaa taa
75330250PRTUnknownObtained from an environmental sample. 30Met Gly
Asn Gly Ala Ala Val Gly Ser Asn Asp Asn Gly Arg Glu Glu1 5 10 15Ser
Val Tyr Val Leu Ser Val Ile Ala Cys Asn Val Tyr Tyr Leu Gln 20 25
30Lys Cys Glu Gly Gly Ala Ser Arg Asp Ser Val Ile Arg Glu Ile Asn
35 40 45Ser Gln Thr Gln Pro Leu Gly Tyr Glu Ile Val Ala Asp Ser Ile
Arg 50 55 60Asp Gly His Ile Gly Ser Phe Ala Cys Lys Met Ala Val Phe
Arg Asn65 70 75 80Asn Gly Asn Gly Asn Cys Val Leu Ala Ile Lys Gly
Thr Asp Met Asn 85 90 95Asn Ile Asn Asp Leu Val Asn Asp Leu Thr Met
Ile Leu Gly Gly Ile 100 105 110Gly Ser Val Ala Ala Ile Gln Pro Thr
Ile Asn Met Ala Gln Glu Leu 115 120 125Ile Asp Gln Tyr Gly Val Asn
Leu Ile Thr Gly His Ser Leu Gly Gly 130 135 140Tyr Met Thr Glu Ile
Ile Ala Thr Asn Arg Gly Leu Pro Gly Ile Ala145 150 155 160Phe Cys
Ala Pro Gly Ser Asn Gly Pro Ile Val Lys Leu Gly Gly Gln 165 170
175Glu Thr Pro Gly Phe His Asn Val Asn Phe Glu His Asp Pro Ala Gly
180 185 190Asn Val Met Thr Gly Val Tyr Thr His Val Gln Trp Ser Ile
Tyr Val 195 200 205Gly Cys Asp Gly Met Thr His Gly Ile Glu Asn Met
Val Asn Tyr Phe 210 215 220Lys Asp Lys Arg Asp Leu Thr Asn Arg Asn
Ile Gln Gly Arg Ser Glu225 230 235 240Ser His Asn Thr Gly Tyr Tyr
Tyr Pro Lys 245 250311422DNAUnknownObtained from an environmental
sample. 31atgaaaaaga aattatgtac atgggctctc gtaacagcga tatcttctgg
agttgttgcg 60attccaaccg tagcatctgc ttgcggaatg ggtgaagtaa tgaaacagga
ggatcaagag 120cacaaacgtg tgaagagatg gtctgcggag catccgcacc
atgctaatga aagcacgcac 180ttatggattg ctcgaaatgc gattcaaatt
atgagtcgta atcaagataa gacggttcaa 240gaaaatgaat tacaattctt
aaaaatacct gaatataagg agttatttga aagagggctt 300tatgatgccg
attatcttga tgagtttaac gatggaggta caggtacaat cggtattgat
360gggctaatta aaggaggctg gaaatctcat ttctatgatc ctgatacgaa
aaagaactat 420aaaggagaag aagaaccaac agccctttcg caaggggata
aatattttaa attagcagga 480gattatttta agaaagaaga ttggaaacaa
gctttctatt atttaggtgt tgcgacgcat 540tacttcacag atgctactca
gccaatgcat gctgctaatt ttacagctgt cgacatgagt 600gcaataaagt
ttcatagcgc ttttgaaaat tatgtaacga cagttcagac accgtttgaa
660gtgaaggatg ataagggaac atataatttg gtcaattctg atgatccgaa
gcagtggata 720catgaaacag cgaaactcgc aaaagcagaa attatgaata
ttactagtga taatattaaa 780tctcaatata ataaaggaaa caaagatctt
tggcaacaag aagttatgcc agctgtccag 840aggagtttag agaaagcgca
aagaaacacg gcgggattta ttcatttatg gtttaaaaca 900tatgttggca
aaactgcagc tgaagatatt gaaactacac aggtaaaaga ttctaatgga
960gaagcaatac aagaacaaaa aaaatactac gttgtgccta gtgagttttt
aaatagaggt 1020ttgacctttg aggtatatgc ttcgaatgac tacgcactat
tatctaatca cgtagatgat 1080aataaagttc atggtacacc tgttcagttt
gtttttgata aagagaataa cggaattgtt 1140catcggggag aaagtgtact
gctgaaaatg acgcaatcta actatgatga ttatgtattt 1200cttaattact
ctaatatgac aaattggtta catcttgcga aacgaaaaac aaatactgca
1260cagtttaaag tgtatccaaa tccggataac tcatctgaat atttcctata
tacagatgga 1320tacccggtaa attatcaaga aaatggtaat gggaagagct
ggattgagtt aggaaagaaa 1380acggataaac cgaaagcgtg gaaatttcaa
caggcagaat aa 142232473PRTUnknownObtained from an environmental
sample. 32Met Lys Lys Lys Leu Cys Thr Trp Ala Leu Val Thr Ala Ile
Ser Ser1 5 10 15Gly Val Val Ala Ile Pro Thr Val Ala Ser Ala Cys Gly
Met Gly Glu 20 25 30Val Met Lys Gln Glu Asp Gln Glu His Lys Arg Val
Lys Arg Trp Ser 35 40 45Ala Glu His Pro His His Ala Asn Glu Ser Thr
His Leu Trp Ile Ala 50 55 60Arg Asn Ala Ile Gln Ile Met Ser Arg Asn
Gln Asp Lys Thr Val Gln65 70 75 80Glu Asn Glu Leu Gln Phe Leu Lys
Ile Pro Glu Tyr Lys Glu Leu Phe 85 90 95Glu Arg Gly Leu Tyr Asp Ala
Asp Tyr Leu Asp Glu Phe Asn Asp Gly 100 105 110Gly Thr Gly Thr Ile
Gly Ile Asp Gly Leu Ile Lys Gly Gly Trp Lys 115 120 125Ser His Phe
Tyr Asp Pro Asp Thr Lys Lys Asn Tyr Lys Gly Glu Glu 130 135 140Glu
Pro Thr Ala Leu Ser Gln Gly Asp Lys Tyr Phe Lys Leu Ala Gly145 150
155 160Asp Tyr Phe Lys Lys Glu Asp Trp Lys Gln Ala Phe Tyr Tyr Leu
Gly 165 170 175Val Ala Thr His Tyr Phe Thr Asp Ala Thr Gln Pro Met
His Ala Ala 180 185 190Asn Phe Thr Ala Val Asp Met Ser Ala Ile Lys
Phe His Ser Ala Phe 195 200 205Glu Asn Tyr Val Thr Thr Val Gln Thr
Pro Phe Glu Val Lys Asp Asp 210 215 220Lys Gly Thr Tyr Asn Leu Val
Asn Ser Asp Asp Pro Lys Gln Trp Ile225 230 235 240His Glu Thr Ala
Lys Leu Ala Lys Ala Glu Ile Met Asn Ile Thr Ser 245 250 255Asp Asn
Ile Lys Ser Gln Tyr Asn Lys Gly Asn Lys Asp Leu Trp Gln 260 265
270Gln Glu Val Met Pro Ala Val Gln Arg Ser Leu Glu Lys Ala Gln Arg
275 280 285Asn Thr Ala Gly Phe Ile His Leu Trp Phe Lys Thr Tyr Val
Gly Lys 290 295 300Thr Ala Ala Glu Asp Ile Glu Thr Thr Gln Val Lys
Asp Ser Asn Gly305 310 315 320Glu Ala Ile Gln Glu Gln Lys Lys Tyr
Tyr Val Val Pro Ser Glu Phe 325 330 335Leu Asn Arg Gly Leu Thr Phe
Glu Val Tyr Ala Ser Asn Asp Tyr Ala 340 345 350Leu Leu Ser Asn His
Val Asp Asp Asn Lys Val His Gly Thr Pro Val 355 360 365Gln Phe Val
Phe Asp Lys Glu Asn Asn Gly Ile Val His Arg Gly Glu 370 375 380Ser
Val Leu Leu Lys Met Thr Gln Ser Asn Tyr Asp Asp Tyr Val Phe385 390
395 400Leu Asn Tyr Ser Asn Met Thr Asn Trp Leu His Leu Ala Lys Arg
Lys 405 410 415Thr Asn Thr Ala Gln Phe Lys Val Tyr Pro Asn Pro Asp
Asn Ser Ser 420 425 430Glu Tyr Phe Leu Tyr Thr Asp Gly Tyr Pro Val
Asn Tyr Gln Glu Asn 435 440 445Gly Asn Gly Lys Ser Trp Ile Glu Leu
Gly Lys Lys Thr Asp Lys Pro 450 455 460Lys Ala Trp Lys Phe Gln Gln
Ala Glu465 47033792DNAUnknownObtained from an environmental sample.
33atgagagcac tcgtgctggc aggcggtgga gccaagggct cgtttcaagt gggcgtgctg
60cagcggttca cccccgcaga cttcggtctc gtggtgggat gctcggtcgg agctttaaac
120gccgcggggt ttgcccacct gggtagccat ggcatcaaag acctctggca
agggatcagg 180agtcgagatg acatcctgtc ccgtgtctgg tggccgtttg
gctcagacgg gatcttctcg 240cagaagcctc ttgaaaagct cgtctccaaa
gcatgcacgg gtcctgctcg ggtgccggtc 300cacgtggcga cggtctgcct
tgaacgcggc cttgtccact acgggatctc cggggactct 360gactttgaga
agaaagtgct ggcatcggct gcgatcccag gcgtggtgaa gccagttaag
420atccatggcg accactacgt cgacggtggt gtcagagaga tctgtccgct
gcgtcgagcc 480atcgacctgg gcgccacgga gatcacagtc atcatgtgcg
ctccggaata catcccgacc 540tggtcgcgta gttcctcgct gttcccgttt
gtgaacgtga tgatccggtc tctcgacatc 600ctgaccgatg agatcctggt
caacgacatc gccgagtgcg tggcaaagaa caagatgcca 660ggtaaacgtc
acgtaaagct caccatctac cggccgaaga aagagctcat gggcacgctc
720gactttgacc ccaaagccat cgccgcaggg atcaaggcag gcaccgaagc
ccagccaagg 780ttctgggagt aa 79234263PRTUnknownObtained from an
environmental sample. 34Met Arg Ala Leu Val Leu Ala Gly Gly Gly Ala
Lys Gly Ser Phe Gln1 5 10 15Val Gly Val Leu Gln Arg Phe Thr Pro Ala
Asp Phe Gly Leu Val Val 20 25 30Gly Cys Ser Val Gly Ala Leu Asn Ala
Ala Gly Phe Ala His Leu Gly 35 40 45Ser His Gly Ile Lys Asp Leu Trp
Gln Gly Ile Arg Ser Arg Asp Asp 50 55 60Ile Leu Ser Arg Val Trp Trp
Pro Phe Gly Ser Asp Gly Ile Phe Ser65 70 75 80Gln Lys Pro Leu Glu
Lys Leu Val Ser Lys Ala Cys Thr Gly Pro Ala 85 90 95Arg Val Pro Val
His Val Ala Thr Val Cys Leu Glu Arg Gly Leu Val 100 105 110His Tyr
Gly Ile Ser Gly Asp Ser Asp Phe Glu Lys Lys Val Leu Ala 115 120
125Ser Ala Ala Ile Pro Gly Val Val Lys Pro Val Lys Ile His Gly Asp
130 135 140His Tyr Val Asp Gly Gly Val Arg Glu Ile Cys Pro Leu Arg
Arg Ala145 150 155 160Ile Asp Leu Gly Ala Thr Glu Ile Thr Val Ile
Met Cys Ala Pro Glu 165 170 175Tyr Ile Pro Thr Trp Ser Arg Ser Ser
Ser Leu Phe Pro Phe Val Asn 180 185 190Val Met Ile Arg Ser Leu Asp
Ile Leu Thr Asp Glu Ile Leu Val Asn 195 200 205Asp Ile Ala Glu Cys
Val Ala Lys Asn Lys Met Pro Gly Lys Arg His 210 215 220Val Lys Leu
Thr Ile Tyr Arg Pro Lys Lys Glu Leu Met Gly Thr Leu225 230 235
240Asp Phe Asp Pro Lys Ala Ile Ala Ala Gly Ile Lys Ala Gly Thr Glu
245 250 255Ala Gln Pro Arg Phe Trp Glu 260351389DNAUnknownObtained
from an environmental sample. 35atgcccgagc cgcccgccgc atgccgttgc
gattgcgcct gcgagcgcga ccagcacctt 60ttttgcaagg gacccaagcg tatcctcgcg
ctcgacggcg gcggcgtgcg cggcgccgtc 120agcgtcgcat tcctcgaacg
gatcgaggcg gtgctcgagg cccggctcgg acgcaaggtg 180ctgctcggcc
actggttcga cctgatcggc ggcacctcga cgggcgccat catcggcggc
240gcgctggcga tgggattcgc ggccgaggac gtccaaagat tctatcacga
gctcgcgccg 300cgggtgttca ggcatccgct cctgcgcatc ggtctcctgc
gcccgttccg cgcgaaattc 360gacgcccgcc tgctgcgcga ggagatccac
cgcatcatcg gcgacagcac gctcggcgac 420aaagcgctga tgaccgggtt
cgcgctcgtc gccaagcgga tggacaccgg cagcacctgg 480atcctcgcca
acaacaagcg cagcaaatac tgggaagggc gggacggcgt cgtcggcaac
540aaggattatc tcctcggcag cctcattcgc gcgagcacgg cggcgccgct
gtatttcgac 600cccgaggagg tcgtgatcgc ggaggcccgc aaggacatcg
agggcatcag gggcctgttc 660gtcgacggcg gcgtcacgcc gcacaacaat
ccttcgctcg cgatgctgct gctggcgctg 720ctcgacgcct accggctgcg
ctgggaaacg ggaccggaca agctcacggt cgtctcgatc 780ggcactggaa
cgcatcgcga ccgcgtcgtt cccgacacgc tcggcatggg caagaacgcg
840aagatcgcgc tgcgcgccat gagctcgctg atgaacgacg
tgcacgagct cgcgctcacg 900cagatgcagt acctcggtga gacgctcacc
ccgtggcgca tcaacgacga gctcggcgac 960atgcggaccg agcggccgcc
gcaaggcaag ctcttccgct tcctccgcta cgacgtccgg 1020ctggagctcg
attggatcaa cgaggacgag gagcgccggc gcaagatcaa gaacaaattc
1080aagcgcgagc tgaccgagac cgacatgatc cgcctgcgca gcctcgacga
tccgacgacc 1140atcccggacc tctacatgct tgcccaggtc gcggccgagg
agcaggtcaa ggcggagcac 1200tggctcggcg acgtgccgga gtggagcgaa
ggcgcgcgcc cgtgtgcgcc gcgccggcac 1260ctgccgccga cgccgccggg
ccgctccgag gattcggcgc gcttccgggc cgagaaggcc 1320gtcggcgagt
ggctcagttt tgcgcgcgcg aacatcacgc gcctcatgtc gcggaagccg
1380ccgggttga 138936462PRTUnknownObtained from an environmental
sample. 36Met Pro Glu Pro Pro Ala Ala Cys Arg Cys Asp Cys Ala Cys
Glu Arg1 5 10 15Asp Gln His Leu Phe Cys Lys Gly Pro Lys Arg Ile Leu
Ala Leu Asp 20 25 30Gly Gly Gly Val Arg Gly Ala Val Ser Val Ala Phe
Leu Glu Arg Ile 35 40 45Glu Ala Val Leu Glu Ala Arg Leu Gly Arg Lys
Val Leu Leu Gly His 50 55 60Trp Phe Asp Leu Ile Gly Gly Thr Ser Thr
Gly Ala Ile Ile Gly Gly65 70 75 80Ala Leu Ala Met Gly Phe Ala Ala
Glu Asp Val Gln Arg Phe Tyr His 85 90 95Glu Leu Ala Pro Arg Val Phe
Arg His Pro Leu Leu Arg Ile Gly Leu 100 105 110Leu Arg Pro Phe Arg
Ala Lys Phe Asp Ala Arg Leu Leu Arg Glu Glu 115 120 125Ile His Arg
Ile Ile Gly Asp Ser Thr Leu Gly Asp Lys Ala Leu Met 130 135 140Thr
Gly Phe Ala Leu Val Ala Lys Arg Met Asp Thr Gly Ser Thr Trp145 150
155 160Ile Leu Ala Asn Asn Lys Arg Ser Lys Tyr Trp Glu Gly Arg Asp
Gly 165 170 175Val Val Gly Asn Lys Asp Tyr Leu Leu Gly Ser Leu Ile
Arg Ala Ser 180 185 190Thr Ala Ala Pro Leu Tyr Phe Asp Pro Glu Glu
Val Val Ile Ala Glu 195 200 205Ala Arg Lys Asp Ile Glu Gly Ile Arg
Gly Leu Phe Val Asp Gly Gly 210 215 220Val Thr Pro His Asn Asn Pro
Ser Leu Ala Met Leu Leu Leu Ala Leu225 230 235 240Leu Asp Ala Tyr
Arg Leu Arg Trp Glu Thr Gly Pro Asp Lys Leu Thr 245 250 255Val Val
Ser Ile Gly Thr Gly Thr His Arg Asp Arg Val Val Pro Asp 260 265
270Thr Leu Gly Met Gly Lys Asn Ala Lys Ile Ala Leu Arg Ala Met Ser
275 280 285Ser Leu Met Asn Asp Val His Glu Leu Ala Leu Thr Gln Met
Gln Tyr 290 295 300Leu Gly Glu Thr Leu Thr Pro Trp Arg Ile Asn Asp
Glu Leu Gly Asp305 310 315 320Met Arg Thr Glu Arg Pro Pro Gln Gly
Lys Leu Phe Arg Phe Leu Arg 325 330 335Tyr Asp Val Arg Leu Glu Leu
Asp Trp Ile Asn Glu Asp Glu Glu Arg 340 345 350Arg Arg Lys Ile Lys
Asn Lys Phe Lys Arg Glu Leu Thr Glu Thr Asp 355 360 365Met Ile Arg
Leu Arg Ser Leu Asp Asp Pro Thr Thr Ile Pro Asp Leu 370 375 380Tyr
Met Leu Ala Gln Val Ala Ala Glu Glu Gln Val Lys Ala Glu His385 390
395 400Trp Leu Gly Asp Val Pro Glu Trp Ser Glu Gly Ala Arg Pro Cys
Ala 405 410 415Pro Arg Arg His Leu Pro Pro Thr Pro Pro Gly Arg Ser
Glu Asp Ser 420 425 430Ala Arg Phe Arg Ala Glu Lys Ala Val Gly Glu
Trp Leu Ser Phe Ala 435 440 445Arg Ala Asn Ile Thr Arg Leu Met Ser
Arg Lys Pro Pro Gly 450 455 460371329DNAUnknownObtained from an
environmental sample. 37atgagaaatt tcagcaaggg attgaccagt attttgctta
gcatagcgac atccaccagt 60gcgatggcct ttacccagat cggggccggc ggagcgattc
cgatgggcca tgagtggcta 120acccgccgct cggcgctgga actgctgaat
gccgacaatc tggtcggcaa tgacccggcc 180gacccacgct tgggctggag
cgaaggtctc gccaacaatc tcgatctctc gaatgcccag 240aacgaagtgc
agcgcatcaa gagcattacc aagagccacg ccctgtatga gccgcgttac
300gatgacgttt tcgccgccat cgtcggcgag cgctgggttg ataccgccgg
tttcaacgtg 360gccaaggcca ccgtcggcaa gatcgattgc ttcagcgccg
tcgcgcaaga gcccgccgat 420gtgcaacaag accatttcat gcgccgttat
gacgacgtgg gtggacaagg gggcgtgaac 480gctgcccgcc gcgcgcagca
gcgctttatc aatcacttcg tcaacgcagc catggccgaa 540gagaagagca
tcaaggcatg ggatggcggc ggttattctt cgctggaaaa agtcagccac
600aactacttct tgtttggccg cgccgttcat ttgttccagg attctttcag
ccccgaacac 660accgtgcgcc tgcctgaaga caattacgtc aaagtccgtc
aggtcaaggc gtatctctgc 720tctgaaggtg ccgaacagca tacgcacaac
acgcaagatg ccatcaactt caccagcggc 780gatgtcatct ggaaacagaa
cacccgtctg gatgcaggct ggagcaccta caaggccagc 840aacatgaagc
cggtggcatt ggttgccctc gaagccagca aagatttgtg ggccgccttt
900attcgcacca tggccgtttc ccgcgaggag cgtcgcgccg tcgccgaaca
ggaagcgcag 960gctctcgtca atcactggtt gtcgttcgac gaacaggaaa
tgctgaactg gtacgaagaa 1020gaagagcacc gcgatcatac gtacgtcaag
gaacccggcc agagcggccc aggttcgtcg 1080ttattcgatt gcatggttgg
tctgggtgtg gcctcgggca gtcaggcgca acgggtggcg 1140gaactcgatc
agcaacgccg ccaatgtttg ttcaacgtca aggccgctac tggctatggc
1200gatctgaatg atccacacat ggatattccg tacaactggc aatgggtgtc
gtcgacgcaa 1260tggaaaatcc ctgcggccga ctggaaaatc ccgcagctgc
ccgccgattc agggaaatca 1320gtcgtcatc 132938443PRTUnknownObtained
from an environmental sample. 38Met Arg Asn Phe Ser Lys Gly Leu Thr
Ser Ile Leu Leu Ser Ile Ala1 5 10 15Thr Ser Thr Ser Ala Met Ala Phe
Thr Gln Ile Gly Ala Gly Gly Ala 20 25 30Ile Pro Met Gly His Glu Trp
Leu Thr Arg Arg Ser Ala Leu Glu Leu 35 40 45Leu Asn Ala Asp Asn Leu
Val Gly Asn Asp Pro Ala Asp Pro Arg Leu 50 55 60Gly Trp Ser Glu Gly
Leu Ala Asn Asn Leu Asp Leu Ser Asn Ala Gln65 70 75 80Asn Glu Val
Gln Arg Ile Lys Ser Ile Thr Lys Ser His Ala Leu Tyr 85 90 95Glu Pro
Arg Tyr Asp Asp Val Phe Ala Ala Ile Val Gly Glu Arg Trp 100 105
110Val Asp Thr Ala Gly Phe Asn Val Ala Lys Ala Thr Val Gly Lys Ile
115 120 125Asp Cys Phe Ser Ala Val Ala Gln Glu Pro Ala Asp Val Gln
Gln Asp 130 135 140His Phe Met Arg Arg Tyr Asp Asp Val Gly Gly Gln
Gly Gly Val Asn145 150 155 160Ala Ala Arg Arg Ala Gln Gln Arg Phe
Ile Asn His Phe Val Asn Ala 165 170 175Ala Met Ala Glu Glu Lys Ser
Ile Lys Ala Trp Asp Gly Gly Gly Tyr 180 185 190Ser Ser Leu Glu Lys
Val Ser His Asn Tyr Phe Leu Phe Gly Arg Ala 195 200 205Val His Leu
Phe Gln Asp Ser Phe Ser Pro Glu His Thr Val Arg Leu 210 215 220Pro
Glu Asp Asn Tyr Val Lys Val Arg Gln Val Lys Ala Tyr Leu Cys225 230
235 240Ser Glu Gly Ala Glu Gln His Thr His Asn Thr Gln Asp Ala Ile
Asn 245 250 255Phe Thr Ser Gly Asp Val Ile Trp Lys Gln Asn Thr Arg
Leu Asp Ala 260 265 270Gly Trp Ser Thr Tyr Lys Ala Ser Asn Met Lys
Pro Val Ala Leu Val 275 280 285Ala Leu Glu Ala Ser Lys Asp Leu Trp
Ala Ala Phe Ile Arg Thr Met 290 295 300Ala Val Ser Arg Glu Glu Arg
Arg Ala Val Ala Glu Gln Glu Ala Gln305 310 315 320Ala Leu Val Asn
His Trp Leu Ser Phe Asp Glu Gln Glu Met Leu Asn 325 330 335Trp Tyr
Glu Glu Glu Glu His Arg Asp His Thr Tyr Val Lys Glu Pro 340 345
350Gly Gln Ser Gly Pro Gly Ser Ser Leu Phe Asp Cys Met Val Gly Leu
355 360 365Gly Val Ala Ser Gly Ser Gln Ala Gln Arg Val Ala Glu Leu
Asp Gln 370 375 380Gln Arg Arg Gln Cys Leu Phe Asn Val Lys Ala Ala
Thr Gly Tyr Gly385 390 395 400Asp Leu Asn Asp Pro His Met Asp Ile
Pro Tyr Asn Trp Gln Trp Val 405 410 415Ser Ser Thr Gln Trp Lys Ile
Pro Ala Ala Asp Trp Lys Ile Pro Gln 420 425 430Leu Pro Ala Asp Ser
Gly Lys Ser Val Val Ile 435 440391335DNAUnknownObtained from an
environmental sample. 39atggccaacc ccatcgtcat catccacggc tggagcgacg
acttcggctc gttccgcaag 60ctgcgcgact tcctctccac caacctcggc gttccggcga
agatcctcaa gctcggcgac 120tggatctcgc tcgacgacga cgtcggctac
gccgacatcg cgatggcgct ggaacgcgcg 180tggaaggcgg agaaactgcc
gaccgcgccg cgttcggtcg acgtcgtcgt gcacagcacc 240ggcgcgctgg
tggtgcgcga atggatgacg cgctaccacg cgcccgaaac cgtgccgatc
300cagcgcttcc tgcacctggc gccggccaac ttcggctcgc acctcgcgca
caagggccgc 360tcgttcatcg gccgcgcggt gaagggctgg aagaccggct
tcgaaaccgg cacccgcatc 420ctgcgcgggc tggaactcgc ctcgccctac
tcgcgcgcgc tggccgagcg cgacctgttc 480gtggcgccgt cgaagcgctg
gtacggcgcc ggccgcatcc tcgccaccgt gctggtcggc 540aacagcggct
actccggcat ccaggccatc gccaacgagg acggctccga cggcaccgtg
600cgcatcggca ccgccaacct gcaggcggcg cttgcgaagg tggtgttccc
gcccggcccg 660gtcgcgccgg tggtgcagtt ccgcaacatc gcgggcgcca
ccgcgttcgc catcgtcgac 720ggcgacaacc attccgacat caccatgaag
gacaagccgt cgaagaccgg catccgcgag 780gaactgatcc tcggcgcgct
gaaggtgcgc gacgccgact tccccgagaa cgccgacggc 840gcgttcccgt
ggcaggcgaa gctcgacgcg aaggccggtg cggccaaggt gtcttcgccc
900gggcgccaga acaccgtggt gcacctcacc gacagcttcg gcgacgacgt
cgtcgatttc 960ttcttcgagt tctggcgcag cgaacgcagc gacaaggtgt
tcgagcagcg cttctacaag 1020gacgtcatcg acgacgtgca cgtgtacgac
ggcaacggcg cgtggcgctc gctcaacctc 1080gacctcgaca agttcgaggc
gctgcgcaag gacccgaagc tcggcttcga gaaactgctg 1140gtcagcgtgt
tcgcctcgcc cgcgaagaag ggcgacgcca aggtcggcta cagcaccgcc
1200accggccgcg acatcggcgc ctggcacgtc gaaggccgtg acttcgccaa
ggccttcacg 1260ccgcaccgca ccctgttcgt cgacatcgag atcccacgca
tcgtcgacga cgcggtgttc 1320cggttccggg aatag
133540444PRTUnknownObtained from an environmental sample. 40Met Ala
Asn Pro Ile Val Ile Ile His Gly Trp Ser Asp Asp Phe Gly1 5 10 15Ser
Phe Arg Lys Leu Arg Asp Phe Leu Ser Thr Asn Leu Gly Val Pro 20 25
30Ala Lys Ile Leu Lys Leu Gly Asp Trp Ile Ser Leu Asp Asp Asp Val
35 40 45Gly Tyr Ala Asp Ile Ala Met Ala Leu Glu Arg Ala Trp Lys Ala
Glu 50 55 60Lys Leu Pro Thr Ala Pro Arg Ser Val Asp Val Val Val His
Ser Thr65 70 75 80Gly Ala Leu Val Val Arg Glu Trp Met Thr Arg Tyr
His Ala Pro Glu 85 90 95Thr Val Pro Ile Gln Arg Phe Leu His Leu Ala
Pro Ala Asn Phe Gly 100 105 110Ser His Leu Ala His Lys Gly Arg Ser
Phe Ile Gly Arg Ala Val Lys 115 120 125Gly Trp Lys Thr Gly Phe Glu
Thr Gly Thr Arg Ile Leu Arg Gly Leu 130 135 140Glu Leu Ala Ser Pro
Tyr Ser Arg Ala Leu Ala Glu Arg Asp Leu Phe145 150 155 160Val Ala
Pro Ser Lys Arg Trp Tyr Gly Ala Gly Arg Ile Leu Ala Thr 165 170
175Val Leu Val Gly Asn Ser Gly Tyr Ser Gly Ile Gln Ala Ile Ala Asn
180 185 190Glu Asp Gly Ser Asp Gly Thr Val Arg Ile Gly Thr Ala Asn
Leu Gln 195 200 205Ala Ala Leu Ala Lys Val Val Phe Pro Pro Gly Pro
Val Ala Pro Val 210 215 220Val Gln Phe Arg Asn Ile Ala Gly Ala Thr
Ala Phe Ala Ile Val Asp225 230 235 240Gly Asp Asn His Ser Asp Ile
Thr Met Lys Asp Lys Pro Ser Lys Thr 245 250 255Gly Ile Arg Glu Glu
Leu Ile Leu Gly Ala Leu Lys Val Arg Asp Ala 260 265 270Asp Phe Pro
Glu Asn Ala Asp Gly Ala Phe Pro Trp Gln Ala Lys Leu 275 280 285Asp
Ala Lys Ala Gly Ala Ala Lys Val Ser Ser Pro Gly Arg Gln Asn 290 295
300Thr Val Val His Leu Thr Asp Ser Phe Gly Asp Asp Val Val Asp
Phe305 310 315 320Phe Phe Glu Phe Trp Arg Ser Glu Arg Ser Asp Lys
Val Phe Glu Gln 325 330 335Arg Phe Tyr Lys Asp Val Ile Asp Asp Val
His Val Tyr Asp Gly Asn 340 345 350Gly Ala Trp Arg Ser Leu Asn Leu
Asp Leu Asp Lys Phe Glu Ala Leu 355 360 365Arg Lys Asp Pro Lys Leu
Gly Phe Glu Lys Leu Leu Val Ser Val Phe 370 375 380Ala Ser Pro Ala
Lys Lys Gly Asp Ala Lys Val Gly Tyr Ser Thr Ala385 390 395 400Thr
Gly Arg Asp Ile Gly Ala Trp His Val Glu Gly Arg Asp Phe Ala 405 410
415Lys Ala Phe Thr Pro His Arg Thr Leu Phe Val Asp Ile Glu Ile Pro
420 425 430Arg Ile Val Asp Asp Ala Val Phe Arg Phe Arg Glu 435
440411419DNAUnknownObtained from an environmental sample.
41atgacgctcc gatcaacgga ctatgcgctg ctggcgcagg agagctacca cgacagccag
60gtggacgccg acgtcaagct ggatggcgtg gcgtataaag tcttcgccac caccagcgac
120gggctcaccg gattccaggc cacggcctac cagcgccagg acaccggcga
ggtagtgatt 180gcgtaccgcg gcacggagtt tgatcgcgag cccgtccgcg
acggcggcgt cgatgcgggc 240atggtgctgc tcggtgtcaa cgcacaggca
ccagcgtcgg aagtgttcac ccggcaagtg 300atcgagaagg cgaaacacga
agccgagctc aacgaccgcg aaccgcagat caccgtcacc 360ggccattccc
tcggcggcac cctcgccgag atcaacgccg cgaagtacgg cctccatggc
420gaaaccttca acgcctacgg cgcagccagc ctcaagggta ttccggaggg
cggcgatacc 480gtcatcgacc acgtccgtgc cggcgatctc gtcagcgcgg
ccagccccca ctacgggcag 540gtacgcgtct acgcggcgca gcaggacatc
gatacgctgc aacacgccgg ttaccgcgat 600gacagcggca tcctcagctt
gcgcaacccg atcaaggcca cggatttcga tgcccatgcc 660atcgataact
tcgtgcccaa cagcaagctg ctcggtcagt cgatcatcgc gccggaaaac
720gtggcgcgtt acgatgccca caaaggcatg gtcgaccgtt accgcgatga
cgtggccgat 780atccgcaagg gcatctcggc gccctgggaa atccccaagg
ccatcggcga gctgaaggac 840accctggagc acgaagcctt cgaactcgcc
ggcaagggca ttctcgcggt ggagcacggc 900ttcgaacatc tcaaggagga
gatcggcgaa ggcatccacg ccgtggagga gaaagcttcc 960agcgcgtggc
ataccctcac ccatcccaag gaatggttcg agcacgataa acccaaggtg
1020accctggacc acccggacca ccccgaccat gccctgttca agcaggcgca
gggcgcggtg 1080cacacagtcg atgcctcgca cggccgcacc cctgacaaga
ccagcgacca gatcgccggc 1140tcgctggtgg tatcggcacg ccgtgacggc
cttgagcggg tagaccgcgc tgtactcagc 1200gatgacgcca accgcctgta
cggtgtgcag ggtgcggtgg actcgccgct gaagcaggtc 1260accgaagtga
acaccgccac cgccgcgcag acatcgctcc agcagagcag cgtggcctgg
1320cagcaacagg cagaaatcgc gcgtcagaac caggcggcaa gccaggctca
gcgcatggac 1380cagcaggtgc cgccgcaggc acccgcgcac ggcatgtaa
141942472PRTUnknownObtained from an environmental sample. 42Met Thr
Leu Arg Ser Thr Asp Tyr Ala Leu Leu Ala Gln Glu Ser Tyr1 5 10 15His
Asp Ser Gln Val Asp Ala Asp Val Lys Leu Asp Gly Val Ala Tyr 20 25
30Lys Val Phe Ala Thr Thr Ser Asp Gly Leu Thr Gly Phe Gln Ala Thr
35 40 45Ala Tyr Gln Arg Gln Asp Thr Gly Glu Val Val Ile Ala Tyr Arg
Gly 50 55 60Thr Glu Phe Asp Arg Glu Pro Val Arg Asp Gly Gly Val Asp
Ala Gly65 70 75 80Met Val Leu Leu Gly Val Asn Ala Gln Ala Pro Ala
Ser Glu Val Phe 85 90 95Thr Arg Gln Val Ile Glu Lys Ala Lys His Glu
Ala Glu Leu Asn Asp 100 105 110Arg Glu Pro Gln Ile Thr Val Thr Gly
His Ser Leu Gly Gly Thr Leu 115 120 125Ala Glu Ile Asn Ala Ala Lys
Tyr Gly Leu His Gly Glu Thr Phe Asn 130 135 140Ala Tyr Gly Ala Ala
Ser Leu Lys Gly Ile Pro Glu Gly Gly Asp Thr145 150 155 160Val Ile
Asp His Val Arg Ala Gly Asp Leu Val Ser Ala Ala Ser Pro 165 170
175His Tyr Gly Gln Val Arg Val Tyr Ala Ala Gln Gln Asp Ile Asp Thr
180 185 190Leu Gln His Ala Gly Tyr Arg Asp Asp Ser Gly Ile Leu Ser
Leu Arg 195 200 205Asn Pro Ile Lys Ala Thr Asp Phe Asp Ala His Ala
Ile Asp Asn Phe 210 215 220Val Pro Asn Ser Lys Leu Leu Gly Gln Ser
Ile Ile Ala Pro Glu Asn225 230 235 240Val Ala Arg Tyr Asp Ala His
Lys Gly Met Val Asp Arg Tyr Arg Asp 245 250 255Asp Val Ala Asp Ile
Arg Lys Gly Ile Ser Ala Pro Trp Glu Ile Pro 260 265 270Lys Ala Ile
Gly Glu Leu Lys Asp Thr Leu Glu His Glu Ala Phe Glu 275 280 285Leu
Ala
Gly Lys Gly Ile Leu Ala Val Glu His Gly Phe Glu His Leu 290 295
300Lys Glu Glu Ile Gly Glu Gly Ile His Ala Val Glu Glu Lys Ala
Ser305 310 315 320Ser Ala Trp His Thr Leu Thr His Pro Lys Glu Trp
Phe Glu His Asp 325 330 335Lys Pro Lys Val Thr Leu Asp His Pro Asp
His Pro Asp His Ala Leu 340 345 350Phe Lys Gln Ala Gln Gly Ala Val
His Thr Val Asp Ala Ser His Gly 355 360 365Arg Thr Pro Asp Lys Thr
Ser Asp Gln Ile Ala Gly Ser Leu Val Val 370 375 380Ser Ala Arg Arg
Asp Gly Leu Glu Arg Val Asp Arg Ala Val Leu Ser385 390 395 400Asp
Asp Ala Asn Arg Leu Tyr Gly Val Gln Gly Ala Val Asp Ser Pro 405 410
415Leu Lys Gln Val Thr Glu Val Asn Thr Ala Thr Ala Ala Gln Thr Ser
420 425 430Leu Gln Gln Ser Ser Val Ala Trp Gln Gln Gln Ala Glu Ile
Ala Arg 435 440 445Gln Asn Gln Ala Ala Ser Gln Ala Gln Arg Met Asp
Gln Gln Val Pro 450 455 460Pro Gln Ala Pro Ala His Gly Met465
470431287DNAUnknownObtained from an environmental sample.
43atgtcgatta ccgtttaccg gaagccctcc ggcgggtttg gagcgatagt tcctcaagcg
60aaaattgaga accttgtttt cgagggcggc ggaccaaagg gcctggtcta tgtcggcgcg
120gtcgaggttc tcggtgaaag gggactgctg gaagggatcg caaatgtcgg
cggcgcttca 180gcaggcgcca tgaccgctct agccgtcggt ctgggactga
gccccaggga aattcgcgcg 240gtcgtcttta accagaacat tgcggacctc
accgatatcg agaagaccgt cgagccgtcc 300tccgggatca caggcatgtt
caagagcgtg ttcaagaagg gttggcaggc ggtgcgcaac 360gtaaccggca
cctctgacga gcgcgggcgc gggctctatc gcggcgagaa gttgcgagcc
420tggatcagag acctgattgc acagcgagtc gaggcagggc gctcagaggt
gctgagccga 480gccgacgccg acgggcggaa cttctatgag aaagccgccg
caaagaaggg cgccctgaca 540tttgccgaac ttgatcgggt ggcgcaaatg
gcgccgggcc tgcggcttcg ccgcctggcc 600ttcaccggaa ccaacttcac
gtcgaagaag ctcgaagtgt tcagtctgca cgagaccccg 660gacatgccga
tcgacgtcgc ggtacgcatc tcggcatcgt tgccatggtt tttcaaatcc
720gtgaaatgga acggctccga atacatagat ggcggatgcc tgtcgaactt
cccaatgccg 780atattcgacg tcgatcccta tcgtggcgac gcatcgtcga
agatccggct cggcatcttc 840ggccagaacc tcgcgacgct cggcttcaag
gtcgacagcg aggaggagat ccgcgacatc 900ctctggcgta gccccgagag
cacgagcgac ggctttttcc aaggcatcct gtcaagcgtg 960aaagcctcgg
cagaacactg ggtcgtcggc atcgatgtcg agggcgccac ccgcgcgtcg
1020aacgtggccg ttcacggcaa gtatgctcag cgaacgatcc agataccgga
cctcggatat 1080agcacgttca agttcgatct ctcagacgcg gacaaggagc
gcatggccga ggccggcgca 1140aaggccacgc gggaatggct ggcgctgtac
ttcgacgacg ccggaataga ggtcgaattt 1200tctgatccga acgaattgcg
cggccagttg tccgacgccg cattcgcaga cctcgaggat 1260tcgtttcgag
ccttgatcgc ggcctag 128744428PRTUnknownObtained from an
environmental sample. 44Met Ser Ile Thr Val Tyr Arg Lys Pro Ser Gly
Gly Phe Gly Ala Ile1 5 10 15Val Pro Gln Ala Lys Ile Glu Asn Leu Val
Phe Glu Gly Gly Gly Pro 20 25 30Lys Gly Leu Val Tyr Val Gly Ala Val
Glu Val Leu Gly Glu Arg Gly 35 40 45Leu Leu Glu Gly Ile Ala Asn Val
Gly Gly Ala Ser Ala Gly Ala Met 50 55 60Thr Ala Leu Ala Val Gly Leu
Gly Leu Ser Pro Arg Glu Ile Arg Ala65 70 75 80Val Val Phe Asn Gln
Asn Ile Ala Asp Leu Thr Asp Ile Glu Lys Thr 85 90 95Val Glu Pro Ser
Ser Gly Ile Thr Gly Met Phe Lys Ser Val Phe Lys 100 105 110Lys Gly
Trp Gln Ala Val Arg Asn Val Thr Gly Thr Ser Asp Glu Arg 115 120
125Gly Arg Gly Leu Tyr Arg Gly Glu Lys Leu Arg Ala Trp Ile Arg Asp
130 135 140Leu Ile Ala Gln Arg Val Glu Ala Gly Arg Ser Glu Val Leu
Ser Arg145 150 155 160Ala Asp Ala Asp Gly Arg Asn Phe Tyr Glu Lys
Ala Ala Ala Lys Lys 165 170 175Gly Ala Leu Thr Phe Ala Glu Leu Asp
Arg Val Ala Gln Met Ala Pro 180 185 190Gly Leu Arg Leu Arg Arg Leu
Ala Phe Thr Gly Thr Asn Phe Thr Ser 195 200 205Lys Lys Leu Glu Val
Phe Ser Leu His Glu Thr Pro Asp Met Pro Ile 210 215 220Asp Val Ala
Val Arg Ile Ser Ala Ser Leu Pro Trp Phe Phe Lys Ser225 230 235
240Val Lys Trp Asn Gly Ser Glu Tyr Ile Asp Gly Gly Cys Leu Ser Asn
245 250 255Phe Pro Met Pro Ile Phe Asp Val Asp Pro Tyr Arg Gly Asp
Ala Ser 260 265 270Ser Lys Ile Arg Leu Gly Ile Phe Gly Gln Asn Leu
Ala Thr Leu Gly 275 280 285Phe Lys Val Asp Ser Glu Glu Glu Ile Arg
Asp Ile Leu Trp Arg Ser 290 295 300Pro Glu Ser Thr Ser Asp Gly Phe
Phe Gln Gly Ile Leu Ser Ser Val305 310 315 320Lys Ala Ser Ala Glu
His Trp Val Val Gly Ile Asp Val Glu Gly Ala 325 330 335Thr Arg Ala
Ser Asn Val Ala Val His Gly Lys Tyr Ala Gln Arg Thr 340 345 350Ile
Gln Ile Pro Asp Leu Gly Tyr Ser Thr Phe Lys Phe Asp Leu Ser 355 360
365Asp Ala Asp Lys Glu Arg Met Ala Glu Ala Gly Ala Lys Ala Thr Arg
370 375 380Glu Trp Leu Ala Leu Tyr Phe Asp Asp Ala Gly Ile Glu Val
Glu Phe385 390 395 400Ser Asp Pro Asn Glu Leu Arg Gly Gln Leu Ser
Asp Ala Ala Phe Ala 405 410 415Asp Leu Glu Asp Ser Phe Arg Ala Leu
Ile Ala Ala 420 425451038DNAUnknownObtained from an environmental
sample. 45atgacaaccc aatttagaaa cttgatattt gaaggcggcg gtgtaaaagg
tgttgcttac 60attggcgcca tgcagattct cgaaaatcgt ggcgtgttgc aagatattca
ccgagtcgga 120gggtgcagtg cgggtgcgat taatgcgctg atttttgcgc
tgggttacac ggttcgtgag 180caaaaagaga tcttacaagc caccgatttt
aaccagttta tggataactc ttggggtgtt 240attcgtgata ttcgcaggct
tgctcgagac tttggctgga ataagggtga tttctttagt 300agctggatag
gtgatttgat tcatcgtcgt ttggggaatc gccgagcgac gttcaaagat
360ctgcaaaatg ccaagcttcc tgatctttat gtcatcggta ctaatctgtc
tacagggttt 420gcagaggttt tttctgccga aagacacccc gatatggagc
tggcgacagc ggtgcgtatc 480tccatgtcga taccgctgtt ctttgcagcc
gtgcgtcacg gtgatcgaca agatgtgtat 540gtcgatgggg gtgttcaact
taactatccg attaaactgt ttgatcggga gcgttacatt 600gatctggcca
aagatcccgg tgctgttcgg cgaacgggtt attacaacaa agaaaacgct
660cgctttcagc ttgagcggcc cggtcatagc ccctatgttt acaatcgcca
gaccttgggt 720ttgcgtcttg atagtcgcga gcagataggg ctctttcgtt
atgacgaacc cctcaagggc 780aaacccatta agtccttcac tgactacgct
cgacaacttt tcggtgcgtt gatgaatgca 840caggaaaaga ttcatctaca
tggcgatgat tggcaacgca cggtctatat cgatacattg 900gatgtgggta
cgacggactt caatctttct gatgcaacta agcaagcact gattgagcaa
960ggaattaacg gcaccgaaaa ttatttcgag tggtttgata atccgttaga
gaagcccgtg 1020aatagagtgg agtcatag 103846345PRTUnknownObtained from
an environmental sample. 46Met Thr Thr Gln Phe Arg Asn Leu Ile Phe
Glu Gly Gly Gly Val Lys1 5 10 15Gly Val Ala Tyr Ile Gly Ala Met Gln
Ile Leu Glu Asn Arg Gly Val 20 25 30Leu Gln Asp Ile His Arg Val Gly
Gly Cys Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ala Leu Gly
Tyr Thr Val Arg Glu Gln Lys Glu Ile 50 55 60Leu Gln Ala Thr Asp Phe
Asn Gln Phe Met Asp Asn Ser Trp Gly Val65 70 75 80Ile Arg Asp Ile
Arg Arg Leu Ala Arg Asp Phe Gly Trp Asn Lys Gly 85 90 95Asp Phe Phe
Ser Ser Trp Ile Gly Asp Leu Ile His Arg Arg Leu Gly 100 105 110Asn
Arg Arg Ala Thr Phe Lys Asp Leu Gln Asn Ala Lys Leu Pro Asp 115 120
125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ala Glu Val Phe
130 135 140Ser Ala Glu Arg His Pro Asp Met Glu Leu Ala Thr Ala Val
Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala Ala Val
Arg His Gly Asp Arg 165 170 175Gln Asp Val Tyr Val Asp Gly Gly Val
Gln Leu Asn Tyr Pro Ile Lys 180 185 190Leu Phe Asp Arg Glu Arg Tyr
Ile Asp Leu Ala Lys Asp Pro Gly Ala 195 200 205Val Arg Arg Thr Gly
Tyr Tyr Asn Lys Glu Asn Ala Arg Phe Gln Leu 210 215 220Glu Arg Pro
Gly His Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230 235
240Leu Arg Leu Asp Ser Arg Glu Gln Ile Gly Leu Phe Arg Tyr Asp Glu
245 250 255Pro Leu Lys Gly Lys Pro Ile Lys Ser Phe Thr Asp Tyr Ala
Arg Gln 260 265 270Leu Phe Gly Ala Leu Met Asn Ala Gln Glu Lys Ile
His Leu His Gly 275 280 285Asp Asp Trp Gln Arg Thr Val Tyr Ile Asp
Thr Leu Asp Val Gly Thr 290 295 300Thr Asp Phe Asn Leu Ser Asp Ala
Thr Lys Gln Ala Leu Ile Glu Gln305 310 315 320Gly Ile Asn Gly Thr
Glu Asn Tyr Phe Glu Trp Phe Asp Asn Pro Leu 325 330 335Glu Lys Pro
Val Asn Arg Val Glu Ser 340 345471476DNAUnknownObtained from an
environmental sample. 47atgtcaacaa aagtagtatt tgtacatgga tggagcgtta
ccaacctaaa tacatatggc 60gaacttccgt tgagattaaa ggccgaagca ataagcagga
acctgaacat cgaagtaaat 120gaaattttcc tgggccgtta tatcagcttt
aatgataaca ttacattaga tgacgtttcg 180cgggctttta atacggccat
tagcgaacag ttagacaata cagacaggtt tatatgtatt 240acacattcta
ccggagggcc ggttattcgc gaatggttaa ataaatacta ttataatgaa
300cgtccaccac taagtcattt aataatgctt gcaccggcca attttggttc
ggcattggct 360cgtttaggga aaagtaaatt aagccgtatt aaaagttggt
ttgaaggtgt agaaccaggg 420cagaaaattt tagactggct ggagtgtgga
agcaaccaat cgtggttact aaataaagac 480tggatcgaca atggcaattt
tcagattggc gctgataagt atttcccgtt tgttatcatt 540ggccagtcga
ttgatcgtaa actttacgat catcttaact catataccgg cgagcttggg
600tccgatggtg tagttcgcac ctcaggagct aatcttaatt cgcggtatat
taagcttgtt 660caggacagaa atacaatagc taatggaaat atttccagta
cattacgaat tgccgaatat 720agagaagctt gtgcaacgcc catacgggta
gttagaggta aatcgcattc gggcgatgaa 780atgggtatca tgaaaagtgt
taaaaaagaa attactgatg ccggaagcaa ggaaacaata 840aatgccatat
tcgagtgtat tgaagttaca aacaacgaac aatatcaatc cttaattact
900aaatttgata acgaaacagc acaggtacaa aaggatgagc tgattgaaac
ggaaacagaa 960ttatttttaa tgcaccgtca tttcattcac gaccgctttt
cgcaattcat ttttaaagta 1020actgactcag aagggcaacc tgttacagat
tatgatttaa tttttacagc cgggccacaa 1080aacgatgcga accacttacc
ggaaggattt gccattgaca ggcaacaaaa ttcaaataat 1140aacgaaacca
ttacgtatta ttttaattac gatgtattga aaggggctcc cgcaaatgtt
1200taccgggacg cattaccagg tatttctatg ctggggctaa ccataaaccc
aaggccggac 1260gaaggttttg taagatatat cccatgcagc attaaagcca
attccgagtt gatggaaaaa 1320gcctttaaac caaattctac taccttggtc
gatattgtta ttcaacgtgt agttagcaaa 1380gaagtttttc ggttggaaaa
gttaactggt agctcaatgc caacagacaa agatgggaat 1440tttaaaaata
ctgaacctgg taacgaaata atatga 147648491PRTUnknownObtained from an
environmental sample. 48Met Ser Thr Lys Val Val Phe Val His Gly Trp
Ser Val Thr Asn Leu1 5 10 15Asn Thr Tyr Gly Glu Leu Pro Leu Arg Leu
Lys Ala Glu Ala Ile Ser 20 25 30Arg Asn Leu Asn Ile Glu Val Asn Glu
Ile Phe Leu Gly Arg Tyr Ile 35 40 45Ser Phe Asn Asp Asn Ile Thr Leu
Asp Asp Val Ser Arg Ala Phe Asn 50 55 60Thr Ala Ile Ser Glu Gln Leu
Asp Asn Thr Asp Arg Phe Ile Cys Ile65 70 75 80Thr His Ser Thr Gly
Gly Pro Val Ile Arg Glu Trp Leu Asn Lys Tyr 85 90 95Tyr Tyr Asn Glu
Arg Pro Pro Leu Ser His Leu Ile Met Leu Ala Pro 100 105 110Ala Asn
Phe Gly Ser Ala Leu Ala Arg Leu Gly Lys Ser Lys Leu Ser 115 120
125Arg Ile Lys Ser Trp Phe Glu Gly Val Glu Pro Gly Gln Lys Ile Leu
130 135 140Asp Trp Leu Glu Cys Gly Ser Asn Gln Ser Trp Leu Leu Asn
Lys Asp145 150 155 160Trp Ile Asp Asn Gly Asn Phe Gln Ile Gly Ala
Asp Lys Tyr Phe Pro 165 170 175Phe Val Ile Ile Gly Gln Ser Ile Asp
Arg Lys Leu Tyr Asp His Leu 180 185 190Asn Ser Tyr Thr Gly Glu Leu
Gly Ser Asp Gly Val Val Arg Thr Ser 195 200 205Gly Ala Asn Leu Asn
Ser Arg Tyr Ile Lys Leu Val Gln Asp Arg Asn 210 215 220Thr Ile Ala
Asn Gly Asn Ile Ser Ser Thr Leu Arg Ile Ala Glu Tyr225 230 235
240Arg Glu Ala Cys Ala Thr Pro Ile Arg Val Val Arg Gly Lys Ser His
245 250 255Ser Gly Asp Glu Met Gly Ile Met Lys Ser Val Lys Lys Glu
Ile Thr 260 265 270Asp Ala Gly Ser Lys Glu Thr Ile Asn Ala Ile Phe
Glu Cys Ile Glu 275 280 285Val Thr Asn Asn Glu Gln Tyr Gln Ser Leu
Ile Thr Lys Phe Asp Asn 290 295 300Glu Thr Ala Gln Val Gln Lys Asp
Glu Leu Ile Glu Thr Glu Thr Glu305 310 315 320Leu Phe Leu Met His
Arg His Phe Ile His Asp Arg Phe Ser Gln Phe 325 330 335Ile Phe Lys
Val Thr Asp Ser Glu Gly Gln Pro Val Thr Asp Tyr Asp 340 345 350Leu
Ile Phe Thr Ala Gly Pro Gln Asn Asp Ala Asn His Leu Pro Glu 355 360
365Gly Phe Ala Ile Asp Arg Gln Gln Asn Ser Asn Asn Asn Glu Thr Ile
370 375 380Thr Tyr Tyr Phe Asn Tyr Asp Val Leu Lys Gly Ala Pro Ala
Asn Val385 390 395 400Tyr Arg Asp Ala Leu Pro Gly Ile Ser Met Leu
Gly Leu Thr Ile Asn 405 410 415Pro Arg Pro Asp Glu Gly Phe Val Arg
Tyr Ile Pro Cys Ser Ile Lys 420 425 430Ala Asn Ser Glu Leu Met Glu
Lys Ala Phe Lys Pro Asn Ser Thr Thr 435 440 445Leu Val Asp Ile Val
Ile Gln Arg Val Val Ser Lys Glu Val Phe Arg 450 455 460Leu Glu Lys
Leu Thr Gly Ser Ser Met Pro Thr Asp Lys Asp Gly Asn465 470 475
480Phe Lys Asn Thr Glu Pro Gly Asn Glu Ile Ile 485
490491257DNAUnknownObtained from an environmental sample.
49atgaattttt ggtcctttct tcttagtata accttaccta tgggggtagg cgttgctcat
60gcacagcccg atacggattt tcaatcggct gagccttatg tctcttctgc gccaatgggg
120cgacaaactt atacttacgt gcgttgttgg tatcgcacca gccacagtac
ggatgatcca 180gcgacagatt ggcagtgggc gagaaactcc gatggtagct
attttacttt gcaaggatac 240tggtggagct cggtaagact aaaaaatatg
ttttacactc aaacctcgca aaatgttatt 300cgtcagcgct gcgaacacac
tttaagcatt aatcatgata atgcggatat tactttttat 360gcggcggata
atcgtttctc attaaaccat acgatttggt cgaatgatcc tgtcatgcag
420gctaatcaaa tcaacaagat tgtcgcgttt ggtgacagct tgtccgatac
cggtaatatt 480tttaatgccg cgcagtggcg ttttcctaat cccaatagtt
ggtttttggg gcatttttct 540aacggtttgg tatggactga gtacttagct
aaacagaaaa acttaccgat atataactgg 600gcggttggtg gcgctgctgg
ggcgaatcaa tatgtggcgt taaccggtgt tacaggccaa 660gtgaactctt
atttacagta catgggtaaa gcgcaaaact atcgtccaca gaataccttg
720tacactttgg tcttcggttt gaatgatttt atgaattata accgtgaggt
tgctgaggtg 780gcggctgatt ttgaaacggc attacagcgt ttaacgcaag
ctggcgcgca aaatatttta 840atgatgacgc taccggatgt gactaaagca
ccacagttta cctactcaac tcaagcggaa 900atcgacttga ttcaaggtaa
aatcaatgcg ttgaacatca agttaaaaca gttgactgcg 960caatatattt
tacaaggcta tgccattcat ctatttgata cttatgagtt atttgattca
1020atggtcgctg aaccggaaaa gcatggcttt gctaatgcca gtgaaccttg
tttgaatctc 1080acccgttctt cagcggcgga ttatttgtac cgtcatccca
ttaccaatac ttgtgctcgt 1140tatggtgcag acaaatttgt attttgggat
gtcacccatc caaccacggc aactcatcgc 1200tatatttcac aaacgctgtt
agcgccgggt aatggattac aatattttaa tttttaa
125750418PRTUnknownObtained from an environmental sample. 50Met Asn
Phe Trp Ser Phe Leu Leu Ser Ile Thr Leu Pro Met Gly Val1 5 10 15Gly
Val Ala His Ala Gln Pro Asp Thr Asp Phe Gln Ser Ala Glu Pro 20 25
30Tyr Val Ser Ser Ala Pro Met Gly Arg Gln Thr Tyr Thr Tyr Val Arg
35 40 45Cys Trp Tyr Arg Thr Ser His Ser Thr Asp Asp Pro Ala Thr Asp
Trp 50 55 60Gln Trp Ala Arg Asn Ser Asp Gly Ser Tyr Phe Thr Leu Gln
Gly Tyr65 70 75 80Trp Trp Ser Ser Val Arg Leu Lys Asn Met Phe Tyr
Thr Gln Thr Ser 85 90 95Gln Asn Val Ile Arg Gln Arg Cys Glu His Thr
Leu Ser Ile Asn His 100 105 110Asp Asn Ala Asp Ile Thr Phe Tyr
Ala
Ala Asp Asn Arg Phe Ser Leu 115 120 125Asn His Thr Ile Trp Ser Asn
Asp Pro Val Met Gln Ala Asn Gln Ile 130 135 140Asn Lys Ile Val Ala
Phe Gly Asp Ser Leu Ser Asp Thr Gly Asn Ile145 150 155 160Phe Asn
Ala Ala Gln Trp Arg Phe Pro Asn Pro Asn Ser Trp Phe Leu 165 170
175Gly His Phe Ser Asn Gly Leu Val Trp Thr Glu Tyr Leu Ala Lys Gln
180 185 190Lys Asn Leu Pro Ile Tyr Asn Trp Ala Val Gly Gly Ala Ala
Gly Ala 195 200 205Asn Gln Tyr Val Ala Leu Thr Gly Val Thr Gly Gln
Val Asn Ser Tyr 210 215 220Leu Gln Tyr Met Gly Lys Ala Gln Asn Tyr
Arg Pro Gln Asn Thr Leu225 230 235 240Tyr Thr Leu Val Phe Gly Leu
Asn Asp Phe Met Asn Tyr Asn Arg Glu 245 250 255Val Ala Glu Val Ala
Ala Asp Phe Glu Thr Ala Leu Gln Arg Leu Thr 260 265 270Gln Ala Gly
Ala Gln Asn Ile Leu Met Met Thr Leu Pro Asp Val Thr 275 280 285Lys
Ala Pro Gln Phe Thr Tyr Ser Thr Gln Ala Glu Ile Asp Leu Ile 290 295
300Gln Gly Lys Ile Asn Ala Leu Asn Ile Lys Leu Lys Gln Leu Thr
Ala305 310 315 320Gln Tyr Ile Leu Gln Gly Tyr Ala Ile His Leu Phe
Asp Thr Tyr Glu 325 330 335Leu Phe Asp Ser Met Val Ala Glu Pro Glu
Lys His Gly Phe Ala Asn 340 345 350Ala Ser Glu Pro Cys Leu Asn Leu
Thr Arg Ser Ser Ala Ala Asp Tyr 355 360 365Leu Tyr Arg His Pro Ile
Thr Asn Thr Cys Ala Arg Tyr Gly Ala Asp 370 375 380Lys Phe Val Phe
Trp Asp Val Thr His Pro Thr Thr Ala Thr His Arg385 390 395 400Tyr
Ile Ser Gln Thr Leu Leu Ala Pro Gly Asn Gly Leu Gln Tyr Phe 405 410
415Asn Phe511482DNAUnknownObtained from an environmental sample.
51atgacaatcc gctcaacgga ctatgcgctg ctcgcgcagg agagctacca cgacagccag
60gtcgatgccg acgtcaaact cgatggcatc gcctacaagg tcttcgccac caccgatgac
120ccgctcacgg ggttccaggc caccgcgtac cagcgccagg acaccggcga
agtcgtcatc 180gcctatcgtg gtacggaatt cgaccgcgag cccgttcgcg
acggcggcgt cgatgccggc 240atggtgctgc tgggggtgaa tgcccagtcg
cctgcctccg agctatttac ccgcgaagtg 300atcgagaagg cgacgcacga
agccgaactc aatgaccgcg agccccggat caccgtgact 360ggccactccc
tcggcggcac cctcgccgaa atcaacgcgg ccaagtacgg cctgcacggc
420gaaaccttca acgcatacgg tgcggccagc ctcaagggca tcccggaagg
cggcaatacc 480gtgatcgacc acgtgcgcgc tggcgacctc gtcagcgccg
ccagcccgca ttacgggcag 540gtgcgcgtct acgcggccca gcaggatatc
gacaccttgc agcatgccgg ctaccgcgac 600gacagcggca tccttagcct
gcgcaacccg atcaaggcca cggatttcga cgcgcacgcc 660atcgacaact
tcgtgccgaa cagcaaactg cttggccagt cgatcatcgc gccggaaaac
720gaagcccgtt acgaagccca caagggcatg gtcgaccgct accgcgatga
cgtggctgac 780atccgcatgc tcgtctccgc tcccctgaac atcccgcgca
ccatcggcga tatcaaggat 840gccgtggaac gcgaggcatt tgagctggct
ggcaagggca tcctcgccgt tgaacacggc 900atcgaagagg tcgtgcacga
ggcaaaggaa ggcttcgagc acctcaagga aggctttgag 960cacctgaagg
aagaagtcag cgagggcttc catgccttcg aggaaaaggc ctccagcgcg
1020tggcatacgc tgacccatcc caaggaatgg ttcgagcacg acaagccgca
ggtcgccctg 1080aaccacccac agcacccgga caacgaactg ttcaagaagg
tgctcgaagg cgtgcaccag 1140gttgatgcga agcagggtcg ttcacccgac
cagctcagtg agaacctggc cgcatcgctt 1200accgttgccg cacgcaagga
aggcctggac aaggtcaacc acgtgctgct cgacgacccc 1260ggcattcgca
cctacgccgt gcagggtgag ctcaactcgc cgttgaagca ggtctccagt
1320gtcgataacg cccaggcggt cgccacaccg gtggcccaga gcagcgcgca
atggcagcag 1380gctgccgagg cgcggcaggc acagcacaat gaggcgcttg
cgcagcagca ggcgcaacag 1440cagcagaaca accggcccaa ccatggggtt
gccggcccgt ga 148252493PRTUnknownObtained from an environmental
sample. 52Met Thr Ile Arg Ser Thr Asp Tyr Ala Leu Leu Ala Gln Glu
Ser Tyr1 5 10 15His Asp Ser Gln Val Asp Ala Asp Val Lys Leu Asp Gly
Ile Ala Tyr 20 25 30Lys Val Phe Ala Thr Thr Asp Asp Pro Leu Thr Gly
Phe Gln Ala Thr 35 40 45Ala Tyr Gln Arg Gln Asp Thr Gly Glu Val Val
Ile Ala Tyr Arg Gly 50 55 60Thr Glu Phe Asp Arg Glu Pro Val Arg Asp
Gly Gly Val Asp Ala Gly65 70 75 80Met Val Leu Leu Gly Val Asn Ala
Gln Ser Pro Ala Ser Glu Leu Phe 85 90 95Thr Arg Glu Val Ile Glu Lys
Ala Thr His Glu Ala Glu Leu Asn Asp 100 105 110Arg Glu Pro Arg Ile
Thr Val Thr Gly His Ser Leu Gly Gly Thr Leu 115 120 125Ala Glu Ile
Asn Ala Ala Lys Tyr Gly Leu His Gly Glu Thr Phe Asn 130 135 140Ala
Tyr Gly Ala Ala Ser Leu Lys Gly Ile Pro Glu Gly Gly Asn Thr145 150
155 160Val Ile Asp His Val Arg Ala Gly Asp Leu Val Ser Ala Ala Ser
Pro 165 170 175His Tyr Gly Gln Val Arg Val Tyr Ala Ala Gln Gln Asp
Ile Asp Thr 180 185 190Leu Gln His Ala Gly Tyr Arg Asp Asp Ser Gly
Ile Leu Ser Leu Arg 195 200 205Asn Pro Ile Lys Ala Thr Asp Phe Asp
Ala His Ala Ile Asp Asn Phe 210 215 220Val Pro Asn Ser Lys Leu Leu
Gly Gln Ser Ile Ile Ala Pro Glu Asn225 230 235 240Glu Ala Arg Tyr
Glu Ala His Lys Gly Met Val Asp Arg Tyr Arg Asp 245 250 255Asp Val
Ala Asp Ile Arg Met Leu Val Ser Ala Pro Leu Asn Ile Pro 260 265
270Arg Thr Ile Gly Asp Ile Lys Asp Ala Val Glu Arg Glu Ala Phe Glu
275 280 285Leu Ala Gly Lys Gly Ile Leu Ala Val Glu His Gly Ile Glu
Glu Val 290 295 300Val His Glu Ala Lys Glu Gly Phe Glu His Leu Lys
Glu Gly Phe Glu305 310 315 320His Leu Lys Glu Glu Val Ser Glu Gly
Phe His Ala Phe Glu Glu Lys 325 330 335Ala Ser Ser Ala Trp His Thr
Leu Thr His Pro Lys Glu Trp Phe Glu 340 345 350His Asp Lys Pro Gln
Val Ala Leu Asn His Pro Gln His Pro Asp Asn 355 360 365Glu Leu Phe
Lys Lys Val Leu Glu Gly Val His Gln Val Asp Ala Lys 370 375 380Gln
Gly Arg Ser Pro Asp Gln Leu Ser Glu Asn Leu Ala Ala Ser Leu385 390
395 400Thr Val Ala Ala Arg Lys Glu Gly Leu Asp Lys Val Asn His Val
Leu 405 410 415Leu Asp Asp Pro Gly Ile Arg Thr Tyr Ala Val Gln Gly
Glu Leu Asn 420 425 430Ser Pro Leu Lys Gln Val Ser Ser Val Asp Asn
Ala Gln Ala Val Ala 435 440 445Thr Pro Val Ala Gln Ser Ser Ala Gln
Trp Gln Gln Ala Ala Glu Ala 450 455 460Arg Gln Ala Gln His Asn Glu
Ala Leu Ala Gln Gln Gln Ala Gln Gln465 470 475 480Gln Gln Asn Asn
Arg Pro Asn His Gly Val Ala Gly Pro 485 490531491DNAUnknownObtained
from an environmental sample. 53atgcgtcagg ttacattagt atttgttcat
ggctacagcg ttacaaacat cgacacttat 60ggtgaaatgc cactcaggct ccgcaacgaa
ggagccacac gtgatataga aataaaaatt 120gagaacattt tcctggggcg
ctacatcagc tttaatgatg atgtgagatt aaatgatgtt 180tccagagcat
tggaaacagc cgtacaacaa cagattgcac cgggaaataa aaacaattcc
240cgttacgtat tcatcaccca ctctaccggc ggaccggtag tgagaaactg
gtgggatctg 300tactataaaa acagcacgaa acaatgccct atgagccacc
tcattatgct ggctcctgcc 360aattttggct cggcactggc acaactggga
aaaagcaaac taagccgcat taaatcctgg 420ttcgatggtg tggaacccgg
acagaatgta ttgaattggc tggaactggg aagcgcggaa 480gcatggaagc
taaacaccga ctggattaag agtgatggaa gtcagatctc ggcacagggt
540atttttcctt ttgtgatcat aggtcaggac attgaccgca aattatacga
tcatttaaac 600tcctacaccg gtgagctggg ttccgacggc gtggtgcgtt
cggccgcagc caatttaaat 660gctacttatg taaaactcac acaacctaaa
cccaccttgg taaatggaaa actggtaaca 720ggtaatctgg aaataggaga
agtaaaacaa gcgccttata cacccatgcg catcgtctca 780aaaaaatcgc
attccaacaa ggatatggga attatgagaa gtgtactgaa atcaacaaat
840gatgccaaca gcgccgaaac ggtaaacgcc atttttgact gcattaatgt
gaaaacctta 900accgattacc agagcattgc cacacagttt gattcgcaaa
caaaagacgt gcaggaaaat 960tcaattattg aaagggaaaa aacgcccttt
ggaactaaaa actatattca cgaccgtttc 1020tcccaggtca ttttcagagt
aacagacagt gaaggttacc cggttaccag ttttgatctg 1080atcctcaccg
gcggcgaaaa aaatgatccc aacgccttgc ctcagggctt ttttgtggac
1140agacaatgca acagtgtcaa taaatcgacc attacttatt ttttaaatta
cgatattatg 1200aacggcacac cagctatagc aggtataaga ccggcatcca
aaggcatgga aaaactgggt 1260ctgatcatta acccaaggcc tgaagaaggc
tttgtgcgtt acattccctg caaaataaac 1320acatcgcccg atttgtttga
cgccgctctg aaacccaacg ccacaacgct tattgatatt 1380gtattgcaac
gcgtggtaag taccgaagta ttccgctttg aaggaacaga cggggtaacg
1440ccgcctaaaa aagatttctc gaaagtgaaa cccggaacgg atattatttg a
149154496PRTUnknownObtained from an environmental sample. 54Met Arg
Gln Val Thr Leu Val Phe Val His Gly Tyr Ser Val Thr Asn1 5 10 15Ile
Asp Thr Tyr Gly Glu Met Pro Leu Arg Leu Arg Asn Glu Gly Ala 20 25
30Thr Arg Asp Ile Glu Ile Lys Ile Glu Asn Ile Phe Leu Gly Arg Tyr
35 40 45Ile Ser Phe Asn Asp Asp Val Arg Leu Asn Asp Val Ser Arg Ala
Leu 50 55 60Glu Thr Ala Val Gln Gln Gln Ile Ala Pro Gly Asn Lys Asn
Asn Ser65 70 75 80Arg Tyr Val Phe Ile Thr His Ser Thr Gly Gly Pro
Val Val Arg Asn 85 90 95Trp Trp Asp Leu Tyr Tyr Lys Asn Ser Thr Lys
Gln Cys Pro Met Ser 100 105 110His Leu Ile Met Leu Ala Pro Ala Asn
Phe Gly Ser Ala Leu Ala Gln 115 120 125Leu Gly Lys Ser Lys Leu Ser
Arg Ile Lys Ser Trp Phe Asp Gly Val 130 135 140Glu Pro Gly Gln Asn
Val Leu Asn Trp Leu Glu Leu Gly Ser Ala Glu145 150 155 160Ala Trp
Lys Leu Asn Thr Asp Trp Ile Lys Ser Asp Gly Ser Gln Ile 165 170
175Ser Ala Gln Gly Ile Phe Pro Phe Val Ile Ile Gly Gln Asp Ile Asp
180 185 190Arg Lys Leu Tyr Asp His Leu Asn Ser Tyr Thr Gly Glu Leu
Gly Ser 195 200 205Asp Gly Val Val Arg Ser Ala Ala Ala Asn Leu Asn
Ala Thr Tyr Val 210 215 220Lys Leu Thr Gln Pro Lys Pro Thr Leu Val
Asn Gly Lys Leu Val Thr225 230 235 240Gly Asn Leu Glu Ile Gly Glu
Val Lys Gln Ala Pro Tyr Thr Pro Met 245 250 255Arg Ile Val Ser Lys
Lys Ser His Ser Asn Lys Asp Met Gly Ile Met 260 265 270Arg Ser Val
Leu Lys Ser Thr Asn Asp Ala Asn Ser Ala Glu Thr Val 275 280 285Asn
Ala Ile Phe Asp Cys Ile Asn Val Lys Thr Leu Thr Asp Tyr Gln 290 295
300Ser Ile Ala Thr Gln Phe Asp Ser Gln Thr Lys Asp Val Gln Glu
Asn305 310 315 320Ser Ile Ile Glu Arg Glu Lys Thr Pro Phe Gly Thr
Lys Asn Tyr Ile 325 330 335His Asp Arg Phe Ser Gln Val Ile Phe Arg
Val Thr Asp Ser Glu Gly 340 345 350Tyr Pro Val Thr Ser Phe Asp Leu
Ile Leu Thr Gly Gly Glu Lys Asn 355 360 365Asp Pro Asn Ala Leu Pro
Gln Gly Phe Phe Val Asp Arg Gln Cys Asn 370 375 380Ser Val Asn Lys
Ser Thr Ile Thr Tyr Phe Leu Asn Tyr Asp Ile Met385 390 395 400Asn
Gly Thr Pro Ala Ile Ala Gly Ile Arg Pro Ala Ser Lys Gly Met 405 410
415Glu Lys Leu Gly Leu Ile Ile Asn Pro Arg Pro Glu Glu Gly Phe Val
420 425 430Arg Tyr Ile Pro Cys Lys Ile Asn Thr Ser Pro Asp Leu Phe
Asp Ala 435 440 445Ala Leu Lys Pro Asn Ala Thr Thr Leu Ile Asp Ile
Val Leu Gln Arg 450 455 460Val Val Ser Thr Glu Val Phe Arg Phe Glu
Gly Thr Asp Gly Val Thr465 470 475 480Pro Pro Lys Lys Asp Phe Ser
Lys Val Lys Pro Gly Thr Asp Ile Ile 485 490
495551041DNAUnknownObtained from an environmental sample.
55atggcttcac aattcagaaa tctggttttt gaaggaggcg gtgtgaaggg catcgcctat
60atcggcgcca tgcaggtgct ggagcagcgg ggactgctca aggatattgt ccgggtggga
120ggtaccagtg caggcgccat caacgcgctg atcttttcgc tgggctttac
catcaaagag 180cagcaggata ttctcaactc caccaacttc agggagttta
tggacagctc gttcgggttc 240atccgaaact tccggaggtt atggagcgaa
ttcggttgga accgcggcga tgtattttcg 300gactgggccg gggagctggt
gaaagagaag ctcggcaaaa agaacgccac gttcggcgat 360ctgaaaaagg
cgaaacgtcc cgatctgtac gtgatcggca ccaatctctc tacggggttt
420tccgagacct tttcgcacga acgccacgcc gacatgcctc tggtagatgc
ggtgcggata 480agcatgtcga tcccgctctt ttttgctgca cggaggctgg
gaaaacgtaa ggatgtgtat 540gtggatggcg gggtgatgct caactatccc
gtgaagctgt tcgacaggga gaagtatatc 600gatttggaga aagagaatga
ggcggcccgc tatgtggagt actacaatca agagaatgcc 660cggtttctgc
tcgagcggcc cggccgaagc ccttatgtgt ataaccggca gactctcggt
720ctgcggctcg acacgcagga agagatcggc ctgttccgtt acgatgagcc
gctgaagggc 780aagcagatca accgtttccc cgaatacgcc agagccctga
tcggctcgct gatgcaggta 840caggagaaca tccacctgaa aagtgacgac
tggcagcgaa cgctctacat caacacgctg 900gatgtgggca ccaccgattt
cgacattacc gacgagaaga aaaaagtgct ggtgaatgag 960gggatcaagg
gagcggagac ctatttccgc tggtttgagg atcccgaaga aaaaccggtg
1020aataaggtga atcttgtctg a 104156346PRTUnknownObtained from an
environmental sample. 56Met Ala Ser Gln Phe Arg Asn Leu Val Phe Glu
Gly Gly Gly Val Lys1 5 10 15Gly Ile Ala Tyr Ile Gly Ala Met Gln Val
Leu Glu Gln Arg Gly Leu 20 25 30Leu Lys Asp Ile Val Arg Val Gly Gly
Thr Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ser Leu Gly Phe
Thr Ile Lys Glu Gln Gln Asp Ile 50 55 60Leu Asn Ser Thr Asn Phe Arg
Glu Phe Met Asp Ser Ser Phe Gly Phe65 70 75 80Ile Arg Asn Phe Arg
Arg Leu Trp Ser Glu Phe Gly Trp Asn Arg Gly 85 90 95Asp Val Phe Ser
Asp Trp Ala Gly Glu Leu Val Lys Glu Lys Leu Gly 100 105 110Lys Lys
Asn Ala Thr Phe Gly Asp Leu Lys Lys Ala Lys Arg Pro Asp 115 120
125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ser Glu Thr Phe
130 135 140Ser His Glu Arg His Ala Asp Met Pro Leu Val Asp Ala Val
Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala Ala Arg
Arg Leu Gly Lys Arg 165 170 175Lys Asp Val Tyr Val Asp Gly Gly Val
Met Leu Asn Tyr Pro Val Lys 180 185 190Leu Phe Asp Arg Glu Lys Tyr
Ile Asp Leu Glu Lys Glu Asn Glu Ala 195 200 205Ala Arg Tyr Val Glu
Tyr Tyr Asn Gln Glu Asn Ala Arg Phe Leu Leu 210 215 220Glu Arg Pro
Gly Arg Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230 235
240Leu Arg Leu Asp Thr Gln Glu Glu Ile Gly Leu Phe Arg Tyr Asp Glu
245 250 255Pro Leu Lys Gly Lys Gln Ile Asn Arg Phe Pro Glu Tyr Ala
Arg Ala 260 265 270Leu Ile Gly Ser Leu Met Gln Val Gln Glu Asn Ile
His Leu Lys Ser 275 280 285Asp Asp Trp Gln Arg Thr Leu Tyr Ile Asn
Thr Leu Asp Val Gly Thr 290 295 300Thr Asp Phe Asp Ile Thr Asp Glu
Lys Lys Lys Val Leu Val Asn Glu305 310 315 320Gly Ile Lys Gly Ala
Glu Thr Tyr Phe Arg Trp Phe Glu Asp Pro Glu 325 330 335Glu Lys Pro
Val Asn Lys Val Asn Leu Val 340 345571413DNAUnknownObtained from an
environmental sample. 57atgcaattag tgttcgtaca cgggtggagt gttacccata
ccaataccta tggtgaatta 60cccgaaagtt tggcggcagg cgccgcgaca cacggcctgc
agatcgatat caggcacgtt 120tttctcggca agtacatcag ctttcacgat
gaggtgactc tggatgatat agcacgtgcc 180ttcgacaagg cgctgagaga
catgtcgggt gatggtgaca cggtctcgcc tttctcctgt 240atcacgcatt
cgaccggcgg ccctgtcgtt cggcactgga ttaacaaatt ctacggcgcg
300cgagggctat cgaaactgcc gctggagcat ttggttatgc tggcgcctgc
caaccacggc 360tccagcctgg cggtactcgg caagcaacgt cttggtcgca
tcaagtcctg gttcgatggc 420gtggagcccg gacaaaaagt gctcgactgg
ctatcgctgg gcagcaatgg gcaatgggcg 480ctcaacaggg attttttgag
ctaccgcccg gccaaacatg gcttcttccc ttttgttctg 540acgggccagg
gtatagacac aaaattctac gattttttga acagctacct tgtggagccc
600ggcagtgacg gtgtggttcg cgtggcgggt gccaatatgc attttcgcta
cctctccctg 660gtacaatctg
agaccgtatt acacaccccg ggcaaggtgc tacagctgga atataacgag
720cggcgccccg tgaagtcccc acaagcggta ccgatgggcg tcttctccca
atttagccac 780tctggcgaca agatggggat tatggcagtc aagcgcaaga
aagacgcgca tcaaatgatc 840gtaacggaag tgctgaagtg tctctgcgta
tcggacagcg atgaatatca gcaaagaggc 900cttgaacttg cagaactgac
cgccagcgaa cagcgcaagc ccatcgaaga ccaggacaag 960attatcagcc
gctatagcat gctggtattt agagtgcgcg accaggcggg caatacgatc
1020ggagtgcacg atttcgatat cctcttactg gccggagata cctatagccc
cgacaaactg 1080ccagaggggt tcttcatgga taaacaggcc aatagagatg
ccggctcact gatctactat 1140gtggatgccg acaaaatgtc cgagatgaaa
gatggctgct acggactgcg ggtggtcgtg 1200cggccggaga aagggttttc
ctattacaca acaggtgagt tcaggtcaga gggtatcccc 1260gtggaccgtg
tatttgcagc aaacgaaacc acctatattg atatcaccat gaaccgaagt
1320gtcgatcaaa atgtattccg gttttcgcct gcaacagagc cacctgaaag
cttcaaaaga 1380accacgccct caggtaccga tatcccttca tag
141358470PRTUnknownObtained from an environmental sample. 58Met Gln
Leu Val Phe Val His Gly Trp Ser Val Thr His Thr Asn Thr1 5 10 15Tyr
Gly Glu Leu Pro Glu Ser Leu Ala Ala Gly Ala Ala Thr His Gly 20 25
30Leu Gln Ile Asp Ile Arg His Val Phe Leu Gly Lys Tyr Ile Ser Phe
35 40 45His Asp Glu Val Thr Leu Asp Asp Ile Ala Arg Ala Phe Asp Lys
Ala 50 55 60Leu Arg Asp Met Ser Gly Asp Gly Asp Thr Val Ser Pro Phe
Ser Cys65 70 75 80Ile Thr His Ser Thr Gly Gly Pro Val Val Arg His
Trp Ile Asn Lys 85 90 95Phe Tyr Gly Ala Arg Gly Leu Ser Lys Leu Pro
Leu Glu His Leu Val 100 105 110Met Leu Ala Pro Ala Asn His Gly Ser
Ser Leu Ala Val Leu Gly Lys 115 120 125Gln Arg Leu Gly Arg Ile Lys
Ser Trp Phe Asp Gly Val Glu Pro Gly 130 135 140Gln Lys Val Leu Asp
Trp Leu Ser Leu Gly Ser Asn Gly Gln Trp Ala145 150 155 160Leu Asn
Arg Asp Phe Leu Ser Tyr Arg Pro Ala Lys His Gly Phe Phe 165 170
175Pro Phe Val Leu Thr Gly Gln Gly Ile Asp Thr Lys Phe Tyr Asp Phe
180 185 190Leu Asn Ser Tyr Leu Val Glu Pro Gly Ser Asp Gly Val Val
Arg Val 195 200 205Ala Gly Ala Asn Met His Phe Arg Tyr Leu Ser Leu
Val Gln Ser Glu 210 215 220Thr Val Leu His Thr Pro Gly Lys Val Leu
Gln Leu Glu Tyr Asn Glu225 230 235 240Arg Arg Pro Val Lys Ser Pro
Gln Ala Val Pro Met Gly Val Phe Ser 245 250 255Gln Phe Ser His Ser
Gly Asp Lys Met Gly Ile Met Ala Val Lys Arg 260 265 270Lys Lys Asp
Ala His Gln Met Ile Val Thr Glu Val Leu Lys Cys Leu 275 280 285Cys
Val Ser Asp Ser Asp Glu Tyr Gln Gln Arg Gly Leu Glu Leu Ala 290 295
300Glu Leu Thr Ala Ser Glu Gln Arg Lys Pro Ile Glu Asp Gln Asp
Lys305 310 315 320Ile Ile Ser Arg Tyr Ser Met Leu Val Phe Arg Val
Arg Asp Gln Ala 325 330 335Gly Asn Thr Ile Gly Val His Asp Phe Asp
Ile Leu Leu Leu Ala Gly 340 345 350Asp Thr Tyr Ser Pro Asp Lys Leu
Pro Glu Gly Phe Phe Met Asp Lys 355 360 365Gln Ala Asn Arg Asp Ala
Gly Ser Leu Ile Tyr Tyr Val Asp Ala Asp 370 375 380Lys Met Ser Glu
Met Lys Asp Gly Cys Tyr Gly Leu Arg Val Val Val385 390 395 400Arg
Pro Glu Lys Gly Phe Ser Tyr Tyr Thr Thr Gly Glu Phe Arg Ser 405 410
415Glu Gly Ile Pro Val Asp Arg Val Phe Ala Ala Asn Glu Thr Thr Tyr
420 425 430Ile Asp Ile Thr Met Asn Arg Ser Val Asp Gln Asn Val Phe
Arg Phe 435 440 445Ser Pro Ala Thr Glu Pro Pro Glu Ser Phe Lys Arg
Thr Thr Pro Ser 450 455 460Gly Thr Asp Ile Pro Ser465
470591038DNAUnknownObtained from an environmental sample.
59atgacaacac aatttagaaa cttgatcttt gaaggcggcg gtgtaaaagg cgttgcttac
60attggcgcca tgcagattct tgaaaatcgt ggcgtgttgc aagatattcg ccgagtcgga
120gggtgcagtg cgggtgcgat taacgcgctg atttttgcgc tgggttacac
ggtccgtgag 180caaaaagaga tcttacaagc caccgatttt aaccagttta
tggataactc ttggggggtt 240attcgtgata ttcgcaggct tgctcgagac
tttggctgga ataagggtga tttctttagt 300agctggatag gtgatttgat
tcatcgtcgt ttggggaatc gccgagcgac gttcaaagat 360ctgcaaaagg
ccaagcttcc tgatctttat gtcatcggta ctaatctgtc tacagggttt
420gcagaggtgt tttctgccga aagacacccc gatatggagc tggcgacagc
ggtgcgtatc 480tccatgtcga taccgctgtt ctttgcggca gtgcgtcatg
gtgatcgaca agatgtgtat 540gtcgatgggg gtgttcaact taactatccg
attaaactgt ttgatcggga gcgttatatt 600gatctggcca aagatcccgg
tgccgttcgg cgaacgggtt attacaacaa agaaaacgct 660cgctttcagc
ttgatcggcc gggccatagc ccctatgttt acaatcgcca gaccttgggt
720ttgcgactgg atagtcgcga ggagataggg ctctttcgtt atgacgaacc
cctcaagggc 780aaacccatta agtccttcac tgactacgct cgacaacttt
tcggtgcgct gatgaatgca 840caggaaaaga ttcatctaca tggcgatgat
tggcaacgca cggtctatat cgatacactc 900gatgtgggta cgacggactt
caatctttct gatgcaacca agcaagcact gattgagcaa 960ggaattaacg
gcaccgaaaa ttatttcgac tggtttgata atccgttaga gaagcctgtg
1020aatagagtgg agtcatag 103860345PRTUnknownObtained from an
environmental sample. 60Met Thr Thr Gln Phe Arg Asn Leu Ile Phe Glu
Gly Gly Gly Val Lys1 5 10 15Gly Val Ala Tyr Ile Gly Ala Met Gln Ile
Leu Glu Asn Arg Gly Val 20 25 30Leu Gln Asp Ile Arg Arg Val Gly Gly
Cys Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ala Leu Gly Tyr
Thr Val Arg Glu Gln Lys Glu Ile 50 55 60Leu Gln Ala Thr Asp Phe Asn
Gln Phe Met Asp Asn Ser Trp Gly Val65 70 75 80Ile Arg Asp Ile Arg
Arg Leu Ala Arg Asp Phe Gly Trp Asn Lys Gly 85 90 95Asp Phe Phe Ser
Ser Trp Ile Gly Asp Leu Ile His Arg Arg Leu Gly 100 105 110Asn Arg
Arg Ala Thr Phe Lys Asp Leu Gln Lys Ala Lys Leu Pro Asp 115 120
125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ala Glu Val Phe
130 135 140Ser Ala Glu Arg His Pro Asp Met Glu Leu Ala Thr Ala Val
Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala Ala Val
Arg His Gly Asp Arg 165 170 175Gln Asp Val Tyr Val Asp Gly Gly Val
Gln Leu Asn Tyr Pro Ile Lys 180 185 190Leu Phe Asp Arg Glu Arg Tyr
Ile Asp Leu Ala Lys Asp Pro Gly Ala 195 200 205Val Arg Arg Thr Gly
Tyr Tyr Asn Lys Glu Asn Ala Arg Phe Gln Leu 210 215 220Asp Arg Pro
Gly His Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230 235
240Leu Arg Leu Asp Ser Arg Glu Glu Ile Gly Leu Phe Arg Tyr Asp Glu
245 250 255Pro Leu Lys Gly Lys Pro Ile Lys Ser Phe Thr Asp Tyr Ala
Arg Gln 260 265 270Leu Phe Gly Ala Leu Met Asn Ala Gln Glu Lys Ile
His Leu His Gly 275 280 285Asp Asp Trp Gln Arg Thr Val Tyr Ile Asp
Thr Leu Asp Val Gly Thr 290 295 300Thr Asp Phe Asn Leu Ser Asp Ala
Thr Lys Gln Ala Leu Ile Glu Gln305 310 315 320Gly Ile Asn Gly Thr
Glu Asn Tyr Phe Asp Trp Phe Asp Asn Pro Leu 325 330 335Glu Lys Pro
Val Asn Arg Val Glu Ser 340 345611257DNAUnknownObtained from an
environmental sample. 61atgacattaa aactctccct gctgatcgcg agcctgagcg
ccgtgtctcc agcagtcttg 60gcaaacgacg tcaatccagc gccactcatg gcgccgtccg
aagcggattc cgcgcagacg 120ctgggcagtc tgacgtacac ctatgttcgc
tgctggtatc gtccggctgc gacgcataat 180gatccttaca ccacctggga
gtgggcgaag aacgcggacg gcagtgattt caccattgat 240ggctattggt
ggtcatcggt gagttacaaa aacatgttct ataccgatac tcagcccgat
300accatcatgc agcgctgtgc agagacgttg gggttaaccc acgataccgc
tgacatcacc 360tatgccgcgg ccgatacccg tttctcctac aaccacacca
tctggagcaa cgatgtcgcc 420aacgcgccga gcaaaatcaa taaggtgatc
gcctttggtg acagcctgtc agacacgggc 480aacattttta acgcctcgca
atggcgcttc ccgaacccga actcctggtt tgtcggccac 540ttctcaaacg
ggtttgtctg gaccgagtat ctggcgcaag gtttggggct gcccctctac
600aactgggccg tgggcggcgc ggcggggcgc aatcaatact gggcgctgac
tggcgtgaat 660gaacaggtca gttcgtacct gacctacatg gagatggcgc
cgaattaccg tgcggagaac 720acgctgttta cactcgaatt cggtctgaat
gattttatga actacgaccg ttcactggca 780gacgtcaaag cagattacag
ctcggcgctg attcgtctgg tggaagccgg agcgaaaaat 840atggtgctgt
tgaccctacc ggatgccacg cgcgcgccgc agttccaata ttcaacgcaa
900gaacacatcg acgaggtgcg cgccaaagtg attggcatga acgcgttcat
tcgtgagcag 960gcacgctact tccagatgca gggcatcaac atttcgctgt
ttgacgccta cacgctgttt 1020gatcagatga tcgccgaccc agccgcgcac
ggctttgata atgccagcgc gccatgtctt 1080gatattcagc gcagctctgc
ggcggactat ctctacacgc atgctctggc agccgagtgt 1140gcctcatccg
gttcagaccg ctttgtgttc tgggatgtga ctcacccaac cacggcaacg
1200catcgctaca tcgccgacca cattctggct accggtgttg cgcagttccc gcgttaa
125762418PRTUnknownObtained from an environmental sample. 62Met Thr
Leu Lys Leu Ser Leu Leu Ile Ala Ser Leu Ser Ala Val Ser1 5 10 15Pro
Ala Val Leu Ala Asn Asp Val Asn Pro Ala Pro Leu Met Ala Pro 20 25
30Ser Glu Ala Asp Ser Ala Gln Thr Leu Gly Ser Leu Thr Tyr Thr Tyr
35 40 45Val Arg Cys Trp Tyr Arg Pro Ala Ala Thr His Asn Asp Pro Tyr
Thr 50 55 60Thr Trp Glu Trp Ala Lys Asn Ala Asp Gly Ser Asp Phe Thr
Ile Asp65 70 75 80Gly Tyr Trp Trp Ser Ser Val Ser Tyr Lys Asn Met
Phe Tyr Thr Asp 85 90 95Thr Gln Pro Asp Thr Ile Met Gln Arg Cys Ala
Glu Thr Leu Gly Leu 100 105 110Thr His Asp Thr Ala Asp Ile Thr Tyr
Ala Ala Ala Asp Thr Arg Phe 115 120 125Ser Tyr Asn His Thr Ile Trp
Ser Asn Asp Val Ala Asn Ala Pro Ser 130 135 140Lys Ile Asn Lys Val
Ile Ala Phe Gly Asp Ser Leu Ser Asp Thr Gly145 150 155 160Asn Ile
Phe Asn Ala Ser Gln Trp Arg Phe Pro Asn Pro Asn Ser Trp 165 170
175Phe Val Gly His Phe Ser Asn Gly Phe Val Trp Thr Glu Tyr Leu Ala
180 185 190Gln Gly Leu Gly Leu Pro Leu Tyr Asn Trp Ala Val Gly Gly
Ala Ala 195 200 205Gly Arg Asn Gln Tyr Trp Ala Leu Thr Gly Val Asn
Glu Gln Val Ser 210 215 220Ser Tyr Leu Thr Tyr Met Glu Met Ala Pro
Asn Tyr Arg Ala Glu Asn225 230 235 240Thr Leu Phe Thr Leu Glu Phe
Gly Leu Asn Asp Phe Met Asn Tyr Asp 245 250 255Arg Ser Leu Ala Asp
Val Lys Ala Asp Tyr Ser Ser Ala Leu Ile Arg 260 265 270Leu Val Glu
Ala Gly Ala Lys Asn Met Val Leu Leu Thr Leu Pro Asp 275 280 285Ala
Thr Arg Ala Pro Gln Phe Gln Tyr Ser Thr Gln Glu His Ile Asp 290 295
300Glu Val Arg Ala Lys Val Ile Gly Met Asn Ala Phe Ile Arg Glu
Gln305 310 315 320Ala Arg Tyr Phe Gln Met Gln Gly Ile Asn Ile Ser
Leu Phe Asp Ala 325 330 335Tyr Thr Leu Phe Asp Gln Met Ile Ala Asp
Pro Ala Ala His Gly Phe 340 345 350Asp Asn Ala Ser Ala Pro Cys Leu
Asp Ile Gln Arg Ser Ser Ala Ala 355 360 365Asp Tyr Leu Tyr Thr His
Ala Leu Ala Ala Glu Cys Ala Ser Ser Gly 370 375 380Ser Asp Arg Phe
Val Phe Trp Asp Val Thr His Pro Thr Thr Ala Thr385 390 395 400His
Arg Tyr Ile Ala Asp His Ile Leu Ala Thr Gly Val Ala Gln Phe 405 410
415Pro Arg631242DNAUnknownObtained from an environmental sample.
63atgaaaaata cgttaatttt ggctggctgt atattggcag ctccagccgt cgcagatgac
60ctaacaatca cccctgaaac tataagtgtg cgctacgcgt ctgaggtgca gaacaaacaa
120acatacactt atgttcgctg ctggtatcgt ccagcgcaga accatgacga
cccttccact 180gagtgggaat gggctcgtga cgacaatggc gattacttca
ctatcgatgg gtactggtgg 240tcgtctgtct ccttcaaaaa catgttctat
accaataccc cgcaaacaga aattgaaaac 300cgctgtaaag aaacactagg
ggttaatcat gatagtgccg atcttcttta ctatgcatca 360gacaatcgtt
tctcctacaa ccatagtatt tggacaaacg acaacgcagt aaacaacaaa
420atcaatcgta ttgtcgcatt cggtgatagc ctgtctgaca ccggtaatct
gtacaatgga 480tcccaatggg tattccccaa ccgtaattct tggtttctcg
gtcacttttc aaacggtttg 540gtgtggactg aatacttagc gcaaaacaaa
aacgtaccac tgtacaactg ggcggtcggt 600ggcgccgccg gcaccaacca
atacgtcgca ttgacaggca tttatgacca agtgacgtct 660tatcttacgt
acatgaagat ggcaaagaac tacaacccaa acaacagttt gatgacgctg
720gaatttggcc taaatgattt catgaattac ggccgagaag tggcggacgt
gaaagctgac 780ttaagtagcg cattgattcg cttgaccgaa tcaggcgcaa
gcaacattct actcttcacg 840ttaccggacg caacaaaggc accgcagttt
aaatattcga ctcaggagga aattgagacc 900gttcgagcta agattcttga
gttcaacact tttattgaag aacaagcgtt actctatcaa 960gctaaaggac
tgaatgtggc cctctacgat gctcatagca tctttgatca gctgacatcc
1020aatcctaaac aacacggttt tgagaactca acagatgcct gtctgaacat
caaccgcagt 1080tcctctgtcg actaccttta cagtcatgag ctaactaacg
attgtgcgta tcatagctct 1140gataaatatg tgttctgggg agtcactcac
ccaaccacag caacacataa atacattgcc 1200gaccaaatca ttcagaccaa
gctagaccag ttcaatttct aa 124264413PRTUnknownObtained from an
environmental sample. 64Met Lys Asn Thr Leu Ile Leu Ala Gly Cys Ile
Leu Ala Ala Pro Ala1 5 10 15Val Ala Asp Asp Leu Thr Ile Thr Pro Glu
Thr Ile Ser Val Arg Tyr 20 25 30Ala Ser Glu Val Gln Asn Lys Gln Thr
Tyr Thr Tyr Val Arg Cys Trp 35 40 45Tyr Arg Pro Ala Gln Asn His Asp
Asp Pro Ser Thr Glu Trp Glu Trp 50 55 60Ala Arg Asp Asp Asn Gly Asp
Tyr Phe Thr Ile Asp Gly Tyr Trp Trp65 70 75 80Ser Ser Val Ser Phe
Lys Asn Met Phe Tyr Thr Asn Thr Pro Gln Thr 85 90 95Glu Ile Glu Asn
Arg Cys Lys Glu Thr Leu Gly Val Asn His Asp Ser 100 105 110Ala Asp
Leu Leu Tyr Tyr Ala Ser Asp Asn Arg Phe Ser Tyr Asn His 115 120
125Ser Ile Trp Thr Asn Asp Asn Ala Val Asn Asn Lys Ile Asn Arg Ile
130 135 140Val Ala Phe Gly Asp Ser Leu Ser Asp Thr Gly Asn Leu Tyr
Asn Gly145 150 155 160Ser Gln Trp Val Phe Pro Asn Arg Asn Ser Trp
Phe Leu Gly His Phe 165 170 175Ser Asn Gly Leu Val Trp Thr Glu Tyr
Leu Ala Gln Asn Lys Asn Val 180 185 190Pro Leu Tyr Asn Trp Ala Val
Gly Gly Ala Ala Gly Thr Asn Gln Tyr 195 200 205Val Ala Leu Thr Gly
Ile Tyr Asp Gln Val Thr Ser Tyr Leu Thr Tyr 210 215 220Met Lys Met
Ala Lys Asn Tyr Asn Pro Asn Asn Ser Leu Met Thr Leu225 230 235
240Glu Phe Gly Leu Asn Asp Phe Met Asn Tyr Gly Arg Glu Val Ala Asp
245 250 255Val Lys Ala Asp Leu Ser Ser Ala Leu Ile Arg Leu Thr Glu
Ser Gly 260 265 270Ala Ser Asn Ile Leu Leu Phe Thr Leu Pro Asp Ala
Thr Lys Ala Pro 275 280 285Gln Phe Lys Tyr Ser Thr Gln Glu Glu Ile
Glu Thr Val Arg Ala Lys 290 295 300Ile Leu Glu Phe Asn Thr Phe Ile
Glu Glu Gln Ala Leu Leu Tyr Gln305 310 315 320Ala Lys Gly Leu Asn
Val Ala Leu Tyr Asp Ala His Ser Ile Phe Asp 325 330 335Gln Leu Thr
Ser Asn Pro Lys Gln His Gly Phe Glu Asn Ser Thr Asp 340 345 350Ala
Cys Leu Asn Ile Asn Arg Ser Ser Ser Val Asp Tyr Leu Tyr Ser 355 360
365His Glu Leu Thr Asn Asp Cys Ala Tyr His Ser Ser Asp Lys Tyr Val
370 375 380Phe Trp Gly Val Thr His Pro Thr Thr Ala Thr His Lys Tyr
Ile Ala385 390 395 400Asp Gln Ile Ile Gln Thr Lys Leu Asp Gln Phe
Asn Phe 405 410651164DNAUnknownObtained from an environmental
sample. 65atgaaccctt ttcttgaaga taaaattaaa tcctccggtc ccaagaaaat
cctcgcctgc 60gatggcggag gtattttggg tttgatgagc gttgaaatcc tagcaaaaat
tgaagcggat 120ttacgcacta agttaggtaa agaccagaac ttcgtgctcg
cggattattt cgattttgtc 180tgcggcacca gcaccggcgc gattatcgct
gcctgtattt ctagtggcat gtcgatggct 240aaaatacgcc aattctatct
cgacagtggg aagcaaatgt
tcgataaggc ctccttgctt 300aagcgcttgc aatacagtta tgacgatgag
ccattggcga ggcagttgcg tgcagccttt 360gatgagcaac tgaaggaaac
cgatgccaag ctgggtagtg cgcacctaaa aacgctgttg 420atgatggtga
tgcgtaacca cagcaccgac tcaccttggc cggtttccaa taacccttac
480gcaaaataca ataatatcgc ccgaaaggat tgcaacctca acctgccttt
atggcaattg 540gtccgtgcca gcaccgccgc tccgacgtat ttcccaccgg
aagtcatcac tttcgcagat 600ggcacacccg aagaatacaa cttcatcttc
gtcgacggtg gcgtgaccac ctacaacaac 660ccagcatatc ttgctttcct
aatggccact gccaagcctt atgccctcaa ctggccgaca 720ggcagcaacc
agttattgat cgtttccgta ggcaccggaa gtgccgccaa tgtccgacct
780aatctggacg tggatgatat gaacctgatc cattttgcca aaaacatccc
ttcagccctg 840atgaatgccg catctgccgg ttgggatatg acctgccggg
tattgggtga atgccgccat 900ggtggcatgt tagatcggga gtttggtgac
atggtgatgc ccgcgtcaag agatcttaat 960tttaccggcc ctaagctttt
tacttatatg cgttatgatc ccgatgtttc ctttgagggc 1020ttgaagacta
tcggtatatc agatatcgat ccagccaaaa tgcagcaaat ggattccgtc
1080aataatattc cagatataca acgggtaggt atcgaatatg ccaaacgcca
tgttgataca 1140gctcattttg aggggtttaa ataa
116466387PRTUnknownObtained from an environmental sample. 66Met Asn
Pro Phe Leu Glu Asp Lys Ile Lys Ser Ser Gly Pro Lys Lys1 5 10 15Ile
Leu Ala Cys Asp Gly Gly Gly Ile Leu Gly Leu Met Ser Val Glu 20 25
30Ile Leu Ala Lys Ile Glu Ala Asp Leu Arg Thr Lys Leu Gly Lys Asp
35 40 45Gln Asn Phe Val Leu Ala Asp Tyr Phe Asp Phe Val Cys Gly Thr
Ser 50 55 60Thr Gly Ala Ile Ile Ala Ala Cys Ile Ser Ser Gly Met Ser
Met Ala65 70 75 80Lys Ile Arg Gln Phe Tyr Leu Asp Ser Gly Lys Gln
Met Phe Asp Lys 85 90 95Ala Ser Leu Leu Lys Arg Leu Gln Tyr Ser Tyr
Asp Asp Glu Pro Leu 100 105 110Ala Arg Gln Leu Arg Ala Ala Phe Asp
Glu Gln Leu Lys Glu Thr Asp 115 120 125Ala Lys Leu Gly Ser Ala His
Leu Lys Thr Leu Leu Met Met Val Met 130 135 140Arg Asn His Ser Thr
Asp Ser Pro Trp Pro Val Ser Asn Asn Pro Tyr145 150 155 160Ala Lys
Tyr Asn Asn Ile Ala Arg Lys Asp Cys Asn Leu Asn Leu Pro 165 170
175Leu Trp Gln Leu Val Arg Ala Ser Thr Ala Ala Pro Thr Tyr Phe Pro
180 185 190Pro Glu Val Ile Thr Phe Ala Asp Gly Thr Pro Glu Glu Tyr
Asn Phe 195 200 205Ile Phe Val Asp Gly Gly Val Thr Thr Tyr Asn Asn
Pro Ala Tyr Leu 210 215 220Ala Phe Leu Met Ala Thr Ala Lys Pro Tyr
Ala Leu Asn Trp Pro Thr225 230 235 240Gly Ser Asn Gln Leu Leu Ile
Val Ser Val Gly Thr Gly Ser Ala Ala 245 250 255Asn Val Arg Pro Asn
Leu Asp Val Asp Asp Met Asn Leu Ile His Phe 260 265 270Ala Lys Asn
Ile Pro Ser Ala Leu Met Asn Ala Ala Ser Ala Gly Trp 275 280 285Asp
Met Thr Cys Arg Val Leu Gly Glu Cys Arg His Gly Gly Met Leu 290 295
300Asp Arg Glu Phe Gly Asp Met Val Met Pro Ala Ser Arg Asp Leu
Asn305 310 315 320Phe Thr Gly Pro Lys Leu Phe Thr Tyr Met Arg Tyr
Asp Pro Asp Val 325 330 335Ser Phe Glu Gly Leu Lys Thr Ile Gly Ile
Ser Asp Ile Asp Pro Ala 340 345 350Lys Met Gln Gln Met Asp Ser Val
Asn Asn Ile Pro Asp Ile Gln Arg 355 360 365Val Gly Ile Glu Tyr Ala
Lys Arg His Val Asp Thr Ala His Phe Glu 370 375 380Gly Phe
Lys385671419DNAUnknownObtained from an environmental sample.
67atggtcattg tcttcgtcca cggatggagc gtgcgcaaca ccaacacgta cgggcagctg
60cccttgcgtc tcaagaagag cttcaaagcc gccgggaaac agattcaggt cgagaacatc
120tacctgggcg agtacgtgag ctttgacgac caggtaacag tcgacgacat
cgcccgcgca 180ttcgattgcg cactgcggga aaaactatac gatccggcga
cgaagcagtg gacgaagttc 240gcctgcatca ctcattccac cggcggcccg
gtcgcgcgct tgtggatgga tctctactac 300ggcgccgcca gactggccga
gtgcccgatg tcccacctcg tgatgctcgc cccggccaat 360catggctcgg
cccttgccca gctcggcaag agccgcctca gccgcatcaa gagcttcttc
420gagggtgtcg aaccgggcca gcgcgtcctc gactggctcg aactcggcag
tgagctgagt 480tgggccctca acacgagatg gctcgactac gactgccgcg
ccgccgcctg ctgggtcttc 540accctcaccg gccagcgcat cgaccggagt
ttgtacgacc atctcaacag ctataccggt 600gagcagggat cggatggcgt
cgtgcgcgtc gccgcggcca acatgaacac caagctgctg 660acctttgaac
agaaggggcg caagctcgtg ttcacaggcc agaagaagac cgccgacacc
720ggccttggcg tcgtgccggg ccggtcgcac tccggccgcg acatgggcat
catcgccagc 780gtgcgcggca ccggcgacca tcccaccctg gaatgggtga
ctcgttgcct ggccgtcacc 840gacgtcaaca cgtacgatgc cgtctgtaag
gatctggacg ctctcaccgc ccagacccag 900aaggatgaaa aggtggaaga
ggtcaaaggc ctgctgcgga cggtcagata ccagacggac 960cgctacgtca
tgctcgtctt ccgcctgaag aacgaccgcg gcgactacct ctccgattac
1020gatctcctgc tcaccgccgg acccaactac tcgcccgacg acctgcccga
aggcttcttc 1080gtcgaccgcc aacggaacca gcggaacccg ggcaagctca
cttactacct gaactacgac 1140gccatggcca aattgaaagg taagaccgcc
gagggccgtc tgggcttcaa gatcctggcg 1200cgcccggtga aaggcggcct
cgtctactat gaggttgcgg agttccagtc cgacgtgggc 1260ggcgtcagca
gcatgctgca gcccaacgca acagtgatga tcgacatcac cctcaatcgc
1320aacgtcgacg cgcgcgtctt ccggttcacc gagaatctgc ccacgggtga
ccagggcgag 1380gaaatcagcg gcgtcccgct ggggcagaac gtcccgtag
141968472PRTUnknownObtained from an environmental sample. 68Met Val
Ile Val Phe Val His Gly Trp Ser Val Arg Asn Thr Asn Thr1 5 10 15Tyr
Gly Gln Leu Pro Leu Arg Leu Lys Lys Ser Phe Lys Ala Ala Gly 20 25
30Lys Gln Ile Gln Val Glu Asn Ile Tyr Leu Gly Glu Tyr Val Ser Phe
35 40 45Asp Asp Gln Val Thr Val Asp Asp Ile Ala Arg Ala Phe Asp Cys
Ala 50 55 60Leu Arg Glu Lys Leu Tyr Asp Pro Ala Thr Lys Gln Trp Thr
Lys Phe65 70 75 80Ala Cys Ile Thr His Ser Thr Gly Gly Pro Val Ala
Arg Leu Trp Met 85 90 95Asp Leu Tyr Tyr Gly Ala Ala Arg Leu Ala Glu
Cys Pro Met Ser His 100 105 110Leu Val Met Leu Ala Pro Ala Asn His
Gly Ser Ala Leu Ala Gln Leu 115 120 125Gly Lys Ser Arg Leu Ser Arg
Ile Lys Ser Phe Phe Glu Gly Val Glu 130 135 140Pro Gly Gln Arg Val
Leu Asp Trp Leu Glu Leu Gly Ser Glu Leu Ser145 150 155 160Trp Ala
Leu Asn Thr Arg Trp Leu Asp Tyr Asp Cys Arg Ala Ala Ala 165 170
175Cys Trp Val Phe Thr Leu Thr Gly Gln Arg Ile Asp Arg Ser Leu Tyr
180 185 190Asp His Leu Asn Ser Tyr Thr Gly Glu Gln Gly Ser Asp Gly
Val Val 195 200 205Arg Val Ala Ala Ala Asn Met Asn Thr Lys Leu Leu
Thr Phe Glu Gln 210 215 220Lys Gly Arg Lys Leu Val Phe Thr Gly Gln
Lys Lys Thr Ala Asp Thr225 230 235 240Gly Leu Gly Val Val Pro Gly
Arg Ser His Ser Gly Arg Asp Met Gly 245 250 255Ile Ile Ala Ser Val
Arg Gly Thr Gly Asp His Pro Thr Leu Glu Trp 260 265 270Val Thr Arg
Cys Leu Ala Val Thr Asp Val Asn Thr Tyr Asp Ala Val 275 280 285Cys
Lys Asp Leu Asp Ala Leu Thr Ala Gln Thr Gln Lys Asp Glu Lys 290 295
300Val Glu Glu Val Lys Gly Leu Leu Arg Thr Val Arg Tyr Gln Thr
Asp305 310 315 320Arg Tyr Val Met Leu Val Phe Arg Leu Lys Asn Asp
Arg Gly Asp Tyr 325 330 335Leu Ser Asp Tyr Asp Leu Leu Leu Thr Ala
Gly Pro Asn Tyr Ser Pro 340 345 350Asp Asp Leu Pro Glu Gly Phe Phe
Val Asp Arg Gln Arg Asn Gln Arg 355 360 365Asn Pro Gly Lys Leu Thr
Tyr Tyr Leu Asn Tyr Asp Ala Met Ala Lys 370 375 380Leu Lys Gly Lys
Thr Ala Glu Gly Arg Leu Gly Phe Lys Ile Leu Ala385 390 395 400Arg
Pro Val Lys Gly Gly Leu Val Tyr Tyr Glu Val Ala Glu Phe Gln 405 410
415Ser Asp Val Gly Gly Val Ser Ser Met Leu Gln Pro Asn Ala Thr Val
420 425 430Met Ile Asp Ile Thr Leu Asn Arg Asn Val Asp Ala Arg Val
Phe Arg 435 440 445Phe Thr Glu Asn Leu Pro Thr Gly Asp Gln Gly Glu
Glu Ile Ser Gly 450 455 460Val Pro Leu Gly Gln Asn Val Pro465
470691038DNAUnknownObtained from an environmental sample.
69atgacaacac aatttagaaa cttgatattt gaaggcggcg gtgtaaaagg tgttgcttac
60attggcgcca tgcagattct cgaaaatcgt ggcgtgttgc aagatattcg ccgagtcgga
120gggtgcagtg cgggtgcgat caacgcgctg atttttgcgc tgggttacac
tgtccgtgag 180caaaaagaga tcttacaagc cacggatttt aaccagttta
tggataactc ttggggtgtt 240attcgtgata ttcgcaggct tgctcgagac
tttggctggc acaagggtga cttctttaat 300agctggatag gtgatttgat
tcatcgtcgt ttggggaatc gccgagcgac gttcaaagat 360ctgcaaaagg
ccaagcttcc tgatctttat gtcatcggta ctaatctgtc tacggggtat
420gcagaggttt tttcagccga aagacacccc gatatggagc tagcgacagc
ggtgcgtatc 480tccatgtcga taccgctgtt ctttgcggcc gtgcgccacg
gtgaccgaca agatgtgtat 540gtcgatgggg gtgttcaact taactatccg
attaaacttt ttgatcggga gcgttacatt 600gatctggcca aagatcccgg
tgccgttcgg cgaacgggct attacaacaa agaaaacgct 660cgctttcagc
ttgagcggcc gggctatagc ccctatgttt acaatcgcca gaccttgggt
720ttgcgactag atagtcgaga ggagataggg ctctttcgtt atgacgaacc
cctcaagggc 780aaacccatta agtccttcac tgactacgct cgacaacttt
tcggtgcgtt gatgaatgca 840caggaaaaga ttcatctaca tggcgatgat
tggcagcgca cggtctatat cgatacattg 900gatgtgggta cgacggactt
caatctttct gatgcaacta agcaagcact gattgaacag 960ggaattaacg
gcaccgaaaa ttatttcgag tggtttgata atccgttgga gaagcctgtt
1020aatagagtgg agtcatag 103870345PRTUnknownObtained from an
environmental sample. 70Met Thr Thr Gln Phe Arg Asn Leu Ile Phe Glu
Gly Gly Gly Val Lys1 5 10 15Gly Val Ala Tyr Ile Gly Ala Met Gln Ile
Leu Glu Asn Arg Gly Val 20 25 30Leu Gln Asp Ile Arg Arg Val Gly Gly
Cys Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ala Leu Gly Tyr
Thr Val Arg Glu Gln Lys Glu Ile 50 55 60Leu Gln Ala Thr Asp Phe Asn
Gln Phe Met Asp Asn Ser Trp Gly Val65 70 75 80Ile Arg Asp Ile Arg
Arg Leu Ala Arg Asp Phe Gly Trp His Lys Gly 85 90 95Asp Phe Phe Asn
Ser Trp Ile Gly Asp Leu Ile His Arg Arg Leu Gly 100 105 110Asn Arg
Arg Ala Thr Phe Lys Asp Leu Gln Lys Ala Lys Leu Pro Asp 115 120
125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Tyr Ala Glu Val Phe
130 135 140Ser Ala Glu Arg His Pro Asp Met Glu Leu Ala Thr Ala Val
Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala Ala Val
Arg His Gly Asp Arg 165 170 175Gln Asp Val Tyr Val Asp Gly Gly Val
Gln Leu Asn Tyr Pro Ile Lys 180 185 190Leu Phe Asp Arg Glu Arg Tyr
Ile Asp Leu Ala Lys Asp Pro Gly Ala 195 200 205Val Arg Arg Thr Gly
Tyr Tyr Asn Lys Glu Asn Ala Arg Phe Gln Leu 210 215 220Glu Arg Pro
Gly Tyr Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230 235
240Leu Arg Leu Asp Ser Arg Glu Glu Ile Gly Leu Phe Arg Tyr Asp Glu
245 250 255Pro Leu Lys Gly Lys Pro Ile Lys Ser Phe Thr Asp Tyr Ala
Arg Gln 260 265 270Leu Phe Gly Ala Leu Met Asn Ala Gln Glu Lys Ile
His Leu His Gly 275 280 285Asp Asp Trp Gln Arg Thr Val Tyr Ile Asp
Thr Leu Asp Val Gly Thr 290 295 300Thr Asp Phe Asn Leu Ser Asp Ala
Thr Lys Gln Ala Leu Ile Glu Gln305 310 315 320Gly Ile Asn Gly Thr
Glu Asn Tyr Phe Glu Trp Phe Asp Asn Pro Leu 325 330 335Glu Lys Pro
Val Asn Arg Val Glu Ser 340 345713264DNAUnknownObtained from an
environmental sample. 71atgtcgctat catcaccgcc cgaaaccccc gaaccccccg
aacccccgtc acccggcgcg 60cgatcgctcc ggggaggatg gagccgccgg gtggccggcc
tgctggccct ggtgctgctc 120accgggctcc tccagatcgt cgtgccgctc
gcacggcccg ccgcggcggc cgtacagcag 180cccgcgatga cgtggaacct
gcatggggcc aagaagaccg cggaactggt tcccgatctg 240atgcgtaacc
ataacgtcac cgtcgcggcc ctccaggaag tggccaacgg caacttcctg
300ggcctcactc ccacagagca cgacgtgccc tacctcaagc cggacggcac
gacctcgact 360ccgccggatc cgcagaaatg gcgggtcgag aagtacaacc
tcgccaagga cgatgcaacc 420gctttcgtga tccggaccgg ctccaacaac
cgcgggctcg cgatcgtcac cacccaggac 480gtcggcgatg tctcgcagaa
tgtacacgtc gtcaatgtga ccgaggattg ggaaggcaag 540atgttccccg
ccctgggggt gaagatcgac ggcgcctggt actactccat ccacgcctcc
600accacgccga agcgcgcgaa caacaacgcc ggcactctgg tcgaggacct
ctccaagctg 660cacgagacgg ccgctttcga aggcgactgg gccgcgatgg
gcgactggaa ccggtacccc 720tccgaggact cgaacgccta cgagaaccaa
cggaagcatc tcaaaggcgc catgcggaca 780aactttccgg ataatcaggc
ggcgttgcgc gaagtcctgg agttcgagtc cgacgaacgc 840gtcatctggc
agggtgcgag gacccacgac cacggcgccg agctcgacta catggtggcc
900aagggagccg gtaacgacta caaggccagc cgatcgacgt cgaagcacgg
ctccgatcac 960tacccggtgt tcttcggtat tggggacgat tcggacacct
gcatgggcgg cacggcgccg 1020gtggcggcga acgcgccgcg tgcggccgcc
accgagtcct gtcccctgga cgacgatctg 1080ccggccgtca tcgtctcgat
gggggacagc tatatctccg gcgagggagg gcgctggcag 1140ggcaacgcca
acacctcctc cgggggcgac tcctggggca ccgaccgggc cgccgacggc
1200acggaggtct acgagaagaa ctccgaaggc agcgatgcct gtcaccgctc
cgacgtcgcg 1260gagatcaagc gcgccgacat cgccgacatc ccggcggaac
gcaggatcaa catcgcctgc 1320tcgggcgccg agaccaagca cctgctcacc
gagaccttca agggtgaaaa gccccagatc 1380gagcagctcg ccgacgtcgc
cgaaacccac cgggtggaca cgatcgtggt ctccatcggc 1440ggcaacgacc
tcgagttcgc cgacatcgtg agccagtgcg ccacggcctt catgctcggg
1500gaaggcgcgt gtcacacgga cgtcgacgat acccttgata gccggttggg
cgatgtgagc 1560agatccgtct ccgaggttct ggccgccatc cgcgacacca
tgatcgaggc cgggcaggac 1620gataccagct acaagctcgt tctccagtcc
taccctgccc cgttgcccgc gtcggatgag 1680atgcggtaca cgggcgatca
ctacgaccgg tacaccgagg gcggctgccc cttctatgac 1740gtcgacctgg
actggacgcg cgacgtcctc atcaaaaaga tcgaagccac gctgcgcggg
1800gtggccaaga gtgcggatgc ggccttcctc aacctgacgg acacgttcac
ggggcacgag 1860ctgtgctcga agcacacccg acaggcggag tccggcgaat
cgctggcgaa tccaatactg 1920gaacacgagg ccgagtgggt gcgcttcgta
ccaggtctca ccacgccggg tgacacggcc 1980gaagccatcc atccgaatgc
gttcggccag cacgccctca gtagctgcct cagccaggcc 2040gtccggacga
tggacgattc ggaccagagg tacttcgagt gcgacgggcg ggacaccgga
2100aatccccgcc tcgtgtggcc acgcagttcg cccatcgacg ccgtcgtgga
gaccgcggac 2160ggttggcagg gcgacgactt ccggctcgcc gaccactaca
tgttccagcg cggcgtctac 2220gcccgcttca acccggacgc ggaccggagc
ggcgcgatcg atccgggccg aatcaccttc 2280ggccaaaccg acggatggct
cggtgaggtg aaggacactt cgaactggcc gagcctgagt 2340ggaaccgact
tcgtcgacgg catcgacgcc gccgccgagg cacgcaccag caccggtcac
2400cagctgctgc tgttccacag cggcgttgag gacaaccagt acgtgcgggt
cgagatggcg 2460ccgggcacca ctgacgacca gctcgtcagg ggccccgtgc
ccatcacgag gtactggccc 2520ctcttccagg acaccccttt cgaatggggc
gtggatgccg ccgcggggga ccagctgaac 2580cgggcgatgg tcttcaggca
cggctatgtg gggctggtgc aggtctccct cgacgctctc 2640agcgacgaat
ggctcgtgga accgacgttg atcggctcgg cgattccggc gctggagggc
2700accccgttcg agacaggggt ggacgcggcg atcgtgcggc accagcaacc
gacggccatg 2760tgggtcgacc tgatcagcgg tacgcaggtg gtgacgctgc
tggtggactt ggacgatctg 2820tcgaagagca cgtacatgac gagcatcgtg
gagatcacga cgatgtggcc gagcctgcgc 2880ggcagcatct tcgactggac
cggcggagag gcgtggaagc cggagaagat gcagatcaag 2940accggcgcgg
gcgatcccta cgacatggac gccgacgacc ggcaggccaa gcctgcggtg
3000tcgggctcgc acgagcagtg ccgtccggag ggactagcgc agacccccgg
cgtgaacacg 3060ccgtactgcg aggtgtacga caccgacggc cgcgaatggc
tgggcgggaa cgggcacgac 3120aggcgggtca tcggctactt caccggctgg
cgcaccggtg agaacgacca gccgcgctac 3180ctggtgccga acatcccgtg
gtcgaaggtg acccacatca actacgcgtt cgcgaaagtc 3240gacgacgaca
acaagatcca aaga 3264721088PRTUnknownObtained from an environmental
sample. 72Met Ser Leu Ser Ser Pro Pro Glu Thr Pro Glu Pro Pro Glu
Pro Pro1 5 10 15Ser Pro Gly Ala Arg Ser Leu Arg Gly Gly Trp Ser Arg
Arg Val Ala 20 25 30Gly Leu Leu Ala Leu Val Leu Leu Thr Gly Leu Leu
Gln Ile Val Val 35 40 45Pro Leu Ala Arg Pro Ala Ala Ala Ala Val Gln
Gln Pro Ala Met Thr 50 55 60Trp Asn Leu His Gly Ala Lys Lys Thr Ala
Glu Leu Val Pro Asp Leu65 70 75 80Met Arg Asn His Asn Val Thr Val
Ala Ala Leu Gln Glu Val Ala Asn 85 90 95Gly Asn Phe Leu Gly Leu Thr
Pro Thr Glu His Asp Val Pro Tyr Leu 100 105
110Lys Pro Asp Gly Thr Thr Ser Thr Pro Pro Asp Pro Gln Lys Trp Arg
115 120 125Val Glu Lys Tyr Asn Leu Ala Lys Asp Asp Ala Thr Ala Phe
Val Ile 130 135 140Arg Thr Gly Ser Asn Asn Arg Gly Leu Ala Ile Val
Thr Thr Gln Asp145 150 155 160Val Gly Asp Val Ser Gln Asn Val His
Val Val Asn Val Thr Glu Asp 165 170 175Trp Glu Gly Lys Met Phe Pro
Ala Leu Gly Val Lys Ile Asp Gly Ala 180 185 190Trp Tyr Tyr Ser Ile
His Ala Ser Thr Thr Pro Lys Arg Ala Asn Asn 195 200 205Asn Ala Gly
Thr Leu Val Glu Asp Leu Ser Lys Leu His Glu Thr Ala 210 215 220Ala
Phe Glu Gly Asp Trp Ala Ala Met Gly Asp Trp Asn Arg Tyr Pro225 230
235 240Ser Glu Asp Ser Asn Ala Tyr Glu Asn Gln Arg Lys His Leu Lys
Gly 245 250 255Ala Met Arg Thr Asn Phe Pro Asp Asn Gln Ala Ala Leu
Arg Glu Val 260 265 270Leu Glu Phe Glu Ser Asp Glu Arg Val Ile Trp
Gln Gly Ala Arg Thr 275 280 285His Asp His Gly Ala Glu Leu Asp Tyr
Met Val Ala Lys Gly Ala Gly 290 295 300Asn Asp Tyr Lys Ala Ser Arg
Ser Thr Ser Lys His Gly Ser Asp His305 310 315 320Tyr Pro Val Phe
Phe Gly Ile Gly Asp Asp Ser Asp Thr Cys Met Gly 325 330 335Gly Thr
Ala Pro Val Ala Ala Asn Ala Pro Arg Ala Ala Ala Thr Glu 340 345
350Ser Cys Pro Leu Asp Asp Asp Leu Pro Ala Val Ile Val Ser Met Gly
355 360 365Asp Ser Tyr Ile Ser Gly Glu Gly Gly Arg Trp Gln Gly Asn
Ala Asn 370 375 380Thr Ser Ser Gly Gly Asp Ser Trp Gly Thr Asp Arg
Ala Ala Asp Gly385 390 395 400Thr Glu Val Tyr Glu Lys Asn Ser Glu
Gly Ser Asp Ala Cys His Arg 405 410 415Ser Asp Val Ala Glu Ile Lys
Arg Ala Asp Ile Ala Asp Ile Pro Ala 420 425 430Glu Arg Arg Ile Asn
Ile Ala Cys Ser Gly Ala Glu Thr Lys His Leu 435 440 445Leu Thr Glu
Thr Phe Lys Gly Glu Lys Pro Gln Ile Glu Gln Leu Ala 450 455 460Asp
Val Ala Glu Thr His Arg Val Asp Thr Ile Val Val Ser Ile Gly465 470
475 480Gly Asn Asp Leu Glu Phe Ala Asp Ile Val Ser Gln Cys Ala Thr
Ala 485 490 495Phe Met Leu Gly Glu Gly Ala Cys His Thr Asp Val Asp
Asp Thr Leu 500 505 510Asp Ser Arg Leu Gly Asp Val Ser Arg Ser Val
Ser Glu Val Leu Ala 515 520 525Ala Ile Arg Asp Thr Met Ile Glu Ala
Gly Gln Asp Asp Thr Ser Tyr 530 535 540Lys Leu Val Leu Gln Ser Tyr
Pro Ala Pro Leu Pro Ala Ser Asp Glu545 550 555 560Met Arg Tyr Thr
Gly Asp His Tyr Asp Arg Tyr Thr Glu Gly Gly Cys 565 570 575Pro Phe
Tyr Asp Val Asp Leu Asp Trp Thr Arg Asp Val Leu Ile Lys 580 585
590Lys Ile Glu Ala Thr Leu Arg Gly Val Ala Lys Ser Ala Asp Ala Ala
595 600 605Phe Leu Asn Leu Thr Asp Thr Phe Thr Gly His Glu Leu Cys
Ser Lys 610 615 620His Thr Arg Gln Ala Glu Ser Gly Glu Ser Leu Ala
Asn Pro Ile Leu625 630 635 640Glu His Glu Ala Glu Trp Val Arg Phe
Val Pro Gly Leu Thr Thr Pro 645 650 655Gly Asp Thr Ala Glu Ala Ile
His Pro Asn Ala Phe Gly Gln His Ala 660 665 670Leu Ser Ser Cys Leu
Ser Gln Ala Val Arg Thr Met Asp Asp Ser Asp 675 680 685Gln Arg Tyr
Phe Glu Cys Asp Gly Arg Asp Thr Gly Asn Pro Arg Leu 690 695 700Val
Trp Pro Arg Ser Ser Pro Ile Asp Ala Val Val Glu Thr Ala Asp705 710
715 720Gly Trp Gln Gly Asp Asp Phe Arg Leu Ala Asp His Tyr Met Phe
Gln 725 730 735Arg Gly Val Tyr Ala Arg Phe Asn Pro Asp Ala Asp Arg
Ser Gly Ala 740 745 750Ile Asp Pro Gly Arg Ile Thr Phe Gly Gln Thr
Asp Gly Trp Leu Gly 755 760 765Glu Val Lys Asp Thr Ser Asn Trp Pro
Ser Leu Ser Gly Thr Asp Phe 770 775 780Val Asp Gly Ile Asp Ala Ala
Ala Glu Ala Arg Thr Ser Thr Gly His785 790 795 800Gln Leu Leu Leu
Phe His Ser Gly Val Glu Asp Asn Gln Tyr Val Arg 805 810 815Val Glu
Met Ala Pro Gly Thr Thr Asp Asp Gln Leu Val Arg Gly Pro 820 825
830Val Pro Ile Thr Arg Tyr Trp Pro Leu Phe Gln Asp Thr Pro Phe Glu
835 840 845Trp Gly Val Asp Ala Ala Ala Gly Asp Gln Leu Asn Arg Ala
Met Val 850 855 860Phe Arg His Gly Tyr Val Gly Leu Val Gln Val Ser
Leu Asp Ala Leu865 870 875 880Ser Asp Glu Trp Leu Val Glu Pro Thr
Leu Ile Gly Ser Ala Ile Pro 885 890 895Ala Leu Glu Gly Thr Pro Phe
Glu Thr Gly Val Asp Ala Ala Ile Val 900 905 910Arg His Gln Gln Pro
Thr Ala Met Trp Val Asp Leu Ile Ser Gly Thr 915 920 925Gln Val Val
Thr Leu Leu Val Asp Leu Asp Asp Leu Ser Lys Ser Thr 930 935 940Tyr
Met Thr Ser Ile Val Glu Ile Thr Thr Met Trp Pro Ser Leu Arg945 950
955 960Gly Ser Ile Phe Asp Trp Thr Gly Gly Glu Ala Trp Lys Pro Glu
Lys 965 970 975Met Gln Ile Lys Thr Gly Ala Gly Asp Pro Tyr Asp Met
Asp Ala Asp 980 985 990Asp Arg Gln Ala Lys Pro Ala Val Ser Gly Ser
His Glu Gln Cys Arg 995 1000 1005Pro Glu Gly Leu Ala Gln Thr Pro
Gly Val Asn Thr Pro Tyr Cys Glu 1010 1015 1020Val Tyr Asp Thr Asp
Gly Arg Glu Trp Leu Gly Gly Asn Gly His Asp1025 1030 1035 1040Arg
Arg Val Ile Gly Tyr Phe Thr Gly Trp Arg Thr Gly Glu Asn Asp 1045
1050 1055Gln Pro Arg Tyr Leu Val Pro Asn Ile Pro Trp Ser Lys Val
Thr His 1060 1065 1070Ile Asn Tyr Ala Phe Ala Lys Val Asp Asp Asp
Asn Lys Ile Gln Arg 1075 1080 108573753DNAUnknownObtained from an
environmental sample. 73atgggaaacg gtgcagcagt tggttccaat gataatggta
gagaagaaag tgtttacgta 60ctttctgtga tcgcctgtaa tgtttattat ttacagaagt
gtgaaggtgg ggcatcgcgt 120gatagcgtga ttagagaaat taatagccaa
actcaacctt taggatatga gattgtagca 180gattctattc gtgatggtca
tattggttct tttgcctgta agatggcagt ctttagaaat 240aatggtaatg
gcaattgtgt tttagcgatc aaagggacag atatgaataa tatcaatgac
300ttggtgaatg atctaaccat gatattagga ggcattggtt ctgttgctgc
aatccaacca 360acgattaaca tggcacaaga actcatcgac caatatggag
tgaatttgat tactggtcac 420tcccttggag gctacatgac tgaaatcatc
gctaccaatc gtggactacc aggtattgca 480ttttgcgcac caggttcaaa
tggtccaatt gtaaaattag gtggacaaga gacacctggc 540tttcacaatg
ttaactttga acatgatcca gcaggtaacg ttatgactgg ggtttatact
600catgtccaat ggagtattta tgtaggatgt gatggtatga ctcatggtat
tgaaaatatg 660gtgaattatt ttaaagataa aagagattta accaatcgca
atattcaagg aagaagtgaa 720agtcataata cgggttatta ttacccaaaa taa
75374250PRTUnknownObtained from an environmental sample. 74Met Gly
Asn Gly Ala Ala Val Gly Ser Asn Asp Asn Gly Arg Glu Glu1 5 10 15Ser
Val Tyr Val Leu Ser Val Ile Ala Cys Asn Val Tyr Tyr Leu Gln 20 25
30Lys Cys Glu Gly Gly Ala Ser Arg Asp Ser Val Ile Arg Glu Ile Asn
35 40 45Ser Gln Thr Gln Pro Leu Gly Tyr Glu Ile Val Ala Asp Ser Ile
Arg 50 55 60Asp Gly His Ile Gly Ser Phe Ala Cys Lys Met Ala Val Phe
Arg Asn65 70 75 80Asn Gly Asn Gly Asn Cys Val Leu Ala Ile Lys Gly
Thr Asp Met Asn 85 90 95Asn Ile Asn Asp Leu Val Asn Asp Leu Thr Met
Ile Leu Gly Gly Ile 100 105 110Gly Ser Val Ala Ala Ile Gln Pro Thr
Ile Asn Met Ala Gln Glu Leu 115 120 125Ile Asp Gln Tyr Gly Val Asn
Leu Ile Thr Gly His Ser Leu Gly Gly 130 135 140Tyr Met Thr Glu Ile
Ile Ala Thr Asn Arg Gly Leu Pro Gly Ile Ala145 150 155 160Phe Cys
Ala Pro Gly Ser Asn Gly Pro Ile Val Lys Leu Gly Gly Gln 165 170
175Glu Thr Pro Gly Phe His Asn Val Asn Phe Glu His Asp Pro Ala Gly
180 185 190Asn Val Met Thr Gly Val Tyr Thr His Val Gln Trp Ser Ile
Tyr Val 195 200 205Gly Cys Asp Gly Met Thr His Gly Ile Glu Asn Met
Val Asn Tyr Phe 210 215 220Lys Asp Lys Arg Asp Leu Thr Asn Arg Asn
Ile Gln Gly Arg Ser Glu225 230 235 240Ser His Asn Thr Gly Tyr Tyr
Tyr Pro Lys 245 250751335DNAUnknownObtained from an environmental
sample. 75atgactacta aaatcttttt aattcacgga tggtctgtca agacaacaca
aacatatcag 60gcgctgcacc ttaagttggc agagcaggga tatcagctgg aagatattta
cctcgggcgg 120tatctgtccc ttgaaaatca tatcgaaata cgggatattg
caaaagcaat gcaccgtgca 180ttgctggaga ggattaccga ctggagtcag
cctttccatt ttattactca cagtacggga 240ggtatggtcg ccaaatattg
gatattgaat cattataaag gaagtattgc aaaacaaaaa 300ccactcaaaa
atgtagtgtt tctggctgca cctaattttg gttcaaggct ggcacaccat
360ggacgtacca tgctgggaga aataatggaa ctgggagaaa cagggaagaa
gattcttgaa 420tctctggagt taggaagtgc tttttcgtgg gatgtgaatg
agcagttttt taatgcgtcc 480aattggaaag ataaagaaat aaagttctat
aacctgatag gagacagggt caaaacggat 540ttttttaaat ccaaaatttt
tccagctgcg tttgaaagcg ggtcagatat ggtgattcgg 600gttgcggcag
gaaatcagaa ctttgtccgg tacaggtacg atagtcagaa agatagcttt
660actgttgtca atgagttgaa aggaattgct tttggtgctc tctaccaata
tacacattcc 720aatgatgatt atggaatcct gaacagcatc aaaaaaagtt
caacccttga aaaccatcag 780gcactcagac taattgtaga atgtctgaag
gtttcgggag ataaagaata tgaaaatgtt 840gttgcacagt tggctgcagc
gacaaaagaa accagagaaa aacgccaggg atatgcacag 900ctggatttcc
gttttcggga tgatgaaggc tttccaatag atgattatgt tgtagagctg
960ggagtaatgg taaatggaaa acctaaacca tctaaaacag tagatgacgt
gcataagaat 1020aaaattacac caaaccatct tactgtattc attaacctga
aagaactgga acctaatctg 1080aagtacttta tcaatattaa atcgatatcg
gaatcctcca tgtatagtta cgatcctgct 1140gtcaggacta tagagcttgc
ttctaacgag attacaaaaa ttatccgtga ggaccataca 1200acacagattg
atgtgatact ttcccggact cctgctaaaa accttttcat gtttcatcgc
1260ggagatgatg aagacctaca tgtgacatgg tcgcggtacg gagaaacaaa
aagtacaaag 1320cagggaataa aataa 133576444PRTUnknownObtained from an
environmental sample. 76Met Thr Thr Lys Ile Phe Leu Ile His Gly Trp
Ser Val Lys Thr Thr1 5 10 15Gln Thr Tyr Gln Ala Leu His Leu Lys Leu
Ala Glu Gln Gly Tyr Gln 20 25 30Leu Glu Asp Ile Tyr Leu Gly Arg Tyr
Leu Ser Leu Glu Asn His Ile 35 40 45Glu Ile Arg Asp Ile Ala Lys Ala
Met His Arg Ala Leu Leu Glu Arg 50 55 60Ile Thr Asp Trp Ser Gln Pro
Phe His Phe Ile Thr His Ser Thr Gly65 70 75 80Gly Met Val Ala Lys
Tyr Trp Ile Leu Asn His Tyr Lys Gly Ser Ile 85 90 95Ala Lys Gln Lys
Pro Leu Lys Asn Val Val Phe Leu Ala Ala Pro Asn 100 105 110Phe Gly
Ser Arg Leu Ala His His Gly Arg Thr Met Leu Gly Glu Ile 115 120
125Met Glu Leu Gly Glu Thr Gly Lys Lys Ile Leu Glu Ser Leu Glu Leu
130 135 140Gly Ser Ala Phe Ser Trp Asp Val Asn Glu Gln Phe Phe Asn
Ala Ser145 150 155 160Asn Trp Lys Asp Lys Glu Ile Lys Phe Tyr Asn
Leu Ile Gly Asp Arg 165 170 175Val Lys Thr Asp Phe Phe Lys Ser Lys
Ile Phe Pro Ala Ala Phe Glu 180 185 190Ser Gly Ser Asp Met Val Ile
Arg Val Ala Ala Gly Asn Gln Asn Phe 195 200 205Val Arg Tyr Arg Tyr
Asp Ser Gln Lys Asp Ser Phe Thr Val Val Asn 210 215 220Glu Leu Lys
Gly Ile Ala Phe Gly Ala Leu Tyr Gln Tyr Thr His Ser225 230 235
240Asn Asp Asp Tyr Gly Ile Leu Asn Ser Ile Lys Lys Ser Ser Thr Leu
245 250 255Glu Asn His Gln Ala Leu Arg Leu Ile Val Glu Cys Leu Lys
Val Ser 260 265 270Gly Asp Lys Glu Tyr Glu Asn Val Val Ala Gln Leu
Ala Ala Ala Thr 275 280 285Lys Glu Thr Arg Glu Lys Arg Gln Gly Tyr
Ala Gln Leu Asp Phe Arg 290 295 300Phe Arg Asp Asp Glu Gly Phe Pro
Ile Asp Asp Tyr Val Val Glu Leu305 310 315 320Gly Val Met Val Asn
Gly Lys Pro Lys Pro Ser Lys Thr Val Asp Asp 325 330 335Val His Lys
Asn Lys Ile Thr Pro Asn His Leu Thr Val Phe Ile Asn 340 345 350Leu
Lys Glu Leu Glu Pro Asn Leu Lys Tyr Phe Ile Asn Ile Lys Ser 355 360
365Ile Ser Glu Ser Ser Met Tyr Ser Tyr Asp Pro Ala Val Arg Thr Ile
370 375 380Glu Leu Ala Ser Asn Glu Ile Thr Lys Ile Ile Arg Glu Asp
His Thr385 390 395 400Thr Gln Ile Asp Val Ile Leu Ser Arg Thr Pro
Ala Lys Asn Leu Phe 405 410 415Met Phe His Arg Gly Asp Asp Glu Asp
Leu His Val Thr Trp Ser Arg 420 425 430Tyr Gly Glu Thr Lys Ser Thr
Lys Gln Gly Ile Lys 435 440771026DNAUnknownObtained from an
environmental sample. 77atggcttatc actttaaaaa cttggtcttc gaaggcggtg
gcgtgaaagg catcgcctac 60gtgggtgctc ttgaagtact tgagagagaa ggcattctga
aagacatcaa acgcgtggct 120ggtacttcgg ctggagcgct ggttgccgtc
ttaatcagtt tgggctatac cgcccaagaa 180ttgaaggaca tcctatggaa
aatcaatttc caaaactttt tggacagctc gtggggcttg 240gtgcgcaaca
cggcacgttt cattgaggat tacggttggt acaaaggtga gtttttccgc
300gaattggttg ccggctacat caaggaaaaa acgggcaata gtgaaagcac
tttcaaggat 360ctggccaaat caaaagattt ccgtggcctc agccttattg
gtagcgatct gtccacagga 420tactcaaagg tgttcagcaa cgaattcacc
ccaaacgtca aagtagctga tgcagcccgc 480atctccatgt cgatacccct
gtttttcaaa gccgttcgcg gtgtaaacgg tgatggacac 540atttacgtcg
atggtggact gttagacaac tatgccatca aggtgttcga ccgcgtcaat
600tacgtaaaga ataagaacaa cgtacggtac accgagtatt atgaaaagac
caacaagtcg 660ctgaaaagca aaaacaagct gaccaacgaa tacgtctaca
ataaagaaac tttgggcttc 720cgattggatg ccaaagaaca gattgagatg
tttctcgacc atagtataga accaaaggca 780aaggacattg actcactatt
ctcttacacg aaggctttgg tcaccaccct catcgacttt 840caaaacaatg
tacatttgca tagtgacgac tggcaacgca cagtctatat cgactcttta
900ggtatcagtt ccactgactt cggcatctct gactctaaaa aacagaaact
cgtcgattca 960ggcattttgc atacgcaaaa atacctggat tggtataaca
acgacgaaga gaaagccaac 1020aaatag 102678341PRTUnknownObtained from
an environmental sample. 78Met Ala Tyr His Phe Lys Asn Leu Val Phe
Glu Gly Gly Gly Val Lys1 5 10 15Gly Ile Ala Tyr Val Gly Ala Leu Glu
Val Leu Glu Arg Glu Gly Ile 20 25 30Leu Lys Asp Ile Lys Arg Val Ala
Gly Thr Ser Ala Gly Ala Leu Val 35 40 45Ala Val Leu Ile Ser Leu Gly
Tyr Thr Ala Gln Glu Leu Lys Asp Ile 50 55 60Leu Trp Lys Ile Asn Phe
Gln Asn Phe Leu Asp Ser Ser Trp Gly Leu65 70 75 80Val Arg Asn Thr
Ala Arg Phe Ile Glu Asp Tyr Gly Trp Tyr Lys Gly 85 90 95Glu Phe Phe
Arg Glu Leu Val Ala Gly Tyr Ile Lys Glu Lys Thr Gly 100 105 110Asn
Ser Glu Ser Thr Phe Lys Asp Leu Ala Lys Ser Lys Asp Phe Arg 115 120
125Gly Leu Ser Leu Ile Gly Ser Asp Leu Ser Thr Gly Tyr Ser Lys Val
130 135 140Phe Ser Asn Glu Phe Thr Pro Asn Val Lys Val Ala Asp Ala
Ala Arg145 150 155 160Ile Ser Met Ser Ile Pro Leu Phe Phe Lys Ala
Val Arg Gly Val Asn 165 170 175Gly Asp Gly His Ile Tyr Val Asp Gly
Gly Leu Leu Asp Asn Tyr Ala 180 185 190Ile Lys Val Phe Asp Arg Val
Asn Tyr Val Lys Asn Lys Asn Asn Val 195 200 205Arg Tyr Thr Glu Tyr
Tyr Glu Lys Thr Asn Lys Ser Leu Lys Ser Lys 210 215 220Asn Lys Leu
Thr Asn
Glu Tyr Val Tyr Asn Lys Glu Thr Leu Gly Phe225 230 235 240Arg Leu
Asp Ala Lys Glu Gln Ile Glu Met Phe Leu Asp His Ser Ile 245 250
255Glu Pro Lys Ala Lys Asp Ile Asp Ser Leu Phe Ser Tyr Thr Lys Ala
260 265 270Leu Val Thr Thr Leu Ile Asp Phe Gln Asn Asn Val His Leu
His Ser 275 280 285Asp Asp Trp Gln Arg Thr Val Tyr Ile Asp Ser Leu
Gly Ile Ser Ser 290 295 300Thr Asp Phe Gly Ile Ser Asp Ser Lys Lys
Gln Lys Leu Val Asp Ser305 310 315 320Gly Ile Leu His Thr Gln Lys
Tyr Leu Asp Trp Tyr Asn Asn Asp Glu 325 330 335Glu Lys Ala Asn Lys
340791701DNAUnknownObtained from an environmental sample.
79atgagaaatt tcagcaaggg attgaccagt attttgctta gcatagcgac atccaccagt
60gcgatggcct ttacccagat cggggccggc ggagcgattc cgatgggcca tgagtggcta
120acccgccgct cggcgctgga actgctgaat gccgacaatc tggtcggcaa
tgacccggcc 180gacccacgct tgggctggag cgaaggtctc gccaacaatc
tcgatctctc gaatgcccag 240aacgaagtgc agcgcatcaa gagcattacc
aagagccacg ccctgtatga gccgcgttac 300gatgacgttt tcgccgccat
cgtcggcgag cgctgggttg ataccgccgg tttcaacgtg 360gccaaggcca
ccgtcggcaa gatcgattgc ttcagcgccg tcgcgcaaga gcccgccgat
420gtgcaacaag accatttcat gcgccgttat gacgacgtgg gtggacaagg
gggcgtgaac 480gctgcccgcc gcgcgcagca gcgctttatc aatcacttcg
tcaacgcagc catggccgaa 540gagaagagca tcaaggcatg ggatggcggc
ggttattctt cgctggaaaa agtcagccac 600aactacttct tgtttggccg
cgccgttcat ttgttccagg attctttcag ccccgaacac 660accgtgcgcc
tgcctgaaga caattacgtc aaagtccgtc aggtcaaggc gtatctctgc
720tctgaaggtg ccgaacagca tacgcacaac acgcaagatg ccatcaactt
caccagcggc 780gatgtcatct ggaaacagaa cacccgtctg gatgcaggct
ggagcaccta caaggccagc 840aacatgaagc cggtggcatt ggttgccctc
gaagccagca aagatttgtg ggccgccttt 900attcgcacca tggccgtttc
ccgcgaggag cgtcgcgccg tcgccgaaca ggaagcgcag 960gctctcgtca
atcactggtt gtcgttcgac gaacaggaaa tgctgaactg gtacgaagaa
1020gaagagcacc gcgatcatac gtacgtcaag gaacccggcc agagcggccc
aggttcgtcg 1080ttattcgatt gcatggttgg tctgggtgtg gcctcgggca
gtcaggcgca acgggtggcg 1140gaactcgatc agcaacgccg ccaatgtttg
ttcaacgtca aggccgctac tggctatggc 1200gatctgaatg atccacacat
ggatattccg tacaactggc aatgggtgtc gtcgacgcaa 1260tggaaaatcc
ctgcggccga ctggaaaatc ccgcagctgc ccgccgattc agggaaatca
1320gtcgtcatca agaattcgat caatggcgat ccgctggtgg cacctgccgg
gctcaagcac 1380aacaccgatg tttacggtgc accgggtgag gcgattgaat
tcattttcgt cggtgatttc 1440aaccatgagg cgtatttccg caccaaggac
aacgcggatc tgttcctgag ttacagcgcg 1500gtatcgggca agggcttgct
gtacaacacg cccaaccagg ccggttatcg tgttcagcct 1560tatggtgtgc
tgtggacgat tgagaatacc tactggaatg atttcctctg gtacaacagc
1620tcgaacgacc gcatctatgt cagcggcacc ggcgctgcca acaagtcaca
ctcccagtgg 1680attattgacg gcttgcagtg a 170180566PRTUnknownObtained
from an environmental sample. 80Met Arg Asn Phe Ser Lys Gly Leu Thr
Ser Ile Leu Leu Ser Ile Ala1 5 10 15Thr Ser Thr Ser Ala Met Ala Phe
Thr Gln Ile Gly Ala Gly Gly Ala 20 25 30Ile Pro Met Gly His Glu Trp
Leu Thr Arg Arg Ser Ala Leu Glu Leu 35 40 45Leu Asn Ala Asp Asn Leu
Val Gly Asn Asp Pro Ala Asp Pro Arg Leu 50 55 60Gly Trp Ser Glu Gly
Leu Ala Asn Asn Leu Asp Leu Ser Asn Ala Gln65 70 75 80Asn Glu Val
Gln Arg Ile Lys Ser Ile Thr Lys Ser His Ala Leu Tyr 85 90 95Glu Pro
Arg Tyr Asp Asp Val Phe Ala Ala Ile Val Gly Glu Arg Trp 100 105
110Val Asp Thr Ala Gly Phe Asn Val Ala Lys Ala Thr Val Gly Lys Ile
115 120 125Asp Cys Phe Ser Ala Val Ala Gln Glu Pro Ala Asp Val Gln
Gln Asp 130 135 140His Phe Met Arg Arg Tyr Asp Asp Val Gly Gly Gln
Gly Gly Val Asn145 150 155 160Ala Ala Arg Arg Ala Gln Gln Arg Phe
Ile Asn His Phe Val Asn Ala 165 170 175Ala Met Ala Glu Glu Lys Ser
Ile Lys Ala Trp Asp Gly Gly Gly Tyr 180 185 190Ser Ser Leu Glu Lys
Val Ser His Asn Tyr Phe Leu Phe Gly Arg Ala 195 200 205Val His Leu
Phe Gln Asp Ser Phe Ser Pro Glu His Thr Val Arg Leu 210 215 220Pro
Glu Asp Asn Tyr Val Lys Val Arg Gln Val Lys Ala Tyr Leu Cys225 230
235 240Ser Glu Gly Ala Glu Gln His Thr His Asn Thr Gln Asp Ala Ile
Asn 245 250 255Phe Thr Ser Gly Asp Val Ile Trp Lys Gln Asn Thr Arg
Leu Asp Ala 260 265 270Gly Trp Ser Thr Tyr Lys Ala Ser Asn Met Lys
Pro Val Ala Leu Val 275 280 285Ala Leu Glu Ala Ser Lys Asp Leu Trp
Ala Ala Phe Ile Arg Thr Met 290 295 300Ala Val Ser Arg Glu Glu Arg
Arg Ala Val Ala Glu Gln Glu Ala Gln305 310 315 320Ala Leu Val Asn
His Trp Leu Ser Phe Asp Glu Gln Glu Met Leu Asn 325 330 335Trp Tyr
Glu Glu Glu Glu His Arg Asp His Thr Tyr Val Lys Glu Pro 340 345
350Gly Gln Ser Gly Pro Gly Ser Ser Leu Phe Asp Cys Met Val Gly Leu
355 360 365Gly Val Ala Ser Gly Ser Gln Ala Gln Arg Val Ala Glu Leu
Asp Gln 370 375 380Gln Arg Arg Gln Cys Leu Phe Asn Val Lys Ala Ala
Thr Gly Tyr Gly385 390 395 400Asp Leu Asn Asp Pro His Met Asp Ile
Pro Tyr Asn Trp Gln Trp Val 405 410 415Ser Ser Thr Gln Trp Lys Ile
Pro Ala Ala Asp Trp Lys Ile Pro Gln 420 425 430Leu Pro Ala Asp Ser
Gly Lys Ser Val Val Ile Lys Asn Ser Ile Asn 435 440 445Gly Asp Pro
Leu Val Ala Pro Ala Gly Leu Lys His Asn Thr Asp Val 450 455 460Tyr
Gly Ala Pro Gly Glu Ala Ile Glu Phe Ile Phe Val Gly Asp Phe465 470
475 480Asn His Glu Ala Tyr Phe Arg Thr Lys Asp Asn Ala Asp Leu Phe
Leu 485 490 495Ser Tyr Ser Ala Val Ser Gly Lys Gly Leu Leu Tyr Asn
Thr Pro Asn 500 505 510Gln Ala Gly Tyr Arg Val Gln Pro Tyr Gly Val
Leu Trp Thr Ile Glu 515 520 525Asn Thr Tyr Trp Asn Asp Phe Leu Trp
Tyr Asn Ser Ser Asn Asp Arg 530 535 540Ile Tyr Val Ser Gly Thr Gly
Ala Ala Asn Lys Ser His Ser Gln Trp545 550 555 560Ile Ile Asp Gly
Leu Gln 565811422DNAUnknownObtained from an environmental sample.
81atgaaaaaga aattatgtac aatggctctt gtaacagcaa tatcttctgg tgttgttacg
60attccaacag aagcacaagc ttgtggaata ggcgaagtaa tgaaacagga gaaccaagag
120cacaaacgtg tgaaaagatg gtctgcggag catccgcatc attcaaatga
aagtacacat 180ttatggattg cacgaaatgc gattcaaatt atgagtcgta
atcaagataa gacggttcaa 240gaaaatgaat tacaattttt aaatactcct
gaatataagg agttatttga aagaggtctt 300tatgatgctg attaccttga
tgaatttaac gatggaggta caggtacaat cggcattgat 360gggctaatta
gaggagggtg gaaatctcat ttttacgatc ccgatacaag aaagaactat
420aaaggggaag aagaaccaac agctctttca caaggagata aatattttaa
attagcaggt 480gaatacttta agaagggcga ccaaaaacaa gctttttatt
atttaggtgt tgcaacgcat 540tactttacag atgctactca accaatgcat
gctgctaatt ttacagccgt cgacacgagt 600gctttaaagt ttcatagcgc
ttttgaaaat tatgtgacga caattcagac acagtatgaa 660gtatctgatg
gtgagggcgt atataattta gtgaattcta atgatccaaa acagtggatc
720catgaaacag cgagactcgc aaaagtggaa atcgggaaca ttaccaatga
cgagattaaa 780tctcactata ataaaggaaa caatgctctt tggcaacaag
aagttatgcc agctgtccag 840aggagtttag agaacgcaca aagaaacacg
gcgggattta ttcatttatg gtttaaaaca 900tttgttggca atactgccgc
tgaagaaatt gaaaatactg tagtgaaaga ttctaaagga 960gaagcaatac
aagataataa aaaatacttc gtagtgccaa gtgagtttct aaatagaggt
1020ttgacctttg aagtatatgc aaggaatgac tatgcactat tatctaatta
cgtagatgat 1080agtaaagttc atggtacgcc agttcagttt gtatttgata
aagataataa cggtatcctt 1140catcgaggag aaagtgtact gctgaaaatg
acgcaatcta actatgataa ttacgtattt 1200ctaaactact ctaacttgac
aaactgggta catcttgcgc aacaaaaaac aaatactgca 1260cagtttaaag
tgtatccaaa tccgaataac ccatctgaat attacctata tacagatgga
1320tacccagtaa attatcaaga aaatggtaac ggaaagagct ggattgtgtt
aggaaagaaa 1380acagatacac caaaagcttg gaaatttata caggctgaat ag
142282473PRTUnknownObtained from an environmental sample. 82Met Lys
Lys Lys Leu Cys Thr Met Ala Leu Val Thr Ala Ile Ser Ser1 5 10 15Gly
Val Val Thr Ile Pro Thr Glu Ala Gln Ala Cys Gly Ile Gly Glu 20 25
30Val Met Lys Gln Glu Asn Gln Glu His Lys Arg Val Lys Arg Trp Ser
35 40 45Ala Glu His Pro His His Ser Asn Glu Ser Thr His Leu Trp Ile
Ala 50 55 60Arg Asn Ala Ile Gln Ile Met Ser Arg Asn Gln Asp Lys Thr
Val Gln65 70 75 80Glu Asn Glu Leu Gln Phe Leu Asn Thr Pro Glu Tyr
Lys Glu Leu Phe 85 90 95Glu Arg Gly Leu Tyr Asp Ala Asp Tyr Leu Asp
Glu Phe Asn Asp Gly 100 105 110Gly Thr Gly Thr Ile Gly Ile Asp Gly
Leu Ile Arg Gly Gly Trp Lys 115 120 125Ser His Phe Tyr Asp Pro Asp
Thr Arg Lys Asn Tyr Lys Gly Glu Glu 130 135 140Glu Pro Thr Ala Leu
Ser Gln Gly Asp Lys Tyr Phe Lys Leu Ala Gly145 150 155 160Glu Tyr
Phe Lys Lys Gly Asp Gln Lys Gln Ala Phe Tyr Tyr Leu Gly 165 170
175Val Ala Thr His Tyr Phe Thr Asp Ala Thr Gln Pro Met His Ala Ala
180 185 190Asn Phe Thr Ala Val Asp Thr Ser Ala Leu Lys Phe His Ser
Ala Phe 195 200 205Glu Asn Tyr Val Thr Thr Ile Gln Thr Gln Tyr Glu
Val Ser Asp Gly 210 215 220Glu Gly Val Tyr Asn Leu Val Asn Ser Asn
Asp Pro Lys Gln Trp Ile225 230 235 240His Glu Thr Ala Arg Leu Ala
Lys Val Glu Ile Gly Asn Ile Thr Asn 245 250 255Asp Glu Ile Lys Ser
His Tyr Asn Lys Gly Asn Asn Ala Leu Trp Gln 260 265 270Gln Glu Val
Met Pro Ala Val Gln Arg Ser Leu Glu Asn Ala Gln Arg 275 280 285Asn
Thr Ala Gly Phe Ile His Leu Trp Phe Lys Thr Phe Val Gly Asn 290 295
300Thr Ala Ala Glu Glu Ile Glu Asn Thr Val Val Lys Asp Ser Lys
Gly305 310 315 320Glu Ala Ile Gln Asp Asn Lys Lys Tyr Phe Val Val
Pro Ser Glu Phe 325 330 335Leu Asn Arg Gly Leu Thr Phe Glu Val Tyr
Ala Arg Asn Asp Tyr Ala 340 345 350Leu Leu Ser Asn Tyr Val Asp Asp
Ser Lys Val His Gly Thr Pro Val 355 360 365Gln Phe Val Phe Asp Lys
Asp Asn Asn Gly Ile Leu His Arg Gly Glu 370 375 380Ser Val Leu Leu
Lys Met Thr Gln Ser Asn Tyr Asp Asn Tyr Val Phe385 390 395 400Leu
Asn Tyr Ser Asn Leu Thr Asn Trp Val His Leu Ala Gln Gln Lys 405 410
415Thr Asn Thr Ala Gln Phe Lys Val Tyr Pro Asn Pro Asn Asn Pro Ser
420 425 430Glu Tyr Tyr Leu Tyr Thr Asp Gly Tyr Pro Val Asn Tyr Gln
Glu Asn 435 440 445Gly Asn Gly Lys Ser Trp Ile Val Leu Gly Lys Lys
Thr Asp Thr Pro 450 455 460Lys Ala Trp Lys Phe Ile Gln Ala Glu465
470831290DNAUnknownObtained from an environmental sample.
83atgaaaaaga tagtgattta ttcatttgta gcaggggtta tgacatcagg cggcgtattt
60gccgccagtg acaatattgt ggagacgtcg accccaccac agcatcaggc cccaagcaga
120caggacaggg cattattcgc gggtgataca acaacctata taaaatgtgt
ctacaaagtg 180gatggccagg atgacagcaa tccatcctca tcttggttat
gggcgaaagt gggtagcaac 240tatgcgaagc tgaaggggta ttggtataat
tcaatgccgc tggcaaacat gttttacact 300gaagtaccct atgcagaggt
gatggacttg tgtaatagca ccctgaaggc ggtaggtgcc 360aactccactc
ttgttattcc atatgcatcg gattacaccc tgtcctatta ctatgtgatt
420tggaatcaag gggctaacca gccggttatc aacgttggcg gcagagagct
tgaccgtatg 480gtggtctttg gtgacagctt gagcgatacc gtcaatgtct
ataacggctc gtacggtacc 540gtgccgaata gtacctcctg gttattgggc
catttctcta acggaaagct ttggcatgaa 600tacctttcca cggtattgaa
tctgcctagc tatgtgtggg cgactggcaa tgcggagagt 660ggagagaaac
ccttctttaa cggattcagt aagcaggtgg attctttcag ggattatcac
720gctcgcacta aaggctacga tattagcaag acgttgttta ccgttctgtt
tggtggaaat 780gattttataa cggggggaaa aagcgccgat gaggtcattg
agcaatatac ggtgtcattg 840aactacttgg ctcaactagg ggcgaagcag
gttgcaattt tccgcttgcc agatttttca 900gtgataccca gcgtttcaac
gtggacagag gctgataagg acaaactgag agagaatagt 960gttcagttta
atgaccaagc cgagaagctg atcgctaaac taaacgcggc acatccccaa
1020acgacgtttt atacgctgag gttggatgac gcttttaagc aggtgttgga
aaacagcgac 1080caatacggct ttgttaataa gactgatacc tgcctggata
tttcccaagg cggatacaac 1140tatgccattg gggcccgcgc gaaaacggca
tgtaagagca gcaatgcggc gtttgtattc 1200tgggacaata tgcatccgac
caccaaaaca cacggattgt tggccgatct tttaaaagat 1260gatgtggtac
gcggcctcgc tgcgccatga 129084429PRTUnknownObtained from an
environmental sample. 84Met Lys Lys Ile Val Ile Tyr Ser Phe Val Ala
Gly Val Met Thr Ser1 5 10 15Gly Gly Val Phe Ala Ala Ser Asp Asn Ile
Val Glu Thr Ser Thr Pro 20 25 30Pro Gln His Gln Ala Pro Ser Arg Gln
Asp Arg Ala Leu Phe Ala Gly 35 40 45Asp Thr Thr Thr Tyr Ile Lys Cys
Val Tyr Lys Val Asp Gly Gln Asp 50 55 60Asp Ser Asn Pro Ser Ser Ser
Trp Leu Trp Ala Lys Val Gly Ser Asn65 70 75 80Tyr Ala Lys Leu Lys
Gly Tyr Trp Tyr Asn Ser Met Pro Leu Ala Asn 85 90 95Met Phe Tyr Thr
Glu Val Pro Tyr Ala Glu Val Met Asp Leu Cys Asn 100 105 110Ser Thr
Leu Lys Ala Val Gly Ala Asn Ser Thr Leu Val Ile Pro Tyr 115 120
125Ala Ser Asp Tyr Thr Leu Ser Tyr Tyr Tyr Val Ile Trp Asn Gln Gly
130 135 140Ala Asn Gln Pro Val Ile Asn Val Gly Gly Arg Glu Leu Asp
Arg Met145 150 155 160Val Val Phe Gly Asp Ser Leu Ser Asp Thr Val
Asn Val Tyr Asn Gly 165 170 175Ser Tyr Gly Thr Val Pro Asn Ser Thr
Ser Trp Leu Leu Gly His Phe 180 185 190Ser Asn Gly Lys Leu Trp His
Glu Tyr Leu Ser Thr Val Leu Asn Leu 195 200 205Pro Ser Tyr Val Trp
Ala Thr Gly Asn Ala Glu Ser Gly Glu Lys Pro 210 215 220Phe Phe Asn
Gly Phe Ser Lys Gln Val Asp Ser Phe Arg Asp Tyr His225 230 235
240Ala Arg Thr Lys Gly Tyr Asp Ile Ser Lys Thr Leu Phe Thr Val Leu
245 250 255Phe Gly Gly Asn Asp Phe Ile Thr Gly Gly Lys Ser Ala Asp
Glu Val 260 265 270Ile Glu Gln Tyr Thr Val Ser Leu Asn Tyr Leu Ala
Gln Leu Gly Ala 275 280 285Lys Gln Val Ala Ile Phe Arg Leu Pro Asp
Phe Ser Val Ile Pro Ser 290 295 300Val Ser Thr Trp Thr Glu Ala Asp
Lys Asp Lys Leu Arg Glu Asn Ser305 310 315 320Val Gln Phe Asn Asp
Gln Ala Glu Lys Leu Ile Ala Lys Leu Asn Ala 325 330 335Ala His Pro
Gln Thr Thr Phe Tyr Thr Leu Arg Leu Asp Asp Ala Phe 340 345 350Lys
Gln Val Leu Glu Asn Ser Asp Gln Tyr Gly Phe Val Asn Lys Thr 355 360
365Asp Thr Cys Leu Asp Ile Ser Gln Gly Gly Tyr Asn Tyr Ala Ile Gly
370 375 380Ala Arg Ala Lys Thr Ala Cys Lys Ser Ser Asn Ala Ala Phe
Val Phe385 390 395 400Trp Asp Asn Met His Pro Thr Thr Lys Thr His
Gly Leu Leu Ala Asp 405 410 415Leu Leu Lys Asp Asp Val Val Arg Gly
Leu Ala Ala Pro 420 425851038DNAUnknownObtained from an
environmental sample. 85atgacaacac aatttagaaa cttgatattt gaaggcggcg
gtgtaaaagg tgttgcttac 60attggcgcca tgcagattct tgaaaatcgt ggcgtgttgc
aagatattcg ccgagtcgga 120gggtgcagtg cgggtgcgat taacgcgctg
atttttgcgc taggttacac ggtccgtgaa 180caaaaagaga tcttacaagc
caccgatttt aaccagttta tggataactc ttggggggtt 240attcgtgata
ttcgcaggct tgctcgagac tttggctgga ataagggtga tttctttagt
300agctggatag gtgatttgat tcatcgtcgt ttggggaatc gccgagcgac
gttcaaagat 360ctgcaaaagg ccaagcttcc tgatctttat gtcatcggta
ctaatctgtc tacagggttt 420gcagaggtgt tttctgccga aagacacccc
gatatggagc tggcgacagc ggtgcgtatc 480tccatgtcga taccgctgtt
ctttgcggcc gtgcgtcacg gtgatcgaca agatgtgtat 540gtcgatgggg
gtgttcaact taactatccg attaaactgt ttgatcggga
gcgttacatt 600gatttggcca aagatcccgg tgccgttcgg cgaacgggtt
attacaacaa agaaaacgct 660cgctttcagc ttgatcggcc gggccatagc
ccctatgttt acaatcgcca gaccttgggt 720ttgcgactgg atagtcgcga
ggagataggg ctctttcgtt atgacgaacc cctcaagggc 780aaacccatta
agtccttcac tgactacgct cgacaacttt tcggtgcgtt gatgaatgca
840caggaaaaga ttcatctaca tggcgatgat tggcaacgca cgatctatat
cgatacattg 900gatgtgggta cgacggactt caatctttct gatgcaacta
agcaagcact gattgagcaa 960ggaattaacg gcaccgaaaa ttatttcgag
tggtttgata atccgttaga gaagcctgtg 1020aatagagtgg agtcatag
103886345PRTUnknownObtained from an environmental sample. 86Met Thr
Thr Gln Phe Arg Asn Leu Ile Phe Glu Gly Gly Gly Val Lys1 5 10 15Gly
Val Ala Tyr Ile Gly Ala Met Gln Ile Leu Glu Asn Arg Gly Val 20 25
30Leu Gln Asp Ile Arg Arg Val Gly Gly Cys Ser Ala Gly Ala Ile Asn
35 40 45Ala Leu Ile Phe Ala Leu Gly Tyr Thr Val Arg Glu Gln Lys Glu
Ile 50 55 60Leu Gln Ala Thr Asp Phe Asn Gln Phe Met Asp Asn Ser Trp
Gly Val65 70 75 80Ile Arg Asp Ile Arg Arg Leu Ala Arg Asp Phe Gly
Trp Asn Lys Gly 85 90 95Asp Phe Phe Ser Ser Trp Ile Gly Asp Leu Ile
His Arg Arg Leu Gly 100 105 110Asn Arg Arg Ala Thr Phe Lys Asp Leu
Gln Lys Ala Lys Leu Pro Asp 115 120 125Leu Tyr Val Ile Gly Thr Asn
Leu Ser Thr Gly Phe Ala Glu Val Phe 130 135 140Ser Ala Glu Arg His
Pro Asp Met Glu Leu Ala Thr Ala Val Arg Ile145 150 155 160Ser Met
Ser Ile Pro Leu Phe Phe Ala Ala Val Arg His Gly Asp Arg 165 170
175Gln Asp Val Tyr Val Asp Gly Gly Val Gln Leu Asn Tyr Pro Ile Lys
180 185 190Leu Phe Asp Arg Glu Arg Tyr Ile Asp Leu Ala Lys Asp Pro
Gly Ala 195 200 205Val Arg Arg Thr Gly Tyr Tyr Asn Lys Glu Asn Ala
Arg Phe Gln Leu 210 215 220Asp Arg Pro Gly His Ser Pro Tyr Val Tyr
Asn Arg Gln Thr Leu Gly225 230 235 240Leu Arg Leu Asp Ser Arg Glu
Glu Ile Gly Leu Phe Arg Tyr Asp Glu 245 250 255Pro Leu Lys Gly Lys
Pro Ile Lys Ser Phe Thr Asp Tyr Ala Arg Gln 260 265 270Leu Phe Gly
Ala Leu Met Asn Ala Gln Glu Lys Ile His Leu His Gly 275 280 285Asp
Asp Trp Gln Arg Thr Ile Tyr Ile Asp Thr Leu Asp Val Gly Thr 290 295
300Thr Asp Phe Asn Leu Ser Asp Ala Thr Lys Gln Ala Leu Ile Glu
Gln305 310 315 320Gly Ile Asn Gly Thr Glu Asn Tyr Phe Glu Trp Phe
Asp Asn Pro Leu 325 330 335Glu Lys Pro Val Asn Arg Val Glu Ser 340
34587870DNAUnknownObtained from an environmental sample.
87atgtcaaaga aactcgtaat atcggtagcg ggcggcggag cactcggaat cggaccactc
60gcattcctgt gcaagattga acagatgctg ggaaagaaga taccccaggt tgcgcaggca
120tacgccggca cttcaaccgg agcaataatt gcggcaggac tggccgaagg
ctactccgcg 180catgaactgt tcgacctata caaatcaaat ctcagcaaga
tattcaccaa atacagctgg 240tacaaacgcc tgcagccaac gtgtcctaca
tatgacaaca gtaacctaaa gaaattactg 300aaggacaaat tcaagggcaa
ggtcggcgac tggaaaactc ccgtatacat cccggcaaca 360cacatgaacg
gccaatccgt agaaaaggtg tgggacttgg gtgacaagaa tgttgacaag
420tggtttgcca ttctgacaag taccgcggca ccaacctatt tcgactgcat
atacgacgac 480gagaagaact gctacatcga tggtggcatg tggtgcaacg
caccaatcga tgtgcttaat 540gcaggcctga tcaagtccgg ctggtccaac
tacaaggtcc tggacctgga gaccggcatg 600gacacaccga atacggaaag
cggaaacaag acacttctcg gatgggggga atacatcata 660agcaactggg
tagcccgttc cagcaagtcc ggcgaatacg aggtaaaggc cataatcggg
720gaagacaatg tatgtgttgc ccgtccatac gtaagcaaga aaccgaagat
ggatgacgtg 780gacagcaaga cgctggatga agtcgtggat atctgggaaa
actacttcta cgccaagcag 840aaagacatcg catcgtggct gaaaatctag
87088289PRTUnknownObtained from an environmental sample. 88Met Ser
Lys Lys Leu Val Ile Ser Val Ala Gly Gly Gly Ala Leu Gly1 5 10 15Ile
Gly Pro Leu Ala Phe Leu Cys Lys Ile Glu Gln Met Leu Gly Lys 20 25
30Lys Ile Pro Gln Val Ala Gln Ala Tyr Ala Gly Thr Ser Thr Gly Ala
35 40 45Ile Ile Ala Ala Gly Leu Ala Glu Gly Tyr Ser Ala His Glu Leu
Phe 50 55 60Asp Leu Tyr Lys Ser Asn Leu Ser Lys Ile Phe Thr Lys Tyr
Ser Trp65 70 75 80Tyr Lys Arg Leu Gln Pro Thr Cys Pro Thr Tyr Asp
Asn Ser Asn Leu 85 90 95Lys Lys Leu Leu Lys Asp Lys Phe Lys Gly Lys
Val Gly Asp Trp Lys 100 105 110Thr Pro Val Tyr Ile Pro Ala Thr His
Met Asn Gly Gln Ser Val Glu 115 120 125Lys Val Trp Asp Leu Gly Asp
Lys Asn Val Asp Lys Trp Phe Ala Ile 130 135 140Leu Thr Ser Thr Ala
Ala Pro Thr Tyr Phe Asp Cys Ile Tyr Asp Asp145 150 155 160Glu Lys
Asn Cys Tyr Ile Asp Gly Gly Met Trp Cys Asn Ala Pro Ile 165 170
175Asp Val Leu Asn Ala Gly Leu Ile Lys Ser Gly Trp Ser Asn Tyr Lys
180 185 190Val Leu Asp Leu Glu Thr Gly Met Asp Thr Pro Asn Thr Glu
Ser Gly 195 200 205Asn Lys Thr Leu Leu Gly Trp Gly Glu Tyr Ile Ile
Ser Asn Trp Val 210 215 220Ala Arg Ser Ser Lys Ser Gly Glu Tyr Glu
Val Lys Ala Ile Ile Gly225 230 235 240Glu Asp Asn Val Cys Val Ala
Arg Pro Tyr Val Ser Lys Lys Pro Lys 245 250 255Met Asp Asp Val Asp
Ser Lys Thr Leu Asp Glu Val Val Asp Ile Trp 260 265 270Glu Asn Tyr
Phe Tyr Ala Lys Gln Lys Asp Ile Ala Ser Trp Leu Lys 275 280 285Ile
891422DNAUnknownObtained from an environmental sample. 89atgaaaaaga
aattatgtac actggctttt gtaacagcaa tatcttctat cgctatcaca 60attccaacag
aagcacaagc ttgtggaata ggcgaagtaa tgaaacagga gaaccaagag
120cacaaacgtg tgaagagatg gtctgcggaa catccacatc atcctaatga
aagtacgcac 180ttatggattg cgcgaaatgc aattcaaata atggcccgta
atcaagataa gacggttcaa 240gaaaatgaat tacaattttt aaatactcct
gaatataagg agttatttga aagaggtctt 300tatgatgctg attaccttga
tgaatttaac gatggaggta caggtacaat cggcattgat 360gggctaatta
aaggagggtg gaaatctcat ttttacgatc ccgatacgag aaagaactat
420aaaggggaag aagaaccaac agctctctct caaggagata aatattttaa
attagcaggc 480gattacttta agaaagagga ttggaaacaa gctttctatt
atttaggtgt tgcgacgcac 540tacttcacag atgctactca gccaatgcat
gctgctaatt ttacagccgt cgacacgagt 600gctttaaagt ttcatagcgc
ttttgaaaat tatgtgacga caattcagac acagtatgaa 660gtatctgatg
gtgagggcgt atataattta gtgaattcta atgatccaaa acagtggatc
720catgaaacag cgagactcgc aaaagtggaa atcgggaaca ttaccaatga
cgagattaaa 780tctcactata ataaaggaaa caatgctctt tggcaacaag
aagttatgcc agctgtccag 840aggagtttag agaacgcaca aagaaacacg
gcgggattta ttcatttatg gtttaaaaca 900tttgttggca atactgccgc
tgaagaaatt gaaaatactg tagtgaaaga ttctaaagga 960gaagcaatac
aagataataa aaaatacttc gtagtgccaa gtgagtttct aaatagaggt
1020ttgacctttg aagtatatgc aaggaatgac tatgcactat tatctaatta
cgtagatgat 1080agtaaagttc atggtacgcc agttcagttt gtatttgata
aagataataa cggtatcctt 1140catcgaggag aaagtatact gctgaaaatg
acgcaatcta actatgataa ttacgtattt 1200ctaaactact ctaacttgac
aaactgggta catcttgcgc aacaaaaaac aaatactgca 1260cagtttaaag
tgtatccaaa tccgaataac ccatctgaat attacctata tacagatgga
1320tacccagtaa attatcaaga aaatggtaac ggaaagagct ggattgtgtt
aggaaagaaa 1380acagatacac caaaagcttg gaaatttata caggctgaat ag
142290473PRTUnknownObtained from an environmental sample. 90Met Lys
Lys Lys Leu Cys Thr Leu Ala Phe Val Thr Ala Ile Ser Ser1 5 10 15Ile
Ala Ile Thr Ile Pro Thr Glu Ala Gln Ala Cys Gly Ile Gly Glu 20 25
30Val Met Lys Gln Glu Asn Gln Glu His Lys Arg Val Lys Arg Trp Ser
35 40 45Ala Glu His Pro His His Pro Asn Glu Ser Thr His Leu Trp Ile
Ala 50 55 60Arg Asn Ala Ile Gln Ile Met Ala Arg Asn Gln Asp Lys Thr
Val Gln65 70 75 80Glu Asn Glu Leu Gln Phe Leu Asn Thr Pro Glu Tyr
Lys Glu Leu Phe 85 90 95Glu Arg Gly Leu Tyr Asp Ala Asp Tyr Leu Asp
Glu Phe Asn Asp Gly 100 105 110Gly Thr Gly Thr Ile Gly Ile Asp Gly
Leu Ile Lys Gly Gly Trp Lys 115 120 125Ser His Phe Tyr Asp Pro Asp
Thr Arg Lys Asn Tyr Lys Gly Glu Glu 130 135 140Glu Pro Thr Ala Leu
Ser Gln Gly Asp Lys Tyr Phe Lys Leu Ala Gly145 150 155 160Asp Tyr
Phe Lys Lys Glu Asp Trp Lys Gln Ala Phe Tyr Tyr Leu Gly 165 170
175Val Ala Thr His Tyr Phe Thr Asp Ala Thr Gln Pro Met His Ala Ala
180 185 190Asn Phe Thr Ala Val Asp Thr Ser Ala Leu Lys Phe His Ser
Ala Phe 195 200 205Glu Asn Tyr Val Thr Thr Ile Gln Thr Gln Tyr Glu
Val Ser Asp Gly 210 215 220Glu Gly Val Tyr Asn Leu Val Asn Ser Asn
Asp Pro Lys Gln Trp Ile225 230 235 240His Glu Thr Ala Arg Leu Ala
Lys Val Glu Ile Gly Asn Ile Thr Asn 245 250 255Asp Glu Ile Lys Ser
His Tyr Asn Lys Gly Asn Asn Ala Leu Trp Gln 260 265 270Gln Glu Val
Met Pro Ala Val Gln Arg Ser Leu Glu Asn Ala Gln Arg 275 280 285Asn
Thr Ala Gly Phe Ile His Leu Trp Phe Lys Thr Phe Val Gly Asn 290 295
300Thr Ala Ala Glu Glu Ile Glu Asn Thr Val Val Lys Asp Ser Lys
Gly305 310 315 320Glu Ala Ile Gln Asp Asn Lys Lys Tyr Phe Val Val
Pro Ser Glu Phe 325 330 335Leu Asn Arg Gly Leu Thr Phe Glu Val Tyr
Ala Arg Asn Asp Tyr Ala 340 345 350Leu Leu Ser Asn Tyr Val Asp Asp
Ser Lys Val His Gly Thr Pro Val 355 360 365Gln Phe Val Phe Asp Lys
Asp Asn Asn Gly Ile Leu His Arg Gly Glu 370 375 380Ser Ile Leu Leu
Lys Met Thr Gln Ser Asn Tyr Asp Asn Tyr Val Phe385 390 395 400Leu
Asn Tyr Ser Asn Leu Thr Asn Trp Val His Leu Ala Gln Gln Lys 405 410
415Thr Asn Thr Ala Gln Phe Lys Val Tyr Pro Asn Pro Asn Asn Pro Ser
420 425 430Glu Tyr Tyr Leu Tyr Thr Asp Gly Tyr Pro Val Asn Tyr Gln
Glu Asn 435 440 445Gly Asn Gly Lys Ser Trp Ile Val Leu Gly Lys Lys
Thr Asp Thr Pro 450 455 460Lys Ala Trp Lys Phe Ile Gln Ala Glu465
470911035DNAUnknownObtained from an environmental sample.
91atgacaaccc aatttagaaa cctgatcttt gagggcggcg gtgtaaaggg cattgcttac
60gtcggagcaa tgcagattct tgaaaatcgt ggtgtattac aagatattca ccgagtcgga
120ggttgtagtg cgggtgcgat taacgcgctg atttttgcgc tgggttacac
agtccgtgag 180caaaaagaga tcttacaaat taccgatttt aaccagttta
tggataactc gtggggtgtt 240attcgggata ttcgcaggct tgcgagagaa
tttggctgga ataagggtaa cttctttaat 300acctggatag gtgatttgat
tcatcgtcgt ttgggtaatc gccgagccac gttcaaagat 360ctgcaaaagg
caaagcttcc tgatctttat gtcatcggta ctaatctgtc tacagggttt
420gcagaggttt tttctgccga aagacacccc gatatggagc tggcgacagc
ggtgcgtatc 480tccatgtcga taccgctgtt ctttgcggcc gtgcgtcacg
gtgatcgaca agatgtgtat 540gtcgatgggg gtgtgcagct taactacccg
atcaagctgt ttgatcgaac tcgttatatt 600gacctcgcca aagatccggg
tgctgctcgc cacacgggtt attacaataa agagaatgct 660cgttttcagc
ttgagcgacc gggccacagt ccttatgtgt acaatcgcca aacattaggc
720ttgcgtcttg acagtcgtga agagatagcg ctgtttcgtt acgacgaacc
tcttcagggt 780aaacccatta agtccttcac tgactacgct cgacaacttt
ttggtgcgct gaagaatgca 840caggaaaaca ttcacctaca tggcgatgat
tggcagcgca cggtctatat cgatacattg 900gatgtgggta cgacggattt
caatctttct gatgcaacca agcaagcact gattgaacag 960ggaattaacg
gcaccgaaaa ttatttcgag tggtttgata atccgtttga gaagcctgtg
1020aatagagtgg agtaa 103592344PRTUnknownObtained from an
environmental sample. 92Met Thr Thr Gln Phe Arg Asn Leu Ile Phe Glu
Gly Gly Gly Val Lys1 5 10 15Gly Ile Ala Tyr Val Gly Ala Met Gln Ile
Leu Glu Asn Arg Gly Val 20 25 30Leu Gln Asp Ile His Arg Val Gly Gly
Cys Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ala Leu Gly Tyr
Thr Val Arg Glu Gln Lys Glu Ile 50 55 60Leu Gln Ile Thr Asp Phe Asn
Gln Phe Met Asp Asn Ser Trp Gly Val65 70 75 80Ile Arg Asp Ile Arg
Arg Leu Ala Arg Glu Phe Gly Trp Asn Lys Gly 85 90 95Asn Phe Phe Asn
Thr Trp Ile Gly Asp Leu Ile His Arg Arg Leu Gly 100 105 110Asn Arg
Arg Ala Thr Phe Lys Asp Leu Gln Lys Ala Lys Leu Pro Asp 115 120
125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ala Glu Val Phe
130 135 140Ser Ala Glu Arg His Pro Asp Met Glu Leu Ala Thr Ala Val
Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala Ala Val
Arg His Gly Asp Arg 165 170 175Gln Asp Val Tyr Val Asp Gly Gly Val
Gln Leu Asn Tyr Pro Ile Lys 180 185 190Leu Phe Asp Arg Thr Arg Tyr
Ile Asp Leu Ala Lys Asp Pro Gly Ala 195 200 205Ala Arg His Thr Gly
Tyr Tyr Asn Lys Glu Asn Ala Arg Phe Gln Leu 210 215 220Glu Arg Pro
Gly His Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230 235
240Leu Arg Leu Asp Ser Arg Glu Glu Ile Ala Leu Phe Arg Tyr Asp Glu
245 250 255Pro Leu Gln Gly Lys Pro Ile Lys Ser Phe Thr Asp Tyr Ala
Arg Gln 260 265 270Leu Phe Gly Ala Leu Lys Asn Ala Gln Glu Asn Ile
His Leu His Gly 275 280 285Asp Asp Trp Gln Arg Thr Val Tyr Ile Asp
Thr Leu Asp Val Gly Thr 290 295 300Thr Asp Phe Asn Leu Ser Asp Ala
Thr Lys Gln Ala Leu Ile Glu Gln305 310 315 320Gly Ile Asn Gly Thr
Glu Asn Tyr Phe Glu Trp Phe Asp Asn Pro Phe 325 330 335Glu Lys Pro
Val Asn Arg Val Glu 34093963DNAUnknownObtained from an
environmental sample. 93gtgattactt tgataaaaaa atgtttatta gtattgacga
tgactctatt atcaggggtt 60ttcgtaccgc tgcagccatc atatgctact gaaaattatc
caaatgattt taaactgttg 120caacataatg tatttttatt gcctgaatca
gtttcttatt ggggtcagga cgaacgtgca 180gattatatga gtaatgcaga
ttactttaag ggacatgatg ctctgctctt aaatgagctt 240tttgacaatg
gaaattcgaa cgtgctgcta atgaacttat ccaaggaata tacatatcaa
300acgccagtgc ttggccgttc gatgagtgga tgggatgaaa ctagaggaag
ctattctaat 360tttgtacccg aagatggtgg tgtagcaatt atcagtaaat
ggccaatcgt ggagaaaata 420cagcatgttt acgcgaatgg ttgcggtgca
gactattatg caaataaagg atttgtttat 480gcaaaagtac aaaaagggga
taaattctat catcttatca gcactcatgc tcaagccgaa 540gataccgggt
gtgatcaggg tgaaggagca gaaattcgtc attcacagtt tcaagaaatc
600aacgacttta ttaaaaataa aaacattccg aaagatgaag tggtatttat
tggtggtgac 660tttaatgtga tgaagagtga cacaacagag tacaatagca
tgttatcaac attaaatgtc 720aatgcgccta ccgaatattt agggcataac
tctacttggg acccagaaac gaacagcatt 780acaggttaca attaccctga
ttatgcgcca cagcatttag attatatttt tgtggaaaaa 840gatcataaac
aaccaagttc atgggtaaat gaaacgatta ctccgaagtc tccaacttgg
900aaggcaatct atgagtataa tgattattcc gatcactatc ctgttaaagc
atacgtaaaa 960taa 96394320PRTUnknownObtained from an environmental
sample. 94Met Ile Thr Leu Ile Lys Lys Cys Leu Leu Val Leu Thr Met
Thr Leu1 5 10 15Leu Ser Gly Val Phe Val Pro Leu Gln Pro Ser Tyr Ala
Thr Glu Asn 20 25 30Tyr Pro Asn Asp Phe Lys Leu Leu Gln His Asn Val
Phe Leu Leu Pro 35 40 45Glu Ser Val Ser Tyr Trp Gly Gln Asp Glu Arg
Ala Asp Tyr Met Ser 50 55 60Asn Ala Asp Tyr Phe Lys Gly His Asp Ala
Leu Leu Leu Asn Glu Leu65 70 75 80Phe Asp Asn Gly Asn Ser Asn Val
Leu Leu Met Asn Leu Ser Lys Glu 85 90 95Tyr Thr Tyr Gln Thr Pro Val
Leu Gly Arg Ser Met Ser Gly Trp Asp 100 105 110Glu Thr Arg Gly Ser
Tyr Ser Asn Phe Val Pro Glu Asp Gly Gly Val 115 120 125Ala Ile Ile
Ser Lys Trp Pro Ile Val Glu Lys Ile Gln His Val Tyr 130 135 140Ala
Asn Gly Cys Gly
Ala Asp Tyr Tyr Ala Asn Lys Gly Phe Val Tyr145 150 155 160Ala Lys
Val Gln Lys Gly Asp Lys Phe Tyr His Leu Ile Ser Thr His 165 170
175Ala Gln Ala Glu Asp Thr Gly Cys Asp Gln Gly Glu Gly Ala Glu Ile
180 185 190Arg His Ser Gln Phe Gln Glu Ile Asn Asp Phe Ile Lys Asn
Lys Asn 195 200 205Ile Pro Lys Asp Glu Val Val Phe Ile Gly Gly Asp
Phe Asn Val Met 210 215 220Lys Ser Asp Thr Thr Glu Tyr Asn Ser Met
Leu Ser Thr Leu Asn Val225 230 235 240Asn Ala Pro Thr Glu Tyr Leu
Gly His Asn Ser Thr Trp Asp Pro Glu 245 250 255Thr Asn Ser Ile Thr
Gly Tyr Asn Tyr Pro Asp Tyr Ala Pro Gln His 260 265 270Leu Asp Tyr
Ile Phe Val Glu Lys Asp His Lys Gln Pro Ser Ser Trp 275 280 285Val
Asn Glu Thr Ile Thr Pro Lys Ser Pro Thr Trp Lys Ala Ile Tyr 290 295
300Glu Tyr Asn Asp Tyr Ser Asp His Tyr Pro Val Lys Ala Tyr Val
Lys305 310 315 320951038DNAUnknownObtained from an environmental
sample. 95atggcttcac aattcaggaa tctggtattt gaaggaggtg gtgtaaaagg
gattgcgtac 60ataggtgcga tgcaggtgct ggatcagcgc ggttatttgg gtgataacat
caaacgcgtt 120ggtggaacca gtgcaggtgc cataaatgcg ctgatttatt
cgttaggata tgacatccac 180gaacaacaag agatactgaa ctctacagat
tttaaaaagt ttatggataa ctcttttgga 240tttgtgaggg atttcagaag
gctatggaat gaatttggat ggaatagagg agactttttt 300cttaaatggt
caggtgagct gatcaaaaat aaattgggca cctcaaaagc cacctttcag
360gatttgaagg atgccggtca gccagatttg tatgtaattg gaacaaattt
atcgacgggg 420ttttccgaga ctttttcata tgaacgtcac cccgatatga
ctcttgcaga agccgtaaga 480atcagtatgt cgcttccgct gtttttcagg
gctgtgcggt tgggcgacag gaatgatgta 540tatgtggatg gtggggttca
gctcaattac ccggtaaaac tatttgatcg tgaaaaatat 600attgatatgg
ataatgaggc ggctgcagca cgatttactg attattacaa caaagaaaat
660gccagatttt cgctccagcg gcctggacga agcccctatg tatataatcg
tcaaaccctt 720ggtttgagac tggatacagc cgaagaaatt gcgcttttca
ggtacgatga acccattcag 780gggaaagaga tcaaacggtt tccggaatat
gcaaaggctc tgatcggcgc actaatgcag 840gtgcaggaaa acatacatct
ccacagtgac gactggcagc gtacgctgta tatcaatacc 900ctggatgtaa
aaaccacaga ttttgaatta accgatgaga aaaaaaagga actggtagaa
960cagggaatcc ttggcgcgga aacctatttc aaatggtttg aagacaggga
tgaagtagtt 1020gtaaaccgcc ttgcttag 103896345PRTUnknownObtained from
an environmental sample. 96Met Ala Ser Gln Phe Arg Asn Leu Val Phe
Glu Gly Gly Gly Val Lys1 5 10 15Gly Ile Ala Tyr Ile Gly Ala Met Gln
Val Leu Asp Gln Arg Gly Tyr 20 25 30Leu Gly Asp Asn Ile Lys Arg Val
Gly Gly Thr Ser Ala Gly Ala Ile 35 40 45Asn Ala Leu Ile Tyr Ser Leu
Gly Tyr Asp Ile His Glu Gln Gln Glu 50 55 60Ile Leu Asn Ser Thr Asp
Phe Lys Lys Phe Met Asp Asn Ser Phe Gly65 70 75 80Phe Val Arg Asp
Phe Arg Arg Leu Trp Asn Glu Phe Gly Trp Asn Arg 85 90 95Gly Asp Phe
Phe Leu Lys Trp Ser Gly Glu Leu Ile Lys Asn Lys Leu 100 105 110Gly
Thr Ser Lys Ala Thr Phe Gln Asp Leu Lys Asp Ala Gly Gln Pro 115 120
125Asp Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ser Glu Thr
130 135 140Phe Ser Tyr Glu Arg His Pro Asp Met Thr Leu Ala Glu Ala
Val Arg145 150 155 160Ile Ser Met Ser Leu Pro Leu Phe Phe Arg Ala
Val Arg Leu Gly Asp 165 170 175Arg Asn Asp Val Tyr Val Asp Gly Gly
Val Gln Leu Asn Tyr Pro Val 180 185 190Lys Leu Phe Asp Arg Glu Lys
Tyr Ile Asp Met Asp Asn Glu Ala Ala 195 200 205Ala Ala Arg Phe Thr
Asp Tyr Tyr Asn Lys Glu Asn Ala Arg Phe Ser 210 215 220Leu Gln Arg
Pro Gly Arg Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu225 230 235
240Gly Leu Arg Leu Asp Thr Ala Glu Glu Ile Ala Leu Phe Arg Tyr Asp
245 250 255Glu Pro Ile Gln Gly Lys Glu Ile Lys Arg Phe Pro Glu Tyr
Ala Lys 260 265 270Ala Leu Ile Gly Ala Leu Met Gln Val Gln Glu Asn
Ile His Leu His 275 280 285Ser Asp Asp Trp Gln Arg Thr Leu Tyr Ile
Asn Thr Leu Asp Val Lys 290 295 300Thr Thr Asp Phe Glu Leu Thr Asp
Glu Lys Lys Lys Glu Leu Val Glu305 310 315 320Gln Gly Ile Leu Gly
Ala Glu Thr Tyr Phe Lys Trp Phe Glu Asp Arg 325 330 335Asp Glu Val
Val Val Asn Arg Leu Ala 340 345971422DNAUnknownObtained from an
environmental sample. 97atgaaaagga aactatgtac atgggctctc gtaacagcaa
tagcttctag tactgcggta 60attccaacag cagcagaagc ttgtggatta ggagaagtaa
tcaaacaaga gaatcaagag 120cacaaacgtg tgaaaagatg gtctgcggag
catccgcatc attcacatga aagtacccat 180ttatggattg cacaaaatgc
gattcaaatt atgagccgta atcaagataa gacggttcaa 240gaaaatgaat
tacaattttt aaatacccct gaatataagg agttatttga aagaggtctt
300tatgatgctg attaccttga tgaatttaac gatggaggta caggtataat
cggcattgat 360gggctaattc gaggagggtg gaaatctcat ttctacgatc
ccgatacaag aaagaactat 420aaaggggagg aagaaccaac agctctttct
caaggagata aatattttaa attagcaggt 480gaatacttta agaagaatga
ttggaaacag gctttctatt atttaggtgt tgcgacgcac 540tactttacag
atgctactca gccaatgcat gctgctaatt ttacagctgt cgacaggagt
600gctataaagt ttcatagtgc ttttgaagat tatgtgacga caattcagga
acagtttaaa 660gtatcagatg gagagggaaa atataattta gtaaattcta
atgatccgaa acagtggatc 720catgaaacag cgagactcgc aaaagtggaa
atcgggaaca ttaccaatga tgtgattaaa 780tctcactata ataaaggaaa
caatgctctt tggcagcaag aagttatgcc agctgttcag 840agaagtttag
aacaagccca aagaaatacg gcgggattta ttcatttatg gtttaaaaca
900tatgttggaa aaacagctgc tgaagatatt gaaaatacta tagtgaaaga
ttctagggga 960gaagcaatac aagagaataa aaaatacttt gtagtaccaa
gtgagttttt aaatagaggc 1020ttaacatttg aagtgtatgc tgcttatgac
tatgcgttat tatctaacca tgtggatgat 1080aataatattc atggtacacc
ggttcaaatt gtatttgata aagaaaataa tgggatcctt 1140catcaaggag
aaagtgcatt gttaaagatg acacaatcca actacgataa ttatgtattt
1200ctaaattatt ctatcataac aaattgggta catcttgcaa aaagagaaaa
caatactgca 1260cagtttaaag tgtatccaaa tccaaataat ccaactgaat
atttcatata tacagatggc 1320tatccagtta attatcaaga aaaaggtaaa
gagaaaagct ggattgtttt aggaaagaaa 1380acggataaac caaaagcatg
gaaatttata caggcggaat aa 142298473PRTUnknownObtained from an
environmental sample. 98Met Lys Arg Lys Leu Cys Thr Trp Ala Leu Val
Thr Ala Ile Ala Ser1 5 10 15Ser Thr Ala Val Ile Pro Thr Ala Ala Glu
Ala Cys Gly Leu Gly Glu 20 25 30Val Ile Lys Gln Glu Asn Gln Glu His
Lys Arg Val Lys Arg Trp Ser 35 40 45Ala Glu His Pro His His Ser His
Glu Ser Thr His Leu Trp Ile Ala 50 55 60Gln Asn Ala Ile Gln Ile Met
Ser Arg Asn Gln Asp Lys Thr Val Gln65 70 75 80Glu Asn Glu Leu Gln
Phe Leu Asn Thr Pro Glu Tyr Lys Glu Leu Phe 85 90 95Glu Arg Gly Leu
Tyr Asp Ala Asp Tyr Leu Asp Glu Phe Asn Asp Gly 100 105 110Gly Thr
Gly Ile Ile Gly Ile Asp Gly Leu Ile Arg Gly Gly Trp Lys 115 120
125Ser His Phe Tyr Asp Pro Asp Thr Arg Lys Asn Tyr Lys Gly Glu Glu
130 135 140Glu Pro Thr Ala Leu Ser Gln Gly Asp Lys Tyr Phe Lys Leu
Ala Gly145 150 155 160Glu Tyr Phe Lys Lys Asn Asp Trp Lys Gln Ala
Phe Tyr Tyr Leu Gly 165 170 175Val Ala Thr His Tyr Phe Thr Asp Ala
Thr Gln Pro Met His Ala Ala 180 185 190Asn Phe Thr Ala Val Asp Arg
Ser Ala Ile Lys Phe His Ser Ala Phe 195 200 205Glu Asp Tyr Val Thr
Thr Ile Gln Glu Gln Phe Lys Val Ser Asp Gly 210 215 220Glu Gly Lys
Tyr Asn Leu Val Asn Ser Asn Asp Pro Lys Gln Trp Ile225 230 235
240His Glu Thr Ala Arg Leu Ala Lys Val Glu Ile Gly Asn Ile Thr Asn
245 250 255Asp Val Ile Lys Ser His Tyr Asn Lys Gly Asn Asn Ala Leu
Trp Gln 260 265 270Gln Glu Val Met Pro Ala Val Gln Arg Ser Leu Glu
Gln Ala Gln Arg 275 280 285Asn Thr Ala Gly Phe Ile His Leu Trp Phe
Lys Thr Tyr Val Gly Lys 290 295 300Thr Ala Ala Glu Asp Ile Glu Asn
Thr Ile Val Lys Asp Ser Arg Gly305 310 315 320Glu Ala Ile Gln Glu
Asn Lys Lys Tyr Phe Val Val Pro Ser Glu Phe 325 330 335Leu Asn Arg
Gly Leu Thr Phe Glu Val Tyr Ala Ala Tyr Asp Tyr Ala 340 345 350Leu
Leu Ser Asn His Val Asp Asp Asn Asn Ile His Gly Thr Pro Val 355 360
365Gln Ile Val Phe Asp Lys Glu Asn Asn Gly Ile Leu His Gln Gly Glu
370 375 380Ser Ala Leu Leu Lys Met Thr Gln Ser Asn Tyr Asp Asn Tyr
Val Phe385 390 395 400Leu Asn Tyr Ser Ile Ile Thr Asn Trp Val His
Leu Ala Lys Arg Glu 405 410 415Asn Asn Thr Ala Gln Phe Lys Val Tyr
Pro Asn Pro Asn Asn Pro Thr 420 425 430Glu Tyr Phe Ile Tyr Thr Asp
Gly Tyr Pro Val Asn Tyr Gln Glu Lys 435 440 445Gly Lys Glu Lys Ser
Trp Ile Val Leu Gly Lys Lys Thr Asp Lys Pro 450 455 460Lys Ala Trp
Lys Phe Ile Gln Ala Glu465 470991053DNAUnknownObtained from an
environmental sample. 99atggcaaagc gttttattct ttcgatcgat ggtggtggca
ttcgcgggat catcccggcg 60gccatcctgg tggagctggc caagcggttg gaggggctgc
cgcttcacaa ggcattcgac 120atgatcgccg ggacatccac cggcggcatc
attgcggcgg ggctgacatg cccgcatcct 180gacgatgagg agacggcggc
gtgcacgccg accgatcttc tcaagcttta tgtcgatcac 240ggcggcaaga
tcttcgagaa aaacccgatc ctcggcctca tcaacccatt cggcctcaac
300gatccgcgct accagccaga tgagctggaa aacaggctga aggcgcagct
cggcttgacg 360gcgacgctcg ataaagggct caccaaggtg ctgatcacgg
cctatgatat ccagcagcgg 420caggcgctgt tcatggcaaa caccgacaac
gagaacagca atttccgcta ctgggaggca 480gcgcgggcga catcggccgc
acccacctat tttccgccgg cgctgatcga aagggttggc 540gagaagaaca
aggacaagcg cttcgtgcca ttgatcgacg gcggcgtctt cgccaacgat
600cctatccttg ccgcctatgt ggaggcgcga aagcagaaat ggggcaatga
cgagctcgtt 660ttcctgtcgc ttggtaccgg ccagcaaaac cgcccgatcg
cctatcagga ggccaagggc 720tggggcattt taggctggat gcagccgtct
catgacacgc cgctgatctc gatcctgatg 780cagggacagg cgagcaccgc
ctcctatcag gccaatgcgc tgctcaatcc gcccggcacc 840aagatcgact
attcgaccgt ggtgacgaag gacaacgcgg cttcgctcag ctatttccgt
900ctcgaccggc agctgagctc gaaggagaac gacgcgctgg acgacgcatc
gcccgaaaac 960atcagggcgc tgaaggcaat cgccgcgcaa atcatcaagg
ataacgcgcc ggcgctcgac 1020gaaatcgcca aacgcatcct ggccaaccaa taa
1053100350PRTUnknownObtained from an environmental sample. 100Met
Ala Lys Arg Phe Ile Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly1 5 10
15Ile Ile Pro Ala Ala Ile Leu Val Glu Leu Ala Lys Arg Leu Glu Gly
20 25 30Leu Pro Leu His Lys Ala Phe Asp Met Ile Ala Gly Thr Ser Thr
Gly 35 40 45Gly Ile Ile Ala Ala Gly Leu Thr Cys Pro His Pro Asp Asp
Glu Glu 50 55 60Thr Ala Ala Cys Thr Pro Thr Asp Leu Leu Lys Leu Tyr
Val Asp His65 70 75 80Gly Gly Lys Ile Phe Glu Lys Asn Pro Ile Leu
Gly Leu Ile Asn Pro 85 90 95Phe Gly Leu Asn Asp Pro Arg Tyr Gln Pro
Asp Glu Leu Glu Asn Arg 100 105 110Leu Lys Ala Gln Leu Gly Leu Thr
Ala Thr Leu Asp Lys Gly Leu Thr 115 120 125Lys Val Leu Ile Thr Ala
Tyr Asp Ile Gln Gln Arg Gln Ala Leu Phe 130 135 140Met Ala Asn Thr
Asp Asn Glu Asn Ser Asn Phe Arg Tyr Trp Glu Ala145 150 155 160Ala
Arg Ala Thr Ser Ala Ala Pro Thr Tyr Phe Pro Pro Ala Leu Ile 165 170
175Glu Arg Val Gly Glu Lys Asn Lys Asp Lys Arg Phe Val Pro Leu Ile
180 185 190Asp Gly Gly Val Phe Ala Asn Asp Pro Ile Leu Ala Ala Tyr
Val Glu 195 200 205Ala Arg Lys Gln Lys Trp Gly Asn Asp Glu Leu Val
Phe Leu Ser Leu 210 215 220Gly Thr Gly Gln Gln Asn Arg Pro Ile Ala
Tyr Gln Glu Ala Lys Gly225 230 235 240Trp Gly Ile Leu Gly Trp Met
Gln Pro Ser His Asp Thr Pro Leu Ile 245 250 255Ser Ile Leu Met Gln
Gly Gln Ala Ser Thr Ala Ser Tyr Gln Ala Asn 260 265 270Ala Leu Leu
Asn Pro Pro Gly Thr Lys Ile Asp Tyr Ser Thr Val Val 275 280 285Thr
Lys Asp Asn Ala Ala Ser Leu Ser Tyr Phe Arg Leu Asp Arg Gln 290 295
300Leu Ser Ser Lys Glu Asn Asp Ala Leu Asp Asp Ala Ser Pro Glu
Asn305 310 315 320Ile Arg Ala Leu Lys Ala Ile Ala Ala Gln Ile Ile
Lys Asp Asn Ala 325 330 335Pro Ala Leu Asp Glu Ile Ala Lys Arg Ile
Leu Ala Asn Gln 340 345 350101996DNABacteria 101ttgtcgctcg
tcgcgtcgct ccgccgcgcc cccggcgccg ccctggccct cgcgcttgcc 60gccgccaccc
tggccgtgac cgcgcagggc gcgaccgccg cccccgccgc ggccgccgcc
120gaggccccgc ggctcaaggt gctcacgtac aacacgttcc tgttctcgaa
gacgctctac 180ccgaactggg gccaggacca ccgggccaag gcgatcccca
ccgccccctt ctaccagggc 240caggacgtcg tggtcctcca ggaggccttc
gacaactccg cgtcggacgc cctcaaggcg 300aactccgccg gccagtaccc
ctaccagacc cccgtcgtgg gccgcggcac cggcggctgg 360gacgccaccg
gcgggtccta ctcctcgacc acccccgagg acggcggcgt gacgatcctc
420agcaagtggc cgatcgtccg caaggagcag tacgtctaca aggacgcgtg
cggcgccgac 480tggtggtcca acaagggctt cgcctacgtc gtgctcaacg
tgaacggcag caaggtgcac 540gtcctcggca cccacgccca gtccaccgac
ccgggctgct cggcgggcga ggcggtgcag 600atgcggagcc gccagttcaa
ggcgatcgac gccttcctcg acgccaagaa catcccggcg 660ggcgagcagg
tgatcgtcgc cggcgacatg aacgtcgact cgcgcacgcc cgagtacggc
720accatgctcg ccgacgccgg tctggcggcg gccgacgcgc gcaccggcca
cccgtactcc 780ttcgacaccg agctgaactc gatcgcctcc gagcgctacc
cggacgaccc gcgcgaggac 840ctcgattacg tcctctaccg cgccgggaac
gcccgccccg ccaactggac caacaacgtg 900gtcctggaga agagcgcccc
gtggaccgtc tccagctggg gcaagagcta cacctacacc 960aacctctccg
accactaccc ggtcaccggc ttctga 996102331PRTBacteriaSIGNAL(1)...(39)
102Leu Ser Leu Val Ala Ser Leu Arg Arg Ala Pro Gly Ala Ala Leu Ala1
5 10 15Leu Ala Leu Ala Ala Ala Thr Leu Ala Val Thr Ala Gln Gly Ala
Thr 20 25 30Ala Ala Pro Ala Ala Ala Ala Ala Glu Ala Pro Arg Leu Lys
Val Leu 35 40 45Thr Tyr Asn Thr Phe Leu Phe Ser Lys Thr Leu Tyr Pro
Asn Trp Gly 50 55 60Gln Asp His Arg Ala Lys Ala Ile Pro Thr Ala Pro
Phe Tyr Gln Gly65 70 75 80Gln Asp Val Val Val Leu Gln Glu Ala Phe
Asp Asn Ser Ala Ser Asp 85 90 95Ala Leu Lys Ala Asn Ser Ala Gly Gln
Tyr Pro Tyr Gln Thr Pro Val 100 105 110Val Gly Arg Gly Thr Gly Gly
Trp Asp Ala Thr Gly Gly Ser Tyr Ser 115 120 125Ser Thr Thr Pro Glu
Asp Gly Gly Val Thr Ile Leu Ser Lys Trp Pro 130 135 140Ile Val Arg
Lys Glu Gln Tyr Val Tyr Lys Asp Ala Cys Gly Ala Asp145 150 155
160Trp Trp Ser Asn Lys Gly Phe Ala Tyr Val Val Leu Asn Val Asn Gly
165 170 175Ser Lys Val His Val Leu Gly Thr His Ala Gln Ser Thr Asp
Pro Gly 180 185 190Cys Ser Ala Gly Glu Ala Val Gln Met Arg Ser Arg
Gln Phe Lys Ala 195 200 205Ile Asp Ala Phe Leu Asp Ala Lys Asn Ile
Pro Ala Gly Glu Gln Val 210 215 220Ile Val Ala Gly Asp Met Asn Val
Asp Ser Arg Thr Pro Glu Tyr Gly225 230 235 240Thr Met Leu Ala Asp
Ala Gly Leu Ala Ala Ala Asp Ala Arg Thr Gly 245 250 255His Pro Tyr
Ser Phe Asp Thr Glu Leu Asn Ser Ile Ala Ser Glu Arg 260 265 270Tyr
Pro Asp Asp Pro Arg Glu Asp Leu Asp Tyr Val Leu Tyr Arg Ala 275 280
285Gly Asn Ala Arg Pro Ala Asn Trp Thr Asn Asn Val Val Leu Glu Lys
290 295 300Ser Ala Pro Trp Thr Val Ser Ser Trp Gly Lys Ser Tyr Thr
Tyr
Thr305 310 315 320Asn Leu Ser Asp His Tyr Pro Val Thr Gly Phe 325
3301032205DNAUnknownObtained from an environmental sample.
103atgagcgaga agaaggagat tcgcgttgcg ttgatcatgg ggggtggcgt
cagcctcggc 60agtttttcgg gtggtgcgct tctcaagacc atcgagctgc tgcagcacac
tgcccgcggt 120ccggcgaaga tcgatgtcgt gaccggtgcc tcggcgggaa
gcatgacgct gggcgtagtc 180atctaccacc tcatgcgggg atcgtcgacc
gatgagattc tccgcgatct gaggcggtcg 240tgggtggaaa tgatctcgtt
cgacggcctc tgtccgccga acctgtcccg tcacgacaag 300ccgagcctgt
tttccgatga gatcgtccgg aagatcgcgg ccaccgtcat cgatatgggg
360cgcaagctcg aggcggctcc tcatccgctt ttcgccgacg aactcgtagc
ctcgttcgca 420ctgacgaacc tgaacggcat ccccgcccgt acggagggcc
agctcatccg gcaggcaaag 480ggaggcggag ggtccgagaa gggctcgaaa
tccgttttcg ccgacgccgt gcagactacc 540tttcaccacg acgtgatgcg
attcgtggtg cggcgcgatc acaacgggca aggcagcctg 600ttcgacagcc
gttaccgggc acgcatactc cctccatgga atgttgggaa gggcggcgat
660gcatgggaag cctttcgcac ggcggctgtt gcctcggggg cgtttccggc
cgcatttcct 720cccgtcgaga tcagccgcaa ccgcgacgaa ttcaacatct
ggcccgatcg catcgaggac 780cagaaggcat ttacgttcga ttacgtggac
ggcggggtac ttcgcaacga acccctccgg 840gaggcgattc acctggccgc
gctgcgcgat gagggagcga cggacatcga gcgtgtgttc 900atcctcatcg
acccgaacat cagcggcacc ggcgaggtct tcccgctctc ctataaccag
960cagatgcgga tcaagccgaa ctacgattcc aacggcgacg tccgacagta
cgatctcgat 1020gtgccggact acaccggcaa tctgatcggg gcgatcggtc
ggctgggttc ggtgatcgtc 1080gggcaggcga cgttccgcga ctggctcaag
gctgccaaag tgaacagcca gatcgagtgg 1140cgacgggaat tgctgcccat
tctccgcgac ctgaacccga accccgggga ggaggcgcgc 1200aggggcgtga
acgggatgat cgacaagatc taccggcaaa agtatcagcg cgccctcgag
1260tcaaagagcg ttccggtcga ggaggtggaa cggcgcgttg ccgaagacat
cgaacgggac 1320ctggcgcggc gccgttcgga ggccggcgac aacgacttca
ttgcccggct cctcctgctc 1380gtcgacctga tcggcaacct gcgtgagaag
cagaagctga acatggtggc gatcaccccc 1440gcttccgcgc cgcacaacga
cgggcgcccc ttgccgctgg ccggcaattt tatgttcagc 1500ttcggggggt
tcttcaggga ggagtacagg caatacgact tctcggtcgg cgaattcgca
1560gcatggaacg tcctgagcac gccggcctcc gagacgccct ttcttgccga
gaccgccccg 1620aaaccgcccg cccgacctcc ccagccgccg gcaatcaatc
ctacctaccg ctcactcggc 1680ccgcccatcc agcagcggtt cgaggagttc
gttcgtgggc acgttcgcgc ctttatcgct 1740tcggtcgctc cgctgggaac
gagagggatc gtcacgggca agattggcgg aaagcttcga 1800acgatgctga
tggcctcgcg caacgggaaa tcagagtact tccggcttcg cctctccggc
1860gttgacgggc tctacctccg aggctccaag ggccgcaacc tgagggcggt
taacggatcg 1920atcgacacgg tcgtcggcgt ctatatcgac gaggaagatc
agcaccgcga tgagtttttc 1980ggtccccatg tcttcggcgc gaacggctca
ggctttacga tggaactatg ggagtcccgc 2040ggttttttcg ggcgtgatcg
tcgcgtcgct gtgatcgagt tggagaacaa ccccggcggg 2100ttcgcaatcg
ccgccggatg caggcggcgg cccggcgtgg tgctggatat ggccaggcgt
2160aacgggcagc cactgcggac ggtggatgtg atggaatttg cgtga
2205104734PRTUnknownObtained from an environmental sample. 104Met
Ser Glu Lys Lys Glu Ile Arg Val Ala Leu Ile Met Gly Gly Gly1 5 10
15Val Ser Leu Gly Ser Phe Ser Gly Gly Ala Leu Leu Lys Thr Ile Glu
20 25 30Leu Leu Gln His Thr Ala Arg Gly Pro Ala Lys Ile Asp Val Val
Thr 35 40 45Gly Ala Ser Ala Gly Ser Met Thr Leu Gly Val Val Ile Tyr
His Leu 50 55 60Met Arg Gly Ser Ser Thr Asp Glu Ile Leu Arg Asp Leu
Arg Arg Ser65 70 75 80Trp Val Glu Met Ile Ser Phe Asp Gly Leu Cys
Pro Pro Asn Leu Ser 85 90 95Arg His Asp Lys Pro Ser Leu Phe Ser Asp
Glu Ile Val Arg Lys Ile 100 105 110Ala Ala Thr Val Ile Asp Met Gly
Arg Lys Leu Glu Ala Ala Pro His 115 120 125Pro Leu Phe Ala Asp Glu
Leu Val Ala Ser Phe Ala Leu Thr Asn Leu 130 135 140Asn Gly Ile Pro
Ala Arg Thr Glu Gly Gln Leu Ile Arg Gln Ala Lys145 150 155 160Gly
Gly Gly Gly Ser Glu Lys Gly Ser Lys Ser Val Phe Ala Asp Ala 165 170
175Val Gln Thr Thr Phe His His Asp Val Met Arg Phe Val Val Arg Arg
180 185 190Asp His Asn Gly Gln Gly Ser Leu Phe Asp Ser Arg Tyr Arg
Ala Arg 195 200 205Ile Leu Pro Pro Trp Asn Val Gly Lys Gly Gly Asp
Ala Trp Glu Ala 210 215 220Phe Arg Thr Ala Ala Val Ala Ser Gly Ala
Phe Pro Ala Ala Phe Pro225 230 235 240Pro Val Glu Ile Ser Arg Asn
Arg Asp Glu Phe Asn Ile Trp Pro Asp 245 250 255Arg Ile Glu Asp Gln
Lys Ala Phe Thr Phe Asp Tyr Val Asp Gly Gly 260 265 270Val Leu Arg
Asn Glu Pro Leu Arg Glu Ala Ile His Leu Ala Ala Leu 275 280 285Arg
Asp Glu Gly Ala Thr Asp Ile Glu Arg Val Phe Ile Leu Ile Asp 290 295
300Pro Asn Ile Ser Gly Thr Gly Glu Val Phe Pro Leu Ser Tyr Asn
Gln305 310 315 320Gln Met Arg Ile Lys Pro Asn Tyr Asp Ser Asn Gly
Asp Val Arg Gln 325 330 335Tyr Asp Leu Asp Val Pro Asp Tyr Thr Gly
Asn Leu Ile Gly Ala Ile 340 345 350Gly Arg Leu Gly Ser Val Ile Val
Gly Gln Ala Thr Phe Arg Asp Trp 355 360 365Leu Lys Ala Ala Lys Val
Asn Ser Gln Ile Glu Trp Arg Arg Glu Leu 370 375 380Leu Pro Ile Leu
Arg Asp Leu Asn Pro Asn Pro Gly Glu Glu Ala Arg385 390 395 400Arg
Gly Val Asn Gly Met Ile Asp Lys Ile Tyr Arg Gln Lys Tyr Gln 405 410
415Arg Ala Leu Glu Ser Lys Ser Val Pro Val Glu Glu Val Glu Arg Arg
420 425 430Val Ala Glu Asp Ile Glu Arg Asp Leu Ala Arg Arg Arg Ser
Glu Ala 435 440 445Gly Asp Asn Asp Phe Ile Ala Arg Leu Leu Leu Leu
Val Asp Leu Ile 450 455 460Gly Asn Leu Arg Glu Lys Gln Lys Leu Asn
Met Val Ala Ile Thr Pro465 470 475 480Ala Ser Ala Pro His Asn Asp
Gly Arg Pro Leu Pro Leu Ala Gly Asn 485 490 495Phe Met Phe Ser Phe
Gly Gly Phe Phe Arg Glu Glu Tyr Arg Gln Tyr 500 505 510Asp Phe Ser
Val Gly Glu Phe Ala Ala Trp Asn Val Leu Ser Thr Pro 515 520 525Ala
Ser Glu Thr Pro Phe Leu Ala Glu Thr Ala Pro Lys Pro Pro Ala 530 535
540Arg Pro Pro Gln Pro Pro Ala Ile Asn Pro Thr Tyr Arg Ser Leu
Gly545 550 555 560Pro Pro Ile Gln Gln Arg Phe Glu Glu Phe Val Arg
Gly His Val Arg 565 570 575Ala Phe Ile Ala Ser Val Ala Pro Leu Gly
Thr Arg Gly Ile Val Thr 580 585 590Gly Lys Ile Gly Gly Lys Leu Arg
Thr Met Leu Met Ala Ser Arg Asn 595 600 605Gly Lys Ser Glu Tyr Phe
Arg Leu Arg Leu Ser Gly Val Asp Gly Leu 610 615 620Tyr Leu Arg Gly
Ser Lys Gly Arg Asn Leu Arg Ala Val Asn Gly Ser625 630 635 640Ile
Asp Thr Val Val Gly Val Tyr Ile Asp Glu Glu Asp Gln His Arg 645 650
655Asp Glu Phe Phe Gly Pro His Val Phe Gly Ala Asn Gly Ser Gly Phe
660 665 670Thr Met Glu Leu Trp Glu Ser Arg Gly Phe Phe Gly Arg Asp
Arg Arg 675 680 685Val Ala Val Ile Glu Leu Glu Asn Asn Pro Gly Gly
Phe Ala Ile Ala 690 695 700Ala Gly Cys Arg Arg Arg Pro Gly Val Val
Leu Asp Met Ala Arg Arg705 710 715 720Asn Gly Gln Pro Leu Arg Thr
Val Asp Val Met Glu Phe Ala 725 730105756DNAUnknownObtained from an
environmental sample. 105atgaaccgtt gtcggaactc actcaacctc
caacttcgcg cggtgaccgt ggcggcgttg 60gtagtcgtcg catcctcggc cgcgctggcg
tgggacagcg cctcgcgcaa tccgacccat 120cccacccaca gctacctcac
cgaatacgcc atcgatcagc ttggggtggc gcggccggag 180ctccggcaat
accgcaagca gatcatcgag ggcgccaaca ccgagctgca cgaactgcca
240gtcaagggga cggcctatgg cctcgacctc gacgccaagc ggcgggaaca
ccgcggcacc 300aatgccggga cagacgacat cgccggctgg tgggcggaaa
gcctccaagc ctatcgcgcc 360ggtgccaagg aacgcgccta cttcgtgctg
ggggtggtgc tgcacatggt cgaggacatg 420ggcgtgccgg cgcacgcgaa
cggcgtctac caccagggca acctgactga attcgacaat 480ttcgagttca
tgggactgtc gaactggaag ccctctttcg ccgacatcaa ccggaccgat
540ccgggctacg ccgacccgtc gcgctactac gagttcagcc gagattggac
ggcggcagac 600gcacccggct atcgcgaccg cgacagcttc tcgaagacct
gggttctcgc cagcccggcc 660gaacgtcagc tgcttcagaa ccgccagggc
cggaccgcca cggtcgccat gtgggcgtta 720cggagcgcga cgaaggcgtt
cgccgggaaa ccctag 756106251PRTUnknownObtained from an environmental
sample. 106Met Asn Arg Cys Arg Asn Ser Leu Asn Leu Gln Leu Arg Ala
Val Thr1 5 10 15Val Ala Ala Leu Val Val Val Ala Ser Ser Ala Ala Leu
Ala Trp Asp 20 25 30Ser Ala Ser Arg Asn Pro Thr His Pro Thr His Ser
Tyr Leu Thr Glu 35 40 45Tyr Ala Ile Asp Gln Leu Gly Val Ala Arg Pro
Glu Leu Arg Gln Tyr 50 55 60Arg Lys Gln Ile Ile Glu Gly Ala Asn Thr
Glu Leu His Glu Leu Pro65 70 75 80Val Lys Gly Thr Ala Tyr Gly Leu
Asp Leu Asp Ala Lys Arg Arg Glu 85 90 95His Arg Gly Thr Asn Ala Gly
Thr Asp Asp Ile Ala Gly Trp Trp Ala 100 105 110Glu Ser Leu Gln Ala
Tyr Arg Ala Gly Ala Lys Glu Arg Ala Tyr Phe 115 120 125Val Leu Gly
Val Val Leu His Met Val Glu Asp Met Gly Val Pro Ala 130 135 140His
Ala Asn Gly Val Tyr His Gln Gly Asn Leu Thr Glu Phe Asp Asn145 150
155 160Phe Glu Phe Met Gly Leu Ser Asn Trp Lys Pro Ser Phe Ala Asp
Ile 165 170 175Asn Arg Thr Asp Pro Gly Tyr Ala Asp Pro Ser Arg Tyr
Tyr Glu Phe 180 185 190Ser Arg Asp Trp Thr Ala Ala Asp Ala Pro Gly
Tyr Arg Asp Arg Asp 195 200 205Ser Phe Ser Lys Thr Trp Val Leu Ala
Ser Pro Ala Glu Arg Gln Leu 210 215 220Leu Gln Asn Arg Gln Gly Arg
Thr Ala Thr Val Ala Met Trp Ala Leu225 230 235 240Arg Ser Ala Thr
Lys Ala Phe Ala Gly Lys Pro 245 250107990DNAUnknownObtained from an
environmental sample 107atgagcaata agaagtttat tttgaaatta ttcatatgta
gtactatact tagcacattt 60gtatttgctt tcaatgataa gcaagcagtt gctgctagcg
ctggtaatgg gcttgaaaac 120tggtcaaaat ggatgcaacc tatacccgat
aacgtaccgt tagcacgaat ttcaattcca 180ggaacacatg atagtggaac
gttcaagttg caaaatccga taaagcaagt atggggaatg 240acgcaagaat
ataattttcg ttaccaaatg gatcacggag ctagaatttt tgatattaga
300gggcgtttaa cagatgataa tacgatagtt cttcatcatg gaccattata
tctttatgta 360acattgcatg aatttataaa tgaagcgaaa caatttttaa
aagataatcc aagtgaaacg 420attattatgt ctttaaaaaa agagtatgag
gatatgaaag gggcagaaga ttcatttagt 480agtacgtttg aaaaaaaata
ttttcctgat cctatctttt taaaaacaga agggaatata 540agacttggag
atgctcgagg aaaaattgtg ctactaaaaa gatacagtgg tagtaatgaa
600tctggaggat ataataattt ttattggcca gataatgaca cgtttacgac
aactgtaaat 660caaaatgtaa atgtaacagt acaagataaa tataaggtga
gttatgatga gaaagtaaca 720tctattaaag atacgataaa tgaaacgatt
aacaacagtg aagattgtaa tcatctatat 780attaatttta caagcttgtc
ttctggtggt acagcatgga atagtccata ttattacgcg 840tcctacataa
atcctgaaat tgcaaactat atgaagcaaa agaatcctac gagagtgggc
900tgggtaattc aagattatat aaatgaaaaa tggtccccaa tactttatga
agaagttata 960agagcgaata agtcacttgt aaaagagtaa
990108329PRTUnknownObtained from an environmental sample 108Met Ser
Asn Lys Lys Phe Ile Leu Lys Leu Phe Ile Cys Ser Thr Ile1 5 10 15Leu
Ser Thr Phe Val Phe Ala Phe Asn Asp Lys Gln Ala Val Ala Ala 20 25
30Ser Ala Gly Asn Gly Leu Glu Asn Trp Ser Lys Trp Met Gln Pro Ile
35 40 45Pro Asp Asn Val Pro Leu Ala Arg Ile Ser Ile Pro Gly Thr His
Asp 50 55 60Ser Gly Thr Phe Lys Leu Gln Asn Pro Ile Lys Gln Val Trp
Gly Met65 70 75 80Thr Gln Glu Tyr Asn Phe Arg Tyr Gln Met Asp His
Gly Ala Arg Ile 85 90 95Phe Asp Ile Arg Gly Arg Leu Thr Asp Asp Asn
Thr Ile Val Leu His 100 105 110His Gly Pro Leu Tyr Leu Tyr Val Thr
Leu His Glu Phe Ile Asn Glu 115 120 125Ala Lys Gln Phe Leu Lys Asp
Asn Pro Ser Glu Thr Ile Ile Met Ser 130 135 140Leu Lys Lys Glu Tyr
Glu Asp Met Lys Gly Ala Glu Asp Ser Phe Ser145 150 155 160Ser Thr
Phe Glu Lys Lys Tyr Phe Pro Asp Pro Ile Phe Leu Lys Thr 165 170
175Glu Gly Asn Ile Arg Leu Gly Asp Ala Arg Gly Lys Ile Val Leu Leu
180 185 190Lys Arg Tyr Ser Gly Ser Asn Glu Ser Gly Gly Tyr Asn Asn
Phe Tyr 195 200 205Trp Pro Asp Asn Asp Thr Phe Thr Thr Thr Val Asn
Gln Asn Val Asn 210 215 220Val Thr Val Gln Asp Lys Tyr Lys Val Ser
Tyr Asp Glu Lys Val Thr225 230 235 240Ser Ile Lys Asp Thr Ile Asn
Glu Thr Ile Asn Asn Ser Glu Asp Cys 245 250 255Asn His Leu Tyr Ile
Asn Phe Thr Ser Leu Ser Ser Gly Gly Thr Ala 260 265 270Trp Asn Ser
Pro Tyr Tyr Tyr Ala Ser Tyr Ile Asn Pro Glu Ile Ala 275 280 285Asn
Tyr Met Lys Gln Lys Asn Pro Thr Arg Val Gly Trp Val Ile Gln 290 295
300Asp Tyr Ile Asn Glu Lys Trp Ser Pro Ile Leu Tyr Glu Glu Val
Ile305 310 315 320Arg Ala Asn Lys Ser Leu Val Lys Glu
325109990DNAUnknownObtained from an environmental sample
109atgagcaata agaagtttat tttgaaatta ttcatatgta gtactatact
tagcacattt 60gtatttgctt tcaatgataa gcaagcagtt gctgctagcg ctggtaatgg
gcttgaaaac 120tggtcaaaat ggatgcaacc tatacccgat aacgtaccgt
tagcacgaat ttcaattcca 180ggaacacatg atagtggaac gttcaagttg
caaaatccga taaagcaagt atggggaatg 240acgcaagaat ataattttcg
ttaccaaatg gatcacggag ctagaatttt tgatattaga 300gggcgtttaa
cagatgataa tacgatagtt cttcatcatg ggccattata tctttatgta
360acattgcatg aatttataaa tgaagcgaaa caatttttaa aagataatcc
aagtgaaacg 420attattatgt ctttaaaaaa agagtatgag gatatgaaag
gggcagaaga ttcatttagt 480agtacgtttg aaaaaaaata ttttcctgat
cctatctttt taaaaacaga agggaatata 540agacttggag atgctcgagg
aaaaattgtg ctactaaaaa gatacagtgg tagtaatgaa 600tctggaggat
ataataattt ttattggcca gataatgaga cgtttacgac aactgtaaat
660caaaatgtaa atgtaacagt acaagataaa tataaagtga gttatgatga
gaaagtaaaa 720tctattaaag atacgataaa tgaaacgatt aacaacagtg
aagattgtaa tcatctatat 780attaatttta caagcttgtc ttctggtggt
acagcatgga atagtccata ttattatgcg 840tcctacataa atcctgaaat
tgcaaactat atgaagcaaa agaatcctat gagagtgggc 900tgggtaattc
aagattatat aaatgaaaaa tggtccccaa tactttatga agaagttata
960agagcgaata agtcacttgt aaaagagtaa 990110329PRTUnknownObtained
from an environmental sample 110Met Ser Asn Lys Lys Phe Ile Leu Lys
Leu Phe Ile Cys Ser Thr Ile1 5 10 15Leu Ser Thr Phe Val Phe Ala Phe
Asn Asp Lys Gln Ala Val Ala Ala 20 25 30Ser Ala Gly Asn Gly Leu Glu
Asn Trp Ser Lys Trp Met Gln Pro Ile 35 40 45Pro Asp Asn Val Pro Leu
Ala Arg Ile Ser Ile Pro Gly Thr His Asp 50 55 60Ser Gly Thr Phe Lys
Leu Gln Asn Pro Ile Lys Gln Val Trp Gly Met65 70 75 80Thr Gln Glu
Tyr Asn Phe Arg Tyr Gln Met Asp His Gly Ala Arg Ile 85 90 95Phe Asp
Ile Arg Gly Arg Leu Thr Asp Asp Asn Thr Ile Val Leu His 100 105
110His Gly Pro Leu Tyr Leu Tyr Val Thr Leu His Glu Phe Ile Asn Glu
115 120 125Ala Lys Gln Phe Leu Lys Asp Asn Pro Ser Glu Thr Ile Ile
Met Ser 130 135 140Leu Lys Lys Glu Tyr Glu Asp Met Lys Gly Ala Glu
Asp Ser Phe Ser145 150 155 160Ser Thr Phe Glu Lys Lys Tyr Phe Pro
Asp Pro Ile Phe Leu Lys Thr 165 170 175Glu Gly Asn Ile Arg Leu Gly
Asp Ala Arg Gly Lys Ile Val Leu Leu 180 185 190Lys Arg Tyr Ser Gly
Ser Asn Glu Ser Gly Gly Tyr Asn Asn Phe Tyr 195 200 205Trp Pro Asp
Asn Glu Thr Phe Thr Thr Thr Val Asn Gln Asn Val Asn 210 215 220Val
Thr Val Gln Asp Lys Tyr Lys Val Ser Tyr Asp Glu Lys Val Lys225 230
235 240Ser Ile Lys Asp Thr Ile Asn Glu Thr Ile Asn Asn Ser Glu Asp
Cys
245 250 255Asn His Leu Tyr Ile Asn Phe Thr Ser Leu Ser Ser Gly Gly
Thr Ala 260 265 270Trp Asn Ser Pro Tyr Tyr Tyr Ala Ser Tyr Ile Asn
Pro Glu Ile Ala 275 280 285Asn Tyr Met Lys Gln Lys Asn Pro Met Arg
Val Gly Trp Val Ile Gln 290 295 300Asp Tyr Ile Asn Glu Lys Trp Ser
Pro Ile Leu Tyr Glu Glu Val Ile305 310 315 320Arg Ala Asn Lys Ser
Leu Val Lys Glu 325111828DNABacteria 111gtgggtgccg gggcgatcct
tctcaccggg gcccccaccg cctcggccgt ggacacgcgc 60gcgtggatgg ggggacacgg
ggacggcacg ccgctccagc ggctcaccat ccccggcacc 120cacgactccg
gcgcccggtt cggcgggccc tggtcggagt gccagaacac caccatcgcc
180cagcagctgg acagcgggat ccggttcctg gacgtccggt gccgggtcac
cggcgggtcc 240ttcgccatcc accacggggc ctcctaccag aacatgatgt
tcggcgacgt cctcgtcgcc 300tgccgcgact tcctcgccgc gcacccctcc
gagaccgtcc tcatgcgggt caagcaggag 360tactcgaccg actccgacgc
caccttccgg gccgtcttcg acgactacct cgacgcgcgc 420ggctggcgct
ccctgttccg catcggcgac ggggtcccgc tgctcggcga ggcccgcggc
480cgggtcgtgc tcatcgccga caacggcgga ctgccgggcg gtctgcgctg
gggcgacggc 540tcggccctcg ccatccagga cgactggaac gcgctgcccg
accccaagta cgccaagatc 600gaggcgcact tccgtaccgc cgtcgcccag
ccgggccggc tgtacgtgaa cttcgtcagc 660acctccgcct acctgccgcc
ccgctggaac tccgacaacc tcaacccgcg cgtgcaccgc 720tacctcgaca
gcgcggccgc cgcgggcgcg aagggcctcg ggatcgtccc catggacttc
780cccaacaccc gctcgggtct ggtcgaggcg ctgctccggc acaactga
828112275PRTBacteriaSIGNAL(1)...(16)DOMAIN(34)...(168)Phosphatidylinosito-
l-specific phospholipase C, X domain 112Met Gly Ala Gly Ala Ile Leu
Leu Thr Gly Ala Pro Thr Ala Ser Ala1 5 10 15Val Asp Thr Arg Ala Trp
Met Gly Gly His Gly Asp Gly Thr Pro Leu 20 25 30Gln Arg Leu Thr Ile
Pro Gly Thr His Asp Ser Gly Ala Arg Phe Gly 35 40 45Gly Pro Trp Ser
Glu Cys Gln Asn Thr Thr Ile Ala Gln Gln Leu Asp 50 55 60Ser Gly Ile
Arg Phe Leu Asp Val Arg Cys Arg Val Thr Gly Gly Ser65 70 75 80Phe
Ala Ile His His Gly Ala Ser Tyr Gln Asn Met Met Phe Gly Asp 85 90
95Val Leu Val Ala Cys Arg Asp Phe Leu Ala Ala His Pro Ser Glu Thr
100 105 110Val Leu Met Arg Val Lys Gln Glu Tyr Ser Thr Asp Ser Asp
Ala Thr 115 120 125Phe Arg Ala Val Phe Asp Asp Tyr Leu Asp Ala Arg
Gly Trp Arg Ser 130 135 140Leu Phe Arg Ile Gly Asp Gly Val Pro Leu
Leu Gly Glu Ala Arg Gly145 150 155 160Arg Val Val Leu Ile Ala Asp
Asn Gly Gly Leu Pro Gly Gly Leu Arg 165 170 175Trp Gly Asp Gly Ser
Ala Leu Ala Ile Gln Asp Asp Trp Asn Ala Leu 180 185 190Pro Asp Pro
Lys Tyr Ala Lys Ile Glu Ala His Phe Arg Thr Ala Val 195 200 205Ala
Gln Pro Gly Arg Leu Tyr Val Asn Phe Val Ser Thr Ser Ala Tyr 210 215
220Leu Pro Pro Arg Trp Asn Ser Asp Asn Leu Asn Pro Arg Val His
Arg225 230 235 240Tyr Leu Asp Ser Ala Ala Ala Ala Gly Ala Lys Gly
Leu Gly Ile Val 245 250 255Pro Met Asp Phe Pro Asn Thr Arg Ser Gly
Leu Val Glu Ala Leu Leu 260 265 270Arg His Asn
275113981DNAUnknownObtained from an environmental sample
113atgagcaata agaagtttat tttgaaatta ttcatatgta gtactatact
tagcacattt 60gtatttgctt tcaatgataa gcaagcagtt gctgctagcg ctggtaatgg
gcttgaaaac 120tggtcaaaat ggatgcaacc tatacccgat aacgtaccgt
tagcacgaat ttcaattcca 180ggaacacatg atagtggaac gttcaagttg
caaaatccga taaagcaagt atggggaatg 240acgcaagaat ataattttcg
ttaccaaatg gatcacggag ctagaatttt tgatattaga 300gggcgtttaa
cagatgataa tacgatagtt cttcatcatg ggccattata tctttatgta
360acattgcatg aatttataaa tgaagcgaaa caatttttaa aagataatcc
aagtgaaacg 420attattatgt ctttaaaaaa agagtatgag gatatgaaag
gggcagaaga ttcatttagt 480agtacgtttg aaaaaaaata ttttcctgat
cctatctttt taaaaacaga agggaatata 540agacttggag atgctcgagg
aaaaattgtg ctactaaaaa gatacagtgg tagtaatgaa 600tctggaggat
ataataattt ttattggcca gataatgaga cgtttacgac aactgtaaat
660caaaatgtaa atgtaacagt acaagataaa tataaagtga gttatgatga
gaaagtaaaa 720tctattaaag atacgataaa tgaaacgatt aacaacagtg
aagattgtaa tcatctatat 780attaatttta caagcttgtc ttctggtggt
acagcatgga atagtccata ttattatgcg 840tcctacataa atcctgaaat
tgcaaactat atgaagcaaa agaatcctat gagagtgggc 900tgggtaattc
aagattatat aaatgaaaaa tggtccccaa tactttatga agaagttata
960agagcgaata agtcactgta a 981114326PRTUnknownObtained from an
environmental sample 114Met Ser Asn Lys Lys Phe Ile Leu Lys Leu Phe
Ile Cys Ser Thr Ile1 5 10 15Leu Ser Thr Phe Val Phe Ala Phe Asn Asp
Lys Gln Ala Val Ala Ala 20 25 30Ser Ala Gly Asn Gly Leu Glu Asn Trp
Ser Lys Trp Met Gln Pro Ile 35 40 45Pro Asp Asn Val Pro Leu Ala Arg
Ile Ser Ile Pro Gly Thr His Asp 50 55 60Ser Gly Thr Phe Lys Leu Gln
Asn Pro Ile Lys Gln Val Trp Gly Met65 70 75 80Thr Gln Glu Tyr Asn
Phe Arg Tyr Gln Met Asp His Gly Ala Arg Ile 85 90 95Phe Asp Ile Arg
Gly Arg Leu Thr Asp Asp Asn Thr Ile Val Leu His 100 105 110His Gly
Pro Leu Tyr Leu Tyr Val Thr Leu His Glu Phe Ile Asn Glu 115 120
125Ala Lys Gln Phe Leu Lys Asp Asn Pro Ser Glu Thr Ile Ile Met Ser
130 135 140Leu Lys Lys Glu Tyr Glu Asp Met Lys Gly Ala Glu Asp Ser
Phe Ser145 150 155 160Ser Thr Phe Glu Lys Lys Tyr Phe Pro Asp Pro
Ile Phe Leu Lys Thr 165 170 175Glu Gly Asn Ile Arg Leu Gly Asp Ala
Arg Gly Lys Ile Val Leu Leu 180 185 190Lys Arg Tyr Ser Gly Ser Asn
Glu Ser Gly Gly Tyr Asn Asn Phe Tyr 195 200 205Trp Pro Asp Asn Glu
Thr Phe Thr Thr Thr Val Asn Gln Asn Val Asn 210 215 220Val Thr Val
Gln Asp Lys Tyr Lys Val Ser Tyr Asp Glu Lys Val Lys225 230 235
240Ser Ile Lys Asp Thr Ile Asn Glu Thr Ile Asn Asn Ser Glu Asp Cys
245 250 255Asn His Leu Tyr Ile Asn Phe Thr Ser Leu Ser Ser Gly Gly
Thr Ala 260 265 270Trp Asn Ser Pro Tyr Tyr Tyr Ala Ser Tyr Ile Asn
Pro Glu Ile Ala 275 280 285Asn Tyr Met Lys Gln Lys Asn Pro Met Arg
Val Gly Trp Val Ile Gln 290 295 300Asp Tyr Ile Asn Glu Lys Trp Ser
Pro Ile Leu Tyr Glu Glu Val Ile305 310 315 320Arg Ala Asn Lys Ser
Leu 325115987DNAUnknownObtained from an environmental sample
115atgaacaata agaagtttat tttgaagtta ttcatatgta gtatggtact
tagcgccttt 60gtatttgctt tcaatgataa gaaaaccgtt gcagctagct ctattaatga
gcttgaaaat 120tggtctagat ggatgaaacc tataaatgat gacataccgt
tagcacgaat ttcaattcca 180ggaacacatg atagtggaac gttcaagttg
caaaatccga taaagcaagt gtggggaatg 240acgcaagaat atgattttcg
ttatcaaatg gatcatggag ctagaatttt tgatataaga 300gggcgtttaa
cagatgataa tacgatagtt cttcatcatg ggccattata tctttatgta
360acactgcacg aatttataaa cgaagcgaaa caatttttaa aagataatcc
aagtgaaacg 420attattatgt ctttaaaaaa agagtatgag gatatgaaag
gggcggaaag ctcatttagt 480agtacgtttg agaaaaatta ttttcgtgat
ccaatctttt taaaaacaga agggaatata 540aagcttggag atgctcgtgg
gaaaattata ttactaaaac gatatagtgg tagtaatgaa 600tctgggggat
ataataattt ctattggcca gacaatgaga cgtttacctc aactataaat
660caaaatgtaa atgtcacagt acaagataaa tataaagtga gttatgatga
gaaagtaaac 720gctattaaag atacattaaa tgaaacgatt aacaatagtg
aagatgttaa tcatctatat 780attaatttta taagcttgtc ttctggtggt
acagcatgga atagtccata ttattatgcg 840tcctacataa atcctgaaat
tgcaaattat atgaagcaaa agaatcctac gagagtgggc 900tggataatac
aagattatat aaatgaaaaa tggtcaccat tactttatca agaagttata
960agagcgaata agtcacttgt aaaatag 987116328PRTUnknownObtained from
an environmental sample 116Met Asn Asn Lys Lys Phe Ile Leu Lys Leu
Phe Ile Cys Ser Met Val1 5 10 15Leu Ser Ala Phe Val Phe Ala Phe Asn
Asp Lys Lys Thr Val Ala Ala 20 25 30Ser Ser Ile Asn Glu Leu Glu Asn
Trp Ser Arg Trp Met Lys Pro Ile 35 40 45Asn Asp Asp Ile Pro Leu Ala
Arg Ile Ser Ile Pro Gly Thr His Asp 50 55 60Ser Gly Thr Phe Lys Leu
Gln Asn Pro Ile Lys Gln Val Trp Gly Met65 70 75 80Thr Gln Glu Tyr
Asp Phe Arg Tyr Gln Met Asp His Gly Ala Arg Ile 85 90 95Phe Asp Ile
Arg Gly Arg Leu Thr Asp Asp Asn Thr Ile Val Leu His 100 105 110His
Gly Pro Leu Tyr Leu Tyr Val Thr Leu His Glu Phe Ile Asn Glu 115 120
125Ala Lys Gln Phe Leu Lys Asp Asn Pro Ser Glu Thr Ile Ile Met Ser
130 135 140Leu Lys Lys Glu Tyr Glu Asp Met Lys Gly Ala Glu Ser Ser
Phe Ser145 150 155 160Ser Thr Phe Glu Lys Asn Tyr Phe Arg Asp Pro
Ile Phe Leu Lys Thr 165 170 175Glu Gly Asn Ile Lys Leu Gly Asp Ala
Arg Gly Lys Ile Ile Leu Leu 180 185 190Lys Arg Tyr Ser Gly Ser Asn
Glu Ser Gly Gly Tyr Asn Asn Phe Tyr 195 200 205Trp Pro Asp Asn Glu
Thr Phe Thr Ser Thr Ile Asn Gln Asn Val Asn 210 215 220Val Thr Val
Gln Asp Lys Tyr Lys Val Ser Tyr Asp Glu Lys Val Asn225 230 235
240Ala Ile Lys Asp Thr Leu Asn Glu Thr Ile Asn Asn Ser Glu Asp Val
245 250 255Asn His Leu Tyr Ile Asn Phe Ile Ser Leu Ser Ser Gly Gly
Thr Ala 260 265 270Trp Asn Ser Pro Tyr Tyr Tyr Ala Ser Tyr Ile Asn
Pro Glu Ile Ala 275 280 285Asn Tyr Met Lys Gln Lys Asn Pro Thr Arg
Val Gly Trp Ile Ile Gln 290 295 300Asp Tyr Ile Asn Glu Lys Trp Ser
Pro Leu Leu Tyr Gln Glu Val Ile305 310 315 320Arg Ala Asn Lys Ser
Leu Val Lys 325117987DNAUnknownObtained from an environmental
sample 117atgaacaata agaagtttat tttgaagtta ttcatatgta gtatggtact
tagcgccttt 60gtatttgctt tcaatgataa gaaaaccgtt gcagctagct ctattaatga
gcttgaaaat 120tggtctagat ggatgaaacc tataaatgat gacataccgt
tagcacgaat ttcaattcca 180ggaacacatg atagtggaac gttcaagttg
caaaatccga taaagcaagt gtggggaatg 240acgcaagaat atgattttcg
ttatcaaatg gatcatggag ctagaatttt tgatataaga 300gggcgtttaa
cagatgataa tacgatagtt cttcatcatg ggccattata tctttatgta
360acactgcacg aatttataaa cgaagcgaaa caatttttaa aagataatcc
aagtgaaacg 420attattatgt ctttaaaaaa agagtatgag gatatgaaag
gggcggaaag ctcatttagt 480agtacgtttg agaaaaatta ttttcgtgat
ccaatctttt taaaaacaga aggaaatata 540aagcttggag atgctcgtgg
gaaaattgta ttactaaaaa gatatagtgg tagtaatgaa 600tctgggggat
ataataattt ctattggcca gacaatgaga cgtttacctc aactataaat
660caaaatgtaa atgtaacagt acaagataaa tataaagtga gttatgatga
gaaaataaac 720gctattaaag atacattaaa tgaaacgatt aacaatagtg
aagatgttaa tcatctatat 780attaatttta caagcttgtc ttctggtggt
acagcatgga atagtccata ttattatgcg 840tcctacataa atcctgaaat
tgcaaattat atgaagcaaa agaatcctac gagagtgggc 900tggataatac
aagattatat aaatgaaaaa tggtcaccat tactttatca agaagttata
960agagcgaata agtcacttgt aaaatag 987118328PRTUnknownObtained from
an environmental sample 118Met Asn Asn Lys Lys Phe Ile Leu Lys Leu
Phe Ile Cys Ser Met Val1 5 10 15Leu Ser Ala Phe Val Phe Ala Phe Asn
Asp Lys Lys Thr Val Ala Ala 20 25 30Ser Ser Ile Asn Glu Leu Glu Asn
Trp Ser Arg Trp Met Lys Pro Ile 35 40 45Asn Asp Asp Ile Pro Leu Ala
Arg Ile Ser Ile Pro Gly Thr His Asp 50 55 60Ser Gly Thr Phe Lys Leu
Gln Asn Pro Ile Lys Gln Val Trp Gly Met65 70 75 80Thr Gln Glu Tyr
Asp Phe Arg Tyr Gln Met Asp His Gly Ala Arg Ile 85 90 95Phe Asp Ile
Arg Gly Arg Leu Thr Asp Asp Asn Thr Ile Val Leu His 100 105 110His
Gly Pro Leu Tyr Leu Tyr Val Thr Leu His Glu Phe Ile Asn Glu 115 120
125Ala Lys Gln Phe Leu Lys Asp Asn Pro Ser Glu Thr Ile Ile Met Ser
130 135 140Leu Lys Lys Glu Tyr Glu Asp Met Lys Gly Ala Glu Ser Ser
Phe Ser145 150 155 160Ser Thr Phe Glu Lys Asn Tyr Phe Arg Asp Pro
Ile Phe Leu Lys Thr 165 170 175Glu Gly Asn Ile Lys Leu Gly Asp Ala
Arg Gly Lys Ile Val Leu Leu 180 185 190Lys Arg Tyr Ser Gly Ser Asn
Glu Ser Gly Gly Tyr Asn Asn Phe Tyr 195 200 205Trp Pro Asp Asn Glu
Thr Phe Thr Ser Thr Ile Asn Gln Asn Val Asn 210 215 220Val Thr Val
Gln Asp Lys Tyr Lys Val Ser Tyr Asp Glu Lys Ile Asn225 230 235
240Ala Ile Lys Asp Thr Leu Asn Glu Thr Ile Asn Asn Ser Glu Asp Val
245 250 255Asn His Leu Tyr Ile Asn Phe Thr Ser Leu Ser Ser Gly Gly
Thr Ala 260 265 270Trp Asn Ser Pro Tyr Tyr Tyr Ala Ser Tyr Ile Asn
Pro Glu Ile Ala 275 280 285Asn Tyr Met Lys Gln Lys Asn Pro Thr Arg
Val Gly Trp Ile Ile Gln 290 295 300Asp Tyr Ile Asn Glu Lys Trp Ser
Pro Leu Leu Tyr Gln Glu Val Ile305 310 315 320Arg Ala Asn Lys Ser
Leu Val Lys 325119987DNAUnknownObtained from an environmental
sample 119atgaacaata agaagtttat tttgaagtta ttcatatgta gtatggtact
tagcgccttt 60gtatttgctt tcaatgataa gaaaaccgtt gcagctagct ctattaatgt
gcttgaaaat 120tggtctagat ggatgaaacc tataaatgat gacataccgt
tagcacgaat ttcaattcca 180ggaacacatg atagtggaac gttcaagttg
caaaatccga taaagcaagt gtggggaatg 240acgcaagaat atgattttcg
ttatcaaatg gatcatggag ctagaatttt tgatataaga 300gggcgtttaa
cagatgataa tacgatagtt cttcatcatg ggccattata tctttatgta
360acactgcacg aatttataaa cgaagcgaaa caatttttaa aagataatcc
aagtgaaacg 420attattatgt ctttaaaaaa agagtatgag gatatgaaag
gggcggaaag ctcatttagt 480agtacgtttg agaaaaatta ttttcgtgat
ccaatctttt taaaaacaga agggaatata 540aagcttggag atgctcgtgg
gaaaattgta ttactaaaaa gatatagtgg tagtaatgaa 600tctgggggat
ataataattt ctattggcca gacaatgaga cgtttacctc aactataaat
660caaaatgtaa atgtaacagt acaagataaa tataaagtga gttatgatga
gaaaataaac 720gctattaaag atacattaaa tgaaacgatt aacaatagtg
aagatgttaa tcatctatat 780attaatttta caagcttgtc ttctggtggt
acagcatgga atagtccata ttattatgcg 840tcctacataa atcctgaaat
tgcaaattat atgaagcaaa agaatcctac gagagtgggc 900tggataatac
aagattatat aaatgaaaaa tggtcaccat tactttatca agaagttata
960agagcgaata agtcacttgt aaaatag 987120328PRTUnknownObtained from
an environmental sample 120Met Asn Asn Lys Lys Phe Ile Leu Lys Leu
Phe Ile Cys Ser Met Val1 5 10 15Leu Ser Ala Phe Val Phe Ala Phe Asn
Asp Lys Lys Thr Val Ala Ala 20 25 30Ser Ser Ile Asn Val Leu Glu Asn
Trp Ser Arg Trp Met Lys Pro Ile 35 40 45Asn Asp Asp Ile Pro Leu Ala
Arg Ile Ser Ile Pro Gly Thr His Asp 50 55 60Ser Gly Thr Phe Lys Leu
Gln Asn Pro Ile Lys Gln Val Trp Gly Met65 70 75 80Thr Gln Glu Tyr
Asp Phe Arg Tyr Gln Met Asp His Gly Ala Arg Ile 85 90 95Phe Asp Ile
Arg Gly Arg Leu Thr Asp Asp Asn Thr Ile Val Leu His 100 105 110His
Gly Pro Leu Tyr Leu Tyr Val Thr Leu His Glu Phe Ile Asn Glu 115 120
125Ala Lys Gln Phe Leu Lys Asp Asn Pro Ser Glu Thr Ile Ile Met Ser
130 135 140Leu Lys Lys Glu Tyr Glu Asp Met Lys Gly Ala Glu Ser Ser
Phe Ser145 150 155 160Ser Thr Phe Glu Lys Asn Tyr Phe Arg Asp Pro
Ile Phe Leu Lys Thr 165 170 175Glu Gly Asn Ile Lys Leu Gly Asp Ala
Arg Gly Lys Ile Val Leu Leu 180 185 190Lys Arg Tyr Ser Gly Ser Asn
Glu Ser Gly Gly Tyr Asn Asn Phe Tyr 195 200 205Trp Pro Asp Asn Glu
Thr Phe Thr Ser Thr Ile Asn Gln Asn Val Asn 210 215 220Val Thr Val
Gln Asp Lys Tyr Lys Val Ser Tyr Asp Glu Lys Ile Asn225 230 235
240Ala Ile Lys Asp Thr Leu Asn Glu Thr Ile Asn Asn Ser Glu Asp Val
245 250 255Asn His Leu Tyr Ile Asn Phe Thr Ser Leu Ser Ser Gly Gly
Thr Ala 260 265 270Trp Asn Ser Pro Tyr Tyr Tyr Ala Ser Tyr Ile Asn
Pro Glu Ile Ala 275 280 285Asn Tyr Met Lys Gln Lys Asn Pro Thr Arg
Val Gly Trp Ile Ile Gln 290 295 300Asp Tyr Ile Asn Glu Lys Trp Ser
Pro Leu Leu Tyr Gln Glu Val Ile305 310 315 320Arg Ala Asn Lys Ser
Leu Val Lys 325121990DNAUnknownObtained from an environmental
sample 121atgcgtaata agaagtttat tttgaaatta ttaatatgta gtacggtact
tagcaccttt 60gtatttgctt tcaatgataa gcaaactgtt gcagctagct ctattaatga
actcgaaaat 120tggtctagat ggatgcagcc tatacctgat gacatgccgt
tagcaagaat ttcaattcca 180ggaacacatg atagtggaac gttcaaactg
caaaatccga taaagcaagt atggggaatg 240acgcaagaat atgattttcg
ttaccaaatg gatcatgggg ctagaatttt tgatataaga 300gggcgtttaa
cagatgataa tacgatagtc cttcatcatg ggccattata tctttatgta
360acactgaacg aatttataaa tgaagcgaaa caatttttaa aagataaccc
aagtgaaacg 420attattatgt ctttaaagaa agagtatgag gatatgaaag
gggcagaaaa ttcatttagt 480agtacgtttg aaaaaaaata ttttcttgat
cctatctttt taaaaacaga agggaatata 540aaacttggag atgctcgtgg
gaaaattgta ctactaaaaa gatatagtgg tagtaatgaa 600tctggaggat
ataataattt ttattggcca gataacgaga cgtttacgac aactgtaaat
660caaaatgtaa atgtaacagt acaagataaa tataaagtga gttatgatga
gaaagtaaaa 720tctattaaag atacgataaa tgaaacgatt aacaatagtg
aagattttaa tcatctatat 780attaatttta caagcttgtc ttctggtggt
acagcatgga atagtccata ttattatgca 840tcctacataa atcctgaaat
tgcaaaccat atgaagcaaa agaatcctac gagagtgggc 900tgggtaattc
aagattatat aaatgaaaaa tggtcaccaa tactttatca agaagttata
960agagcgaata agtcacttat aaaagagtag 990122329PRTUnknownObtained
from an environmental sample 122Met Arg Asn Lys Lys Phe Ile Leu Lys
Leu Leu Ile Cys Ser Thr Val1 5 10 15Leu Ser Thr Phe Val Phe Ala Phe
Asn Asp Lys Gln Thr Val Ala Ala 20 25 30Ser Ser Ile Asn Glu Leu Glu
Asn Trp Ser Arg Trp Met Gln Pro Ile 35 40 45Pro Asp Asp Met Pro Leu
Ala Arg Ile Ser Ile Pro Gly Thr His Asp 50 55 60Ser Gly Thr Phe Lys
Leu Gln Asn Pro Ile Lys Gln Val Trp Gly Met65 70 75 80Thr Gln Glu
Tyr Asp Phe Arg Tyr Gln Met Asp His Gly Ala Arg Ile 85 90 95Phe Asp
Ile Arg Gly Arg Leu Thr Asp Asp Asn Thr Ile Val Leu His 100 105
110His Gly Pro Leu Tyr Leu Tyr Val Thr Leu Asn Glu Phe Ile Asn Glu
115 120 125Ala Lys Gln Phe Leu Lys Asp Asn Pro Ser Glu Thr Ile Ile
Met Ser 130 135 140Leu Lys Lys Glu Tyr Glu Asp Met Lys Gly Ala Glu
Asn Ser Phe Ser145 150 155 160Ser Thr Phe Glu Lys Lys Tyr Phe Leu
Asp Pro Ile Phe Leu Lys Thr 165 170 175Glu Gly Asn Ile Lys Leu Gly
Asp Ala Arg Gly Lys Ile Val Leu Leu 180 185 190Lys Arg Tyr Ser Gly
Ser Asn Glu Ser Gly Gly Tyr Asn Asn Phe Tyr 195 200 205Trp Pro Asp
Asn Glu Thr Phe Thr Thr Thr Val Asn Gln Asn Val Asn 210 215 220Val
Thr Val Gln Asp Lys Tyr Lys Val Ser Tyr Asp Glu Lys Val Lys225 230
235 240Ser Ile Lys Asp Thr Ile Asn Glu Thr Ile Asn Asn Ser Glu Asp
Phe 245 250 255Asn His Leu Tyr Ile Asn Phe Thr Ser Leu Ser Ser Gly
Gly Thr Ala 260 265 270Trp Asn Ser Pro Tyr Tyr Tyr Ala Ser Tyr Ile
Asn Pro Glu Ile Ala 275 280 285Asn His Met Lys Gln Lys Asn Pro Thr
Arg Val Gly Trp Val Ile Gln 290 295 300Asp Tyr Ile Asn Glu Lys Trp
Ser Pro Ile Leu Tyr Gln Glu Val Ile305 310 315 320Arg Ala Asn Lys
Ser Leu Ile Lys Glu 325123849DNAUnknownObtained from an
environmental sample 123atgaaaaaga aagtattagc actagcagct atggttgctt
tagctgcacc agttcaaagt 60gtagtgtttg cgcaaacaaa taatagtgaa agtcctgcac
cgatcttaag atggtcagct 120gaggacaagc ataatgaggg agttagtact
catttgtgga ttgtaaatcg tgcaattgac 180atcatgtctc gtaatacagc
gattgtgaag ccaaatgaaa ctgctttatt aaatgagtgg 240cgtactgatt
tagaaaatgg tatttattct gctgattacg agaatcctta ttatgataat
300agtacatatg cttctcattt ttacgatccg gatactggaa aaacatatat
tccttttgcg 360aaacaggcaa aagaaacagg tacaaaatat tttaaacttg
ctggtgaagc atacaaaaat 420caagatatga aacaggcatt cttctattta
ggattatcac ttcattattt aggagatgta 480aatcagccaa tgcatgcagc
aaactttacg aatctttctt atccaatggg tttccattct 540aaatatgaaa
attttgttga tacaataaaa aataactata tagtttcaga tagtagtgga
600tattggaatt ggaaaggggc aaacccagaa gattggattc aaggagcagc
agtagcggct 660aaacaagatt atcctggtat tgtgaacgat acgacaaaag
attggtttgt aaaagcagct 720gtatctcaag catatgcaga taaatggcgt
gcagaagtaa caccggtgac aggaaaacgc 780ttaatggagg cacagcgcgt
tacagctggt tatattcatt tatggtttga tacgtatgta 840aatcactaa
849124282PRTUnknownObtained from an environmental sample 124Met Lys
Lys Lys Val Leu Ala Leu Ala Ala Met Val Ala Leu Ala Ala1 5 10 15Pro
Val Gln Ser Val Val Phe Ala Gln Thr Asn Asn Ser Glu Ser Pro 20 25
30Ala Pro Ile Leu Arg Trp Ser Ala Glu Asp Lys His Asn Glu Gly Val
35 40 45Ser Thr His Leu Trp Ile Val Asn Arg Ala Ile Asp Ile Met Ser
Arg 50 55 60Asn Thr Ala Ile Val Lys Pro Asn Glu Thr Ala Leu Leu Asn
Glu Trp65 70 75 80Arg Thr Asp Leu Glu Asn Gly Ile Tyr Ser Ala Asp
Tyr Glu Asn Pro 85 90 95Tyr Tyr Asp Asn Ser Thr Tyr Ala Ser His Phe
Tyr Asp Pro Asp Thr 100 105 110Gly Lys Thr Tyr Ile Pro Phe Ala Lys
Gln Ala Lys Glu Thr Gly Thr 115 120 125Lys Tyr Phe Lys Leu Ala Gly
Glu Ala Tyr Lys Asn Gln Asp Met Lys 130 135 140Gln Ala Phe Phe Tyr
Leu Gly Leu Ser Leu His Tyr Leu Gly Asp Val145 150 155 160Asn Gln
Pro Met His Ala Ala Asn Phe Thr Asn Leu Ser Tyr Pro Met 165 170
175Gly Phe His Ser Lys Tyr Glu Asn Phe Val Asp Thr Ile Lys Asn Asn
180 185 190Tyr Ile Val Ser Asp Ser Ser Gly Tyr Trp Asn Trp Lys Gly
Ala Asn 195 200 205Pro Glu Asp Trp Ile Gln Gly Ala Ala Val Ala Ala
Lys Gln Asp Tyr 210 215 220Pro Gly Ile Val Asn Asp Thr Thr Lys Asp
Trp Phe Val Lys Ala Ala225 230 235 240Val Ser Gln Ala Tyr Ala Asp
Lys Trp Arg Ala Glu Val Thr Pro Val 245 250 255Thr Gly Lys Arg Leu
Met Glu Ala Gln Arg Val Thr Ala Gly Tyr Ile 260 265 270His Leu Trp
Phe Asp Thr Tyr Val Asn His 275 2801251710DNAUnknownObtained from
an environmental sample 125atggctgaca acgagttgcc cctggcgcgg
cccagggaga cgccgccgtg ccgccccggc 60acgttcgagc ttgggctcgc cctggctggc
gcggtctctg gcggcgccta cgccgcggga 120gtcctggatt tcttctacga
agccctcgag cattggtacg aggccaggga ggcgggagcg 180ccggtgccca
accacgacgt gctcctccgg atcatctcgg gtgcgtcggc gggcagtatc
240aatggcgtgc tttcgggcat cgcgctgccg taccgttttc cccacgtgca
cagcgggccc 300gcgcccgagg gtgccacggg caaccccttc tacgacgcct
gggtgaagcg catcgacgtg 360cgcgaactgt tgggcaacga agacctggcc
gatcccacgc agccggtggc atccctgctc 420gacgccacct gcctggatac
gatcgcgaag gacatgctcg gcttctcggc ggcgccggcc 480acccggccgt
acgtcgctaa tccgctgaaa tgcgtgttca cggtgaccaa cctgcgtggc
540gttccttacg tcgtgcagtt caagggaaac ccggagatcc ccggccacgg
catgatggcc 600cacgccgact ggctgcgctt cgccgtcgac accgggcagg
gcgaccggga tggggaatgg 660atgttccccg atgaacggct cgtcagcggg
ccgagccatg cgcggactcc ggcctggcaa 720ggtttcatgg aggcggcgct
cgcttcgtcg gcgttcccgg ccggcttgcg tttccgcgaa 780gtcgcccggc
cctggagcga ttacgaccag cgcgtcgtgg tggtgcccaa ccaggcgggg
840gccgcggtcc cggtcccgct cccgccggcc tgggcggagg gcgagggcag
cgatggggac 900taccggttcg tcgcggtgga tggtggcgcg atggacaacg
agccgttcga acttgcccgt 960accgagctgg cgggcacgct cggccgcaat
ccacgcgaag ggaaccgggt caaccgcatc 1020gtgatcatgc tcgatccgtt
tcccgaggcc gaggcgccgg gacccgcgga agccgcgagc 1080acgaatctcg
tcgaggcgat ggcctcgctg tttggtgcgt ggaaacagca ggcacggttc
1140aagccggagg aagtggcgct cgccctggat tcgaccgtgt acagccgctt
catgatcgcg 1200cccagccggc cgtgcatgga gggcgggcca cggtggatcg
gtgggcgagc gctcgccgcg 1260ggtgcgctgg gtggcttctc ggggttcctg
gcggaggcat acaggcacca cgatttcctc 1320ctgggacgcc gcaactgcca
acgcttcctc gccgagcgcc tgttgatccc cgcggacaat 1380ccgatcttcg
ccggctggat cgacgatccc tccctgcagg gctacatccg cgagatcgat
1440ggcgtgcgtt acgccccggt catcccgctg gtgggcggct gccagggctt
gcgcgagccg 1500ttgcccacgt ggccgcgtgg tgcattcgac ctggactcgc
tcatgccgct ggtcgagcgc 1560cgcatgcagc gcctgtattc ggcggctacc
gcgacgctcg gtggccgctt cgccacctgg 1620ctggcgtggc gcttctacct
gcgccgcaag ctcctcgacc tggtctcaag ccgtatccgt 1680agcgcattga
gggacttcgg cctttggtga 1710126569PRTUnknownObtained from an
environmental sample 126Met Ala Asp Asn Glu Leu Pro Leu Ala Arg Pro
Arg Glu Thr Pro Pro1 5 10 15Cys Arg Pro Gly Thr Phe Glu Leu Gly Leu
Ala Leu Ala Gly Ala Val 20 25 30Ser Gly Gly Ala Tyr Ala Ala Gly Val
Leu Asp Phe Phe Tyr Glu Ala 35 40 45Leu Glu His Trp Tyr Glu Ala Arg
Glu Ala Gly Ala Pro Val Pro Asn 50 55 60His Asp Val Leu Leu Arg Ile
Ile Ser Gly Ala Ser Ala Gly Ser Ile65 70 75 80Asn Gly Val Leu Ser
Gly Ile Ala Leu Pro Tyr Arg Phe Pro His Val 85 90 95His Ser Gly Pro
Ala Pro Glu Gly Ala Thr Gly Asn Pro Phe Tyr Asp 100 105 110Ala Trp
Val Lys Arg Ile Asp Val Arg Glu Leu Leu Gly Asn Glu Asp 115 120
125Leu Ala Asp Pro Thr Gln Pro Val Ala Ser Leu Leu Asp Ala Thr Cys
130 135 140Leu Asp Thr Ile Ala Lys Asp Met Leu Gly Phe Ser Ala Ala
Pro Ala145 150 155 160Thr Arg Pro Tyr Val Ala Asn Pro Leu Lys Cys
Val Phe Thr Val Thr 165 170 175Asn Leu Arg Gly Val Pro Tyr Val Val
Gln Phe Lys Gly Asn Pro Glu 180 185 190Ile Pro Gly His Gly Met Met
Ala His Ala Asp Trp Leu Arg Phe Ala 195 200 205Val Asp Thr Gly Gln
Gly Asp Arg Asp Gly Glu Trp Met Phe Pro Asp 210 215 220Glu Arg Leu
Val Ser Gly Pro Ser His Ala Arg Thr Pro Ala Trp Gln225 230 235
240Gly Phe Met Glu Ala Ala Leu Ala Ser Ser Ala Phe Pro Ala Gly Leu
245 250 255Arg Phe Arg Glu Val Ala Arg Pro Trp Ser Asp Tyr Asp Gln
Arg Val 260 265 270Val Val Val Pro Asn Gln Ala Gly Ala Ala Val Pro
Val Pro Leu Pro 275 280 285Pro Ala Trp Ala Glu Gly Glu Gly Ser Asp
Gly Asp Tyr Arg Phe Val 290 295 300Ala Val Asp Gly Gly Ala Met Asp
Asn Glu Pro Phe Glu Leu Ala Arg305 310 315 320Thr Glu Leu Ala Gly
Thr Leu Gly Arg Asn Pro Arg Glu Gly Asn Arg 325 330 335Val Asn Arg
Ile Val Ile Met Leu Asp Pro Phe Pro Glu Ala Glu Ala 340 345 350Pro
Gly Pro Ala Glu Ala Ala Ser Thr Asn Leu Val Glu Ala Met Ala 355 360
365Ser Leu Phe Gly Ala Trp Lys Gln Gln Ala Arg Phe Lys Pro Glu Glu
370 375 380Val Ala Leu Ala Leu Asp Ser Thr Val Tyr Ser Arg Phe Met
Ile Ala385 390 395 400Pro Ser Arg Pro Cys Met Glu Gly Gly Pro Arg
Trp Ile Gly Gly Arg 405 410 415Ala Leu Ala Ala Gly Ala Leu Gly Gly
Phe Ser Gly Phe Leu Ala Glu 420 425 430Ala Tyr Arg His His Asp Phe
Leu Leu Gly Arg Arg Asn Cys Gln Arg 435 440 445Phe Leu Ala Glu Arg
Leu Leu Ile Pro Ala Asp Asn Pro Ile Phe Ala 450 455 460Gly Trp Ile
Asp Asp Pro Ser Leu Gln Gly Tyr Ile Arg Glu Ile Asp465 470 475
480Gly Val Arg Tyr Ala Pro Val Ile Pro Leu Val Gly Gly Cys Gln Gly
485 490 495Leu Arg Glu Pro Leu Pro Thr Trp Pro Arg Gly Ala Phe Asp
Leu Asp 500 505 510Ser Leu Met Pro Leu Val Glu Arg Arg Met Gln Arg
Leu Tyr Ser Ala 515 520 525Ala Thr Ala Thr Leu Gly Gly Arg Phe Ala
Thr Trp Leu Ala Trp Arg 530 535 540Phe Tyr Leu Arg Arg Lys Leu Leu
Asp Leu Val Ser Ser Arg Ile Arg545 550 555 560Ser Ala Leu Arg Asp
Phe Gly Leu Trp 5651271038DNAUnknownObtained from an environmental
sample 127atgacaaccc aatttagaaa cttgatcttt gaaggtggtg gtgtaaaagg
agttgcttac 60attggcgcca tgcagattct tgaaaatcgt ggcgtgttgc aagatattca
ccgagtcgga 120ggttgcagtg ccggtgcgat taacgcgctg atttttgcgc
tgggttacac ggttcgtgag 180caaaaagaga tcttacaagc caccgatttt
aaccagttta tggataactc ttggggggtt 240attcgtgata ttcgcaggct
tgctcgagac tttggctgga ataagggtgg cttctttaat 300agctggatag
gtgatttgat tcatcgtcgt ttggggaatc gccgagcgac gttcaaggat
360ctgcaaaagg ccaagcttcc tgatctttat gtcatcggta ctaatctgtc
tacagggttt 420gcagaggttt tttctgccga aagacacccc gatatggagc
tagcgacagc ggtgcgcatc 480tccatgtcga taccgctgtt ctttgcggcc
gtgcgccacg gtgatcgaca agatgtgtat 540gtcgatggag gtgttcaact
taactatccg attaaactgt ttgatcggga gcgttatatt 600gatctggcca
aagatcccgg tgccgttcgg cgaacgggtt attacaataa agaaaacgct
660cgctttcagc ttgaacggcc gggccatagc ccctatgttt acaatcgcca
gaccttgggt 720ttgcgactgg atagtcgaga ggagataggg ctttttcgtt
atgacgaacc cctcaagggc 780aaaccgatta agtccttcac tgactacgct
cgacaacttt tcggtgcgtt gatgaatgcg 840caggaaaaca ttcatctaca
tggcgatgat tggcagcgca cggtctatat cgacacactg 900gatgtgagta
cgacggactt caatctttct gatgcaacca agcaagcact gattgagcaa
960ggaattaacg gcaccgaaaa ttatttcgag tggtttgata atccgttaga
gaagcctgtg 1020aatagagtgg agtcatag 1038128345PRTUnknownObtained
from an environmental sample 128Met Thr Thr Gln Phe Arg Asn Leu Ile
Phe Glu Gly Gly Gly Val Lys1 5 10 15Gly Val Ala Tyr Ile Gly Ala Met
Gln Ile Leu Glu Asn Arg Gly Val 20 25 30Leu Gln Asp Ile His Arg Val
Gly Gly Cys Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ala Leu
Gly Tyr Thr Val Arg Glu Gln Lys Glu Ile 50 55 60Leu Gln Ala Thr Asp
Phe Asn Gln Phe Met Asp Asn Ser Trp Gly Val65 70 75 80Ile Arg Asp
Ile Arg Arg Leu Ala Arg Asp Phe Gly Trp Asn Lys Gly 85 90 95Gly Phe
Phe Asn Ser Trp Ile Gly Asp Leu Ile His Arg Arg Leu Gly 100 105
110Asn Arg Arg Ala Thr Phe Lys Asp Leu Gln Lys Ala Lys Leu Pro Asp
115 120 125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ala Glu
Val Phe 130 135 140Ser Ala Glu Arg His Pro Asp Met Glu Leu Ala Thr
Ala Val Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala
Ala Val Arg His Gly Asp Arg 165 170 175Gln Asp Val Tyr Val Asp Gly
Gly Val Gln Leu Asn Tyr Pro Ile Lys 180 185 190Leu Phe Asp Arg Glu
Arg Tyr Ile Asp Leu Ala Lys Asp Pro Gly Ala 195 200 205Val Arg Arg
Thr Gly Tyr Tyr Asn Lys Glu Asn Ala Arg Phe Gln Leu 210 215 220Glu
Arg Pro Gly His Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230
235 240Leu Arg Leu Asp Ser Arg Glu Glu Ile Gly Leu Phe Arg Tyr Asp
Glu 245 250 255Pro Leu Lys Gly Lys Pro Ile Lys Ser Phe Thr Asp Tyr
Ala Arg Gln 260 265 270Leu Phe Gly Ala Leu Met Asn Ala Gln Glu Asn
Ile His Leu His Gly 275 280 285Asp Asp Trp Gln Arg Thr Val Tyr Ile
Asp Thr Leu Asp Val Ser Thr 290 295 300Thr Asp Phe Asn Leu Ser Asp
Ala Thr Lys Gln Ala Leu Ile Glu Gln305 310 315 320Gly Ile Asn Gly
Thr Glu Asn Tyr Phe Glu Trp Phe Asp Asn Pro Leu 325 330 335Glu Lys
Pro Val Asn Arg Val Glu Ser 340 3451291434DNAUnknownObtained from
an environmental sample 129atgaaaaaga aaatatgtac attggctctt
gtatcagcaa taacttctgg agttgtgacg
60attccaacgg tagcatctgc ttgcagaata ggcgaagaag taatgaaaca ggagaaacag
120gataatcaag agcacaaacg tgtgaaaaga tggtctgcgg agcatccgca
tcattctaat 180gaaagcacgc acttatggat tgctcgaaat gcgattcaaa
ttatgagtcg taatcaagat 240aacacggtcc aaaacaatga attacagttc
ttaaatattc ctgaatataa ggagttattt 300gaaagaggac tttatgatgc
tgattacctt gatgaattta acgatggcgg tacaggtaca 360atcggcattg
atgggctaat taaaggaggg tggaaatctc atttttatga tccagatacg
420aaaaagaatt ataaaggaga agaagctcca acagccctta cgcaaggaga
taaatatttt 480aaattagcag gagactattt taagaaagag gatttgaaac
aagctttcta ctatttaggt 540gttgcgactc actatttcac agatgctact
cagccaatgc atgctgctaa ttttacagct 600gtcgacatga gtgcgataaa
gtttcatagc gcttttgaaa attatgtaac gacaattcag 660acgccatttg
aagtgaagga tgataaagga acctataatt tggttgattc taatgatccg
720aagcagtgga tacatgaaac agcgaaactc gcaaaagcgg aaattatgaa
tattactaat 780gatactatta aatctcaata taataaaggg aacaatgatc
tttggcaaca aggagttatg 840ccagctgttc agagaagtct ggaaacagca
caaaggaaca cggcaggatt tattcattta 900tggtttaaaa catatgttgg
caaaactgct gctgaagata ttgaaaatac acaagtaaaa 960gattctaacg
gagaagcaat acaagaaaat aaaaaatact acgttgtacc gagtgagttt
1020ttaaatagag gtttgacctt tgaggtatat gctgcaaatg actacgcact
attagctaat 1080cacgtagatg ataataaagt tcatggtaca cctgttcagt
ttgtttttga taaagacaat 1140aacggaattc ttcatcgggg agaaagtgca
ctgatgaaaa tgacgcaatc taactatgct 1200gattatgtat ttctcaatta
ctctaatatg acaaattggg tacatcttgc gaaacgaaaa 1260acaaatactt
cacagtttaa agtgtatcca aatccggata actcatctga atatttctta
1320tatacagatg gatacccggt aaattatcaa gaaaatggta acggaaagag
ctggattgtg 1380ttaggaaaga aaacggataa accaaaagcg tggaaattta
tacaggcgga ataa 1434130477PRTUnknownObtained from an environmental
sample 130Met Lys Lys Lys Ile Cys Thr Leu Ala Leu Val Ser Ala Ile
Thr Ser1 5 10 15Gly Val Val Thr Ile Pro Thr Val Ala Ser Ala Cys Arg
Ile Gly Glu 20 25 30Glu Val Met Lys Gln Glu Lys Gln Asp Asn Gln Glu
His Lys Arg Val 35 40 45Lys Arg Trp Ser Ala Glu His Pro His His Ser
Asn Glu Ser Thr His 50 55 60Leu Trp Ile Ala Arg Asn Ala Ile Gln Ile
Met Ser Arg Asn Gln Asp65 70 75 80Asn Thr Val Gln Asn Asn Glu Leu
Gln Phe Leu Asn Ile Pro Glu Tyr 85 90 95Lys Glu Leu Phe Glu Arg Gly
Leu Tyr Asp Ala Asp Tyr Leu Asp Glu 100 105 110Phe Asn Asp Gly Gly
Thr Gly Thr Ile Gly Ile Asp Gly Leu Ile Lys 115 120 125Gly Gly Trp
Lys Ser His Phe Tyr Asp Pro Asp Thr Lys Lys Asn Tyr 130 135 140Lys
Gly Glu Glu Ala Pro Thr Ala Leu Thr Gln Gly Asp Lys Tyr Phe145 150
155 160Lys Leu Ala Gly Asp Tyr Phe Lys Lys Glu Asp Leu Lys Gln Ala
Phe 165 170 175Tyr Tyr Leu Gly Val Ala Thr His Tyr Phe Thr Asp Ala
Thr Gln Pro 180 185 190Met His Ala Ala Asn Phe Thr Ala Val Asp Met
Ser Ala Ile Lys Phe 195 200 205His Ser Ala Phe Glu Asn Tyr Val Thr
Thr Ile Gln Thr Pro Phe Glu 210 215 220Val Lys Asp Asp Lys Gly Thr
Tyr Asn Leu Val Asp Ser Asn Asp Pro225 230 235 240Lys Gln Trp Ile
His Glu Thr Ala Lys Leu Ala Lys Ala Glu Ile Met 245 250 255Asn Ile
Thr Asn Asp Thr Ile Lys Ser Gln Tyr Asn Lys Gly Asn Asn 260 265
270Asp Leu Trp Gln Gln Gly Val Met Pro Ala Val Gln Arg Ser Leu Glu
275 280 285Thr Ala Gln Arg Asn Thr Ala Gly Phe Ile His Leu Trp Phe
Lys Thr 290 295 300Tyr Val Gly Lys Thr Ala Ala Glu Asp Ile Glu Asn
Thr Gln Val Lys305 310 315 320Asp Ser Asn Gly Glu Ala Ile Gln Glu
Asn Lys Lys Tyr Tyr Val Val 325 330 335Pro Ser Glu Phe Leu Asn Arg
Gly Leu Thr Phe Glu Val Tyr Ala Ala 340 345 350Asn Asp Tyr Ala Leu
Leu Ala Asn His Val Asp Asp Asn Lys Val His 355 360 365Gly Thr Pro
Val Gln Phe Val Phe Asp Lys Asp Asn Asn Gly Ile Leu 370 375 380His
Arg Gly Glu Ser Ala Leu Met Lys Met Thr Gln Ser Asn Tyr Ala385 390
395 400Asp Tyr Val Phe Leu Asn Tyr Ser Asn Met Thr Asn Trp Val His
Leu 405 410 415Ala Lys Arg Lys Thr Asn Thr Ser Gln Phe Lys Val Tyr
Pro Asn Pro 420 425 430Asp Asn Ser Ser Glu Tyr Phe Leu Tyr Thr Asp
Gly Tyr Pro Val Asn 435 440 445Tyr Gln Glu Asn Gly Asn Gly Lys Ser
Trp Ile Val Leu Gly Lys Lys 450 455 460Thr Asp Lys Pro Lys Ala Trp
Lys Phe Ile Gln Ala Glu465 470 475131927DNAUnknownObtained from an
environmental sample 131atgccgagcc caaaaagtaa tattgatgtt atcagcatcg
atggtggtgg aatacgtgga 60gtattctccg ttacattatt ggatagatta tgtaagacct
atcccaatct tcttaagaaa 120acatatctgt ttgctggaac atctacaggt
gggatcattg ccttaggatt agcaaacaac 180atgacacctc ttgagataag
agccttgtac gagaagaacg gttcaaagat atttcataaa 240tctgtgtggg
aaggcgttaa agatttaggt ggaaccatag gtgcaaagta tagtaacaag
300aatcttaaat ccgttttgaa aaaatacttt ggttcattga agttaaaaga
tttatctaaa 360aaagtactaa tacctacttt tgatttacac tcagacaaag
aagaaggcta tccaatgtgg 420aagcctaagt tctatcacaa ctttgatgga
gaaacggaag atatagaaaa gctcgttctt 480gatgtagcta tgatgacatc
agcagcgccc actttcttcc ctacatacaa cgggcatatt 540gatggcggtg
ttgtagccaa caatccatcg atggccgcat tagcccagat tatggatgaa
600agatatggca tcaatgcctc tgaagttcat attcttaata taggaacagg
ttttaaccct 660gcttatgtta agatgaatcc aggggaagag aaagactggg
gtgaacttca gtggataaaa 720cctttaatca atcttctagt cgatggctct
atggatgttt ctacttatta ttgtaagcaa 780gtcttacgtg ataattttta
tagggttaac atgaaattac ctaagaacgt agaaatggat 840gatcctaatt
ctattcctta tttaattgaa cttgcaaact cagttgatct aactgaatgt
900atcaactggc ttaattcgag gtggtaa 927132308PRTUnknownObtained from
an environmental sample 132Met Pro Ser Pro Lys Ser Asn Ile Asp Val
Ile Ser Ile Asp Gly Gly1 5 10 15Gly Ile Arg Gly Val Phe Ser Val Thr
Leu Leu Asp Arg Leu Cys Lys 20 25 30Thr Tyr Pro Asn Leu Leu Lys Lys
Thr Tyr Leu Phe Ala Gly Thr Ser 35 40 45Thr Gly Gly Ile Ile Ala Leu
Gly Leu Ala Asn Asn Met Thr Pro Leu 50 55 60Glu Ile Arg Ala Leu Tyr
Glu Lys Asn Gly Ser Lys Ile Phe His Lys65 70 75 80Ser Val Trp Glu
Gly Val Lys Asp Leu Gly Gly Thr Ile Gly Ala Lys 85 90 95Tyr Ser Asn
Lys Asn Leu Lys Ser Val Leu Lys Lys Tyr Phe Gly Ser 100 105 110Leu
Lys Leu Lys Asp Leu Ser Lys Lys Val Leu Ile Pro Thr Phe Asp 115 120
125Leu His Ser Asp Lys Glu Glu Gly Tyr Pro Met Trp Lys Pro Lys Phe
130 135 140Tyr His Asn Phe Asp Gly Glu Thr Glu Asp Ile Glu Lys Leu
Val Leu145 150 155 160Asp Val Ala Met Met Thr Ser Ala Ala Pro Thr
Phe Phe Pro Thr Tyr 165 170 175Asn Gly His Ile Asp Gly Gly Val Val
Ala Asn Asn Pro Ser Met Ala 180 185 190Ala Leu Ala Gln Ile Met Asp
Glu Arg Tyr Gly Ile Asn Ala Ser Glu 195 200 205Val His Ile Leu Asn
Ile Gly Thr Gly Phe Asn Pro Ala Tyr Val Lys 210 215 220Met Asn Pro
Gly Glu Glu Lys Asp Trp Gly Glu Leu Gln Trp Ile Lys225 230 235
240Pro Leu Ile Asn Leu Leu Val Asp Gly Ser Met Asp Val Ser Thr Tyr
245 250 255Tyr Cys Lys Gln Val Leu Arg Asp Asn Phe Tyr Arg Val Asn
Met Lys 260 265 270Leu Pro Lys Asn Val Glu Met Asp Asp Pro Asn Ser
Ile Pro Tyr Leu 275 280 285Ile Glu Leu Ala Asn Ser Val Asp Leu Thr
Glu Cys Ile Asn Trp Leu 290 295 300Asn Ser Arg
Trp3051331053DNAUnknownObtained from an environmental sample
133atgactacac agtttcgcaa tctcgttttc gaaggaggcg gcgtcagggg
tatagcctat 60gtgggggcaa tgcaggttct tgagcaacgg ggaatgctca ggaacataga
ccgtgcaggc 120ggcacgagcg ccggtgcgat taacgcactc atcttttcac
tcggctatga cataaggtct 180cagctcgaaa tactccattc taccgacttt
agaaatttta tggatagttc cttcgggata 240atcagggata tccgccgtct
tgcacgggat ttcggatggt acaagggtga tttcttcaca 300ggctggattg
gcaagcttat aaaagacagg ctcggtagcg agaaagcaac tttccgtgac
360cttgcagaat cagattgtcc cgatctgtat gtgatcggca ccaacctctc
aaccggcttc 420gccgaggtat tctcagccga gagacatccc gatatgcctc
ttgcaacggc tgtccgtatc 480agcatgtcga tccctctatt ttttgctgca
atgcgttatg gtccgaggga agacgtattt 540gtagacggtg gggtagtact
caactatcct gtaaagctgt ttgacaggtt gaaatacatt 600gaaagcgggg
agacggagga agccgcacgc tataccgaat attataacag ggagaacgca
660cggttccttc tcaaaagtcc cgaccgcagt ccctatgttt ataaccgtca
gacactgggt 720ttgcgtctcg atacgcgtga ggagattgca catttccgtt
atgacgagcc cctggagggt 780aaaaaaatca tacgctttac ggattatgca
cgggcactcg tttcaacctt gcttcaggtt 840caggaaaacc agcatctgca
cagtgacgac tggcagcgta cagtttacat tgacacactg 900gatgtgaaga
cgactgattt tgatatcacg gataagcaga aggacatcct gataaagcag
960ggaattaacg gagcggagaa ctatttgggt tggtttgaag acccgtatga
aaaacccgcc 1020aaccgcctgc ccggtggcag caagtctgac tga
1053134350PRTUnknownObtained from an environmental sample 134Met
Thr Thr Gln Phe Arg Asn Leu Val Phe Glu Gly Gly Gly Val Arg1 5 10
15Gly Ile Ala Tyr Val Gly Ala Met Gln Val Leu Glu Gln Arg Gly Met
20 25 30Leu Arg Asn Ile Asp Arg Ala Gly Gly Thr Ser Ala Gly Ala Ile
Asn 35 40 45Ala Leu Ile Phe Ser Leu Gly Tyr Asp Ile Arg Ser Gln Leu
Glu Ile 50 55 60Leu His Ser Thr Asp Phe Arg Asn Phe Met Asp Ser Ser
Phe Gly Ile65 70 75 80Ile Arg Asp Ile Arg Arg Leu Ala Arg Asp Phe
Gly Trp Tyr Lys Gly 85 90 95Asp Phe Phe Thr Gly Trp Ile Gly Lys Leu
Ile Lys Asp Arg Leu Gly 100 105 110Ser Glu Lys Ala Thr Phe Arg Asp
Leu Ala Glu Ser Asp Cys Pro Asp 115 120 125Leu Tyr Val Ile Gly Thr
Asn Leu Ser Thr Gly Phe Ala Glu Val Phe 130 135 140Ser Ala Glu Arg
His Pro Asp Met Pro Leu Ala Thr Ala Val Arg Ile145 150 155 160Ser
Met Ser Ile Pro Leu Phe Phe Ala Ala Met Arg Tyr Gly Pro Arg 165 170
175Glu Asp Val Phe Val Asp Gly Gly Val Val Leu Asn Tyr Pro Val Lys
180 185 190Leu Phe Asp Arg Leu Lys Tyr Ile Glu Ser Gly Glu Thr Glu
Glu Ala 195 200 205Ala Arg Tyr Thr Glu Tyr Tyr Asn Arg Glu Asn Ala
Arg Phe Leu Leu 210 215 220Lys Ser Pro Asp Arg Ser Pro Tyr Val Tyr
Asn Arg Gln Thr Leu Gly225 230 235 240Leu Arg Leu Asp Thr Arg Glu
Glu Ile Ala His Phe Arg Tyr Asp Glu 245 250 255Pro Leu Glu Gly Lys
Lys Ile Ile Arg Phe Thr Asp Tyr Ala Arg Ala 260 265 270Leu Val Ser
Thr Leu Leu Gln Val Gln Glu Asn Gln His Leu His Ser 275 280 285Asp
Asp Trp Gln Arg Thr Val Tyr Ile Asp Thr Leu Asp Val Lys Thr 290 295
300Thr Asp Phe Asp Ile Thr Asp Lys Gln Lys Asp Ile Leu Ile Lys
Gln305 310 315 320Gly Ile Asn Gly Ala Glu Asn Tyr Leu Gly Trp Phe
Glu Asp Pro Tyr 325 330 335Glu Lys Pro Ala Asn Arg Leu Pro Gly Gly
Ser Lys Ser Asp 340 345 3501351710DNAUnknownObtained from an
environmental sample 135atggctgaca acgagttacc cctggcccgc cccagggaaa
cccctccgtg ccgtcccggc 60acgttcgagc tggggctggc gctcgccggc gcggtatcgg
gcggcgccta cgccgcgggc 120gtgctggatt tcttctacga ggcgctggag
cactggtacg acgcgaaggc gaacggtgcg 180cccgtgccga gccacgacgt
gctgctacgg atcatttcag gcgcctccgc gggcagcatc 240aacggcgtgc
tttccggcat cgcgttgccg taccgcttcc cgcacgtgca cagcggaccc
300gcgccccggc aggcgacggg aaaccccttc tacgacgcgt gggtgaggcg
catcgatgta 360cgcgagctgc tgggcgaggc cgacctggct aacccggcgc
ggccgatcac ctcgctgctt 420gattccagca gcctggatac gatcgcgaag
gacatgctcg gctacgccgg cgtgccggcc 480gcgcgccctt acatcgcgaa
cccgctgaaa tgcgtgttca ccgtgacgaa tcttcgcggc 540gtgccctacg
tggtgcagtt caagggcaac cccgagattc ccggccacgg catgatggcg
600cacgccgatt ggctgcgctt cgccatcgac tcggggcagg gcgaacgcga
tggcgcatgg 660atgttccccg acgagcgcat cgtcagcggc ccgagccatg
cgcgcagccc ggcctggcat 720gcgctcatgg aggcggccct ggcgtcgtcc
gcgttcccgg ccggcctgcg cttccgcgag 780gtggcccggc cgtggagcga
ttacgaccag cgcgtggttg tcgtgcccgg tcaggatggc 840atggcggtgc
cggtaccgct gccaccagcg tggggcgaag gggagggtgg gaagggcgac
900taccgctttg tcgccgtgga tggtggcgcc atggataacg aaccgttcga
gctggcccgc 960acggagcttg cgggcacgat gggccgcaac ccgcgtgaag
gtacccgggt gaatcgtatc 1020gtgattatgc tcgatccgtt tccggaggcc
gaggcgcccg gcccctcgga ggcggcgtcg 1080acgaacctgg tggaagcgat
ggcgtcgctg ttcggtgcat ggaagcagca ggcgcggttc 1140aagcccgagg
aagtggcgct ggccctcgat agcacggtgt acagccgctt catgatcgcg
1200cctagccgcc cctgcacgga tggcggcccg cggtggatcg gcggccgcgc
gctcaccgcg 1260ggcgcactgg gtggcttctc ggggttcctg gccgaggatt
accgccacca cgatttcctc 1320ctgggccggc gtaactgcca gcggtttctc
gccgagcggc tgctcgttcc cgcaacgaac 1380ccgatcttcg ctggatggat
cgacgatccc gcactgcagg gctacgtgcg tgagatcgat 1440ggtgagcgct
ttgcccccgt gattccccta gtgggcggct gccaggccct gcaagagccc
1500ttgccggcgt ggccgcgtgg ggcgttcgac atggatgcgc tcatgcccct
ggtcgagaag 1560cgcatgcagg ccctgtacac ggcggccacc acgaagctgg
gtggccgctt cgccatgtgg 1620ctcgcgtggc gcttcttcat ccgccgcaaa
ctcctcgaca tcgtctcaag ccgtatccgc 1680aatgcgctga aagacttcgg
cctttggtga 1710136569PRTUnknownObtained from an environmental
sample 136Met Ala Asp Asn Glu Leu Pro Leu Ala Arg Pro Arg Glu Thr
Pro Pro1 5 10 15Cys Arg Pro Gly Thr Phe Glu Leu Gly Leu Ala Leu Ala
Gly Ala Val 20 25 30Ser Gly Gly Ala Tyr Ala Ala Gly Val Leu Asp Phe
Phe Tyr Glu Ala 35 40 45Leu Glu His Trp Tyr Asp Ala Lys Ala Asn Gly
Ala Pro Val Pro Ser 50 55 60His Asp Val Leu Leu Arg Ile Ile Ser Gly
Ala Ser Ala Gly Ser Ile65 70 75 80Asn Gly Val Leu Ser Gly Ile Ala
Leu Pro Tyr Arg Phe Pro His Val 85 90 95His Ser Gly Pro Ala Pro Arg
Gln Ala Thr Gly Asn Pro Phe Tyr Asp 100 105 110Ala Trp Val Arg Arg
Ile Asp Val Arg Glu Leu Leu Gly Glu Ala Asp 115 120 125Leu Ala Asn
Pro Ala Arg Pro Ile Thr Ser Leu Leu Asp Ser Ser Ser 130 135 140Leu
Asp Thr Ile Ala Lys Asp Met Leu Gly Tyr Ala Gly Val Pro Ala145 150
155 160Ala Arg Pro Tyr Ile Ala Asn Pro Leu Lys Cys Val Phe Thr Val
Thr 165 170 175Asn Leu Arg Gly Val Pro Tyr Val Val Gln Phe Lys Gly
Asn Pro Glu 180 185 190Ile Pro Gly His Gly Met Met Ala His Ala Asp
Trp Leu Arg Phe Ala 195 200 205Ile Asp Ser Gly Gln Gly Glu Arg Asp
Gly Ala Trp Met Phe Pro Asp 210 215 220Glu Arg Ile Val Ser Gly Pro
Ser His Ala Arg Ser Pro Ala Trp His225 230 235 240Ala Leu Met Glu
Ala Ala Leu Ala Ser Ser Ala Phe Pro Ala Gly Leu 245 250 255Arg Phe
Arg Glu Val Ala Arg Pro Trp Ser Asp Tyr Asp Gln Arg Val 260 265
270Val Val Val Pro Gly Gln Asp Gly Met Ala Val Pro Val Pro Leu Pro
275 280 285Pro Ala Trp Gly Glu Gly Glu Gly Gly Lys Gly Asp Tyr Arg
Phe Val 290 295 300Ala Val Asp Gly Gly Ala Met Asp Asn Glu Pro Phe
Glu Leu Ala Arg305 310 315 320Thr Glu Leu Ala Gly Thr Met Gly Arg
Asn Pro Arg Glu Gly Thr Arg 325 330 335Val Asn Arg Ile Val Ile Met
Leu Asp Pro Phe Pro Glu Ala Glu Ala 340 345 350Pro Gly Pro Ser Glu
Ala Ala Ser Thr Asn Leu Val Glu Ala Met Ala 355 360 365Ser Leu Phe
Gly Ala Trp Lys Gln Gln Ala Arg Phe Lys Pro Glu Glu 370 375 380Val
Ala Leu Ala Leu Asp Ser Thr Val Tyr Ser Arg Phe Met Ile Ala385 390
395 400Pro Ser Arg Pro Cys Thr Asp Gly Gly Pro Arg Trp Ile Gly Gly
Arg 405 410 415Ala Leu Thr Ala Gly Ala Leu Gly Gly Phe Ser Gly Phe
Leu Ala Glu 420 425 430Asp Tyr Arg His His Asp Phe Leu Leu Gly
Arg Arg Asn Cys Gln Arg 435 440 445Phe Leu Ala Glu Arg Leu Leu Val
Pro Ala Thr Asn Pro Ile Phe Ala 450 455 460Gly Trp Ile Asp Asp Pro
Ala Leu Gln Gly Tyr Val Arg Glu Ile Asp465 470 475 480Gly Glu Arg
Phe Ala Pro Val Ile Pro Leu Val Gly Gly Cys Gln Ala 485 490 495Leu
Gln Glu Pro Leu Pro Ala Trp Pro Arg Gly Ala Phe Asp Met Asp 500 505
510Ala Leu Met Pro Leu Val Glu Lys Arg Met Gln Ala Leu Tyr Thr Ala
515 520 525Ala Thr Thr Lys Leu Gly Gly Arg Phe Ala Met Trp Leu Ala
Trp Arg 530 535 540Phe Phe Ile Arg Arg Lys Leu Leu Asp Ile Val Ser
Ser Arg Ile Arg545 550 555 560Asn Ala Leu Lys Asp Phe Gly Leu Trp
5651371038DNAUnknownObtained from an environmental sample
137atgacaacac aatttagaaa cttgatattt gaaggcggcg gtgtaaaagg
tgttgcttac 60attggcgcca tgcagattct tgaaaatcgt ggcgtgttgc aagatattcg
ccgagtcgga 120gggtgcagtg cgggtgcgat taacgcgctg atttttgcgc
taggttacac ggtccgtgaa 180caaaaagaga tcttacaagc caccgatttt
aaccagttta tggataactc ttggggggtt 240attcgtgata ttcgcaggct
tgctcgagac tttggctgga ataagggtga tttctttagt 300agctggatag
gtgatttgat tcatcgtcgt ttggggaatc gccgagcgac gttcaaagat
360ctgcaaaagg ccaagcttcc tgatctttat gtcatcggta ctaatctgtc
tacagggttt 420gcagaggtgt tttctgccga aagacacccc gatatggagc
tggcgacagc ggtgcgtatc 480tccatgtcga taccgctgtt ctttgcggcc
gtgcgtcacg gtgatcgaca agatgtgtat 540gtcgatgggg gtgttcaact
taactatccg attaaactgt ttgatcggga gcgttacatt 600gatttggcca
aagatcccgg tgccgttcgg cgaacgggtt attacaacaa agaaaacgct
660cgctttcagc ttgatcggcc gggccatagc ccctatgttt acaatcgcca
gaccttgggt 720ttgcgactgg atagtcgcga ggagataggg ctctttcgtt
atgacgaacc cctcaagggc 780aaacccatta agtccttcac tgactacgct
cgacaacttt tcggtgcgtt gatgaatgca 840caggaaaaga ttcatctaca
tggcgatgat tggcaacgca cgatctatat cgatacattg 900gatgtgggta
cgacggactt caatctttct gatgcaacta agcaagcact gattgagcaa
960ggaattaacg gcaccgaaaa ttatttcgag tggtttgata atccgttaga
gaagcctgtg 1020aatagagtgg agtcatag 1038138345PRTUnknownObtained
from an environmental sample 138Met Thr Thr Gln Phe Arg Asn Leu Ile
Phe Glu Gly Gly Gly Val Lys1 5 10 15Gly Val Ala Tyr Ile Gly Ala Met
Gln Ile Leu Glu Asn Arg Gly Val 20 25 30Leu Gln Asp Ile Arg Arg Val
Gly Gly Cys Ser Ala Gly Ala Ile Asn 35 40 45Ala Leu Ile Phe Ala Leu
Gly Tyr Thr Val Arg Glu Gln Lys Glu Ile 50 55 60Leu Gln Ala Thr Asp
Phe Asn Gln Phe Met Asp Asn Ser Trp Gly Val65 70 75 80Ile Arg Asp
Ile Arg Arg Leu Ala Arg Asp Phe Gly Trp Asn Lys Gly 85 90 95Asp Phe
Phe Ser Ser Trp Ile Gly Asp Leu Ile His Arg Arg Leu Gly 100 105
110Asn Arg Arg Ala Thr Phe Lys Asp Leu Gln Lys Ala Lys Leu Pro Asp
115 120 125Leu Tyr Val Ile Gly Thr Asn Leu Ser Thr Gly Phe Ala Glu
Val Phe 130 135 140Ser Ala Glu Arg His Pro Asp Met Glu Leu Ala Thr
Ala Val Arg Ile145 150 155 160Ser Met Ser Ile Pro Leu Phe Phe Ala
Ala Val Arg His Gly Asp Arg 165 170 175Gln Asp Val Tyr Val Asp Gly
Gly Val Gln Leu Asn Tyr Pro Ile Lys 180 185 190Leu Phe Asp Arg Glu
Arg Tyr Ile Asp Leu Ala Lys Asp Pro Gly Ala 195 200 205Val Arg Arg
Thr Gly Tyr Tyr Asn Lys Glu Asn Ala Arg Phe Gln Leu 210 215 220Asp
Arg Pro Gly His Ser Pro Tyr Val Tyr Asn Arg Gln Thr Leu Gly225 230
235 240Leu Arg Leu Asp Ser Arg Glu Glu Ile Gly Leu Phe Arg Tyr Asp
Glu 245 250 255Pro Leu Lys Gly Lys Pro Ile Lys Ser Phe Thr Asp Tyr
Ala Arg Gln 260 265 270Leu Phe Gly Ala Leu Met Asn Ala Gln Glu Lys
Ile His Leu His Gly 275 280 285Asp Asp Trp Gln Arg Thr Ile Tyr Ile
Asp Thr Leu Asp Val Gly Thr 290 295 300Thr Asp Phe Asn Leu Ser Asp
Ala Thr Lys Gln Ala Leu Ile Glu Gln305 310 315 320Gly Ile Asn Gly
Thr Glu Asn Tyr Phe Glu Trp Phe Asp Asn Pro Leu 325 330 335Glu Lys
Pro Val Asn Arg Val Glu Ser 340 3451391692DNAUnknownObtained from
an environmental sample 139atgaaaataa agccgctcac gttttctttt
ggattagcag tcactagctc ggtgcaagcc 60ttcactcaat ttggcggaca aggcgttatg
ccgatgggtc acgaatggtt aacgcgcacc 120gctgctctcg aggtacttaa
tgcagagcat atcatcgaag cggatccgaa tgacccaaga 180tatacttggc
aggacggact tgctaaaaac cttgaactta ataccgccca atctgaaatc
240acgcgcttac aatctcattt aaataataac ccgctctatg agccgagata
cgacggtata 300aactcagcca tcgttggtga acgctgggtc gatattgcag
ggtttaacgt cacaacagcc 360agcgcagacc cgactggccc taattgcttt
agcgcagttt cacaagagcc cgcagatatt 420cagcaagacc actttatgcg
ccgctatgat gatattggag gtcaaggtgg agttgatgct 480gcttatcgcg
cacagcaacg atttgtgcaa cactttgtgg atgcggccat ggccgaaaaa
540aaacgactaa aagtatggga cggtggtggc cattctgcgt tagcagaggt
agatcataat 600tactttttat ttggtcgtgc ggttcaccta tttcaagact
catttagtcc agaacacacg 660gtacggctcc ctcaagataa ctacgaaaaa
gtttggcagg ttaaggcata tctttgctca 720gagggggctg agcaacattc
acacgatacc aaagacgtgc tcaactttgc cagtggcgat 780gttatttggc
aacctcaaac ccgactagaa gcaggctggc aatcttacca gatcagcagt
840atgaagcccg ttgctattgt ggcccttgaa gccagtaaag atctttgggc
tgcgtttatt 900cgcaccatgg cgaccccaaa agcacagaga cgtaacgtgg
caacgcaaga agcccaacaa 960cttgtacaaa actggttgtc ttttgatgag
gcccagatgc tgacttggta tcaagatgag 1020aataagcgtg accatactta
tgtgcttgcc cccaatgaaa cgggaaaagg aaaatctctg 1080gaagcctgta
tgacagagct aaaggtaggc actagcagtc aagcagaacg ggttgcgcaa
1140ctggaagccg agcgtaatca atgcctatac aacattgagg cggaacctgg
ctttgcagac 1200ttaaacgatc cacacctcga tattccatat aactggcgct
ggaagtctct gacttggcaa 1260acgcctccta gtggctggac atacccacaa
ctaaatgcag ataccggcga gcaagtcgcc 1320attaaatcgc cgataaataa
tcagtattta tctgcacaaa ctctaagtaa cgacaccccg 1380atcactctga
gtcaagcaca tccaatttcc ttgatccaag tgacgaatgc acagggccag
1440cactatttta ggagcgctca agccccttca ctatttctgg gttatagcaa
caaaattgca 1500ggctacctca agcttgtaga ttcacccaag caagccctat
atacgttgat ttatcaaggt 1560ggtctttgga atatccaaaa tgaattttgg
caacagtata tctggttaaa tcaagacaaa 1620gagcggccgg aattaaatcg
ccatggtgag cctagccaat taaacgctca gtggatggtc 1680gaacacttat aa
1692140563PRTUnknownObtained from an environmental sample 140Met
Lys Ile Lys Pro Leu Thr Phe Ser Phe Gly Leu Ala Val Thr Ser1 5 10
15Ser Val Gln Ala Phe Thr Gln Phe Gly Gly Gln Gly Val Met Pro Met
20 25 30Gly His Glu Trp Leu Thr Arg Thr Ala Ala Leu Glu Val Leu Asn
Ala 35 40 45Glu His Ile Ile Glu Ala Asp Pro Asn Asp Pro Arg Tyr Thr
Trp Gln 50 55 60Asp Gly Leu Ala Lys Asn Leu Glu Leu Asn Thr Ala Gln
Ser Glu Ile65 70 75 80Thr Arg Leu Gln Ser His Leu Asn Asn Asn Pro
Leu Tyr Glu Pro Arg 85 90 95Tyr Asp Gly Ile Asn Ser Ala Ile Val Gly
Glu Arg Trp Val Asp Ile 100 105 110Ala Gly Phe Asn Val Thr Thr Ala
Ser Ala Asp Pro Thr Gly Pro Asn 115 120 125Cys Phe Ser Ala Val Ser
Gln Glu Pro Ala Asp Ile Gln Gln Asp His 130 135 140Phe Met Arg Arg
Tyr Asp Asp Ile Gly Gly Gln Gly Gly Val Asp Ala145 150 155 160Ala
Tyr Arg Ala Gln Gln Arg Phe Val Gln His Phe Val Asp Ala Ala 165 170
175Met Ala Glu Lys Lys Arg Leu Lys Val Trp Asp Gly Gly Gly His Ser
180 185 190Ala Leu Ala Glu Val Asp His Asn Tyr Phe Leu Phe Gly Arg
Ala Val 195 200 205His Leu Phe Gln Asp Ser Phe Ser Pro Glu His Thr
Val Arg Leu Pro 210 215 220Gln Asp Asn Tyr Glu Lys Val Trp Gln Val
Lys Ala Tyr Leu Cys Ser225 230 235 240Glu Gly Ala Glu Gln His Ser
His Asp Thr Lys Asp Val Leu Asn Phe 245 250 255Ala Ser Gly Asp Val
Ile Trp Gln Pro Gln Thr Arg Leu Glu Ala Gly 260 265 270Trp Gln Ser
Tyr Gln Ile Ser Ser Met Lys Pro Val Ala Ile Val Ala 275 280 285Leu
Glu Ala Ser Lys Asp Leu Trp Ala Ala Phe Ile Arg Thr Met Ala 290 295
300Thr Pro Lys Ala Gln Arg Arg Asn Val Ala Thr Gln Glu Ala Gln
Gln305 310 315 320Leu Val Gln Asn Trp Leu Ser Phe Asp Glu Ala Gln
Met Leu Thr Trp 325 330 335Tyr Gln Asp Glu Asn Lys Arg Asp His Thr
Tyr Val Leu Ala Pro Asn 340 345 350Glu Thr Gly Lys Gly Lys Ser Leu
Glu Ala Cys Met Thr Glu Leu Lys 355 360 365Val Gly Thr Ser Ser Gln
Ala Glu Arg Val Ala Gln Leu Glu Ala Glu 370 375 380Arg Asn Gln Cys
Leu Tyr Asn Ile Glu Ala Glu Pro Gly Phe Ala Asp385 390 395 400Leu
Asn Asp Pro His Leu Asp Ile Pro Tyr Asn Trp Arg Trp Lys Ser 405 410
415Leu Thr Trp Gln Thr Pro Pro Ser Gly Trp Thr Tyr Pro Gln Leu Asn
420 425 430Ala Asp Thr Gly Glu Gln Val Ala Ile Lys Ser Pro Ile Asn
Asn Gln 435 440 445Tyr Leu Ser Ala Gln Thr Leu Ser Asn Asp Thr Pro
Ile Thr Leu Ser 450 455 460Gln Ala His Pro Ile Ser Leu Ile Gln Val
Thr Asn Ala Gln Gly Gln465 470 475 480His Tyr Phe Arg Ser Ala Gln
Ala Pro Ser Leu Phe Leu Gly Tyr Ser 485 490 495Asn Lys Ile Ala Gly
Tyr Leu Lys Leu Val Asp Ser Pro Lys Gln Ala 500 505 510Leu Tyr Thr
Leu Ile Tyr Gln Gly Gly Leu Trp Asn Ile Gln Asn Glu 515 520 525Phe
Trp Gln Gln Tyr Ile Trp Leu Asn Gln Asp Lys Glu Arg Pro Glu 530 535
540Leu Asn Arg His Gly Glu Pro Ser Gln Leu Asn Ala Gln Trp Met
Val545 550 555 560Glu His Leu
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