U.S. patent application number 14/550579 was filed with the patent office on 2015-03-19 for starch process.
The applicant listed for this patent is NOVOZYMES A/S. Invention is credited to Carsten Andersen, Carsten Hjort, Sven Pedersen, Anders Vikso-Nielsen.
Application Number | 20150079628 14/550579 |
Document ID | / |
Family ID | 33542435 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079628 |
Kind Code |
A1 |
Vikso-Nielsen; Anders ; et
al. |
March 19, 2015 |
STARCH PROCESS
Abstract
The present invention relates to a process for enzymatic
hydrolysis of granular starch into a soluble starch hydrolysate at
a temperature below the initial gelatinization temperature of said
granular starch.
Inventors: |
Vikso-Nielsen; Anders;
(Slangerup, DK) ; Andersen; Carsten; (Vaerloese,
DK) ; Pedersen; Sven; (Gentofte, DK) ; Hjort;
Carsten; (Vaerloese, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES A/S |
Bagsvaerd |
|
DK |
|
|
Family ID: |
33542435 |
Appl. No.: |
14/550579 |
Filed: |
November 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13333286 |
Dec 21, 2011 |
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14550579 |
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10561671 |
Dec 20, 2005 |
8105801 |
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PCT/DK2004/004563 |
Jun 25, 2004 |
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13333286 |
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60482589 |
Jun 25, 2003 |
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60514854 |
Oct 27, 2003 |
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Current U.S.
Class: |
435/43 ; 127/67;
435/110; 435/136; 435/137; 435/139; 435/144; 435/162; 435/64;
435/95; 435/99 |
Current CPC
Class: |
C12N 9/2428 20130101;
C12P 7/06 20130101; C07K 2319/00 20130101; C12N 9/2408 20130101;
C12N 9/2414 20130101; C12P 19/16 20130101; C12N 9/2411 20130101;
Y02E 50/17 20130101; C13K 11/00 20130101; C12N 9/242 20130101; C12P
19/14 20130101; C12P 19/22 20130101; C12P 19/02 20130101; Y02E
50/10 20130101; C12N 9/2425 20130101 |
Class at
Publication: |
435/43 ; 435/99;
435/144; 435/110; 435/137; 435/139; 435/136; 435/64; 435/162;
435/95; 127/67 |
International
Class: |
C12P 19/14 20060101
C12P019/14; C12P 19/22 20060101 C12P019/22; C13K 11/00 20060101
C13K011/00; C12P 19/02 20060101 C12P019/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 25, 2003 |
DK |
PA 2003 00949 |
Oct 24, 2003 |
DK |
PA 2003 01568 |
Claims
1. A process for producing a soluble starch hydrolyzate, comprising
subjecting an aqueous granular starch slurry at a temperature below
the initial gelatinization temperature of said granular starch to
the action of a first enzyme and a second enzyme, wherein (a) the
first enzyme (i) is a member of Glycoside Hydrolase Family 13; (ii)
has alpha-1,4-glucosidic hydrolysis activity; and (iii) comprises a
functional carbohydrate-binding module (CBM) belonging to CBM
Family 20, which has an amino acid sequence having at least 80%
homology to the amino acid sequence of SEQ ID NO: 2; and (b) the
second enzyme is a fungal alpha-amylase (EC 3.2.1.1), a
beta-amylase (E.C. 3.2.1.2), or a glucoamylase (E.C.3.2.1.3).
2. The process of claim 1, wherein the CBM has an amino acid
sequence having at least 85% homology to the amino acid sequence of
SEQ ID NO: 2.
3. The process of claim 1, wherein the first enzyme is an
alpha-amylase.
4. The process of claim 1, wherein the first enzyme comprises an
amino acid sequence having at least 95% homology to the amino acid
sequence of SEQ ID NO: 19.
5. The process of claim 1, wherein the first enzyme is a hybrid
alpha-amylase.
6. The process of claim 1, wherein the first enzyme is an
alpha-amylase which comprises a catalytic domain which has an amino
acid sequence having at least 90% homology to the amino acid
sequence of SEQ ID NO: 4.
7. The process of claim 1, wherein the first enzyme is an
alpha-amylase which comprises a catalytic domain which has an amino
acid sequence having at least 95% homology to the amino acid
sequence of SEQ ID NO: 4.
8. The process of claim 1, wherein the second enzyme is a fungal
alpha-amylase.
9. The process of claim 1, wherein the second enzyme is a
beta-amylase.
10. The process of claim 1, wherein the second enzyme is a
glucoamylase.
11. The process of claim 1, wherein the starch slurry has 20-55%
dry solids granular starch.
12. The process of claim 1, wherein at least 85% of the dry solids
of the granular starch is converted into the soluble starch
hydrolyzate.
13. The process of claim 1, further comprising subjecting the
granular starch slurry to the action of an isoamylase and/or a
pullulanase.
14. The process of claim 1, which is conducted at a temperature of
at least 58.degree. C.
15. The process of claim 1, which is conducted at a pH of 3-7.
16. The process of claim 1, wherein the soluble starch hydrolyzate
has a DX of at least 94.5%.
17. The process of claim 1, wherein the granular starch is obtained
from tubers, roots, stems, or whole grain.
18. The process of claim 1, wherein the granular starch is obtained
from cereals.
19. The process of claim 1, wherein the granular starch is obtained
from corn, cobs, wheat, barley, rye, milo, sago, cassava, tapioca,
sorghum, rice or potatoes.
20. The process of claim 1, wherein the granular starch is obtained
from dry milling of whole grain or from wet milling of whole grain
or from milled corn grits.
21. The process of claim 1, which is conducted in an
ultrafiltration system wherein the retentate is held under
recirculation in the presence of enzymes, raw starch and water and
the permeate is the soluble starch hydrolyzate.
22. The process of claim 1, which is conducted in a continuous
membrane reactor with ultrafiltration membranes and wherein the
retentate is held under recirculation in the presence of enzymes,
raw starch and water and the permeate is the soluble starch
hydrolyzate.
23. The process of claim 1, which is conducted in a continuous
membrane reactor with microfiltration membranes and wherein the
retentate is held under recirculation in presence of enzymes, raw
starch and water and the permeate is the soluble starch
hydrolyzate.
24. A process for production of high fructose starch-based syrup
(HFSS), comprising subjecting a soluble starch hydrolyzate produced
by the process of claim 1 to conversion into high fructose
starch-based syrup (HFSS).
25. A process for production of a fermentation product, comprising
fermenting a soluble starch hydrolyzate produced by the process of
claim 1.
26. The process of claim 1, wherein the fermentation product is
citric acid, monosodium glutamate, gluconic acid, sodium gluconate,
calcium gluconate, potassium gluconate, glucono delta lactone,
sodium erythorbate, itaconic acid, lactic acid, gluconic acid;
ketones; amino acids, glutamic acid (sodium monoglutaminate),
penicillin, tetracyclin; enzymes; vitamins or hormones.
27. A process for production of fuel or potable ethanol, comprising
fermenting a soluble starch hydrolyzate produced by the process of
claim 1.
28. The process of claim 27, wherein the fermentation step is
carried out simultaneously or separately/sequential to the
hydrolysis of the granular starch.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 13/333,286 filed Dec. 21, 2011, which is a divisional of U.S.
Ser. No. 10/561,671 filed on Dec. 20, 2005, now U.S. Pat. No.
8,105,801, which is a 35 U.S.C. 371 national application of
PCT/DK2004/0004563 filed Jun. 25, 2004, which claims priority or
the benefit under 35 U.S.C. 119 of Danish application nos. PA 2003
00949 and PA 2003 01568 filed Jun. 25, 2003 and Oct. 24, 2003,
respectively, and U.S. provisional application Nos. 60/482,589 and
60/514,854 filed Jun. 25, 2003 and Oct. 27, 2003, respectively, the
contents of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a process for hydrolysis of
granular starch into a soluble starch hydrolysate at a temperature
below the initial gelatinization temperature of said granular
starch.
BACKGROUND OF THE INVENTION
[0003] A large number of processes have been described for
converting starch to starch hydrolysates, such as maltose, glucose
or specialty syrups, either for use as sweeteners or as precursors
for other saccharides such as fructose. Glucose may also be
fermented to ethanol or other fermentation products, such as citric
acid, monosodium glutamate, gluconic acid, sodium gluconate,
calcium gluconate, potassium gluconate, glucono delta lactone,
sodium erythorbate, itaconic acid, lactic acid, gluconic acid;
ketones; amino acids, glutamic acid (sodium monoglutaminate),
penicillin, tetracyclin; enzymes; vitamins, such as riboflavin,
B12, beta-carotene or hormones.
[0004] Starch is a high molecular-weight polymer consisting of
chains of glucose units. It usually consists of about 80%
amylopectin and 20% amylose. Amylopectin is a branched
polysaccharide in which linear chains of alpha-1,4 D-glucose
residues are joined by alpha-1,6 glucosidic linkages.
[0005] Amylose is a linear polysaccharide built up of
D-glucopyranose units linked together by alpha-1,4 glucosidic
linkages. In the case of converting starch into a soluble starch
hydrolysate, the starch is depolymerized. The conventional
depolymerization process consists of a gelatinization step and two
consecutive process steps, namely a liquefaction process and a
saccharification process.
[0006] Granular starch consists of microscopic granules, which are
insoluble in water at room temperature. When an aqueous starch
slurry is heated, the granules swell and eventually burst,
dispersing the starch molecules into the solution. During this
"gelatinization" process there is a dramatic increase in viscosity.
As the solids level is 30-40% in a typical industrial process, the
starch has to be thinned or "liquefied" so that it can be handled.
This reduction in viscosity is today mostly obtained by enzymatic
degradation. During the liquefaction step, the long-chained starch
is degraded into smaller branched and linear units (maltodextrins)
by an alpha-amylase. The liquefaction process is typically carried
out at about 105-110.degree. C. for about 5 to 10 minutes followed
by about 1-2 hours at about 95.degree. C. The temperature is then
lowered to 60.degree. C., a glucoamylase or a beta-amylase and
optionally a debranching enzyme, such as an isoamylase or a
pullulanase are added, and the saccharification process proceeds
for about 24 to 72 hours.
[0007] It will be apparent from the above discussion that the
conventional starch conversion process is very energy consuming due
to the different requirements in terms of temperature during the
various steps. It is thus desirable to be able to select the
enzymes used in the process so that the overall process can be
performed without having to gelatinize the starch. Such processes
are the subject for the patents U.S. Pat. No. 4,591,560, U.S. Pat.
No. 4,727,026 and U.S. Pat. No. 4,009,074 and EP 0171218.
[0008] The present invention relates to a one-step process for
converting granular starch into soluble starch hydrolysate at a
temperature below initial gelatinization temperature of the
starch.
SUMMARY OF THE INVENTION
[0009] In a first aspect the invention provides a process for
producing a soluble starch hydrolysate, the process comprising
subjecting a aqueous granular starch slurry at a temperature below
the initial gelatinization temperature of said granular starch to
the action of a first enzyme, which enzyme; is a member of the
Glycoside Hydrolase Family 13; has alpha-1.4-glucosidic hydrolysis
activity, and; comprises a functional Carbohydrate-Binding Module
(CBM) belonging to CBM Family 20, which CBM has an amino acid
sequence having at least 60% homology to an amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and
SEQ ID NO:3; and which second enzyme is selected from the list
comprising a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase
(E.C. 3.2.1.2), and a glucoamylase (E.C.3.2.1.3).
[0010] The process of the first aspect of the invention may be
performed as a one step process and/or as a process comprising one
or more steps.
[0011] In a second aspect the invention provides a process for
production of high fructose starch-based syrup (HFSS), the process
comprising producing a soluble starch hydrolysate by the process of
the first aspect of the invention, and further comprising a step
for conversion of the soluble starch hydrolysate into a high
fructose starch-based syrup (HFSS).
[0012] In a third aspect the invention provides a process for
production of fuel or potable ethanol; comprising producing a
soluble starch hydrolysate by the process of the first aspect of
the invention, and further comprising a step for fermentation of
the soluble starch hydrolysate into ethanol, wherein the
fermentation step is carried out simultaneously or
separately/sequential to the hydrolysis of the granular starch.
[0013] In a fourth aspect the invention provides a use of an enzyme
having alpha-amylase activity in a process for hydrolysis of
starch, said enzyme comprising a functional CBM having an amino
acid sequence having at least 60% homology to an amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, and SEQ ID NO:3.
[0014] In a fifth aspect the invention provides a use of an enzyme
having alpha-amylase activity in a process for hydrolysis of
granular starch, said enzyme comprising an amino acid sequence
having at least 75%, least 80%, at least 85%, at least 90%, least
95%, at least 98%, such as at least 99% homology to an amino acid
sequence selected from the group consisting of SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.
[0015] In a sixth aspect the invention provides a use of an enzyme
having alpha-amylase activity and a functional CBM in a process for
hydrolysis of granular starch, said enzyme comprising an amino acid
sequence having at least 75%, at least 80%, at least 85%, at least
90%, least 95%, at least 98%, such as at least 99% homology to an
amino acid sequence selected from the group consisting of SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0016] The term "granular starch" is understood as raw uncooked
starch, i.e. starch that has not been subjected to a
gelatinization. Starch is formed in plants as tiny granules
insoluble in water. These granules are preserved in starches at
temperatures below the initial gelatinization temperature. When put
in cold water, the grains may absorb a small amount of the liquid.
Up to 50.degree. C. to 70.degree. C. the swelling is reversible,
the degree of reversibility being dependent upon the particular
starch. With higher temperatures an irreversible swelling called
gelatinization begins.
[0017] The term "initial gelatinization temperature" is understood
as the lowest temperature at which gelatinization of the starch
commences. Starch heated in water begins to gelatinize between
50.degree. C. and 75.degree. C.; the exact temperature of
gelatinization depends on the specific starch and can readily be
determined by the skilled artisan. Thus, the initial gelatinization
temperature may vary according to the plant species, to the
particular variety of the plant species as well as with the growth
conditions. In the context of this invention the initial
gelatinization temperature of a given starch is the temperature at
which birefringence is lost in 5% of the starch granules using the
method described by Gorinstein and Lii, 1992, Starch/Starke 44(12):
461-466.
[0018] The term "soluble starch hydrolysate" is understood as the
soluble products of the processes of the invention and may comprise
mono- di-, and oligosaccharides, such as glucose, maltose,
maltodextrins, cyclodextrins and any mixture of these. Preferably
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% of the dry
solids of the granular starch is converted into a soluble starch
hydrolysate.
[0019] The term "Speciality Syrups", is an in the art recognised
term and is characterised according to DE and carbohydrate spectrum
(See the article "New Speciality Glucose Syrups", p. 50+, in the
textbook "Molecular Structure and Function of Food Carbohydrate",
Edited by G. G. Birch and L. F. Green, Applied Science Publishers
LTD., London). Typically Speciality Syrups have a DE in the range
from 35 to 45.
[0020] The "Glycoside Hydrolase Family 13" is in the context of
this invention defined as the group of hydrolases comprising a
catalytic module having a (beta/alpha).sub.8 or TIM barrel
structure and acting on starch and related substrates through an
alpha-retaining reacting mechanism (Koshland, 1953, Biol. Rev.
Camp. Philos. Soc. 28: 416-436).
[0021] The enzymes having "alpha-1.4-glucosidic hydrolysis
activity" is in the context of this invention defined as comprising
the group of enzymes which catalyze the hydrolysis and/or synthesis
of alpha-1,4-glucosidic bonds as defined by Takata (Takata et al.,
1992, J. Biol. Chem. 267: 18447-18452) and by Koshland (Koshland,
1953, Biol. Rev. Camp. Philos. Soc. 28: 416-436).
[0022] The "Carbohydrate-Binding Module of Family 20" or a CBM-20
module is in the context of this invention defined as a sequence of
approximately 100 amino acids having at least 45% homology to the
Carbohydrate-Binding Module (CBM) of the polypeptide disclosed in
FIG. 1 by Joergensen et al., 1997, Biotechnol. Lett. 19: 1027-1031.
The CBM comprises the last 102 amino acids of the polypeptide, i.e.
the subsequence from amino acid 582 to amino acid 683. The
numbering of Glycoside Hydrolase Families applied in this
disclosure follows the concept of Coutinho, P. M. & Henrissat,
B. (1999) CAZy-Carbohydrate-Active Enzymes server at URL:
afmb.cnrs-mrs.fr/.about.cazy/CAZY/index.html or alternatively
Coutinho, P. M. & Henrissat, B. 1999; The modular structure of
cellulases and other carbohydrate-active enzymes: an integrated
database approach. In "Genetics, Biochemistry and Ecology of
Cellulose Degradation", K. Ohmiya, K. Hayashi, K. Sakka, Y.
Kobayashi, S. Karita and T. Kimura eds., Uni Publishers Co., Tokyo,
pp. 15-23, and Bourne, Y. & Henrissat, B. 2001; Glycoside
hydrolases and glycosyltransferases: families and functional
modules, Current Opinion in Structural Biology 11:593-600.
[0023] A carbohydrate-binding module (CBM) is a polypeptide amino
acid sequence which binds preferentially to a poly- or
oligosaccharide (carbohydrate), frequently--but not necessarily
exclusively--to a water-insoluble (including crystalline) form
thereof.
[0024] Although a number of types of CBMs have been described in
the patent and scientific literature, the majority thereof--many of
which derive from cellulolytic enzymes (cellulases)--are commonly
referred to as "cellulose-binding modules"; a typical
cellulose-binding module will thus be a CBM which occurs in a
cellulase. Likewise, other sub-classes of CBMs would embrace, e.g.,
chitin-binding modules (CBMs which typically occur in chitinases),
xylan-binding modules (CBMs which typically occur in xylanases),
mannan-binding modules (CBMs which typically occur in mannanases),
starch-binding modules (CBMs which may occur in certain amylolytic
enzymes, such as certain glucoamylases, or in enzymes such as
cyclodextrin glucanotransferases), or in alpha-amylases.
[0025] CBMs are found as integral parts of large polypeptides or
proteins consisting of two or more polypeptide amino acid sequence
regions, especially in hydrolytic enzymes (hydrolases) which
typically comprise a catalytic module containing the active site
for substrate hydrolysis and a carbohydrate-binding module (CBM)
for binding to the carbohydrate substrate in question. Such enzymes
can comprise more than one catalytic module and one, two or three
CBMs, and optionally further comprise one or more polypeptide amino
acid sequence regions linking the CBM(s) with the catalytic
module(s), a region of the latter type usually being denoted a
"linker". Examples of hydrolytic enzymes comprising a CBM--some of
which have already been mentioned above--are cellulases, xylanases,
mannanases, arabinofuranosidases, acetylesterases and chitinases.
CBMs have also been found in algae, e.g. in the red alga Porphyra
purpurea in the form of a non-hydrolytic polysaccharide-binding
protein.
[0026] In proteins/polypeptides in which CBMs occur (e.g. enzymes,
typically hydrolytic enzymes), a CBM may be located at the N or C
terminus or at an internal position.
[0027] That part of a polypeptide or protein (e.g. hydrolytic
enzyme) which constitutes a CBM per se typically consists of more
than about 30 and less than about 250 amino acid residues.
[0028] Preferred for the invention are enzymes comprising a CBM
comprising an amino acid sequence selected from the group
consisting of amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and
SEQ ID NO:3 as well as enzymes comprising a CBM comprising an amino
acid sequence having at least 50% homology to an amino acid
sequence selected from the group consisting of amino acid sequences
SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
[0029] The polypeptide "homology" referred to in this disclosure is
understood as the degree of identity between two sequences
indicating a derivation of the first sequence from the second. The
homology may suitably be determined by means of computer programs
known in the art such as GAP provided in the GCG program package
(Program Manual for the Wisconsin Package, Version 8, August 1994,
Genetics Computer Group, 575 Science Drive, Madison, Wis., USA
53711) (Needleman and Wunsch, 1970, Journal of Molecular Biology
48: 443-453. The following settings for amino acid sequence
comparison are used: GAP creation penalty of 3.0 and GAP extension
penalty of 0.1.
[0030] The enzyme to be used as a first enzyme of the present
invention is a four module alpha-amylase consisting of a three
module amylase core and a separate carbohydrate binding module of
family 20. The alpha-amylase may be a wild type alpha-amylase
derived from bacterial or fungal sources, or it may be mutants,
protein engineered variants, or other variants of such wild types,
or it may be hybrids of variants or wild types.
[0031] Preferably the alpha-amylase is a wild type enzyme. More
preferably the alpha-amylase is a variant and/or hybrid of the
above alpha-amylases comprising amino acid modifications leading to
increased activity, increased protein stability at low pH, and/or
at high pH, increased stability towards calcium depletion, and/or
increased stability at elevated temperature.
[0032] The term "Enzyme hybrids" referred to in this disclosure is
understood as modified enzymes comprising an amino acid sequence of
an amylolytic enzyme [which in the context of the present invention
may be, e.g., an alpha-amylase (EC 3.2.1.1), an isoamylase (EC
3.2.1.68) or a pullulanase (EC 3.2.1.41)] linked (i.e. covalently
bound) to an amino acid sequence comprising a CBM. The CBM is
preferably but not exclusively fused to the N-terminal. The hybrid
may comprise more than one CBM.
[0033] CBM-containing enzyme hybrids, as well as detailed
descriptions of the preparation and purification thereof, are known
in the art [see, e.g., WO 90/00609, WO 94/24158 and WO 95/16782, as
well as Greenwood et al., 1994, Biotechnology and Bioengineering
44: 1295-1305]. They may, e.g., be prepared by transforming into a
host cell a DNA construct comprising at least a fragment of DNA
encoding the cellulose-binding module ligated, with or without a
linker, to a DNA sequence encoding the enzyme of interest, and
growing the transformed host cell to express the fused gene.
[0034] The construction of a hybrid protein between a carbohydrate
binding module (CBM) and an alpha-amylase requires one or more of
the following steps to obtain a stable, expressible and applicable
enzyme.
[0035] 1) Aligning the CBM-donor molecule with the donor of the
catalytic modules using conventional methods is often required to
identify possible crossing points. If the homology is relatively
high there might be several possible crossing point. If however the
homology is low or if only the sequence of the catalytic module and
the CBM are available, respectively, the CBM can be attached as an
elongation to the catalytic module either in the beginning of the
sequence, i.e. in the N-terminal inserted after an eventually
signal sequence; or in the C-terminal prior to the termination
signal. Regardless if the CBM is located in the N- or in the
C-terminal it might be beneficial to either delete a few amino
acids or insert a number of amino acid as a linker to obtain an
expressible and application stable enzyme.
[0036] 2) Construction the DNA hybrid of the genes coding for the
CBM and the amylolytic module according to the considerations made
under 1) can be made by methods known to persons skilled in the
art. These methods include among others, PCR reactions using
primers designed to hybridize over the resulting DNA crossing
point, DNA digesting followed by ligation or in-vivo combination
for example by yeast.
[0037] 3) A simple attachment of a CBM to an amylolytic module
often results in a hybrid protein that is expressed poorly due to
folding or stability problems or in a hybrid protein lacking
sufficient stability and/or activity under a given application. To
overcome such problems the hybrid protein may be subjected to
protein engineering either by site directed mutagenesis methods or
by more random approaches. This includes both the amino acids in
the modules of the CBM and in the amylolytic modules as well as
optimizing the transition from amylolytic module to CBM, with
respect to length and amino acid sequence.
[0038] Preferred as a first enzyme for the present invention are
hybrid enzymes comprising a CBM comprising an amino acid sequence
selected from the group consisting of amino acid sequences SEQ ID
NO:1, SEQ ID NO:2, and SEQ ID NO:3 as well as enzymes comprising an
amino acid sequence having at least 50% at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, least 80%, at least 85%,
at least 90%, least 95%, at least 98%, such as at least 99%
homology to an amino acid sequence selected from the group
consisting of the amino acid sequences SEQ ID NO:1, SEQ ID NO:2,
and SEQ ID NO:3.
[0039] Also preferred as a first enzyme for the present invention
are hybrid enzymes comprising an amino acid sequence having
alpha-amylolytic activity and comprising an amino acid sequence
selected from the group consisting of amino acid sequences SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID
NO:18 as well as enzymes comprising an amino acid sequence having
at least 70%, at least 75%, least 80%, at least 85%, at least 90%,
least 95%, at least 98%, such as at least 99% homology to an amino
acid sequence selected from the group consisting of amino acid
sequences SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ
ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,
and SEQ ID NO:18.
[0040] Preferably the first enzyme of the present invention
comprises a CBM and/or an alpha-amylolytic sequence derived from a
fungi, such as from a strain belonging to a Talaromyces sp., or
from a strain belonging to an Aspergillus sp. such as A. awamori,
A. kawachii, A. niger, A. oryzae etc. or from a bacteria, such as
from a strain belonging to Bacillus sp, such as from a strain
belonging to B. amyloliquefaciens, B. flavothermus, B.
licheniformis or B. stearothermophilus.
[0041] More preferred as a first enzyme of the present invention is
a four module alpha-amylase consisting of a three module amylase
core and a separate carbohydrate binding module of family 20. Most
preferred is a four module alpha-amylase comprising an amino acid
sequence having at least 70%, at least 75%, least 80%, at least
85%, at least 90%, least 95%, at least 98%, such as at least 99%
homology to an amino acid sequence selected from the group
consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID
NO:22.
[0042] Preferably the first enzyme of the present invention is a
four module alpha-amylase isolated from a fungus or a bacteria,
such as from a species of Bacillus sp, such as the polypeptides
shown in SEQ ID NO:20, and SEQ ID NO:21, or from a strain of
Bacillus flavothermus, such as the polypeptide shown in SEQ ID
NO:19, or from a strain of Aspergillus kawachii such as the
polypeptide shown in SEQ ID NO:22.
[0043] Most preferred as a first of the present invention is an
alpha-amylase comprising an amino acid sequence having at least
70%, at least 75%, least 80%, at least 85%, at least 90%, least
95%, at least 98%, such as at least 99% homology to an amino acid
sequence selected from the group consisting of SEQ ID NO:19, SEQ ID
NO:20, SEQ ID NO:21 and SEQ ID NO:22.
[0044] The above alpha-amylases may be added in an amount of
0.001-1.0 KNU/g DS, preferably from 0.002-0.5 KNU/g DS, preferably
0.02-0.1 KNU/g DS.
Fungal Alpha-Amylase
[0045] A particular enzyme to be used as a second enzyme in the
processes of the invention is a fungal alpha-amylase (EC 3.2.1.1),
such as a fungamyl-like alpha-amylase. In the present disclosure,
the term "fungamyl-like alpha-amylase" indicates an alpha-amylase
which exhibits a high homology, i.e. more than 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85% or even 90% homology to the amino acid sequence
shown in SEQ ID No. 10 in WO96/23874. Fungal alpha-amylases may be
added in an amount of 0.001-1.0 AFAU/g DS, preferably from
0.002-0.5 AFAU/g DS, preferably 0.02-0.1 AFAU/g DS.
Beta-Amylase
[0046] Another particular enzyme to be used as a second enzyme in
the processes of the invention may be a beta-amylase (E.0 3.2.1.2).
Beta-amylase is the name traditionally given to exo-acting
maltogenic amylases, which catalyze the hydrolysis of
1,4-alpha-glucosidic linkages in amylose, amylopectin and related
glucose polymers thereby releasing maltose.
[0047] Beta-amylases have been isolated from various plants and
microorganisms (Fogarty and Kelly, 1979, Progress in Industrial
Microbiology 15: 112-115). These beta-amylases are characterized by
having optimum temperatures in the range from 40.degree. C. to
65.degree. C. and optimum pH in the range from 4.5 to 7.0.
Contemplated beta-amylase include the beta-amylase from barley
Spezyme.RTM. BBA 1500, Spezyme.RTM. DBA and Optimalt.TM. ME,
Optimalt.TM. BBA from Genencor Int. as well as Novozym.TM. WBA from
Novozymes A/S.
Glucoamylase
[0048] A further particular enzyme to be used as a second enzyme in
the processes of the invention may also be a glucoamylase
(E.C.3.2.1.3) derived from a microorganism or a plant. Preferred is
glucoamylases of fungal or bacterial origin selected from the group
consisting of Aspergillus glucoamylases, in particular A. niger G1
or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102), or
variants thereof, such as disclosed in WO 92/00381 and WO 00/04136;
the A. awamori glucoamylase (WO 84/02921), A. oryzae (Agric. Biol.
Chem. 55(4): 941-949 (1991)), or variants or fragments thereof.
[0049] Other contemplated Aspergillus glucoamylase variants include
variants to enhance the thermal stability: G137A and G139A (Chen et
al., 1996, Prot. Engng. 9: 499-505); D257E and D293E/Q (Chen et
al., 1995, Prot. Engng. 8: 575-582); N182 (Chen et al., 1994,
Biochem. J. 301: 275-281); disulphide bonds, A246C (Fierobe et al.,
1996, Biochemistry 35: 8698-8704; and introduction of Pro residues
in position A435 and S436 (Li et al., 1997, Protein Engng. 10:
1199-1204. Other contemplated glucoamylases include Talaromyces
glucoamylases, in particular derived from Talaromyces emersonii (WO
99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153),
Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No.
4,587,215). Bacterial glucoamylases contemplated include
glucoamylases from the genus Clostridium, in particular C.
thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO
86/01831). Preferred glucoamylases include the glucoamylases
derived from Aspergillus oryzae, such as a glucoamylase having 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85% or even 90% homology to the amino
acid sequence shown in SEQ ID NO:2 in WO 00/04136. Also
contemplated are the commercial products AMG 200L; AMG 300 L;
SAN.TM. SUPER and AMG.TM. E (from Novozymes); OPTIDEX.TM. 300 (from
Genencor Int.); AMIGASE.TM. and AMIGASE.TM. PLUS (from DSM);
G-ZYME.TM. G900 (from Enzyme Bio-Systems); G-ZYME.TM. G990 ZR (A.
niger glucoamylase and low protease content).
[0050] Glucoamylases may be added in an amount of 0.02-2.0 AGU/g
DS, preferably 0.1-1.0 AGU/g DS, such as 0.2 AGU/g DS.
Additional Enzymes
[0051] The processes of the invention may be carried out in the
presence of a third enzyme. A particular third enzyme may be a
Bacillus alpha-amylase (often referred to as "Termamyl-like
alpha-amylases"). Well-known Termamyl-like alpha-amylases include
alpha-amylase derived from a strain of B. licheniformis
(commercially available as Termamyl), B. amyloliquefaciens, and B.
stearothermophilus alpha-amylase. Other Termamyl-like
alpha-amylases include alpha-amylase derived from a strain of the
Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of
which are described in detail in WO 95/26397, and the alpha-amylase
described by Tsukamoto et al., 1988, Biochemical and Biophysical
Research Communications 151: pp. 25-31. In the context of the
present invention a Termamyl-like alpha-amylase is an alpha-amylase
as defined in WO99/19467 on page 3, line 18 to page 6, line 27.
Contemplated variants and hybrids are described in WO 96/23874, WO
97/41213, and WO 99/19467. Specifically contemplated is a
recombinant B. stearothermophilus alpha-amylase variant with the
mutations: 1181*+G182*+N193F. Bacillus alpha-amylases may be added
in effective amounts well known to the person skilled in the
art.
[0052] Another particular third enzyme of the process may be a
debranching enzyme, such as an isoamylase (E.C. 3.2.1.68) or a
pullulanases (E.C. 3.2.1.41). Isoamylase hydrolyses
alpha-1,6-D-glucosidic branch linkages in amylopectin and
beta-limit dextrins and can be distinguished from pullulanases by
the inability of isoamylase to attack pullulan, and by the limited
action on alpha-limit dextrins. Debranching enzyme may be added in
effective amounts well known to the person skilled in the art.
Embodiments of the Invention
[0053] The starch slurry to be subjected to the processes of the
invention may have 20-55% dry solids granular starch, preferably
25-40% dry solids granular starch, more preferably 30-35% dry
solids granular starch.
[0054] After being subjected to the process of the first aspect of
the invention at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or preferably at least 99% of the dry solids of the
granular starch is converted into a soluble starch hydrolysate.
[0055] According to the invention the processes of the first and
second aspect is conducted at a temperature below the initial
gelatinization temperature. Preferably the temperature at which the
processes are conducted is at least 30.degree. C., at least
31.degree. C., at least 32.degree. C., at least 33.degree. C., at
least 34.degree. C., at least 35.degree. C., at least 36.degree.
C., at least 37.degree. C., at least 38.degree. C., at least
39.degree. C., at least 40.degree. C., at least 41.degree. C., at
least 42.degree. C., at least 43.degree. C., at least 44.degree.
C., at least 45.degree. C., at least 46.degree. C., at least
47.degree. C., at least 48.degree. C., at least 49.degree. C., at
least 50.degree. C., at least 51.degree. C., at least 52.degree.
C., at least 53.degree. C., at least 54.degree. C., at least
55.degree. C., at least 56.degree. C., at least 57.degree. C., at
least 58.degree. C., at least 59.degree. C., or preferably at least
60.degree. C.
[0056] The pH at which the process of the first aspect of the
invention is conducted may in be in the range of 3.0 to 7.0,
preferably from 3.5 to 6.0, or more preferably from 4.0-5.0.
[0057] The exact composition of the products of the process of the
first aspect of the invention, the soluble starch hydrolysate,
depends on the combination of enzymes applied as well as the type
of granular starch processed. Preferably the soluble hydrolysate is
maltose with a purity of at least 85%, at least 90%, at least
95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least
97.0%, at least 97.5%, at least 98.0%, at least 98.5, at least
99.0% or at least 99.5%. Even more preferably the soluble starch
hydrolysate is glucose, and most preferably the starch hydrolysate
has a DX (glucose percent of total solubilised dry solids) of at
least 94.5%, at least 95.0%, at least 95.5%, at least 96.0%, at
least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at
least 98.5, at least 99.0% or at least 99.5%. Equally contemplated,
however, is the process wherein the product of the process of the
invention, the soluble starch hydrolysate, is a speciality syrup,
such as a speciality syrup containing a mixture of glucose,
maltose, DP3 and DPn for use in the manufacture of ice creams,
cakes, candies, canned fruit.
[0058] The granular starch to be processed in the processes of the
invention may in particular be obtained from tubers, roots, stems,
legumes, cereals or whole grain. More specifically the granular
starch may be obtained from corns, corn grits, cobs, wheat, barley,
rye, milo, sago, cassava, tapioca, sorghum, rice, peas, bean,
banana or potatoes. Specially contemplated are both waxy and
non-waxy types of corn and barley. The granular starch to be
processed may be a highly refined starch quality, preferably at
least 90%, at least 95%, at least 97% or at least 99.5% pure or it
may be a more crude starch containing material comprising milled
whole grain including non-starch fractions such as germ residues
and fibres. The raw material, such as whole grain, is milled in
order to open up the structure and allowing for further processing.
Two milling processes are preferred according to the invention: wet
and dry milling. In dry milling the whole kernel is milled and
used. Wet milling gives a good separation of germ and meal (starch
granules and protein) and is with a few exceptions applied at
locations where the starch hydrolysate is used in production of
syrups. Both dry and wet milling are well known in the art of
starch processing and are equally contemplated for the processes of
the invention. The process of the first aspect of the invention may
be conducted in an ultrafiltration system where the retentate is
held under recirculation in presence of enzymes, raw starch and
water and where the permeate is the soluble starch hydrolysate.
Equally contemplated is the process conducted in a continuous
membrane reactor with ultrafiltration membranes and where the
retentate is held under recirculation in presence of enzymes, raw
starch and water and where the permeate is the soluble starch
hydrolysate. Also contemplated is the process conducted in a
continuous membrane reactor with microfiltration membranes and
where the retentate is held under recirculation in presence of
enzymes, raw starch and water and where the permeate is the soluble
starch hydrolysate.
[0059] In the process of the second aspect of the invention the
soluble starch hydrolysate of the process of the first aspect of
the invention is subjected to conversion into high fructose
starch-based syrup (HFSS), such as high fructose corn syrup (HFCS).
This conversion is preferably achieved using a glucose isomerase,
and more preferably by an immobilized glucose isomerase supported
on a solid support. Contemplated isomerases comprises the
commercial products Sweetzyme.TM. IT from Novozymes A/S, G-zyme.TM.
IMGI and G-zyme.TM. G993, Ketomax.TM. and G-zyme.TM. G993 from
Rhodia, G-zyme.TM. G993 liquid and GenSweet.TM. IGI from Genencor
Int.
[0060] In the process of the third aspect of the invention the
soluble starch hydrolysate of the process of the first aspect of
the invention is used for production of fuel or potable ethanol. In
the process of the third aspect the fermentation may be carried out
simultaneously or separately/sequential to the hydrolysis of the
granular starch slurry. When the fermentation is performed
simultaneous to the hydrolysis the temperature is preferably
between 30.degree. C. and 35.degree. C., and more preferably
between 31.degree. C. and 34.degree. C. The process of the third
aspect of the invention may be conducted in an ultrafiltration
system where the retentate is held under recirculation in presence
of enzymes, raw starch, yeast, yeast nutrients and water and where
the permeate is an ethanol containing liquid. Equally contemplated
is the process conducted in a continuous membrane reactor with
ultrafiltration membranes and where the retentate is held under
recirculation in presence of enzymes, raw starch, yeast, yeast
nutrients and water and where the permeate is an ethanol containing
liquid.
[0061] The soluble starch hydrolysate of the process of the first
aspect of the invention may also be used for production of a
fermentation product comprising fermenting the treated starch into
a fermentation product, such as citric acid, monosodium glutamate,
gluconic acid, sodium gluconate, calcium gluconate, potassium
gluconate, glucono delta lactone, or sodium erythorbate.
[0062] In another embodiment the starch slurry is being contacted
with a polypeptide comprising a CBM, but no amylolytic module, i.e.
application of loose CBMs. The loose CBMs may be starch binding
modules, cellulose-binding modules, chitin-binding modules,
xylan-binding modules, mannan-binding modules, and other binding
modules. Preferred CBMs in the present context are microbial CBMs,
particularly bacterial or fungal CBMs. Particularly preferred are
the starch binding modules shown in the present disclosure as the
polypeptide sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 or
the starch binding modules disclosed in U.S. provisional
application No. 60/511,044 as SEQ ID NO:12; the CBM of the
glucoamylase from Hormoconis sp. such as from Hormoconis resinae
(Syn. Creosote fungus or Amorphotheca resinae) (SWISSPROT:Q03045),
SEQ ID NO:13; the CBM from Lentinula sp. such as from Lentinula
edodes (shiitake mushroom) (SPTREMBL:Q9P4C5), SEQ ID NO:14; the CBM
from Neurospora sp. such as from Neurospora
crassa(SWISSPROT:P14804), SEQ ID NO:15; the CBM from Talaromyces
sp. such as from Talaromyces byssochlamydioides, SEQ ID NO:16; the
CBM from Geosmithia sp. such as from Geosmithia cylindrospora, SEQ
ID NO:17: the CBM from Scorias sp. such as from Scorias spongiosa,
SEQ ID NO:18; the CBM from Eupenicillium sp. such as from
Eupenicillium ludwigii, SEQ ID NO:19; the CBM from Aspergillus sp.
such as from Aspergillus japonicus, SEQ ID NO:20; the CBM from
Penicillium sp. such as from Penicillium cf. miczynskii, SEQ ID
NO:21; the CBM from Mz1 Penicillium sp., SEQ ID NO:22; the CBM from
Thysanophora sp., SEQ ID NO:23; the CBM from Humicola sp. such as
from Humicola grisea var. thermoidea. Most preferred CBMs include
the CBMs disclosed in U.S. provisional application No. 60/511,044
as SEQ ID NO:24; the CBM of the glucoamylase from Aspergillus sp.
such as from Aspergillus niger, and as SEQ ID NO:25; the CBM of the
glucoamylase from Athelia sp. such as from Athelia rolfsii. Also
preferred for the invention is the application of any CBM having at
least 50%, 60%, 70%, 80% or even at least 90% homology to any of
the afore mentioned CBM amino acid sequences.
[0063] The loose CBMs may be applied to the granular starch slurry
in effective amounts.
Materials and Methods
Alpha-Amylase Activity (KNU)
[0064] The amylolytic activity may be determined using potato
starch as substrate. This method is based on the break-down of
modified potato starch by the enzyme, and the reaction is followed
by mixing samples of the starch/enzyme solution with an iodine
solution. Initially, a blackish-blue colour is formed, but during
the break-down of the starch the blue colour gets weaker and
gradually turns into a reddish-brown, which is compared to a
coloured glass standard.
[0065] One Kilo Novo alpha amylase Unit (KNU) is defined as the
amount of enzyme which, under standard conditions (i.e. at
37.degree. C.+/-0.05; 0.0003 M Ca.sup.2+; and pH 5.6) dextrinizes
5.26 g starch dry substance Merck Amylum solubile.
[0066] A folder AF 9/6 describing this analytical method in more
detail is available upon request to Novozymes A/S, Denmark, which
folder is hereby included by reference.
Glucoamylase Activity (AGU)
[0067] The Novo Glucoamylase Unit (AGU) is defined as the amount of
enzyme, which hydrolyzes 1 micromole maltose per minute at
37.degree. C. and pH 4.3.
[0068] The activity is determined as AGU/ml by a method modified
after (AEL-SM-0131, available on request from Novozymes) using the
Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard:
AMG-standard, batch 7-1195, 195 AGU/ml. 375 microL substrate (1%
maltose in 50 mM Sodium acetate, pH 4.3) is incubated 5 minutes at
37.degree. C. 25 microL enzyme diluted in sodium acetate is added.
The reaction is stopped after 10 minutes by adding 100 microL 0.25
M NaOH. 20 microL is transferred to a 96 well microtitre plate and
200 microL GOD-Perid solution (124036, Boehringer Mannheim) is
added. After 30 minutes at room temperature, the absorbance is
measured at 650 nm and the activity calculated in AGU/ml from the
AMG-standard. A folder (AEL-SM-0131) describing this analytical
method in more detail is available on request from Novozymes A/S,
Denmark, which folder is hereby included by reference.
Fungal Alpha-Amylase Activity (FAU)
[0069] Fungal alpha-amylase activity may be measured in FAU (Fungal
Alpha-Amylase Units). One (1) FAU is the amount of enzyme which
under standard conditions (i.e. at 37.degree. C. and pH 4.7) breaks
down 5260 mg solid starch (Amylum solubile, Merck) per hour. A
folder AF 9.1/3, describing this FAU assay in more details, is
available upon request from Novozymes A/S, Denmark, which folder is
hereby included by reference.
Acid Alpha-Amylase Activity (AFAU)
[0070] Acid alpha-amylase activity may be measured in AFAU (Acid
Fungal Alpha-amylase Units), which are determined relative to an
enzyme standard.
[0071] The standard used is AMG 300 L (from Novozymes A/S,
glucoamylase wildtype Aspergillus niger G1, also disclosed in Boel
et al., 1984, EMBO J. 3(5): 1097-1102 and in WO 92/00381). The
neutral alpha-amylase in this AMG falls after storage at room
temperature for 3 weeks from approx. 1 FAU/mL to below 0.05
FAU/mL.
[0072] The acid alpha-amylase activity in this AMG standard is
determined in accordance with the following description. In this
method 1 AFAU is defined as the amount of enzyme, which degrades
5.26 mg starch dry solids per hour under standard conditions.
[0073] Iodine forms a blue complex with starch but not with its
degradation products. The intensity of colour is therefore directly
proportional to the concentration of starch. Amylase activity is
determined using reverse colorimetry as a reduction in the
concentration of starch under specified analytic conditions.
##STR00001##
[0074] Standard Conditions/Reaction Conditions: (Per Minute)
[0075] Substrate: starch, approx. 0.17 g/L
[0076] Buffer: Citate, approx. 0.03 M
[0077] Iodine (1.sub.2): 0.03 g/L
[0078] CaCl.sub.2: 1.85 mM
[0079] pH: 2.50-0.05
[0080] Incubation temperature: 40.degree. C.
[0081] Reaction time: 23 seconds
[0082] Wavelength: lambda=590 nm
[0083] Enzyme concentration: 0.025 AFAU/mL
[0084] Enzyme working range: 0.01-0.04 AFAU/mL
[0085] If further details are preferred these can be found in
EB-SM-0259.02/01 available on request from Novozymes A/S, and
incorporated by reference.
Beta-Amylase Activity)(DP.degree.)
[0086] The activity of SPEZYME.RTM. BBA 1500 is expressed in Degree
of Diastatic Power)(DP.degree.. It is the amount of enzyme
contained in 0.1 ml of a 5% solution of the sample enzyme
preparation that will produce sufficient reducing sugars to reduce
5 ml of Fehling's solution when the sample is incubated with 100 ml
of substrate for 1 hour at 20.degree. C.
Pullulanase Activity (New Pullulanase Unit Novo (NPUN)
[0087] Pullulanase activity may be determined relative to a
pullulan substrate. Pullulan is a linear D-glucose polymer
consisting essentially of maltotriosyl units joined by
1,6-alpha-links. Endo-pullulanases hydrolyze the 1,6-alpha-links at
random, releasing maltotriose,
6.sup.3-alpha-maltotriosyl-maltotriose,
6.sup.3-alpha-(6.sup.3-alpha-maltotriosyl-maltotriosyl)-maltotriose.
[0088] One new Pullulanase Unit Novo (NPUN) is a unit of
endo-pullulanase activity and is measured relative to a Novozymes
A/S Promozyme D standard. Standard conditions are 30 minutes
reaction time at 40.degree. C. and pH 4.5; and with 0.7% pullulan
as substrate. The amount of red substrate degradation product is
measured spectrophotometrically at 510 nm and is proportional to
the endo-pullulanase activity in the sample. A folder
(EB-SM.0420.02/01) describing this analytical method in more detail
is available upon request to Novozymes A/S, Denmark, which folder
is hereby included by reference.
[0089] Under the standard conditions one NPUN is approximately
equal to the amount of enzyme which liberates reducing carbohydrate
with a reducing power equivalent to 2.86 micromole glucose per
minute.
Determination of Sugar Profile and Solubilised Dry Solids
[0090] The sugar composition of the starch hydrolysates was
determined by HPLC and glucose yield was subsequently calculated as
DX. .degree. BRIX, solubilised (soluble) dry solids of the starch
hydrolysate were determined by refractive index measurement.
Materials
[0091] The following enzyme activities were used. A bacterial
alpha-amylase with a CBD having the sequence depicted in SEQ ID
NO:19 and the same bacterial alpha-amylase but without the CBD
module (SEQ ID NO:4). A glucoamylase derived from Aspergillus niger
having the amino acid sequence shown in WO 00/04136 as SEQ ID No: 2
or one of the disclosed variants. An acid fungal alpha-amylase
derived from Aspergillus niger.
[0092] Wheat starch (S-5127) was obtained from Sigma-Aldrich.
Example 1
[0093] This example illustrates the conversion of granular wheat
starch into glucose using a bacterial four module alpha-amylase and
a glucoamylase and an acid fungal amylase. A slurry with 33% dry
solids (DS) granular starch was prepared by adding 247.5 g of wheat
starch under stirring to 502.5 ml of water. The pH was adjusted
with HCl to 4.5. The granular starch slurry was distributed to 100
ml blue cap flasks with 75 g in each flask. The flasks were
incubated with magnetic stirring in a 60.degree. C. water bath. At
zero hours the enzyme activities given in table 1 were dosed to the
flasks. Samples were withdrawn after 24, 48, 72, and 96 hours.
TABLE-US-00001 TABLE 1 The enzyme activity levels used. Bacterial
alpha- Acid fungal amylase Glucoamylase alpha-amylase KNU/kg DS
AGU/kg DS AFAU/kg DS 100.0 200 50
[0094] Total dry solids starch was determined using the following
method. The starch was completely hydrolyzed by adding an excess
amount of alpha-amylase (300 KNU/Kg dry solids) and placing the
sample in an oil bath at 95.degree. C. for 45 minutes. Subsequently
the samples were cooled to 60.degree. C. and an excess amount of
glucoamylase (600 AGU/kg DS) was added followed by incubation for 2
hours at 60.degree. C.
[0095] Soluble dry solids in the starch hydrolysate were determined
by refractive index measurement on samples after filtering through
a 0.22 microM filter. The sugar profile was determined by HPLC. The
amount of glucose was calculated as DX. The results are shown in
tables 2 and 3.
TABLE-US-00002 TABLE 2 Soluble dry solids as percentage of total
dry substance at 100 KNU/kg DS alpha-amylase dosage. KNU/kg DS 24
hours 48 hours 72 hours 96 hours 100.0 92.5 96 97.3 99.2
TABLE-US-00003 TABLE 3 The DX of the soluble hydrolysate at 100
KNU/kg DS alpha-amylase dosage. KNU/kg DS 24 hours 48 hours 72
hours 96 hours 100.0 88.4 92.4 93.7 95.3
Example 2
[0096] This example illustrates the only partial conversion of
granular starch into glucose using a glucoamylase and an acid
fungal alpha-amylase.
[0097] Flasks with 33% DS granular starch were prepared and
incubated as described in example 1. At zero hours the enzyme
activities given in table 4 were dosed to the flasks. Samples were
withdrawn after 24, 48, 72, and 96 hours. The samples were analyzed
as described in examples 1. The results are shown in tables 5 and
6.
TABLE-US-00004 TABLE 4 The enzyme activity level used. Glucoamylase
Acid fungal alpha-amylase AGU/kg DS AFAU/kg DS 200 50
TABLE-US-00005 TABLE 5 Soluble dry solids as percentage of total
dry substance. 24 hours 48 hours 72 hours 96 hours 28.5 36.3 41.6
45.7
TABLE-US-00006 TABLE 6 DX of the soluble hydrolysate. 24 hours 48
hours 72 hours 96 hours 27.7 34.9 39.2 42.2
Example 3
[0098] In example 3 conversion of granular wheat starch into
glucose was performed using a glucoamylase (200 AGU/kg DS), an acid
fungal amylase (50 AFAU/kg DS) and either the intact bacterial four
module alpha-amylase (SEQ ID NO:19) also used in example 1 or the
same bacterial four module alpha-amylase but without the CBD module
(SEQ ID NO:4) (100 KNU/kg DS). A slurry with 33% dry solids (DS)
granular starch was prepared and incubated as described in example
1. Samples were withdrawn after 24, 46, 70, and 90 hours.
[0099] Total dry solids starch was determined as described in
example 1. Soluble dry solids in the starch hydrolysate and the
sugar profile were determined as described in example 1. The
results are shown in tables 7 and 8.
TABLE-US-00007 TABLE 7 Soluble dry solids as percentage of total
dry substance. Enzymes: glucoamylase, fungal acid amylase and
bacterial alpha-amylase with the CBD module (SEQ ID NO: 19) or
without the CBD module (SEQ ID NO: 4). 24 hours 46 hours 70 hours
90 hours Without CBD 89.7 92.4 92.4 92.5 With CBD 94.1 95.2 96.9
97.1
TABLE-US-00008 TABLE 8 The DX of the soluble hydrolysate: Enzymes:
glucoamylase, fungal acid amylase and bacterial alpha-amylase with
the CBD module (SEQ ID NO: 19) or without the CBD module (SEQ ID
NO: 4). 24 hours 46 hours 70 hours 90 hours Without CBD 85.9 88.7
89.0 89.0 With CBD 89.9 93.3 93.0 93.2
Sequence CWU 1
1
221102PRTBacillus flavothermus 1Ile Ser Thr Thr Ser Gln Ile Thr Phe
Thr Val Asn Asn Ala Thr Thr 1 5 10 15 Val Trp Gly Gln Asn Val Tyr
Val Val Gly Asn Ile Ser Gln Leu Gly 20 25 30 Asn Trp Asp Pro Val
His Ala Val Gln Met Thr Pro Ser Ser Tyr Pro 35 40 45 Thr Trp Thr
Val Thr Ile Pro Leu Leu Gln Gly Gln Asn Ile Gln Phe 50 55 60 Lys
Phe Ile Lys Lys Asp Ser Ala Gly Asn Val Ile Trp Glu Asp Ile 65 70
75 80 Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala Ser Gly Ala Tyr
Thr 85 90 95 Ala Ser Trp Asn Val Pro 100 299PRTBacillus sp. 2Thr
Ser Asn Val Thr Phe Thr Val Asn Asn Ala Thr Thr Val Tyr Gly 1 5 10
15 Gln Asn Val Tyr Val Val Gly Asn Ile Pro Glu Leu Gly Asn Trp Asn
20 25 30 Ile Ala Asn Ala Ile Gln Met Thr Pro Ser Ser Tyr Pro Thr
Trp Lys 35 40 45 Thr Thr Val Ser Leu Pro Gln Gly Lys Ala Ile Glu
Phe Lys Phe Ile 50 55 60 Lys Lys Asp Ser Ala Gly Asn Val Ile Trp
Glu Asn Ile Ala Asn Arg 65 70 75 80 Thr Tyr Thr Val Pro Phe Ser Ser
Thr Gly Ser Tyr Thr Ala Asn Trp 85 90 95 Asn Val Pro
3102PRTAlcaliphilic Bacillus 3Thr Ser Thr Thr Ser Gln Ile Thr Phe
Thr Val Asn Asn Ala Thr Thr 1 5 10 15 Val Trp Gly Gln Asn Val Tyr
Val Val Gly Asn Ile Ser Gln Leu Gly 20 25 30 Asn Trp Asp Pro Val
Asn Ala Val Gln Met Thr Pro Ser Ser Tyr Pro 35 40 45 Thr Trp Val
Val Thr Val Pro Leu Pro Gln Ser Gln Asn Ile Gln Phe 50 55 60 Lys
Phe Ile Lys Lys Asp Gly Ser Gly Asn Val Ile Trp Glu Asn Ile 65 70
75 80 Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala Ser Gly Ala Tyr
Thr 85 90 95 Ala Asn Trp Asn Val Pro 100 4484PRTBacillus
flavothermus 4Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp Tyr 1 5 10 15 Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val
Ala Asn Asn Ala Gln 20 25 30 Ser Leu Ala Asn Leu Gly Ile Thr Ala
Leu Trp Leu Pro Pro Ala Tyr 35 40 45 Lys Gly Thr Ser Ser Ser Asp
Val Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Lys Thr
Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala Gly 85 90 95 Met
Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp 100 105
110 Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125 Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys
Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys
Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Glu
Ser Arg Lys Leu Asn Arg Ile 165 170 175 Tyr Lys Phe Arg Gly Thr Gly
Lys Ala Trp Asp Trp Glu Val Asp Thr 180 185 190 Glu Asn Gly Asn Tyr
Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp 195 200 205 His Pro Glu
Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val 210 215 220 Thr
Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile 225 230
235 240 Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln
Thr 245 250 255 Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr
Asp Ile Ser 260 265 270 Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly
Ser Met Ser Leu Phe 275 280 285 Asp Ala Pro Leu His Asn Asn Phe Tyr
Ile Ala Ser Lys Ser Gly Gly 290 295 300 Tyr Phe Asp Met Arg Thr Leu
Leu Asn Asn Thr Leu Met Lys Asp Gln 305 310 315 320 Pro Thr Leu Ala
Val Thr Leu Val Asp Asn His Asp Thr Glu Pro Gly 325 330 335 Gln Ser
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr 340 345 350
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly 355
360 365 Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser
Lys 370 375 380 Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr
Gly Thr Gln 385 390 395 400 His Asp Tyr Ile Asp Ser Ala Asp Ile Ile
Gly Trp Thr Arg Glu Gly 405 410 415 Val Ala Glu Lys Ala Asn Ser Gly
Leu Ala Ala Leu Ile Thr Asp Gly 420 425 430 Pro Gly Gly Ser Lys Trp
Met Tyr Val Gly Lys Gln His Ala Gly Lys 435 440 445 Thr Phe Tyr Asp
Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn 450 455 460 Ala Asp
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile 465 470 475
480 Trp Val Pro Lys 5485PRTBacillus sp 5Ala Asn Thr Ala Pro Val Asn
Gly Thr Met Met Gln Tyr Phe Glu Trp 1 5 10 15 Asp Leu Pro Asn Asp
Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala 20 25 30 Ser Ser Leu
Ser Ala Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala 35 40 45 Tyr
Lys Gly Thr Ser Gln Ala Asp Val Gly Tyr Gly Val Tyr Asp Leu 50 55
60 Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr
65 70 75 80 Gly Thr Lys Thr Gln Tyr Leu Gln Ala Ile Gln Ala Ala Lys
Ser Ala 85 90 95 Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His
Lys Ala Gly Ala 100 105 110 Asp Ser Thr Glu Trp Val Asp Ala Val Glu
Val Asn Pro Ser Asn Arg 115 120 125 Asn Gln Glu Thr Ser Gly Thr Tyr
Gln Ile Gln Ala Trp Thr Lys Phe 130 135 140 Asp Phe Pro Gly Arg Gly
Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp 145 150 155 160 Tyr His Phe
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg 165 170 175 Ile
Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185
190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met
195 200 205 Asp His Pro Glu Val Val Ala Glu Leu Lys Asn Trp Gly Lys
Trp Tyr 210 215 220 Val Asn Thr Thr Asn Val Asp Gly Phe Arg Leu Asp
Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Phe Pro Asp Trp
Leu Ser Tyr Val Arg Asn Gln 245 250 255 Thr Gly Lys Asn Leu Phe Ala
Val Gly Glu Phe Trp Gly Tyr Asp Val 260 265 270 Asn Lys Leu His Asn
Tyr Ile Thr Lys Thr Asn Gly Ala Met Ser Leu 275 280 285 Phe Asp Ala
Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Ser 290 295 300 Gly
Tyr Phe Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp 305 310
315 320 Gln Pro Ala Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln
Pro 325 330 335 Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys
Pro Leu Ala 340 345 350 Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr
Pro Cys Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr
Asn Ile Pro Gly Leu Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Ile
Ala Arg Arg Asp Tyr Ala Tyr Gly Thr 385 390 395 400 Gln Arg Asp Tyr
Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Ile
Asp Ala Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp 420 425 430
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Arg His Ala Gly 435
440 445 Lys Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr
Ile 450 455 460 Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly
Ser Val Ser 465 470 475 480 Ile Trp Val Ala Lys 485
6484PRTAlkaliphilic bacillus 6Gly Ser Val Pro Val Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asp Asp Gly Thr Leu
Trp Thr Lys Val Ala Asn Asn Ala Gln 20 25 30 Ser Leu Ala Asn Leu
Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr 35 40 45 Lys Gly Thr
Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55 60 Asp
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70
75 80 Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala
Gly 85 90 95 Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala
Gly Ala Asp 100 105 110 Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn
Pro Ser Asp Arg Asn 115 120 125 Gln Glu Ile Ser Gly Thr Tyr Gln Ile
Gln Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr
Tyr Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile 165 170 175 Tyr Lys
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr 180 185 190
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp 195
200 205 His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr
Val 210 215 220 Ile Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val
Lys His Ile 225 230 235 240 Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser
Tyr Leu Arg Thr Gln Thr 245 250 255 Gln Lys Pro Leu Phe Ala Val Gly
Glu Phe Trp Ser Tyr Asp Ile Asn 260 265 270 Lys Leu His Asn Tyr Ile
Thr Lys Thr Asn Gly Ser Met Ser Leu Phe 275 280 285 Asp Ala Pro Leu
His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly Gly 290 295 300 Tyr Phe
Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Glu Gln 305 310 315
320 Pro Thr Leu Ser Val Thr Leu Val Asp Asn His Asp Thr Glu Pro Gly
325 330 335 Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu
Ala Tyr 340 345 350 Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys
Val Phe Tyr Gly 355 360 365 Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile
Pro Ala Leu Lys Ser Lys 370 375 380 Leu Asp Pro Leu Leu Ile Ala Arg
Arg Asp Tyr Ala Tyr Gly Thr Gln 385 390 395 400 His Asp Tyr Ile Asp
Asn Ala Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415 Val Ala Glu
Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly 420 425 430 Pro
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys 435 440
445 Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
450 455 460 Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val
Ser Ile 465 470 475 480 Trp Val Pro Lys 7517PRTBacillus sp. 7Met
Ser Leu Phe Lys Lys Ile Phe Pro Trp Ile Leu Ser Leu Leu Leu 1 5 10
15 Leu Phe Leu Phe Ile Ala Pro Phe Ser Ile Gln Thr Glu Lys Val Arg
20 25 30 Ala Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp 35 40 45 Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val
Ala Asn Asn Ala 50 55 60 Gln Ser Leu Ala Asn Leu Gly Ile Thr Ala
Leu Trp Leu Pro Pro Ala 65 70 75 80 Tyr Lys Gly Thr Ser Ser Ser Asp
Val Gly Tyr Gly Val Tyr Asp Leu 85 90 95 Tyr Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr 100 105 110 Gly Thr Lys Thr
Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala 115 120 125 Gly Met
Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala 130 135 140
Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg 145
150 155 160 Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr
Lys Phe 165 170 175 Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe
Lys Trp Arg Trp 180 185 190 Tyr His Phe Asp Gly Thr Asp Trp Asp Glu
Ser Arg Lys Leu Asn Arg 195 200 205 Ile Tyr Lys Phe Arg Gly Thr Gly
Lys Ala Trp Asp Trp Glu Val Asp 210 215 220 Thr Glu Asn Gly Asn Tyr
Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met 225 230 235 240 Asp His Pro
Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr 245 250 255 Val
Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His 260 265
270 Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln
275 280 285 Thr Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr
Asp Ile 290 295 300 Ser Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly
Ser Met Ser Leu 305 310 315 320 Phe Asp Ala Pro Leu His Asn Asn Phe
Tyr Ile Ala Ser Lys Ser Gly 325 330 335 Gly Tyr Phe Asp Met Arg Thr
Leu Leu Asn Asn Thr Leu Met Lys Asp 340 345 350 Gln Pro Thr Leu Ala
Val Thr Leu Val Asp Asn His Asp Thr Glu Pro 355 360 365 Gly Gln Ser
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala 370 375 380 Tyr
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr 385 390
395 400 Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys
Ser 405 410 415 Lys Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala
Tyr Gly Thr 420 425 430 Gln His Asp Tyr Ile Asp Ser Ala Asp Ile Ile
Gly Trp Thr Arg Glu 435 440 445 Gly Val Ala Glu Lys Ala Asn Ser Gly
Leu Ala Ala Leu Ile Thr Asp 450 455 460 Gly Pro Gly Gly Ser Lys Trp
Met Tyr Val Gly Lys Gln His Ala Gly 465 470 475 480 Lys Thr Phe Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile 485 490 495 Asn Ala
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser 500 505 510
Ile Trp Val Pro Lys 515 8550PRTUnknownbacterial 8Met Ser Leu Phe
Lys Lys Ile Phe Pro Trp Ile Val Ser Leu Leu Leu 1 5 10
15 Leu Phe Ser Phe Ile Ala Pro Phe Ser Ile Gln Thr Glu Lys Val Arg
20 25 30 Ala Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp 35 40 45 Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val
Ala Asn Asn Ala 50 55 60 Gln Ser Leu Ala Asn Leu Gly Ile Thr Ala
Leu Trp Leu Pro Pro Ala 65 70 75 80 Tyr Lys Gly Thr Ser Ser Ser Asp
Val Gly Tyr Gly Val Tyr Asp Leu 85 90 95 Tyr Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr 100 105 110 Gly Thr Lys Thr
Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala 115 120 125 Gly Met
Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala 130 135 140
Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg 145
150 155 160 Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr
Lys Phe 165 170 175 Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe
Lys Trp Arg Trp 180 185 190 Tyr His Phe Asp Gly Thr Asp Trp Asp Glu
Ser Arg Lys Leu Asn Arg 195 200 205 Ile Tyr Lys Phe Arg Gly Thr Gly
Lys Ala Trp Asp Trp Glu Val Asp 210 215 220 Thr Glu Asn Gly Asn Tyr
Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met 225 230 235 240 Asp His Pro
Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr 245 250 255 Val
Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His 260 265
270 Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln
275 280 285 Thr Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr
Asp Ile 290 295 300 Asn Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly
Ser Met Ser Leu 305 310 315 320 Phe Asp Ala Pro Leu His Asn Asn Phe
Tyr Ile Ala Ser Lys Ser Gly 325 330 335 Gly Tyr Phe Asp Met Arg Thr
Leu Leu Asn Asn Thr Leu Met Lys Asp 340 345 350 Gln Pro Thr Leu Ser
Val Thr Leu Val Asp Asn His Asp Thr Glu Pro 355 360 365 Gly Gln Ser
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala 370 375 380 Tyr
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Ile Phe Tyr 385 390
395 400 Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys
Ser 405 410 415 Lys Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala
Tyr Gly Thr 420 425 430 Gln His Asp Tyr Ile Asp Asn Ala Asp Ile Ile
Gly Trp Thr Arg Glu 435 440 445 Gly Val Ala Glu Lys Ala Asn Ser Gly
Leu Ala Ala Leu Ile Thr Asp 450 455 460 Gly Pro Gly Gly Ser Lys Trp
Met Tyr Val Gly Lys Gln His Ala Gly 465 470 475 480 Lys Thr Phe Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile 485 490 495 Asn Ala
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser 500 505 510
Ile Trp Val Pro Lys Thr Ser Thr Thr Ser Gln Ile Thr Phe Thr Val 515
520 525 Asn Asn Ala Thr Thr Val Trp Gly Gln Asn Val Tyr Val Val Gly
Asn 530 535 540 Ile Ser Gln Leu Gly Asn 545 550
9482PRTUnknownbacterial 9Ala Pro Val Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Asp Leu Pro 1 5 10 15 Asn Asp Gly Thr Leu Trp Thr Lys
Val Lys Asn Glu Ala Thr Asn Leu 20 25 30 Ser Ser Leu Gly Ile Thr
Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ser
Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu
Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80
Ala Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly Met Gln 85
90 95 Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Gly
Thr 100 105 110 Glu Phe Val Asp Ala Val Glu Val Asn Pro Ser Asn Arg
Asn Gln Glu 115 120 125 Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr
Lys Phe Asp Phe Pro 130 135 140 Gly Arg Gly Asn Thr Tyr Ser Ser Phe
Lys Trp Arg Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Arg Gly Thr
Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn 180 185 190 Gly Asn
Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro 195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr 210
215 220 Thr Asn Val Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
Tyr 225 230 235 240 Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn
Gln Thr Gly Lys 245 250 255 Asn Leu Phe Ala Val Gly Glu Phe Trp Ser
Tyr Asp Val Asn Lys Leu 260 265 270 His Asn Tyr Ile Thr Lys Thr Asn
Gly Ser Met Ser Leu Phe Asp Ala 275 280 285 Pro Leu His Asn Asn Phe
Tyr Ile Ala Ser Lys Ser Ser Gly Tyr Phe 290 295 300 Asp Met Arg Tyr
Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser 305 310 315 320 Leu
Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser 325 330
335 Leu Gln Ser Trp Val Glu Ala Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350 Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly
Asp Tyr 355 360 365 Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys
Ser Lys Ile Asp 370 375 380 Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala
Tyr Gly Thr Gln Arg Asp 385 390 395 400 Tyr Ile Asp His Gln Asp Ile
Ile Gly Trp Thr Arg Glu Gly Ile Asp 405 410 415 Ala Lys Pro Asn Ser
Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 420 425 430 Gly Ser Lys
Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe 435 440 445 Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455
460 Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val
465 470 475 480 Ala Lys 10482PRTUnknownbacterial 10Ala Pro Val Asn
Gly Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro 1 5 10 15 Asn Asp
Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Thr Asn Leu 20 25 30
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly 35
40 45 Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
Leu 50 55 60 Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr
Gly Thr Lys 65 70 75 80 Thr Gln Tyr Ile Gln Ala Ile Gln Thr Ala Gln
Ala Ala Gly Met Gln 85 90 95 Val Tyr Ala Asp Val Val Phe Asn His
Lys Ala Gly Ala Asp Ser Thr 100 105 110 Glu Phe Val Asp Ala Val Glu
Val Asn Pro Ser Asn Arg Asn Gln Glu 115 120 125 Thr Ser Gly Thr Tyr
Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro 130 135 140 Gly Arg Gly
Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe 145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165
170 175 Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
Asn 180 185 190 Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met
Asp His Pro 195 200 205 Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr
Trp Tyr Val Asn Thr 210 215 220 Thr Asn Ile Asp Gly Phe Arg Leu Asp
Ala Val Lys His Ile Lys Tyr 225 230 235 240 Ser Phe Phe Pro Asp Trp
Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys 245 250 255 Asn Leu Phe Ala
Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu 260 265 270 His Asn
Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe Asp Ala 275 280 285
Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe 290
295 300 Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro
Ser 305 310 315 320 Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln
Pro Gly Gln Ser 325 330 335 Leu Gln Ser Trp Val Glu Pro Trp Phe Lys
Gln Leu Ala Tyr Ala Phe 340 345 350 Ile Leu Thr Arg Gln Glu Gly Tyr
Pro Cys Val Phe Tyr Gly Asp Tyr 355 360 365 Tyr Gly Ile Pro Lys Tyr
Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp 370 375 380 Pro Leu Leu Ile
Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp 385 390 395 400 Tyr
Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp 405 410
415 Ala Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430 Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys
Val Phe 435 440 445 Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr
Ile Asn Ala Asp 450 455 460 Gly Trp Gly Glu Phe Lys Val Asn Gly Gly
Ser Val Ser Ile Trp Val 465 470 475 480 Ala Lys
11482PRTUnknownbacterial 11Ala Pro Val Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Asp Leu Pro 1 5 10 15 Asn Asp Gly Thr Leu Trp Thr Lys
Val Lys Asn Glu Ala Ser Ser Leu 20 25 30 Ser Ser Leu Gly Ile Thr
Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Gly
Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu
Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80
Thr Gln Tyr Leu Gln Ala Ile Gln Ala Ala Lys Ser Ala Gly Met Gln 85
90 95 Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Ser
Thr 100 105 110 Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asn Arg
Asn Gln Glu 115 120 125 Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr
Lys Phe Asp Phe Pro 130 135 140 Gly Arg Gly Asn Thr Tyr Ser Ser Phe
Lys Trp Arg Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Arg Gly Thr
Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn 180 185 190 Gly Asn
Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro 195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr 210
215 220 Thr Asn Val Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
Tyr 225 230 235 240 Ser Phe Phe Pro Asp Trp Leu Thr His Val Arg Ser
Gln Thr Arg Lys 245 250 255 Asn Leu Phe Ala Val Gly Glu Phe Trp Ser
Tyr Asp Val Asn Lys Leu 260 265 270 His Asn Tyr Ile Thr Lys Thr Ser
Gly Thr Met Ser Leu Phe Asp Ala 275 280 285 Pro Leu His Asn Asn Phe
Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe 290 295 300 Asp Met Arg Tyr
Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser 305 310 315 320 Leu
Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser 325 330
335 Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350 Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly
Asp Tyr 355 360 365 Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys
Ser Lys Ile Asp 370 375 380 Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala
Tyr Gly Thr Gln Arg Asp 385 390 395 400 Tyr Ile Asp His Gln Asp Ile
Ile Gly Trp Thr Arg Glu Gly Ile Asp 405 410 415 Ser Lys Pro Asn Ser
Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 420 425 430 Gly Ser Lys
Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe 435 440 445 Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455
460 Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val
465 470 475 480 Ala Lys 12482PRTUnknownbacterial 12Ala Pro Val Asn
Gly Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro 1 5 10 15 Asn Asp
Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ser Ser Leu 20 25 30
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly 35
40 45 Thr Ser Gln Gly Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
Leu 50 55 60 Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr
Gly Thr Lys 65 70 75 80 Thr Gln Tyr Leu Gln Ala Ile Gln Ala Ala Lys
Ser Ala Gly Met Gln 85 90 95 Val Tyr Ala Asp Val Val Phe Asn His
Lys Ala Gly Ala Asp Ser Thr 100 105 110 Glu Trp Val Asp Ala Val Glu
Val Asn Pro Ser Asn Arg Asn Gln Glu 115 120 125 Thr Ser Gly Thr Tyr
Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro 130 135 140 Asp Arg Gly
Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe 145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165
170 175 Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
Asn 180 185 190 Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met
Asp His Pro 195 200 205 Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr
Trp Tyr Val Asn Thr 210 215 220 Thr Asn Val Asp Gly Phe Arg Leu Asp
Ala Val Lys His Ile Lys Tyr 225 230 235 240 Ser Phe Phe Pro Asp Trp
Leu Thr Tyr Val Arg Ser Gln Thr Gln Lys 245 250 255 Asn Leu Phe Ala
Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu 260 265 270 His Asn
Tyr Ile Thr Lys Thr Ser Gly Thr Met Ser Leu Phe Asp Ala 275 280 285
Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe 290
295 300 Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro
Ser 305 310
315 320 Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln
Ser 325 330 335 Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
Tyr Ala Phe 340 345 350 Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val
Phe Tyr Gly Asp Tyr 355 360 365 Tyr Gly Ile Pro Lys Tyr Asn Ile Pro
Gly Leu Lys Ser Lys Ile Asp 370 375 380 Pro Leu Leu Ile Ala Arg Arg
Asp Tyr Ala Tyr Gly Thr Gln Arg Asp 385 390 395 400 Tyr Ile Asp His
Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp 405 410 415 Ser Lys
Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe 435
440 445 Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala
Asp 450 455 460 Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
Ile Trp Val 465 470 475 480 Ala Lys 13483PRTBacillus licheniformis
13Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1
5 10 15 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr
Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala
Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr
Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys Gly Thr Val
Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile
Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val
Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val
Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile
Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135
140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
Ile Tyr Lys 165 170 175 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser
Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile
Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu Ile Lys Arg Trp
Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 Leu Asp Gly Phe Arg
Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg
Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260
265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro
Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly
Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys
His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp
Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp
Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys
Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385
390 395 400 Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu
Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile
Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg
Gln Asn Ala Gly Glu Thr 435 440 445 Trp His Asp Ile Thr Gly Asn Arg
Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly Trp Gly Glu Phe
His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Arg
14483PRTBacillus amyloliquefacience 14Val Asn Gly Thr Leu Met Gln
Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly Gln His Trp Lys
Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30 Ile Gly Ile
Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45 Gln
Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55
60 Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu
65 70 75 80 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln
Val Tyr 85 90 95 Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp
Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu Val Asn Pro Ala Asn
Arg Asn Gln Glu Thr Ser 115 120 125 Glu Glu Tyr Gln Ile Lys Ala Trp
Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140 Gly Asn Thr Tyr Ser Asp
Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150 155 160 Ala Asp Trp
Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165 170 175 Gly
Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185
190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val
195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu
Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile
Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln Ala Val Arg
Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp
Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr
Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe Asn
Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg
Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310
315 320 Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu
Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala
Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr
Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr Ser Pro Lys Glu Ile
Pro Ser Leu Lys Asp Asn Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg
Lys Glu Tyr Ala Tyr Gly Pro Gln His 385 390 395 400 Asp Tyr Ile Asp
His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser
Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430
Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435
440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly
Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val
Ser Ile Tyr 465 470 475 480 Val Gln Lys 15483PRTBacillus
stearothermophilus 15Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys
Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly Ile Thr
Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser
Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu
Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys
Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90
95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg
Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr
Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe
Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp
Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile
Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215
220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser
Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser
Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asn
Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe
Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr
Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu
Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340
345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly
Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys
Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala
Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile
Ile Gly Trp Thr Arg Glu Gly Val 405 410 415 Thr Glu Lys Pro Gly Ser
Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys
Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 Phe Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465
470 475 480 Val Pro Arg 16485PRTUnknownbacterial 16His His Asn Gly
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro
Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30
Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35
40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu
Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr
Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala
Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met
Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala
Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly
Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly
Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165
170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val
Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp
Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn
Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe
Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr
Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn
Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala
Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290
295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln
Arg 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His
Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu
Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu
Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile
Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro
Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 Gln
Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410
415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys
Ala Gly 435 440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly
Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val
Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485
17484PRTUnknownbacterial 17Gly Ser Val Pro Val Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asp Asp Gly Thr Leu Trp
Thr Lys Val Ala Asn Asn Ala Gln 20 25 30 Ser Leu Ala Asn Leu Gly
Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr 35 40 45 Lys Gly Thr Ser
Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55 60 Asp Leu
Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80
Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala Gly 85
90 95 Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala
Asp 100 105 110 Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser
Asp Arg Asn 115 120 125 Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala
Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser
Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp
Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile 165 170 175 Tyr Lys Phe Arg
Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr 180 185
190 Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp
195 200 205 His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp
Tyr Val 210 215 220 Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala
Val Lys His Ile 225 230 235 240 Lys Tyr Ser Phe Phe Pro Asp Trp Leu
Ser Tyr Val Arg Thr Gln Thr 245 250 255 Gln Lys Pro Leu Phe Ala Val
Gly Glu Phe Trp Ser Tyr Asp Ile Asn 260 265 270 Lys Leu His Asn Tyr
Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe 275 280 285 Asp Ala Pro
Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly Gly 290 295 300 Tyr
Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln 305 310
315 320 Pro Thr Leu Ser Val Thr Leu Val Asp Asn His Asp Thr Glu Pro
Gly 325 330 335 Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro
Leu Ala Tyr 340 345 350 Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro
Cys Ile Phe Tyr Gly 355 360 365 Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn
Ile Pro Ala Leu Lys Ser Lys 370 375 380 Leu Asp Pro Leu Leu Ile Ala
Arg Arg Asp Tyr Ala Tyr Gly Thr Gln 385 390 395 400 His Asp Tyr Ile
Asp Asn Ala Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415 Val Ala
Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly 420 425 430
Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys 435
440 445 Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
Asn 450 455 460 Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser
Val Ser Ile 465 470 475 480 Trp Val Pro Lys
18485PRTUnknownbacterial 18Ala Asn Thr Ala Pro Ile Asn Glu Thr Met
Met Gln Tyr Phe Glu Trp 1 5 10 15 Asp Leu Pro Asn Asp Gly Thr Leu
Trp Thr Lys Val Lys Asn Glu Ala 20 25 30 Ala Asn Leu Ser Ser Leu
Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala 35 40 45 Tyr Lys Gly Thr
Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu 50 55 60 Tyr Asp
Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr 65 70 75 80
Gly Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala 85
90 95 Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly
Ala 100 105 110 Asp Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro
Ser Asn Arg 115 120 125 Asn Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln
Ala Trp Thr Lys Phe 130 135 140 Asp Phe Pro Gly Arg Gly Asn Thr Tyr
Ser Ser Phe Lys Trp Arg Trp 145 150 155 160 Tyr His Phe Asp Gly Thr
Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg 165 170 175 Ile Tyr Lys Phe
Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu
Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met 195 200 205
Asp His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr 210
215 220 Val Asn Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys
His 225 230 235 240 Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Thr Tyr
Val Arg Asn Gln 245 250 255 Thr Gly Lys Asn Leu Phe Ala Val Gly Glu
Phe Trp Ser Tyr Asp Val 260 265 270 Asn Lys Leu His Asn Tyr Ile Thr
Lys Thr Asn Gly Ser Met Ser Leu 275 280 285 Phe Asp Ala Pro Leu His
Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser 290 295 300 Gly Tyr Phe Asp
Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp 305 310 315 320 Gln
Pro Ser Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro 325 330
335 Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
340 345 350 Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val
Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro
Gly Leu Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Ile Ala Arg Arg
Asp Tyr Ala Tyr Gly Thr 385 390 395 400 Gln Arg Asp Tyr Ile Asp His
Gln Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Ile Asp Thr Lys
Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp 420 425 430 Gly Pro Gly
Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly 435 440 445 Lys
Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile 450 455
460 Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
465 470 475 480 Ile Trp Val Ala Lys 485 19619PRTBacillus
flavothermus 19Met Ser Leu Phe Lys Lys Ser Phe Pro Trp Ile Leu Ser
Leu Leu Leu 1 5 10 15 Leu Phe Ser Phe Ile Ala Pro Phe Ser Ile Gln
Thr Glu Lys Val Arg 20 25 30 Ala Gly Ser Val Pro Val Asn Gly Thr
Met Met Gln Tyr Phe Glu Trp 35 40 45 Tyr Leu Pro Asp Asp Gly Thr
Leu Trp Thr Lys Val Ala Asn Asn Ala 50 55 60 Gln Ser Leu Ala Asn
Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala 65 70 75 80 Tyr Lys Gly
Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu 85 90 95 Tyr
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr 100 105
110 Gly Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala
115 120 125 Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala
Gly Ala 130 135 140 Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn
Pro Ser Asp Arg 145 150 155 160 Asn Gln Glu Ile Ser Gly Thr Tyr Gln
Ile Gln Ala Trp Thr Lys Phe 165 170 175 Asp Phe Pro Gly Arg Gly Asn
Thr Tyr Ser Ser Phe Lys Trp Arg Trp 180 185 190 Tyr His Phe Asp Gly
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg 195 200 205 Ile Tyr Lys
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 210 215 220 Thr
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met 225 230
235 240 Asp His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp
Tyr 245 250 255 Val Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala
Val Lys His 260 265 270 Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser
Tyr Val Arg Thr Gln 275 280 285 Thr Gln Lys Pro Leu Phe Ala Val Gly
Glu Phe Trp Ser Tyr Asp Ile 290 295 300 Ser Lys Leu His Asn Tyr Ile
Thr Lys Thr Asn Gly Ser Met Ser Leu 305 310 315 320 Phe Asp Ala Pro
Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly 325 330 335 Gly Tyr
Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp 340 345 350
Gln Pro Thr Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Glu Pro 355
360 365 Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu
Ala 370 375 380 Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys
Val Phe Tyr 385 390 395 400 Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn
Ile Pro Ala Leu Lys Ser 405 410 415 Lys Leu Asp Pro Leu Leu Ile Ala
Arg Arg Asp Tyr Ala Tyr Gly Thr 420 425 430 Gln His Asp Tyr Ile Asp
Ser Ala Asp Ile Ile Gly Trp Thr Arg Glu 435 440 445 Gly Val Ala Glu
Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp 450 455 460 Gly Pro
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly 465 470 475
480 Lys Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
485 490 495 Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser
Val Ser 500 505 510 Ile Trp Val Pro Lys Ile Ser Thr Thr Ser Gln Ile
Thr Phe Thr Val 515 520 525 Asn Asn Ala Thr Thr Val Trp Gly Gln Asn
Val Tyr Val Val Gly Asn 530 535 540 Ile Ser Gln Leu Gly Asn Trp Asp
Pro Val His Ala Val Gln Met Thr 545 550 555 560 Pro Ser Ser Tyr Pro
Thr Trp Thr Val Thr Ile Pro Leu Leu Gln Gly 565 570 575 Gln Asn Ile
Gln Phe Lys Phe Ile Lys Lys Asp Ser Ala Gly Asn Val 580 585 590 Ile
Trp Glu Asp Ile Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala 595 600
605 Ser Gly Ala Tyr Thr Ala Ser Trp Asn Val Pro 610 615
20613PRTBacillus 20Met Ser Tyr Leu Lys Lys Val Trp Leu Tyr Tyr Thr
Ile Ile Ala Thr 1 5 10 15 Leu Ile Ile Ser Phe Phe Thr Pro Phe Ser
Thr Ala Gln Ala Asn Thr 20 25 30 Ala Pro Val Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Asp Leu Pro 35 40 45 Asn Asp Gly Thr Leu Trp
Thr Lys Val Lys Asn Glu Ala Ser Ser Leu 50 55 60 Ser Ala Leu Gly
Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly 65 70 75 80 Thr Ser
Gln Ala Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu 85 90 95
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys 100
105 110 Thr Gln Tyr Leu Gln Ala Ile Gln Ala Ala Lys Ser Ala Gly Met
Gln 115 120 125 Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala
Asp Ser Thr 130 135 140 Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser
Asn Arg Asn Gln Glu 145 150 155 160 Thr Ser Gly Thr Tyr Gln Ile Gln
Ala Trp Thr Lys Phe Asp Phe Pro 165 170 175 Gly Arg Gly Asn Thr Tyr
Ser Ser Phe Lys Trp Arg Trp Tyr His Phe 180 185 190 Asp Gly Thr Asp
Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 195 200 205 Phe Arg
Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn 210 215 220
Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro 225
230 235 240 Glu Val Val Ala Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val
Asn Thr 245 250 255 Thr Asn Val Asp Gly Phe Arg Leu Asp Ala Val Lys
His Ile Lys Tyr 260 265 270 Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val
Arg Asn Gln Thr Gly Lys 275 280 285 Asn Leu Phe Ala Val Gly Glu Phe
Trp Gly Tyr Asp Val Asn Lys Leu 290 295 300 His Asn Tyr Ile Thr Lys
Thr Asn Gly Ala Met Ser Leu Phe Asp Ala 305 310 315 320 Pro Leu His
Asn Asn Phe Tyr Ile Ala Ser Lys Ser Ser Gly Tyr Phe 325 330 335 Asp
Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ala 340 345
350 Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser
355 360 365 Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr
Ala Phe 370 375 380 Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe
Tyr Gly Asp Tyr 385 390 395 400 Tyr Gly Ile Pro Lys Tyr Asn Ile Pro
Gly Leu Lys Ser Lys Ile Asp 405 410 415 Pro Leu Leu Ile Ala Arg Arg
Asp Tyr Ala Tyr Gly Thr Gln Arg Asp 420 425 430 Tyr Ile Asp His Gln
Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp 435 440 445 Ala Lys Pro
Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 450 455 460 Gly
Ser Lys Trp Met Tyr Val Gly Lys Arg His Ala Gly Lys Val Phe 465 470
475 480 Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala
Asp 485 490 495 Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
Ile Trp Val 500 505 510 Ala Lys Thr Ser Asn Val Thr Phe Thr Val Asn
Asn Ala Thr Thr Val 515 520 525 Tyr Gly Gln Asn Val Tyr Val Val Gly
Asn Ile Pro Glu Leu Gly Asn 530 535 540 Trp Asn Ile Ala Asn Ala Ile
Gln Met Thr Pro Ser Ser Tyr Pro Thr 545 550 555 560 Trp Lys Thr Thr
Val Ser Leu Pro Gln Gly Lys Ala Ile Glu Phe Lys 565 570 575 Phe Ile
Lys Lys Asp Ser Ala Gly Asn Val Ile Trp Glu Asn Ile Ala 580 585 590
Asn Arg Thr Tyr Thr Val Pro Phe Ser Ser Thr Gly Ser Tyr Thr Ala 595
600 605 Asn Trp Asn Val Pro 610 21619PRTAlkaliphilic bacillus 21Met
Ser Leu Phe Lys Lys Ile Phe Pro Trp Ile Leu Ser Leu Leu Leu 1 5 10
15 Leu Phe Ser Phe Ile Ala Pro Phe Ser Ile Gln Thr Glu Lys Val Arg
20 25 30 Ala Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp 35 40 45 Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val
Ala Asn Asn Ala 50 55 60 Gln Ser Leu Ala Asn Leu Gly Ile Thr Ala
Leu Trp Leu Pro Pro Ala 65 70 75 80 Tyr Lys Gly Thr Ser Ser Ser Asp
Val Gly Tyr Gly Val Tyr Asp Leu 85 90 95 Tyr Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr 100 105 110 Gly Thr Lys Thr
Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala 115 120 125 Gly Met
Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala 130 135 140
Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg 145
150 155 160 Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr
Lys Phe 165 170 175 Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe
Lys Trp Arg Trp 180 185 190 Tyr His Phe Asp Gly Thr Asp Trp Asp Glu
Ser Arg Lys Leu Asn Arg 195 200 205 Ile Tyr Lys Phe Arg Gly Thr Gly
Lys Ala Trp Asp Trp Glu Val Asp 210 215 220 Thr Glu Asn Gly Asn Tyr
Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met 225 230 235 240 Asp His Pro
Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr 245 250 255 Val
Ile Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His 260 265
270 Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Leu Arg Thr
Gln
275 280 285 Thr Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr
Asp Ile 290 295 300 Asn Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly
Ser Met Ser Leu 305 310 315 320 Phe Asp Ala Pro Leu His Asn Asn Phe
Tyr Ile Ala Ser Lys Ser Gly 325 330 335 Gly Tyr Phe Asp Met Arg Thr
Leu Leu Asn Asn Thr Leu Met Lys Glu 340 345 350 Gln Pro Thr Leu Ser
Val Thr Leu Val Asp Asn His Asp Thr Glu Pro 355 360 365 Gly Gln Ser
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala 370 375 380 Tyr
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr 385 390
395 400 Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys
Ser 405 410 415 Lys Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala
Tyr Gly Thr 420 425 430 Gln His Asp Tyr Ile Asp Asn Ala Asp Ile Ile
Gly Trp Thr Arg Glu 435 440 445 Gly Val Ala Glu Lys Ala Asn Ser Gly
Leu Ala Ala Leu Ile Thr Asp 450 455 460 Gly Pro Gly Gly Ser Lys Trp
Met Tyr Val Gly Lys Gln His Ala Gly 465 470 475 480 Lys Thr Phe Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile 485 490 495 Asn Ala
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser 500 505 510
Ile Trp Val Pro Lys Thr Ser Thr Thr Ser Gln Ile Thr Phe Thr Val 515
520 525 Asn Asn Ala Thr Thr Val Trp Gly Gln Asn Val Tyr Val Val Gly
Asn 530 535 540 Ile Ser Gln Leu Gly Asn Trp Asp Pro Val Asn Ala Val
Gln Met Thr 545 550 555 560 Pro Ser Ser Tyr Pro Thr Trp Val Val Thr
Val Pro Leu Pro Gln Ser 565 570 575 Gln Asn Ile Gln Phe Lys Phe Ile
Lys Lys Asp Gly Ser Gly Asn Val 580 585 590 Ile Trp Glu Asn Ile Ser
Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala 595 600 605 Ser Gly Ala Tyr
Thr Ala Asn Trp Asn Val Pro 610 615 22640PRTAspergillus kawachii
22Met Arg Val Ser Thr Ser Ser Ile Ala Leu Ala Val Ser Leu Phe Gly 1
5 10 15 Lys Leu Ala Leu Gly Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser
Ile 20 25 30 Tyr Phe Leu Leu Thr Asp Arg Phe Gly Arg Thr Asp Asn
Ser Thr Thr 35 40 45 Ala Thr Cys Asn Thr Gly Asp Gln Ile Tyr Cys
Gly Gly Ser Trp Gln 50 55 60 Gly Ile Ile Asn His Leu Asp Tyr Ile
Gln Gly Met Gly Phe Thr Ala 65 70 75 80 Ile Trp Ile Ser Pro Ile Thr
Glu Gln Leu Pro Gln Asp Thr Ser Asp 85 90 95 Gly Glu Ala Tyr His
Gly Tyr Trp Gln Gln Lys Ile Tyr Tyr Val Asn 100 105 110 Ser Asn Phe
Gly Thr Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu 115 120 125 His
Ala Arg Gly Met Tyr Leu Met Val Asp Val Val Pro Asn His Met 130 135
140 Gly Tyr Ala Gly Asn Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro
145 150 155 160 Phe Asp Ser Ser Ser Tyr Phe His Pro Tyr Cys Leu Ile
Thr Asp Trp 165 170 175 Asp Asn Leu Thr Met Val Gln Asp Cys Trp Glu
Gly Asp Thr Ile Val 180 185 190 Ser Leu Pro Asp Leu Asn Thr Thr Glu
Thr Ala Val Arg Thr Ile Trp 195 200 205 Tyr Asp Trp Val Ala Asp Leu
Val Ser Asn Tyr Ser Val Asp Gly Leu 210 215 220 Arg Ile Asp Ser Val
Glu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr 225 230 235 240 Gln Glu
Ala Ala Gly Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn 245 250 255
Pro Ala Leu Asp Cys Pro Tyr Gln Lys Tyr Leu Asp Gly Val Leu Asn 260
265 270 Tyr Pro Ile Tyr Trp Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser
Gly 275 280 285 Ser Ile Ser Asn Leu Tyr Asn Met Ile Lys Ser Val Ala
Ser Asp Cys 290 295 300 Ser Asp Pro Thr Leu Leu Gly Asn Phe Ile Glu
Asn His Asp Asn Pro 305 310 315 320 Arg Phe Ala Ser Tyr Thr Ser Asp
Tyr Ser Gln Ala Lys Asn Val Leu 325 330 335 Ser Tyr Ile Phe Leu Ser
Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu 340 345 350 Glu Gln His Tyr
Ser Gly Gly Asp Val Pro Tyr Asn Arg Glu Ala Thr 355 360 365 Trp Leu
Ser Gly Tyr Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala 370 375 380
Thr Thr Asn Ala Ile Arg Lys Leu Ala Ile Ser Ala Asp Ser Asp Tyr 385
390 395 400 Ile Thr Tyr Lys Asn Asp Pro Ile Tyr Thr Asp Ser Asn Thr
Ile Ala 405 410 415 Met Arg Lys Gly Thr Ser Gly Ser Gln Ile Ile Thr
Val Leu Ser Asn 420 425 430 Lys Gly Ser Ser Gly Ser Ser Tyr Thr Leu
Thr Leu Ser Gly Ser Gly 435 440 445 Tyr Thr Ser Gly Thr Lys Leu Ile
Glu Ala Tyr Thr Cys Thr Ser Val 450 455 460 Thr Val Asp Ser Asn Gly
Asp Ile Pro Val Pro Met Ala Ser Gly Leu 465 470 475 480 Pro Arg Val
Leu Leu Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys 485 490 495 Gly
Gly Ser Gly Asn Thr Thr Thr Thr Thr Thr Ala Ala Thr Ser Thr 500 505
510 Ser Lys Ala Thr Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr
515 520 525 Ser Ser Ser Cys Thr Ala Thr Ser Thr Thr Leu Pro Ile Thr
Phe Glu 530 535 540 Glu Leu Val Thr Thr Thr Tyr Gly Glu Glu Val Tyr
Leu Ser Gly Ser 545 550 555 560 Ile Ser Gln Leu Gly Glu Trp His Thr
Ser Asp Ala Val Lys Leu Ser 565 570 575 Ala Asp Asp Tyr Thr Ser Ser
Asn Pro Glu Trp Ser Val Thr Val Ser 580 585 590 Leu Pro Val Gly Thr
Thr Phe Glu Tyr Lys Phe Ile Lys Val Asp Glu 595 600 605 Gly Gly Ser
Val Thr Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val 610 615 620 Pro
Glu Cys Gly Ser Gly Ser Gly Glu Thr Val Val Asp Thr Trp Arg 625 630
635 640
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