U.S. patent application number 10/124880 was filed with the patent office on 2003-02-06 for novel rhamnogalacturonan hydrolases.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Andersen, Lene Nonboe, Dela, Hanne, Jorgensen, Per Lina, Outtrup, Helle, Schnorr, Kirk, Schulein, Martin.
Application Number | 20030026810 10/124880 |
Document ID | / |
Family ID | 26064305 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030026810 |
Kind Code |
A1 |
Jorgensen, Per Lina ; et
al. |
February 6, 2003 |
Novel rhamnogalacturonan hydrolases
Abstract
Novel isolated enzymes exhibiting rhamnogalacturonan hydrolase
activity and belonging to a glycosyl hydrolase family other than
family 28 are obtained and cloned from bacterial strains, for
example, Bacillus licheniformis, Bacillus subtilis, Bacillus
halodurans, Bacillus agaradhaerens, Caldicellulosiruptor,
Streptomyces and Sorangium.
Inventors: |
Jorgensen, Per Lina;
(Copenhagen K, DK) ; Schnorr, Kirk; (Copenhagen
N., DK) ; Andersen, Lene Nonboe; (Allerod, DK)
; Schulein, Martin; (Copenhagen, DK) ; Dela,
Hanne; (Copenhagen, DK) ; Outtrup, Helle;
(Ballerup, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
26064305 |
Appl. No.: |
10/124880 |
Filed: |
April 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10124880 |
Apr 19, 2002 |
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09311626 |
May 13, 1999 |
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6399347 |
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09311626 |
May 13, 1999 |
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PCT/DK99/00244 |
May 3, 1999 |
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60084358 |
May 5, 1998 |
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Current U.S.
Class: |
424/190.1 ;
435/200; 435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/2402 20130101;
C13B 20/002 20130101; A23K 20/189 20160501; C12Y 302/01171
20130101; A23L 2/84 20130101; C11D 3/38636 20130101; A23K 30/18
20160501; C13K 1/06 20130101; C11B 1/025 20130101 |
Class at
Publication: |
424/190.1 ;
435/200; 435/69.1; 435/252.3; 536/23.2; 435/320.1 |
International
Class: |
A61K 039/02; C12N
009/24; C07H 021/04; C12P 021/02; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
May 1, 1998 |
DK |
0608/98 |
Claims
What is claimed is:
1. An isolated enzyme exhibiting rhamnogalacturonan hydrolase
activity wherein the enzyme belongs to a glycosyl hydrolase family
other than family 28.
2. The enzyme according to claim 2, which is obtained from a
microbial strain belonging to Bacteria.
3. The enzyme according to claim 1 which i) comprises a polypeptide
comprising an amino acid sequence having amino acids 1-621 of the
sequence of SEQ ID NO:2, or ii) comprises an analogue of the
polypeptide defined in i) which is at least 75% homologous with the
polypeptide, or iii) comprises a mutant or variant of the
polypeptide which is derived from the polypeptide by substitution,
deletion or addition of one or several amino acids or iv) is
immunologically reactive with a polyclonal antibody raised against
the polypeptide in purified form.
4. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 3.
5. The enzyme according to claim 1 which i) comprises a polypeptide
comprising an amino acid sequence having amino acids 1-620 of the
sequence of SEQ ID NO:4, or ii) comprises an analogue of the
polypeptide defined in i) which is at least 75% homologous with the
polypeptide, or iii) comprises a mutant or variant of the
polypeptide which is derived from the polypeptide by substitution,
deletion or addition of one or several amino acids or iv) is
immunologically reactive with a polyclonal antibody raised against
the polypeptide in purified form.
6. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 5.
7. The enzyme according to claim 1 which i) comprises a polypeptide
comprising an amino acid sequence having amino acids 1-620 of the
sequence of SEQ ID NO:6, or ii) comprises an analogue of the
polypeptide defined in i) which is at least 75% homologous with the
polypeptide, or iii) comprises a mutant or variant of the
polypeptide which is derived from the polypeptide by substitution,
deletion or addition of one or several amino acids or iv) is
immunologically reactive with a polyclonal antibody raised against
the polypeptide in purified form.
8. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 7.
9. The enzyme according to claim 1 which i) comprises a polypeptide
comprising an amino acid sequence comprising amino acids 1-471 of
the sequence of SEQ ID NO:8, or ii) comprises an analogue of the
polypeptide defined in i) of which a part is at least 75%
homologous with the polypeptide, or iii) comprises a mutant or
variant of the polypeptide which is derived from the polypeptide by
substitution, deletion or addition of one or several amino acids or
iv) is immunologically reactive with a polyclonal antibody raised
against the polypeptide in purified form.
10. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 9.
11. The enzyme according to claim 1 which i) comprises a
polypeptide comprising an amino acid sequence comprising amino
acids 1-170 of the sequence of SEQ ID NO:10, or ii) comprises an
analogue of the polypeptide defined in i) of which a part is at
least 75% homologous with the polypeptide, or iii) comprises a
mutant or variant of the polypeptide which is derived from the
polypeptide by substitution, deletion or addition of one or several
amino acids or iv) is immunologically reactive with a polyclonal
antibody raised against the polypeptide in purified form.
12. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 11.
13. The enzyme according to claim 1 which i) comprises a
polypeptide comprising an amino acid sequence comprising amino
acids 1-112 of the sequence of SEQ ID NO:12, or ii) comprises an
analogue of the polypeptide defined in i) of which a part is at
least 75% homologous with the polypeptide, or iii) comprises a
mutant or variant of the polypeptide which is derived from the
polypeptide by substitution, deletion or addition of one or several
amino acids or iv) is immunologically reactive with a polyclonal
antibody raised against the polypeptide in purified form.
14. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 13.
15. The enzyme according to claim 1 which i) comprises a
polypeptide comprising an amino acid sequence having amino acids
1-655 of the sequence of SEQ ID NO:14, or ii) comprises an analogue
of the polypeptide defined in i) which is at least 75% homologous
with the polypeptide, or iii) comprises a mutant or variant of the
polypeptide which is derived from the polypeptide by substitution,
deletion or addition of one or several amino acids or iv) is
immunologically reactive with a polyclonal antibody raised against
the polypeptide in purified form.
16. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 15.
17. The enzyme according to claim 1 which i) comprises a
polypeptide comprising an amino acid sequence having amino acids
1-631 of the sequence of SEQ ID NO:16, or ii) comprises an analogue
of the polypeptide defined in i) which is at least 75% homologous
with the polypeptide, or iii) comprises a mutant or variant of the
polypeptide which is derived from the polypeptide by substitution,
deletion or addition of one or several amino acids or iv) is
immunologically reactive with a polyclonal antibody raised against
the polypeptide in purified form.
18. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 17.
19. The enzyme according to claim 1 which i) comprises a
polypeptide comprising an amino acid sequence having amino acids
1-389 of the sequence of SEQ ID NO:18, or ii) comprises an analogue
of the polypeptide defined in i) which is at least 75% homologous
with the polypeptide, or iii) comprises a mutant or variant of the
polypeptide which is derived from the polypeptide by substitution,
deletion or addition of one or several amino acids or iv) is
immunologically reactive with a polyclonal antibody raised against
the polypeptide in purified form.
20. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 19.
21. The enzyme according to claim 1 which i) comprises a
polypeptide comprising an amino acid sequence comprising amino
acids 1-169 of the sequence of SEQ ID NO:20, or ii) comprises an
analogue of the polypeptide defined in i) of which a part is at
least 75% homologous with the polypeptide, or iii) comprises a
mutant or variant of the polypeptide which is derived from the
polypeptide by substitution, deletion or addition of one or several
amino acids or iv) is immunologically reactive with a polyclonal
antibody raised against the polypeptide in purified form.
22. An isolated polynucleotide molecule encoding a polypeptide
exhibiting rhamnogalacturonan hydrolase activity of claim 21.
23. An isolated enzyme exhibiting rhamnogalacturonan hydrolase
activity, exhibiting the characteristics of: a) cleaving a
rhamnogalacturonan backbone, wherein the rhamnoses are left as the
non-reducing ends; and b) a relative activity towards hairy regions
from apples of at least 30% at pH 8.
24. An isolated enzyme exhibiting rhamnogalacturonan hydrolase
activity, exhibiting the characteristics of: a) cleaving a
rhamnogalacturonan backbone, wherein the rhamnoses are left as the
non-reducing ends; and b) a relative activity towards hairy regions
from apples of at least 30% at pH 9.
25. A method of reducing the viscosity of a plant material, which
method comprises treating the plant material with the enzyme of
claim 1 under suitable conditions for the viscosity to be
reduced.
26. An animal feed composition comprising plant material rich in
pectic substance and the rhamnogalacturonan hydrolase of claim
1.
27. A method for increasing the digestibility of an animal feed,
comprising adding the rhamnogalacturonan hydrolase of claim 1 to
one or more of soy, pea or rapeseed, or other material derived from
Fabales or Cruciferaceae.
28. A method for reducing the viscosity of an animal feed,
comprising adding the rhamnogalacturonan hydrolase of claim 1 to
one or more of soy, pea or rapeseed, or other material derived from
Fabales or Cruciferaceae.
29. A detergent composition comprising the enzyme according to
claim 1 and a surfactant.
30. The enzyme according to claim 1 which comprises at least one
amino acid sequence segment selected from the group of amino acid
sequence segments consisting of a) NIRAGAHTQF(M or L)VYD(F or
L)DGDGKAE; b) YGNRVDRFLAG; c) YGNRVDRFLAGXAYLDG; d) AGQGNH(N or
S)LS(I or V)ADVDGDGKDEII; and e) AGQGNH(N or S)L(S or A)(I or
V)ADVDGDGKDEII.
31. The enzyme according to claim 1 which comprises at least one
amino acid sequence segment selected from the group of amino acid
sequence segments consisting of a) EVRDATIGLL; b) NNYVVGNPI; and c)
DADRTNRA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of
application Ser. No. 09/311,626, filed on May 13, 1999(now
allowed), which is a continuation of PCT/DK99/00244 filed May 3,
1999 and claims priority under 35 U.S.C. 119 of U.S. provisional
application No. 60/084,358 filed May 5, 1998 and Danish application
no. 0608/98 filed May 1, 1998, the contents of which are fully
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to microbial enzymes capable
of degrading the rhamnogalacturonan backbone in hairy regions of
pectins, more specifically to novel families of microbial
rhamnogalacturonan hydrolases and the genes encoding such enzymes;
to a method of producing such enzymes; and to methods for using
such enzymes in the textile, detergent, animal feed and cellulose
fiber processing industries.
[0004] 2. Description of the Related Art
[0005] Pectic polysaccharides constitute the major matrix
polysaccharides in the middle lamella and primary cell wall of
dicotyledonous plants (Carpita and Gibeaut, 1993). The main
backbone in pectins can be divided into linear homogalacturonan
(smooth) regions of up to 200 residues of (1,4)-linked
alpha-D-galacturonic acid (GalUA), and highly branched
rhamnogalacturonan (hairy) regions consisting of a backbone of
repeating alpha-(1,2)-L-Rha-alpha-(1,4)-D-GalUA disaccharide units
(Carpita and Gibeaut, 1993; O'Neill et al., 1990; Thibault et al.,
1993). The hydroxyl at the C-4 position of the rhamnose residues
serves as the attachment point for the side chains (hairs),
consisting mainly of neutral oligosaccharides, such as arabinan,
galactan and/or arabinogalactan (Carpita and Gibeaut, 1993; O'Neill
et al., 1990; Schols et al., 1990). In addition, the GalUA residues
in the backbone may be acetyl esterified at the C-2 or C-3 position
or methyl esterified at the carboxy group (Carpita and Gibeaut,
1993; Schols et al., 1990).
[0006] The distribution and composition of the side chains vary
considerably between different cell types and physiological states,
but in general about half of the rhamnosyl units in the
rhamnogalacturonan regions have side chains attached. The galactan
side chains are in most plants type 1 galactans, which are composed
of .beta.-1,4 linked galactopyranose with some branching points and
a length of up to 60 saccharide units (DP60). Arabinofuranose
residues or short arabinan oligomers can be attached to the
galactan chain at the O-3 of the galactosyl unit, thus named
arabinogalactan. Galactans (or arabinogalactans) have an important
function in the primary cell wall, where they interact with other
structural components of the cell wall such as xyloglucans or
arabinoxylans. Thus they possibly serve to anchor the pectic matrix
in the cell wall. (Carpita & Gibeaut, 1993, Plant J., 3, 1-30;
O'Neill et al., 1990, Methods in Plant Biochemistry, 415-441;
Selvendran, 1983, The Chemistry of Plant Cell Walls. Dietary
Fibers; Hwang et al., Food Hydrocolloids, 7, 39-53; Fry, 1988, The
growing Plant Cell Wall: Chemical and Metabolic Analysis).
[0007] Sugar beet debranched arabinan and potato galactan from
Megazyme (Ireland, http://www.megazyme.com/Purchase/index.html)
contain rhamnose and galacturonic acid indicating that these
substrates and their AZCL derivatives contain some
rhamnogalacturonan.
[0008] The biological degradation of pectic substances is a complex
process involving several enzymes produced by a wide variety of
saprophytic, plant pathogenic fungi and bacteria (Pilnik and
Rombouts, 1979). For example, the hydrolysis of smooth,
homogalacturonan regions of pectin by polygalacturonases is
dependent upon demethylation of the homogalacturonan backbone by
pectin methylesterase (Christgau et al., 1996; Pilnik and Rombouts,
1979). Several microbial polygalacturonases, pectate lyases, pectin
methylesterases and pectin lyases active within the smooth regions
of pectin have been described in literature.
[0009] A number of enzymes capable of hydrolyzing arabinan,
galactan or arabinogalactan side chains in the hairy regions have
been characterized. By contrast, only few enzymes capable of
degrading the rhamnogalacturonan backbone have been reported. A
rhamnogalacturonan hydrolase belonging to family 28 of glycosyl
hydrolases and a rhamnogalacturonan lyase belonging to lyase family
4, both from Aspergillus aculeatus, have been cloned and
characterized (Kofod et al. (1994) Journal of Biological Chemistry
Vol. 269 (46) pp. 29182-29189; Kauppinen et al. (1995) Journal of
Biological Chemistry Vol. 270 (45) pp. 27172-27178; Azadi et al.
(1995) Glycobiology Vol. 5 (8) pp. 783-789; Mutter et al. (1996)
Plant Physiology Vol. 110 (1) pp. 73-77; Mutter et al. (1998) Plant
Physiology Vol. 117 (1) pp. 141-152; Mutter et al. (1998)
Carbohydrate Research Vol. 311 (3) pp. 155-164). The sequence
families can be found on http://afmb.cnr-smrs.fr/.-
about.pedro/CAZY/db.html.
[0010] Degradation of rhamnogalacturonan by the Aspergillus
rhamnogalacturonan hydrolase and lyase is enhanced by removal of
acetyl groups from the backbone (Kofod et al., 1994; Schols et al.,
1990). A rhamnogalacturonan acetylesterase (RGAE) cloned and
characterized from Aspergillus aculeatus specifically removes
acetyl groups from hairy regions and acts in synergy with the
rhamnogalacturonases in degradation of apple pectin
rhamnogalacturonan (Kauppinen et al., 1995).
[0011] JP 10-033169 discloses a method for purification of
rhamnogalacturonase from enzyme preparations containing numerous
enzymes or liquid cultures of Aspergillus, Bacillus or Erwinia.
[0012] The object of the present invention is to provide a novel
rhamnogalacturonan hydrolase enzyme which can degrade the backbone
of hairy regions of pectin in an effective manner useful in a
number of different industrial applications.
SUMMARY OF THE INVENTION
[0013] The inventors have now found a number of novel bacterial
enzymes exhibiting rhamnogalacturonan hydrolase activity which are
believed to be members of two hitherto unidentified families of
glycosyl hydrolases according to the classification based on
hydrophobic cluster analysis (Henrissat, B. et al.). The novel
enzymes have no amino acid sequence homology to known
rhamnogalacturonan hydrolases from family 28 or rhamnogalacturonan
lyases from lyase family 4. More specifically, novel families of
enzymes degrading rhamnogalacturonan by hydrolysis has been found.
The enzymes are of bacterial origin, with few or no cystein bridges
and with a potential of being expressed in high yields in Gram
positive bacterial hosts. The rhamnogalacturonase enzymes of this
invention show enzymatic activity at neutral and alkaline
conditions and, accordingly, they are very useful in a number of
industrial applications.
[0014] The novel enzymes exhibit catalytic activity on
rhamnogalacturonan as well as on the Megazyme products AZCL potato
galactan and AZCL debranched arabinan. This activity can be
explained by the presence of rhamnogalacturonan in the two AZCL
carbohydrate polymers.
[0015] The inventors have succeeded in identifying either partial
or full length DNA sequences encoding the novel enzymes. The DNA
sequences are listed in the appended sequence listing as SEQ ID
NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, and the deduced amino
acid sequences are listed in the sequence listing as SEQ ID NOS: 2,
4, 6, 8, 10, 12, 14, 16, 18 and 20, respectively.
[0016] In a first aspect, the present invention relates to an
enzyme exhibiting rhamnogalacturonan hydrolase activity wherein the
enzyme belongs to a glycosyl hydrolase family other than family 28.
In a preferred embodiment, the enzyme of the invention is obtained
from a microbial strain belonging to Bacteria, preferably to
Firmicutes or Proteobacteria, more preferably to Actinobacteria,
Myxobacteria or the Bacillus/Clostridium group.
[0017] In second and third aspects, the invention relates to an
enzyme comprising at least one amino acid sequence segment selected
from the group of amino acid sequence segments consisting of
NIRAGAHTQF(M or L)VYD(F or L)DGDGKAE; YGNRVDRFLAG;
YGNRVDRFLAGXAYLDG; AGQGNH(N or S)LS(I or V)ADVDGDGKDEII; and
AGQGNH(N or S)L(S or A)(I or V)ADVDGDGKDEII; and to an enzyme
comprising at least one amino acid sequence segment selected from
the group of amino acid sequence segments consisting of EVRDATIGLL;
NNYVVGNPI; and DADRTNRA.
[0018] In further aspects, the invention relates to a
rhamnogalacturonan hydrolase enzyme which is i) a polypeptide
produced by a strain selected from the group consisting of Bacillus
licheniformis, Bacillus halodurans, Bacillus subtilis, Bacillus
agaradhaerens, Bacillus sp. AA386, Sorangium cellulosum,
Streptomyces coelicolor and Caldicellulosiruptor sp.; or ii) a
polypeptide comprising an amino acid sequence as shown in positions
1-621 of SEQ ID NO:2 or in positions 1-620 of SEQ ID NO:4, or in
positions 1-620 of SEQ ID NO:6 or in positions 1-471 of SEQ ID NO:8
or in positions 1-170 of SEQ ID NO:10 or in positions 1-112 of SEQ
ID NO:12 or in positions 1-655 of SEQ ID NO:14 or in positions
1-631 of SEQ ID NO:16 or in positions 1-389 of SEQ ID NO:18 or in
positions 1-169 of SEQ ID NO:20; or iii) an analogue of the
polypeptide defined in i) or ii) which is at least 75% homologous
with said polypeptide and can be derived from said polypeptide by
substitution, deletion or insertion of one or several amino
acids.
[0019] Within other aspects, the present invention provides an
isolated polynucleotide molecule selected from the group consisting
of (a) polynucleotide molecules encoding a rhamnogalacturonase and
comprising a sequence of nucleotides selected from the group
consisting of the nucleotide sequences shown in SEQ ID NO:1 from
nucleotide 1 to nucleotide 1863, SEQ ID NO:3 from nucleotide 1 to
nucleotide 1863, and SEQ ID NO:5 from nucleotide 1 to nucleotide
1863, in SEQ ID NO: 7 from nucleotide 1 to nucleotide 1413, in SEQ
ID NO: 9 from nucleotide 1 to nucleotide 512, in SEQ ID NO: 11 from
nucleotide 1 to nucleotide 336, in SEQ ID NO: 13 from nucleotide 1
to nucleotide 1965, in SEQ ID NO: 15 from nucleotide 1 to
nucleotide 1896, in SEQ ID NO: 17 from nucleotide 1 to nucleotide
1168, in SEQ ID NO: 19 from nucleotide 1 to nucleotide 507; (b)
species homologs of (a); (c) polynucleotide molecules that encode a
polypeptide which can degrade the rhamnogalacturonan backbone of
hairy regions of pectin and which is at least 75% identical to the
amino acid sequence of SEQ ID NO: 2 from amino acid residue 1 to
amino acid residue 621, SEQ ID NO: 4 from amino acid residue 1 to
amino acid residue 620, or SEQ ID NO: 6 from amino acid residue 1
to amino acid residue 620, SEQ ID NO: 8 from amino acid residue 1
to amino acid residue 471, SEQ ID NO: 10 from amino acid residue 1
to amino acid residue 170, SEQ ID NO: 12 from amino acid residue 1
to amino acid residue 112, SEQ ID NO: 14 from amino acid residue 1
to amino acid residue 655, SEQ ID NO: 16 from amino acid residue 1
to amino acid residue 631, SEQ ID NO: 18 from amino acid residue 1
to amino acid residue 389, SEQ ID NO: 20 from amino acid residue 1
to amino acid residue 169; (d) molecules complementary to (a), (b)
or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or
(d).
[0020] The E. coli plasmids comprising the polynucleotide molecules
(the DNA sequences corresponding to SEQ ID NOS: 1, 3, 9, 13, 17
respectively) encoding an enzyme of the present invention has been
transformed into a strain of the Escherichia coli which was
deposited by the inventors according to the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124
Braunschweig, Federal Republic of Germany, on Apr. 24, 1998 under
the deposition numbers DSM 12123 and DSM 12122, on Sep. 8, 1998
under the deposition number DSM 12405, on Apr. 24, 1998 under the
deposition number DSM 12124, and on May 29, 1998 under the
deposition number DSM 12202, respectively.
[0021] Within another aspect of the invention there is provided an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment selected from the
group consisting of (a) polynucleotide molecules encoding a
rhamnogalacturonan hydrolase and comprising a sequence of
nucleotides as shown in SEQ ID NO: 1 from nucleotide 1 to
nucleotide 1863, SEQ ID NO:3 from nucleotide 1 to nucleotide 1863,
and SEQ ID NO:5 from nucleotide 1 to nucleotide 1863, in SEQ ID NO:
7 from nucleotide 1 to nucleotide 1413, in SEQ ID NO: 9 from
nucleotide 1 to nucleotide 512, in SEQ ID NO: 11 from nucleotide 1
to nucleotide 336, in SEQ ID NO: 13 from nucleotide 1 to nucleotide
1965, in SEQ ID NO: 15 from nucleotide 1 to nucleotide 1896, in SEQ
ID NO: 17 from nucleotide 1 to nucleotide 1168, in SEQ ID NO: 19
from nucleotide 1 to nucleotide 507; (b) species homologs of (a);
(c) polynucleotide molecules that encode a polypeptide which can
degrade rhamnogalacturonan backbone of hairy regions of pectin and
which is at least 75% identical to the amino acid sequence of SEQ
ID NO: 2 from amino acid residue 1 to amino acid residue 621, SEQ
ID NO: 4 from amino acid residue 1 to amino acid residue 620, or
SEQ ID NO: 6 from amino acid residue 1 to amino acid residue 620,
SEQ ID NO: 8 from amino acid residue 1 to amino acid residue 471,
SEQ ID NO: 10 from amino acid residue 1 to amino acid residue 170,
SEQ ID NO: 12 from amino acid residue 1 to amino acid residue 1 12,
SEQ ID NO: 1 4 from amino acid residue 1 to amino acid residue 655,
SEQ ID NO: 16 from amino acid residue 1 to amino acid residue 631,
SEQ ID NO: 18 from amino acid residue 1 to amino acid residue 389,
SEQ ID NO: 20 from amino acid residue 1 to amino acid residue 169;
and (d) degenerate nucleotide sequences of (a), (b), or (c); and a
transcription terminator.
[0022] Within yet another aspect of the present invention there is
provided a cultured cell into which has been introduced an
expression vector as disclosed above, wherein said cell expresses
the polypeptide encoded by the DNA segment.
[0023] Within another aspect of the present invention there is
provided a composition comprising a purified polypeptide according
to the invention, i.e. an enzyme, in combination with other
polypeptides exhibiting enzymatic activity.
[0024] At present it is contemplated that the novel enzyme of the
present invention is useful for the treatment of cellulosic
material, especially cellulose-containing fiber, yarn, woven or
non-woven fabric. The treatment can be carried out during the
processing of cellulosic material into a material ready for garment
manufacture or fabric manufacture, e.g. in the desizing or scouring
step; or during industrial or household laundering of such fabric
or garment.
[0025] Accordingly, in further aspects the present invention
relates to a detergent composition comprising a rhamnoglacturonan
hydrolase enzyme or an enzyme capable of degrading
rhamnogalacturonan backbone of hairy regions of pectin; and to use
of the enzyme of the invention for the treatment of
cellulose-containing fibers, yarn, woven or non-woven fabric.
[0026] It is also contemplated that the enzyme of the invention is
effective for use in an enzymatic scouring process in the
preparation of cellulosic material e.g. for proper response in
subsequent dyeing operations. Further, it is contemplated that
detergent compositions comprising the novel enzyme are capable of
removing or bleaching certain soils or stains present on laundry,
e.g. soils and spots resulting from food, plants, and the like
containing pectic substances. It is also contemplated that
treatment with detergent compositions comprising the novel enzyme
can prevent binding of certain soils to the cellulosic
material.
DRAWINGS
[0027] In the attached drawings,
[0028] FIG. 1 shows high performance size exclusion chromatography
(HPSEC) of hairy regions from apples (MHR) degraded by the
rhamnogalacturonan hydrolase from Bacillus sp. AA 386 (BXR1).
[0029] FIG. 2 shows high performance size exclusion chromatography
(HPSEC) of MHR degraded by the rhamnogalacturonan hydrolase from
Bacillus licheniformis (BLR3).
[0030] FIG. 3 shows high performance size exclusion chromatography
(HPSEC) of MHR degraded by the rhamnogalacturonan hydrolase from
Bacillus subtilis (BSR5).
[0031] FIG. 4 shows high performance size exclusion chromatography
(HPSEC) of MHR degraded by the rhamnogalacturonan hydrolase from
Bacillus halodurans KJ59 (BXA15).
[0032] FIG. 5 shows high performance size exclusion chromatography
(HPSEC) of saponified hairy regions from apples (MHR-S) degraded by
the rhamnogalacturonan hydrolase from Bacillus sp. AA 386
(BXR1).
[0033] FIG. 6 shows high performance size exclusion chromatography
(HPSEC) of MHR-S degraded by the rhamnogalacturonan hydrolase from
Bacillus licheniformis (BLR3).
[0034] FIG. 7 shows high performance size exclusion chromatography
(HPSEC) of MHR-S degraded by the rhamnogalacturonan hydrolase from
Bacillus subtilis (BSR5).
[0035] FIG. 8 shows high performance size exclusion chromatography
(HPSEC) of MHR-S degraded by the rhamnogalacturonan hydrolase from
Bacillus halodurans KJ59 (BXA15).
[0036] FIG. 9 shows high performance size exclusion chromatography
(HPSEC) of rhamnogalacturonan obtained from Megazyme (RG) degraded
by the rhamnogalacturonan hydrolase from Bacillus sp. AA 386
(BXR1).
[0037] FIG. 10 shows high performance size exclusion chromatography
(HPSEC) of RG degraded by the rhamnogalacturonan hydrolase from
Bacillus licheniformis (BLR3).
[0038] FIG. 11 shows high performance size exclusion chromatography
(HPSEC) of RG degraded by the rhamnogalacturonan hydrolase from
Bacillus halodurans KJ59 (BXA15).
[0039] FIG. 12 shows high performance size exclusion chromatography
(HPSEC) of saponified RG (RG-S) degraded by the rhamnogalacturonan
hydrolase from Bacillus sp. AA 386 (BXR1).
[0040] FIG. 13 shows high performance size exclusion chromatography
(HPSEC) of RG-S degraded by the rhamnogalacturonan hydrolase from
Bacillus licheniformis (BLR3).
[0041] FIG. 14 shows high performance size exclusion chromatography
(HPSEC) of RG-S degraded by the rhamnogalacturonan hydrolase from
Bacillus halodurans KJ59 (BXA15).
DEFINITIONS
[0042] Prior to discussing this invention in further detail, the
following terms will first be defined.quadrature.
[0043] The term "ortholog" (or "species homolog") denotes a
polypeptide or protein obtained from one species that has homology
to an analogous polypeptide or protein from a different
species.
[0044] The term "paralog" denotes a polypeptide or protein obtained
from a given species that has homology to a distinct polypeptide or
protein from that same species.
[0045] The term "expression vector" denotes a DNA molecule, linear
or circular, that comprises a segment encoding a polypeptide of
interest operably linked to additional segments that provide for
its transcription. Such additional segments may include promoter
and terminator sequences, and may optionally include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, and the like. Expression
vectors are generally derived from plasmid or viral DNA, or may
contain elements of both. The expression vector of the invention
may be any expression vector that is conveniently subjected to
recombinant DNA procedures, and the choice of vector will often
depend on the host cell into which the vector it is to be
introduced. Thus, the vector may be an autonomously replicating
vector, i.e. a vector which exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication,
e.g. a plasmid. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0046] The term "recombinant expressed" or "recombinantly
expressed" used herein in connection with expression of a
polypeptide or protein is defined according to the standard
definition in the art. Recombinantly expression of a protein is
generally performed by using an expression vector as described
immediately above.
[0047] The term "isolated", when applied to a polynucleotide
molecule, denotes that the polynucleotide has been removed from its
natural genetic milieu and is thus free of other extraneous or
unwanted coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985). The term "an isolated
polynucleotide" may alternatively be termed "a cloned
polynucleotide".
[0048] When applied to a protein/polypeptide, the term "isolated"
indicates that the protein is found in a condition other than its
native environment. In a preferred form, the isolated protein is
substantially free of other proteins, particularly other homologous
proteins (i.e. "homologous impurities" (see below)). It is
preferred to provide the protein in a greater than 40% pure form,
more preferably greater than 60% pure form.
[0049] Even more preferably it is preferred to provide the protein
in a highly purified form, i.e., greater than 80% pure, more
preferably greater than 95% pure, and even more preferably greater
than 99% pure, as determined by SDS-PAGE.
[0050] The term "isolated protein/polypeptide may alternatively be
termed "purified protein/polypeptide".
[0051] The term "homologous impurities" means any impurity (e.g.
another polypeptide than the polypeptide of the invention) which
originate from the homologous cell where the polypeptide of the
invention is originally obtained from.
[0052] The term "obtained from" as used herein in connection with a
specific microbial source, means that the polynucleotide and/or
polypeptide produced by the specific source, or by a cell in which
a gene from the source have been inserted.
[0053] The term "operably linked", when referring to DNA segments,
denotes that the segments are arranged so that they function in
concert for their intended purposes, e.g. transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0054] The term "polynucleotide" denotes a single- or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. Polynucleotides include RNA
and NA, and may be isolated from natural sources, synthesized in
vitro, or prepared from a combination of natural and synthetic
molecules.
[0055] The term "complements of polynucleotide molecules" denotes
polynucleotide molecules having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5'
CCCGTGCAT 3'.
[0056] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0057] The term "promoter" denotes a portion of a gene containing
DNA sequences that provide for the binding of RNA polymerase and
initiation of transcription. Promoter sequences are commonly, but
not always, found in the 5' non-coding regions of genes.
[0058] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger peptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0059] The term "pectin" denotes pectate, polygalacturonic acid,
and pectin which may be esterified to a higher or lower degree.
[0060] The term "hairy regions of pectins" denotes the highly
branched rhamnogalacturonan regions of pectins consisting of
repeating alfa-1,2-L-Rha-alfa-1,4-D-GalUA disaccharide units with
side chains mainly consisting of neutral oligosaccharides such as
arabinan, galactan and/or arabinogalactan and wherein the GalUA
residues may be acetyl esterified or methyl esterified.
[0061] The term "pectic substance" denotes pectin and hairy regions
of pectins.
[0062] The term "rhamnogalacturonase" denotes an enzyme capable of
degrading the rhamnogalacturonan backbone of hairy regions of
pectins.
[0063] The term "rhamnogalacturonan hydrolase" denotes an enzyme
belonging to the enzyme class of glycosyl hydrolases (EC 3.2.X.X
according to the IUB Enzyme Nomenclature). The rhamnogalaturonan
hydrolase enzyme exhibits activity towards rhamnogalacturonan
backbone of hairy regions of pectin.
[0064] The term "glycosyl hydrolase family" has been descibed
in:
[0065] 1. Henrissat, B. "A classification of glycosyl hydrolases
based of amino-acid sequence similarities." Biochem. J. 280:
309-316 (1991).
[0066] 2. Henrissat, B., Bairoch, A. "New families in the
classification of glycosyl hydrolases based on amino-acid sequence
similarities. Biochem. J. 293: 781-788 (1993).
[0067] 3. Henrissat, B., Bairoch, A. "Updating the sequence-based
classification of glycosyl hydrolases." Biochem. J. 316: 695-696
(1996).
[0068] 4. Davies, G., Henrissat, B. "Structures and mechanisms of
glycosyl hydrolases." Structure 3: 853-859 (1995).
[0069] Public available data from:
http://afmb.cnrs-mrs.fr/.about.pedro/CA- ZY/db.html
DETAILED DESCRIPTION OF THE INVENTION
[0070] How to Use a Sequence of the Invention to get Other Related
Sequences:
[0071] The disclosed sequence information herein relating to a
polynucleotide sequence encoding an enzyme of the invention can be
used as a tool to identify other homologous enzymes exhibiting the
same enzymatic activity. For instance, polymerase chain reaction
(PCR) can be used to amplify sequences encoding other homologous
enzymes from a variety of microbial sources, in particular of
different Bacillus species.
[0072] Assay for Activity Test
[0073] A polypeptide of the invention capable of degrading the
rhamnogalacturonan backbone of hairy regions of pectins may be
tested for this activity according to test procedures known in the
art, such as by applying a solution to be tested to 4 mm diameter
holes punched out in agar plates containing 0.5% AZCL potato
galactan (Megazyme, Ireland) and 0.5% AZCL debranched arabinan
(Megazyme), respectively.quadrature. The enzyme of the invention
shows activity on both substrates.
[0074] Polynucleotides:
[0075] Within preferred embodiments of the invention an isolated
polynucleotide of the invention will hybridize to similar sized
regions of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19,
respectively, or a sequence complementary thereto, under at least
medium stringency conditions.
[0076] In particular polynucleotides of the invention will
hybridize to a double-stranded DNA probe comprising a selected
sequence from the group shown in: SEQ ID NO: 1 from nucleotide 1 to
nucleotide 1863, SEQ ID NO:3 from nucleotide 1 to nucleotide 1863,
SEQ ID NO:5 from nucleotide 1 to nucleotide 1863, SEQ ID NO: 7 from
nucleotide 1 to nucleotide 1413, SEQ ID NO: 9 from nucleotide 1 to
nucleotide 512, SEQ ID NO: 11 from nucleotide 1 to nucleotide 336,
SEQ ID NO: 13 from nucleotide 1 to nucleotide 1965, SEQ ID NO: 15
from nucleotide 1 to nucleotide 1896, SEQ ID NO: 17 from nucleotide
1 to nucleotide 1168, and SEQ ID NO: 19 from nucleotide 1 to
nucleotide 507, under at least medium stringency conditions, but
preferably at high stringency conditions as described in detail
below.
[0077] Suitable experimental conditions for determining
hybridization at medium, or high stringency between a nucleotide
probe and a homologous DNA or RNA sequence involves presoaking of
the filter containing the DNA fragments or RNA to hybridize in
5.times. SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989)
for 10 min, and prehybridization of the filter in a solution of
5.times. SSC, 5.times. Denhardt's solution (Sambrook et al. 1989),
0.5% SDS and 100 .mu.g/ml of denatured sonicated salmon sperm DNA
(Sambrook et al. 1989), followed by hybridization in the same
solution containing a concentration of 10 ng/ml of a random-primed
(Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem.
132:6-13), 32P-dCTP-labeled (specific activity >1.times.10.sup.9
cpm/.mu.g) probe for 12 hours at ca. 45.degree. C. The filter is
then washed twice for 30 minutes in 2.times. SSC, 0.5% SDS at least
60.degree. C. (medium stringency), still more preferably at least
65.degree. C. (medium/high stringency), even more preferably at
least 70.degree. C. (high stringency), and even more preferably at
least 75.degree. C. (very high stringency).
[0078] Molecules to which the oligonucleotide probe hybridizes
under these conditions are detected using a x-ray film.
[0079] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for isolating DNA
and RNA are well known in the art. DNA and RNA encoding genes of
interest can be cloned in Gene Banks or DNA libraries by means of
methods known in the art.
[0080] Polynucleotides encoding polypeptides of the invention
capable of degrading rhamnogalacturonan backbones of hairy regions
of pectins, ie rhamnogalacturonases, are then identified and
isolated by, for example, hybridization or PCR.
[0081] The present invention further provides counterpart
polypeptides and polynucleotides from different bacterial strains
(orthologs or paralogs). Of particular interest are polypeptides
from gram-positive strains, including species of Bacillus such as
Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus
stearothermophilus, Bacillus agaradhaerens, Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans,
Bacillus halodurans, Bacillus lautus, Bacillus thuringiensis,
Bacillus clausii or Bacillus licheniformis; and polypeptides from
Thermoanaerobacter group, including species of
Caldicellulosiruptor; and polypeptides from Actinobacteria,
preferably from Actinomycetales, more preferably from
Streptomycetaceae, including species of Streptomyces, in particular
Streptomyces coelicolor; and polypeptides from Proteobacteria,
preferably from Myxobacteria, including species of Sorangiaceae,
especially Sorangium, for example Sorangium cellulosum.
[0082] Based on their findings of novel enzymes encoded by the DNA
sequences listed as SEQ ID NOS: 1 and 3 respectively, the inventors
have searched publicly available databases for genes highly
homologous therewith and have succeeded in identifying the DNA
sequence corresponding to the gene known as the YesW gene from
Bacillus subtilis, the "YesW" name implying to the skilled person
that this DNA sequence has an unknown open reading frame and does
not have any significant homology to any known gene, i.e. that the
function of the gene/the polypeptide possibly encoded by the gene
is unknown. The YesW gene is listed as SEQ ID NO:5 and the derived
amino acid sequence is listed as SEQ ID NO:6. The present inventors
have succeeded in cloning and expressing the YesW gene in E.coli as
well as in B. subtilis, cf. the examples below, and have
demonstrated the enzymatic activity of the expressed polypeptide.
In a similar manner, YesW type genes have been identified from
Streptomyces coelicolor and Sorangium cellulosum.
[0083] Species homologues of a polypeptide of the invention can be
cloned using information and compositions provided by the present
invention in combination with conventional cloning techniques. For
example, DNA can be cloned using chromosomal DNA obtained from a
cell type that expresses the protein. Suitable sources of DNA can
be identified by probing Southern or Northern blots with probes
designed from the sequences disclosed herein. A library is then
prepared from chromosomal DNA of a positive cell line. A DNA
encoding a polypeptide of the invention can then be isolated by a
variety of methods, such as by probing with a complete or partial
DNA sequence or gene, or with one or more sets of degenerate probes
based on the disclosed sequences. A DNA can also be cloned using
the polymerase chain reaction, or PCR (Mullis, U.S. Pat. No.
4,683,202), using primers designed from the sequences disclosed
herein. Within an additional method, the DNA library can be used to
transform or transfect host cells, and expression of the DNA of
interest can be detected with an antibody (monoclonal or
polyclonal) raised against the enzyme of the invention (e.g. BXR1,
BSR5, BLR3, BXA15), expressed and possibly purified as described in
the examples below or by a rhamnogalacturonase activity test or
another test relating to a polypeptide capable of degrading
rhamnogalacturonan backbone of hairy regions of pectins.
[0084] Polypeptides:
[0085] The sequence of amino acids no. 1-621 of SEQ ID No 2 is a
full length enzyme sequence. The sequence of amino acids no. 1-620
of SEQ ID No 4 is a full length enzyme sequence. The sequence of
amino acids no. 1-620 of SEQ ID No 6 is a full length enzyme
sequence. The sequence of amino acids no. 1-655 of SEQ ID No 14 is
a full length enzyme sequence. The sequence of amino acids no.
1-631 of SEQ ID No 16 is believed to be a full length enzyme
sequence. The sequence of amino acids no. 1-389 of SEQ ID No 18 is
a full length enzyme sequence. The sequence of amino acids no.
1-471 of SEQ ID No 8 is part of a full length enzyme sequence. The
sequence of amino acids no. 1-170 of SEQ ID No 10 is part of a full
length enzyme sequence. The sequence of amino acids no. 1-112 of
SEQ ID No 12 is part of a full length enzyme sequence. The sequence
of amino acids no. 1-169 of SEQ ID No 20 is part of a full length
enzyme sequence (corrected for reading frame skips).
[0086] The present invention also provides polypeptides that are
substantially homologous to the polypeptides of SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, 18 or 20 and their species homologs (paralogs or
orthologs). The term "substantially homologous" is used herein to
denote polypeptides having 45%, preferably at least 50%, more
preferably at least 55%, more preferably at least 60%, more
preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, preferably at least 80%, more preferably
at least 85%, and even more preferably at least 90%, sequence
identity to the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16, 18 or 20 or their orthologs or paralogs. Such polypeptides
will more preferably be at least 95% identical, and most preferably
98% or more identical to the sequence shown in SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, 18 or 20 or their orthologs or paralogs. Percent
sequence identity is determined by conventional methods, by means
of computer programs known in the art such as GAP provided in the
GCG program package (Wisconsin Package Version 9.1, Genetics
Computer Group (GCG), Madison, Wisc.) which is disclosed in
Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular
Biology, 48, 443-453, this citation is hereby incorporated by
reference in its entirety). GAP is used with the following settings
for polypeptide sequence comparison: The standard PAM table
blosum62 with a gap creation penalty of 12 and a gap extension
penalty of 4 was employed throughout.
[0087] Substantially homologous proteins and polypeptides are
characterized as having one or more amino acid substitutions,
deletions or additions. These changes are preferably of a minor
nature, that is conservative amino acid substitutions (see Table 2)
and other substitutions that do not significantly affect the
folding or activity of the protein or polypeptide; small deletions,
typically of one to about 30 amino acids; and small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue, a small linker peptide of up to about 20-25 residues, or a
small extension that facilitates purification (an affinity tag),
such as a poly-histidine tract, protein A (Nilsson et al., EMBO J.
4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991. See, in
general Ford et al., Protein Expression and Purification 2: 95-107,
1991, which is incorporated herein by reference. DNAs encoding
affinity tags are available from commercial suppliers (e.g.,
Pharmacia Biotech, Piscataway, N.J.; New England Biolabs, Beverly,
Mass.).
[0088] However, even though the changes described above preferably
are of a minor nature, such changes may also be of a larger nature
such as fusion of larger polypeptides of up to 300 amino acids or
more both as amino- or carboxyl-terminal extensions to the
polypeptide of the invention.
1TABLE 1 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serine threonine methionine
[0089] In addition to the 20 standard amino acids, non-standard
amino acids (such as 4-hydroxyproline, 6-N-methyl lysine,
2-aminoisobutyric acid, isovaline and a-methyl serine) may be
substituted for amino acid residues of a polypeptide according to
the invention. A limited number of non-conservative amino acids,
amino acids that are not encoded by the genetic code, and unnatural
amino acids may be substituted for amino acid residues. "Unnatural
amino acids" have been modified after protein synthesis, and/or
have a chemical structure in their side chain(s) different from
that of the standard amino acids. Unnatural amino acids can be
chemically synthesized, or preferably, are commercially available,
and include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and
3,3-dimethylproline.
[0090] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989).
In the latter technique, single alanine mutations are introduced at
every residue in the molecule, and the resultant mutant molecules
are tested for biological activity (i.e activity towards galactan
and arabinan) to identify amino acid residues that are critical to
the activity of the molecule. See also, Hilton et al., J. Biol.
Chem. 271:4699-4708, 1996. The active site of the enzyme or other
biological interaction can also be determined by physical analysis
of structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., Science 255:306-312,
1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et
al., FEBS Lett. 309:59-64, 1992. The identities of essential amino
acids can also be inferred from analysis of homologies with
polypeptides which are related to a polypeptide according to the
invention.
[0091] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis, recombination and/or shuffling
followed by a relevant screening procedure, such as those disclosed
by Reidhaar-Olson and Sauer (Science 241:53-57, 1988), Bowie and
Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989), WO95/17413,
or WO 95/22625. Briefly, these authors disclose methods for
simultaneously randomizing two or more positions in a polypeptide,
or recombination/shuffling of different mutations (WO95/17413,
WO95/22625), followed by selecting for functional a polypeptide,
and then sequencing the mutagenized polypeptides to determine the
spectrum of allowable substitutions at each position. Other methods
that can be used include phage display (e.g., Lowman et al.,
Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No.
5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed
mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA
7:127, 1988).
[0092] Mutagenesis/shuffling methods as disclosed above can be
combined with high-throughput, automated screening methods to
detect activity of cloned, mutagenized polypeptides in host cells.
Mutagenized DNA molecules that encode active polypeptides can be
recovered from the host cells and rapidly sequenced using modern
equipment. These methods allow the rapid determination of the
importance of individual amino acid residues in a polypeptide of
interest, and can be applied to polypeptides of unknown
structure.
[0093] Using the methods discussed above, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptides that
are substantially homologous to the peptides disclosed herein and
retain the enzymatic activity of the wild-type protein.
[0094] Identification of Other Novel Carbohydrases in these Two
Subfamilies
[0095] Two basic approaches can be used.
[0096] 1) Screening with assay plates (AZCL debranched arabinan and
AZCL potato galactan for example) other libraries of bacterial
species.
[0097] 2) This class of enzyme has a high level of sequence
identity at the amino acid level as shown in the following
alignment.
[0098] Multiple Sequence Alignment of Subfamily A Performed by
Clustal Analysis (DNAstar, Megalign ver. 3.1.7)
[0099] BXR1: Bacillus sp. AA386 (SEQ ID NO: 2)
[0100] BLR3: B. licheniformis (SEQ ID NO:4)
[0101] BSR5: B. subtilis (SEQ ID NO:6)
[0102] BXR9: B. halodurans C4538 (SEQ ID NO:14)
[0103] SCR6: Sorangium cellulosum (SEQ ID NO:8)
[0104] STR12: Streptomyces coelicolor (SEQ ID NO:16)
[0105] XXR7: Caldicellulosiruptor sp. (SEQ ID NO:10)
[0106] XXR13: Caldicellulosiruptor sp. (SEQ ID NO:12)
2 1 60 Blr3.pro
MR....RSCLMIRRRKRMFTAVTLLVLLVMGTSVCPV.....KAEGAAR.QMEALNRGLV
Bsr5.pro MR....RSCLMIRRRKRMFTAVTLLVLLVMGTSVCPV.....KAEGAAR.QMEALNR-
GLV bxr1.PRO
M..........FSKRLHHFWRV.MLGLVVVVSTIGSVFLPVSTASAAPR.QAEN- ISRGLV
Bxr9.pro M..............................................LR.Q-
KEQLDRGLV SCR6.pro
................................................- ............
Str12.pro VRHPHTRPHAPHPHRRRPRALAAALAAAGLLGAGLTTLAPDTAE-
AATAR.QVEALDRGVV Xxr13.pro
........................................- ....................
Xxr7.PRO MRK.........KKIYRSWLGIVVIILWVIYCVFNPY-
NLAIKNVKGAVSSQVEKLKRGLI 61 120 B1r3.pro
AVKTDGGIFVSWRFLGTENASVLFNVYRDG- QKLNAAPV.KTTNYVDKNGSAGSTYTVRAV
Bsr5.pro AVKTDGGIFVSWRFLGTENASVLFNVY-
RDGQKLNAAPV.KTTNYVDKNGSAGSTYTVRAV bxr1.PRO
AVKVSSGVFISWRLLGTEQLSTSF- NVYRNGTKVNAAPITNSTNLLDTAGTTSSTYTVRAV
Bxr9.pro
AVKRADGVFLSWRLLGTEHPLTVFHVYRDGEKITKAGLQEGTNFVDADGMTDSVYQIKAV
SCR6.pro
............................................................
Str12.pro
SVHTGDGNLVSWRWLGTDPDNVAFNVYRAGTKVNSSPVTGSTTYFHSGAPSHADYTVRAV
Xxr13.pro ........................................................-
.... Xxr7.PRO
AIKVNNGVYLTWRMFGSDPADIGFNIYRNGQKINQIPIQVSTNYLDTGGNTTS- KYFIRPV 121
180 Blr3.pro VNGTEQPASEKASVWAQPYHSVPLDKPAGGTTPKGESYTYSANDAS-
VGDVDGDGQYELIL Bsr5.pro
VNGTEQPASEKASVWAQPYHSVPLDKPAGGTTPKGESYTYSAN- DASVGDVDGDGQYELIL
bxr1.PRO VGGVEQPASPAVRVWANNYLDVPIQAPPGGRTPDGVNYTY-
SANDASIGDLDGDGEYEIVL Bxr9.pro
.AGKDEDMSNPVSVWDDEYLAIPLDKPEGGVTPDGVS- YEYTANDASVGDGDGQYEIIL
Str12.pro VNGTEQGDSVHAIQFRAGYKDVPISPPSGGTTPDG-
VSYTYEANDASVGDLDGDGALDLVL Xxr13.pro
...............................- .............................
Xxr7.PRO INGHEIENSEEVSVLPTNYIEIKLNRPP-
..TSPLGA..IYSPNDASVGDLDGDGEYEIVL 181 240 Blr3.pro
KWDPSNSKDNSQDGYTGDVLIDAYKLDGTKLWRINLGKNIRAGAHYTQFMVYDLDGDGKA
Bsr5.pro
KWDPSNSKDNSQDGYTGDVLIDAYKLDGTKLWRINLGKNIRAGAHYTQFMVYDLDGDGKA
bxr1.PRO
KWDPTNSKDNSQGGYTGNVYLDAYKLNGTRLWRIDLGRNIRAGAHYTQFLVYDFDGDGKA
Bxr9.pro KWDPTNSKDNSRSGYTGNVYLDAYKLDGTKLWRLDLGRNIRAGAHYSQFLVYDFDGN-
GRS SCR6.pro
KWDPSNLKDNSQAGRTGKTYLDAYSLEGERLWRIDLGVNIRAGAHYSPFLVYDL- DGDGKA
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..............................................- ..............
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...DYRNSSGYILDGPEYLTIFSGETGEALDTI SCR6.pro
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............................................................
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............................................................ 301
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..............................- .............................. 361
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............................................................
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............................................................ 481
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.....................................- .......................
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............... Xxr13.pro
.........................................- ...................
Xxr7.PRO ......................................-
......................
[0107] Multiple Sequence Alignment of Subfamily B Performed by
Clustal Analysis (DNAstar, Megalign ver. 3.1.7)
[0108] BXA1: B. halodurans KJ59 (SEQ ID NO: 18)
[0109] BLR3: B. agaradhaerens (SEQ ID NO: 20)
3 1 60 Bxr15.pro
MKLGMWFSGLILVVGLLVGGNEAKANEVVNARDFGATPGVATSQTN.ALHAAMRHFYDR
Bar16.pro RD...FWDRG............................PGVSGKAKRMPLHAAMRY-
FYDR 61 120 Bxa15.pro
GVQG.TVYIPAGTYSIDEALRFHSGVNIVGDGMGRTILKKTGNSNNYV- VGNPIMRGSN.N
Bar16.pro GVRGKTVYLPAGTYSVDSALRFHQGVNLVGDGVGRTIIKKVGSQ-
NNYVVGNPIFRGGTTN 121 180 Bxa15.pro
LNVTVSNLTIDADRTNRAQRGLGQVGGM..NLDADV- SNLTLERVEVRDATIGLLLRRLKN
Bar16.pro LNVTVSHITFDADRTNRASQGLGQVGGTGEQF-
TALVSNLTLEHIEVRDATIGLLVRRXR 181 240 Bxa15.pro
SVVRDSVIDNTTGHGIAFGHENHPI- GDVRNNLITGNRITNSTGGSGINLSRATYTTVTHN
Bar16.pro
SVISDSLIDRTSWHGIATGSE....................................... 241
300 Bxa15.pro
QVINDRQQDDSYGGIRIPNGGEHNTVEYNTIRNYPRGIFVLSGARHNQINHNTVIDSRIH
Bar16.pro
........................................................... 301 360
Bxa15.pro GVLIQADHNTLRENRIQQLNSSLNPESVVRIAPGSNNSILNNNIQAHSNFRN-
IGIRVTGD Bar16.pro
................................................- ........... 361
394 Bxa15.pro SNNNVIRNNRIGTQGTLVSIEGGRNNVNEGNVRQ Bar16.pro
..................................
[0110] Therefore three approaches can be used to take advantage of
this:
[0111] a) Design degenerate PCR primer sets deduced from consensus
amino acid sequences from the disclosed rhamnogalacturonases from
subfamily A or subfamily B. Perform PCR on DNA from bacterial
species, eg Bacillus, and determine which species give a specific
band of approximately the correct size. Clone and sequence or
sequence directly this PCR fragment. This sequence information can
be used to design specific primers facing outwards and one of
several hybrid PCR approaches may be used to obtain the rest of the
sequence (Sakamoto et al., 1997)). These methods are known by those
skilled in the art.
[0112] Accordingly, it is contemplated that the novel enzyme of the
present invention can be identified by amino acid consensus domains
common to all amino acid sequences in this class which are provided
herein (as seen in above alignment). Preferred consensus domains
can be selected from the following groups:
[0113] Subfamily A:
[0114] SANDAS
[0115] LKWDP(S or T)NSKDN
[0116] DAYKL(D or N)GT
[0117] NIRAGAHTQF(M or L)VYD(F or L)DGDGKAE
[0118] KTADGT
[0119] LSGPE(Y or F)LTV
[0120] YGNRVDRFLAG
[0121] YGNRVDRFLAGXAYLDG
[0122] AGQGNH(N or S)LS(I or V)ADVDGDGKDEII
[0123] AGQGNH(N or S)L(S or A) (I or V)ADVDGDGKDEII
[0124] LRIYTTT
[0125] YTLMHD
[0126] (Y or P)TLMHD(P or S)VYRL(S or G)IAWQN
[0127] VYRL(S or G)IAWQN
[0128] Subfamily B:
[0129] EVRDATIGLL
[0130] NNYVVGNPI
[0131] DADRTNRA
[0132] b) Cloned PCR fragment can also be labelled and used to
probe a genomic DNA library as either colony or phage. In this way
a full length clone can be obtained. Screening plasmid or phage
libraries is well known by those skilled in the art. Generally,
screening in these cases is done under high stringency to avoid
false positives. Screening is usually performed with hybridization
conditions as follows: 6.times. SSC and 68.theta.C for
hybridization of the probe to the filters. Then final washes at
0.2.times. SSC and 68.theta.C.
[0133] c) Heterologous probing of a genomic DNA library may also be
performed to clone genes homologous to rhamnogalactuonases of the
invention. DNA sequence homology appears to be quite high in this
family so this method is a generally useful one. Hybridization is
then made under low stringency the lowest range normally used being
6.times. SCC and 42.theta.C for the hybridization, and washing with
2.times. SSC and typically 42.theta.C. Additional washes may be
necessary at higher stringency after monitoring with X-ray
film.
[0134] Protein Production:
[0135] The polypeptides of the present invention, including
full-length proteins, fragments thereof and fusion proteins, can be
produced in genetically engineered host cells according to
conventional techniques. Suitable host cells are those cell types
that can be transformed or transfected with exogenous DNA and grown
in culture, and include bacteria, fungal cells, and cultured higher
eukaryotic cells. Bacterial cells, particularly cultured cells of
gram-positive organisms, are preferred. Gram-positive cells from
the genus of Bacillus are especially preferred, such as Bacillus
subtilis, Bacillus lentus, Bacillus brevis, Bacillus
stearothermophilus, Bacillus agaradhaerens, Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulars,
Bacillus halodurans, Bacillus lautus, Bacillus thuringiensis,
Bacillus clausii, or in particular Bacillus licheniformis.
[0136] Techniques for manipulating cloned DNA molecules and
introducing exogenous DNA into a variety of host cells are
disclosed by Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; Ausubel et al. (eds.), Current Protocols in
Molecular Biology, John Wiley and Sons, Inc., NY, 1987; and
(Bacillus subtilis and Other Gram-Positive Bacteria, Sonensheim et
al., 1993, American Society for Microbiology, Washington D.C.),
which are incorporated herein by reference.
[0137] In general, a DNA sequence encoding an enzyme of the present
invention is operably linked to other genetic elements required for
its expression, generally including a transcription promoter and
terminator within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0138] To direct a polypeptide into the secretory pathway of a host
cell, a secretory signal sequence (also known as a leader sequence,
prepro sequence or pre sequence) is provided in the expression
vector. The secretory signal sequence may be that of the
polypeptide, or may be derived from another secreted protein or
synthesized de novo. Numerous suitable secretory signal sequences
are known in the art and reference is made to (Bacillus subtilis
and Other Gram-Positive Bacteria, Sonensheim et al., 1993, American
Society for Microbiology, Washington D.C.; and Cutting, S. M.(eds.)
"Molecular Biological Methods for Bacillus". John Wiley and Sons,
1990) for further description of suitable secretory signal
sequences especially for secretion in a Bacillus host cell. The
secretory signal sequence is joined to the DNA sequence in the
correct reading frame. Secretory signal sequences are commonly
positioned 5' to the DNA sequence encoding the polypeptide of
interest, although certain signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.
5,143,830).
[0139] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell.
[0140] Protein Isolation
[0141] When the expressed recombinant polypeptide is secreted the
polypeptide may be purified from the growth media. Preferably the
expression host cells are removed from the media before
purification of the polypeptide (e.g. by centrifugation).
[0142] When the expressed recombinant polypeptide is not secreted
from the host cell, the host cell are preferably disrupted and the
polypeptide released into an aqueous "extract" which is the first
stage of such purification techniques. Preferably the expression
host cells are removed from the media before the cell disruption
(e.g. by centrifugation).
[0143] The cell disruption may be performed by conventional
techniques such as by lysozyme digestion or by forcing the cells
through high pressure. See (Robert K. Scobes, Protein Purification,
Second edition, Springer-Verlag) for further description of such
cell disruption techniques.
[0144] Whether or not the expressed recombinant polypeptides (or
chimeric polypeptides) is secreted or not it can be purified using
fractionation and/or conventional purification methods and
media.
[0145] Ammonium sulfate precipitation and acid or chaotrope
extraction may be used for fractionation of samples. Exemplary
purification steps may include hydroxyapatite, size exclusion, FPLC
and reverse-phase high performance liquid chromatography. Suitable
anion exchange media include derivatized dextrans, agarose,
cellulose, polyacrylamide, specialty silicas, and the like. PEI,
DEAE, QAE and Q derivatives are preferred, with DEAE Fast-Flow
Sepharose (Pharmacia, Piscataway, N.J.) being particularly
preferred. Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers.
[0146] Selection of a particular method is a matter of routine
design and is determined in part by the properties of the chosen
support. See, for example, Affinity Chromatography: Principles
& Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,
1988.
[0147] Polypeptides of the invention or fragments thereof may also
be prepared through chemical synthesis. Polypeptides of the
invention may be monomers or multimers; glycosylated or
non-glycosylated; pegylated or non-pegylated; and may or may not
include an initial methionine amino acid residue.
[0148] In the present context, the term "enzyme preparation" is
intended to mean either be a conventional enzymatic fermentation
product, possibly isolated and purified, from a single species of a
microorganism, such preparation usually comprising a number of
different enzymatic activities; or a mixture of monocomponent
enzymes, preferably enzymes derived from bacterial or fungal
species by using conventional recombinant techniques, which enzymes
have been fermented and possibly isolated and purified separately
and which may originate from different species, preferably fungal
or bacterial species; or the fermentation product of a
microorganism which acts as a host cell for expression of a
recombinant enzyme of the present invention, but which
microorganism simultaneously produces other enzymes, e.g.
galactanases, arabinases, proteases, or cellulases, being naturally
occurring fermentation products of the microorganism, i.e. the
enzyme complex conventionally produced by the corresponding
naturally occurring microorganism.
[0149] The enzyme preparation of the invention may further comprise
one or more enzymes selected from the group consisting of
proteases, cellulases (endo-E-1,4-glucanases), E-glucanases
(endo-E-1,3(4)-glucanases), lipases, cutinases, peroxidases,
laccases, amylases, glucoamylases, pectinases, reductases,
oxidases, phenoloxidases, ligninases, pullulanases, arabinanases,
galactanases, hemicellulases, mannanases, xyloglucanases,
xylanases, pectin acetyl esterases, rhamnogalacturonan acetyl
esterases, polygalacturonases, rhamnogalacturonases, pectin lyases,
pectate lyases, pectin methylesterases, cellobiohydrolases,
transglutaminases; or mixtures thereof. In a preferred embodiment,
one or more or all enzymes in the preparation is produced by using
recombinant techniques, i.e. the enzyme(s) is/are mono-component
enzyme(s) which is/are mixed with the other enzyme(s) to form an
enzyme preparation with the desired enzyme blend.
[0150] In another aspect, the present invention also relates to a
method of producing the enzyme preparation of the invention, the
method comprising culturing a microorganism capable of producing
the enzyme of the invention under conditions permitting the
production of the enzyme, and recovering the enzyme from the
culture. Culturing may be carried out using conventional
fermentation techniques, e.g. culturing in shake flasks or
fermenters with agitation to ensure sufficient aeration on a growth
medium inducing production of the enzyme of the present invention.
The growth medium may contain a conventional N-source such as
peptone, yeast extract or casamino acids, a reduced amount of a
conventional C-source such as dextrose or sucrose, and an inducer
such as hairy regions from pectin or composite plant substrates
such as apple pulp. The recovery may be carried out using
conventional techniques, e.g. separation of bio-mass and
supernatant by centrifugation or filtration, recovery of the
supernatant or disruption of cells if the enzyme of interest is
intracellular, perhaps followed by further purification as
described in EP 0 406 314 or by crystallization as described in WO
97/15660.
[0151] Examples of useful bacteria producing the enzyme or the
enzyme preparation of the invention are Gram positive bacteria,
preferably from the Bacillus/Lactobacillus subdivision, preferably
a strain from the genus Bacillus, especially a strain of Bacillus
licheniformis, Bacillus halodurans, Bacillus agaradhaerens, or
Bacillus subtilis. ATCC 14580 is the type strain of Bacillus
licheniformis. DSM 8721 is the type strain of Bacillus
agaradhaerens.
[0152] In yet another aspect, the present invention relates to an
isolated enzyme capable of degrading rhamnogalacturonan backbones
of hairy regions of pectins having the properties described above
and which is free from homologous impurities, and is produced using
conventional recombinant techniques.
[0153] Use in the Detergent Industry
[0154] In further aspects, the present invention relates to a
detergent composition comprising the enzyme or enzyme preparation
of the invention, and to a process for machine treatment of fabrics
comprising treating fabric during a washing cycle of a machine
washing process with a washing solution containing the enzyme or
enzyme preparation of the invention.
[0155] Typically, the detergent composition of the invention
comprises conventional ingredients such as surfactants (anionic,
nonionic, zwitterionic, amphoteric), builders, and other
ingredients, e.g. as described in WO 97/01629 which is hereby
incorporated by reference.
[0156] Use in the Textile and Cellulosic Fiber Processing
Industries
[0157] The enzyme of the present invention can be used in
combination with other carbohydrate degrading enzymes (for instance
galactanase, arabinanase, xyloglucanase, pectinase) for
biopreparation of fibers or for cleaning of fibers in combination
with detergents. Cotton fibers consist of a primary cell wall layer
containing pectin and a secondary layer containing mainly
cellulose. Under cotton preparation or cotton refining part of the
primary cell wall will be removed. The present invention relates to
either help during cotton refining by removal of hairy regions of
the primary cell wall. Or during cleaning of the cotton to remove
residual pectic substances and prevent graying of the textile.
[0158] In the present context, the term "cellulosic material" is
intended to mean fibers, sewn and unsewn fabrics, including knits,
wovens, denims, yarns, and toweling, made from cotton, cotton
blends or natural or manmade cellulosics (e.g. originating from
xylan-containing cellulose fibers such as from wood pulp) or blends
thereof. Examples of blends are blends of cotton or rayon/viscose
with one or more companion material such as wool, synthetic fibers
(e.g. polyamide fibers, acrylic fibers, polyester fibers, polyvinyl
alcohol fibers, polyvinyl chloride fibers, polyvinylidene chloride
fibers, polyurethane fibers, polyurea fibers, aramid fibers), and
cellulose-containing fibers (e.g. rayon/viscose, ramie, hemp,
flax/linen, jute, cellulose acetate fibers, lyocell).
[0159] The preparation of the present invention is useful in the
cellulosic fiber processing industry for the pretreatment or
retting of fibers from hemp, flax or linen.
[0160] The processing of cellulosic material for the textile
industry, as for example cotton fiber, into a material ready for
garment manufacture involves several steps: spinning of the fiber
into a yarn; construction of woven or knit fabric from the yarn and
subsequent preparation, dyeing and finishing operations. Woven
goods are constructed by weaving a filling yarn between a series of
warp yarns; the yarns could be two different types. Knitted goods
are constructed by forming a network of interlocking loops from one
continuous length of yarn. The cellulosic fibers can also be used
for non-woven fabric.
[0161] The preparation process prepares the textile for the proper
response in dyeing operations. The sub-steps involved in
preparation are desizing (for woven goods), scouring and bleaching.
A one step combined scour/bleach process is also used by the
industry. Although preparation processes are most commonly employed
in the fabric state; scouring, bleaching and dyeing operations can
also be done at the fiber or yarn stage.
[0162] The processing regime can be either batch or continuous with
the fabric being contacted by the liquid processing stream in open
width or rope form. Continuous operations generally use a saturator
whereby an approximate equal weight of chemical bath per weight of
fabric is applied to the fabric, followed by a heated dwell chamber
where the chemical reaction takes place. A washing section then
prepares the fabric for the next processing step. Batch processing
generally takes place in one processing bath whereby the fabric is
contacted with approximately 8-15 times its weight in chemical
bath. After a reaction period, the chemicals are drained, fabric
rinsed and the next chemical is applied. Discontinuous pad-batch
processing involves a saturator whereby an approximate equal weight
of chemical bath per weight of fabric is applied to the fabric,
followed by a dwell period which in the case of cold pad-batch
might be one or more days.
[0163] Woven goods are the prevalent form of textile fabric
construction. The weaving process demands a "sizing" of the warp
yarn to protect it from abrasion. Starch, polyvinyl alcohol (PVA),
carboxymethyl cellulose, waxes and acrylic binders are examples of
typical sizing chemicals used because of availability and cost. The
size must be removed after the weaving process as the first step in
preparing the woven goods. The sized fabric in either rope or open
width form is brought in contact with the processing liquid
containing the desizing agents. The desizing agent employed depends
upon the type of size to be removed. For PVA sizes, hot water or
oxidative processes are often used. The most common sizing agent
for cotton fabric is based upon starch. Therefore most often, woven
cotton fabrics are desized by a combination of hot water, the
enzyme A-amylase to hydrolyze the starch and a wetting agent or
surfactant. The cellulosic material is allowed to stand with the
desizing chemicals for a "holding period" sufficiently long to
accomplish the desizing. The holding period is dependent upon the
type of processing regime and the temperature and can vary from 15
minutes to 2 hours, or in some cases, several days. Typically, the
desizing chemicals are applied in a saturator bath which generally
ranges from about 15.theta.C to about 55.theta.C. The fabric is
then held in equipment such as a "J-box" which provides sufficient
heat, usually between about 55.theta.C and about 100.theta.C, to
enhance the activity of the desizing agents. The chemicals,
including the removed sizing agents, are washed away from the
fabric after the termination of the holding period.
[0164] In order to ensure a high whiteness or a good wettability
and resulting dyeability, the size chemicals and other applied
chemicals must be thoroughly removed. It is generally believed that
an efficient desizing is of crucial importance to the following
preparation processes: scouring and bleaching.
[0165] The scouring process removes much of the non-cellulosic
compounds naturally found in cotton. In addition to the natural
non-cellulosic impurities, scouring can remove dirt, soils and
residual manufacturing introduced materials such as spinning,
coning or slashing lubricants. The scouring process employs sodium
hydroxide or related causticizing agents such as sodium carbonate,
potassium hydroxide or mixtures thereof. Generally an alkali stable
surfactant is added to the process to enhance solubilization of
hydrophobic compounds and/or prevent their redeposition back on the
fabric. The treatment is generally at a high temperature,
80.theta.C-100.theta.C, employing strongly alkaline solutions, pH
13-14, of the scouring agent. Due to the non-specific nature of
chemical processes not only are the impurities but the cellulose
itself is attacked, leading to damages in strength or other
desirable fabric properties. The softness of the cellulosic fabric
is a function of residual natural cotton waxes. The non-specific
nature of the high temperature strongly alkaline scouring process
cannot discriminate between the desirable natural cotton lubricants
and the manufacturing introduced lubricants. Furthermore, the
conventional scouring process can cause environmental problems due
to the highly alkaline effluent from these processes. The scouring
stage prepares the fabric for the optimal response in bleaching. An
inadequately scoured fabric will need a higher level of bleach
chemical in the subsequent bleaching stages.
[0166] The bleaching step decolorizes the natural cotton pigments
and removes any residual natural woody cotton trash components not
completely removed during ginning, carding or scouring. The main
process in use today is an alkaline hydrogen peroxide bleach. In
many cases, especially when a very high whiteness is not needed,
bleaching can be combined with scouring.
[0167] It is contemplated that the scouring step can be carried out
using the enzyme or enzyme preparation of the present invention in
combination with a few other enzyme activities at a temperature of
about 5.theta.C-80.theta.C and a pH of about 7-11, thus
substituting or supplementing the highly causticizing agents.
[0168] Degradation or Modification of Plant Material
[0169] The enzyme or enzyme preparation according to the invention
is preferably used as an agent for degradation or modification of
plant cell walls or any pectin-containing material originating from
plant cells walls due to the high plant cell wall degrading
activity of the enzyme of the invention.
[0170] The enzyme of the present invention can be used in
combination with other pectinolytic or hemicellulytic enzymes to
degrade hairy regions of pectins.
[0171] The enzyme of the present invention may be used alone or
together with other enzymes like glucanases, pectinases and/or
hemicellulases to improve the extraction of oil from oil-rich plant
material, like soy-bean oil from soy-beans, olive-oil from olives
or rapeseed-oil from rape-seed or sunflower oil from sunflower.
[0172] The enzyme of the present invention may be used for
separation of components of plant cell materials. of particular
interest is the separation of sugar or starch rich plant material
into components of considerable commercial interest (like sucrose
from sugar beet or starch from potato) and components of low
interest (like pulp or hull fractions). Also, of particular
interest is the separation of protein-rich or oil-rich crops into
valuable protein and oil and invaluable hull fractions, The
separation process may be performed by use of methods known in the
art.
[0173] The enzyme of the invention may also be used in the
preparation of fruit or vegetable juice in order to increase yield,
and in the enzymatic hydrolysis of various plant cell wall-derived
materials or waste materials, e.g. from wine or juice production,
or agricultural residues such as vegetable hulls, bean hulls, sugar
beet pulp, olive pulp, potato pulp, and the like.
[0174] The plant material may be degraded in order to improve
different kinds of processing, facilitate purification or
extraction like purification of pectins from citrus, improve the
feed value, decrease the water binding capacity, improve the
degradability in waste water plants, improve the conversion of
plant material to ensilage, etc.
[0175] By means of an enzyme preparation of the invention it is
possible to regulate the consistency and appearance of processed
fruit or vegetables. The consistency and appearance has been shown
to be a product of the actual combination of enzymes used for
processing, i.e. the specificity of the enzymes with which the
enzyme of the invention is combined. Examples include the
production of clear juice e.g. from apples, pears or berries; cloud
stable juice e.g. from apples, pears, berries, citrus or tomatoes;
and purees e.g. from carrots and tomatoes.
[0176] The enzyme of the invention may be used in modifying the
viscosity of plant cell wall derived material. The viscosity
reduction may be obtained by treating the pectin containing plant
material with an enzyme preparation of the invention under suitable
conditions for full or partial degradation of the pectin containing
material.
[0177] The enzyme can be used e.g. in combination with other
enzymes for the removal of pectic substances from plant fibres.
This removal is essential e.g. in the production of textile fibres
or other cellulosic materials. For this purpose plant fibre
material is treated with a suitable amount of the enzyme of the
invention under suitable conditions for obtaining full or partial
degradation of pectic substances associated with the plant fibre
material.
[0178] Animal Feed Additive
[0179] The enzyme of the present invention may be used for
modification of animal feed and may exert their effect either in
vitro (by modifying components of the feed) or in vivo. The enzyme
is particularly suited for addition to animal feed compositions
containing high amounts of pectic substances, e.g. feed containing
plant material from soy bean, rape seed, lupin etc. When added to
the feed the enzyme may significantly improve the in vivo
break-down of plant cell wall material, whereby a better
utilization of the plant nutrients by the animal is achieved.
Thereby, the growth rate and/or feed conversion ratio (i.e. the
weight of ingested feed relative to weight gain) of the animal is
improved. Also, by the degradation of pectin the enzyme may improve
the digestibility and uptake of non-carbohydrate feed constituents
such as protein, fat and minerals.
[0180] For further description reference is made to PCT/DK
96/00443.
[0181] Wine and Juice Processing
[0182] The enzyme or enzyme preparation of the invention may be
used for de-pectinization and viscosity reduction in vegetable or
fruit juice, especially in apple or pear juice. This may be
accomplished by treating the fruit or vegetable juice with an
enzyme preparation of the invention in an amount effective for
degrading pectin-containing material contained in the fruit or
vegetable juice.
[0183] The enzyme or enzyme preparation may be used in the
treatment of mash from fruits and vegetables in order to improve
the extractability or degradability of the mash. For instance, the
enzyme preparation may be used in the treatment of mash from apples
and pears for juice production, and in the mash treatment of grapes
for wine production.
[0184] Materials and Methods
[0185] Deposited organisms and donor strains:
[0186] E.coli (BLR3) comprises the plasmid containing the DNA from
B.licheniformis ATCC 14580 encoding the enzyme of the invention
(represented by SEQ ID NO 3) and is deposited as DSM 12122 on Apr.
24, 1998.
[0187] E. coli (clone BXR1) comprises the plasmid containing DNA
from Bacillus. sp. AA386 encoding the enzyme of the invention
(represented by SEQ ID NO 1) and is deposited as DSM 12123 on Apr.
24, 1998.
[0188] E. coli (clone BXR9) comprises the plasmid containing DNA
from B. halodurans C4538 encoding the enzyme of the invention
(represented by SEQ ID NO 13) and is deposited as DSM 12124 on Apr.
24, 1998.
[0189] E. coli (clone BXA15) comprises the plasmid containing DNA
from B. halodurans KJ59 encoding the enzyme of the invention
(represented by SEQ ID NO 17) and is deposited as DSM 12202 on May
29, 1998.
[0190] E. coli (clone XXR7) comprises the plasmid containing DNA
from Caldocellulosiruptor sp. 124 encoding the enzyme of the
invention (represented by partial SEQ ID NO 9) and is deposited as
DSM 12405 on Sep. 8, 1998.
[0191] All of the above deposits were made according to the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure at the Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg
1b, D-38124 Braunschweig, Federal Republic of Germany.
[0192] The donor strain Bacillus licheniformis is publicly
available, for example from the deposit ATCC 14580.
[0193] The donor strain Bacillus agaradhaerens is publicly
available, for example from the type strain deposit DSM 8721.
[0194] The donor strain Sorangium cellulosum is disclosed in U.S.
Pat. No. 5,716,849 which is hereby incorporated by reference in its
entirety. The YesW homolog gene of Sorangium cellulosum disclosed
in this U.S. patent is incomplete in that it lacks about 170 amino
acids from the N terminus; a subsequence is specifically disclosed
in this U.S. patent but with unknown functionality. The partial DNA
sequence of SEQ ID NO:7 is a sequence which has not been cloned by
the present inventors but which is believed to encode for a
rhamnogalacturonase of the present invention, since it shows
sequence similarity to the enzymes identified by the present
inventors.
[0195] The donor strain Streptomyces coelicolor comprises a YesW
homolog gene which was submitted on Sep. 4, 1998 to the
EMBL/GeneBank/DDBJ databases (Streptomyces coelicolor sequencing
project, Sanger Centre, Wellcome Trust Genome Campus, Hinxton,
Cambridge CB10 1SA, E-mail: barrell@sanger.ac.uk Cosmids supplied
by Prof. David A. Hopwood, [3] John Innes Centre, Norwich Research
Park, Colney, Norwich, Norfolk NR4 7UH, UK) but with hitherto
unknown functionality (see SEQ ID NO:15 of the sequences listing
herein).
[0196] The donor strain Bacillus subtilis comprises a YesW homolog
gene available in the databases (TREMBL 031527; GeneBank Z99107)
but with hitherto unknown functionality (see SEQ ID NO:5 of the
sequences listing herein).
[0197] Other Strains
[0198] E. coli strains:
[0199] Cells of E. coli SJ2 (Diderichsen, B., Wedsted, U.,
Hedegaard, L., Jensen, B. R., Sj.o slashed.holm, C. (1990) Cloning
of alde, which encodes alpha-acetolactate decarboxylase, an
exoenzyme from Bacillus brevis. J. Bacteriol., 172, 4315-4321),
were prepared for and transformed by electroporation using a Gene
Pulser.TM. electroporator from BIO-RAD as described by the
supplier. XL1-Blue MRF.sup.- and XLOLR E. coli strains were
provided by Stratagene inc. (USA) and used according to the
manufacturer's instructions.
[0200] B.subtilis strains:
[0201] DN1885. (Diderichsen, B., Wedsted, U., Hedegaard, L.,
Jensen, B. R., Sj.o slashed.holm, C. (1990) Cloning of aldb, which
encodes alpha-acetolactate decarboxylase, an exoenzyme from
Bacillus brevis. J. Bacteriol., 172, 4315-4321). PL1801 (B.subtilis
DN1885 where the two major proteases have been inactivated).
[0202] Competent cells were prepared and transformed as described
by Yasbin, R. E., Wilson, G. A. and Young, F. E. (1975)
Transformation and transfection in lysogenic strains of Bacillus
subtilis evidence for selective induction of prophage in competent
cells. J. Bacteriol, 121:296-304.
[0203] Plasmids
[0204] pSJ1678: See International Patent Application published as
WO 94/19454 which is hereby incorporated by reference in its
entirety.
[0205] PUC19: See publication Yanisch-Perron et al., (1985) Gene
33:103-109 which is hereby incorporated by reference in its
entirety).
[0206] pBK-CAMV: Stratagene inc. La Jolla Calif., USA.
[0207] Bacteriophage ZAP Express: Stratagene inc. La Jolla Calif.,
USA.
[0208] pMOL944: This plasmid is a pUB110 derivative essentially
containing elements making the plasmid propagatable in Bacillus
subtilis, kanamycin resistance gene and having a strong promoter
and signal peptide cloned from the amyL gene of B.licheniformis
ATCC14580. The signal peptide contains a SacII site making it
convenient to clone the DNA encoding the mature part of a protein
in-fusion with the signal peptide. This results in the expression
of a Pre-protein which is directed towards the exterior of the
cell.
[0209] The plasmid was constructed by means of ordinary genetic
engineering and is briefly described in the following.
[0210] Construction of pMOL944:
[0211] The pUB110 plasmid (McKenzie, T. et al., 1986, Plasmid
15:93-103) was digested with the unique restriction enzyme NciI. A
PCR fragment amplified from the amyL promoter encoded on the
plasmid pDN1981 (P. L. J.o slashed.rgensen et al.,1990, Gene, 96,
p37-41.) was digested with NciI and inserted in the NciI digested
pUB110 to give the plasmid pSJ2624.
[0212] The two PCR primers used have the following sequences:
[0213] # LWN5494
5'-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC-3'
[0214] # LWN5495 5'-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAA
TGAGGCAGCAAGAAGAT-3'
[0215] The primer #LWN5494 inserts a NotI site in the plasmid.
[0216] The plasmid pSJ2624 was then digested with SacI and NotI and
a new PCR fragment amplified on amyL promoter encoded on the
pDN1981 was digested with SacI and NotI and this DNA fragment was
inserted in the SacI-NotI digested pSJ2624 to give the plasmid
pSJ2670.
[0217] This cloning replaces the first amyL promoter cloning with
the same promoter but in the opposite direction. The two primers
used for PCR amplification have the following sequences:
[0218] #LWN5938 5'-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATG
AGGCAGCAAGAAGAT-3'
[0219] #LWN5939 5'-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC-3'
[0220] The plasmid pSJ2670 was digested with the restriction
enzymes PstI and BclI and a PCR fragment amplified from a cloned
DNA sequence encoding the alkaline amylase SP722 (disclosed in
International Patent Application published as WO 95/26397 which is
hereby incorporated by reference in its entirety) was digested with
PstI and BclI and inserted to give the plasmid pMOL944. The two
primers used for PCR amplification have the following sequence:
[0221] #LWN7864 5'-AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3'
[0222] #LWN7901 5'-AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG-3'
[0223] The primer #LWN7901 inserts a SacII site in the plasmid.
[0224] Determination of Rhamnogalacturonase Activity
[0225] Activity was determined by the release of blue color after
incubation at 40.theta.C with a 0.2% slurry of AZCL-substrate on an
Eppendorf thermomixer for 20 minutes followed by centrifugation and
determination by spectroscopy at 620 nm.
[0226] Substrates:
[0227] AZCL-potato galactan (Megazyme). The manufacturer Megazyme
does not describe the composition of this substrate but the soluble
potato galactan has the following composition: Galactose:
Arabinose: Rhamnose: Galacturonic acid=91:2:1.7:0.35. This
indicates that the substrate contains rhamnogalacturonan.
[0228] AZCL debranced arabinan from Megazyme. The composition of
the soluble arabinan from sugar beets is Arabinose: Galactose:
Rhamnose: Galacturonic acid=88:3:2:7. This indicates that the
substrate contains rhamnogalacturonan.
[0229] Media
[0230] TY (as described in Ausubel, F. M. et al. (eds.) "Current
protocols in Molecular Biology". John Wiley and Sons, 1995). LB
agar (as described in Ausubel, F. M. et al. (eds.) "Current
protocols in Molecular Biology". John Wiley and Sons, 1995).
[0231] BPX media is described in EP 0 506 780 (WO 91/09129).
[0232] LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M
potassium phosphate, pH 7.0
[0233] The following examples illustrate the invention.
EXAMPLE 1
[0234] Cloning of Rhamnogalacturonase Encoding Genes from Bacillus
Species and from Caldicellulosiruptor sp.
[0235] Genomic DNA Preparation
[0236] The strains B. licheniformis, ATCC 14580, and Bacillus sp.
AA386 respectively, were propagated in liquid TY medium. After 16
hours incubation at 30.degree. C. and 300 rpm, the cells were
harvested, and genomic DNA isolated by the method described by
Pitcher et al. (1989). The alkalophilic strains B. halodurans KJ59
and C4538, was grown in TY with pH adjusted to approximately pH 9.7
by the addition of 50 ml of 1 M Sodium-Sesquicarbonat per 500 ml
TY. After 24 hours incubation at 30.degree. C. and 300 rpm, the
cells were harvested, and genomic DNA was isolated by the method
described by Pitcher et al. (1989).
[0237] Caldicellulosiruptor sp. 124 was grown on DSMZ medium 640
according to the described method for growing Caldicellulosiruptor
saccharolyticus (Rainey, F. A., Donnison, A. M., Janssen, P. H.,
Saul, D., Rodrigo, A., Bergquist, P. L., Daniel, R. M.,
Stackebrandt, E., Morgan, H. W., 1994, Description of
Caldicellulosiruptor saccharolyticus gen. nov., sp. nov.: an
obligately anaerobic, extremely thermophilic, cellulolytic
bacterium. FEMS Microbiol. Lett.
[0238] 120:263-266). Cells were harvested and genomic DNA isolated
by the method described by Pitcher et al. (1989).
[0239] Genomic Library Construction
[0240] Libraries for B. licheniformis, ATCC 14580, and Bacillus sp.
AA386, and Bacillus halodurans C4538
[0241] Genomic DNA was partially digested with restriction enzyme
Sau3A, and size-fractionated by electrophoresis on a 0.7% agarose
gel. Fragments between 2 and 10 kb in size was isolated by
electrophoresis onto DEAE-cellulose paper (Dretzen et al.
(1981))
[0242] Isolated DNA fragments were ligated to BamHI digested
pSJ1678 plasmid DNA, and the ligation mixture was used to transform
E. coli SJ2.
[0243] Libraries for Bacillus halodurans KJ59 and
Caldicellulosiruptor sp. 124
[0244] The Bacillus halodurans KJ59 and the Caldicellulosiruptor
sp. 124 libraries were screened as mass excised plasmid versions of
the ZAP express phage libraries. The ZAP Express cloning kit used
was with BamHI digested and dephosphorylated arms from Stratagene.
Genomic DNA was isolated by the method of Pitcher et al., 1989.
Isolated DNA was partially digested with Sau3A and size
fractionated on a 1% agarose gel. DNA was excised from the agarose
gel between 2 and 6 Kb and purified using Qiaspin DNA fragment
purification procedure (Qiagen GmBH). 100 ng of purified,
fractionated DNA was ligated with 1 ug of BamHI dephosphorylated
ZAPexpress vector arms (4 degrees overnight). Ligation reaction was
packaged directly with GigaPackIII Gold according to the
manufacturers instructions (Stratagene). Phage libraries were
titered with XL1blue mrf.sup.- (Stratagene). Mass excised libraries
were made of the phage libraries according to the manufacturers
instructions. The excised plasmids were screened in XLOLR cells
(Stratagene) by adding kanamycin (50 ug/ml) to the selection medium
described below instead of chloramphenicol.
[0245] Identification of Rhamnogalacturonase Positive Clones
[0246] The B. licheniformis, ATCC 14580, Bacillus sp. AA386,
Bacillus halodurans C4538 and Bacillus halodurans KJ59 DNA
libraries in E. coli, constructed as described above, were screened
on LB agar plates containing 0.1% AZCL-debranched arabinan
(Megazyme) and 9 .mu.g/ml Chloramphenicol or 50 .mu.g/ml kanamycin
and incubated overnight at 37.degree. C. The libraries were also
screened on LB agar plates containing 0.1% AZCL-Galactan (potato,
Megazyme). Clones expressing rhamnogalacturonase activity appeared
with blue diffusion halos on both indicator plates. The plasmids of
these positive clones were isolated by Qiagen plasmid spin preps on
1 ml of overnight culture broth (cells incubated at 37.degree. C.
in LB with 9 .mu.g/ml Chloramphenicol or 50 .mu.g/ml kanamycin and
shaking at 250 rpm).
[0247] The positive clones were further characterised by DNA
sequencing of the cloned genomic DNA fragments.
[0248] Identification of Rhamnogalacturonase Gene Fragments from
Caldicellulosiruptor sp. I24.
[0249] Partial sequences with similarity to rhamnogalacturonase
genes of the invention, were identified by sequencing recombinant
plasmids of the Caldicellulosiruptor sp. library.
[0250] Nucleotide Sequence Analysis
[0251] The nucleotide sequences of the genomic rhamnogalacturonan
clones were determined from both strands by the dideoxy
chain-termination method (Sanger, F., Nicklen, S., and Coulson, A.
R. (1977) Proc. Natl. Acad. Sci. U.S. A. 74, 5463-5467) using 500
ng of Qiagen-purified template (Qiagen, USA), the Taq
deoxy-terminal cycle sequencing kit (Perkin-Elmer, USA),
fluorescent labelled terminators and 5 pmol of vector polylinker
primers (Stratagene, USA) or synthetic oligonucleotide primers.
[0252] Analysis of the sequence data was performed with the DNA
Star analysis package (WWW.dnastar.com) or with the GCG-Unix
software package (Wisconsin Package Version 9.1, Genetics Computer
Group (GCG), Madison, Wis.) according to Devereux et al., 1984.
[0253] Based on this sequence analysis it was found that the
rhamnogalacturonase enzymes of the present invention represents two
novel families of rhamnogalacturonases. In this context, one family
is denoted Subfamily A and another family is denoted Subfamily B.
In the attached sequence listings the mature enzyme protein
represented by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16,
respectively, belongs to Subfamily A; and SEQ ID NOS: 18 and 20
represent Subfamily B.
[0254] The sequence of the B. licheniformis (BLR3) clone encoding
the mature enzyme protein is shown in SEQ ID NO:3. The derived
protein sequence is shown in SEQ ID NO:4. The sequence of the
Bacillus sp. AA386 clone (BXR1) encoding the mature protein is
shown in SEQ ID NO:1. The derived protein sequence is shown in SEQ
ID NO:2. The sequence of the Bacillus halodurans C4538 clone
encoding the mature protein is shown in SEQ ID NO:13. The derived
protein sequence is shown in SEQ ID NO:14. The sequence of the
Bacillus halodurans KJ59 clone encoding the mature protein is shown
in SEQ ID NO:17. The derived protein sequence is shown in SEQ ID
NO:18. The partial sequence of a Caldocellulosiruptor sp. gene is
shown in SEQ ID NO:9. The derived partial protein sequence is shown
in SEQ ID NO:10. The partial sequence of another
Caldocellulosiruptor sp. gene is shown in SEQ ID NO:11. The derived
partial protein sequence is shown in SEQ ID NO:12. The partial
sequence of the Bacillus agaradhaerens gene is shown in SEQ ID
NO:19. The derived partial protein sequence is shown in SEQ ID
NO:20 (Corrected for reading frame skips).
[0255] Based on sequence analysis, the following known genes of
hitherto unknown functionality were identified: the Bacillus
subtilis YesW gene (TREMBL: 031526; GeneBank: Z99107) represented
by the DNA sequence of SEQ ID NO:5 and the derived protein sequence
of SEQ ID NO:6; and the Streptomyces coelicolor YesW gene
(E1319264; GeneBank: AL031515) represented by the DNA sequence of
SEQ ID NO:15 (GTG is apparent start codon) and the derived protein
sequence of SEQ ID NO:16 (Valine is first amino acid); and the
Sorangium cellulosum gene represented by the partial sequence shown
in SEQ ID NO:7. The derived partial protein sequence is shown in
SEQ ID NO:8.
[0256] Subcloning and Expression in E. coli of the YesW Gene from
Bacillus subtilis Encoding the Enzyme of the Invention
[0257] The YesW encoding DNA sequence was PCR amplified using the
PCR primer set consisting of two oligo nucleotides:
[0258] YesW upper EcoRI primer:
[0259] 5'-TCGCCGGAATTCGTGCAGTGTCCGAAATAGGCAGATGC-3'
[0260] YesW lower SphI primer:
[0261] 5'-TCGCCGGCATGCGTTCTGTCTGTACCGCAATCAAACC-3'
[0262] Restriction sites EcoRI and SphI are underlined.
[0263] Chromosomal DNA isolated from B. subtilis DN1885 as
described (Pitcher et al., 1989) was used as template in a PCR
reaction using Amplitaq DNA Polymerase (Perkin Elmer) according to
manufacturers instructions. The PCR reaction was set up in PCR
buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01%
(w/v) gelatin) containing 200 .mu.M of each dNTP, 2.5 units of
AmpliTaq polymerase (Perkin-Elmer, Cetus, USA) and 100 pmol of each
primer.
[0264] The PCR reactions was performed using a DNA thermal cycler
(Landgraf, Germany). One incubation at 94.degree. C. for 1 min
followed by thirty cycles of PCR performed using a cycle profile of
denaturation at 94.degree. C. for 30 sec. annealing at 60.degree.
C. for 1 min, and extension at 72.degree. C. for 2 min. Five-.mu.l
aliquots of the amplification product was analysed by
electrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance
of a DNA fragment size 2.06 kb indicated proper amplification of
the gene segment.
[0265] Subcloning of PCR Fragment:
[0266] Fortyfive-.mu.l aliquots of the PCR products generated as
described above were purified using QIAquick PCR purification kit
(Qiagen, USA) according to the manufacturer's instructions. The
purified DNA was eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5. 5
.mu.g of pUC19 and twentyfive-.mu.l of the purified PCR fragment
was digested with EcoRI and SphI, electrophoresed in 0.8% low
gelling temperature agarose (SeaPlaque GTG, FMC) gels, the relevant
fragments were excised from the gels, and purified using QIAquick
Gel extraction Kit (Qiagen, USA) according to the manufacturer's
instructions. The isolated PCR DNA fragment was then ligated to the
EcoRI-SphI digested and purified pUC19. The ligation was performed
overnight at 16.degree. C. using 0.5 .mu.g of each DNA fragment, 1
U of T4 DNA ligase and T4 ligase buffer (Boehringer Mannheim,
Germany).
[0267] The ligation mixture was used to transform E.coli SJ2 by
electroporation as described above. The transformed cells were
plated onto LBPG with 50 .mu.g/ml of ampicillin. After 18 hours
incubation at 37.degree. C. colonies were seen on plates. Several
clones were analyzed by isolating plasmid DNA from overnight
culture broth.
[0268] One clone called PL3142 contained the expected plasmid
pPL3142 confirmed by restriction analysis and DNA sequencing.
PL3142 was re-streaked onto two sets of LB agar plates; one
containing 0.5% AZCL-debranched arabinan (Megazyme), the other
containing 0.5% AZCL-potato galactan (Megazyme). The clone PL3142
showed activity on both plates.
[0269] Sequence Similarities
[0270] The deduced amino acid sequences of the rhamnogalacturonan
hydrolases of the present invention show the following similarity
when compared to the Bacillus licheniformis rhamnogalacturonan
hydrolase (SEQ ID NO:4) in the case of Subfamily A members and
Bacillus halodurans KJ59 (SEQ ID NO:18) in the case of Subfamily B
members:
4 SEQ ID NO: 4 SEQ ID NO: 18 SEQ ID NO: 2 67.3/71.7 SEQ ID NO: 20
73.8/80.0 SEQ ID NO: 6 77.4/81.8 SEQ ID NO: 8 58.0/64.0 SEQ ID NO:
10 64.0/68.5 SEQ ID NO: 12 48.0/57.2 SEQ ID NO: 14 62.7/69.8 SEQ ID
NO: 16 59.5/65.5
[0271] Values are in percent identity/percent similarity
respectively compared to the Bacillus licheniformis
rhamnogalacturonan hydrolase (SEQ ID NO:4) and Bacillus halodurans
KJ59 (SEQ ID NO:18), respectively. Only the core region (amino acid
sequence minus the secretion signal, if present) was analyzed. The
GAP program in the GCG package ver. 9.1 (Devereux et al., 1984) was
used. The standard PAM table blosum62 with a gap creation penalty
of 12 and a gap extension penalty of 4 was employed throughout.
EXAMPLE 2
[0272] Subcloning and Expression in B. subtilis of the YesW Gene
from Bacillus subtilis Encoding the Enzyme of the Invention;
Purification and Characterisation of the Enzyme
[0273] The YesW encoding DNA sequence was PCR amplified using the
PCR primer set consisting of these two oligo nucleotides:
[0274] YesW .upper.SacII
[0275] 5'-GCAGCCGCGGCAGAAGGGGCAGCGCGGCAGATGG-3'
[0276] YesW .lower.NotI
[0277] 5'-TCGCCGGCGGCCGCGTTCTGTCTGTACCGCAATCAAACC-3'
[0278] Restriction Sites SacII and NotI are Underlined
[0279] Chromosomal DNA isolated from B. subtilis DN1885 as
described above was used as template in a PCR reaction using
Amplitaq DNA Polymerase (Perkin Elmer) according to manufacturers
instructions. The PCR reaction was set up in PCR buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% (w/v)
gelatin) containing 200 .mu.M of each DNTP, 2.5 units of AmpliTaq
polymerase (Perkin-Elmer, Cetus, USA) and 100 pmol of each
primer.
[0280] The PCR reaction was performed using a DNA thermal cycler
(Landgraf, Germany). One incubation at 94.degree. C. for 1 min
followed by thirty cycles of PCR performed using a cycle profile of
denaturation at 94.degree. C. for 30 sec, annealing at 60.degree.
C. for 1 min, and extension at 72.degree. C. for 2 min. Five-.mu.l
aliquots of the amplification product was analysed by
electrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance
of a DNA fragment size 1.866 kb indicated proper amplification of
the gene segment.
[0281] Subcloning of PCR Fragment:
[0282] Fortyfive-.mu.l aliquots of the PCR products generated as
described above were purified using QIAquick PCR purification kit
(Qiagen, USA) according to the manufacturer's instructions. The
purified DNA was eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5.
[0283] 5 .mu.g of pMOL944 and twentyfive-.mu.l of the purified PCR
fragment was digested with SacII and NotI, electrophoresed in 0.8
low gelling temperature agarose (SeaPlaque GTG, FMC) gels, the
relevant fragments were excised from the gels, and purified using
QIAquick Gel extraction Kit (Qiagen, USA) according to the
manufacturer's instructions. The isolated PCR DNA fragment was then
ligated to the SacII-NotI digested and purified pMOL944. The
ligation was performed overnight at 16.degree. C. using 0.5 .mu.g
of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer
(Boehringer Mannheim, Germany).
[0284] The ligation mixture was used to transform competent B.
subtilis PL1801 cells. The transformed cells were plated onto LBPG
with 10 .mu.g/ml of kanamycin. After 18 hours incubation at
37.degree. C. colonies were seen on plates. Several clones were
analyzed by isolating plasmid DNA from overnight culture broth.
[0285] One such positive clone was restreaked several times on agar
plates as used above, this clone was called PL3151. The clone
PL3151 was grown overnight in TY-10 .mu.g/ml Kanamycin at
37.degree. C., and next day 1 ml of cells were used to isolate
plasmid from the cells using the Qiaprep Spin Plasmid Miniprep Kit
#27106 according to the manufacturers recommendations for
B.subtilis plasmid preparations. This DNA was sequenced and
confirmed that the sequence corresponds to the mature part of the
B. subtilis YesW gene (SEQ ID NO:5).
[0286] PL3151 was grown in 25.times.200 ml BPX media with 10
.mu.g/ml of Kanamycin in 500 ml two baffled shakeflasks for 5 days
at 37.degree. C. at 300 rpm.
[0287] Purification and Characterisation:
[0288] 600 ml of culture broth was purified as follows: The pH was
first adjusted to 5.5, using acetic acid and then 5 ml of cationic
agent (C521 10%) and 5 ml of anionic agent (A130 0.1%) were added
during agitation for flocculation. The flocculated material was
separated by centrifugation using a Sorval RC 3B centrifuge at
10000 rpm for 30 min at 6.degree. C. The resulting supernatant
contained substantial amounts of a 67 kDa protein visualized on
SDS-PAGE.
[0289] The supernatant was clarified using Whatman glass filters
GF/D and C before final concentration on a filtron UF membrane with
a cut off of 10 kDa. The total volume of 200 ml was adjusted to pH
8.5 using NaOH. The enzyme solution was applied to a 50 ml HPQ
Sepharose column. All the 67 kDa peptide ran through the column
that had been equilibrated with 25 mM Tris pH 8.5. The HPQ
sepharose purification step was repeated and the fractions
containing the 67 kDa protein were pooled and concentrated
resulting in a rhamnogalacturonan hydrolase enzyme that was
approximately 90% pure but which also contained some Bacillus
subtilis rhamnogalacturonase (0.1 mg/ml). The preparation contained
4.7 mg/ml of the 67 kDa rhamnogalacturonan hydrolase enzyme.
[0290] The purified rhamnogalacturonase enzyme has activity on AZCL
galactan and AZCL debranched arabinan as well as on
rhamnogalacturonan. Based on the DNA sequence SEQ ID NO. 5, the
following data was obtained:
[0291] Molar extinction coefficient: 116780
[0292] Molecular weight: 63566 Dalton
[0293] pI (estimated): 5.2.
EXAMPLE 3
[0294] Subcloning and expression in B. subtilis of the YesW
Rhamnogalacturonase Gene from Bacillus sp. AA386 (E. coli Clone
Deposition No. DSM 12123); Purification and Characterisation of the
Enzyme
[0295] The YesW encoding DNA sequence was PCR amplified using the
PCR primer set consisting of these two oligo nucleotides:
[0296] YesW .upper.PstI
[0297] 5'-CTCGCTGCAGCAGCGGCGGCACCCAGACAGGCGGAGAACATTAGC-3'
[0298] YesW .lower.NotI
[0299] 5'-CGACGACGTGCGGCCGCCATTATGCGCCTGCTCTTCG-3'
[0300] Restriction Sites PstI and NotI are Underlined
[0301] Plasmid DNA isolated from E. coli clone BXR1 (DSM 12123) as
described above was used as template in a PCR reaction using
Amplitaq DNA Polymerase (Perkin Elmer) according to manufacturers
instructions. The PCR reaction was set up in PCR buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% (w/v)
gelatin) containing 200 .mu.M of each DNTP, 2.5 units of AmpliTaq
polymerase (Perkin-Elmer, Cetus, USA) and 100 pmol of each
primer.
[0302] The PCR reaction was performed using a DNA thermal cycler
(Landgraf, Germany). One incubation at 94.degree. C. for 1 min
followed by thirty cycles of PCR performed using a cycle profile of
denaturation at 94.degree. C. for 30 sec, annealing at 60.degree.
C. for 1 min, and extension at 72.degree. C. for 2 min. Five-.mu.l
aliquots of the amplification product was analysed by
electrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance
of a DNA fragment size 1.941 kb indicated proper amplification of
the gene segment.
[0303] Subcloning of PCR Fragment:
[0304] Fortyfive-.mu.l aliquots of the PCR products generated as
described above were purified using QIAquick PCR purification kit
(Qiagen, USA) according to the manufacturer's instructions. The
purified DNA was eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5.
[0305] 5 .mu.g of pMOL944 was digested with PstI and NotI and
twentyfive-.mu.l of the purified PCR fragment was digested with
PstI (partial digest) and NotI, electrophoresed in 0.8% low gelling
temperature agarose (SeaPlaque GTG, FMC) gels, the relevant
fragments were excised from the gels, and purified using QIAquick
Gel extraction Kit (Qiagen, USA) according to the manufacturer's
instructions. The isolated PCR DNA fragment was then ligated to the
PstI-NotI digested and purified pMOL944. The ligation was performed
overnight at 16.degree. C. using 0.5 .mu.g of each DNA fragment, 1
U of T4 DNA ligase and T4 ligase buffer (Boehringer Mannheim,
Germany).
[0306] The ligation mixture was used to transform competent
B.subtilis PL1801 cells. The transformed cells were plated onto
LBPG-10 .mu.g/ml of Kanamycin-agar plates. After 18 hours
incubation at 37.degree. C. colonies were seen on plates. Several
clones were analyzed by isolating plasmid DNA from overnight
culture broth.
[0307] One such positive clone was restreaked several times on agar
plates as used above, this clone was called PL2988. The clone
PL2988 was grown overnight in TY-10 .mu.g/ml Kanamycin at
37.degree. C., and next day 1 ml of cells were used to isolate
plasmid from the cells using the Qiaprep Spin Plasmid Miniprep Kit
#27106 according to the manufacturers recommendations for B.
subtilis plasmid preparations. This DNA was sequenced and confirmed
that the sequence corresponds to the mature part of the Bacillus
sp. AA386 YesW gene (SEQ ID NO:1).
[0308] PL2988 was grown in 25.times.200 ml BPX media with 10
.mu.g/ml of Kanamycin in 500 ml two baffled shakeflasks for 5 days
at 37.degree. C. at 300 rpm.
[0309] Purification and Characterisation
[0310] 600 ml of culture broth was purified as follows: The pH was
adjusted to 5.5, using acetic acid and 5 ml of cationic agent (C521
10%) and 5 ml of anionic agent (A130 0.1%) was added during
agitation for flocculation. The flocculated material was separated
by centrifugation using a Sorval RC 3B centrifuge at 10000 rpm for
30 min at 6.degree. C. The resulting supernatant contained a
substantial amount of a 63 kDa peptide as visualized by
SDS-PAGE.
[0311] The supernatant was clarified using Whatman glass filters
GF/D and C before final concentration on a filtron UF membrane with
a cut off of 10 kDa. The total volume of 140 ml was adjusted to pH
8.5 with NaOH and was applied to a 50 ml HPQ Sepharose column. All
the 63 kDa peptide ran through the column equilibrated with 25 mM
Tris pH 8.5. The experiment was repeated and some of the activity
ran through once again, however, a pure rhamnogalacturonan
hydrolase was eluted from the column by using a NaCl gradient. The
active eluted fractions containing the 63 kDa protein, as
visualized in SDS-PAGE, was pooled and concentrated resulting in an
essentially pure protein which contained no Bacillus subtilis
rhamnogalacturonase. The enzyme preparation contained 8 mg/ml of
the 63 kDa rhamnogalacturonase.
[0312] The pure rhamnogalacturonase enzyme has activity on AZCL
Galactan and AZCL debranched arabinan as well as on
rhamnogalacturonan.
[0313] Based on the DNA sequence, SEQ ID NO. 1, the following data
was obtained:
[0314] Molar extinction coefficient: 128280
[0315] Molelcular weight: 63453 Dalton
[0316] pI (estimated): 5.2.
EXAMPLE 4
[0317] Subcloning and Expression in B. subtilis of the YesW
Rhamnogalacturonase Gene from Bacillus licheniformis (E. coli Clone
Deposition No. DSM 12122); Purification and Characterisation of the
Enzyme
[0318] The YesW encoding DNA sequence was PCR amplified using the
PCR primer set consisting of these two oligo nucleotides:
[0319] YesW .upper.SacII
[0320] 5'-GCAGCCGCGGCAGACGGGCGGACGGCTGCGCAGG-3'
[0321] YesW .lower.NotI
[0322] 5'-GTGGGCGGCCGCGCCTGAGAAAATCCGTAGCCAGCACC-3'
[0323] Restriction Sites SacII and NotI are Underlined
[0324] Plasmid DNA isolated from E.coli clone BLR3 (DSM 12122) as
described above was used as template in a PCR reaction using
Amplitaq DNA Polymerase (Perkin Elmer) according to manufacturers
instructions. The PCR reaction was set up in PCR buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% (w/v)
gelatin) containing 200 .mu.M of each dNTP, 2.5 units of AmpliTaq
polymerase (Perkin-Elmer, Cetus, USA) and 100 pmol of each
primer.
[0325] The PCR reaction was performed using a DNA thermal cycler
(Landgraf, Germany). One incubation at 94.degree. C. for 1 min
followed by thirty cycles of PCR performed using a cycle profile of
denaturation at 94.degree. C. for 30 sec, annealing at 60.degree.
C. for 1 min, and extension at 72.degree. C. for 2 min. Five-.mu.l
aliquots of the amplification product was analysed by
electrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance
of a DNA fragment size 2.028 kb indicated proper amplification of
the gene segment.
[0326] Subcloning of PCR Fragment:
[0327] Fortyfive-.mu.l aliquots of the PCR products generated as
described above were purified using QIAquick PCR purification kit
(Qiagen, USA) according to the manufacturer's instructions. The
purified DNA was eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5.
[0328] 5 .mu.g of pMOL944 and twentyfive-.mu.l of the purified PCR
fragment was digested with SacII and NotI, electrophoresed in 0.8%
low gelling temperature agarose (SeaPlaque GTG, FMC) gels, the
relevant fragments were excised from the gels, and purified using
QIAquick Gel extraction Kit (Qiagen, USA) according to the
manufacturer's instructions. The isolated PCR DNA fragment was then
ligated to the SacII-NotI digested and purified pMOL944. The
ligation was performed overnight at 16.degree. C. using 0.5 .mu.g
of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer
(Boehringer Mannheim, Germany).
[0329] The ligation mixture was used to transform competent
B.subtilis PL1801 cells. The transformed cells were plated onto
LBPG-10 .mu.g/ml of Kanamycin-agar plates. After 18 hours
incubation at 37.degree. C. colonies were seen on plates. Several
clones were analyzed by isolating plasmid DNA from overnight
culture broth.
[0330] One such positive clone was restreaked several times on agar
plates as used above, this clone was called PL3149. The clone
PL3149 was grown overnight in TY-10 .mu.g/ml Kanamycin at
37.degree. C., and next day 1 ml of cells were used to isolate
plasmid from the cells using the Qiaprep Spin Plasmid Miniprep Kit
#27106 according to the manufacturers recommendations for
B.subtilis plasmid preparations. This DNA was sequenced and shown
to correspond to the mature part of the B. licheniformis YesW gene
(SEQ ID NO:3).
[0331] PL3149 was grown in 25.times.200 ml BPX media with 10
.mu.g/ml of Kanamycin in 500 ml two baffled shakeflasks for 5 days
at 37.degree. C. at 300 rpm.
[0332] Purification and Characterisation
[0333] 600 ml of culture broth was purified as follows: The pH was
adjusted to 5.5, using acetic acid. 5 ml of cationic agent (C521
10%) and 5 ml of anionic agent (A130 0.1%) were added during
agitation for flocculation. The flocculated material was separated
by centrifugation using a Sorval RC 3B centrifuge at 10000 rpm for
30 min at 6.degree. C. The resulting supernatant contained
substantial amounts of a 64 kDa peptide as visualized by
SDS-PAGE.
[0334] The supernatant was clarified using Whatman glass filters
GF/D and C before final concentration on a filtron UF membrane with
a cut off of 10 kDa. The total volume of 140 ml was adjusted to pH
8.5 with NaOH. The enzyme solution was applied to a 50 ml HPQ
Sepharose column. All the 64 kDa peptide ran through the column
equilibrated with 25 mM Tris pH 8.5. The experiment was repeated
and some of the activity ran through once again however, a pure
rhamnogalaturonase was eluted from the column by using a NaCl
gradient. The active eluted fractions containing the 64 kDa
protein, as visualized by SDS-PAGE, was pooled and concentrated
which resulted in an essentially pure enzyme containing no Bacillus
subtilis rhamnogalacturonase. The enzyme preparation contained 2.7
mg/ml of the 64 kDa rhamnogalacturonase.
[0335] The pure rhamnogalaturonase enzyme has activity on AZCL
Galactan and AZCL debranched arabinan as well as on
rhamnogalacturonan.
[0336] Based on the DNA sequence, SEQ ID NO. 3, the following data
was obtained:
[0337] Molar extinction coefficient: 120620
[0338] Moelcular weight: 64287 Dalton
[0339] pI (estimated): 5.4.
EXAMPLE 5
[0340] Subcloning and Expression in B. subtilis of the
Rhamnogalacturonase Gene from Bacillus halodurans KJ59;
Purification and Characterisation of the Enzyme
[0341] The rhamnogalacturonase encoding DNA sequence was PCR
amplified using the PCR primer set consisting of these two oligo
nucleotides:
[0342] YesW .upper.PstI
[0343] 5'-GCGCTCTGCAGCAGCGGCGAAATGAAGTGGTGAATGCAAGGGATTTTGG-3'
[0344] YesW .lower.NotI
[0345] 5'-GTCAGGCGTGCGGCCGCGGTGTAGAGGTGCGATGATGGATGGG-3'
[0346] Restriction Sites SacII and NotI are Underlined.
[0347] Plasmid DNA isolated from E.coli clone BXA15 (DSM 12202) as
described above was used as template in a PCR reaction using
Amplitaq DNA Polymerase (Perkin Elmer) according to manufacturers
instructions. The PCR reaction was set up in PCR buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% (w/v)
gelatin) containing 200 .mu.M of each dNTP, 2.5 units of AmpliTaq
polymerase (Perkin-Elmer, Cetus, USA) and 100 pmol of each
primer.
[0348] The PCR reaction was performed using a DNA thermal cycler
(Landgraf, Germany). One incubation at 94.degree. C. for 1 min
followed by thirty cycles of PCR performed using a cycle profile of
denaturation at 94.degree. C. for 30 sec, annealing at 60.degree.
C. for 1 min, and extension at 72.degree. C. for 2 min. Five-.mu.l
aliquots of the amplification product was analysed by
electrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance
of a DNA fragment size 1.778 kb indicated proper amplification of
the gene segment.
[0349] Subcloning of PCR Fragment:
[0350] Fortyfive-.mu.l aliquots of the PCR products generated as
described above were purified using QIAquick PCR purification kit
(Qiagen, USA) according to the manufacturer's instructions. The
purified DNA was eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5.
[0351] 5 .mu.g of pMOL944 and twentyfive-.mu.l of the purified PCR
fragment was digested with PstI and NotI, electrophoresed in 0.8%
low gelling temperature agarose (SeaPlaque GTG, FMC) gels, the
relevant fragments were excised from the gels, and purified using
QIAquick Gel extraction Kit (Qiagen, USA) according to the
manufacturer's instructions. The isolated PCR DNA fragment was then
ligated to the PstI-NotI digested and purified pMOL944. The
ligation was performed overnight at 16.degree. C. using 0.5 .mu.g
of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer
(Boehringer Mannheim, Germany).
[0352] The ligation mixture was used to transform competent
B.subtilis PL1801 cells. The transformed cells were plated onto
LBPG-10 .mu.g/ml of Kanamycin-agar plates. After 18 hours
incubation at 37.degree. C. colonies were seen on plates. Several
clones were analyzed by isolating plasmid DNA from overnight
culture broth.
[0353] One such positive clone was restreaked several times on agar
plates as used above, this clone was called PL2990. The clone
PL2990 was grown overnight in TY-10 .mu.g/ml Kanamycin at
37.degree. C., and next day 1 ml of cells were used to isolate
plasmid from the cells using the Qiaprep Spin Plasmid Miniprep Kit
#27106 according to the manufacturers recommendations for
B.subtilis plasmid preparations. This plasmid DNA was sequenced and
confirmed the sequence corresponds to the mature part of the B.
halodurans KJ59 gene (SEQ ID NO:17).
[0354] PL2990 was grown in 25.times.200 ml BPX media with 10
.mu.g/ml of Kanamycin in 500 ml two baffled shakeflasks for 5 days
at 37.degree. C. at 300 rpm.
[0355] Purification and Characterisation:
[0356] 600 ml of culture broth was purified as follows: The pH was
adjusted to 5.5, using acetic acid. 5 ml of cationic agent (C521
10%) and 5 ml of anionic agent (A130 0.1%) were added during
agitation for flocculation. The flocculated material was separated
by centrifugation using a Sorval RC 3B centrifuge at 10000 rpm for
30 min at 6.degree. C. The resulting supernatant contained
substantial amounts of a 42 kDa peptide as visualized by
SDS-PAGE.
[0357] The supernatant was clarified using Whatman glass filters
GF/D and C before final concentration on a filtron UF membrane with
a cut off of 10 kDa. The total volume of 140 ml was washed with
deionized water until the conductivity was below 1 mSi. The enzyme
solution was applied to a 50 ml HPS Sepharose column equilibrated
with 25 mM sodium acetate buffer pH 5.5. All the 42 kDa protein
bound to the column and the enzyme was eluted using a sodium
chloride gradient. The active eluted fractions containing the 42
kDa protein, as visualized by SDS-PAGE, were pooled and
concentrated resulting in approximately a 25% pure enzyme
preparation with trace amounts of Bacillus subtilis
rhamnogalacturonase. The preparation contained 0.4 mg/ml of the 42
kDa rhamnogalacturonase.
[0358] The partially pure rhamnogalaturonase enzyme has activity on
AZCL Galactan and AZCL debranched arabinan as well as on
rhamnogalacturonan.
[0359] Based on the DNA sequence SEQ. ID NO. 17 the following data
was obtained:
[0360] Molar extinction coefficient: 15930.
EXAMPLE 6
[0361] Degradation of Hairy Regions from Apples and
Rhamnogalacturonan Obtained from Megazyme by the Enzyme of the
Invention
[0362] Hairy regions from apples (MHR), which mainly contain
arabinan side chains on the rhamnogalacturonan backbone, were
extracted essentially as described (Schols, H. A. et al (1990)).
Rhamnogalacturonan (RG) prepared from soy bean pectin was obtained
from Megazyme.
[0363] MHR and RG were saponified (MHR-S and RG-S, respectively)
according to Kofod et al (1994).
[0364] The rhamnogalacturonan hydrolase enzymes from Bacillus sp.
AA386 (BXR1), B. licheniformis (BLR3), B. subtilis (BSR5) and B.
halodurans KJ59 (BXA15) were produced and purified as described in
examples 2-5. Enzyme activity was determined using 0.2% AZCL potato
galactan in 0.1 M glycin pH 9 buffer and incubation at 40.degree.
C. for 15 min. OD.sub.620 was measured on the supernatant. For this
experiment, 1 unit was defined as the amount of enzyme giving an
increase in OD.sub.620 of 0.35.
[0365] 1 ml aliquots of a 0.75% solution of MHR, MHR-S, RG and
RG-S, respectively, in 0.1 M glycin pH 9 buffer were incubated in
thermomixers with the enzymes BXR1, BLR3, BSR5 and BXA15 at
30.degree. C. for 1, 2, 4 and 24 hours. The enzyme dose was based
on the activity on AZCL potato galactan i.e. 1 unit per ml
substrate.
[0366] Degradation products were analyzed by high performance size
exclusion chromatography (HPSEC) as described in Kauppinen et al
(1995). The HPSEC analysis of MHR incubated with the enzymes showed
that MHR was depolymerized to some extent by all four enzymes
(FIGS. 1, 2, 3, 4). Further, the HPSEC analysis showed that the
enzymes were capable of extensively degrading the MHR-S substrate
resulting in the formation of oligomers (FIGS. 5, 6, 7, 8). In
MHR-S both methyl and acetyl esters are removed, and therefore, it
cannot be concluded whether both or only one type of ester linkages
inhibit the enzyme action.
[0367] RG and RG-S were both degraded by the enzymes BXR1, BLR3 and
BXA15 and to the same extent (FIGS. 9, 10, 11, 12, 13, 14). These
substrates were not depolymerized as much as the MHR-S substrate,
probably, because the RG substrate has a high content of xylose and
arabinose.
EXAMPLE 7
[0368] Determination of Hydrolase vs Lyase Activity
[0369] Determination of Enzyme Activity by Measuring Reducing
Ends
[0370] The assay was carried out in a solution of 0.5% saponified
hairy regions from apples (MHR-S) in 100 mM Tris buffer pH 8.
Enzymes, purified as described in Example 2 and 5, respectively,
were dosed as follow:
5 Enzyme conc. in assay B. subtilis (BSR5) 0.24 mg/ml B. halodurans
KJ59 (BXA15) 0.02 mg/ml
[0371] The assay mix was incubated at 40.degree. C. for 15 minutes
followed by a 10 fold dilution before determination of reducing
sugars by the PHBAH method (Lever, M. 1972, A new reaction for
colormetric determination of carbohydrates. Anal. Biochem.
47:273-279). Substrate without addition of enzyme was used as
control in this experiment. Galacturonic acid was used as
standard.
[0372] The following data were obtained and show that new reducing
ends were formed by action of the enzymes:
6 OD.sub.410 Control 0.94 BSR5 2.1 BXA15 1.7
[0373] Lyase Assay
[0374] Cleavage of rhamnogalacturonan by a lyase, i.e. by
beta-elimination, generates a new reducing end and a new
non-reducing end with a double bond between C-4 and C-5 of the
sugar residue. This double bond gives a characteristic absorption
maximum at 235 nm, the extinction coefficient is 5.5
mM.sup.-1.times.cm.sup.-1 (Albersheim, P. 1966, Methods in
Enzymology, vol 8 p. 628).
[0375] For determination of beta-elimination an assay measuring the
increase in absorbency at 235 nm was carried out using a solution
of 0.1% saponified hairy regions from apples (MHR-S) in 50 mM Tris
buffer pH 8. Enzymes, purified as described in Example 2 and 5,
respectively, were dosed as follow:
7 Enzyme konc. in assay B. subtilis (BSR5) 0.047 mg/ml B.
halodurans KJ59 (BXA15) 0.004 mg/ml
[0376] The assay was performed using a 0.5 ml cuvette with a 1 cm
light path on a HP diode array spectrophotometer in a temperature
controlled cuvette holder with continuous measurement of the
absorbency at 235 nm. The temperature was 40.degree. C. and
absorbency was followed for 15 minutes. Substrate without addition
of enzyme was used as control in this experiment.
[0377] The following data were obtained showing that the enzymes
have no lyase activity:
8 Increase in OD.sub.235 (15 min.) Control 0.4 BSR5 0.4 BXA15
0.4
[0378] Conclusion: The enzymes BSR5 (B. subtilis) and BXA15 (B.
halodurans KJ59) are both hydrolases.
LITERATURE
[0379] Lever, M. (1972) A new reaction for colormetric
determination of carbohydrates. Anal. Biochem. 47, 273-279.
[0380] N. C. Carpita and D. M. Gibeaut (1993) The Plant Journal
3:1-30.
[0381] Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucleic
Acids Res. 12, 387-395.
[0382] Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B. R.,
Sj.o slashed.holm, C. (1990) Cloning of aldb, which encodes
alpha-acetolactate decarboxylase, an exoenzyme from Bacillus
brevis. J. Bacteriol. 172:4315-4321.
[0383] Dretzen, G., Bellard, M., Sassone-Corsi, P., Chambon, P.
(1981) A reliable method for the recovery of DNA fragments from
agarose and acrylamide gels. Anal. Biochem., 112, 295-298.
[0384] Kauppinen et al (1995) J. Biol. Chem. 270:27172-27178.
[0385] Kofod et al (1994) J. Biol. Chem. 269:29182-29189.
[0386] Pitcher, D. G., Saunders, N. A., Owen, R. J. (1989). Rapid
extraction of bacterial genomic DNA with guanidium thiocyanate.
Lett. Appl. Microbiol., 8, 151-156.
[0387] Schols, H. A. et al (1990) Carbohydr. Res. 206:117-129.
Sequence CWU 1
1
61 1 1863 DNA Bacillus sp. AA 386 1 atgtttagta agcggttaca
tcatttctgg cgagtgatgc tggggctggt tgttgttgta 60 tccacgatcg
ggtcggtgtt tctcccggtg tccacagcgt cggctgcacc cagacaggcg 120
gagaacatta gccgcggcct ggtagcggtg aaggtaagca gcggcgtgtt catcagctgg
180 cggctgcttg ggacggagca gctctcgact tccttcaatg tatatcggaa
cggaactaaa 240 gtgaacgccg ccccgattac gaacagcacg aacctgctgg
atactgcggg cacgacttct 300 tccacttata cggtccgggc cgtcgtcggc
ggcgtggagc agccggcctc ccccgccgtc 360 cgcgtgtggg caaacaacta
cctggatgtc ccgatccagg cgcctcccgg cgggagaaca 420 ccggatgggg
tgaactatac ctacagcgcc aatgatgcca gcatcggaga cctagatggc 480
gatggggaat acgagatcgt gctcaaatgg gaccctacga actccaagga caattcccaa
540 ggcggctata cgggcaatgt ttacctggat gcctacaagc ttaatggcac
gcggctgtgg 600 cgaatcgacc tgggtcgaaa cattcgggcc ggcgcgcatt
acacacagtt tctcgtttat 660 gattttgacg gagacgggaa ggcggagatc
gtctgcaaga cggcggacgg caccgttgac 720 ggcacgggca tcaccatcgg
caatgccaat gccgatcatc gcaacgcgaa tggttatgtg 780 ctctccgggc
ccgagttcct taccgtcttc tccggtcaga caggcaaagc cttaacgacc 840
atcgactatg ttccgccaag aggaaatgta tccagttggg gcgacaatta cggcaaccgt
900 gtggaccgct tcctggccgg agtagcttat cttgatggcg tccaccccag
catcattatg 960 gcacgcgggt attatacccg gaccgttgtt gttgcctatg
actggaatgg ccgcgcactg 1020 acccgaagat ggacatttga cagcaacagc
tccaccaatc ccggaacagc cggacaaggc 1080 aaccacagct taagcgtcgc
cgatgtcgac ggggatggca aggacgaaat catctacggc 1140 gctctgacca
tcaacgacaa cggggccaca ctgtacaaca ccagactcgg gcatggcgat 1200
gcgctgcatg taggagattt caatccaaac cggcctggac ttgaagtatt caaagttatg
1260 gaggatgcca atgcacctta cggtgctgct gtatgggatg ccgctaccgg
gcagattctg 1320 tggggggtgc gtaccggcag ggacaccggc cggggtatgg
ccgcggatat tgacccgaac 1380 catccagggg tagaggtatg ggccagcggc
ggcgtcgggc tgtattccat tacaggcacc 1440 aaaatcagca ataacacgcc
ttcgataaac tttggcatct ggtgggacgg cgatctgtcc 1500 cgagagctgc
tggacgatat tcggatcgac aagtggaatt acaacaacaa caccatgtac 1560
aatctgctaa ccggctctgg cgtcgcctcc aacaatggca ccaaagccac gccaacgctg
1620 caggccgatc tgatcggcga ttggcgcgag gaggtcatct ggagaaaatc
cgacaacacc 1680 gcgcttcgca tctacacaac taccgatctg accaatcata
agatatacac gctgatgcat 1740 gatccggtat accggctgag catcgcctgg
cagaacgtcg cctacaacca gcctcctcat 1800 acaggcttct tcctggggag
cggtatgggg ccggttacga aaccggatat ctatgtcgtt 1860 cca 1863 2 621 PRT
Bacillus sp. AA 386 2 Met Phe Ser Lys Arg Leu His His Phe Trp Arg
Val Met Leu Gly Leu 1 5 10 15 Val Val Val Val Ser Thr Ile Gly Ser
Val Phe Leu Pro Val Ser Thr 20 25 30 Ala Ser Ala Ala Pro Arg Gln
Ala Glu Asn Ile Ser Arg Gly Leu Val 35 40 45 Ala Val Lys Val Ser
Ser Gly Val Phe Ile Ser Trp Arg Leu Leu Gly 50 55 60 Thr Glu Gln
Leu Ser Thr Ser Phe Asn Val Tyr Arg Asn Gly Thr Lys 65 70 75 80 Val
Asn Ala Ala Pro Ile Thr Asn Ser Thr Asn Leu Leu Asp Thr Ala 85 90
95 Gly Thr Thr Ser Ser Thr Tyr Thr Val Arg Ala Val Val Gly Gly Val
100 105 110 Glu Gln Pro Ala Ser Pro Ala Val Arg Val Trp Ala Asn Asn
Tyr Leu 115 120 125 Asp Val Pro Ile Gln Ala Pro Pro Gly Gly Arg Thr
Pro Asp Gly Val 130 135 140 Asn Tyr Thr Tyr Ser Ala Asn Asp Ala Ser
Ile Gly Asp Leu Asp Gly 145 150 155 160 Asp Gly Glu Tyr Glu Ile Val
Leu Lys Trp Asp Pro Thr Asn Ser Lys 165 170 175 Asp Asn Ser Gln Gly
Gly Tyr Thr Gly Asn Val Tyr Leu Asp Ala Tyr 180 185 190 Lys Leu Asn
Gly Thr Arg Leu Trp Arg Ile Asp Leu Gly Arg Asn Ile 195 200 205 Arg
Ala Gly Ala His Tyr Thr Gln Phe Leu Val Tyr Asp Phe Asp Gly 210 215
220 Asp Gly Lys Ala Glu Ile Val Cys Lys Thr Ala Asp Gly Thr Val Asp
225 230 235 240 Gly Thr Gly Ile Thr Ile Gly Asn Ala Asn Ala Asp His
Arg Asn Ala 245 250 255 Asn Gly Tyr Val Leu Ser Gly Pro Glu Phe Leu
Thr Val Phe Ser Gly 260 265 270 Gln Thr Gly Lys Ala Leu Thr Thr Ile
Asp Tyr Val Pro Pro Arg Gly 275 280 285 Asn Val Ser Ser Trp Gly Asp
Asn Tyr Gly Asn Arg Val Asp Arg Phe 290 295 300 Leu Ala Gly Val Ala
Tyr Leu Asp Gly Val His Pro Ser Ile Ile Met 305 310 315 320 Ala Arg
Gly Tyr Tyr Thr Arg Thr Val Val Val Ala Tyr Asp Trp Asn 325 330 335
Gly Arg Ala Leu Thr Arg Arg Trp Thr Phe Asp Ser Asn Ser Ser Thr 340
345 350 Asn Pro Gly Thr Ala Gly Gln Gly Asn His Ser Leu Ser Val Ala
Asp 355 360 365 Val Asp Gly Asp Gly Lys Asp Glu Ile Ile Tyr Gly Ala
Leu Thr Ile 370 375 380 Asn Asp Asn Gly Ala Thr Leu Tyr Asn Thr Arg
Leu Gly His Gly Asp 385 390 395 400 Ala Leu His Val Gly Asp Phe Asn
Pro Asn Arg Pro Gly Leu Glu Val 405 410 415 Phe Lys Val Met Glu Asp
Ala Asn Ala Pro Tyr Gly Ala Ala Val Trp 420 425 430 Asp Ala Ala Thr
Gly Gln Ile Leu Trp Gly Val Arg Thr Gly Arg Asp 435 440 445 Thr Gly
Arg Gly Met Ala Ala Asp Ile Asp Pro Asn His Pro Gly Val 450 455 460
Glu Val Trp Ala Ser Gly Gly Val Gly Leu Tyr Ser Ile Thr Gly Thr 465
470 475 480 Lys Ile Ser Asn Asn Thr Pro Ser Ile Asn Phe Gly Ile Trp
Trp Asp 485 490 495 Gly Asp Leu Ser Arg Glu Leu Leu Asp Asp Ile Arg
Ile Asp Lys Trp 500 505 510 Asn Tyr Asn Asn Asn Thr Met Tyr Asn Leu
Leu Thr Gly Ser Gly Val 515 520 525 Ala Ser Asn Asn Gly Thr Lys Ala
Thr Pro Thr Leu Gln Ala Asp Leu 530 535 540 Ile Gly Asp Trp Arg Glu
Glu Val Ile Trp Arg Lys Ser Asp Asn Thr 545 550 555 560 Ala Leu Arg
Ile Tyr Thr Thr Thr Asp Leu Thr Asn His Lys Ile Tyr 565 570 575 Thr
Leu Met His Asp Pro Val Tyr Arg Leu Ser Ile Ala Trp Gln Asn 580 585
590 Val Ala Tyr Asn Gln Pro Pro His Thr Gly Phe Phe Leu Gly Ser Gly
595 600 605 Met Gly Pro Val Thr Lys Pro Asp Ile Tyr Val Val Pro 610
615 620 3 1869 DNA Bacillus licheniformis 3 atgaaaaaag gaaagaaaag
gtggaagaac ctgttggccg cgtcatctct tttattaatc 60 acgctagtga
ccggcttctc ggagcaagct gaggcagacg ggcggacggc tgcgcaggca 120
aggcaaatgg aatcgcttaa cagggggctt gtcgctgtta aaacggggaa cggtgtcttt
180 gtcagctggc ggcttctggg aaccgaaccg tcttctgttt cacttaatgt
gtatcgaaac 240 ggaaagaagc tgaacggttc tccgattaca tcgagcacaa
actatcagga tgcaggcggg 300 gatttgaacg ccgtttacca ggtgcgcgcc
gttttgaacg gcagggagca ggctccttct 360 gaatccgtcg gcgtattgaa
taaacaatat aaatctgttc cgctgcaaaa accggccgga 420 ggaaaaacgc
ctgatggggt gtcatacaca tacagcgcca atgatgcgag cttaggcgac 480
cttgttggag acgcccaata tgaaatcttt ctcaagtggg atccttccaa ttcaaaggat
540 aattcacagg acggatacac gggagatgtg ctgattgacg catacaagct
tgacggcacc 600 atgatgtgga gaatcaacct tggcaaaaat attcgcgccg
gcgcccatta tacgcagttt 660 ctcgtctatg actttgacgg cgatggaaaa
gcggaaatcg ccatgaagac ggcagacggg 720 acgaaggacg gcaaagggaa
ggtgatcggc aatgcaaacg ccgattaccg caatgcccaa 780 ggccgaattt
tgtcagggcc tgagtatttg acggttttta aaggcgatac aggcgctgag 840
cttacaacgg tcaactacga acctgcccgg ggaaatgtag ccgattgggg agacagctac
900 ggcaacaggg ttgaccgctt tctggccggt gtcgcatacc ttgacgggga
gcggccgagt 960 tttgtcatgg cacgcggtta ttacacgaga acagtgctag
tcgcttacaa cttcagaggc 1020 ggaaagctga ccaagctgtg gacgttcgat
tcggatgctc ccggaaatgg cgcctatgcc 1080 ggtcaaggca accacagttt
gagcgtcgcc gacgttgacg gagatggaaa ggacgagatc 1140 atatacggag
cgatggctgt cgatcatgac ggaaaaggcc tctactcaac cggctgggga 1200
catggggatg ccatgcatac agggaacctg gacccgtcaa ggcctggact ggaagtcttc
1260 caagtccatg aaaacagcaa ttctccttat ggcttgtcct tccgcgatgc
gaaaacagga 1320 aagatcatct ggggagttca cgcaggtaaa gatgtcggac
gcggaatggc cgctgatatc 1380 gatccgcgct acgaaggagc ggaagtatgg
gcgaacggca gtctttatac ggcaaaaggc 1440 gtaaaaatcg gaaacacatt
gccttcatca acgaacttcg gcatctggtg ggacggcgat 1500 ctccaaagag
agcttctgga cagcaacaga attgataaat gggattatca aaattcgcga 1560
accgtcaact tgctgacagc gtccggagct tcggcaaata acggaacaaa agcgacgccg
1620 tccctgcagg cggacattct cggagactgg cgcgaagaag tggtctggcg
agcggaggac 1680 agcagcgaac tgcgcatcta cacgacgaca gacgtgacgg
agcaccgcat gtatacgctg 1740 atgcatgatg cagtctatcg cctcggtatc
gcctggcaga atgtcggcta caaccagcct 1800 ccgcacaccg gcttttattt
aggcgaaggc atgcagacac cggagaagcc gaacatttat 1860 acacgctga 1869 4
622 PRT Bacillus licheniformis 4 Met Lys Lys Gly Lys Lys Arg Trp
Lys Asn Leu Leu Ala Ala Ser Ser 1 5 10 15 Leu Leu Leu Ile Thr Leu
Val Thr Gly Phe Ser Glu Gln Ala Glu Ala 20 25 30 Asp Gly Arg Thr
Ala Ala Gln Ala Arg Gln Met Glu Ser Leu Asn Arg 35 40 45 Gly Leu
Val Ala Val Lys Thr Gly Asn Gly Val Phe Val Ser Trp Arg 50 55 60
Leu Leu Gly Thr Glu Pro Ser Ser Val Ser Leu Asn Val Tyr Arg Asn 65
70 75 80 Gly Lys Lys Leu Asn Gly Ser Pro Ile Thr Ser Ser Thr Asn
Tyr Gln 85 90 95 Asp Ala Gly Gly Asp Leu Asn Ala Val Tyr Gln Val
Arg Ala Val Leu 100 105 110 Asn Gly Arg Glu Gln Ala Pro Ser Glu Ser
Val Gly Val Leu Asn Lys 115 120 125 Gln Tyr Lys Ser Val Pro Leu Gln
Lys Pro Ala Gly Gly Lys Thr Pro 130 135 140 Asp Gly Val Ser Tyr Thr
Tyr Ser Ala Asn Asp Ala Ser Leu Gly Asp 145 150 155 160 Leu Val Gly
Asp Ala Gln Tyr Glu Ile Phe Leu Lys Trp Asp Pro Ser 165 170 175 Asn
Ser Lys Asp Asn Ser Gln Asp Gly Tyr Thr Gly Asp Val Leu Ile 180 185
190 Asp Ala Tyr Lys Leu Asp Gly Thr Met Met Trp Arg Ile Asn Leu Gly
195 200 205 Lys Asn Ile Arg Ala Gly Ala His Tyr Thr Gln Phe Leu Val
Tyr Asp 210 215 220 Phe Asp Gly Asp Gly Lys Ala Glu Ile Ala Met Lys
Thr Ala Asp Gly 225 230 235 240 Thr Lys Asp Gly Lys Gly Lys Val Ile
Gly Asn Ala Asn Ala Asp Tyr 245 250 255 Arg Asn Ala Gln Gly Arg Ile
Leu Ser Gly Pro Glu Tyr Leu Thr Val 260 265 270 Phe Lys Gly Asp Thr
Gly Ala Glu Leu Thr Thr Val Asn Tyr Glu Pro 275 280 285 Ala Arg Gly
Asn Val Ala Asp Trp Gly Asp Ser Tyr Gly Asn Arg Val 290 295 300 Asp
Arg Phe Leu Ala Gly Val Ala Tyr Leu Asp Gly Glu Arg Pro Ser 305 310
315 320 Phe Val Met Ala Arg Gly Tyr Tyr Thr Arg Thr Val Leu Val Ala
Tyr 325 330 335 Asn Phe Arg Gly Gly Lys Leu Thr Lys Leu Trp Thr Phe
Asp Ser Asp 340 345 350 Ala Pro Gly Asn Gly Ala Tyr Ala Gly Gln Gly
Asn His Ser Leu Ser 355 360 365 Val Ala Asp Val Asp Gly Asp Gly Lys
Asp Glu Ile Ile Tyr Gly Ala 370 375 380 Met Ala Val Asp His Asp Gly
Lys Gly Leu Tyr Ser Thr Gly Trp Gly 385 390 395 400 His Gly Asp Ala
Met His Thr Gly Asn Leu Asp Pro Ser Arg Pro Gly 405 410 415 Leu Glu
Val Phe Gln Val His Glu Asn Ser Asn Ser Pro Tyr Gly Leu 420 425 430
Ser Phe Arg Asp Ala Lys Thr Gly Lys Ile Ile Trp Gly Val His Ala 435
440 445 Gly Lys Asp Val Gly Arg Gly Met Ala Ala Asp Ile Asp Pro Arg
Tyr 450 455 460 Glu Gly Ala Glu Val Trp Ala Asn Gly Ser Leu Tyr Thr
Ala Lys Gly 465 470 475 480 Val Lys Ile Gly Asn Thr Leu Pro Ser Ser
Thr Asn Phe Gly Ile Trp 485 490 495 Trp Asp Gly Asp Leu Gln Arg Glu
Leu Leu Asp Ser Asn Arg Ile Asp 500 505 510 Lys Trp Asp Tyr Gln Asn
Ser Arg Thr Val Asn Leu Leu Thr Ala Ser 515 520 525 Gly Ala Ser Ala
Asn Asn Gly Thr Lys Ala Thr Pro Ser Leu Gln Ala 530 535 540 Asp Ile
Leu Gly Asp Trp Arg Glu Glu Val Val Trp Arg Ala Glu Asp 545 550 555
560 Ser Ser Glu Leu Arg Ile Tyr Thr Thr Thr Asp Val Thr Glu His Arg
565 570 575 Met Tyr Thr Leu Met His Asp Ala Val Tyr Arg Leu Gly Ile
Ala Trp 580 585 590 Gln Asn Val Gly Tyr Asn Gln Pro Pro His Thr Gly
Phe Tyr Leu Gly 595 600 605 Glu Gly Met Gln Thr Pro Glu Lys Pro Asn
Ile Tyr Thr Arg 610 615 620 5 1863 DNA Bacillus subtilis 5
atgagaagga gctgtctgat gattagacga aggaaacgca tgtttaccgc tgttacgttg
60 ctggtcttgt tggtgatggg aacctctgta tgtcctgtga aagctgaagg
ggcagcgcgg 120 cagatggaag cgctgaaccg ggggcttgta gcggtcaaga
cggacggggg catttttgtc 180 agctggcggt ttcttggaac cgaaaacgca
tctgttttgt tcaatgtgta cagagacggg 240 caaaaactga atgctgcgcc
tgtcaaaaca acgaactatg tggataaaaa cggttcggcg 300 ggctcaacgt
atacggttcg ggctgttgta aacggtaccg aacagccggc ttctgaaaaa 360
gcctccgtat gggcgcagcc gtatcattcc gtcccgctgg ataaaccggc tggcggcacg
420 acgccaaagg gtgaatctta cacgtacagc gctaatgacg caagtgttgg
cgatgtggat 480 ggtgacgggc aatacgagct gatcctgaaa tgggacccgt
ccaactcaaa agacaattca 540 caggatggct atacgggtga cgtgctgatt
gacgcgtata aactggacgg cacaaagtta 600 tggcggatca atctcggcaa
aaacatcaga gcgggcgcgc actacaccca gtttatggtg 660 tatgaccttg
atggtgacgg aaaagcagaa gtggcaatga aaacggcaga cgggacaaaa 720
gacggcacgg gcaaagtaat tggaaatgcc aatgcagatt acagaaatga acaggggcgt
780 gtgctttcag gccctgaata tctcactgtg tttcaaggtt caaccgggaa
agagcttgtc 840 accgcaaatt ttgaaccggc gcgcggcaat gtgtcggatt
ggggagacag ctacggcaac 900 cgtgttgacc gttttctcgc cggcattgcc
taccttgatg gacagcggcc gagcctgatc 960 atgaccagag ggtattacgc
taaaaccatg ctagttgcct ataacttcag ggacggaaag 1020 ctgtcaaagc
tttggacgct ggactcctca aagtcaggaa atgaagcgtt tgccggacag 1080
gggaatcaca acctgagcat cgcggacgtt gacggggatg gaaaagatga gattattttc
1140 ggctcaatgg ctgttgatca tgacgggaaa ggcatgtact cgaccggctt
aggccatggg 1200 gatgccctcc atacaggaga tcttgatccg ggccggccgg
ggcttgaggt gtttcaagtt 1260 catgaggaca aaaatgcaaa atacggctta
tctttccggg atgctgcaac tggaaaaatc 1320 ctttggggcg tttatgccgg
caaggatgta ggccggggaa tggctgctga tattgacccg 1380 cgttatccgg
gacaggaggt gtgggcaaac ggttctctct actcagcgaa aggggtcaaa 1440
atcggaagcg gggttccgtc ctcgaccaac ttcggcatct ggtgggacgg cgatctgctc
1500 cgggaacagc tggacagcaa ccgaattgat aagtgggatt atcaaaacgg
cgtatcgaaa 1560 aatatgctga ctgcatcagg cgcagcggct aacaacggca
caaaagcaac accaacgctt 1620 caggctgatc tgctcggtga ctggcgcgag
gaagtggtgt ggagaacgga ggacagcagt 1680 gctctgcgca tttacacgac
gaccattccg actgagcaca ggctgtatac gctgatgcac 1740 gatccggtgt
accggcttgg catcgcctgg caaaatatcg cctataacca gccgccgcac 1800
acaagcttct ttttaggaga cggcatggcg gaacagccaa aaccaaatat gtatacgcct
1860 taa 1863 6 620 PRT Bacillus subtilis 6 Met Arg Arg Ser Cys Leu
Met Ile Arg Arg Arg Lys Arg Met Phe Thr 1 5 10 15 Ala Val Thr Leu
Leu Val Leu Leu Val Met Gly Thr Ser Val Cys Pro 20 25 30 Val Lys
Ala Glu Gly Ala Ala Arg Gln Met Glu Ala Leu Asn Arg Gly 35 40 45
Leu Val Ala Val Lys Thr Asp Gly Gly Ile Phe Val Ser Trp Arg Phe 50
55 60 Leu Gly Thr Glu Asn Ala Ser Val Leu Phe Asn Val Tyr Arg Asp
Gly 65 70 75 80 Gln Lys Leu Asn Ala Ala Pro Val Lys Thr Thr Asn Tyr
Val Asp Lys 85 90 95 Asn Gly Ser Ala Gly Ser Thr Tyr Thr Val Arg
Ala Val Val Asn Gly 100 105 110 Thr Glu Gln Pro Ala Ser Glu Lys Ala
Ser Val Trp Ala Gln Pro Tyr 115 120 125 His Ser Val Pro Leu Asp Lys
Pro Ala Gly Gly Thr Thr Pro Lys Gly 130 135 140 Glu Ser Tyr Thr Tyr
Ser Ala Asn Asp Ala Ser Val Gly Asp Val Asp 145 150 155 160 Gly Asp
Gly Gln Tyr Glu Leu Ile Leu Lys Trp Asp Pro Ser Asn Ser 165 170 175
Lys Asp Asn Ser Gln Asp Gly Tyr Thr Gly Asp Val Leu Ile Asp Ala 180
185 190 Tyr Lys Leu Asp Gly Thr Lys Leu Trp Arg Ile Asn Leu Gly Lys
Asn 195 200 205 Ile Arg Ala Gly Ala His Tyr Thr Gln Phe Met Val Tyr
Asp Leu Asp 210 215 220 Gly Asp Gly Lys Ala Glu Val Ala Met Lys Thr
Ala Asp Gly Thr Lys 225 230 235 240 Asp Gly Thr Gly Lys Val
Ile Gly Asn Ala Asn Ala Asp Tyr Arg Asn 245 250 255 Glu Gln Gly Arg
Val Leu Ser Gly Pro Glu Tyr Leu Thr Val Phe Gln 260 265 270 Gly Ser
Thr Gly Lys Glu Leu Val Thr Ala Asn Phe Glu Pro Ala Arg 275 280 285
Gly Asn Val Ser Asp Trp Gly Asp Ser Tyr Gly Asn Arg Val Asp Arg 290
295 300 Phe Leu Ala Gly Ile Ala Tyr Leu Asp Gly Gln Arg Pro Ser Leu
Ile 305 310 315 320 Met Thr Arg Gly Tyr Tyr Ala Lys Thr Met Leu Val
Ala Tyr Asn Phe 325 330 335 Arg Asp Gly Lys Leu Ser Lys Leu Trp Thr
Leu Asp Ser Ser Lys Ser 340 345 350 Gly Asn Glu Ala Phe Ala Gly Gln
Gly Asn His Asn Leu Ser Ile Ala 355 360 365 Asp Val Asp Gly Asp Gly
Lys Asp Glu Ile Ile Phe Gly Ser Met Ala 370 375 380 Val Asp His Asp
Gly Lys Gly Met Tyr Ser Thr Gly Leu Gly His Gly 385 390 395 400 Asp
Ala Leu His Thr Gly Asp Leu Asp Pro Gly Arg Pro Gly Leu Glu 405 410
415 Val Phe Gln Val His Glu Asp Lys Asn Ala Lys Tyr Gly Leu Ser Phe
420 425 430 Arg Asp Ala Ala Thr Gly Lys Ile Leu Trp Gly Val Tyr Ala
Gly Lys 435 440 445 Asp Val Gly Arg Gly Met Ala Ala Asp Ile Asp Pro
Arg Tyr Pro Gly 450 455 460 Gln Glu Val Trp Ala Asn Gly Ser Leu Tyr
Ser Ala Lys Gly Val Lys 465 470 475 480 Ile Gly Ser Gly Val Pro Ser
Ser Thr Asn Phe Gly Ile Trp Trp Asp 485 490 495 Gly Asp Leu Leu Arg
Glu Gln Leu Asp Ser Asn Arg Ile Asp Lys Trp 500 505 510 Asp Tyr Gln
Asn Gly Val Ser Lys Asn Met Leu Thr Ala Ser Gly Ala 515 520 525 Ala
Ala Asn Asn Gly Thr Lys Ala Thr Pro Thr Leu Gln Ala Asp Leu 530 535
540 Leu Gly Asp Trp Arg Glu Glu Val Val Trp Arg Thr Glu Asp Ser Ser
545 550 555 560 Ala Leu Arg Ile Tyr Thr Thr Thr Ile Pro Thr Glu His
Arg Leu Tyr 565 570 575 Thr Leu Met His Asp Pro Val Tyr Arg Leu Gly
Ile Ala Trp Gln Asn 580 585 590 Ile Ala Tyr Asn Gln Pro Pro His Thr
Ser Phe Phe Leu Gly Asp Gly 595 600 605 Met Ala Glu Gln Pro Lys Pro
Asn Met Tyr Thr Pro 610 615 620 7 1413 DNA Sorangium cellulosum 7
ctggacggcg acgggcggta cgagatcatc gtcaagtggg acccgtcgaa cctcaaggac
60 aactcgcagg ctggccgcac cggcaagacg tacctcgacg cctactcgct
cgagggcgag 120 cggctctggc gcatcgacct cggcgtgaac atccgggccg
gagcgcacta ctcgccgttc 180 ctcgtctacg atctcgacgg cgacgggaag
gcggaggtgg ccgtcaagac ggcgccgggg 240 acacgcgacg gcacgggcga
gcccctcagc aaggggcccg cggcgaacga cgacgacagc 300 cgggactacc
gcaacaacga cggctacatc ctgaccggcc cggagtacct caccgtgttc 360
tccggggaga ccggcgccga gctcgcgacg accgacttcg tggtggggcg cggcgacccg
420 tgcagctggg gcaacaacga gtgttacggc aatcgcgtcg accgcttcgt
cggcacggtc 480 gcgttcctcg acgacaccgg tcgtccgagc gtggtgttcg
gccgcggcta ctacgcgcgc 540 accacgctgt cggcgtggaa ctaccgcgac
ggcgcgctca cgaacctctg gacgttcgac 600 tccagctcga gccgcgacaa
cggggcgtac gccggcatgg gcacccactc catcagcgtc 660 gccaacgtgg
atgacgatcc gcagcaggag atcatcaacg ggggcgccac gttcgacaac 720
gacggcaagg gcctgtgcgc cgtggactac tacggtcacg gcgacgcgct gcacgtcacg
780 gatcacatcc tgtcgcgccc cggcctcgag gtgttccagc cgtacgaggg
cggggactca 840 cccgcctatg ccatgcgcga cgcgcgcacg tgcgaggtcc
tctggcgggg gccgggcaac 900 ggcggcgagg agggccccgg ccgcggcgtg
gcggccgacg tcgatccgcg caacccgggc 960 agcgaggcgt gggtcaatag
cagccagctc ctgagcggcg cggacggcga cgccatcggg 1020 aaccgccccg
cgtcgtccaa cttcctcatc tggtgggacg cggatctgag ccgggagctg 1080
ctcgacggca acagcatccg ccaggccgac ggcgagggaa gcaacttcgc ggccgagggc
1140 tgcaccgcga acaacggctc gaagagcaac ccgaccctca gcgccgatat
cctcggcgac 1200 tggcgcgaag aggtgatctt ccgctgcggc agctcgattc
gtatcttcac cacgaaccgc 1260 gtcgccacga gccggatcca caccctgatg
cacgatccgc agtaccgcgt ggccatctcg 1320 tggcagaacg gcgcctacaa
ccagccgcct cacccgagct tccacatcgg ggaggggatg 1380 gcgccggtcc
cgaagccgga catccacgtc cgc 1413 8 471 PRT Sorangium cellulosum 8 Leu
Asp Gly Asp Gly Arg Tyr Glu Ile Ile Val Lys Trp Asp Pro Ser 1 5 10
15 Asn Leu Lys Asp Asn Ser Gln Ala Gly Arg Thr Gly Lys Thr Tyr Leu
20 25 30 Asp Ala Tyr Ser Leu Glu Gly Glu Arg Leu Trp Arg Ile Asp
Leu Gly 35 40 45 Val Asn Ile Arg Ala Gly Ala His Tyr Ser Pro Phe
Leu Val Tyr Asp 50 55 60 Leu Asp Gly Asp Gly Lys Ala Glu Val Ala
Val Lys Thr Ala Pro Gly 65 70 75 80 Thr Arg Asp Gly Thr Gly Glu Pro
Leu Ser Lys Gly Pro Ala Ala Asn 85 90 95 Asp Asp Asp Ser Arg Asp
Tyr Arg Asn Asn Asp Gly Tyr Ile Leu Thr 100 105 110 Gly Pro Glu Tyr
Leu Thr Val Phe Ser Gly Glu Thr Gly Ala Glu Leu 115 120 125 Ala Thr
Thr Asp Phe Val Val Gly Arg Gly Asp Pro Cys Ser Trp Gly 130 135 140
Asn Asn Glu Cys Tyr Gly Asn Arg Val Asp Arg Phe Val Gly Thr Val 145
150 155 160 Ala Phe Leu Asp Asp Thr Gly Arg Pro Ser Val Val Phe Gly
Arg Gly 165 170 175 Tyr Tyr Ala Arg Thr Thr Leu Ser Ala Trp Asn Tyr
Arg Asp Gly Ala 180 185 190 Leu Thr Asn Leu Trp Thr Phe Asp Ser Ser
Ser Ser Arg Asp Asn Gly 195 200 205 Ala Tyr Ala Gly Met Gly Thr His
Ser Ile Ser Val Ala Asn Val Asp 210 215 220 Asp Asp Pro Gln Gln Glu
Ile Ile Asn Gly Gly Ala Thr Phe Asp Asn 225 230 235 240 Asp Gly Lys
Gly Leu Cys Ala Val Asp Tyr Tyr Gly His Gly Asp Ala 245 250 255 Leu
His Val Thr Asp His Ile Leu Ser Arg Pro Gly Leu Glu Val Phe 260 265
270 Gln Pro Tyr Glu Gly Gly Asp Ser Pro Ala Tyr Ala Met Arg Asp Ala
275 280 285 Arg Thr Cys Glu Val Leu Trp Arg Gly Pro Gly Asn Gly Gly
Glu Glu 290 295 300 Gly Pro Gly Arg Gly Val Ala Ala Asp Val Asp Pro
Arg Asn Pro Gly 305 310 315 320 Ser Glu Ala Trp Val Asn Ser Ser Gln
Leu Leu Ser Gly Ala Asp Gly 325 330 335 Asp Ala Ile Gly Asn Arg Pro
Ala Ser Ser Asn Phe Leu Ile Trp Trp 340 345 350 Asp Ala Asp Leu Ser
Arg Glu Leu Leu Asp Gly Asn Ser Ile Arg Gln 355 360 365 Ala Asp Gly
Glu Gly Ser Asn Phe Ala Ala Glu Gly Cys Thr Ala Asn 370 375 380 Asn
Gly Ser Lys Ser Asn Pro Thr Leu Ser Ala Asp Ile Leu Gly Asp 385 390
395 400 Trp Arg Glu Glu Val Ile Phe Arg Cys Gly Ser Ser Ile Arg Ile
Phe 405 410 415 Thr Thr Asn Arg Val Ala Thr Ser Arg Ile His Thr Leu
Met His Asp 420 425 430 Pro Gln Tyr Arg Val Ala Ile Ser Trp Gln Asn
Gly Ala Tyr Asn Gln 435 440 445 Pro Pro His Pro Ser Phe His Ile Gly
Glu Gly Met Ala Pro Val Pro 450 455 460 Lys Pro Asp Ile His Val Arg
465 470 9 512 DNA Caldocellulosiruptor sp. 9 atgagaaaaa agaaaattta
taggtcctgg ttgggtatag tggttattat tttgtgggtt 60 atatattgtg
tatttaatcc gtataattta gcaataaaga atgttaaggg tgcagtttca 120
agtcaagttg agaagttaaa gaggggactg attgcaatta aagttaataa tggtgtttat
180 cttacgtgga ggatgtttgg ttcagatcct gctgatattg gcttcaatat
ataccgaaat 240 gggcaaaaaa taaaccaaat tcctattcaa gttagcacaa
attatcttga tacaggaggg 300 aatactactt caaaatactt cattaggcca
gttataaatg gccatgaaat agagaattca 360 gaagaagttt cagtcttacc
taccaactat attgaaatta aattaaacag accacctacc 420 tcacctttgg
gagcaatata ttctccgaat gacgcaagtg taggagattt agatggtgat 480
ggagaatacg aaatagtcct taaatgggat cc 512 10 170 PRT
Caldocellusiruptor sp. 10 Met Arg Lys Lys Lys Ile Tyr Arg Ser Trp
Leu Gly Ile Val Val Ile 1 5 10 15 Ile Leu Trp Val Ile Tyr Cys Val
Phe Asn Pro Tyr Asn Leu Ala Ile 20 25 30 Lys Asn Val Lys Gly Ala
Val Ser Ser Gln Val Glu Lys Leu Lys Arg 35 40 45 Gly Leu Ile Ala
Ile Lys Val Asn Asn Gly Val Tyr Leu Thr Trp Arg 50 55 60 Met Phe
Gly Ser Asp Pro Ala Asp Ile Gly Phe Asn Ile Tyr Arg Asn 65 70 75 80
Gly Gln Lys Ile Asn Gln Ile Pro Ile Gln Val Ser Thr Asn Tyr Leu 85
90 95 Asp Thr Gly Gly Asn Thr Thr Ser Lys Tyr Phe Ile Arg Pro Val
Ile 100 105 110 Asn Gly His Glu Ile Glu Asn Ser Glu Glu Val Ser Val
Leu Pro Thr 115 120 125 Asn Tyr Ile Glu Ile Lys Leu Asn Arg Pro Pro
Thr Ser Pro Leu Gly 130 135 140 Ala Ile Tyr Ser Pro Asn Asp Ala Ser
Val Gly Asp Leu Asp Gly Asp 145 150 155 160 Gly Glu Tyr Glu Ile Val
Leu Lys Trp Asp 165 170 11 336 DNA Caldocellusiruptor sp. 11
gatcttacaa gagaattatt ggacaaaact aatatatata aatgggatta taatactaac
60 tcatctaaaa ccatttttac agcaagtggg tgttcagcta ataatggtac
gaaggcaact 120 ccatgtttga gtgcagatat attgggtgac tggcgcgagg
aagttatatt ccgtacttct 180 gacaattcag ctattaggat atatatgact
actatgcaga catcatacaa aattccaaca 240 ttgatgcata atcgtcaata
cagagtgtca atagcatggc aaaacgtagc ttacaaccaa 300 ccgccccaca
caaattttta ttttggagaa ggtatg 336 12 112 PRT Caldocellulosiruptor
sp. 12 Asp Leu Thr Arg Glu Leu Leu Asp Lys Thr Asn Ile Tyr Lys Trp
Asp 1 5 10 15 Tyr Asn Thr Asn Ser Ser Lys Thr Ile Phe Thr Ala Ser
Gly Cys Ser 20 25 30 Ala Asn Asn Gly Thr Lys Ala Thr Pro Cys Leu
Ser Ala Asp Ile Leu 35 40 45 Gly Asp Trp Arg Glu Glu Val Ile Phe
Arg Thr Ser Asp Asn Ser Ala 50 55 60 Ile Arg Ile Tyr Met Thr Thr
Met Gln Thr Ser Tyr Lys Ile Pro Thr 65 70 75 80 Leu Met His Asn Arg
Gln Tyr Arg Val Ser Ile Ala Trp Gln Asn Val 85 90 95 Ala Tyr Asn
Gln Pro Pro His Thr Asn Phe Tyr Phe Gly Glu Gly Met 100 105 110 13
1965 DNA Bacillus halodurans C4539 13 atgcttaggc aaaaggagca
gctagacaga gggcttgtcg ccgtaaaacg ggcggatgga 60 gtgtttttaa
gctggcgtct actcgggacg gagcatccgc ttacggtctt tcatgtgtac 120
cgtgatggag aaaaaatcac gaaagctggg ctgcaagaag ggaccaattt tgtcgacgct
180 gacgggatga ctgactctgt ctatcaaata aaggcggtag ctgggaaaga
tgaagatatg 240 tccaatcctg tatcggtatg ggatgatgaa tatcttgcca
tccctcttga taagccagaa 300 ggaggagtca ctccggatgg tgtttcctat
gaatatacag ccaatgatgc aagtgttgga 360 gatttagatg gggacgggca
gtacgaaatt attttgaaat gggatccaac aaattcaaaa 420 gataactcac
ggtctggtta tacagggaac gtttatcttg atgcgtataa attagatgga 480
acgaagcttt ggcgtctaga tttgggacga aacattcgag ctggagccca ttatagccag
540 tttctcgtct atgattttga tggaaacggt cgttcagaag tagtcttaaa
aacagcagac 600 ggaacgattg atggagttgg caacgtgata ggggatcaag
acgccgatta tcgcaactca 660 tcaggttaca ttttagatgg tcctgaatac
ttgacgatct tttctgggga aacaggcgaa 720 gcgctagaca cgattgacta
tgttccgcca cgtgggaatg tcagtgattg gggcgacaat 780 tatggtaatc
gtgtagaccg cttcctagca ggtgtggctt atttagacgg agaaagacca 840
agctttgtaa tggctagagg ctattatacg cgcacggtgc tagctgctta tcaatgggac
900 gatgggaaaa taaaagagca atgggtgttt gatagcaatg atccaggaaa
tgaacgctat 960 gcagggcaag gtaaccatag tctagcaatc gcggatgtag
acggtgatgg caaggatgag 1020 attatctatg gtgctatggt ggtggatcac
gatggcactg ggctttattc gactggctgg 1080 ggccatggag atgctaacca
tgtgagcaac ctaaatccga atcgcaaagg gttggagatt 1140 tttcagcctc
atgaagactc gcgctctcct gtgggctacg gtattcggga tgcagagacg 1200
ggtgagctgc tctggggtga atttacaggg accgacgttg gacgggcgtt ggcagccgat
1260 attgatccgc gttttgatgg ggcagagcta tgggcatctg ctcaatggga
tgggcgcgaa 1320 ggaagtggcc tattttccgt tgaaggcgaa tccattacga
caaaaacccc acaatcggtt 1380 aattttgcga tttggtggac gggtgatttg
ctgcgtgagc tccttgatca ttcctttgac 1440 ccgagcaaag atccgcatgg
ggttggaaaa atcgagaagt gggattggga aaaggaagag 1500 ctagtggaga
ttttcgttcc agaagggaca aggtcaaaca actggacgaa aggtaaccca 1560
tccttacaag ccgacttgtt tggggattgg cgggaggaag ttatttggcc atctgctgat
1620 agtaacgagc tacgaatcta taccacgacc gaagaaacag agcaccgtat
cccaacactt 1680 atgcatgact ctgtctatcg tttgagtgtt gcttggcaaa
atgtcggata taatcagcca 1740 ccgcatacga gctacttcct cggccacggc
atgaaggaag ccccgctacc aaaagtgcat 1800 gcaggacaag tagtaccagt
tgagctgaaa gcaaatcagc aagggaaaaa gaagctatcg 1860 gttcaagtga
gattcgattc accaacggcg ggagaatccc tcgtatcatc atctgtcaga 1920
ttattcgtca atggggaaac aatccaagca gagaaagtac acagg 1965 14 655 PRT
Bacillus halodurans C4539 14 Met Leu Arg Gln Lys Glu Gln Leu Asp
Arg Gly Leu Val Ala Val Lys 1 5 10 15 Arg Ala Asp Gly Val Phe Leu
Ser Trp Arg Leu Leu Gly Thr Glu His 20 25 30 Pro Leu Thr Val Phe
His Val Tyr Arg Asp Gly Glu Lys Ile Thr Lys 35 40 45 Ala Gly Leu
Gln Glu Gly Thr Asn Phe Val Asp Ala Asp Gly Met Thr 50 55 60 Asp
Ser Val Tyr Gln Ile Lys Ala Val Ala Gly Lys Asp Glu Asp Met 65 70
75 80 Ser Asn Pro Val Ser Val Trp Asp Asp Glu Tyr Leu Ala Ile Pro
Leu 85 90 95 Asp Lys Pro Glu Gly Gly Val Thr Pro Asp Gly Val Ser
Tyr Glu Tyr 100 105 110 Thr Ala Asn Asp Ala Ser Val Gly Asp Leu Asp
Gly Asp Gly Gln Tyr 115 120 125 Glu Ile Ile Leu Lys Trp Asp Pro Thr
Asn Ser Lys Asp Asn Ser Arg 130 135 140 Ser Gly Tyr Thr Gly Asn Val
Tyr Leu Asp Ala Tyr Lys Leu Asp Gly 145 150 155 160 Thr Lys Leu Trp
Arg Leu Asp Leu Gly Arg Asn Ile Arg Ala Gly Ala 165 170 175 His Tyr
Ser Gln Phe Leu Val Tyr Asp Phe Asp Gly Asn Gly Arg Ser 180 185 190
Glu Val Val Leu Lys Thr Ala Asp Gly Thr Ile Asp Gly Val Gly Asn 195
200 205 Val Ile Gly Asp Gln Asp Ala Asp Tyr Arg Asn Ser Ser Gly Tyr
Ile 210 215 220 Leu Asp Gly Pro Glu Tyr Leu Thr Ile Phe Ser Gly Glu
Thr Gly Glu 225 230 235 240 Ala Leu Asp Thr Ile Asp Tyr Val Pro Pro
Arg Gly Asn Val Ser Asp 245 250 255 Trp Gly Asp Asn Tyr Gly Asn Arg
Val Asp Arg Phe Leu Ala Gly Val 260 265 270 Ala Tyr Leu Asp Gly Glu
Arg Pro Ser Phe Val Met Ala Arg Gly Tyr 275 280 285 Tyr Thr Arg Thr
Val Leu Ala Ala Tyr Gln Trp Asp Asp Gly Lys Ile 290 295 300 Lys Glu
Gln Trp Val Phe Asp Ser Asn Asp Pro Gly Asn Glu Arg Tyr 305 310 315
320 Ala Gly Gln Gly Asn His Ser Leu Ala Ile Ala Asp Val Asp Gly Asp
325 330 335 Gly Lys Asp Glu Ile Ile Tyr Gly Ala Met Val Val Asp His
Asp Gly 340 345 350 Thr Gly Leu Tyr Ser Thr Gly Trp Gly His Gly Asp
Ala Asn His Val 355 360 365 Ser Asn Leu Asn Pro Asn Arg Lys Gly Leu
Glu Ile Phe Gln Pro His 370 375 380 Glu Asp Ser Arg Ser Pro Val Gly
Tyr Gly Ile Arg Asp Ala Glu Thr 385 390 395 400 Gly Glu Leu Leu Trp
Gly Glu Phe Thr Gly Thr Asp Val Gly Arg Ala 405 410 415 Leu Ala Ala
Asp Ile Asp Pro Arg Phe Asp Gly Ala Glu Leu Trp Ala 420 425 430 Ser
Ala Gln Trp Asp Gly Arg Glu Gly Ser Gly Leu Phe Ser Val Glu 435 440
445 Gly Glu Ser Ile Thr Thr Lys Thr Pro Gln Ser Val Asn Phe Ala Ile
450 455 460 Trp Trp Thr Gly Asp Leu Leu Arg Glu Leu Leu Asp His Ser
Phe Asp 465 470 475 480 Pro Ser Lys Asp Pro His Gly Val Gly Lys Ile
Glu Lys Trp Asp Trp 485 490 495 Glu Lys Glu Glu Leu Val Glu Ile Phe
Val Pro Glu Gly Thr Arg Ser 500 505 510 Asn Asn Trp Thr Lys Gly Asn
Pro Ser Leu Gln Ala Asp Leu Phe Gly 515 520 525 Asp Trp Arg Glu Glu
Val Ile Trp Pro Ser Ala Asp Ser Asn Glu Leu 530 535 540 Arg Ile Tyr
Thr Thr Thr Glu Glu Thr Glu His Arg Ile Pro Thr Leu 545 550 555 560
Met His Asp Ser Val Tyr Arg Leu Ser Val Ala Trp Gln Asn Val Gly 565
570 575 Tyr Asn Gln Pro
Pro His Thr Ser Tyr Phe Leu Gly His Gly Met Lys 580 585 590 Glu Ala
Pro Leu Pro Lys Val His Ala Gly Gln Val Val Pro Val Glu 595 600 605
Leu Lys Ala Asn Gln Gln Gly Lys Lys Lys Leu Ser Val Gln Val Arg 610
615 620 Phe Asp Ser Pro Thr Ala Gly Glu Ser Leu Val Ser Ser Ser Val
Arg 625 630 635 640 Leu Phe Val Asn Gly Glu Thr Ile Gln Ala Glu Lys
Val His Arg 645 650 655 15 1896 DNA Streptomyces coelicolor 15
gtgaggcacc cccacacccg cccccacgcc ccccacccgc accgcagacg cccacgcgcc
60 ctggccgcgg ccctcgccgc cgccgggctc ctcggcgcgg gcctgacgac
gctggccccc 120 gacaccgccg aggccgccac ggcacgccag gtcgaggccc
tggaccgggg cgtcgtcagc 180 gtccacaccg gcgacgggaa cctggtcagc
tggcgctggc tgggcaccga cccggacaac 240 gtcgcgttca acgtctaccg
ggccggtacg aaggtcaact ccagccccgt caccggctcc 300 accacctact
tccactccgg cgcgccctcc cacgccgact acaccgtccg cgcggtcgtg 360
aacggcacgg agcagggcga ctccgtccac gcgatccagt tccgggccgg ctacaaggac
420 gtaccgatca gcccgccctc cggcggcacc acccccgacg gcgtctccta
cacctacgag 480 gccaacgacg cctccgtcgg cgacctcgac ggcgacggcg
ccctcgacct cgtcctcaag 540 tggcagccga ccaacgccaa ggacaactcc
cagtccggct acaccggcaa cacggtcgtc 600 gacggcatca agctcgacgg
cacccgcctg tggcgcgtcg acctgggccg caacatccgc 660 tccggcgccc
actacaccca gttccaggtg tacgactacg acggcgacgg ccgggccgag 720
gtcgccatga agaccgccga cggcaccaag gacggcaccg gcgcggtcat cggcaactcc
780 tcggcggatc accgcaactc gagcggctac gtcctctccg gccccgaata
cctcaccatg 840 ttcaacggcc ggaccggcac cgcgatgggg accgtcgact
acgtcccggc ccgcggctcg 900 gtctcctcct ggggcgactc ctacggcaac
cgcgtcgacc ggttcctggc gggcacggcg 960 tacctggacg gctcccgccc
ctccgtgatt atggcgcgcg ggtactacac gcgcacggtg 1020 atcgcggcct
gggactggcg ggacggccgg ttcacccgcc gctggacctt cgacaccaac 1080
tcctccacca acagcggcaa gggctacgac ggccagggca accaccagct ctccgtcgcg
1140 gacgtggacg gtgacggccg ggacgagatc gtctacggcg cgatggccgt
cgacgacaac 1200 ggctacgccc tgtggaccac caggaacggc cacggcgacg
ccatgcacgt cggcgacctc 1260 gacccgtccc gggcgggcct ggaggagttc
aaggtcgacg aggacggctc gaagccctcg 1320 tcgtacctgg cggacgcccg
cacgggccag atcctctggt ccaccggcgc gagcggcgac 1380 aacggccgcg
gtgtctccgg ggacatctgg tcgggcagcg cgggcgccga gtcctggtcg 1440
tccgcggaga gcggcatccg caaccccaag ggcaccgtcg tcggcagccg caagccctcc
1500 agcgccaact tcctttcctg gtgggacggc gacaccgtcc gtgaactcct
cgacggcacc 1560 cacgtcgaca agtacggcac ctcgggcgac acccgcctgc
tcaccggctc cggcgtcgcc 1620 tccaacaacg gcaccaaggc caccccggtc
ctggccggcg acatcctcgg cgactggcgc 1680 gaggaggtcg tctggcgcac
gtcgaacaac acggccctgc gcatctactc caccccctac 1740 gacacggaca
cccgcatcac gaccctcctc cacgacaccc agtaccgcac cgcactggcc 1800
tggcagaaca ccgcctacaa ccagccaccg cacccgagct tcttcctcgg aagcgggatg
1860 ccgacggccc cccggccgtc ggtccacacg ccctga 1896 16 631 PRT
Streptomyces coelicolor 16 Val Arg His Pro His Thr Arg Pro His Ala
Pro His Pro His Arg Arg 1 5 10 15 Arg Pro Arg Ala Leu Ala Ala Ala
Leu Ala Ala Ala Gly Leu Leu Gly 20 25 30 Ala Gly Leu Thr Thr Leu
Ala Pro Asp Thr Ala Glu Ala Ala Thr Ala 35 40 45 Arg Gln Val Glu
Ala Leu Asp Arg Gly Val Val Ser Val His Thr Gly 50 55 60 Asp Gly
Asn Leu Val Ser Trp Arg Trp Leu Gly Thr Asp Pro Asp Asn 65 70 75 80
Val Ala Phe Asn Val Tyr Arg Ala Gly Thr Lys Val Asn Ser Ser Pro 85
90 95 Val Thr Gly Ser Thr Thr Tyr Phe His Ser Gly Ala Pro Ser His
Ala 100 105 110 Asp Tyr Thr Val Arg Ala Val Val Asn Gly Thr Glu Gln
Gly Asp Ser 115 120 125 Val His Ala Ile Gln Phe Arg Ala Gly Tyr Lys
Asp Val Pro Ile Ser 130 135 140 Pro Pro Ser Gly Gly Thr Thr Pro Asp
Gly Val Ser Tyr Thr Tyr Glu 145 150 155 160 Ala Asn Asp Ala Ser Val
Gly Asp Leu Asp Gly Asp Gly Ala Leu Asp 165 170 175 Leu Val Leu Lys
Trp Gln Pro Thr Asn Ala Lys Asp Asn Ser Gln Ser 180 185 190 Gly Tyr
Thr Gly Asn Thr Val Val Asp Gly Ile Lys Leu Asp Gly Thr 195 200 205
Arg Leu Trp Arg Val Asp Leu Gly Arg Asn Ile Arg Ser Gly Ala His 210
215 220 Tyr Thr Gln Phe Gln Val Tyr Asp Tyr Asp Gly Asp Gly Arg Ala
Glu 225 230 235 240 Val Ala Met Lys Thr Ala Asp Gly Thr Lys Asp Gly
Thr Gly Ala Val 245 250 255 Ile Gly Asn Ser Ser Ala Asp His Arg Asn
Ser Ser Gly Tyr Val Leu 260 265 270 Ser Gly Pro Glu Tyr Leu Thr Met
Phe Asn Gly Arg Thr Gly Thr Ala 275 280 285 Met Gly Thr Val Asp Tyr
Val Pro Ala Arg Gly Ser Val Ser Ser Trp 290 295 300 Gly Asp Ser Tyr
Gly Asn Arg Val Asp Arg Phe Leu Ala Gly Thr Ala 305 310 315 320 Tyr
Leu Asp Gly Ser Arg Pro Ser Val Ile Met Ala Arg Gly Tyr Tyr 325 330
335 Thr Arg Thr Val Ile Ala Ala Trp Asp Trp Arg Asp Gly Arg Phe Thr
340 345 350 Arg Arg Trp Thr Phe Asp Thr Asn Ser Ser Thr Asn Ser Gly
Lys Gly 355 360 365 Tyr Asp Gly Gln Gly Asn His Gln Leu Ser Val Ala
Asp Val Asp Gly 370 375 380 Asp Gly Arg Asp Glu Ile Val Tyr Gly Ala
Met Ala Val Asp Asp Asn 385 390 395 400 Gly Tyr Ala Leu Trp Thr Thr
Arg Asn Gly His Gly Asp Ala Met His 405 410 415 Val Gly Asp Leu Asp
Pro Ser Arg Ala Gly Leu Glu Glu Phe Lys Val 420 425 430 Asp Glu Asp
Gly Ser Lys Pro Ser Ser Tyr Leu Ala Asp Ala Arg Thr 435 440 445 Gly
Gln Ile Leu Trp Ser Thr Gly Ala Ser Gly Asp Asn Gly Arg Gly 450 455
460 Val Ser Gly Asp Ile Trp Ser Gly Ser Ala Gly Ala Glu Ser Trp Ser
465 470 475 480 Ser Ala Glu Ser Gly Ile Arg Asn Pro Lys Gly Thr Val
Val Gly Ser 485 490 495 Arg Lys Pro Ser Ser Ala Asn Phe Leu Ser Trp
Trp Asp Gly Asp Thr 500 505 510 Val Arg Glu Leu Leu Asp Gly Thr His
Val Asp Lys Tyr Gly Thr Ser 515 520 525 Gly Asp Thr Arg Leu Leu Thr
Gly Ser Gly Val Ala Ser Asn Asn Gly 530 535 540 Thr Lys Ala Thr Pro
Val Leu Ala Gly Asp Ile Leu Gly Asp Trp Arg 545 550 555 560 Glu Glu
Val Val Trp Arg Thr Ser Asn Asn Thr Ala Leu Arg Ile Tyr 565 570 575
Ser Thr Pro Tyr Asp Thr Asp Thr Arg Ile Thr Thr Leu Leu His Asp 580
585 590 Thr Gln Tyr Arg Thr Ala Leu Ala Trp Gln Asn Thr Ala Tyr Asn
Gln 595 600 605 Pro Pro His Pro Ser Phe Phe Leu Gly Ser Gly Met Pro
Thr Ala Pro 610 615 620 Arg Pro Ser Val His Thr Pro 625 630 17 1168
DNA Bacillus halodurans KJ59 17 atgaacaagt tagggatgtg gttctctgga
ttgatcttag tagttggtct attggtcgga 60 gggaacgaag cgaaggcaaa
tgaagtggtg aatgcaaggg attttggtgc gactccaggg 120 gtagcaacct
cacaaacaaa tgcgcttcat gcggcgatgc gtcattttta tgatcgcggg 180
gtgcaaggaa cggtctatat tccggcggga acttattcaa ttgacgaagc gctcaggttt
240 cactcaggtg taaacatcgt tggtgatggg atgggaagga ccattttaaa
gaagacagga 300 aacagtaaca attatgtggt tggcaatccc attatgagag
ggtcgaacaa cctcaatgtg 360 acggtttcta atttaacgat tgatgccgat
cgaacgaacc gagcgcagcg gggattaggg 420 caagtcgggg gcatgaatct
tgacgcggat gtgagcaatt taacgttaga gcgtgtcgaa 480 gtacgtgatg
ccacgattgg gcttttatta agacggttaa aaaactcggt tgttagagat 540
agtgtgatcg acaatacgac tggtcatggg atcgcatttg gtcatgaaaa tcatccgatt
600 ggagatgttc gcaacaacct tattacagga aaccgaatta cgaattctac
tggcggtagt 660 gggattaacc tgtcacgggc cacgtatacg actgttaccc
ataaccaagt gataaatgat 720 cgacaacagg atgactcgta tggtggcatt
cgaattccaa atggtgggga gcataatacg 780 gttgaatata atacgattcg
aaactatcca cgaggaatct ttgtattaag cggtgcaagg 840 cataaccaaa
tcaatcataa caccgtcatt gattcgagaa ttcatggagt gttgatccaa 900
gctgatcata acacgcttcg tgaaaaccgg attcagcagc ttaacagctc gttaaatccg
960 gagtcggtgg ttcgcatcgc cccgggtagc aataattcca ttctcaataa
caacatccaa 1020 gcccattcga actttcgaaa tatcgggatt cgggtgacgg
gggattcgaa caacaatgtg 1080 attcgcaata accgaatcgg cacccaaggc
acacttgtaa gtatagaagg tgggcgtaac 1140 aatgtgaatg aagggaacgt
acgacaat 1168 18 389 PRT Bacillus halodurans KJ59 18 Met Asn Lys
Leu Gly Met Trp Phe Ser Gly Leu Ile Leu Val Val Gly 1 5 10 15 Leu
Leu Val Gly Gly Asn Glu Ala Lys Ala Asn Glu Val Val Asn Ala 20 25
30 Arg Asp Phe Gly Ala Thr Pro Gly Val Ala Thr Ser Gln Thr Asn Ala
35 40 45 Leu His Ala Ala Met Arg His Phe Tyr Asp Arg Gly Val Gln
Gly Thr 50 55 60 Val Tyr Ile Pro Ala Gly Thr Tyr Ser Ile Asp Glu
Ala Leu Arg Phe 65 70 75 80 His Ser Gly Val Asn Ile Val Gly Asp Gly
Met Gly Arg Thr Ile Leu 85 90 95 Lys Lys Thr Gly Asn Ser Asn Asn
Tyr Val Val Gly Asn Pro Ile Met 100 105 110 Arg Gly Ser Asn Asn Leu
Asn Val Thr Val Ser Asn Leu Thr Ile Asp 115 120 125 Ala Asp Arg Thr
Asn Arg Ala Gln Arg Gly Leu Gly Gln Val Gly Gly 130 135 140 Met Asn
Leu Asp Ala Asp Val Ser Asn Leu Thr Leu Glu Arg Val Glu 145 150 155
160 Val Arg Asp Ala Thr Ile Gly Leu Leu Leu Arg Arg Leu Lys Asn Ser
165 170 175 Val Val Arg Asp Ser Val Ile Asp Asn Thr Thr Gly His Gly
Ile Ala 180 185 190 Phe Gly His Glu Asn His Pro Ile Gly Asp Val Arg
Asn Asn Leu Ile 195 200 205 Thr Gly Asn Arg Ile Thr Asn Ser Thr Gly
Gly Ser Gly Ile Asn Leu 210 215 220 Ser Arg Ala Thr Tyr Thr Thr Val
Thr His Asn Gln Val Ile Asn Asp 225 230 235 240 Arg Gln Gln Asp Asp
Ser Tyr Gly Gly Ile Arg Ile Pro Asn Gly Gly 245 250 255 Glu His Asn
Thr Val Glu Tyr Asn Thr Ile Arg Asn Tyr Pro Arg Gly 260 265 270 Ile
Phe Val Leu Ser Gly Ala Arg His Asn Gln Ile Asn His Asn Thr 275 280
285 Val Ile Asp Ser Arg Ile His Gly Val Leu Ile Gln Ala Asp His Asn
290 295 300 Thr Leu Arg Glu Asn Arg Ile Gln Gln Leu Asn Ser Ser Leu
Asn Pro 305 310 315 320 Glu Ser Val Val Arg Ile Ala Pro Gly Ser Asn
Asn Ser Ile Leu Asn 325 330 335 Asn Asn Ile Gln Ala His Ser Asn Phe
Arg Asn Ile Gly Ile Arg Val 340 345 350 Thr Gly Asp Ser Asn Asn Asn
Val Ile Arg Asn Asn Arg Ile Gly Thr 355 360 365 Gln Gly Thr Leu Val
Ser Ile Glu Gly Gly Arg Asn Asn Val Asn Glu 370 375 380 Gly Asn Val
Arg Gln 385 19 507 DNA Bacillus agaradhaerens misc_feature
(1)...(507) n= a, c, g or t 19 agatttttgg gaccgaggcc caggtgtttc
aggcaaagcc aaacggatgc cattacatgc 60 agcgatgcgt tatttctatg
atagaggtgt tagagggaaa ccgtctattt gccggcgggc 120 acatattcag
tggatagcgc cttacgcttt catcaagggg ttaatcttgt gggagatggt 180
gtaggacgaa caatcattaa aaaagtaggc agccaaaata attatgttgt aggtaaccct
240 atttttcgtg gggggacgac taatcttaat gtgacagtct ctcacatcac
ctttgatgca 300 gatcggacaa accgtgcgtc tcaaggtctt ggacaagtag
gtgggacttg gagaacagtt 360 tactgcgctc gtatccaatt taacgttgga
acacatagaa gtaagggatg ccactattgg 420 tntgcttgtt agaaggtaga
gattctgtta tttcagacag ccttantgat cggacgagtn 480 ggcatggtat
tgccacaggg agtgaat 507 20 169 PRT Bacillus agaradhaerens VARIANT
147 Xaa = any amino acid 20 Arg Asp Phe Trp Asp Arg Gly Pro Gly Val
Ser Gly Lys Ala Lys Arg 1 5 10 15 Met Pro Leu His Ala Ala Met Arg
Tyr Phe Tyr Asp Arg Gly Val Arg 20 25 30 Gly Lys Thr Val Tyr Leu
Pro Ala Gly Thr Tyr Ser Val Asp Ser Ala 35 40 45 Leu Arg Phe His
Gln Gly Val Asn Leu Val Gly Asp Gly Val Gly Arg 50 55 60 Thr Ile
Ile Lys Lys Val Gly Ser Gln Asn Asn Tyr Val Val Gly Asn 65 70 75 80
Pro Ile Phe Arg Gly Gly Thr Thr Asn Leu Asn Val Thr Val Ser His 85
90 95 Ile Thr Phe Asp Ala Asp Arg Thr Asn Arg Ala Ser Gln Gly Leu
Gly 100 105 110 Gln Val Gly Gly Thr Gly Glu Gln Phe Thr Ala Leu Val
Ser Asn Leu 115 120 125 Thr Leu Glu His Ile Glu Val Arg Asp Ala Thr
Ile Gly Leu Leu Val 130 135 140 Arg Arg Xaa Arg Ser Val Ile Ser Asp
Ser Leu Ile Asp Arg Thr Ser 145 150 155 160 Trp His Gly Ile Ala Thr
Gly Ser Glu 165 21 22 PRT Microbial VARIANT 11 Xaa= Met or Leu 21
Asn Ile Arg Ala Gly Ala His Thr Gln Phe Xaa Val Tyr Asp Xaa Asp 1 5
10 15 Gly Asp Gly Lys Ala Glu 20 22 11 PRT Microbial 22 Tyr Gly Asn
Arg Val Asp Arg Phe Leu Ala Gly 1 5 10 23 17 PRT Microbial VARIANT
12 Xaa= any amino acid 23 Tyr Gly Asn Arg Val Asp Arg Phe Leu Ala
Gly Xaa Ala Tyr Leu Asp 1 5 10 15 Gly 24 22 PRT Microbial VARIANT 7
Xaa= Asn or Ser 24 Ala Gly Gln Gly Asn His Xaa Leu Ser Xaa Ala Asp
Val Asp Gly Asp 1 5 10 15 Gly Lys Asp Glu Ile Ile 20 25 22 PRT
Microbial VARIANT 7 Xaa= Asn or Ser 25 Ala Gly Gln Gly Asn His Xaa
Leu Xaa Xaa Ala Asp Val Asp Gly Asp 1 5 10 15 Gly Lys Asp Glu Ile
Ile 20 26 10 PRT Microbial 26 Glu Val Arg Asp Ala Thr Ile Gly Leu
Leu 1 5 10 27 9 PRT Microbial 27 Asn Asn Tyr Val Val Gly Asn Pro
Ile 1 5 28 8 PRT Microbial 28 Asp Ala Asp Arg Thr Asn Arg Ala 1 5
29 6 PRT Microbial 29 Ser Ala Asn Asp Ala Ser 1 5 30 11 PRT
Microbial VARIANT 6 Xaa= Ser or Thr 30 Leu Lys Trp Asp Pro Xaa Asn
Ser Lys Asp Asn 1 5 10 31 8 PRT Microbial VARIANT 6 Xaa= Asp or Asn
31 Asp Ala Tyr Lys Leu Xaa Gly Thr 1 5 32 22 PRT Microbial VARIANT
11 Xaa= Met or Leu 32 Asn Ile Arg Ala Gly Ala His Thr Gln Phe Xaa
Val Tyr Asp Xaa Asp 1 5 10 15 Gly Asp Gly Lys Ala Glu 20 33 6 PRT
Microbial 33 Lys Thr Ala Asp Gly Thr 1 5 34 9 PRT Microbial VARIANT
6 Xaa= Tyr or Phe 34 Leu Ser Gly Pro Glu Xaa Leu Thr Val 1 5 35 11
PRT Microbial 35 Tyr Gly Asn Arg Val Asp Arg Phe Leu Ala Gly 1 5 10
36 17 PRT Microbial VARIANT 12 Xaa= any amino acid 36 Tyr Gly Asn
Arg Val Asp Arg Phe Leu Ala Gly Xaa Ala Tyr Leu Asp 1 5 10 15 Gly
37 22 PRT Microbial VARIANT 7 Xaa=Asn or Ser 37 Ala Gly Gln Gly Asn
His Xaa Leu Ser Xaa Ala Asp Val Asp Gly Asp 1 5 10 15 Gly Lys Asp
Glu Ile Ile 20 38 22 PRT Microbial VARIANT 7 Xaa= Asn or Ser 38 Ala
Gly Gln Gly Asn His Xaa Leu Xaa Xaa Ala Asp Val Asp Gly Asp 1 5 10
15 Gly Lys Asp Glu Ile Ile 20 39 7 PRT Mirobial 39 Leu Arg Ile Tyr
Thr Thr Thr 1 5 40 6 PRT Microbial 40 Tyr Thr Leu Met His Asp 1 5
41 17 PRT Microbial VARIANT 1 Xaa= Tyr or Pro 41 Xaa Thr Leu Met
His Asp Xaa Val Tyr Arg Leu Xaa Ile Ala Trp Gln 1 5 10 15 Asn 42 10
PRT Microbial VARIANT 5 Xaa= Ser or Gly 42 Val Tyr Arg Leu Xaa Ile
Ala Trp Gln Asn 1 5 10 43 10 PRT Microbial 43 Glu Val Arg Asp Ala
Thr Ile Gly Leu Leu 1 5 10 44 9 PRT Microbial 44 Asn Asn Tyr Val
Val Gly Asn Pro Ile 1 5 45 8 PRT Microbial 45 Asp Ala Asp Arg Thr
Asn Arg Ala 1 5 46 42 DNA Artificial Sequence Primer 46 gtcgccgggg
cggccgctat caattggtaa ctgtatctca gc 42 47 64 DNA Artificial
Sequence Primer 47 gtcgcccggg agctctgatc aggtaccaag cttgtcgacc
tgcagaatga ggcagcaaga 60 agat 64 48 61 DNA Artificial Sequence
Primer 48 gtcggcggcc gctgatcacg taccaagctt gtcgacctgc agaatgaggc
agcaagaaga 60 t 61 49 35 DNA Artificial Sequence Primer 49
gtcggagctc tatcaattgg taactgtatc tcagc 35 50 35 DNA Artificial
Sequence Primer 50 aacagctgat cacgactgat cttttagctt ggcac 35 51 37
DNA Artificial Sequence Primer 51 aactgcagcc gcggcacatc ataatgggac
aaatggg
37 52 38 DNA Artificial Sequence Primer 52 tcgccggaat tcgtgcagtg
tccgaaatag gcagatgc 38 53 37 DNA Artificial Sequence Primer 53
tcgccggcat gcgttctgtc tgtaccgcaa tcaaacc 37 54 34 DNA Artificial
Sequence Primer 54 gcagccgcgg cagaaggggc agcgcggcag atgg 34 55 39
DNA Artificial Sequence Primer 55 tcgccggcgg ccgcgttctg tctgtaccgc
aatcaaacc 39 56 45 DNA Artificial Sequence Primer 56 ctcgctgcag
cagcggcggc acccagacag gcggagaaca ttagc 45 57 37 DNA Artificial
Sequence Primer 57 cgacgacgtg cggccgccat tatgcgcctg ctcttcg 37 58
34 DNA Artificial Sequence Primer 58 gcagccgcgg cagacgggcg
gacggctgcg cagg 34 59 87 DNA Artificial Sequence Primer 59
gtgggcggcc gcgcctgaga aaatccgtag ccagcaccgc gctctgcagc agcggcgaaa
60 tgaagtggtg aatgcaaggg attttgg 87 60 49 DNA Artificial Sequence
Primer 60 gcgctctgca gcagcggcga aatgaagtgg tgaatgcaag ggattttgg 49
61 43 DNA Artificial Sequence Primer 61 gtcaggcgtg cggccgcggt
gtagaggtgc gatgatggat ggg 43
* * * * *
References