U.S. patent application number 15/884749 was filed with the patent office on 2018-06-21 for serine proteases.
The applicant listed for this patent is DANISCO US INC.. Invention is credited to LILIA MARIA BABE, ANJA HEMMINGSEN KELLETT-SMITH, MARC KOLKMAN, RIE MEJLDAL.
Application Number | 20180171320 15/884749 |
Document ID | / |
Family ID | 54478988 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180171320 |
Kind Code |
A1 |
KOLKMAN; MARC ; et
al. |
June 21, 2018 |
SERINE PROTEASES
Abstract
The present disclosure relates to serine proteases and variants
thereof. Compositions containing the serine proteases are suitable
for use in cleaning fabrics and hard surfaces, as well as in a
variety of industrial applications.
Inventors: |
KOLKMAN; MARC; (OEGSTGEEST,
NL) ; MEJLDAL; RIE; (OSTBIRK, DK) ;
KELLETT-SMITH; ANJA HEMMINGSEN; ( RHUS, DK) ; BABE;
LILIA MARIA; (EMERALD HILLS, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANISCO US INC. |
Palo Alto |
CA |
US |
|
|
Family ID: |
54478988 |
Appl. No.: |
15/884749 |
Filed: |
January 31, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15521386 |
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PCT/US15/57526 |
Oct 27, 2015 |
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15884749 |
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62069184 |
Oct 27, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/76 20130101;
C12N 15/78 20130101; C12N 9/6424 20130101; C12N 9/54 20130101; C12N
15/75 20130101; C12Y 304/21062 20130101; C12N 15/70 20130101; C12N
15/77 20130101; C11D 3/386 20130101 |
International
Class: |
C12N 9/64 20060101
C12N009/64; C12N 15/70 20060101 C12N015/70; C11D 3/386 20060101
C11D003/386; C12N 15/78 20060101 C12N015/78; C12N 15/75 20060101
C12N015/75; C12N 15/77 20060101 C12N015/77; C12N 15/76 20060101
C12N015/76 |
Claims
1. A BspAL03279-clade of subtilisins comprising a subtilisin or
recombinant polypeptide or active fragment thereof comprising a
DLGDXXRFGX.sub.aGLLXXXXAVX (SEQ ID NO:26) motif, wherein X is any
amino acid and X.sub.a is N or S.
2. The BspAL03279-clade of subtilisins according to claim 1,
wherein the subtilisin or recombinant polypeptide or active
fragment thereof further comprises an amino acid sequence having at
least 70% amino acid sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 3, 6, 12, and
15.
3. The BspAL03279-clade of subtilisins according to claim 1 or 2,
wherein X.sub.a is N.
4. The BspAL03279-clade of subtilisins according to claim 1 or 2,
with proviso that the subtilisin or recombinant polypeptide or
active fragment thereof does not comprise ADK62564.
5. The BspAL03279-clade of subtilisins of any of the above claims,
wherein the subtilisin and/or recombinant polypeptide or active
fragment has protease activity.
6. A recombinant polypeptide or active fragment thereof comprising
an amino acid sequence having at least 80% amino acid sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 3, 6, 9, 12, and 15, with the proviso
that the recombinant polypeptide or active fragment thereof does
not comprise ADK62564 or optionally, WP_035392836, WP_038476582,
WP_035392836, or WP_047989534.
7. A recombinant polypeptide or active fragment thereof of the
BspAL03279-clade.
8. The recombinant polypeptide or active fragment thereof of claim
7, wherein the recombinant polypeptide or active fragment thereof
comprises a DLGDXXRFGX.sub.aGLLNXXXAVX (SEQ ID NO:26) motif,
wherein X is any amino acid and X.sub.a is N or S.
9. The recombinant polypeptide or active fragment thereof according
to claim 7 or 8, wherein the recombinant polypeptide or an active
fragment thereof comprises an amino acid sequence having at least
70% amino acid sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 3, 6, 12, and 15.
10. The recombinant polypeptide or active fragment thereof of any
one of claims 7 to 9, wherein X.sub.a is N.
11. The recombinant polypeptide or an active fragment thereof of
any one of claims 7 to 9, with proviso that the subtilisin or
recombinant polypeptide or active fragment thereof does not
comprise ADK62564.
12. The recombinant polypeptide or an active fragment thereof of
any one of claims 6-11, wherein the polypeptide has protease
activity.
13. The recombinant polypeptide or an active fragment thereof of
claim 12, wherein the protease activity is subtilisin protease
activity on a dimethylcasein substrate.
14. A composition comprising a surfactant and a subtilisin or
recombinant polypeptide or active fragment thereof of any one of
claims 1-5, or the recombinant polypeptide or active fragment
thereof of any one of claims 6-13.
15. The composition of claim 14, wherein the surfactant is selected
from the group consisting of an anionic surfactant, a cationic
surfactant, a zwitterionic surfactant, an ampholytic surfactant, a
semi-polar non-ionic surfactant, and a combination thereof.
16. The composition of claim 14 or 15, wherein the composition is a
detergent composition.
17. The composition of claim 16, wherein the detergent composition
is selected from the group consisting of a laundry detergent, a
fabric softening detergent, a dishwashing detergent, and a
hard-surface cleaning detergent.
18. The composition of any one of claims 14-17, wherein said
composition further comprises at least one calcium ion and/or zinc
ion; at least one stabilizer; from about 0.001% to about 1.0 weight
% of said subtilisin or recombinant polypeptide or active fragment
thereof of any one of claims 1-5, or the recombinant polypeptide or
active fragment thereof of any one of claims 6-13; at least one
bleaching agent; at least one adjunct ingredient; and/or one or
more additional enzymes or enzyme derivatives selected from the
group consisting of acyl transferases, alpha-amylases,
beta-amylases, alpha-galactosidases, arabinosidases, aryl
esterases, beta-galactosidases, carrageenases, catalases,
cellobiohydrolases, cellulases, chondroitinases, cutinases,
endo-beta-1,4-glucanases, endo-beta-mannanases, esterases,
exo-mannanases, galactanases, glucoamylases, hemicellulases,
hyaluronidases, keratinases, laccases, lactases, ligninases,
lipases, lipoxygenases, mannanases, oxidases, pectate lyases,
pectin acetyl esterases, pectinases, pentosanases, peroxidases,
phenoloxidases, phosphatases, phospholipases, phytases,
polygalacturonases, proteases, pullulanases, reductases,
rhamnogalacturonases, beta-glucanases, tannases, transglutaminases,
xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases,
metalloproteases, additional serine proteases, and combinations
thereof.
19. The composition of any one of claims 14-18, wherein said
composition contains phosphate or is phosphate-free and/or contains
borate or is borate-free.
20. The composition of any one of claims 14-19, wherein said
composition is a granular, powder, solid, bar, liquid, tablet, gel,
paste or unit dose composition.
21. The composition of any one of claims 14-20, wherein said
composition is formulated at a pH of from about 8 to about 12.
22. A method of cleaning comprising contacting a surface or an item
in need of cleaning with the subtilisin or recombinant polypeptide
or active fragment thereof of any one of claims 1-5, the
recombinant polypeptide or active fragment thereof of any one of
claims 6-13, or the composition of any one of claims 14-21; and
optionally further comprising the step of rinsing said surface or
item after contacting said surface or item with said subtilisin,
recombinant polypeptide, or composition.
23. The method of claim 22, wherein said item is dishware or
fabric.
24. A polynucleotide comprising a nucleic acid sequence encoding
the subtilisin or recombinant polypeptide or active fragment
thereof of any one of claims 1-5, or the recombinant polypeptide or
active fragment thereof of any one of claims 6-13.
25. The polynucleotide of claim 24, wherein said polynucleotide
comprises a nucleic acid sequence having at least 70% identity to
the nucleic acid sequence of SEQ ID NO:1, 4, 10, 13, 16, 18, 22, or
24.
26. An expression vector comprising the polynucleotide of claim 24
or 25.
27. A host cell comprising the vector of claim 26.
28. The host cell of claim 27, wherein the host cell is of a
species selected from Bacillus spp., Streptomyces spp., Escherichia
spp., Aspergillus spp., Trichoderma spp., Pseudomonas spp.,
Corynebacterium spp., Saccharomyces spp., or Pichia spp.
29. The host cell of claim 28, wherein said Bacillus spp. is
Bacillus subtilis.
30. A method for producing the subtilisin or recombinant
polypeptide or active fragment thereof of any one of claims 1-5, or
the recombinant polypeptide or active fragment thereof of any one
of claims 6-13 comprising: (a) stably transforming the host cell of
any one of claims 27-29 with the expression vector of claim 26; (b)
cultivating said transformed host cell under conditions suitable
for said host cell to produce said subtilisin polypeptide; and (c)
recovering said subtilisin or polypeptide.
31. The method of claim 30, wherein said expression vector
comprises a heterologous polynucleotide sequence encoding a
heterologous pro-peptide.
32. The method of claim 30 or 31, wherein said expression vector
comprises one or both of a heterologous promoter and a
polynucleotide sequence encoding a heterologous signal peptide.
33. A textile, leather, or feather processing composition
comprising the subtilisin or recombinant polypeptide or active
fragment thereof of any one of claims 1-5, or the recombinant
polypeptide or active fragment thereof of any one of claims
6-13.
34. A composition comprising the subtilisin or recombinant
polypeptide or active fragment thereof of any one of claims 1-5, or
the recombinant polypeptide or active fragment thereof of any one
of claims 6-13, wherein said composition is an animal feed, contact
lens cleaning, or wound cleaning composition.
Description
[0001] This application is a Continuation of U.S. application Ser.
No. 15/521,386, filed Apr. 24, 2017, which is a 371 of
International Application No. PCT/US15/57526, filed Oct. 27, 2015
and claims the benefit of priority from U.S. Provisional
Application No. 62/069,184 filed Oct. 27, 2014, all of which are
herein incorporated by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named 20180130_NB40679USCNT_SeqLst.txt created on Jan.
30, 2018 and having a size of 103 kilobytes and is filed
concurrently with the specification. The sequence listing contained
in this ASCII formatted document is part of the specification and
is herein incorporated by reference in its entirety.
[0003] The present disclosure relates to serine proteases cloned
from Bacillus spp., and variants thereof. Compositions containing
the serine proteases are suitable for use in cleaning fabrics and
hard surfaces, as well as in a variety of industrial
applications.
[0004] Serine proteases are enzymes (EC No. 3.4.21) possessing an
active site serine that initiates hydrolysis of peptide bonds of
proteins. There are two broad categories of serine proteases, based
on their structure: chymotrypsin-like (trypsin-like) and
subtilisin-like. The prototypical subtilisin (EC No. 3.4.21.62) was
initially obtained from Bacillus subtilis. Subtilisins and their
homologues are members of the S8 peptidase family of the MEROPS
classification scheme. Members of family S8 have a catalytic triad
in the order Asp, His and Ser in their amino acid sequence.
[0005] Although serine proteases have long been known in the art of
industrial enzymes, there remains a need for further serine
proteases that are suitable for particular conditions and uses.
[0006] The present compositions and methods relate to recombinant
serine proteases cloned from Bacillus spp., and variants thereof.
Compositions containing the serine proteases are suitable for use
in cleaning fabrics and hard surfaces, as well as in a variety of
industrial applications.
[0007] In some embodiments, the invention is a BspAL03279-clade of
subtilisins. The BspAL03279-clade of subtilisins is characterized
by a common motif over the sequence that begins with Aspartic acid
(D250) and ends at position 269, according to BspAL03279 numbering.
In some embodiments, the invention is a recombinant polypeptide or
active fragment thereof of a BspAL03279-clade subtilisin. In
further embodiments, the BspAL03279-clade of subtilisins comprises
a subtilisin or recombinant polypeptide or active fragment thereof
comprising a DLGDXXRFGX.sub.aGLLXXXXAVX (SEQ ID NO:26) motif,
wherein X is any amino acid and X.sub.a is N or S ("Motif 1"). In
yet further embodiments, the BspAL03279-clade of subtilisins
comprises a subtilisin or recombinant polypeptide or active
fragment thereof comprising a DLGDXXRFGX.sub.aGLLXXXXAVX (SEQ ID
NO:26) motif, wherein X is any amino acid and X.sub.a is N ("Motif
2"). In yet still further embodiments, the BspAL03279-clade of
subtilisins comprises a subtilisin or recombinant polypeptide or
active fragment thereof comprising a DLGDXXRFGX.sub.aGLLXXXXAVX
(SEQ ID NO:26) motif, wherein X is any amino acid and X.sub.a is S
("Motif 3").
[0008] In some embodiments, the invention is a recombinant
polypeptide or active fragment thereof comprising an amino acid
sequence having at least 70% or 80% identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9,
12, and 15. In some embodiments, the recombinant polypeptide has
cleaning activity in a detergent composition, including an
automatic dish washing detergent and a laundry detergent.
[0009] In some embodiments, the invention is a composition
comprising a surfactant and the recombinant polypeptide stated
above. In some embodiments, the surfactant is selected from the
group consisting of a non-ionic surfactant, an anionic surfactant,
a cationic surfactant, a zwitterionic surfactant, an ampholytic
surfactant, a semi-polar non-ionic surfactant, and a combination
thereof. In some embodiments, the composition is a detergent
composition, such as a laundry detergent, a fabric softening
detergent, a dishwashing detergent, and a hard-surface cleaning
detergent. In some embodiments, the composition further comprises
at least one calcium ion and/or zinc ion, at least one stabilizer,
at least one bleaching agent, phosphate, or borate. In some
embodiments the composition is phosphate-free and/or borate-free.
In some embodiments, the composition is a granular, powder, solid,
bar, liquid, tablet, gel, paste or unit dose composition. In some
embodiments, the composition further comprising one or more
additional enzymes or enzyme derivatives selected from the group
consisting of acyl transferases, alpha-amylases, beta-amylases,
alpha-galactosidases, arabinosidases, aryl esterases,
beta-galactosidases, carrageenases, catalases, cellobiohydrolases,
cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases,
endo-beta-mannanases, esterases, exo-mannanases, galactanases,
glucoamylases, hemicellulases, hyaluronidases, keratinases,
laccases, lactases, ligninases, lipases, lipoxygenases, mannanases,
oxidases, pectate lyases, pectin acetyl esterases, pectinases,
pentosanases, peroxidases, phenoloxidases, phosphatases,
phospholipases, phytases, polygalacturonases, proteases,
pullulanases, reductases, rhamnogalacturonases, beta-glucanases,
tannases, transglutaminases, xylan acetyl-esterases, xylanases,
xyloglucanases, xylosidases, metalloproteases, additional serine
proteases, and combinations thereof.
[0010] In some embodiments, the invention is a method of cleaning,
comprising contacting a surface or an item with a composition
listed above. In some embodiments, the invention is a method for
producing a recombinant polypeptide comprising stably transforming
a host cell with an expression vector comprising a polynucleotide
encoding the recombinant polypeptide above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 provides a plasmid map for expression of BspAL03279
protease.
[0012] FIG. 2A-E provides MUSCLE multiple sequence alignment of
subtilisins including BspAL03279, BspAK01305, Bps02003, Bohn00569,
and Bpan04382.
[0013] FIG. 3 provides a phylogenetic tree of subtilisins including
BspAL03279, BspAK01305, Bps02003, Bohn00569, and Bpan04382.
[0014] Described are compositions and methods relating to
recombinant serine proteases from Bacillus species. The
compositions and methods are based, in part, on the observation
that recombinant BspAL03279, BspAK01305, Bps02003, Bohn00569, and
Bpan04382, among others, have protease activity in the presence of
a surfactant, in basic reaction conditions, and at elevated
temperatures. These features of BspAL03279, BspAK01305, Bps02003,
Bohn00569, and Bpan04382 make these proteases well suited for use
in cleaning fabrics and hard surfaces, as well as in textile,
leather and feather processing. The new proteases are also well
suited to inclusion in compositions for protein degradation,
including but not limited to laundry and dish washing
detergents.
[0015] Prior to describing the present compositions and methods in
detail, the following terms are defined for clarity. Terms and
abbreviations not defined should be accorded their ordinary meaning
as used in the art. Unless defined otherwise herein, all technical
and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art. Unless otherwise
indicated, the practice of the present disclosure involves
conventional techniques commonly used in molecular biology, protein
engineering, and microbiology. Although any methods and materials
similar or equivalent to those described herein find use in the
practice of the present disclosure, some suitable methods and
materials are described herein. The terms defined immediately below
are more fully described by reference to the Specification as a
whole.
[0016] As used herein, the singular "a," "an" and "the" includes
the plural unless the context clearly indicates otherwise. Unless
otherwise indicated, nucleic acid sequences are written left to
right in 5' to 3' orientation; and amino acid sequences are written
left to right in amino to carboxy orientation. It is to be
understood that this disclosure is not limited to the particular
methodology, protocols, and reagents described herein, absent an
indication to the contrary.
[0017] It is intended that every maximum numerical limitation given
throughout this Specification includes every lower numerical
limitation, as if such lower numerical limitations were expressly
written herein. Every minimum numerical limitation given throughout
this Specification will include every higher numerical limitation,
as if such higher numerical limitations were expressly written
herein. Every numerical range given throughout this Specification
will include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0018] As used herein in connection with a numerical value, the
term "about" refers to a range of +/-0.5 of the numerical value,
unless the term is otherwise specifically defined in context. For
instance, the phrase a "pH value of about 6" refers to pH values of
from 5.5 to 6.5, unless the pH value is specifically defined
otherwise.
[0019] As used herein, the terms "protease" and "proteinase" refer
to an enzyme that has the ability to break down proteins and
peptides. A protease has the ability to conduct "proteolysis," by
hydrolysis of peptide bonds that link amino acids together in a
peptide or polypeptide chain forming the protein. This activity of
a protease as a protein-digesting enzyme is referred to as
"proteolytic activity." Many well-known procedures exist for
measuring proteolytic activity. For example, proteolytic activity
may be ascertained by comparative assays that analyze the
respective protease's ability to hydrolyze a suitable substrate.
Exemplary substrates useful in the analysis of protease or
proteolytic activity, include, but are not limited to, di-methyl
casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine
elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111).
Colorimetric assays utilizing these substrates are well known in
the art (See e.g., WO99/34011 and U.S. Pat. No. 6,376,450). The pNA
peptidyl assay (See e.g., Del Mar et al., Anal Biochem, 99:316-320,
1979) also finds use in determining the active enzyme
concentration. This assay measures the rate at which p-nitroaniline
is released as the enzyme hydrolyzes a soluble synthetic substrate,
such as
succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide
(suc-AAPF-pNA). The rate of production of yellow color from the
hydrolysis reaction is measured at 410 nm on a spectrophotometer
and is proportional to the active enzyme concentration. In
addition, absorbance measurements at 280 nanometers (nm) can be
used to determine the total protein concentration in a sample of
purified protein. The activity on substrate/protein concentration
gives the enzyme specific activity.
[0020] The term "variant," with respect to a polypeptide, refers to
a polypeptide that differs from a specified wild-type, parental, or
reference polypeptide in that it includes one or more
naturally-occurring or man-made substitutions, insertions, or
deletions of an amino acid. Similarly, the term "variant," with
respect to a polynucleotide, refers to a polynucleotide that
differs in nucleotide sequence from a specified wild-type,
parental, or reference polynucleotide. The identity of the
wild-type, parental, or reference polypeptide or polynucleotide
will be apparent from context.
[0021] As used herein, "the genus Bacillus" includes all species
within the genus "Bacillus," as known to those of skill in the art,
including but not limited to B. subtilis, B. licheniformis, B.
lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.
coagulans, B. circulans, B. gibsonii, and B. thuringiensis. It is
recognized that the genus Bacillus continues to undergo taxonomical
reorganization. Thus, it is intended that the genus include species
that have been reclassified, including but not limited to such
organisms as Bacillus stearothermophilus, which is now named
"Geobacillus stearothermophilus", or Bacillus polymyxa, which is
now "Paenibacillus polymyxa" The production of resistant endospores
under stressful environmental conditions is considered the defining
feature of the genus Bacillus, although this characteristic also
applies to the recently named Alicyclobacillus, Amphibacillus,
Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus,
Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus,
Thermobacillus, Ureibacillus, and Virgibacillus.
[0022] As used herein, the term "mutation" refers to changes made
to a reference amino acid or nucleic acid sequence. It is intended
that the term encompass substitutions, insertions and
deletions.
[0023] As used herein, the term "vector" refers to a nucleic acid
construct used to introduce or transfer nucleic acid(s) into a
target cell or tissue. A vector is typically used to introduce
foreign DNA into a cell or tissue. Vectors include plasmids,
cloning vectors, bacteriophages, viruses (e.g., viral vector),
cosmids, expression vectors, shuttle vectors, and the like. A
vector typically includes an origin of replication, a multicloning
site, and a selectable marker. The process of inserting a vector
into a target cell is typically referred to as transformation. The
present invention includes, in some embodiments, a vector that
comprises a DNA sequence encoding a serine protease polypeptide
(e.g., precursor or mature serine protease polypeptide) that is
operably linked to a suitable prosequence (e.g., secretory, signal
peptide sequence, etc.) capable of effecting the expression of the
DNA sequence in a suitable host, and the folding and translocation
of the recombinant polypeptide chain.
[0024] As used herein in the context of introducing a nucleic acid
sequence into a cell, the term "introduced" refers to any method
suitable for transferring the nucleic acid sequence into the cell.
Such methods for introduction include but are not limited to
protoplast fusion, transfection, transformation, electroporation,
conjugation, and transduction. Transformation refers to the genetic
alteration of a cell which results from the uptake, optional
genomic incorporation, and expression of genetic material (e.g.,
DNA).
[0025] As used herein, a nucleic acid is "operably linked" with
another nucleic acid sequence when it is placed into a functional
relationship with another nucleic acid sequence. For example, a
promoter or enhancer is operably linked to a nucleotide coding
sequence if the promoter affects the transcription of the coding
sequence. A ribosome binding site may be operably linked to a
coding sequence if it is positioned so as to facilitate translation
of the coding sequence. Typically, "operably linked" DNA sequences
are contiguous. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, synthetic oligonucleotide
adaptors or linkers may be used in accordance with conventional
practice.
[0026] As used herein the term "gene" refers to a polynucleotide
(e.g., a DNA segment), that encodes a polypeptide and includes
regions preceding and following the coding regions. In some
instances a gene includes intervening sequences (introns) between
individual coding segments (exons).
[0027] As used herein, "recombinant" when used with reference to a
cell typically indicates that the cell has been modified by the
introduction of a foreign nucleic acid sequence or that the cell is
derived from a cell so modified. For example, a recombinant cell
may comprise a gene not found in identical form within the native
(non-recombinant) form of the cell, or a recombinant cell may
comprise a native gene (found in the native form of the cell) that
has been modified and re-introduced into the cell. A recombinant
cell may comprise a nucleic acid endogenous to the cell that has
been modified without removing the nucleic acid from the cell; such
modifications include those obtained by gene replacement,
site-specific mutation, and related techniques known to those of
ordinary skill in the art. Recombinant DNA technology includes
techniques for the production of recombinant DNA in vitro and
transfer of the recombinant DNA into cells where it may be
expressed or propagated, thereby producing a recombinant
polypeptide. "Recombination" and "recombining" of polynucleotides
or nucleic acids refer generally to the assembly or combining of
two or more nucleic acid or polynucleotide strands or fragments to
generate a new polynucleotide or nucleic acid.
[0028] A nucleic acid or polynucleotide is said to "encode" a
polypeptide if, in its native state or when manipulated by methods
known to those of skill in the art, it can be transcribed and/or
translated to produce the polypeptide or a fragment thereof. The
anti-sense strand of such a nucleic acid is also said to encode the
sequence.
[0029] The terms "host strain" and "host cell" refer to a suitable
host for an expression vector comprising a DNA sequence of
interest.
[0030] A "protein" or "polypeptide" comprises a polymeric sequence
of amino acid residues. The terms "protein" and "polypeptide" are
used interchangeably herein. The single and 3-letter code for amino
acids as defined in conformity with the IUPAC-IUB Joint Commission
on Biochemical Nomenclature (JCBN) is used throughout this
disclosure. The single letter X refers to any of the twenty amino
acids. It is also understood that a polypeptide may be coded for by
more than one nucleotide sequence due to the degeneracy of the
genetic code. Mutations can be named by the one letter code for the
parent amino acid, followed by a position number and then the one
letter code for the variant amino acid. For example, mutating
glycine (G) at position 87 to serine (S) is represented as "G087S"
or "G87S". When describing modifications, a position followed by
amino acids listed in parentheses indicates a list of substitutions
at that position by any of the listed amino acids. For example,
6(L,I) means position 6 can be substituted with a leucine or
isoleucine. At times, in a sequence, a slash (/) is used to define
substitutions, e.g. F/V, indicates that the particular position may
have a phenylalanine or valine at that position.
[0031] A "prosequence" or "propeptide sequence" refers to an amino
acid sequence between the signal peptide sequence and mature
protease sequence that is necessary for the proper folding and
secretion of the protease; they are sometimes referred to as
intramolecular chaperones. Cleavage of the prosequence or
propeptide sequence results in a mature active protease. Bacterial
serine proteases are often expressed as pro-enzymes.
[0032] The terms "signal sequence" and "signal peptide" refer to a
sequence of amino acid residues that may participate in the
secretion or direct transport of the mature or precursor form of a
protein. The signal sequence is typically located N-terminal to the
precursor or mature protein sequence. The signal sequence may be
endogenous or exogenous. A signal sequence is normally absent from
the mature protein. A signal sequence is typically cleaved from the
protein by a signal peptidase after the protein is transported.
[0033] The term "mature" form of a protein, polypeptide, or peptide
refers to the functional form of the protein, polypeptide, or
peptide without the signal peptide sequence and propeptide
sequence.
[0034] The term "precursor" form of a protein or peptide refers to
a mature form of the protein having a prosequence operably linked
to the amino or carbonyl terminus of the protein. The precursor may
also have a "signal" sequence operably linked to the amino terminus
of the prosequence. The precursor may also have additional
polypeptides that are involved in post-translational activity
(e.g., polypeptides cleaved therefrom to leave the mature form of a
protein or peptide).
[0035] The term "wild-type" in reference to an amino acid sequence
or nucleic acid sequence indicates that the amino acid sequence or
nucleic acid sequence is a native or naturally-occurring sequence.
As used herein, the term "naturally-occurring" refers to anything
(e.g., proteins, amino acids, or nucleic acid sequences) that is
found in nature. Conversely, the term "non-naturally occurring"
refers to anything that is not found in nature (e.g., recombinant
nucleic acids and protein sequences produced in the laboratory or
modification of the wild-type sequence).
[0036] As used herein with regard to amino acid residue positions,
"corresponding to" or "corresponds to" or "corresponds" refers to
an amino acid residue at the enumerated position in a protein or
peptide, or an amino acid residue that is analogous, homologous, or
equivalent to an enumerated residue in a protein or peptide. As
used herein, "corresponding region" generally refers to an
analogous position in a related proteins or a reference
protein.
[0037] The terms "derived from" and "obtained from" refer to not
only a protein produced or producible by a strain of the organism
in question, but also a protein encoded by a DNA sequence isolated
from such strain and produced in a host organism containing such
DNA sequence. Additionally, the term refers to a protein which is
encoded by a DNA sequence of synthetic and/or cDNA origin and which
has the identifying characteristics of the protein in question. To
exemplify, "proteases derived from Bacillus" refers to those
enzymes having proteolytic activity that are naturally produced by
Bacillus, as well as to serine proteases like those produced by
Bacillus sources but which through the use of genetic engineering
techniques are produced by other host cells transformed with a
nucleic acid encoding the serine proteases.
[0038] The term "identical" in the context of two polynucleotide or
polypeptide sequences refers to the nucleic acids or amino acids in
the two sequences that are the same when aligned for maximum
correspondence, as measured using sequence comparison or analysis
algorithms described below and known in the art.
[0039] As used herein, "% identity" or percent identity" or "PID"
refers to protein sequence identity. Percent identity may be
determined using standard techniques known in the art. Useful
algorithms include the BLAST algorithms (See, Altschul et al., J
Mol Biol, 215:403-410, 1990; and Karlin and Altschul, Proc Natl
Acad Sci USA, 90:5873-5787, 1993). The BLAST program uses several
search parameters, most of which are set to the default values. The
NCBI BLAST algorithm finds the most relevant sequences in terms of
biological similarity but is not recommended for query sequences of
less than 20 residues (Altschul et al., Nucleic Acids Res,
25:3389-3402, 1997; and Schaffer et al., Nucleic Acids Res,
29:2994-3005, 2001). Exemplary default BLAST parameters for a
nucleic acid sequence searches include: Neighboring words
threshold=11; E-value cutoff=10; Scoring Matrix=NUC.3.1 (match=1,
mismatch=-3); Gap Opening=5; and Gap Extension=2. Exemplary default
BLAST parameters for amino acid sequence searches include: Word
size=3; E-value cutoff=10; Scoring Matrix=BLOSUM62; Gap Opening=11;
and Gap extension=1. A percent (%) amino acid sequence identity
value is determined by the number of matching identical residues
divided by the total number of residues of the "reference" sequence
including any gaps created by the program for optimal/maximum
alignment. BLAST algorithms refer to the "reference" sequence as
the "query" sequence.
[0040] As used herein, "homologous proteins" or "homologous
proteases" refers to proteins that have distinct similarity in
primary, secondary, and/or tertiary structure. Protein homology can
refer to the similarity in linear amino acid sequence when proteins
are aligned. Homologous search of protein sequences can be done
using BLASTP and PSI-BLAST from NCBI BLAST with threshold (E-value
cut-off) at 0.001 (Altschul S F, Madde T L, Shaffer A A, Zhang J,
Zhang Z, Miller W, Lipman D J. Gapped BLAST and PSI BLAST a new
generation of protein database search programs. Nucleic Acids Res
1997 Set 1; 25(17):3389-402). Using this information, proteins
sequences can be grouped. A phylogenetic tree can be built using
the amino acid sequences. Amino acid sequences can be entered in a
program such as the Vector NTI Advance suite and a Guide Tree can
be created using the Neighbor Joining (NJ) method (Saitou and Nei,
Mol Biol Evol, 4:406-425, 1987). The tree construction can be
calculated using Kimura's correction for sequence distance and
ignoring positions with gaps. A program such as AlignX can display
the calculated distance values in parenthesis following the
molecule name displayed on the phylogenetic tree.
[0041] Another useful algorithm for comparison of multiple protein
sequences is the MUSCLE program from Geneious software (Biomatters
Ltd.) (Robert C. Edgar. MUSCLE: multiple sequence alignment with
high accuracy and high throughput Nucl. Acids Res. (2004) 32 (5):
1792-1797).
[0042] Understanding the homology between molecules can reveal the
evolutionary history of the molecules as well as information about
their function; if a newly sequenced protein is homologous to an
already characterized protein, there is a strong indication of the
new protein's biochemical function. The most fundamental
relationship between two entities is homology; two molecules are
said to be homologous if they have been derived from a common
ancestor. Homologous molecules, or homologs, can be divided into
two classes, paralogs and orthologs. Paralogs are homologs that are
present within one species. Paralogs often differ in their detailed
biochemical functions. Orthologs are homologs that are present
within different species and have very similar or identical
functions. A protein superfamily is the largest grouping (clade) of
proteins for which common ancestry can be inferred. Usually this
common ancestry is based on sequence alignment and mechanistic
similarity. Superfamilies typically contain several protein
families which show sequence similarity within the family. The term
"protein clan" is commonly used for protease superfamilies based on
the MEROPS protease classification system.
[0043] The CLUSTAL W algorithm is another example of a sequence
alignment algorithm (See, Thompson et al., Nucleic Acids Res,
22:4673-4680, 1994). Default parameters for the CLUSTAL W algorithm
include: Gap opening penalty=10.0; Gap extension penalty=0.05;
Protein weight matrix=BLOSUM series; DNA weight matrix=IUB; Delay
divergent sequences %=40; Gap separation distance=8; DNA
transitions weight=0.50; List hydrophilic residues=GPSNDQEKR; Use
negative matrix=OFF; Toggle Residue specific penalties=ON; Toggle
hydrophilic penalties=ON; and Toggle end gap separation
penalty=OFF. In CLUSTAL algorithms, deletions occurring at either
terminus are included. For example, a variant with a five amino
acid deletion at either terminus (or within the polypeptide) of a
polypeptide of 500 amino acids would have a percent sequence
identity of 99% (495/500 identical residues.times.100) relative to
the "reference" polypeptide. Such a variant would be encompassed by
a variant having "at least 99% sequence identity" to the
polypeptide.
[0044] A nucleic acid or polynucleotide is "isolated" when it is at
least partially or completely separated from other components,
including but not limited to for example, other proteins, nucleic
acids, cells, etc. Similarly, a polypeptide, protein or peptide is
"isolated" when it is at least partially or completely separated
from other components, including but not limited to for example,
other proteins, nucleic acids, cells, etc. On a molar basis, an
isolated species is more abundant than are other species in a
composition. For example, an isolated species may comprise at least
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, or about 100% (on a
molar basis) of all macromolecular species present. Preferably, the
species of interest is purified to essential homogeneity (i.e.,
contaminant species cannot be detected in the composition by
conventional detection methods). Purity and homogeneity can be
determined using a number of techniques well known in the art, such
as agarose or polyacrylamide gel electrophoresis of a nucleic acid
or a protein sample, respectively, followed by visualization upon
staining. If desired, a high-resolution technique, such as high
performance liquid chromatography (HPLC) or a similar means can be
utilized for purification of the material.
[0045] The term "purified" as applied to nucleic acids or
polypeptides generally denotes a nucleic acid or polypeptide that
is essentially free from other components as determined by
analytical techniques well known in the art (e.g., a purified
polypeptide or polynucleotide forms a discrete band in an
electrophoretic gel, chromatographic eluate, and/or a media
subjected to density gradient centrifugation). For example, a
nucleic acid or polypeptide that gives rise to essentially one band
in an electrophoretic gel is "purified." A purified nucleic acid or
polypeptide is at least about 50% pure, usually at least about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about
99.7%, about 99.8% or more pure (e.g., percent by weight on a molar
basis). In a related sense, a composition is enriched for a
molecule when there is a substantial increase in the concentration
of the molecule after application of a purification or enrichment
technique. The term "enriched" refers to a compound, polypeptide,
cell, nucleic acid, amino acid, or other specified material or
component that is present in a composition at a relative or
absolute concentration that is higher than a starting
composition.
[0046] As used herein, the term "functional assay" refers to an
assay that provides an indication of a protein's activity. In some
embodiments, the term refers to assay systems in which a protein is
analyzed for its ability to function in its usual capacity. For
example, in the case of a protease, a functional assay involves
determining the effectiveness of the protease to hydrolyze a
proteinaceous substrate.
[0047] The term "cleaning activity" refers to a cleaning
performance achieved by a serine protease polypeptide or reference
protease under conditions prevailing during the proteolytic,
hydrolyzing, cleaning, or other process of the disclosure. In some
embodiments, cleaning performance of a serine protease polypeptide
or reference protease may be determined by using various assays for
cleaning one or more various enzyme sensitive stains on an item or
surface (e.g., a stain resulting from food, grass, blood, ink,
milk, oil, and/or egg protein). Cleaning performance of a variant
or reference protease can be determined by subjecting the stain on
the item or surface to standard wash condition(s) and assessing the
degree to which the stain is removed by using various
chromatographic, spectrophotometric, or other quantitative
methodologies. Exemplary cleaning assays and methods are known in
the art and include, but are not limited to those described in
WO99/34011 and U.S. Pat. No. 6,605,458, both of which are herein
incorporated by reference, as well as those cleaning assays and
methods included in the Examples provided below.
[0048] The term "cleaning effective amount" of a serine protease
polypeptide or reference protease refers to the amount of protease
that achieves a desired level of enzymatic activity in a specific
cleaning composition. Such effective amounts are readily
ascertained by one of ordinary skill in the art and are based on
many factors, such as the particular protease used, the cleaning
application, the specific composition of the cleaning composition,
and whether a liquid or dry (e.g., granular, tablet, bar)
composition is required, etc.
[0049] The term "cleaning adjunct material" refers to any liquid,
solid, or gaseous material included in cleaning composition other
than a serine protease polypeptide of the disclosure. In some
embodiments, the cleaning compositions of the present disclosure
include one or more cleaning adjunct materials. Each cleaning
adjunct material is typically selected depending on the particular
type and form of cleaning composition (e.g., liquid, granule,
powder, bar, paste, spray, tablet, gel, foam, or other
composition). Preferably, each cleaning adjunct material is
compatible with the protease enzyme used in the composition.
[0050] Cleaning compositions and cleaning formulations include any
composition that is suited for cleaning, bleaching, disinfecting,
and/or sterilizing any object, item, and/or surface. Such
compositions and formulations include, but are not limited to for
example, liquid and/or solid compositions, including cleaning or
detergent compositions (e.g., liquid, tablet, gel, bar, granule,
and/or solid laundry cleaning or detergent compositions and fine
fabric detergent compositions; hard surface cleaning compositions
and formulations, such as for glass, wood, ceramic and metal
counter tops and windows; carpet cleaners; oven cleaners; fabric
fresheners; fabric softeners; and textile, laundry booster cleaning
or detergent compositions, laundry additive cleaning compositions,
and laundry pre-spotter cleaning compositions; dishwashing
compositions, including hand or manual dishwashing compositions
(e.g., "hand" or "manual" dishwashing detergents) and automatic
dishwashing compositions (e.g., "automatic dishwashing
detergents"). Single dosage unit forms also find use with the
present invention, including but not limited to pills, tablets,
gelcaps, or other single dosage units such as pre-measured powders
or liquids.
[0051] Cleaning composition or cleaning formulations, as used
herein, include, unless otherwise indicated, granular or
powder-form all-purpose or heavy-duty washing agents, especially
cleaning detergents; liquid, granular, gel, solid, tablet, paste,
or unit dosage form all-purpose washing agents, especially the
so-called heavy-duty liquid (HDL) detergent or heavy-duty dry (HDD)
detergent types; liquid fine-fabric detergents; hand or manual
dishwashing agents, including those of the high-foaming type; hand
or manual dishwashing, automatic dishwashing, or dishware or
tableware washing agents, including the various tablet, powder,
solid, granular, liquid, gel, and rinse-aid types for household and
institutional use; liquid cleaning and disinfecting agents,
including antibacterial hand-wash types, cleaning bars,
mouthwashes, denture cleaners, car shampoos, carpet shampoos,
bathroom cleaners; hair shampoos and/or hair-rinses for humans and
other animals; shower gels and foam baths and metal cleaners; as
well as cleaning auxiliaries, such as bleach additives and
"stain-stick" or pre-treat types. In some embodiments, granular
compositions are in "compact" form; in some embodiments, liquid
compositions are in a "concentrated" form.
[0052] As used herein, "fabric cleaning compositions" include hand
and machine laundry detergent compositions including laundry
additive compositions and compositions suitable for use in the
soaking and/or pretreatment of stained fabrics (e.g., clothes,
linens, and other textile materials).
[0053] As used herein, "non-fabric cleaning compositions" include
non-textile (i.e., non-fabric) surface cleaning compositions,
including, but not limited to for example, hand or manual or
automatic dishwashing detergent compositions, oral cleaning
compositions, denture cleaning compositions, contact lens cleaning
compositions, wound debridement compositions, and personal
cleansing compositions.
[0054] As used herein, the term "detergent composition" or
"detergent formulation" is used in reference to a composition
intended for use in a wash medium for the cleaning of soiled or
dirty objects, including particular fabric and/or non-fabric
objects or items. Such compositions of the present disclosure are
not limited to any particular detergent composition or formulation.
Indeed, in some embodiments, the detergents of the disclosure
comprise at least one serine protease polypeptide of the disclosure
and, in addition, one or more surfactants, transferase(s),
hydrolytic enzymes, oxido reductases, builders (e.g., a builder
salt), bleaching agents, bleach activators, bluing agents,
fluorescent dyes, caking inhibitors, masking agents, enzyme
activators, antioxidants, and/or solubilizers. In some instances, a
builder salt is a mixture of a silicate salt and a phosphate salt,
preferably with more silicate (e.g., sodium metasilicate) than
phosphate (e.g., sodium tripolyphosphate). Some compositions of the
disclosure, such as, but not limited to, cleaning compositions or
detergent compositions, do not contain any phosphate (e.g.,
phosphate salt or phosphate builder).
[0055] As used herein, the term "bleaching" refers to the treatment
of a material (e.g., fabric, laundry, pulp, etc.) or surface for a
sufficient length of time and/or under appropriate pH and/or
temperature conditions to effect a brightening (i.e., whitening)
and/or cleaning of the material. Examples of chemicals suitable for
bleaching include, but are not limited to, for example, ClO.sub.2,
H.sub.2O.sub.2, peracids, NO.sub.2, etc.
[0056] As used herein, "wash performance" of a protease (e.g., a
serine protease polypeptide of the disclosure) refers to the
contribution of a serine protease polypeptide to washing that
provides additional cleaning performance to the detergent as
compared to the detergent without the addition of the serine
protease polypeptide to the composition. Wash performance is
compared under relevant washing conditions. In some test systems,
other relevant factors, such as detergent composition, sud
concentration, water hardness, washing mechanics, time, pH, and/or
temperature, can be controlled in such a way that condition(s)
typical for household application in a certain market segment
(e.g., hand or manual dishwashing, automatic dishwashing, dishware
cleaning, tableware cleaning, fabric cleaning, etc.) are
imitated.
[0057] The term "relevant washing conditions" is used herein to
indicate the conditions, particularly washing temperature, time,
washing mechanics, sud concentration, type of detergent and water
hardness, actually used in households in a hand dishwashing,
automatic dishwashing, or laundry detergent market segment.
[0058] As used herein, the term "disinfecting" refers to the
removal of contaminants from the surfaces, as well as the
inhibition or killing of microbes on the surfaces of items. It is
not intended that the present disclosure be limited to any
particular surface, item, or contaminant(s) or microbes to be
removed.
[0059] The "compact" form of the cleaning compositions herein is
best reflected by density and, in terms of composition, by the
amount of inorganic filler salt. Inorganic filler salts are
conventional ingredients of detergent compositions in powder form.
In conventional detergent compositions, the filler salts are
present in substantial amounts, typically about 17 to about 35% by
weight of the total composition. In contrast, in compact
compositions, the filler salt is present in amounts not exceeding
about 15% of the total composition. In some embodiments, the filler
salt is present in amounts that do not exceed about 10%, or more
preferably, about 5%, by weight of the composition. In some
embodiments, the inorganic filler salts are selected from the
alkali and alkaline-earth-metal salts of sulfates and chlorides. In
some embodiments, the filler salt is sodium sulfate.
[0060] The present disclosure provides novel serine protease
enzymes. The serine protease polypeptides of the present disclosure
include isolated, recombinant, substantially pure, or non-naturally
occurring polypeptides. In some embodiments, the polypeptides are
useful in cleaning applications and can be incorporated into
cleaning compositions that are useful in methods of cleaning an
item or a surface in need thereof.
[0061] In some embodiments, the invention is a BspAL03279-clade of
subtilisins. In other embodiments, the invention is a recombinant
polypeptide or active fragment thereof of a BspAL03279-clade
subtilisin. In further embodiments, the BspAL03279-clade of
subtilisins comprises a subtilisin or recombinant polypeptide or
active fragment thereof comprising a DLGDXXRFGX.sub.aGLLXXXXAVX
(SEQ ID NO:26) motif, wherein X is any amino acid and X.sub.a is N
or S ("Motif 1"). In yet further embodiments, the BspAL03279-clade
of subtilisins comprises a subtilisin or recombinant polypeptide or
active fragment thereof comprising a DLGDXXRFGX.sub.aGLLXXXXAVX
(SEQ ID NO:26) motif, wherein X is any amino acid and X.sub.a is N
("Motif 2"). In yet still further embodiments, the BspAL03279-clade
of subtilisins comprises a subtilisin or recombinant polypeptide or
active fragment thereof comprising a DLGDXXRFGX.sub.aGLLXXXXAVX
(SEQ ID NO:26) motif, wherein X is any amino acid and X.sub.a is S
("Motif 3").
[0062] In still further embodiments, the BspAL03279-clade of
subtilisins comprises a subtilisin or recombinant polypeptide or
active fragment thereof comprising a DLGDXXRFGX.sub.aGLLXXXXAVX
(SEQ ID NO:26) motif, wherein X is any amino acid and X.sub.a is N
or S, with the proviso that the subtilisin or recombinant
polypeptide or active fragment thereof does not comprise ADK62564.
In an even further embodiment, the BspAL03279-clade of subtilisins
comprises a subtilisin or recombinant polypeptide or active
fragment thereof comprising a DLGDXXRFGX.sub.aGLLXXXXAVX (SEQ ID
NO:26) motif, wherein X is any amino acid and X.sub.a is S, with
the proviso that the subtilisin or recombinant polypeptide or
active fragment thereof does not comprise ADK62564.
[0063] In some embodiments, the polypeptide of the present
invention is a polypeptide having a specified degree of amino acid
sequence homology to the exemplified polypeptides, e.g., 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs:3, 6, 9, 12, and 15. In other embodiments,
the recombinant polypeptide or active fragment thereof comprises an
amino acid sequence having at least 75% amino acid sequence
identity to an amino acid sequence of SEQ ID NO:6 or 12, with the
proviso that the recombinant polypeptide or active fragment thereof
does not comprise ADK62564. In other embodiments, the recombinant
polypeptide or active fragment thereof comprises an amino acid
sequence having at least 80% amino acid sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 3, 9, and 15. In other embodiments, the recombinant
polypeptide or active fragment thereof comprises an amino acid
sequence having at least 80% amino acid sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 3, 6, 9, 12, and 15, with the proviso that the recombinant
polypeptide or active fragment thereof does not comprise ADK62564
or optionally WP_035392836, WP_038476582, WP_035392836 or
WP_047989534. In other embodiments, the recombinant polypeptide or
active fragment thereof comprises an amino acid sequence having at
least 97% amino acid sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 3, 9, 12, and
15.
[0064] Homology can be determined by amino acid sequence alignment,
e.g., using a program such as BLAST, ALIGN, MUSCLE, or CLUSTAL, as
described herein. In some embodiments, the polypeptide is an
isolated, recombinant, substantially pure, or non-naturally
occurring enzyme having protease activity, such as subtilisin
activity, or casein hydrolysis activity (for example,
dimethylcasein hydrolysis activity).
[0065] Also provided is a polypeptide enzyme of the present
invention, having protease activity, such as alkaline protease
activity, said enzyme comprising an amino acid sequence which
differs from the amino acid sequence of SEQ ID NOs:3, 6, 9, 12, and
15 by no more than 50, no more than 40, no more than 30, no more
than 25, no more than 20, no more than 15, no more than 10, no more
than 9, no more than 8, no more than 7, no more than 6, no more
than 5, no more than 4, no more than 3, no more than 2, or no more
than 1 amino acid residue(s), when aligned using any of the
previously described alignment methods.
[0066] As noted above, the variant enzyme polypeptides of the
invention have enzymatic activities (e.g., protease activities) and
thus are useful in cleaning applications, including but not limited
to, methods for cleaning dishware items, tableware items, fabrics,
and items having hard surfaces (e.g., the hard surface of a table,
table top, wall, furniture item, floor, ceiling, etc.). Exemplary
cleaning compositions comprising one or more variant serine
protease enzyme polypeptides of the invention are described infra.
The enzymatic activity (e.g., protease enzyme activity) of an
enzyme polypeptide of the invention can be determined readily using
procedures well known to those of ordinary skill in the art. The
Examples presented infra describe methods for evaluating the
enzymatic activity and cleaning performance. The performance of
polypeptide enzymes of the invention in removing stains (e.g., a
protein stain such as blood/milk/ink or egg yolk), cleaning hard
surfaces, or cleaning laundry, dishware or tableware item(s) can be
readily determined using procedures well known in the art and/or by
using procedures set forth in the Examples.
[0067] The serine protease polypeptides of the present invention
can have protease activity over a broad range of pH conditions. In
some embodiments, the serine protease polypeptides have protease
activity on dimethylcasein as a substrate, as demonstrated in
Examples below.
[0068] In some embodiments, the serine protease polypeptides of the
present invention demonstrate cleaning performance in a cleaning
composition. Cleaning compositions often include ingredients
harmful to the stability and performance of enzymes, making
cleaning compositions a harsh environment for enzymes, e.g. serine
proteases, to retain function. Thus, it is not trivial for an
enzyme to be put in a cleaning composition and expect enzymatic
function (e.g. serine protease activity, such as demonstrated by
cleaning performance). In some embodiments, the serine protease
polypeptides of the present invention demonstrate cleaning
performance in automatic dishwashing (ADW) detergent compositions.
In some embodiments, the cleaning performance in ADW detergent
compositions includes cleaning of egg yolk stains. In some
embodiments, the serine protease polypeptides of the present
invention demonstrate cleaning performance in laundry detergent
compositions. In some embodiments, the cleaning performance in
laundry detergent compositions includes cleaning of blood/milk/ink
stains. In each of the cleaning compositions, the serine protease
polypeptides of the present invention demonstrate cleaning
performance with or without a bleach component.
[0069] A polypeptide of the invention can be subject to various
changes, such as one or more amino acid insertions, deletions,
and/or substitutions, either conservative or non-conservative,
including where such changes do not substantially alter the
enzymatic activity of the polypeptide. Similarly, a nucleic acid of
the invention can also be subject to various changes, such as one
or more substitutions of one or more nucleotides in one or more
codons such that a particular codon encodes the same or a different
amino acid, resulting in either a silent variation (e.g., when the
encoded amino acid is not altered by the nucleotide mutation) or
non-silent variation, one or more deletions of one or more nucleic
acids (or codons) in the sequence, one or more additions or
insertions of one or more nucleic acids (or codons) in the
sequence, and/or cleavage of or one or more truncations of one or
more nucleic acids (or codons) in the sequence. Many such changes
in the nucleic acid sequence may not substantially alter the
enzymatic activity of the resulting encoded polypeptide enzyme
compared to the polypeptide enzyme encoded by the original nucleic
acid sequence. A nucleic acid sequence of the invention can also be
modified to include one or more codons that provide for optimum
expression in an expression system (e.g., bacterial expression
system), while, if desired, said one or more codons still encode
the same amino acid(s).
[0070] The invention provides isolated, non-naturally occurring, or
recombinant nucleic acids which may be collectively referred to as
"nucleic acids of the invention" or "polynucleotides of the
invention", which encode polypeptides of the invention. Nucleic
acids of the invention, including all described below, are useful
in recombinant production (e.g., expression) of polypeptides of the
invention, typically through expression of a plasmid expression
vector comprising a sequence encoding the polypeptide of interest
or fragment thereof. As discussed above, polypeptides include
serine protease polypeptides having enzymatic activity (e.g.,
proteolytic activity) which are useful in cleaning applications and
cleaning compositions for cleaning an item or a surface (e.g.,
surface of an item) in need of cleaning.
[0071] In some embodiments, the polynucleotide of the present
invention is a polynucleotide having a specified degree of nucleic
acid homology to the exemplified polynucleotide. In some
embodiments, the polynucleotide comprises a nucleic acid sequence
having at least 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99 or 100% identity to the nucleic acid sequence of SEQ
ID NOs:1, 4, 7, 10, 13, 16, 18, 20, 22, or 24. In some embodiments,
the polynucleotide comprises a nucleic acid sequence selected from
the group consisting of SEQ ID NOs:1, 4, 7, 10, 13, 16, 18, 20, 22,
and 24. In other embodiments, the polynucleotide may also have a
complementary nucleic acid sequence to a nucleic acid sequence
selected from the group consisting of SEQ ID NOs:1, 4, 7, 10, 13,
16, 18, 20, 22, and 24. In some embodiments, the polynucleotide
comprises a nucleic acid sequence encoding a recombinant
polypeptide or an active fragment thereof, comprising an amino acid
sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or
100% amino acid identity to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 3, 6, 9, 12, and 15. In some
embodiments, the polynucleotide comprises a nucleic acid sequence
encoding a recombinant polypeptide or an active fragment thereof,
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs:3, 6, 9, 12, and 15. Homology can be
determined by amino acid sequence alignment, e.g., using a program
such as BLAST, ALIGN, MUSCLE, or CLUSTAL, as described herein.
[0072] In some embodiments, the invention provides an isolated,
recombinant, substantially pure, synthetically derived, or
non-naturally occurring nucleic acid comprising a nucleotide
sequence encoding any polypeptide (including any fusion protein,
etc.) of the invention described above in the section entitled
"Polypeptides of the Invention" and elsewhere herein. The invention
also provides an isolated, recombinant, substantially pure,
synthetically derived, or non-naturally-occurring nucleic acid
comprising a nucleotide sequence encoding a combination of two or
more of any polypeptides of the invention described above and
elsewhere herein. The present invention provides nucleic acids
encoding a serine protease polypeptide of the present invention,
wherein the serine protease polypeptide is a mature form having
proteolytic activity. In some embodiments, the serine protease is
expressed recombinantly with a homologous pro-peptide sequence. In
other embodiments, the serine protease is expressed recombinantly
with a heterologous pro-peptide sequence (e.g., GG36 pro-peptide
sequence.
[0073] Nucleic acids of the invention can be generated by using any
suitable synthesis, manipulation, and/or isolation techniques, or
combinations thereof. For example, a polynucleotide of the
invention may be produced using standard nucleic acid synthesis
techniques, such as solid-phase synthesis techniques that are
well-known to those skilled in the art. In such techniques,
fragments of up to 50 or more nucleotide bases are typically
synthesized, then joined (e.g., by enzymatic or chemical ligation
methods) to form essentially any desired continuous nucleic acid
sequence. The synthesis of the nucleic acids of the invention can
be also facilitated by any suitable method known in the art,
including but not limited to chemical synthesis using the classical
phosphoramidite method (See e.g., Beaucage et al. Tetrahedron
Letters 22:1859-69 [1981]); or the method described by Matthes et
al. (See, Matthes et al., EMBO J. 3:801-805 [1984], as is typically
practiced in automated synthetic methods. Nucleic acids of the
invention also can be produced by using an automatic DNA
synthesizer. Customized nucleic acids can be ordered from a variety
of commercial sources (e.g., The Midland Certified Reagent Company,
the Great American Gene Company, Operon Technologies Inc., and
DNA2.0). Other techniques for synthesizing nucleic acids and
related principles are known in the art (See e.g., Itakura et al.,
Ann. Rev. Biochem. 53:323 [1984]; and Itakura et al., Science
198:1056 [1984]).
[0074] As indicated above, recombinant DNA techniques useful in
modification of nucleic acids are well known in the art. For
example, techniques such as restriction endonuclease digestion,
ligation, reverse transcription and cDNA production, and polymerase
chain reaction (e.g., PCR) are known and readily employed by those
of skill in the art. Nucleotides of the invention may also be
obtained by screening cDNA libraries using one or more
oligonucleotide probes that can hybridize to or PCR-amplify
polynucleotides which encode a serine protease polypeptide
polypeptide(s) of the invention. Procedures for screening and
isolating cDNA clones and PCR amplification procedures are well
known to those of skill in the art and described in standard
references known to those skilled in the art. Some nucleic acids of
the invention can be obtained by altering a naturally occurring
polynucleotide backbone (e.g., that encodes an enzyme or parent
protease) by, for example, a known mutagenesis procedure (e.g.,
site-directed mutagenesis, site saturation mutagenesis, and in
vitro recombination). A variety of methods are known in the art
that are suitable for generating modified polynucleotides of the
invention that encode serine protease polypeptides of the
invention, including, but not limited to, for example,
site-saturation mutagenesis, scanning mutagenesis, insertional
mutagenesis, deletion mutagenesis, random mutagenesis,
site-directed mutagenesis, and directed-evolution, as well as
various other recombinatorial approaches.
[0075] The present invention provides vectors comprising at least
one serine protease polynucleotide of the invention described
herein (e.g., a polynucleotide encoding a serine protease
polypeptide of the invention described herein), expression vectors
or expression cassettes comprising at least one nucleic acid or
polynucleotide of the invention, isolated, substantially pure, or
recombinant DNA constructs comprising at least one nucleic acid or
polynucleotide of the invention, isolated or recombinant cells
comprising at least one polynucleotide of the invention, and
compositions comprising one or more such vectors, nucleic acids,
expression vectors, expression cassettes, DNA constructs, cells,
cell cultures, or any combination or mixtures thereof.
[0076] In some embodiments, the invention provides recombinant
cells comprising at least one vector (e.g., expression vector or
DNA construct) of the invention which comprises at least one
nucleic acid or polynucleotide of the invention. Some such
recombinant cells are transformed or transfected with such at least
one vector, although other methods are available and known in the
art. Such cells are typically referred to as host cells. Some such
cells comprise bacterial cells, including, but are not limited to
Bacillus sp. cells, such as B. subtilis cells. The invention also
provides recombinant cells (e.g., recombinant host cells)
comprising at least one serine protease polypeptide of the
invention.
[0077] In some embodiments, the invention provides a vector
comprising a nucleic acid or polynucleotide of the invention. In
some embodiments, the vector is an expression vector or expression
cassette in which a polynucleotide sequence of the invention which
encodes a serine protease polypeptide of the invention is operably
linked to one or additional nucleic acid segments required for
efficient gene expression (e.g., a promoter operably linked to the
polynucleotide of the invention which encodes a serine protease
polypeptide of the invention). A vector may include a transcription
terminator and/or a selection gene, such as an antibiotic
resistance gene, that enables continuous cultural maintenance of
plasmid-infected host cells by growth in antimicrobial-containing
media.
[0078] An expression vector may be derived from plasmid or viral
DNA, or in alternative embodiments, contains elements of both.
Exemplary vectors include, but are not limited to pC194, pJH101,
pE194, pHP13 (See, Harwood and Cutting [eds.], Chapter 3, Molecular
Biological Methods for Bacillus, John Wiley & Sons [1990];
suitable replicating plasmids for B. subtilis include those listed
on p. 92) See also, Perego, Integrational Vectors for Genetic
Manipulations in Bacillus subtilis, in Sonenshein et al., [eds.]
Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry,
Physiology and Molecular Genetics, American Society for
Microbiology, Washington, D.C. [1993], pp. 615-624), and
p2JM103BBI.
[0079] For expression and production of a protein of interest
(e.g., serine protease polypeptide) in a cell, at least one
expression vector comprising at least one copy of a polynucleotide
encoding the serine protease polypeptide, and in some instances
comprising multiple copies, is transformed into the cell under
conditions suitable for expression of the serine protease. In some
embodiments of the present invention, a polynucleotide sequence
encoding the serine protease polypeptide (as well as other
sequences included in the vector) is integrated into the genome of
the host cell, while in other embodiments, a plasmid vector
comprising a polynucleotide sequence encoding the serine protease
polypeptide remains as autonomous extra-chromosomal element within
the cell. The invention provides both extrachromosomal nucleic acid
elements as well as incoming nucleotide sequences that are
integrated into the host cell genome. The vectors described herein
are useful for production of the serine protease polypeptides of
the invention. In some embodiments, a polynucleotide construct
encoding the serine protease polypeptide is present on an
integrating vector that enables the integration and optionally the
amplification of the polynucleotide encoding the serine protease
polypeptide into the host chromosome. Examples of sites for
integration are well known to those skilled in the art. In some
embodiments, transcription of a polynucleotide encoding a serine
protease polypeptide of the invention is effectuated by a promoter
that is the wild-type promoter for the selected precursor protease.
In some other embodiments, the promoter is heterologous to the
precursor protease, but is functional in the host cell.
Specifically, examples of suitable promoters for use in bacterial
host cells include, but are not limited to, for example, the amyE,
amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters, the
promoter of the B. stearothermophilus maltogenic amylase gene, the
B. amyloliquefaciens (BAN) amylase gene, the B. subtilis alkaline
protease gene, the B. clausii alkaline protease gene the B. pumilis
xylosidase gene, the B. thuringiensis cryIIIA, and the B.
licheniformis alpha-amylase gene. Additional promoters include, but
are not limited to the A4 promoter, as well as phage Lambda PR or
PL promoters, and the E. coli lac, trp or tac promoters.
[0080] Serine protease polypeptides of the present invention can be
produced in host cells of any suitable microorganism, including
bacteria and fungi. In some embodiments, serine protease
polypeptides of the present invention can be produced in
Gram-positive bacteria. In some embodiments, the host cells are
Bacillus spp., Streptomyces spp., Escherichia spp., Aspergillus
spp., Trichoderma spp., Pseudomonas spp., Corynebacterium spp.,
Saccharomyces spp., or Pichia spp. In some embodiments, the serine
protease polypeptides are produced by Bacillus sp. host cells.
Examples of Bacillus sp. host cells that find use in the production
of the serine protease polypeptides of the invention include, but
are not limited to B. licheniformis, B. lentus, B. subtilis, B.
amyloliquefaciens, B. lentus, B. brevis, B. stearothermophilus, B.
alkalophilus, B. coagulans, B. circulans, B. pumilis, B.
thuringiensis, B. clausii, and B. megaterium, as well as other
organisms within the genus Bacillus. In some embodiments, B.
subtilis host cells are used for production of serine protease
polypeptides. U.S. Pat. Nos. 5,264,366 and 4,760,025 (RE 34,606)
describe various Bacillus host strains that can be used for
producing serine protease polypeptide of the invention, although
other suitable strains can be used.
[0081] Several bacterial strains that can be used to produce serine
protease polypeptides of the invention include non-recombinant
(i.e., wild-type) Bacillus sp. strains, as well as variants of
naturally-occurring strains and/or recombinant strains. In some
embodiments, the host strain is a recombinant strain, wherein a
polynucleotide encoding a polypeptide of interest has been
introduced into the host. In some embodiments, the host strain is a
B. subtilis host strain and particularly a recombinant Bacillus
subtilis host strain. Numerous B. subtilis strains are known,
including, but not limited to for example, 1A6 (ATCC 39085), 168
(1A01), SB19, W23, Ts85, B637, PB1753 through PB1758, PB3360,
JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100
(ATCC 39,094), GX4931, PBT 110, and PEP 211 strain (See e.g., Hoch
et al., Genetics 73:215-228 [1973]; See also, U.S. Pat. Nos.
4,450,235 and 4,302,544, and EP0134048, each of which is
incorporated by reference in its entirety). The use of B. subtilis
as an expression host cells is well known in the art (See e.g.,
Palva et al., Gene 19:81-87 [1982]; Fahnestock and Fischer, J.
Bacteriol., 165:796-804 [1986]; and Wang et al., Gene 69:39-47
[1988]).
[0082] In some embodiments, the Bacillus host cell is a Bacillus
sp. that includes a mutation or deletion in at least one of the
following genes, degU, degS, degR and degQ. In some embodiments,
the mutation is in a degU gene, and in some embodiments the
mutation is degU(Hy)32 (See e.g., Msadek et al., J. Bacteriol.
172:824-834 [1990]; and Olmos et al., Mol. Gen. Genet. 253:562-567
[1997]). In some embodiments, the Bacillus host comprises a
mutation or deletion in scoC4 (See e.g., Caldwell et al., J.
Bacteriol. 183:7329-7340 [2001]); spoIIE (See e.g., Arigoni et al.,
Mol. Microbiol. 31:1407-1415 [1999]); and/or oppA or other genes of
the opp operon (See e.g., Perego et al., Mol. Microbiol. 5:173-185
[1991]). Indeed, it is contemplated that any mutation in the opp
operon that causes the same phenotype as a mutation in the oppA
gene will find use in some embodiments of the altered Bacillus
strain of the invention. In some embodiments, these mutations occur
alone, while in other embodiments, combinations of mutations are
present. In some embodiments, an altered Bacillus host cell strain
that can be used to produce a serine protease polypeptide of the
invention is a Bacillus host strain that already includes a
mutation in one or more of the above-mentioned genes. In addition,
Bacillus sp. host cells that comprise mutation(s) and/or deletions
of endogenous protease genes find use. In some embodiments, the
Bacillus host cell comprises a deletion of the aprE and the nprE
genes. In other embodiments, the Bacillus sp. host cell comprises a
deletion of 5 protease genes, while in other embodiments, the
Bacillus sp. host cell comprises a deletion of 9 protease genes
(See e.g., US 2005/0202535, incorporated herein by reference).
[0083] Host cells are transformed with at least one nucleic acid
encoding at least one serine protease polypeptide of the invention
using any suitable method known in the art. Methods for introducing
a nucleic acid (e.g., DNA) into Bacillus cells or E. coli cells
utilizing plasmid DNA constructs or vectors and transforming such
plasmid DNA constructs or vectors into such cells are well known.
In some embodiments, the plasmids are subsequently isolated from E.
coli cells and transformed into Bacillus cells. However, it is not
essential to use intervening microorganisms such as E. coli, and in
some embodiments, a DNA construct or vector is directly introduced
into a Bacillus host.
[0084] Those of skill in the art are well aware of suitable methods
for introducing nucleic acid sequences of the invention into
Bacillus cells (See e.g., Ferrari et al., "Genetics," in Harwood et
al. [eds.], Bacillus, Plenum Publishing Corp. [1989], pp. 57-72;
Saunders et al., J. Bacteriol. 157:718-726 [1984]; Hoch et al., J.
Bacteriol. 93:1925-1937 [1967]; Mann et al., Current Microbiol.
13:131-135 [1986]; Holubova, Folia Microbiol. 30:97 [1985]; Chang
et al., Mol. Gen. Genet. 168:11-115 [1979]; Vorobjeva et al., FEMS
Microbiol. Lett. 7:261-263 [1980]; Smith et al., Appl. Env.
Microbiol. 51:634 [1986]; Fisher et al., Arch. Microbiol.
139:213-217 [1981]; and McDonald, J. Gen. Microbiol. 130:203
[1984]). Indeed, such methods as transformation, including
protoplast transformation and transfection, transduction, and
protoplast fusion are well known and suited for use in the present
invention. Methods known in the art to transform Bacillus cells
include such methods as plasmid marker rescue transformation, which
involves the uptake of a donor plasmid by competent cells carrying
a partially homologous resident plasmid (See, Contente et al.,
Plasmid 2:555-571 [1979]; Haima et al., Mol. Gen. Genet.
223:185-191 [1990]; Weinrauch et al., J. Bacteriol. 154:1077-1087
[1983]; and Weinrauch et al., J. Bacteriol. 169:1205-1211 [1987]).
In this method, the incoming donor plasmid recombines with the
homologous region of the resident "helper" plasmid in a process
that mimics chromosomal transformation.
[0085] In addition to commonly used methods, in some embodiments,
host cells are directly transformed with a DNA construct or vector
comprising a nucleic acid encoding a serine protease polypeptide of
the invention (i.e., an intermediate cell is not used to amplify,
or otherwise process, the DNA construct or vector prior to
introduction into the host cell). Introduction of the DNA construct
or vector of the invention into the host cell includes those
physical and chemical methods known in the art to introduce a
nucleic acid sequence (e.g., DNA sequence) into a host cell without
insertion into the host genome. Such methods include, but are not
limited to calcium chloride precipitation, electroporation, naked
DNA, liposomes and the like. In additional embodiments, DNA
constructs or vector are co-transformed with a plasmid, without
being inserted into the plasmid. In further embodiments, a
selective marker is deleted from the altered Bacillus strain by
methods known in the art (See, Stahl et al., J. Bacteriol.
158:411-418 [1984]; and Palmeros et al., Gene 247:255 -264
[2000]).
[0086] In some embodiments, the transformed cells of the present
invention are cultured in conventional nutrient media. The suitable
specific culture conditions, such as temperature, pH and the like
are known to those skilled in the art and are well described in the
scientific literature. In some embodiments, the invention provides
a culture (e.g., cell culture) comprising at least one serine
protease polypeptide or at least one nucleic acid of the
invention.
[0087] In some embodiments, host cells transformed with at least
one polynucleotide sequence encoding at least one serine protease
polypeptide of the invention are cultured in a suitable nutrient
medium under conditions permitting the expression of the present
protease, after which the resulting protease is recovered from the
culture. In some embodiments, the protease produced by the cells is
recovered from the culture medium by conventional procedures,
including, but not limited to for example, separating the host
cells from the medium by centrifugation or filtration,
precipitating the proteinaceous components of the supernatant or
filtrate by means of a salt (e.g., ammonium sulfate),
chromatographic purification (e.g., ion exchange, gel filtration,
affinity, etc.).
[0088] In some embodiments, a serine protease polypeptide produced
by a recombinant host cell is secreted into the culture medium. A
nucleic acid sequence that encodes a purification facilitating
domain may be used to facilitate purification of proteins. A vector
or DNA construct comprising a polynucleotide sequence encoding a
serine protease polypeptide may further comprise a nucleic acid
sequence encoding a purification facilitating domain to facilitate
purification of the serine protease polypeptide (See e.g., Kroll et
al., DNA Cell Biol. 12:441-53 [1993]). Such purification
facilitating domains include, but are not limited to, for example,
metal chelating peptides such as histidine-tryptophan modules that
allow purification on immobilized metals (See, Porath, Protein
Expr. Purif. 3:263-281 [1992]), protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system. The inclusion
of a cleavable linker sequence such as Factor XA or enterokinase
(e.g., sequences available from Invitrogen, San Diego, Calif.)
between the purification domain and the heterologous protein also
find use to facilitate purification.
[0089] Assays for detecting and measuring the enzymatic activity of
an enzyme, such as a serine protease polypeptide of the invention,
are well known. Various assays for detecting and measuring activity
of proteases (e.g., serine protease polypeptides of the invention),
are also known to those of ordinary skill in the art. In
particular, assays are available for measuring protease activity
that are based on the release of acid-soluble peptides from casein
or hemoglobin, measured as absorbance at 280 nm or colorimetrically
using the Folin method. Other exemplary assays involve the
solubilization of chromogenic substrates (See e.g., Ward,
"Proteinases," in Fogarty (ed.), Microbial Enzymes and
Biotechnology, Applied Science, London, [1983], pp. 251-317). Other
exemplary assays include, but are not limited to
succinyl-Ala-Ala-Pro-Phe-para nitroanilide assay (suc-AAPF-pNA) and
the 2,4,6-trinitrobenzene sulfonate sodium salt assay (TNBS assay).
Numerous additional references known to those in the art provide
suitable methods (See e.g., Wells et al., Nucleic Acids Res.
11:7911-7925 [1983]; Christianson et al., Anal. Biochem.
223:119-129 [1994]; and Hsia et al., Anal Biochem. 242:221-227
[1999]).
[0090] A variety of methods can be used to determine the level of
production of a mature protease (e.g., mature serine protease
polypeptides of the present invention) in a host cell. Such methods
include, but are not limited to, for example, methods that utilize
either polyclonal or monoclonal antibodies specific for the
protease. Exemplary methods include, but are not limited to
enzyme-linked immunosorbent assays (ELISA), radioimmunoassays
(RIA), fluorescent immunoassays (FIA), and fluorescent activated
cell sorting (FACS). These and other assays are well known in the
art (See e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).
[0091] In some other embodiments, the invention provides methods
for making or producing a mature serine protease polypeptide of the
invention. A mature serine protease polypeptide does not include a
signal peptide or a propeptide sequence. Some methods comprise
making or producing a serine protease polypeptide of the invention
in a recombinant bacterial host cell, such as for example, a
Bacillus sp. cell (e.g., a B. subtilis cell). In some embodiments,
the invention provides a method of producing a serine protease
polypeptide of the invention, the method comprising cultivating a
recombinant host cell comprising a recombinant expression vector
comprising a nucleic acid encoding a serine protease polypeptide of
the invention under conditions conducive to the production of the
serine protease polypeptide. Some such methods further comprise
recovering the serine protease polypeptide from the culture.
[0092] In some embodiments the invention provides methods of
producing a serine protease polypeptide of the invention, the
methods comprising: (a) introducing a recombinant expression vector
comprising a nucleic acid encoding a serine protease polypeptide of
the invention into a population of cells (e.g., bacterial cells,
such as B. subtilis cells); and (b) culturing the cells in a
culture medium under conditions conducive to produce the serine
protease polypeptide encoded by the expression vector. Some such
methods further comprise: (c) isolating the serine protease
polypeptide from the cells or from the culture medium.
[0093] Unless otherwise noted, all component or composition levels
provided herein are made in reference to the active level of that
component or composition, and are exclusive of impurities, for
example, residual solvents or by-products, which may be present in
commercially available sources. Enzyme components weights are based
on total active protein. All percentages and ratios are calculated
by weight unless otherwise indicated. All percentages and ratios
are calculated based on the total composition unless otherwise
indicated. Compositions of the invention include cleaning
compositions, such as detergent compositions. In the exemplified
detergent compositions, the enzymes levels are expressed by pure
enzyme by weight of the total composition and unless otherwise
specified, the detergent ingredients are expressed by weight of the
total compositions.
[0094] While not essential for the purposes of the present
invention, the non-limiting list of adjuncts illustrated
hereinafter are suitable for use in the instant cleaning
compositions. In some embodiments, these adjuncts are incorporated
for example, to assist or enhance cleaning performance, for
treatment of the substrate to be cleaned, or to modify the
aesthetics of the cleaning composition as is the case with
perfumes, colorants, dyes or the like. It is understood that such
adjuncts are in addition to the serine protease polypeptides of the
present invention. The precise nature of these additional
components, and levels of incorporation thereof, will depend on the
physical form of the composition and the nature of the cleaning
operation for which it is to be used. Suitable adjunct materials
include, but are not limited to, bleach catalysts, other enzymes,
enzyme stabilizing systems, chelants, optical brighteners, soil
release polymers, dye transfer agents, dispersants, suds
suppressors, dyes, perfumes, colorants, filler salts,
photoactivators, fluorescers, fabric conditioners, hydrolyzable
surfactants, preservatives, anti-oxidants, anti-shrinkage agents,
anti-wrinkle agents, germicides, fungicides, color speckles,
silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity
sources, solubilizing agents, carriers, processing aids, pigments,
and pH control agents, surfactants, builders, chelating agents, dye
transfer inhibiting agents, deposition aids, dispersants,
additional enzymes, and enzyme stabilizers, catalytic materials,
bleach activators, bleach boosters, hydrogen peroxide, sources of
hydrogen peroxide, preformed peracids, polymeric dispersing agents,
clay soil removal/anti-redeposition agents, brighteners, suds
suppressors, dyes, perfumes, structure elasticizing agents, fabric
softeners, carriers, hydrotropes, processing aids and/or pigments.
In addition to the disclosure below, suitable examples of such
other adjuncts and levels of use are found in U.S. Pat. Nos.
5,576,282; 6,306,812; 6,326,348; 6,610,642; 6,605,458; 5,705,464;
5,710,115; 5,698,504; 5,695,679; 5,686,014; and 5,646,101 all of
which are incorporated herein by reference. In embodiments in which
the cleaning adjunct materials are not compatible with the serine
protease polypeptides of the present invention in the cleaning
compositions, then suitable methods of keeping the cleaning adjunct
materials and the protease(s) separated (i.e., not in contact with
each other) until combination of the two components is appropriate
are used. Such separation methods include any suitable method known
in the art (e.g., gelcaps, encapsulation, tablets, physical
separation, etc.). The aforementioned adjunct ingredients may
constitute the balance of the cleaning compositions of the present
invention.
[0095] The cleaning compositions of the present invention are
advantageously employed for example, in laundry applications, hard
surface cleaning applications, dishwashing applications, including
automatic dishwashing and hand dishwashing, as well as cosmetic
applications such as dentures, teeth, hair and skin cleaning. The
enzymes of the present invention are also suited for use in contact
lens cleaning and wound debridement applications. In addition, due
to the unique advantages of increased effectiveness in lower
temperature solutions, the enzymes of the present invention are
ideally suited for laundry applications. Furthermore, the enzymes
of the present invention find use in granular and liquid
compositions.
[0096] The serine protease polypeptides of the present invention
also find use in cleaning additive products. In some embodiments,
low temperature solution cleaning applications find use. In some
embodiments, the present invention provides cleaning additive
products including at least one enzyme of the present invention is
ideally suited for inclusion in a wash process when additional
bleaching effectiveness is desired. Such instances include, but are
not limited to low temperature solution cleaning applications. In
some embodiments, the additive product is in its simplest form, one
or more proteases. In some embodiments, the additive is packaged in
dosage form for addition to a cleaning process. In some
embodiments, the additive is packaged in dosage form for addition
to a cleaning process where a source of peroxygen is employed and
increased bleaching effectiveness is desired. Any suitable single
dosage unit form finds use with the present invention, including
but not limited to pills, tablets, gelcaps, or other single dosage
units such as pre-measured powders or liquids. In some embodiments,
filler(s) or carrier material(s) are included to increase the
volume of such compositions. Suitable filler or carrier materials
include, but are not limited to, various salts of sulfate,
carbonate and silicate as well as talc, clay and the like. Suitable
filler or carrier materials for liquid compositions include, but
are not limited to water or low molecular weight primary and
secondary alcohols including polyols and diols. Examples of such
alcohols include, but are not limited to, methanol, ethanol,
propanol and isopropanol. In some embodiments, the compositions
contain from about 5% to about 90% of such materials. Acidic
fillers find use to reduce pH. Alternatively, in some embodiments,
the cleaning additive includes adjunct ingredients, as more fully
described below.
[0097] The present cleaning compositions and cleaning additives
require an effective amount of at least one of the serine protease
polypeptides provided herein, alone or in combination with other
proteases and/or additional enzymes. The required level of enzyme
is achieved by the addition of one or more serine protease
polypeptides of the present invention. Typically the present
cleaning compositions comprise at least about 0.0001 weight
percent, from about 0.0001 to about 10, from about 0.001 to about
1, or from about 0.01 to about 0.1 weight percent of at least one
of the serine protease polypeptides of the present invention.
[0098] The cleaning compositions herein are typically formulated
such that, during use in aqueous cleaning operations, the wash
water will have a pH of from about 4.0 to about 11.5, or even from
about 5.0 to about 11.5, or even from about 5.0 to about 8.0, or
even from about 7.5 to about 10.5. Liquid product formulations are
typically formulated to have a pH from about 3.0 to about 9.0 or
even from about 3 to about 5. Granular laundry products are
typically formulated to have a pH from about 9 to about 11. In some
embodiments, the cleaning compositions of the present invention can
be formulated to have an alkaline pH under wash conditions, such as
a pH of from about 8.0 to about 12.0, or from about 8.5 to about
11.0, or from about 9.0 to about 11.0. In some embodiments, the
cleaning compositions of the present invention can be formulated to
have a neutral pH under wash conditions, such as a pH of from about
5.0 to about 8.0, or from about 5.5 to about 8.0, or from about 6.0
to about 8.0, or from about 6.0 to about 7.5. In some embodiments,
the neutral pH conditions can be measured when the cleaning
composition is dissolved 1:100 (wt:wt) in de-ionised water at
20.degree. C., measured using a conventional pH meter. Techniques
for controlling pH at recommended usage levels include the use of
buffers, alkalis, acids, etc., and are well known to those skilled
in the art.
[0099] In some embodiments, when the serine protease polypeptide(s)
is/are employed in a granular composition or liquid, it is
desirable for the serine protease polypeptide to be in the form of
an encapsulated particle to protect the serine protease polypeptide
from other components of the granular composition during storage.
In addition, encapsulation is also a means of controlling the
availability of the serine protease polypeptide during the cleaning
process. In some embodiments, encapsulation enhances the
performance of the serine protease polypeptide(s) and/or additional
enzymes. In this regard, the serine protease polypeptides of the
present invention are encapsulated with any suitable encapsulating
material known in the art. In some embodiments, the encapsulating
material typically encapsulates at least part of the serine
protease polypeptide(s) of the present invention. Typically, the
encapsulating material is water-soluble and/or water-dispersible.
In some embodiments, the encapsulating material has a glass
transition temperature (Tg) of 0.degree. C. or higher. Glass
transition temperature is described in more detail in WO 97/11151.
The encapsulating material is typically selected from consisting of
carbohydrates, natural or synthetic gums, chitin, chitosan,
cellulose and cellulose derivatives, silicates, phosphates,
borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes,
and combinations thereof. When the encapsulating material is a
carbohydrate, it is typically selected from monosaccharides,
oligosaccharides, polysaccharides, and combinations thereof. In
some typical embodiments, the encapsulating material is a starch
(See e.g., EP0922499; U.S. Pat. No. 4,977,252; U.S. Pat. No.
5,354,559, and U.S. Pat. No. 5,935,826). In some embodiments, the
encapsulating material is a microsphere made from plastic such as
thermoplastics, acrylonitrile, methacrylonitrile,
polyacrylonitrile, polymethacrylonitrile and mixtures thereof;
commercially available microspheres that find use include, but are
not limited to those supplied by EXPANCEL.RTM. (Stockviksverken,
Sweden), and PM6545, PM6550, PM7220, PM7228, EXTENDOSPHERES.RTM.,
LUXSIL.RTM., Q-CEL.RTM., and SPHERICEL.RTM. (PQ Corp., Valley
Forge, Pa.).
[0100] There are a variety of wash conditions including varying
detergent formulations, wash water volumes, wash water
temperatures, and lengths of wash time, to which proteases involved
in washing are exposed. A low detergent concentration system
includes detergents where less than about 800 ppm of the detergent
components is present in the wash water. A medium detergent
concentration includes detergents where between about 800 ppm and
about 2000 ppm of the detergent components is present in the wash
water. A high detergent concentration system includes detergents
where greater than about 2000 ppm of the detergent components are
present in the wash water. In some embodiments, the "cold water
washing" of the present invention utilizes "cold water detergent"
suitable for washing at temperatures from about 10.degree. C. to
about 40.degree. C., or from about 20.degree. C. to about
30.degree. C., or from about 15.degree. C. to about 25.degree. C.,
as well as all other combinations within the range of about
15.degree. C. to about 35.degree. C., and all ranges within
10.degree. C. to 40.degree. C.
[0101] Different geographies typically have different water
hardness. Water hardness is usually described in terms of the
grains per gallon mixed Ca.sup.2+/Mg.sup.2+. Hardness is a measure
of the amount of calcium (Ca.sup.2+) and magnesium (Mg.sup.2+) in
the water. Most water in the United States is hard, but the degree
of hardness varies. Moderately hard (60-120 ppm) to hard (121-181
ppm) water has 60 to 181 parts per million.
TABLE-US-00001 TABLE I Water Hardness Water Grains per gallon Parts
per million Soft less than 1.0 less than 17 Slightly hard 1.0 to
3.5 17 to 60 Moderately hard 3.5 to 7.0 60 to 120 Hard 7.0 to 10.5
120 to 180 Very hard greater than 10.5 greater than 180
[0102] Accordingly, in some embodiments, the present invention
provides serine protease polypeptides that show surprising wash
performance in at least one set of wash conditions (e.g., water
temperature, water hardness, and/or detergent concentration). In
some embodiments, the serine protease polypeptides of the present
invention are comparable in wash performance to other serine
protease polypeptide proteases. In some embodiments of the present
invention, the serine protease polypeptides provided herein exhibit
enhanced oxidative stability, enhanced thermal stability, enhanced
cleaning capabilities under various conditions, and/or enhanced
chelator stability. In addition, the serine protease polypeptides
of the present invention find use in cleaning compositions that do
not include detergents, again either alone or in combination with
builders and stabilizers.
[0103] In some embodiments of the present invention, the cleaning
compositions comprise at least one serine protease polypeptide of
the present invention at a level from about 0.00001 to about 10% by
weight of the composition and the balance (e.g., about 99.999 to
about 90.0%) comprising cleaning adjunct materials by weight of
composition. In some other embodiments of the present invention,
the cleaning compositions of the present invention comprises at
least one serine protease polypeptide at a level of about 0.0001 to
about 10%, about 0.001 to about 5%, about 0.001 to about 2%, about
0.005 to about 0.5% by weight of the composition and the balance of
the cleaning composition (e.g., about 99.9999 to about 90.0%, about
99.999 to about 98%, about 99.995 to about 99.5% by weight)
comprising cleaning adjunct materials.
[0104] In some embodiments, the cleaning compositions of the
present invention comprise one or more additional detergent
enzymes, which provide cleaning performance and/or fabric care
and/or dishwashing benefits. Examples of suitable enzymes include,
but are not limited to, acyl transferases, alpha-amylases,
beta-amylases, alpha-galactosidases, arabinosidases, aryl
esterases, beta-galactosidases, carrageenases, catalases,
cellobiohydrolases, cellulases, chondroitinases, cutinases,
endo-beta-1,4-glucanases, endo-beta-mannanases, esterases,
exo-mannanases, galactanases, glucoamylases, hemicellulases,
hyaluronidases, keratinases, laccases, lactases, ligninases,
lipases, lipoxygenases, mannanases, oxidases, pectate lyases,
pectin acetyl esterases, pectinases, pentosanases, peroxidases,
phenoloxidases, phosphatases, phospholipases, phytases,
polygalacturonases, proteases, pullulanases, reductases,
rhamnogalacturonases, beta-glucanases, tannases, transglutaminases,
xylan acetyl-esterases, xylanases, xyloglucanases, and xylosidases,
or any combinations or mixtures thereof. In some embodiments, a
combination of enzymes is used (i.e., a "cocktail") comprising
conventional applicable enzymes like protease, lipase, cutinase
and/or cellulase in conjunction with amylase is used.
[0105] In addition to the serine protease polypeptides provided
herein, any other suitable protease finds use in the compositions
of the present invention. Suitable proteases include those of
animal, vegetable or microbial origin. In some embodiments,
microbial proteases are used. In some embodiments, chemically or
genetically modified mutants are included. In some embodiments, the
protease is a serine protease, preferably an alkaline microbial
protease or a trypsin-like protease. Examples of alkaline proteases
include subtilisins, especially those derived from Bacillus (e.g.,
subtilisin, lentus, amyloliquefaciens, subtilisin Carlsberg,
subtilisin 309, subtilisin 147 and subtilisin 168). Additional
examples include those mutant proteases described in US RE 34,606;
U.S. Pat. Nos. 5,955,340; 5,700,676; 6,312,936; and 6,482,628, all
of which are incorporated herein by reference. Additional protease
examples include, but are not limited to trypsin (e.g., of porcine
or bovine origin), and the Fusarium protease described in
WO89/06270. In some embodiments, commercially available protease
enzymes that find use in the present invention include, but are not
limited to MAXATASE.RTM., MAXACAL.TM., MAXAPEM.TM., OPTICLEAN.RTM.,
OPTIMASE.RTM., PROPERASE.RTM., PURAFECT.RTM., PURAFECT.RTM. OXP,
PURAMAX.TM., EXCELLASE.TM., PREFERENZ.TM. proteases (e.g. P100,
P110, P280), EFFECTENZ.TM. proteases (e.g. P1000, P1050, P2000),
EXCELLENZ.TM. proteases (e.g. P1000), ULTIMASE.RTM., and
PURAFAST.TM. (Genencor); ALCALASE.RTM., SAVINASE.RTM.,
PRIMASE.RTM., DURAZYM.TM., POLARZYME.RTM., OVOZYME.RTM.,
KANNASE.RTM., LIQUANASE.RTM., NEUTRASE.RTM., RELASE.RTM. and
ESPERASE.RTM. (Novozymes); BLAP.TM. and BLAP.TM. (Henkel
Kommanditgesellschaft auf Aktien, Duesseldorf, Germany), and KAP
(B. alkalophilus subtilisin; Kao Corp., Tokyo, Japan). Various
proteases are described in WO95/23221, WO92/21760, WO 09/149200,
WO09/149144, WO09/149145, WO11/072099, WO10/056640, WO10/056653, WO
11/140364, WO12/151534, US 2008/0090747, and U.S. Pat. Nos.
5,801,039; 5,340,735; 5,500,364; 5,855,625; RE 34,606; U.S. Pat.
Nos. 5,955,340; 5,700,676; 6,312,936; 6,482,628; 8,530,219; and
various other patents. In some further embodiments, neutral
metalloproteases find use in the present invention, including but
not limited to the neutral metalloproteases described in
WO1999014341, WO1999033960, WO 1999014342, WO1999034003,
WO2007044993, WO2009058303, WO2009058661, WO 2014/071410,
WO2014/194032, WO2014/194034, WO2014/194054, and WO2014/194117.
Exemplary metalloproteases include nprE, the recombinant form of
neutral metalloprotease expressed in B. subtilis (See e.g.,
WO07/044993), and PMN, the purified neutral metalloprotease from B
amyloliquefaciens.
[0106] In addition, any suitable lipase finds use in the present
invention. Suitable lipases include, but are not limited to those
of bacterial or fungal origin. Chemically or genetically modified
mutants are encompassed by the present invention. Examples of
useful lipases include H. lanuginosa lipase (See e.g., EP 258 068,
and EP 305 216), Rhizomucor miehei lipase (See e.g., EP 238 023),
Candida lipase, such as C. antarctica lipase (e.g., C. antarctica
lipase A or B; See e.g., EP214761), Pseudomonas lipases such as P.
alcaligenes lipase and P. pseudoalcaligenes lipase (See e.g., EP
218 272), P. cepacia lipase (See e.g., EP 331 376), P. stutzeri
lipase (See e.g., GB 1,372,034), P. fluorescens lipase, Bacillus
lipase (e.g., B. subtilis lipase [Dartois et al., Biochem. Biophys.
Acta 1131:253-260 [1993]); B. stearothermophilus lipase [See e.g.,
JP 64/744992]; and B. pumilus lipase [See e.g., WO 91/16422]).
[0107] Furthermore, a number of cloned lipases find use in some
embodiments of the present invention, including but not limited to
Penicillium camembertii lipase (See, Yamaguchi et al., Gene
103:61-67 [1991]), Geotricum candidum lipase (See, Schimada et al.,
J. Biochem., 106:383-388 [1989]), and various Rhizopus lipases such
as R. delemar lipase (See, Hass et al., Gene 109:117-113 [1991]), a
R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem.
56:716-719 [1992]) and R. oryzae lipase.
[0108] Other types of lipase polypeptide enzymes such as cutinases
also find use in some embodiments of the present invention,
including but not limited to the cutinase derived from Pseudomonas
mendocina (See, WO88/09367), and the cutinase derived from Fusarium
solani pisi (See, WO90/09446).
[0109] Additional suitable lipases include lipases such as M1
LIPASE.TM., LUMA FAST.TM., and LIPOMAX.TM. (Genencor); LIPEX.RTM.,
LIPOLASE.RTM. and LIPOLASE.RTM. ULTRA (Novozymes); and LIPASE P.TM.
"Amano" (Amano Pharmaceutical Co. Ltd., Japan).
[0110] In some embodiments of the present invention, the cleaning
compositions of the present invention further comprise lipases at a
level from about 0.00001 to about 10% of additional lipase by
weight of the composition and the balance of cleaning adjunct
materials by weight of composition. In some other embodiments of
the present invention, the cleaning compositions of the present
invention also comprise lipases at a level of about 0.0001 to about
10%, about 0.001 to about 5%, about 0.001 to about 2%, about 0.005
to about 0.5% lipase by weight of the composition.
[0111] In some embodiments of the present invention, any suitable
amylase finds use in the present invention. In some embodiments,
any amylase (e.g., alpha and/or beta) suitable for use in alkaline
solutions also find use. Suitable amylases include, but are not
limited to those of bacterial or fungal origin. Chemically or
genetically modified mutants are included in some embodiments.
Amylases that find use in the present invention, include, but are
not limited to .alpha.-amylases obtained from B. licheniformis (See
e.g., GB 1,296,839). Additional suitable amylases include those
found in W09510603, WO9526397, WO9623874, WO9623873, WO9741213, WO
9919467, WO0060060, WO0029560, WO9923211, WO9946399, WO0060058,
WO0060059, WO9942567, WO0114532, WO02092797, WO0166712, WO0188107,
WO0196537, WO 0210355, WO9402597, WO0231124, WO9943793, WO9943794,
WO2004113551, WO 2005001064, WO2005003311, WO0164852, WO2006063594,
WO2006066594, WO 2006066596, WO2006012899, WO2008092919,
WO2008000825, WO2005018336, WO 2005066338, WO2009140504,
WO2005019443, WO2010091221, WO2010088447, WO 0134784, WO2006012902,
WO2006031554, WO2006136161, WO2008101894, WO 2010059413,
WO2011098531, WO2011080352, WO2011080353, WO2011080354, WO
2011082425, WO2011082429, WO2011076123, WO2011087836, WO2011076897,
WO 94183314, WO9535382, WO9909183, WO9826078, WO9902702, WO9743424,
WO9929876, WO9100353, WO9605295, WO9630481, WO9710342,
WO2008088493, WO2009149419, WO2009061381, WO2009100102,
WO2010104675, WO2010117511, and WO2010115021. Commercially
available amylases that find use in the present invention include,
but are not limited to DURAMYL.RTM., TERMAMYL.RTM., FUNGAMYL.RTM.,
STAINZYME.RTM., STAINZYME PLUS.RTM., STAINZYME ULTRA.RTM., and
BAN.TM. (Novozymes), as well as POWERASE.TM., RAPIDASE.RTM. and
MAXAMYL.RTM. P (Genencor).
[0112] In some embodiments of the present invention, the cleaning
compositions of the present invention further comprise amylases at
a level from about 0.00001 to about 10% of additional amylase by
weight of the composition and the balance of cleaning adjunct
materials by weight of composition. In some other embodiments of
the present invention, the cleaning compositions of the present
invention also comprise amylases at a level of about 0.0001 to
about 10%, about 0.001 to about 5%, about 0.001 to about 2%, about
0.005 to about 0.5% amylase by weight of the composition.
[0113] In some further embodiments, any suitable cellulase finds
used in the cleaning compositions of the present invention.
Suitable cellulases include, but are not limited to those of
bacterial or fungal origin. Chemically or genetically modified
mutants are included in some embodiments. Suitable cellulases
include, but are not limited to H. insolens cellulases (See e.g.,
U.S. Pat. No. 4,435,307). Especially suitable cellulases are the
cellulases having color care benefits (See e.g., EP0495257).
Commercially available cellulases that find use in the present
include, but are not limited to CELLUZYME.RTM., CAREZYME.RTM.
(Novozymes), REVITALENZ.TM. 100 (Danisco US Inc) and KAC-500(B).TM.
(Kao Corporation). In some embodiments, cellulases are incorporated
as portions or fragments of mature wild-type or variant cellulases,
wherein a portion of the N-terminus is deleted (See e.g., U.S. Pat.
No. 5,874,276). Additional suitable cellulases include those found
in WO2005054475, WO2005056787, and U.S. Pat. Nos. 7,449,318 and
7,833,773. In some embodiments, the cleaning compositions of the
present invention further comprise cellulases at a level from about
0.00001 to about 10% of additional cellulase by weight of the
composition and the balance of cleaning adjunct materials by weight
of composition. In some other embodiments of the present invention,
the cleaning compositions of the present invention also comprise
cellulases at a level of about 0.0001 to about 10%, about 0.001 to
about 5%, about 0.001 to about 2%, about 0.005 to about 0.5%
cellulase by weight of the composition.
[0114] Any mannanase suitable for use in detergent compositions
also finds use in the present invention. Suitable mannanases
include, but are not limited to those of bacterial or fungal
origin. Chemically or genetically modified mutants are included in
some embodiments. Various mannanases are known which find use in
the present invention (See e.g., U.S. Pat. Nos. 6,566,114;
6,602,842; and 6,440,991, all of which are incorporated herein by
reference). Commercially available mannanases that find use in the
present invention include, but are not limited to MANNASTAR.RTM.,
PURABRITE.TM., and MANNAWAY.RTM.. In some embodiments, the cleaning
compositions of the present invention further comprise mannanases
at a level from about 0.00001 to about 10% of additional mannanase
by weight of the composition and the balance of cleaning adjunct
materials by weight of composition. In some embodiments of the
present invention, the cleaning compositions of the present
invention also comprise mannanases at a level of about 0.0001 to
about 10%, about 0.001 to about 5%, about 0.001 to about 2%, about
0.005 to about 0.5% mannanase by weight of the composition.
[0115] In some embodiments, peroxidases are used in combination
with hydrogen peroxide or a source thereof (e.g., a percarbonate,
perborate or persulfate) in the compositions of the present
invention. In some alternative embodiments, oxidases are used in
combination with oxygen. Both types of enzymes are used for
"solution bleaching" (i.e., to prevent transfer of a textile dye
from a dyed fabric to another fabric when the fabrics are washed
together in a wash liquor), preferably together with an enhancing
agent (See e.g., WO94/12621 and WO95/01426). Suitable
peroxidases/oxidases include, but are not limited to those of
plant, bacterial or fungal origin. Chemically or genetically
modified mutants are included in some embodiments. In some
embodiments, the cleaning compositions of the present invention
further comprise peroxidase and/or oxidase enzymes at a level from
about 0.00001 to about 10% of additional peroxidase and/or oxidase
by weight of the composition and the balance of cleaning adjunct
materials by weight of composition. In some other embodiments of
the present invention, the cleaning compositions of the present
invention also comprise peroxidase and/or oxidase enzymes at a
level of about 0.0001 to about 10%, about 0.001 to about 5%, about
0.001 to about 2%, about 0.005 to about 0.5% peroxidase and/or
oxidase enzymes by weight of the composition.
[0116] In some embodiments, additional enzymes find use, including
but not limited to perhydrolases (See e.g., WO 2005056782,
WO2007106293, WO2008063400, WO2008106214, and WO2008106215). In
addition, in some embodiments, mixtures of the above mentioned
enzymes are encompassed herein, in particular one or more
additional protease, amylase, lipase, mannanase, and/or at least
one cellulase. Indeed, it is contemplated that various mixtures of
these enzymes will find use in the present invention. It is also
contemplated that the varying levels of the serine protease
polypeptide(s) and one or more additional enzymes may both
independently range to about 10%, the balance of the cleaning
composition being cleaning adjunct materials. The specific
selection of cleaning adjunct materials are readily made by
considering the surface, item, or fabric to be cleaned, and the
desired form of the composition for the cleaning conditions during
use (e.g., through the wash detergent use).
[0117] In some embodiments, an effective amount of one or more
serine protease polypeptide(s) provided herein is included in
compositions useful for cleaning a variety of surfaces in need of
proteinaceous stain removal. Such cleaning compositions include
cleaning compositions for such applications as cleaning hard
surfaces, fabrics, and dishes. Indeed, in some embodiments, the
present invention provides fabric cleaning compositions, while in
other embodiments the present invention provides non-fabric
cleaning compositions. Notably, the present invention also provides
cleaning compositions suitable for personal care, including oral
care (including dentrifices, toothpastes, mouthwashes, etc., as
well as denture cleaning compositions), skin, and hair cleaning
compositions. It is intended that the present invention encompass
detergent compositions in any form (i.e., liquid, granular, bar,
semi-solid, gels, emulsions, tablets, capsules, etc.).
[0118] By way of example, several cleaning compositions wherein the
serine protease polypeptides of the present invention find use are
described in greater detail below. In some embodiments in which the
cleaning compositions of the present invention are formulated as
compositions suitable for use in laundry machine washing method(s),
the compositions of the present invention preferably contain at
least one surfactant and at least one builder compound, as well as
one or more cleaning adjunct materials preferably selected from
organic polymeric compounds, bleaching agents, additional enzymes,
suds suppressors, dispersants, lime-soap dispersants, soil
suspension and anti-redeposition agents and corrosion inhibitors.
In some embodiments, laundry compositions also contain softening
agents (i.e., as additional cleaning adjunct materials). The
compositions of the present invention also find use in detergent
additive products in solid or liquid form. Such additive products
are intended to supplement and/or boost the performance of
conventional detergent compositions and can be added at any stage
of the cleaning process. In some embodiments, the density of the
laundry detergent compositions herein ranges from about 400 to
about 1200 g/liter, while in other embodiments it ranges from about
500 to about 950 g/liter of composition measured at 20.degree.
C.
[0119] In embodiments formulated as compositions for use in manual
dishwashing methods, the compositions of the invention preferably
contain at least one surfactant and preferably at least one
additional cleaning adjunct material selected from organic
polymeric compounds, suds enhancing agents, group II metal ions,
solvents, hydrotropes and additional enzymes.
[0120] In some embodiments, various cleaning compositions such as
those provided in U.S. Pat. No. 6,605,458, find use with the serine
protease polypeptides of the present invention. Thus, in some
embodiments, the compositions comprising at least one serine
protease polypeptide of the present invention is a compact granular
fabric cleaning composition, while in other embodiments, the
composition is a granular fabric cleaning composition useful in the
laundering of colored fabrics, in further embodiments, the
composition is a granular fabric cleaning composition which
provides softening through the wash capacity, in additional
embodiments, the composition is a heavy duty liquid fabric cleaning
composition. In some embodiments, the compositions comprising at
least one serine protease polypeptide of the present invention are
fabric cleaning compositions such as those described in U.S. Pat.
Nos. 6,610,642 and 6,376,450. In addition, the serine protease
polypeptides of the present invention find use in granular laundry
detergent compositions of particular utility under European or
Japanese washing conditions (See e.g., U.S. Pat. No.
6,610,642).
[0121] In some alternative embodiments, the present invention
provides hard surface cleaning compositions comprising at least one
serine protease polypeptide provided herein. Thus, in some
embodiments, the compositions comprising at least one serine
protease polypeptide of the present invention is a hard surface
cleaning composition such as those described in U.S. Pat. Nos.
6,610,642; 6,376,450; and 6,376,450.
[0122] In yet further embodiments, the present invention provides
dishwashing compositions comprising at least one serine protease
polypeptide provided herein. Thus, in some embodiments, the
compositions comprising at least one serine protease polypeptide of
the present invention is a hard surface cleaning composition such
as those in U.S. Pat. Nos. 6,610,642 and 6,376,450. In some still
further embodiments, the present invention provides dishwashing
compositions comprising at least one serine protease polypeptide
provided herein. In some further embodiments, the compositions
comprising at least one serine protease polypeptide of the present
invention comprise oral care compositions such as those in U.S.
Pat. Nos. 6,376,450 and 6,376,450. The formulations and
descriptions of the compounds and cleaning adjunct materials
contained in the aforementioned U.S. Pat. Nos. 6,376,450;
6,605,458; 6,605,458; and 6,610,642, find use with the serine
protease polypeptides provided herein.
[0123] The cleaning compositions of the present invention are
formulated into any suitable form and prepared by any process
chosen by the formulator, non-limiting examples of which are
described in U.S. Pat. Nos. 5,879,584; 5,691,297; 5,574,005;
5,569,645; 5,565,422; 5,516,448; 5,489,392; and 5,486,303, all of
which are incorporated herein by reference. When a low pH cleaning
composition is desired, the pH of such composition is adjusted via
the addition of a material such as monoethanolamine or an acidic
material such as HCl.
[0124] In some embodiments, the cleaning compositions according to
the present invention comprise an acidifying particle or an amino
carboxylic builder. Examples of an amino carboxylic builder include
aminocarboxylic acids, salts and derivatives thereof. In some
embodiment, the amino carboxylic builder is an aminopolycarboxylic
builder, such as glycine-N,N-diacetic acid or derivative of general
formula MOOC--CHR--N(CH.sub.2COOM).sub.2 where R is C.sub.1-12alkyl
and M is alkali metal. In some embodiments, the amino carboxylic
builder can be methylglycine diacetic acid (MGDA), GLDA
(glutamic-N,N-diacetic acid), iminodisuccinic acid (IDS),
carboxymethyl inulin and salts and derivatives thereof, aspartic
acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid
(ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic
acid (IDA), N-(2-sulfomethyl) aspartic acid (SMAS),
N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic
acid (SMGL), N-(2-sulfoethyl) glutamic acid (SEGL), IDS
(iminodiacetic acid) and salts and derivatives thereof such as
N-methyliminodiacetic acid (MIDA), alpha-alanine-N,N-diacetic acid
(alpha-ALDA), serine-N,N-diacetic acid (SEDA),
isoserine-N,Ndiacetic acid (ISDA), phenylalanine-N,N-diacetic acid
(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic
acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and
sulfomethyl-N,N-diacetic acid (SMDA) and alkali metal salts and
derivative thereof. In some embodiments, the acidifying particle
has a weight geometric mean particle size of from about 400.mu. to
about 1200.mu. and a bulk density of at least 550 g/L. In some
embodiments, the acidifying particle comprises at least about 5% of
the builder.
[0125] In some embodiments, the acidifying particle can comprise
any acid, including organic acids and mineral acids. Organic acids
can have one or two carboxyls and in some instances up to 15
carbons, especially up to 10 carbons, such as formic, acetic,
propionic, capric, oxalic, succinic, adipic, maleic, fumaric,
sebacic, malic, lactic, glycolic, tartaric and glyoxylic acids. In
some embodiments, the acid is citric acid. Mineral acids include
hydrochloric and sulphuric acid. In some instances, the acidifying
particle of the invention is a highly active particle comprising a
high level of amino carboxylic builder. Sulphuric acid has been
found to further contribute to the stability of the final
particle.
[0126] In some embodiments, the cleaning compositions according to
the present invention comprise at least one surfactant and/or a
surfactant system wherein the surfactant is selected from nonionic
surfactants, anionic surfactants, cationic surfactants, ampholytic
surfactants, zwitterionic surfactants, semi-polar nonionic
surfactants and mixtures thereof. In some embodiments, the
surfactant is present at a level of from about 0.1 to about 60%,
while in alternative embodiments the level is from about 1 to about
50%, while in still further embodiments the level is from about 5
to about 40%, by weight of the cleaning composition.
[0127] In some embodiments, the cleaning compositions of the
present invention comprise one or more detergent builders or
builder systems. In some embodiments incorporating at least one
builder, the cleaning compositions comprise at least about 1%, from
about 3 to about 60% or even from about 5 to about 40% builder by
weight of the cleaning composition. Builders include, but are not
limited to, the alkali metal, ammonium and alkanolammonium salts of
polyphosphates, alkali metal silicates, alkaline earth and alkali
metal carbonates, aluminosilicates, polycarboxylate compounds,
ether hydroxypolycarboxylates, copolymers of maleic anhydride with
ethylene or vinyl methyl ether, 1,3,5-trihydroxy
benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid,
the various alkali metal, ammonium and substituted ammonium salts
of polyacetic acids such as ethylenediamine tetraacetic acid and
nitrilotriacetic acid, as well as polycarboxylates such as mellitic
acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic
acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic
acid, and soluble salts thereof. Indeed, it is contemplated that
any suitable builder will find use in various embodiments of the
present invention.
[0128] In some embodiments, the builders form water-soluble
hardness ion complexes (e.g., sequestering builders), such as
citrates and polyphosphates (e.g., sodium tripolyphosphate and
sodium tripolyphospate hexahydrate, potassium tripolyphosphate, and
mixed sodium and potassium tripolyphosphate, etc.). It is
contemplated that any suitable builder will find use in the present
invention, including those known in the art (See e.g.,
EP2100949).
[0129] In some embodiments, builders for use herein include
phosphate builders and non-phosphate builders. In some embodiments,
the builder is a phosphate builder. In some embodiments, the
builder is a non-phosphate builder. If present, builders are used
in a level of from 0.1 to 80%, or from 5 to 60%, or from 10 to 50%
by weight of the composition. In some embodiments the product
comprises a mixture of phosphate and non-phosphate builders.
Suitable phosphate builders include mono-phosphates, di-phosphates,
tri-polyphosphates or oligomeric-polyphosphates, including the
alkali metal salts of these compounds, including the sodium salts.
In some embodiments, a builder can be sodium tripolyphosphate
(STPP). Additionally, the composition can comprise carbonate and/or
citrate, preferably citrate that helps to achieve a neutral pH
composition of the invention. Other suitable non-phosphate builders
include homopolymers and copolymers of polycarboxylic acids and
their partially or completely neutralized salts, monomeric
polycarboxylic acids and hydroxycarboxylic acids and their salts.
In some embodiments, salts of the above mentioned compounds include
the ammonium and/or alkali metal salts, i.e. the lithium, sodium,
and potassium salts, including sodium salts. Suitable
polycarboxylic acids include acyclic, alicyclic, hetero-cyclic and
aromatic carboxylic acids, wherein in some embodiments, they can
contain at least two carboxyl groups which are in each case
separated from one another by, in some instances, no more than two
carbon atoms.
[0130] In some embodiments, the cleaning compositions of the
present invention contain at least one chelating agent. Suitable
chelating agents include, but are not limited to copper, iron
and/or manganese chelating agents and mixtures thereof. In
embodiments in which at least one chelating agent is used, the
cleaning compositions of the present invention comprise from about
0.1 to about 15% or even from about 3.0 to about 10% chelating
agent by weight of the subject cleaning composition.
[0131] In some still further embodiments, the cleaning compositions
provided herein contain at least one deposition aid. Suitable
deposition aids include, but are not limited to, polyethylene
glycol; polypropylene glycol; polycarboxylate; soil release
polymers such as polytelephthalic acid, clays such as kaolinite,
montmorillonite, atapulgite, illite, bentonite, and halloysite; and
mixtures thereof.
[0132] As indicated herein, in some embodiments, anti-redeposition
agents find use in some embodiments of the present invention. In
some embodiments, non-ionic surfactants find use. For example, in
automatic dishwashing embodiments, non-ionic surfactants find use
for surface modification purposes, in particular for sheeting, to
avoid filming and spotting and to improve shine. These non-ionic
surfactants also find use in preventing the re-deposition of soils.
In some embodiments, the anti-redeposition agent is a non-ionic
surfactant as known in the art (See e.g., EP 2100949). In some
embodiments, the non-ionic surfactant can be ethoxylated nonionic
surfactants, epoxy-capped poly(oxyalkylated) alcohols and amine
oxides surfactants.
[0133] In some embodiments, the cleaning compositions of the
present invention include one or more dye transfer inhibiting
agents. Suitable polymeric dye transfer inhibiting agents include,
but are not limited to, polyvinylpyrrolidone polymers, polyamine
N-oxide polymers, copolymers of N-vinylpyrrolidone and
N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or
mixtures thereof. In embodiments in which at least one dye transfer
inhibiting agent is used, the cleaning compositions of the present
invention comprise from about 0.0001 to about 10%, from about 0.01
to about 5%, or even from about 0.1 to about 3% by weight of the
cleaning composition.
[0134] In some embodiments, silicates are included within the
compositions of the present invention. In some such embodiments,
sodium silicates (e.g., sodium disilicate, sodium metasilicate, and
crystalline phyllosilicates) find use. In some embodiments,
silicates are present at a level of from about 1 to about 20%. In
some embodiments, silicates are present at a level of from about 5
to about 15% by weight of the composition.
[0135] In some still additional embodiments, the cleaning
compositions of the present invention also contain dispersants.
Suitable water-soluble organic materials include, but are not
limited to the homo- or co-polymeric acids or their salts, in which
the polycarboxylic acid comprises at least two carboxyl radicals
separated from each other by not more than two carbon atoms.
[0136] In some further embodiments, the enzymes used in the
cleaning compositions are stabilized by any suitable technique. In
some embodiments, the enzymes employed herein are stabilized by the
presence of water-soluble sources of calcium and/or magnesium ions
in the finished compositions that provide such ions to the enzymes.
In some embodiments, the enzyme stabilizers include
oligosaccharides, polysaccharides, and inorganic divalent metal
salts, including alkaline earth metals, such as calcium salts, such
as calcium formate. It is contemplated that various techniques for
enzyme stabilization will find use in the present invention. For
example, in some embodiments, the enzymes employed herein are
stabilized by the presence of water-soluble sources of zinc (II),
calcium (II) and/or magnesium (II) ions in the finished
compositions that provide such ions to the enzymes, as well as
other metal ions (e.g., barium (II), scandium (II), iron (II),
manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II),
nickel (II), and oxovanadium (IV). Chlorides and sulfates also find
use in some embodiments of the present invention. Examples of
suitable oligosaccharides and polysaccharides (e.g., dextrins) are
known in the art (See e.g., WO07/145964). In some embodiments,
reversible protease inhibitors also find use, such as
boron-containing compounds (e.g., borate, 4-formyl phenyl boronic
acid) and/or a tripeptide aldehyde find use to further improve
stability, as desired.
[0137] In some embodiments, bleach, bleach activators and/or bleach
catalysts are present in the compositions of the present invention.
In some embodiments, the cleaning compositions of the present
invention comprise inorganic and/or organic bleaching compound(s).
Inorganic bleaches include, but are not limited to perhydrate salts
(e.g., perborate, percarbonate, perphosphate, persulfate, and
persilicate salts). In some embodiments, inorganic perhydrate salts
are alkali metal salts. In some embodiments, inorganic perhydrate
salts are included as the crystalline solid, without additional
protection, although in some other embodiments, the salt is coated.
Any suitable salt known in the art finds use in the present
invention (See e.g., EP2100949).
[0138] In some embodiments, bleach activators are used in the
compositions of the present invention. Bleach activators are
typically organic peracid precursors that enhance the bleaching
action in the course of cleaning at temperatures of 60.degree. C.
and below. Bleach activators suitable for use herein include
compounds which, under perhydrolysis conditions, give aliphatic
peroxoycarboxylic acids having preferably from about 1 to about 10
carbon atoms, in particular from about 2 to about 4 carbon atoms,
and/or optionally substituted perbenzoic acid. Additional bleach
activators are known in the art and find use in the present
invention (See e.g., EP2100949).
[0139] In addition, in some embodiments and as further described
herein, the cleaning compositions of the present invention further
comprise at least one bleach catalyst. In some embodiments, the
manganese triazacyclononane and related complexes find use, as well
as cobalt, copper, manganese, and iron complexes. Additional bleach
catalysts find use in the present invention (See e.g., U.S. Pat.
Nos. 4,246,612; 5,227,084; 4,810,410; WO99/06521; and
EP2100949).
[0140] In some embodiments, the cleaning compositions of the
present invention contain one or more catalytic metal complexes. In
some embodiments, a metal-containing bleach catalyst finds use. In
some embodiments, the metal bleach catalyst comprises a catalyst
system comprising a transition metal cation of defined bleach
catalytic activity, (e.g., copper, iron, titanium, ruthenium,
tungsten, molybdenum, or manganese cations), an auxiliary metal
cation having little or no bleach catalytic activity (e.g., zinc or
aluminum cations), and a sequestrate having defined stability
constants for the catalytic and auxiliary metal cations,
particularly ethylenediaminetetraacetic acid, ethylenediaminetetra
(methylenephosphonic acid) and water-soluble salts thereof are used
(See e.g., U.S. Pat. No. 4,430,243). In some embodiments, the
cleaning compositions of the present invention are catalyzed by
means of a manganese compound. Such compounds and levels of use are
well known in the art (See e.g., U.S. Pat. No. 5,576,282). In
additional embodiments, cobalt bleach catalysts find use in the
cleaning compositions of the present invention. Various cobalt
bleach catalysts are known in the art (See e.g., U.S. Pat. Nos.
5,597,936 and 5,595,967) and are readily prepared by known
procedures.
[0141] In some additional embodiments, the cleaning compositions of
the present invention include a transition metal complex of a
macropolycyclic rigid ligand (MRL). As a practical matter, and not
by way of limitation, in some embodiments, the compositions and
cleaning processes provided by the present invention are adjusted
to provide on the order of at least one part per hundred million of
the active MRL species in the aqueous washing medium, and in some
embodiments, provide from about 0.005 to about 25 ppm, more
preferably from about 0.05 to about 10 ppm, and most preferably
from about 0.1 to about 5 ppm of the MRL in the wash liquor.
[0142] In some embodiments, transition-metals in the instant
transition-metal bleach catalyst include, but are not limited to
manganese, iron and chromium. MRLs also include, but are not
limited to special ultra-rigid ligands that are cross-bridged
(e.g., 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane).
Suitable transition metal MRLs are readily prepared by known
procedures (See e.g., WO2000/32601, and U.S. Pat. No.
6,225,464).
[0143] In some embodiments, the cleaning compositions of the
present invention comprise metal care agents. Metal care agents
find use in preventing and/or reducing the tarnishing, corrosion,
and/or oxidation of metals, including aluminum, stainless steel,
and non-ferrous metals (e.g., silver and copper). Suitable metal
care agents include those described in EP2100949, WO9426860 and
WO94/26859). In some embodiments, the metal care agent is a zinc
salt. In some further embodiments, the cleaning compositions of the
present invention comprise from about 0.1 to about 5% by weight of
one or more metal care agent.
[0144] In some embodiments, the cleaning composition is a high
density liquid (HDL) composition having a variant serine protease
polypeptide protease. The HDL liquid laundry detergent can comprise
a detersive surfactant (10-40%) comprising anionic detersive
surfactant (selected from a group of linear or branched or random
chain, substituted or unsubstituted alkyl sulphates, alkyl
sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl
phosphonates, alkyl carboxylates, and/or mixtures thereof); and
optionally non-ionic surfactant (selected from a group of linear or
branched or random chain, substituted or unsubstituted alkyl
alkoxylated alcohol, for example a C.sub.8-C.sub.18alkyl
ethoxylated alcohol and/or C.sub.6-C.sub.12alkyl phenol
alkoxylates), optionally wherein the weight ratio of anionic
detersive surfactant (with a hydrophilic index (HIc) of from 6.0 to
9) to non-ionic detersive surfactant is greater than 1:1.
[0145] The composition can comprise optionally, a surfactancy
boosting polymer consisting of amphiphilic alkoxylated grease
cleaning polymers (selected from a group of alkoxylated polymers
having branched hydrophilic and hydrophobic properties, such as
alkoxylated polyalkylenimines in the range of 0.05 wt %-10 wt %)
and/or random graft polymers (typically comprising of hydrophilic
backbone comprising monomers selected from the group consisting of:
unsaturated C.sub.1-C.sub.6carboxylic acids, ethers, alcohols,
aldehydes, ketones, esters, sugar units, alkoxy units, maleic
anhydride, saturated polyalcohols such as glycerol, and mixtures
thereof; and hydrophobic side chain(s) selected from the group
consisting of: C.sub.4-C.sub.25alkyl group, polypropylene,
polybutylene, vinyl ester of a saturated
C.sub.2-C.sub.6mono-carboxylic acid, C.sub.1-C.sub.6alkyl ester of
acrylic or methacrylic acid, and mixtures thereof.
[0146] The composition can comprise additional polymers such as
soil release polymers (include anionically end-capped polyesters,
for example SRP1, polymers comprising at least one monomer unit
selected from saccharide, dicarboxylic acid, polyol and
combinations thereof, in random or block configuration, ethylene
terephthalate-based polymers and co-polymers thereof in random or
block configuration, for example Repel-o-tex SF, SF-2 and SRP6,
Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325,
Marloquest SL), anti-redeposition polymers (0.1 wt % to 10 wt %,
include carboxylate polymers, such as polymers comprising at least
one monomer selected from acrylic acid, maleic acid (or maleic
anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic
acid, citraconic acid, methylenemalonic acid, and any mixture
thereof, vinylpyrrolidone homopolymer, and/or polyethylene glycol,
molecular weight in the range of from 500 to 100,000 Da);
cellulosic polymer (including those selected from alkyl cellulose,
alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl
carboxyalkyl cellulose examples of which include carboxymethyl
cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl
carboxymethyl cellulose, and mixtures thereof) and polymeric
carboxylate (such as maleate/acrylate random copolymer or
polyacrylate homopolymer).
[0147] The composition can further comprise saturated or
unsaturated fatty acid, preferably saturated or unsaturated
C.sub.12-C.sub.24fatty acid (0 to 10 wt %); deposition aids
(examples for which include polysaccharides, preferably cellulosic
polymers, poly diallyl dimethyl ammonium halides (DADMAC), and
co-polymers of DADMAC with vinyl pyrrolidone, acrylamides,
imidazoles, imidazolinium halides, and mixtures thereof, in random
or block configuration, cationic guar gum, cationic cellulose such
as cationic hydroxyethyl cellulose, cationic starch, cationic
polyacylamides, and mixtures thereof.
[0148] The composition can further comprise dye transfer inhibiting
agents examples of which include manganese phthalocyanine,
peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinyloxazolidones and polyvinylimidazoles and/or mixtures
thereof; chelating agents examples of which include
ethylene-diamine-tetraacetic acid (EDTA); diethylene triamine penta
methylene phosphonic acid (DTPMP); hydroxy-ethane diphosphonic acid
(HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methyl glycine
diacetic acid (MGDA); diethylene triamine penta acetic acid (DTPA);
propylene diamine tetracetic acid (PDTA); 2-hydroxypyridine-N-oxide
(HPNO); or methyl glycine diacetic acid (MGDA); glutamic acid
N,N-diacetic acid (N,N-dicarboxymethyl glutamic acid tetrasodium
salt (GLDA); nitrilotriacetic acid (NTA);
4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any salts
thereof; N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),
triethylenetetraaminehexaacetic acid (TTHA),
N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine
(DHEG), ethylenediaminetetrapropionic acid (EDTP) and derivatives
thereof.
[0149] The composition may comprise an enzyme stabilizer (examples
of which include polyols such as propylene glycol or glycerol,
sugar or sugar alcohol, lactic acid, reversible protease inhibitor,
boric acid, or a boric acid derivative, e.g., an aromatic borate
ester, or a phenyl boronic acid derivative such as 4-formylphenyl
boronic acid).
[0150] The composition can further comprise silicone or fatty-acid
based suds suppressors; hueing dyes, calcium and magnesium cations,
visual signaling ingredients, anti-foam (0.001 wt % to about 4.0 wt
%), and/or structurant/thickener (0.01 wt % to 5 wt %, selected
from the group consisting of diglycerides and triglycerides,
ethylene glycol distearate, microcrystalline cellulose, cellulose
based materials, microfiber cellulose, biopolymers, xanthan gum,
gellan gum, and mixtures thereof).
[0151] Suitable detersive surfactants also include cationic
detersive surfactants (selected from a group of alkyl pyridinium
compounds, alkyl quarternary ammonium compounds, alkyl quarternary
phosphonium compounds, alkyl ternary sulphonium compounds, and/or
mixtures thereof); zwitterionic and/or amphoteric detersive
surfactants (selected from a group of alkanolamine
sulpho-betaines); ampholytic surfactants; semi-polar non-ionic
surfactants and mixtures thereof.
[0152] The composition can be any liquid form, for example a liquid
or gel form, or any combination thereof. The composition may be in
any unit dose form, for example a pouch.
[0153] In some embodiments, the cleaning composition is a high
density powder (HDD) composition having a variant serine protease
polypeptide protease. The HDD powder laundry detergent can comprise
a detersive surfactant including anionic detersive surfactants
(selected from a group of linear or branched or random chain,
substituted or unsubstituted alkyl sulphates, alkyl sulphonates,
alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates,
alkyl carboxylates and/or mixtures thereof), non-ionic detersive
surfactant (selected from a group of linear or branched or random
chain, substituted or unsubstituted C.sub.8-C.sub.18 alkyl
ethoxylates, and/or C.sub.6-C.sub.12 alkyl phenol alkoxylates),
cationic detersive surfactants (selected from a group of alkyl
pyridinium compounds, alkyl quaternary ammonium compounds, alkyl
quaternary phosphonium compounds, alkyl ternary sulphonium
compounds, and mixtures thereof), zwitterionic and/or amphoteric
detersive surfactants (selected from a group of alkanolamine
sulpho-betaines); ampholytic surfactants; semi-polar non-ionic
surfactants and mixtures thereof; builders (phosphate free builders
[for example zeolite builders examples of which include zeolite A,
zeolite X, zeolite P and zeolite MAP in the range of 0 to less than
10 wt %]; phosphate builders [examples of which include sodium
tri-polyphosphate in the range of 0 to less than 10 wt %]; citric
acid, citrate salts and nitrilotriacetic acid or salt thereof in
the range of less than 15 wt %); silicate salt (sodium or potassium
silicate or sodium meta-silicate in the range of 0 to less than 10
wt %, or layered silicate (SKS-6)); carbonate salt (sodium
carbonate and/or sodium bicarbonate in the range of 0 to less than
10 wt %); and bleaching agents (photobleaches, examples of which
include sulfonated zinc phthalocyanines, sulfonated aluminum
phthalocyanines, xanthenes dyes, and mixtures thereof; hydrophobic
or hydrophilic bleach activators (examples of which include
dodecanoyl oxybenzene sulfonate, decanoyl oxybenzene sulfonate,
decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethy hexanoyl
oxybenzene sulfonate, tetraacetyl ethylene diamine-TAED, and
nonanoyloxybenzene sulfonate-NOBS, nitrile quats, and mixtures
thereof; hydrogen peroxide; sources of hydrogen peroxide (inorganic
perhydrate salts examples of which include mono or tetra hydrate
sodium salt of perborate, percarbonate, persulfate, perphosphate,
or persilicate); preformed hydrophilic and/or hydrophobic peracids
(selected from a group consisting of percarboxylic acids and salts,
percarbonic acids and salts, perimidic acids and salts,
peroxymonosulfuric acids and salts) & mixtures thereof and/or
bleach catalyst (such as imine bleach boosters examples of which
include iminium cations and polyions; iminium zwitterions; modified
amines; modified amine oxides; N-sulphonyl imines; N-phosphonyl
imines; N-acyl imines; thiadiazole dioxides; perfluoroimines;
cyclic sugar ketones and mixtures thereof; metal-containing bleach
catalyst for example copper, iron, titanium, ruthenium, tungsten,
molybdenum, or manganese cations along with an auxiliary metal
cations such as zinc or aluminum and a sequestrate such as
ethylenediaminetetraacetic acid,
ethylenediaminetetra(methylenephosphonic acid) and water-soluble
salts thereof).
[0154] The composition can further comprise additional detergent
ingredients including perfume microcapsules, starch encapsulated
perfume accord, hueing agents, additional polymers including fabric
integrity and cationic polymers, dye lock ingredients,
fabric-softening agents, brighteners (for example C.I. Fluorescent
brighteners), flocculating agents, chelating agents, alkoxylated
polyamines, fabric deposition aids, and/or cyclodextrin.
[0155] In some embodiments, the cleaning composition is an
automatic dishwashing (ADW) detergent composition having a serine
protease of the present invention. The ADW detergent composition
can comprise two or more non-ionic surfactants selected from a
group of ethoxylated non-ionic surfactants, alcohol alkoxylated
surfactants, epoxy-capped poly(oxyalkylated) alcohols, or amine
oxide surfactants present in amounts from 0 to 10% by weight;
builders in the range of 5-60% comprising either phosphate
(mono-phosphates, di-phosphates, tri-polyphosphates or
oligomeric-polyphosphates, preferred sodium tripolyphosphate-STPP
or phosphate-free builders [amino acid based compounds, examples of
which include MGDA (methyl-glycine-diacetic acid), and salts and
derivatives thereof, GLDA (glutamic-N,Ndiacetic acid) and salts and
derivatives thereof, IDS (iminodisuccinic acid) and salts and
derivatives thereof, carboxy methyl inulin and salts and
derivatives thereof and mixtures thereof, nitrilotriacetic acid
(NTA), diethylene triamine penta acetic acid (DTPA),
B-alaninediacetic acid (B-ADA) and their salts], homopolymers and
copolymers of poly-carboxylic acids and their partially or
completely neutralized salts, monomeric polycarboxylic acids and
hydroxycarboxylic acids and their salts in the range of 0.5 to 50%
by weight; sulfonated/carboxylated polymers (provide dimensional
stability to the product) in the range of about 0.1 to about 50% by
weight; drying aids in the range of about 0.1 to about 10% by
weight (selected from polyesters, especially anionic polyesters
optionally together with further monomers with 3 to 6
functionalities which are conducive to polycondensation,
specifically acid, alcohol or ester functionalities,
polycarbonate-, polyurethane- and/or polyurea-polyorganosiloxane
compounds or precursor compounds thereof of the reactive cyclic
carbonate and urea type); silicates in the range from about 1 to
about 20% by weight (sodium or potassium silicates for example
sodium disilicate, sodium meta-silicate and crystalline
phyllosilicates); bleach-inorganic (for example perhydrate salts
such as perborate, percarbonate, perphosphate, persulfate and
persilicate salts) and organic (for example organic peroxyacids
including diacyl and tetraacylperoxides, especially
diperoxydodecanedioc acid, diperoxytetradecanedioc acid, and
diperoxyhexadecanedioc acid); bleach activators-organic peracid
precursors in the range from about 0.1 to about 10% by weight;
bleach catalysts (selected from manganese triazacyclononane and
related complexes, Co, Cu, Mn and Fe bispyridylamine and related
complexes, and pentamine acetate cobalt(III) and related
complexes); metal care agents in the range from about 0.1 to 5% by
weight (selected from benzatriazoles, metal salts and complexes,
and/or silicates); enzymes in the range from about 0.01 to 5.0 mg
of active enzyme per gram of automatic dishwashing detergent
composition (acyl transferases, alpha-amylases, beta-amylases,
alpha-galactosidases, arabinosidases, aryl esterases,
beta-galactosidases, carrageenases, catalases, cellobiohydrolases,
cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases,
endo-beta-mannanases, esterases, exo-mannanases, galactanases,
glucoamylases, hemicellulases, hyaluronidases, keratinases,
laccases, lactases, ligninases, lipases, lipoxygenases, mannanases,
oxidases, pectate lyases, pectin acetyl esterases, pectinases,
pentosanases, peroxidases, phenoloxidases, phosphatases,
phospholipases, phytases, polygalacturonases, proteases,
pullulanases, reductases, rhamnogalacturonases, beta-glucanases,
tannases, transglutaminases, xylan acetyl-esterases, xylanases,
xyloglucanases, and xylosidases, and any mixture thereof); and
enzyme stabilizer components (selected from oligosaccharides,
polysaccharides and inorganic divalent metal salts).
[0156] In some embodiments, the cleaning composition is
borate-free. In some embodiments, the cleaning composition is
phosphate-free.
[0157] Representative detergent formulations that beneficially
include a serine protease polypeptide of the present invention
include the detergent formulations found in WO2013063460, pages
78-152, and in particular the tables of pages 94 to 152 are hereby
incorporated by reference. The serine proteases are normally
incorporated into the detergent composition at a level of from
0.00001 to 10% of enzyme protein by weight of the composition. In
some embodiments, the detergent composition comprises more than
0.0001%, 0.001%, 0.01%, or 0.1% of the serine protease by weight of
the composition. In some embodiments, the detergent composition
comprises less than 1%, 0.1%, 0.01%, or 0.001% of the serine
protease by weight of the composition.
[0158] Also provided are compositions and methods of treating
fabrics (e.g., to desize a textile) using a serine protease
polypeptide of the present invention. Fabric-treating methods are
well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For
example, the feel and appearance of a fabric can be improved by a
method comprising contacting the fabric with a serine protease in a
solution. The fabric can be treated with the solution under
pressure.
[0159] A serine protease of the present invention can be applied
during or after the weaving of a textile, or during the desizing
stage, or one or more additional fabric processing steps. During
the weaving of textiles, the threads are exposed to considerable
mechanical strain. Prior to weaving on mechanical looms, warp yarns
are often coated with sizing starch or starch derivatives to
increase their tensile strength and to prevent breaking. A serine
protease of the present invention can be applied during or after
the weaving to remove the sizing starch or starch derivatives.
After weaving, the serine protease can be used to remove the size
coating before further processing the fabric to ensure a
homogeneous and wash-proof result.
[0160] A serine protease of the present invention can be used alone
or with other desizing chemical reagents and/or desizing enzymes to
desize fabrics, including cotton-containing fabrics, as detergent
additives, e.g., in aqueous compositions. An amylase also can be
used in compositions and methods for producing a stonewashed look
on indigo-dyed denim fabric and garments. For the manufacture of
clothes, the fabric can be cut and sewn into clothes or garments,
which are afterwards finished. In particular, for the manufacture
of denim jeans, different enzymatic finishing methods have been
developed. The finishing of denim garment normally is initiated
with an enzymatic desizing step, during which garments are
subjected to the action of proteolytic enzymes to provide softness
to the fabric and make the cotton more accessible to the subsequent
enzymatic finishing steps. The serine protease can be used in
methods of finishing denim garments (e.g., a "bio-stoning
process"), enzymatic desizing and providing softness to fabrics,
and/or finishing process.
[0161] The serine protease polypeptides described herein find
further use in the enzyme aided removal of proteins from animals
and their subsequent degradation or disposal, such as feathers,
skin, hair, hide, and the like. In some instances, immersion of the
animal carcass in a solution comprising a serine protease
polypeptide of the present invention can act to protect the skin
from damage in comparison to the traditional immersion in scalding
water or the defeathering process. In one embodiment, feathers can
be sprayed with an isolated serine protase polypeptide of the
present invention under conditions suitable for digesting or
initiating degradation of the plumage. In some embodiments, a
serine protease of the present invention can be used, as above, in
combination with an oxidizing agent.
[0162] In some embodiments, removal of the oil or fat associated
with raw feathers is assisted by using a serine protease
polypeptide of the present invention. In some embodiments, the
serine protease polypeptides are used in compositions for cleaning
the feathers as well as to sanitize and partially dehydrate the
fibers. In yet other embodiments, the disclosed serine protease
polypeptides find use in recovering protein from plumage. In some
other embodiments, the serine protease polypeptides are applied in
a wash solution in combination with 95% ethanol or other polar
organic solvent with or without a surfactant at about 0.5%
(v/v).
[0163] In a further aspect of the invention, the serine protease
polypeptides of the present invention can be used as a component of
an animal feed composition, animal feed additive and/or pet food
comprising a serine protease and variants thereof. The present
invention further relates to a method for preparing such an animal
feed composition, animal feed additive composition and/or pet food
comprising mixing the serine protease polypeptide with one or more
animal feed ingredients and/or animal feed additive ingredients
and/or pet food ingredients. Furthermore, the present invention
relates to the use of the serine protease polypeptide in the
preparation of an animal feed composition and/or animal feed
additive composition and/or pet food.
[0164] The term "animal" includes all non-ruminant and ruminant
animals. In a particular embodiment, the animal is a non-ruminant
animal, such as a horse and a mono-gastric animal. Examples of
mono-gastric animals include, but are not limited to, pigs and
swine, such as piglets, growing pigs, sows; poultry such as
turkeys, ducks, chicken, broiler chicks, layers; fish such as
salmon, trout, tilapia, catfish and carps; and crustaceans such as
shrimps and prawns. In a further embodiment the animal is a
ruminant animal including, but not limited to, cattle, young
calves, goats, sheep, giraffes, bison, moose, elk, yaks, water
buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and
nilgai.
[0165] In the present context, it is intended that the term "pet
food" is understood to mean a food for a household animal such as,
but not limited to, dogs, cats, gerbils, hamsters, chinchillas,
fancy rats, guinea pigs; avian pets, such as canaries, parakeets,
and parrots; reptile pets, such as turtles, lizards and snakes; and
aquatic pets, such as tropical fish and frogs.
[0166] The terms "animal feed composition," "feedstuff" and
"fodder" are used interchangeably and can comprise one or more feed
materials selected from the group comprising a) cereals, such as
small grains (e.g., wheat, barley, rye, oats and combinations
thereof) and/or large grains such as maize or sorghum; b) by
products from cereals, such as corn gluten meal, Distillers Dried
Grain Solubles (DDGS) (particularly corn based Distillers Dried
Grain Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts,
rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c)
protein obtained from sources such as soya, sunflower, peanut,
lupin, peas, fava beans, cotton, canola, fish meal, dried plasma
protein, meat and bone meal, potato protein, whey, copra, sesame;
d) oils and fats obtained from vegetable and animal sources; and e)
minerals and vitamins.
[0167] The protease polypeptides described herein find further use
in the enzyme aided bleaching of paper pulps such as chemical
pulps, semi-chemical pulps, kraft pulps, mechanical pulps or pulps
prepared by the sulfite method. In general terms, paper pulps are
incubated with a protease polypeptide of the present invention
under conditions suitable for bleaching the paper pulp.
[0168] In some embodiments, the pulps are chlorine free pulps
bleached with oxygen, ozone, peroxide or peroxyacids. In some
embodiments, the protease polypeptides are used in enzyme aided
bleaching of pulps produced by modified or continuous pulping
methods that exhibit low lignin contents. In some other
embodiments, the protease polypeptides are applied alone or
preferably in combination with xylanase and/or endoglucanase and/or
alpha-galactosidase and/or cellobiohydrolase enzymes.
[0169] The protease polypeptides described herein find further use
in the enzyme aided removal of proteins from animals and their
subsequent degradation or disposal, such as feathers, skin, hair,
hide, and the like. In some instances, immersion of the animal
carcass in a solution comprising a protease polypeptide of the
present invention can act to protect the skin from damage in
comparison to the traditional immersion in scalding water or the
defeathering process. In one embodiment, feathers can be sprayed
with an isolated protease polypeptide of the present invention
under conditions suitable for digesting or initiating degradation
of the plumage. In some embodiments, a protease of the present
invention can be used, as above, in combination with an oxidizing
agent.
[0170] In some embodiments, removal of the oil or fat associated
with raw feathers is assisted by using a protease polypeptide of
the present invention. In some embodiments, the protease
polypeptides are used in compositions for cleaning the feathers as
well as to sanitize and partially dehydrate the fibers. In some
other embodiments, the protease polypeptides are applied in a wash
solution in combination with 95% ethanol or other polar organic
solvent with or without a surfactant at about 0.5% (v/v). In yet
other embodiments, the disclosed protease polypeptides find use in
recovering protein from plumage. The disclosed protease
polypeptides may be used alone or in combination in suitable
feather processing and proteolytic methods, such as those disclosed
in PCT/EP2013/065362, PCT/EP2013/065363, and PCT/EP2013/065364,
which are hereby incorporated by reference. In some embodiments,
the recovered protein can be subsequently used in animal or fish
feed.
EXAMPLES
[0171] The following examples are provided to demonstrate and
illustrate certain preferred embodiments and aspects of the present
disclosure and should not be construed as limiting. In the
experimental disclosure which follows, the following abbreviations
apply: ADW (automatic dish washing); BMI (blood/milk/ink); BSA
(bovine serum albumin); CAPS (N-cyclohexyl-3-aminopropanesulfonic
acid); CHES (N-cyclohexyl-2-aminoethanesulfonic acid); DMC
(dimethyl casein); HDD (heavy duty dry/powder); HDL (heavy duty
liquid); HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid); MTP (microtiter plate); ND (not done); OD (optical density);
PCR (polymerase chain reaction); ppm (parts per million); QS
(quantity sufficient); rpm (revolutions per minute); AAPF
(succinyl-Ala-Ala-Pro-Phe-p-nitroanilide); TNBSA
(2,4,6-trinitrobenzene sulfonic acid); v/v (volume to volume); and
w/v (weight to volume).
Example 1
Discovery and Identification of Bacillus Serine Proteases
[0172] Bacillus sp. DSM 8714, Bacillus sp. DSM 8717, B.
pseudalcaliphilus DSM 8725, B. oshimensis NCIMB 14023, and B.
patagoniensis DSM 16117, were all selected as a potential source
for enzymes useful in industrial applications. The DSM strains were
obtained from Leibniz-Institut DSMZ--Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH. Bacillus oshimensis NCIMB
14023 was obtained from NCIMB Ltd, Aberdeen, Scotland. WDG290 and
WDG291 are from the Dupont Culture Collection.
[0173] To identify enzymes produced by these strains and the genes
that encode these enzymes, the genomes of these strains were
sequenced using Illumina.RTM. sequencing by synthesis (SBS)
technology. Genome sequencing and assembly of the sequence data was
performed by BaseClear (Leiden, The Netherlands). Contigs were
annotated by BioXpr (Namur, Belgium). One of genes identified this
way in strain Bacillus sp. DSM 8714 encodes a protein that shows
homology to serine proteases of various other bacteria. The
sequence of this gene, BspAL03279.n, is depicted in SEQ ID
NO:1.
[0174] SEQ ID NO: 1 sets forth the nucleotide sequence of the
BspAL03279.n gene: ATGA
ATCGAAAACCAGTTAAACTAATCGCAGGAACAGCTCTTGTTATGGGCTTTGTCATCA
GTTCATCATCCATATCAACTGCCGAGGAAACAAAAAAGACTTATCTTATTGGCTTTG
ATGCTCAGGAAGAAGTCGAAACATTCACGAATATGGTCGATTCTGAGATAGGGGCT
CTATCTGAAGAAGAAATTGATATTACCTACGAATTTAAAGAAATACCGGTCGTCTCT
GCTGAAATGAGTGAAGAAGAATATGCAGCATTACTAGAAGACCCATCGATATCATA
TATTGAAGAAGACATCGAAGTAACAACAATGGCCCAAGCCATTCCATGGGGAATTA
GTCAAATTAGTGCCCCTGAAGCGCAAATTGCTGGATTTACTGGTGAGGGTGTAAATG
TTGCGGTGCTGGATACTGGAATAGAGGATCACCCCGATTTAAACGTTCAAGGCGGTG
TTAGCTTTGTTCAAGGAGAGCCGGATTATCAGGATGGAAATGGACACGGAACCCAT
GTCGCCGGTACAATCGCTGCCCTTGATAACGACGAAGGCGTAATTGGAGTCGCACC
AAATGCAGATCTTTATGCAGTCAAAGTTCTGGGTGCAAATGGTTCTGGCTCAGTCAG
CTCAATTGCTCAAGGGCTTGAATGGGCAGGAGAAAACGGAATGGACATTGCAAACT
TAAGCTTAGGTAGCTCAGCACCTAGCGCGACACTCGAGCAAGCAGTGGATGAAGCA
ACCGCAAATGGTGTCCTCGTTGTTGCCGCTTCTGGGAACTCTGGTGCAAGTTCCATT
GGTTATCCAGCTCGCTATGATAATGCTATGGCCGTTGGCGCCACCGACCAGTCAGAT
GGCCTAGCTAGCTTTTCTCAGTACGGTGATGGCTTAGACATCGTTGCTCCAGGTGTT
GGCATCGATAGTACCTATCCTGGTAGCTCATACGATAGCTTAAGTGGAACATCAATG
GCAACACCTCATGTTGCTGGTGCCGCAGCATTGGTGAAAGAAAAGAATCCACTTTGG
TCAAATGAACAAATTCGCGCTCATTTAAACGAAACTGCAACTGACCTTGGCGATATG
TATCGTTTTGGTAATGGACTTTTAAACGCACATGCCGCTGTTGAA.
[0175] The preproenzyme encoded by the BspAL03279.n gene is
depicted in SEQ ID NO:2. At the N-terminus, the protein has a
signal peptide with a length of 28 amino acids as predicted by
SignalP-NN. The presence of a signal peptide indicates that this
serine protease is a secreted enzyme. The enzyme has a pro sequence
which is predicted to be 78 amino acids.
[0176] SEQ ID NO:2 sets forth the amino acid sequence of the serine
protease precursor of BspAL03279 (the signal peptide sequence is
underlined and in bold, the prosequence is in italics):
MNRKPVKLIAGTALVMGFVISSSSISTAEETKKTYLIGFDAQEEVETFTNMVDSEI
GALSEEEIDITYEFKEIPVVSAEMSEEEYAALLEDPSISYIEEDIEVTTMAQAIPWGISQISAPEA
QIAGFTGEGVNVAVLDTGIEDHPDLNVQGGVSFVQGEPDYQDGNGHGTHVAGTIAALD
NDEGVIGVAPNADLYAVKVLGANGSGSVSSIAQGLEWAGENGMDIANLSLGSSAPSAT
LEQAVDEATANGVLVVAASGNSGASSIGYPARYDNAMAVGATDQSDGLASFSQYGDG
LDIVAPGVGIDSTYPGSSYDSLSGTSMATPHVAGAAALVKEKNPLWSNEQIRAHLNETA
TDLGDMYRFGNGLLNAHAAVE.
[0177] SEQ ID NO:3 sets forth the amino acid sequence of the
predicted mature protease BspAL03279 (269 amino acids):
AQAIPWGISQISAPEAQIAGFTGEGVNVAVLDTGIEDHPD
LNVQGGVSFVQGEPDYQDGNGHGTHVAGTIAALDNDEGVIGVAPNADLYAVKVLGAN
GSGSVSSIAQGLEWAGENGMDIANLSLGSSAPSATLEQAVDEATANGVLVVAASGNSG
ASSIGYPARYDNAMAVGATDQSDGLASFSQYGDGLDIVAPGVGIDSTYPGSSYDSLSGT
SMATPHVAGAAALVKEKNPLWSNEQIRAHLNETATDLGDMYRFGNGLLNAHAAVE.
[0178] In Bacillus sp. DSM 8717, another gene was identified
encoding a serine protease. The nucleotide sequence of this gene,
BspAK01305.n, is depicted in SEQ ID NO:4:ATGAAGA
AAAGATCAAACGTTTTAATCGCAGGAACAGCGATCGCAACCATTGCTTTAATAGGA
ACACCATCCATTTCAGAAGCTGCAGAGGAAAAAAAATCTTATTTAATTGGCTTTGAT
GAACCTCAAGAAGTTGAGCAATTTACAACAAATTTGGAAGAAGAGATTCGTACACA
AGCAGATGATGCTATTGATGTAACGTACGAGTTTAAAGATATTCCTGTTCTTGCCGT
AGATATGACGGAAGAAGAAATGACTGAACTCAAAAATGAAGAGAGTATTTCCTATA
TTGAAGAAGATCAAGAAGTGACAACGATGGCGCAAAGCATTCCATGGGGAATTGAA
AGAATTGGCACGCCAGCAGCACACGCATCAGGATTCACAGGTAGCGGTGTAAGTGT
CGCGGTCCTTGATACAGGGATTGATCCACATTCTGACTTAAATGTACAAGGGGGGGT
TAGTTTTGTACCAGGCGAAAGTGGAGCAGATGATGGAAATGGACACGGTACTCATG
TAGCAGGAACGATTGCAGCGTTAGATAATGATGAAGGCGTTTTAGGCGTTGCTCCAG
AGGTTGATCTCTTTGCAGTAAAAGTTTTAAGTGCATCTGGATCAGGATCAATTAGTT
CGATTGCGCAAGGTTTAGAGTGGACAGCTGAAAACAACATTGATGTGGCTAATTTA
AGCTTAGGCAGTCCCTCTCCTAGTCAGACGCTAGAACAAGCGGTTAATGACGCCAC
AGATAGTGGTGTGCTTGTAGTAGCAGCAGCAGGGAATTCTGGAACAAGCTCATTAG
GTTATCCAGCTCGTTATGATAATGCAATGGCTGTTGGCGCTACCGACCAATCCGATA
GCCTGGCTAGCTTCTCACAGTATGGCGAGGGTCTTGACTTAGTCGCTCCTGGTGTTG
GTGTAGAAAGCACGTACCCAGGTGGAGGTTATGACAGCTTAAGCGGCACATCTATG
GCTGCTCCACATGTTGCAGGTGCAGCAGCACTCGTTAAACAAAAAAATCCAGGCTG
GACAAACGAACAAATACGAAGCCATTTAAACGATACAGCCAATGATCTTGGCGATT
CGTTCCGCTTCGGTAGTGGCTTATTGAATGCCGAAAATGCCGTTCAA.
[0179] The preproenzyme encoded by the BspAK01305.n gene is
depicted in SEQ ID NO:5. At the N-terminus, the protein has a
signal peptide with a length of 28 amino acids as predicted by
SignalP-NN. The presence of a signal peptide indicates that this
serine protease is a secreted enzyme. The enzyme has a pro sequence
which is predicted to be 78 amino acids.
[0180] SEQ ID NO:5 sets forth the amino acid sequence of the serine
protease precursor of BspAK01305 (the signal peptide sequence is
underlined and in bold, the prosequence is in italics):
MKKRSNVLIAGTAIATIALIGTPSISEAAEEKKSYLIGFDEPQEVEQFTTNLEEEIR
TQADDAIDVTYEFKDIPVLAVDMTEEEMTELKNEESISYIEEDQEVTTMAQSIPWGIERIGTPA
AHASGFTGSGVSVAVLDTGIDPHSDLNVQGGVSFVPGESGADDGNGHGTHVAGTIAAL
DNDEGVLGVAPEVDLFAVKVLSASGSGSISSIAQGLEWTAENNIDVANLSLGSPSPSQTL
EQAVNDATDSGVLVVAAAGNSGTSSLGYPARYDNAMAVGATDQSDSLASFSQYGEGL
DLVAPGVGVESTYPGGGYDSLSGTSMAAPHVAGAAALVKQKNPGWTNEQIRSHLNDT
ANDLGDSFRFGSGLLNAENAVQ.
[0181] SEQ ID NO:6 sets forth the amino acid sequence of the
predicted mature protease BspAK01305 (269 amino acids):
AQSIPWGIERIGTPAAHASGFTGSGVSVAVLDTGIDPHS
DLNVQGGVSFVPGESGADDGNGHGTHVAGTIAALDNDEGVLGVAPEVDLFAVKVLSA
SGSGSISSIAQGLEWTAENNIDVANLSLGSPSPSQTLEQAVNDATDSGVLVVAAAGNSGT
SSLGYPARYDNAMAVGATDQSDSLASFSQYGEGLDLVAPGVGVESTYPGGGYDSLSGT
SMAAPHVAGAAALVKQKNPGWTNEQIRSHLNDTANDLGDSFRFGSGLLNAENAVQ.
[0182] In B. pseudalcaliphilus DSM 8725, another gene was
identified that encodes a serine protease. The nucleotide sequence
of this gene, Bps02003.n, is depicted in SEQ ID NO:7: GTG
AATCAAGGATGGAAAAAACTTCTCACAATGACAGCGGTTGTTTTATTATTTTCATTA
ACAAGTATGACAGTATTGGCAGATGAAGAGAAAAAGACCTATTTAATCGGGTTCCA
TAATCAGCTAGATGTCAACGAATTTATTGAGGAGGATGTAACGAATACAAATGGCG
TGCAATTATATACGTCAGAGGATAAGTCTGCACAGGTACAATTAGAGGTCTTACATG
AATTTGAGCAAATCCCAGTTGTTGCTGTTGAGCTGAGTCCAGCTGATATCAAGGCAT
TAGAGGCAGAGTCAGGTATTGCCTATATTGAAGAAGACTTTGACGTTACGATTGCGA
ACCAAACCGTACCGTGGGGAATCGCTCAGGTACAAGCTCCACAAGCGCATGAATTA
GGCCACAGTGGGTCAGGAACAAAAGTAGCGGTACTTGATACTGGTATTGCTGAGCA
TGCTGATTTATTCATTCATGGAGGAGCAAGCTTTGTTGCAGGTGAGCCAGATTATCA
TGATTTAAATGGGCACGGAACTCACGTAGCAGGAACAATCGCTGCACTTAATGATG
GAGCCGGAGTAATCGGTGTTGCACCAGACGCAGAATTATATGCGGTCAAAGTATTA
GGGGCAAGTGGTAGTGGTTCGGTAAGTTCAATTGCACAAGGTTTAGAATGGGCTGG
TGATAATGGTATGGACGTAGCCAATCTAAGCTTAGGTAGCCCGGTTGGTAGTGATAC
GTTAGAGCAAGCAGTTAATTACGCAACGGATTCAGGGGTTCTTGTTGTGGCTGCTTC
TGGTAATAGTGGGTCAGGGACTGTTTCTTACCCAGCTCGATATGATAACGCATTTGC
TGTTGGTGCAACAGACCAAGTGAATAACCGTGCAAGCTTTTCACAATATGGAACGG
GGTTAGATATTGTCGCACCTGGTGTTGAAGTTGAAAGTACGTACTTAAATGGTGAGT
ATGCGAGCTTGAGTGGTACTTCCATGGCGACACCACATGTCGCGGGGGTCGCGGCGT
TAATAAAAGCTAAAAATCCAATGTTATCTAATGAAGAGATTCGTCAGCAATTAGTTC
AGACAGCTACACCGTTAGGAAGTGCTGATATGTATGGAAGTGGTTTAGTTAATGCAG
AGGTGGCTGTACAA.
[0183] The preproenzyme encoded by the Bps02003.n gene is depicted
in SEQ ID NO:8. At the N-terminus, the protein has a signal peptide
with a length of 27 amino acids as predicted by SignalP-NN
(Emanuelsson et al., Nature Protocols (2007) 2: 953-971). The
presence of a signal peptide indicates that this serine protease is
a secreted enzyme. The enzyme has a pro sequence which is predicted
to be 87 amino acids.
[0184] SEQ ID NO:8 sets forth the amino acid sequence of the serine
protease precursor of Bps02003 (the signal peptide sequence is
underlined and in bold, the prosequence is in italics):
VNQGWKKLLTMTAVVLLFSLTSMTVLADEEKKTYLIGFHNQLDVNEFIEEDVTNTNGV
QLYTSEDKSAQVQLEVLHEFEQIPVVAVELSPADIKALEAESGIAYIEEDFDVTIANQTVPWGI
AQVQAPQAHELGHSGSGTKVAVLDTGIAEHADLFIHGGASFVAGEPDYHDLNGHGTHV
AGTIAALNDGAGVIGVAPDAELYAVKVLGASGSGSVSSIAQGLEWAGDNGMDVANLSL
GSPVGSDTLEQAVNYATDSGVLVVAASGNSGSGTVSYPARYDNAFAVGATDQVNNRA
SFSQYGTGLDIVAPGVEVESTYLNGEYASLSGTSMATPHVAGVAALIKAKNPMLSNEEI
RQQLVQTATPLGSADMYGSGLVNAEVAVQ.
[0185] SEQ ID NO:9 sets forth the amino acid sequence of the
predicted mature protease Bps02003 (269 amino acids):
NQTVPWGIAQVQAPQAHELGHSGSGTKVAVLDTGIAEHAD
LFIHGGASFVAGEPDYHDLNGHGTHVAGTIAALNDGAGVIGVAPDAELYAVKVLGASG
SGSVSSIAQGLEWAGDNGMDVANLSLGSPVGSDTLEQAVNYATDSGVLVVAASGNSGS
GTVSYPARYDNAFAVGATDQVNNRASFSQYGTGLDIVAPGVEVESTYLNGEYASLSGT
SMATPHVAGVAALIKAKNPMLSNEEIRQQLVQTATPLGSADMYGSGLVNAEVAVQ.
[0186] In B. oshimensis NCIMB 14023, another gene was identified
encoding a serine protease. The nucleotide sequence of this gene,
Bohn00569.n, is depicted in SEQ ID NO:10:
ATGAAGAAAAGAACACACGTATTAATTGCAGGAACAGCAGTCGCAACCATTGCTTT
AATAGGAACACCATCCATTTCAGAAGCAGCAGAGGAAAAAAAATCTTATTTAATTG
GCTTTGATGAACCTCAGGAAGTGGAGCAATTTACAACAAATTTAGCAGAAGAGATT
CGCACACAAGCAGATGATGCGATTGATGTAACGTACGAATTTAAGGAGATTCCTGTT
CTTGCAGTAGAAATGACAGAAGAAGAGATGGCTGAACTCAAAAATGAAGAGAGTAT
TTCCTATATTGAAGAGGATCAAGAAGTGACAACGATGGCACAAAGCATTCCATGGG
GAATCGAAAGAATTGGCACGCCAGCTGCACAGGCCTCAGGATTTACAGGCAGTGGT
GTAAGTGTAGCAGTCCTTGATACAGGAATTGATCCACACTCTGACTTAAATATACAA
GGTGGCGTTAGTTTTGTACCAGGCGAAAGTGGGTCAGATGATGGAAATGGACACGG
TACTCATGTAGCAGGTACGATTGCAGCGTTAGATAATGATCAAGGGGTATTGGGTGT
TGCGCCAGACGTTGATCTTTTTGCAGTAAAAGTCTTAAGTGCTTCTGGATCAGGATC
GATTAGTTCGATTGCGCAAGGGTTAGAGTGGACAGCAGAAAACAATATTGATGTAG
CCAATCTAAGTTTAGGAAGCCCCTCTCCTAGTCAGACATTAGAGCAAGCGGTTAATG
ATGCCACAGATAGCGGTGTGCTTGTAGTAGCAGCAGCAGGGAATTCTGGGACAAGT
TCATTAGGATATCCAGCTCGTTATGATCATGCAATGGCTGTTGGCGCTACCGATGAG
TCGGATAGTCTCGCTAGCTTCTCACAGTATGGAGAGGGACTCGATTTAGTCGCACCT
GGCGTTGGTGTAGAAAGTACGTACCCAGGTGGAGGTTATGACAGCTTAAGCGGAAC
ATCTATGGCTGCTCCACATGTTGCAGGTGCCGCAGCACTCGTTAAGCAAAAAAATCC
AAGCTGGACAAACGAACAAATACGAGGCCATTTAAACGATACAGCCAATGATCTTG
GCGATTCGTTCCGCTTTGGTAGTGGCTTACTGAATGTTGAAAATGCCGTTCAA.
[0187] The preproenzyme encoded by the Bohn00569.n gene is depicted
in SEQ ID NO:11. At the N-terminus, the protein has a signal
peptide with a length of 28 amino acids as predicted by SignalP-NN.
The presence of a signal peptide indicates that this serine
protease is a secreted enzyme. The enzyme has a pro sequence which
is predicted to be 78 amino acids.
[0188] SEQ ID NO:11 sets forth the amino acid sequence of the
serine protease precursor of Bohn00569 (the signal peptide sequence
is underlined and in bold, the prosequence is in italics):
MKKRTHVLIAGTAVATIALIGTPSISEAAEEKKSYLIGFDEPQEVEQFTTNLAEEIRTQAD
DAIDVTYEFKEIPVLAVEMTEEEMAELKNEESISYIEEDQEVTTMAQSIPWGIERIGTPAAQAS
GFTGSGVSVAVLDTGIDPHSDLNIQGGVSFVPGESGSDDGNGHGTHVAGTIAALDNDQG
VLGVAPDVDLFAVKVLSASGSGSISSIAQGLEWTAENNIDVANLSLGSPSPSQTLEQAVN
DATDSGVLVVAAAGNSGTSSLGYPARYDHAMAVGATDESDSLASFSQYGEGLDLVAP
GVGVESTYPGGGYDSLSGTSMAAPHVAGAAALVKQKNPSWTNEQIRGHLNDTANDLG
DSFRFGSGLLNVENAVQ.
[0189] SEQ ID NO: 12 sets forth the amino acid sequence of the
predicted mature protease Bohn00569 (269 amino acids):
AQSIPWGIERIGTPAAQASGFTGSGVSVAVLDTGIDPHSDL
NIQGGVSFVPGESGSDDGNGHGTHVAGTIAALDNDQGVLGVAPDVDLFAVKVLSASGS
GSISSIAQGLEWTAENNIDVANLSLGSPSPSQTLEQAVNDATDSGVLVVAAAGNSGTSSL
GYPARYDHAMAVGATDESDSLASFSQYGEGLDLVAPGVGVESTYPGGGYDSLSGTSM
AAPHVAGAAALVKQKNPSWTNEQIRGHLNDTANDLGDSFRFGSGLLNVENAVQ.
[0190] In B. patagoniensis DSM 16117, another gene was identified
encoding a serine protease. The nucleotide sequence of this gene,
Bpan04382.n, is depicted in SEQ ID NO:13:
ATGAATCGAAAACCAGTTAAACTAATCGCAGGAACAGTTCTTGTTATGGGCTTTGTC
ATCAGTTCATCATCCATATCAACTGCCGAGGAAACAAAAAAGACTTATCTTATTGGT
TTTGACGCTCAGGAAGAAGTCGAAACATTCACGAATATCGTTGATTCTGAGATAGGG
GCTTTATCTGAAGAAGATATTGACATTACCTACGAATTTAAAGACATACCGGTCGTC
TCTGCTGAAATGAGTGATGAGGAGTATGCAGCATTACTAGAAGACCCATCGATATC
ATATATTGAAGAAGACATCGAAGTAACAACAATGGCCCAAACCATTCCATGGGGCA
TTAGTCAAATTAGTGCTCCTGAAGCACAAATCGCTGGATTTACTGGTGAGGGCGTAA
ACGTCGCGGTGCTGGATACTGGAATAGAAGATCACCCCGACTTAAACGTTCAAGGC
GGTGTTAGCTTTGTTCAAGGAGAGCCGGATTATCAGGATGGAAATGGACACGGAAC
CCATGTCGCCGGTACAATCGCTGCCCTTGATAACGACGAAGGCGTAATTGGAGTCGC
ACCAAATGCAGATCTTTATGCAGTCAAAGTTCTTGGTGCAAATGGTTCAGGCTCGGT
CAGCTCAATTGCTCAAGGGCTTGAATGGGCAGGAGAAAATGGGATGGACATTGCAA
ACTTAAGCCTAGGTAGCTCTGCACCTAGCGCGACACTCGAGCAAGCAGTGGATGAA
GCAACCGCAAATGGCGTCCTCGTTGTAGCCGCTTCTGGGAACTCGGGTGCAAGTTCT
ATTGGTTATCCGGCTCGCTATGATAACGCTATGGCCGTTGGCGCCACCGACCAGTCA
GACAGCCTAGCTAACTTTTCTCAATATGGCGAAGGCTTAGACATTGTAGCTCCAGGT
GTTGGCATCGATAGTACCTATACTGGCAGCTCATACGACAGCTTAAGTGGAACATCA
ATGGCCACCCCTCATGTTGCTGGATCCGCAGCATTGGTGAAAGAAAAGAATCCACTT
TGGTCAAATGAACAAATTCGTGCTCATTTAAACGAAACTGCAACTGACCTTGGAGAT
ACGTATCGTTTTGGTAATGGGCTTTTAAACGCACATGCCGCTGTTGAA.
[0191] The preproenzyme encoded by the Bpan04382.n gene is depicted
in SEQ ID NO:14. At the N-terminus, the protein has a signal
peptide with a length of 28 amino acids as predicted by SignalP-NN.
The presence of a signal peptide indicates that this serine
protease is a secreted enzyme. The enzyme has a pro sequence which
is predicted to be 78 amino acids.
[0192] SEQ ID NO:14 sets forth the amino acid sequence of the
serine protease precursor of Bpan04382 (the signal peptide sequence
is underlined and in bold, the prosequence is in italics):
MNRKPVKLIAGTVLVMGFVISSSSISTAEETKKTYLIGFDAQEEVETFTNIVDSEIGALSEE
DIDITYEFKDIPMAEMSDEEYAALLEDPSISYIEEDIEVTTMAQTIPWGISQISAPEAQIAGFT
GEGVNVAVLDTGIEDHPDLNVQGGVSFVQGEPDYQDGNGHGTHVAGTIAALDNDEGVI
GVAPNADLYAVKVLGANGSGSVSSIAQGLEWAGENGMDIANLSLGSSAPSATLEQAVD
EATANGVLVVAASGNSGASSIGYPARYDNAMAVGATDQSDSLANFSQYGEGLDIVAPG
VGIDSTYTGSSYDSLSGTSMATPHVAGSAALVKEKNPLWSNEQIRAHLNETATDLGDTY
RFGNGLLNAHAAVE.
[0193] SEQ ID NO:15 sets forth the amino acid sequence of the
predicted mature protease Bpan04382 (269 amino acids):
AQTIPWGISQISAPEAQIAGFT GEGVNVAVLDTGIEDHPDL
NVQGGVSFVQGEPDYQDGNGHGTHVAGTIAALDNDEGVIGVAPNADLYAVKVLGANG
SGSVSSIAQGLEWAGENGMDIANLSLGSSAPSATLEQAVDEATANGVLVVAASGNSGA
SSIGYPARYDNAMAVGATDQSDSLANFSQYGEGLDIVAPGVGIDSTYTGSSYDSLSGTS
MATPHVAGSAALVKEKNPLWSNEQIRAHLNETATDLGDTYRFGNGLLNAHAAVE.
Example 2
Heterologous Expression of Bacillus sp. Serine Proteases
[0194] BspAL03279, BspAK01305, Bps02003, Bohn00569, and Bpan04382
proteases were produced in B. subtilis using an expression cassette
consisting of the B. subtilis aprE promoter, the B. subtilis aprE
signal peptide sequence, the native protease pro-peptides, and the
mature gene of interest protease and a BPN' terminator. The
cassettes were cloned into the pBN based replicating shuttle vector
(Babe' et al. (1998), Biotechnol. Appl. Biochem. 27: 117-124) and a
suitable B. subtilis strain was transformed with the vectors.
[0195] A representative plasmid map of the pBN vector containing
BspAL03279 gene (pBN-BspAL03279) is shown in FIG. 1. To produce
BspAL03279, BspAK01305, Bps02003, Bohn00569, and Bpan04382, B.
subtilis transformants containing pBN-BspAL03279, pBN-BspAK01305,
pBN-Bps02003, pBN-Bohn00569, and pBN-Bpan04382 were cultured in 15
ml Falcon tubes for 16 hours in TSB (broth) with 10 ppm neomycin,
and 300 .mu.l of the pre-cultures were added to a 500 mL flask
filled with 30 mL of cultivation media (described below)
supplemented with 10 ppm neomycin. The flasks were incubated for 48
hours at 32.degree. C. with constant rotational mixing at 180 rpm.
Cultures were harvested by centrifugation at 14500 rpm for 20 min
in conical tubes. The culture supernatants were used for assays.
The cultivation media was an enriched semi-defined media based on
MOPs buffer, with urea as major nitrogen source, glucose as the
main carbon source, and supplemented with 1% soytone for robust
cell growth.
[0196] The nucleotide pro-mature sequence of the BspAL03279 gene in
plasmid pBN-BspAL03279 is depicted in SEQ ID NO:16:
GAGGAAACAAAAAAGACTTATCTTATTG GC
TTTGATGCTCAGGAAGAAGTCGAAACATTCACGAATATGGTCGATTCTGAGATAGG
GGCTCTATCTGAAGAAGAAATTGATATTACCTACGAATTTAAAGAAATACCGGTCGT
CTCTGCTGAAATGAGTGAAGAAGAATATGCAGCATTACTAGAAGACCCATCGATAT
CATATATTGAAGAAGACATCGAAGTAACAACAATGGCCCAAGCCATTCCATGGGGA
ATTAGTCAAATTAGTGCCCCTGAAGCGCAAATTGCTGGATTTACTGGTGAGGGTGTA
AATGTTGCGGTGCTGGATACTGGAATAGAGGATCACCCCGATTTAAACGTTCAAGGC
GGTGTTAGCTTTGTTCAAGGAGAGCCGGATTATCAGGATGGAAATGGACACGGAAC
CCATGTCGCCGGTACAATCGCTGCCCTTGATAACGACGAAGGCGTAATTGGAGTCGC
ACCAAATGCAGATCTTTATGCAGTCAAAGTTCTGGGTGCAAATGGTTCTGGCTCAGT
CAGCTCAATTGCTCAAGGGCTTGAATGGGCAGGAGAAAACGGAATGGACATTGCAA
ACTTATCATTAGGTAGCTCAGCACCTAGCGCGACACTGGAACAAGCAGTGGATGAA
GCAACCGCAAATGGTGTCCTCGTTGTTGCCGCTTCTGGGAACTCTGGTGCAAGTTCC
ATTGGTTATCCAGCTCGCTATGATAATGCTATGGCCGTTGGCGCCACCGACCAGTCA
GATGGCCTAGCATCATTTTCTCAGTACGGTGATGGCTTAGACATCGTTGCTCCAGGT
GTTGGCATCGATAGTACCTATCCTGGTAGCTCATACGATAGCTTAAGTGGAACATCA
ATGGCAACACCTCATGTTGCTGGTGCCGCAGCATTGGTGAAAGAAAAGAATCCACTT
TGGTCAAATGAACAAATTCGCGCTCATTTAAACGAAACTGCAACTGACCTTGGCGAT
ATGTATCGTTTTGGTAATGGACTTTTAAACGCACATGCCGCTGTTGAA.
[0197] The amino acid sequence of the BspAL03279 precursor protein
expressed from plasmid pBN-BspAL03279 is depicted in SEQ ID NO:17
(the predicted pro-peptide is shown in underlined text):
EETKKTYLIGFDAQEEVETFTNMVDSEIGALSEEEIDITYEFKEIPVVSAE
MSEEEYAALLEDPSISYIEEDIEVTTMAQAIPWGISQISAPEAQIAGFTGEGVNVAVLDTGI
EDHPDLNVQGGVSFVQGEPDYQDGNGHGTHVAGTIAALDNDEGVIGVAPNADLYAVK
VLGANGSGSVSSIAQGLEWAGENGMDIANLSLGSSAPSATLEQAVDEATANGVLVVAA
SGNSGASSIGYPARYDNAMAVGATDQSDGLASFSQYGDGLDIVAPGVGIDSTYPGSSYD
SLSGTSMATPHVAGAAALVKEKNPLWSNEQIRAHLNETATDLGDMYRFGNGLLNAHA AVE.
[0198] The nucleotide pro-mature sequence of the BspAK01305 gene in
plasmid pBN-BspAK01305 is depicted in SEQ ID NO:18:
GCAGAGGAAAAAAAATCTTATTTAATTGGC
TTTGATGAACCTCAAGAAGTTGAGCAATTTACAACAAATTTGGAAGAAGAGATTCGT
ACACAAGCAGATGATGCTATTGATGTAACGTACGAGTTTAAAGATATTCCTGTTCTT
GCCGTAGATATGACGGAAGAAGAAATGACTGAACTCAAAAATGAAGAGAGTATTTC
CTATATTGAAGAAGATCAAGAAGTGACAACGATGGCGCAAAGCATTCCATGGGGAA
TTGAAAGAATTGGCACGCCAGCAGCACACGCATCAGGATTCACAGGTAGCGGTGTA
AGTGTCGCGGTCCTTGATACAGGGATTGATCCACATTCTGACTTAAATGTTCAAGGG
GGGGTTAGTTTTGTACCAGGCGAAAGTGGAGCAGATGATGGAAATGGACACGGTAC
TCATGTAGCAGGAACGATTGCAGCGTTAGATAATGATGAAGGCGTTTTAGGCGTTGC
TCCAGAGGTTGATCTCTTTGCAGTAAAAGTTTTAAGTGCATCTGGATCAGGATCAAT
TAGTTCGATTGCGCAAGGTTTAGAGTGGACAGCTGAAAACAACATTGATGTGGCTA
ATTTATCTTTAGGCAGTCCCTCTCCTAGTCAGACGCTAGAACAAGCGGTTAATGACG
CCACAGATAGTGGTGTGCTTGTAGTAGCAGCAGCAGGGAACTCTGGAACAAGCTCA
TTAGGTTATCCAGCTCGTTATGATAATGCAATGGCTGTTGGCGCTACCGACCAATCC
GATAGCCTGGCATCATTCTCACAGTATGGCGAGGGTCTTGACTTAGTCGCTCCTGGT
GTTGGTGTAGAAAGCACGTACCCAGGTGGAGGTTATGACAGCTTAAGCGGCACATC
TATGGCTGCTCCACATGTTGCAGGTGCAGCAGCACTCGTTAAACAAAAAAATCCAG
GCTGGACAAACGAACAAATACGAAGCCATTTAAACGATACAGCCAATGATCTTGGC
GATTCGTTCCGCTTCGGTAGTGGCTTATTGAATGCCGAAAATGCCGTTCAA.
[0199] The amino acid sequence of the BspAK01305 precursor protein
expressed from plasmid pBN-BspAK01305 is depicted in SEQ ID NO:19
(the predicted pro-peptide is shown in underlined text):
AEEKKSYLIGFDEPQEVEQFTTNLEEEIRTQADDAIDVTYEFKDIPVLAV
DMTEEEMTELKNEESISYIEEDQEVTTMAQSIPWGIERIGTPAAHASGFTGSGVSVAVLD
TGIDPHSDLNVQGGVSFVPGESGADDGNGHGTHVAGTIAALDNDEGVLGVAPEVDLFA
VKVLSASGSGSISSIAQGLEWTAENNIDVANLSLGSPSPSQTLEQAVNDATDSGVLVVAA
AGNSGTSSLGYPARYDNAMAVGATDQSDSLASFSQYGEGLDLVAPGVGVESTYPGGG
YDSLSGTSMAAPHVAGAAALVKQKNPGWTNEQIRSHLNDTANDLGDSFRFGSGLLNAE
NAVQ.
[0200] The nucleotide pro-mature sequence of the Bps02003 gene in
plasmid pBN-Bps02003 is depicted in SEQ ID NO:20:
GATGAAGAGAAAAAGACCTATTTAATCGGGTT
CCATAATCAGCTAGATGTCAACGAATTTATTGAGGAGGATGTAACGAATACAAATG
GCGTGCAATTATATACGTCAGAGGATAAGTCTGCACAGGTACAATTAGAGGTCTTAC
ATGAATTTGAGCAAATCCCAGTTGTTGCTGTTGAGCTGAGTCCAGCTGATATCAAGG
CATTAGAGGCAGAGTCAGGTATTGCCTATATTGAAGAAGACTTTGACGTTACGATTG
CGAACCAAACCGTACCGTGGGGAATCGCTCAGGTACAAGCTCCACAAGCGCATGAA
TTAGGCCACAGTGGGTCAGGAACAAAAGTAGCGGTACTTGATACTGGTATTGCTGA
GCATGCTGATTTATTCATTCATGGAGGAGCATCATTTGTTGCAGGTGAGCCAGATTA
TCATGATTTAAATGGGCACGGAACTCACGTAGCAGGAACAATCGCTGCACTTAATG
ATGGAGCCGGAGTAATCGGTGTTGCACCAGACGCAGAATTATATGCGGTCAAAGTA
TTAGGGGCAAGTGGTAGTGGTTCGGTAAGTTCAATTGCACAAGGTTTAGAATGGGCT
GGTGATAATGGTATGGACGTAGCCAATCTATCATTAGGTAGCCCGGTTGGTAGTGAT
ACGTTAGAGCAAGCAGTTAATTACGCAACGGATTCAGGGGTTCTTGTTGTGGCTGCT
TCTGGTAATAGTGGGTCAGGGACTGTTTCTTACCCAGCTCGATATGATAACGCATTT
GCTGTTGGTGCAACAGACCAAGTGAATAACCGTGCATCATTTTCACAATATGGAACG
GGGTTAGATATTGTCGCACCTGGTGTTGAAGTTGAAAGTACGTACTTAAATGGTGAG
TATGCGAGCTTGAGTGGTACTTCCATGGCGACACCACATGTCGCGGGGGTCGCGGCG
TTAATAAAAGCTAAAAATCCAATGTTATCTAATGAAGAGATTCGTCAGCAATTAGTT
CAGACAGCTACACCGTTAGGAAGTGCTGATATGTATGGAAGTGGTTTAGTTAATGCA
GAGGTGGCTGTTCAA.
[0201] The amino acid sequence of the Bps02003 precursor protein
expressed from plasmid pBN-Bps02003 is depicted in SEQ ID NO:21
(the predicted pro-peptide is shown in underlined text):
DEEKKTYLIGFHNQLDVNEFIEEDVTNTNGVQLYTSEDKSAQVQLEVLHEFEQIPV
VAVELSPADIKALEAESGIAYIEEDFDVTIANQTVPWGIAQVQAPQAHELGHSGSGTKVA
VLDTGIAEHADLFIHGGASFVAGEPDYHDLNGHGTHVAGTIAALNDGAGVIGVAPDAEL
YAVKVLGASGSGSVSSIAQGLEWAGDNGMDVANLSLGSPVGSDTLEQAVNYATDSGV
LVVAASGNSGSGTVSYPARYDNAFAVGATDQVNNRASFSQYGTGLDIVAPGVEVESTY
LNGEYASLSGTSMATPHVAGVAALIKAKNPMLSNEEIRQQLVQTATPLGSADMYGSGL
VNAEVAVQ.
[0202] The nucleotide pro-mature sequence of the Bohn00569 gene in
plasmid pBN-Bohn00569 is depicted in SEQ ID NO:22:
GCAGAGGAAAAAAAATCTTATTTAATTGGCT
TTGATGAACCTCAGGAAGTGGAGCAATTTACAACAAATTTAGCAGAAGAGATTCGC
ACACAAGCAGATGATGCGATTGATGTAACGTACGAATTTAAGGAGATTCCTGTTCTT
GCAGTAGAAATGACAGAAGAAGAGATGGCTGAACTCAAAAATGAAGAGAGTATTTC
CTATATTGAAGAGGATCAAGAAGTGACAACGATGGCACAAAGCATTCCATGGGGAA
TCGAAAGAATTGGCACGCCAGCTGCACAGGCCTCAGGATTTACAGGCAGTGGTGTA
AGTGTAGCAGTCCTTGATACAGGAATTGATCCACACTCTGACTTAAATATACAAGGT
GGCGTTAGTTTTGTACCAGGCGAAAGTGGGTCAGATGATGGAAATGGACACGGTAC
TCATGTAGCAGGTACGATTGCAGCGTTAGATAATGATCAAGGGGTATTGGGTGTTGC
GCCAGACGTTGATCTTTTTGCAGTAAAAGTCTTAAGTGCTTCTGGATCAGGATCGAT
TAGTTCGATTGCGCAAGGGTTAGAGTGGACAGCAGAAAACAATATTGATGTAGCCA
ATCTAAGTTTAGGAAGCCCCTCTCCTAGTCAGACATTAGAGCAAGCGGTTAATGATG
CCACAGATAGCGGTGTGCTTGTAGTAGCAGCAGCAGGGAACTCTGGGACAAGTTCA
TTAGGATATCCAGCTCGTTATGATCATGCAATGGCTGTTGGCGCTACCGATGAGTCG
GATAGTCTCGCATCATTCTCACAGTATGGAGAGGGACTCGATTTAGTCGCACCTGGC
GTTGGTGTAGAAAGTACGTACCCAGGTGGAGGTTATGACAGCTTAAGCGGAACATC
TATGGCTGCTCCACATGTTGCAGGTGCCGCAGCACTCGTTAAGCAAAAAAATCCAAG
CTGGACAAACGAACAAATACGAGGCCATTTAAACGATACAGCCAATGATCTTGGCG
ATTCGTTCCGCTTTGGTAGTGGCTTACTGAATGTTGAAAATGCCGTTCAA.
[0203] The amino acid sequence of the Bohn00569 precursor protein
expressed from plasmid pBN-Bohn00569 is depicted in SEQ ID NO:23
(the predicted pro-peptide is shown in underlined text):
AEEKKSYLIGFDEPQEVEQFTTNLAEEIRTQADDAIDVTYEFK EIPVLAV
EMTEEEMAELKNEESISYIEEDQEVTTMAQSIPWGIERIGTPAAQASGFTGSGVSVAVLD
TGIDPHSDLNIQGGVSFVPGESGSDDGNGHGTHVAGTIAALDNDQGVLGVAPDVDLFA
VKVLSASGSGSISSIAQGLEWTAENNIDVANLSLGSPSPSQTLEQAVNDATDSGVLVVAA
AGNSGTSSLGYPARYDHAMAVGATDESDSLASFSQYGEGLDLVAPGVGVESTYPGGGY
DSLSGTSMAAPHVAGAAALVKQKNPSWTNEQIRGHLNDTANDLGDSFRFGSGLLNVEN AVQ.
[0204] The nucleotide pro-mature sequence of the Bpan04382 gene in
plasmid pBN-Bpan04382 is depicted in SEQ ID NO:24:
GAGGAAACAAAAAAGACTTATCTTATTGGTT
TTGACGCTCAGGAAGAAGTCGAAACATTCACGAATATCGTTGATTCTGAGATAGGG
GCTTTATCTGAAGAAGATATTGACATTACCTACGAATTTAAAGACATACCGGTCGTC
TCTGCTGAAATGAGTGATGAGGAGTATGCAGCATTACTAGAAGACCCATCGATATC
ATATATTGAAGAAGACATCGAAGTAACAACAATGGCCCAAACCATTCCATGGGGCA
TTAGTCAAATTAGTGCTCCTGAAGCACAAATCGCTGGATTTACTGGTGAGGGCGTAA
ACGTCGCGGTGCTGGATACTGGAATAGAAGATCACCCCGACTTAAACGTTCAAGGC
GGTGTTAGCTTTGTTCAAGGAGAGCCGGATTATCAGGATGGAAATGGACACGGAAC
CCATGTCGCCGGTACAATCGCTGCCCTTGATAACGACGAAGGCGTAATTGGAGTCGC
ACCAAATGCAGATCTTTATGCAGTCAAAGTTCTTGGTGCAAATGGTTCAGGCTCGGT
CAGCTCAATTGCTCAAGGGCTTGAATGGGCAGGAGAAAATGGGATGGACATTGCAA
ACTTAAGCCTAGGTAGCTCTGCACCTAGCGCGACACTGGAACAAGCAGTGGATGAA
GCAACCGCAAATGGCGTCCTCGTTGTAGCCGCTTCTGGGAACTCGGGTGCAAGTTCT
ATTGGTTATCCGGCTCGCTATGATAACGCTATGGCCGTTGGCGCCACCGACCAGTCA
GACAGCCTAGCTAACTTTTCTCAATATGGCGAAGGCTTAGACATTGTAGCTCCAGGT
GTTGGCATCGATAGTACCTATACTGGCAGCTCATACGACAGCTTAAGTGGAACATCA
ATGGCCACCCCTCATGTTGCTGGCTCAGCAGCATTGGTGAAAGAAAAGAATCCACTT
TGGTCAAATGAACAAATTCGTGCTCATTTAAACGAAACTGCAACTGACCTTGGAGAT
ACGTATCGTTTTGGTAATGGGCTTTTAAACGCACATGCCGCTGTTGAATAA.
[0205] The amino acid sequence of the Bpan04382 precursor protein
expressed from plasmid pBN-Bpan04382 is depicted in SEQ ID NO:25
(the predicted pro-peptide is shown in underlined text):
EETKKTYLIGFDAQEEVETFTNIVDSEIGALSEEDIDITYEFKDIPVVSAEMSDEEYA
ALLEDPSISYIEEDIEVTTMAQTIPWGISQISAPEAQIAGFTGEGVNVAVLDTGIEDHPDLN
VQGGVSFVQGEPDYQDGNGHGTHVAGTIAALDNDEGVIGVAPNADLYAVKVLGANGS
GSVSSIAQGLEWAGENGMDIANLSLGSSAPSATLEQAVDEATANGVLVVAASGNSGAS
SIGYPARYDNAMAVGATDQSDSLANFSQYGEGLDIVAPGVGIDSTYTGSSYDSLSGTSM
ATPHVAGSAALVKEKNPLWSNEQIRAHLNETATDLGDTY.
N and C-Term Analysis
[0206] Bsp02003: The N-terminal is determined as N[1] by the
"Protein N-terminal Acetylation" method. The C-terminal Q[269] is
determined by the ".sub.18O-labelling" method
[0207] Bohn00569: The N-terminal is determined as A[1] by the
"Protein N-terminal Acetylation" method. The C-terminal Q[269] is
determined by the ".sub.18O-labelling" method.
Protein Determination by Stain Free Imager Criterion
[0208] Protein was quantified by the stain-free Imager Criterion
method. The method utilizes stain-free precast PAGE gels, where the
intensity of each band will depend on amount of tryptophan residues
present in the protein of interest. The Criterion.TM. TGX
(Tris-Glycine extended) Stain-Free.TM. precast gels for PAGE
include unique trihalo compounds. This allows rapid fluorescent
detection of proteins with the Gel Doc.TM. EZ imaging system. The
trihalo compounds react with tryptophan residues in a UV-induced
reaction to produce fluorescence, which can be easily detected by
the Gel Doc EZ imager within gels. Reagents used in the assay:
Concentrated (10.times.) Laemmli Sample Buffer (Kem-En-Tec,
Catalogue #42556); either 18 or 26-well Criterion TGX Strain-Free
Precast gels (Bio-Rad, Catalogue #567-8124 and 567-8125,
respectively); and protein markers "Precision Plus Protein
Standards" (Bio-Rad, Catalogue #161-0363). The assay was carried
out as follow: 25 .mu.l protein sample and 25 .mu.l 0.5M HCL was
added to a 96-well-PCR plate on ice for 10 min to inactivate the
protease and prevent self-hydrolysis. 50 .mu.l of the acid protein
mix was added to 50 .mu.L sample buffer containing 0.385 mg DTT in
the 96-well PCR plate. After that, the chamber was filled by
running buffer, gel cassette was set. Then, 10 .mu.L of each sample
together with markers was loaded in each pocket and electrophoresis
was started at 200 V for 35 min. Following electrophoresis, the gel
was transferred to Imager. Image Lab software was used to calculate
the intensity of each band. By knowing the protein amount and the
tryptophan content of the standard sample, the calibration curve
can be made. The amount of experimental sample can be determined by
extrapolation of the band intensity and tryptophan numbers to
protein concentration. The protein quantification method was
employed to prepare samples of the BspAL03279, BspAK01305,
Bps02003, Bohn00569, and Bpan04382 proteases used for assays shown
in subsequent Examples.
N and C Terminal Amino Acid Determination of Bps02003 and
Bohn00569
[0209] In preparation for sequence confirmation, a sample of
isolated protein is subjected to a series of chemical treatments in
a 10 kDa spinfilter. The sample is denatured and reduced by urea
and DTT treatment. A guanidination step was performed to convert
lysines to homoarginines to protect lysine side chains from
acetylation. Acetylation reaction using iodoacetamide then modifies
only the proteins' N-terminal residue. The sample is then mixed
with a buffer containing .sup.18O water and the enzymes trypsin and
chymotrypsin are added for digestion. The resulting peptides will
contain mixtures of .sup.18O and .sup.16O, except for the Carboxyl
terminus which will retain the native .sup.16O. The digestion
products were separated and analyzed using a Proxeon nano-LC system
followed by LTQ Orbitrap (Thermo Fisher) high resolution mass
spectrometer and the amino acid sequence was deduced from the MS/MS
fragment spectrum of the peptides, and the isotopic pattern of the
peptides. A sample of Bps02003 protein expressed from plasmid
pBN-Bps02003 was analyzed as described above. The sequence of the
mature protein was determined to correspond to sequence listed in
SEQ ID NO: 9, consisting of 269 amino acids. A sample of Bohn00569
protein expressed from plasmid pBN-Bohn00569 was analyzed as
described above. The sequence of the mature protein was determined
to correspond to sequence listed in SEQ ID:12, consisting of 269
amino acids.
Example 3
Protease Activity of Bacillus sp. Serine Proteases
[0210] The protease activities of BspAL03279, BspAK01305, Bps02003,
Bohn00569, and Bpan04382 proteases were tested by measuring the
hydrolysis of dimethyl casein (DMC) substrate. The reagent
solutions used for the DMC assay were: 2.5% DMC (Sigma) in 100 mM
Sodium Carbonate pH 9.5, 0.075% TNBSA (2,4,6-trinitrobenzene
sulfonic acid, Thermo Scientific) in Reagent A. Reagent A: 45.4 g
Na.sub.2B.sub.4O.sub.7.10H.sub.20 (Merck) in 15 mL 4N NaOH to reach
a final volume of 1000 mL in MQ water. Dilution Solution: 10 mM
NaCl, 0.1 mM CaCl.sub.2, 0.005% Tween-80. Protease supernatants
were diluted in Dilution Solution to appropriate concentration for
the assay. A 96-well microtiter plate (MTP) was filled with 95
.mu.l DMC substrate followed by the addition of 5 .mu.l diluted
protease supernatant. 100 .mu.l of TNBSA in Reagent A was then
added with slow mixing. Activity was measured at 405 nm over 5 min
using a SpectraMax plate reader in kinetic mode at RT. The
absorbance of a blank containing no protease was subtracted from
the values. The activity was expressed as mOD/min. The protease
activity measured for BspAL03279, BspAK01305, Bps02003, Bohn00569,
and Bpan04382, proteases are shown in Table 1.
TABLE-US-00002 TABLE 1 Protease activity of Bacillus sp. serine
proteases on DMC substrate Activity in DMC assay Host Organism
Protease mOD/min/ppm Bacillus sp. DSM 8714 BspAL03279 84 Bacillus
sp. DSM 8717 BspAK01305 75 B. pseudalcaliphilus DSM 8725 Bps02003
81 B. oshimensis NCIMB 14023 Bohn00569 97 B. patagoniensis DSM
16117 Bpan04382 60 B. lentus GG36 54 B. amyloliquifaciens BPN'
23
[0211] The pH dependence of proteolytic activity of Bacillus sp.
serine proteases was studied using N-suc-AAPF-pNA (AAPF) as
substrate in a 50 mM Acetate/Bis-Tris/HEPES/CHES buffer. The
activity was measured at pH between 4 to 11 with 1 pH unit
increments. For the AAPF assay, the reagent solutions used were: 50
mM Acetate/Bis-Tris/HEPES/CHES buffer and 160 mM suc-AAPF-pNA in
DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a
working solution, 1 mL suc-AAPF-pNA stock solution was added to 100
mL Acetate/Bis-Tris/HEPES/CHES buffer and mixed. An enzyme sample
was added to a MTP (Costar 9017) containing suc-AAPF-pNA working
solution and assayed for activity at 405 nm over 3 min using a
SpectraMax plate reader in kinetic mode at 40.degree. C. The
protease activity was expressed as mOD*min-1. The activity was
converted to percentages of relative activity, by defining the
activity at the optimal pH as 100%. Ranges for which the Bacillus
sp. serine proteases maintain .gtoreq.50% of activity, under the
conditions of this assay are shown in table 2.
TABLE-US-00003 TABLE 2 pH profile of Bacillus sp. serine proteases
pH range for which .gtoreq.50% Host Organism Protease activity is
maintained Bacillus sp. DSM 8714 BspAL03279 8-11 Bacillus sp. DSM
8717 BspAK01305 8-11 B. pseudalcaliphilus DSM 8725 Bps02003 8-11 B.
oshimensis NCIMB 14023 Bohn00569 8-11 B. patagoniensis DSM 16117
Bpan04382 7-11 B. lentus GG36 8-11 B. amyloliquifaciens BPN'Y217L
8-11
Example 4
Comparison of Bacillus sp. Serine Proteases to Related
Molecules
[0212] Related proteins were identified by a BLAST search (Altschul
et al., Nucleic Acids Res, 25:3389-402, 1997) against the NCBI
non-redundant protein database using the amino acid sequences of
BspAL03279 (SEQ ID NO:3), BspAK01305 (SEQ ID NO:6), Bps02003 (SEQ
ID NO:9), Bohn00569 (SEQ ID NO:12), and Bpan04382 (SEQ ID NO:15) as
query sequence and a subset are shown on Tables 2A-6A.
[0213] A similar search was run against the Genome Quest Patent
database with search parameters set to default values using the
amino acid sequences for BspAL03279 (SEQ ID NO: 3), BspAK01305 (SEQ
ID NO:6), Bps02003 (SEQ ID NO:9), Bohn00569 (SEQ ID NO:12), and
Bpan04382 (SEQ ID NO:15), as the query sequence, and a subset are
shown in Tables 2B-6B.
[0214] Percent identity (PID) for both search sets is defined as
the number of identical residues divided by the number of aligned
residues in the pairwise alignment. Value labeled "Sequence length"
on tables corresponds to the length (in amino acids) for the
proteins referenced with the listed Accession numbers, while
"Aligned length" refers to sequence used for alignment and PID
calculation.
TABLE-US-00004 TABLE 2A List of sequences with percent identity to
BspAL03279 protein identified from the NCBI non-redundant protein
database Sequence Alignment Accession # PID Organism Length Length
ADK62564.1 78 Bacillus sp. B001 375 269 WP_035392836 77 Bacillus
sp. JCM 375 269 19047 AAA22212.1 76 Bacillus alkalophilus 380 269
WP_038476582 76 Bacillus lehensis G1 375 269 BAA02442.1 75 Bacillus
sp. 380 267 P29600 75 Bacillus lentus 269 267 BAD63300.1 75
Bacillus clausii 380 267 KSM-K16 WP_042417589 73 Geomicrobium sp.
380 269 JCM 19038 WP_042358689 73 Geomicrobium sp. 380 269 JCM
19055 BAA25184.1 72 Bacillus sp. AprN 379 266 AFK08970.1 72
Bacillus lehensis 378 266 BAA06157.1 71 Bacillus sp. Sendai 382 266
AAA87324.1 71 Bacillus subtilis 378 268 WP_010192403.1 59 Bacillus
sp. m3-13 381 275 ABI26631.1 58 Bacillus clausii 361 269 BAA05540.1
58 Bacillus sp. AprM 361 269 CAJ70731.1 57 Bacillus licheniformis
379 274 AAT75303.1 57 Bacillus mojavensis 379 274
TABLE-US-00005 TABLE 2B List of sequences with percent identity to
BspAL03279 protein identified from the Genome Quest database
Sequence Alignment Patent ID # PID Organism Length Length EP1160327
76.4 Bacillus sp Synthetic 269 267 US6271012 76.03 Bacillus sp;
PB92 269 267 Synthetic WO9402618 76.03 Bacillus novalis 269 267
EP0405901 76.03 Bacillus subtilis; 309 269 267 JP2012524542- 76.03
Bacillus clausii 269 267 0031 JP2012524542- 76.03 Bacillus
alcalophilus 269 267 0047 US7445912-0016 76.03 Bacillus subtilis
269 267 WO9402618 75.66 Bacillus novalis 269 267
TABLE-US-00006 TABLE 3A List of sequences with percent identity to
BspAK01305 protein identified from the NCBI non-redundant protein
database Sequence Alignment Accession # PID Organism Length Length
WP_035392836 100 Bacillus sp. 375 269 JCM 19047 ADK62564.1 99
Bacillus sp. B001 375 269 WP_038476582 96 Bacillus lehensis G1 375
269 WP_042358689 75 Geomicrobium sp. 380 267 JCM 19055 WP_042417589
75 Geomicrobium sp. 380 269 JCM 19038 WP_042398727 73 Geomicrobium
sp. 380 269 JCM 19037 AAA22212.1 72 Bacillus alkalophilus 380 269
P29600 72 Bacillus lentus 269 269 BAD63300.1 72 Bacillus clausii
380 269 KSM-K16 WP_034632645 72 Bacillus okhensis 382 268
BAA06157.1 70 Bacillus sp. Sendai 382 268 BAA25184.1 69 Bacillus
sp. AprN 379 268 AFK08970.1 68 Bacillus lehensis 378 268 AAA87324.1
68 Bacillus subtilis 378 268 AGS78407.1 66 Bacillus gibsonii 375
268 WP_010192403.1 60 Bacillus sp. m3-13 381 275 AAC43580.1 59
Bacillus sp. SprC 378 275
TABLE-US-00007 TABLE 3B List of sequences with percent identity to
BspAK01305 protein identified from the Genome Quest database
Sequence Alignment Patent ID # PID Organism Length Length US5677272
73.2 Synthetic Bacillus 269 269 lentus WO2015044206- 73.2 B.
lentus; DSM 5483 269 269 0010 Synthetic EP1160327 72.9 Bacillus sp
Synthetic 269 269 US8389262-0001 72.5 Bacillus lentus 269 269
US20130217607- 72.5 Bacillus Alkalophilus 269 269 0001 PB92
WO2008010925 72.5 Bacillus sp.; PB92 380 269 JP2012524542- 72.5
Bacillus clausii 382 269 0059
TABLE-US-00008 TABLE 4A List of sequences with percent identity to
Bps02003 protein identified from the NCBI non-redundant protein
database Sequence Alignment Accession # PID Organism Length Length
WP_047989534 100 Bacillus 373 269 pseudalcaliphilus AAA22212.1 76
Bacillus alkalophilus 380 269 BAD63300.1 76 Bacillus clausii 380
268 KSM-K16 P29600 75 Bacillus lentus 269 268 WP_047986748 74
Bacillus 382 269 pseudalcaliphilus WP_034632645 74 Bacillus
okhensis 382 269 BAA06157.1 73 Bacillus sp. Sendai 382 269
BAA25184.1 70 Bacillus sp. AprN 379 268 AFK08970.1 70 Bacillus
lehensis 378 268 AAA87324.1 71 Bacillus subtilis 378 268 AGS78407.1
67 Bacillus gibsonii 375 268 ADK62564.1 67 Bacillus sp. B001 375
268 BAA02442.1 61 Bacillus sp. 361 269 BAA05540.1 61 Bacillus sp.
AprM 361 269 ADC49870.1 60 Bacillus pseudofirmus 374 272 OF4
ABI26631.1 60 Bacillus clausii 361 269 WP_010192403.1 60 Bacillus
sp. m3-13 381 274
TABLE-US-00009 TABLE 4B List of sequences with percent identity to
Bps02003 protein identified from the Genome Quest database Sequence
Alignment Patent ID # PID Organism Length Length EP1160327 76.5
Bacillus sp Synthetic 269 268 DE4224125 76.5 Bacillus alcalophilus;
HA1 DSM 5466 380 268 WO03054185 76.1 Bacillus alkalophilus 268 268
WO9402618 76.1 Bacillus novalis 269 268 EP0415296 76.1 Bacillus
alcalophilus 269 268 US8530218-0013 76.1 Bacillus clausii 269 268
US8530218-0047 76.1 Bacillus alcalophilus 269 268 WO2013188344-0003
76.1 Bacillus clausii 269 268 EP2660309-0001 76.1 Bacillus
alcalophilus 269 268 US20130171717-0006 76.1 Bacillus lentus 269
268 JP1993361428-0006 76.1 Bacillus clausii KSM-K16 269 268
JP2013153763-0002 76.1 B. lentus (subtilisin 309) 273 268 DE4224125
76.1 Bacillus alcalophilus; HA1 DSM 5466 380 268 WO2005118793 76.1
Bacillus sp.; DSM 14390 380 268
TABLE-US-00010 TABLE 5A List of sequences with percent identity to
Bohn00569 protein identified from the NCBI non-redundant protein
database Sequence Alignment Accession # PID Organism Length Length
WP_038476582 100 Bacillus lehensis G1 375 269 WP_035392836 96
Bacillus sp. 375 269 JCM 19047 ADK62564.1 96 Bacillus sp. B001 375
269 WP_042358689 75 Geomicrobium sp. 380 267 JCM 19055 WP_042417589
75 Geomicrobium sp. 380 269 JCM 19038 WP_042398727 72 Geomicrobium
sp. 380 269 JCM 19037 AAA22212.1 71 Bacillus alkalophilus 380 269
P29600 71 Bacillus lentus 269 269 BAD63300.1 71 Bacillus clausii
380 269 KSM-K16 BAA25184.1 69 Bacillus sp. AprN 379 268 BAA06157.1
69 Bacillus sp. Sendai 382 268 AFK08970.1 68 Bacillus lehensis 378
268 AAA87324.1 68 Bacillus subtilis 378 268 AGS78407.1 65 Bacillus
gibsonii 375 268 WP_010192403.1 60 Bacillus sp. m3-13 381 275
AAC43580.1 59 Bacillus sp. SprC 378 275 WP_022628745.1 57 Bacillus
marmarensi 374 273 YP_003972439.1 57 Bacillus atrophaeus 382 275
1942
TABLE-US-00011 TABLE 5B List of sequences with percent identity to
Bohn00569 protein identified from the Genome Quest database
Sequence Alignment Patent ID # PID Organism Length Length
WO2015044206-0010 72.5 Bacillus lentus; DSM 5483 Synthetic 269 269
WO9211348 72.5 Bacillus subtilis Synthetic 269 269 US6312936 72.5
Bacillus lentus Synthetic 269 269 DE4224125 72.1 Bacillus
alcalophilus; HA1 DSM 5466 380 269 WO9402618 71.8 Bacillus novalis
269 269 US20130217607-0001 71.8 Bacillus Alkalophilus PB92 269 269
US8530218-0013 71.8 Bacillus clausii 269 269 US7445912-0016 71.8
Bacillus subtilis 269 269
TABLE-US-00012 TABLE 6A List of sequences with percent identity to
Bpan04382 protein identified from the NCBI non-redundant protein
database Seuqence Alignment Accession # PID Organism Length Length
ADK62564.1 78 Bacillus sp. B001 375 269 WP_035392836 77 Bacillus
sp. JCM 375 269 19047 WP_038476582 75 Bacillus lehensis G1 375 269
AAA22212.1 75 Bacillus alkalophilus 380 267 BAA02442.1 74 Bacillus
sp. 380 267 P29600 74 Bacillus lentus 269 267 BAD63300.1 74
Bacillus clausii 380 267 KSM-K16 WP_042358689 73 Geomicrobium sp.
380 269 JCM 19055 WP_042417589 73 Geomicrobium sp. 380 269 JCM
19038 BAA25184.1 72 Bacillus sp. AprN 379 266 AFK08970.1 72
Bacillus lehensis 378 266 AAA87324.1 71 Bacillus subtilis 378 268
BAA06157.1 71 Bacillus sp. Sendai 382 266 AGS78407.1 67 Bacillus
gibsonii 375 266 ABI26631.1 59 Bacillus clausii 361 269 BAA02443.2
59 Bacillus halodurans 361 269 BAA05540.1 59 Bacillus sp. AprM 361
269 ADD64465.1 58 Bacillus sp. JB99 361 269 WP_010192403.1 58
Bacillus sp. m3-13 381 275 ADC49870.1 58 Bacillus pseudofirmus 374
273 OF4 AAC43580.1 57 Bacillus sp. SprC 378 275 BAD11988.2 57
Bacillus sp. KSM-LD1 376 275 CAJ70731.1 56 Bacillus licheniformis
379 274 AAT75303.1 56 Bacillus mojavensis 379 274 CAA24990.1 54
Bacillus 376 275 amyloliquefaciens
TABLE-US-00013 TABLE 6B List of sequences with percent identity to
Bpan04382 protein identified from the Genome Quest database
Sequence Alignment Patent ID # PID Organism Length Length EP1160327
75.7 Bacillus sp Synthetic 269 267 US6271012 75.3 Bacillus sp; PB92
Synthetic 269 267 WO9402618 75.3 Bacillus novalis 269 267 EP0405901
75.3 Bacillus subtilis; 309 269 267 WO2013188344-0003 75.3 Bacillus
clausii 269 267 US20130217607-0001 75.3 Bacillus Alkalophilus PB92
269 267 WO9402618 75.3 Bacillus novalis 269 267 DE4224125 75.3
Bacillus alcalophilus; HA1 DSM 5466 380 267 DE19530816 74.9
Bacillus lentus; DSM 5483 269 267
Alignment of Homologous Sequences
[0215] An alignment of the mature protein amino acid sequences for
BspAL03279 (SEQ ID NO:3), BspAK01305 (SEQ ID NO:6), Bps02003 (SEQ
ID NO:9), Bohn00569 (SEQ ID NO:12), and Bpan04382 (SEQ ID NO:15)
with the sequences of the mature forms of various subtilisins from
Tables 2A-6A is shown in FIG. 2. The sequences were aligned with
default parameters using the MUSCLE program from Geneious software
(Biomatters Ltd.) (Robert C. Edgar. MUSCLE: multiple sequence
alignment with high accuracy and high throughput Nucl. Acids Res.
(2004) 32 (5): 1792-1797). A phylogenetic tree for amino acid
sequences of the mature forms of the subtilisins from FIG. 2 was
built using the Geneious Tree builder program and is displayed in
FIG. 3.
Example 5
Unique Features of the BspAL03279-Clade of Subtilisins
[0216] The FIG. 2 alignment was reviewed for unique sequence
similarities across the BspAL03279-clade of subtilisins. The
BspAL03279-clade of subtilisins is characterized by a common motif
over the sequence that begins with Aspartic acid (D250) and ends at
position 269, according to BspAL03279 numbering. This motif can be
characterized as DLGDXXRFGX.sub.aGLLXXXXAVX (SEQ ID NO:26), where X
is any amino acid and X.sub.a is N or S. FIG. 2 includes box around
the motif.
[0217] The BspAL03279, BspAK01305, Bpan04382, Bohn00569, and
ADK62564.1 subtilisins, which have been identified as a
BspAL03279-clade of subtilisins based on the shared sequence motif
set forth above, also cluster together in the phylogenetic tree
that was built using various bacterial subtilisins and which is set
forth in FIG. 3.
[0218] The amino acid identity across the mature forms of various
subtilisins from Tables 2A-6A is shown on Table 7 below, wherein
the percent amino acid identity is calculated over, for example,
the 269 residues of the BspAL03279 mature sequence.
[0219] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein can be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
4611125DNAunknownBacillus sp. DSM 8714 1atgaatcgaa aaccagttaa
actaatcgca ggaacagctc ttgttatggg ctttgtcatc 60agttcatcat ccatatcaac
tgccgaggaa acaaaaaaga cttatcttat tggctttgat 120gctcaggaag
aagtcgaaac attcacgaat atggtcgatt ctgagatagg ggctctatct
180gaagaagaaa ttgatattac ctacgaattt aaagaaatac cggtcgtctc
tgctgaaatg 240agtgaagaag aatatgcagc attactagaa gacccatcga
tatcatatat tgaagaagac 300atcgaagtaa caacaatggc ccaagccatt
ccatggggaa ttagtcaaat tagtgcccct 360gaagcgcaaa ttgctggatt
tactggtgag ggtgtaaatg ttgcggtgct ggatactgga 420atagaggatc
accccgattt aaacgttcaa ggcggtgtta gctttgttca aggagagccg
480gattatcagg atggaaatgg acacggaacc catgtcgccg gtacaatcgc
tgcccttgat 540aacgacgaag gcgtaattgg agtcgcacca aatgcagatc
tttatgcagt caaagttctg 600ggtgcaaatg gttctggctc agtcagctca
attgctcaag ggcttgaatg ggcaggagaa 660aacggaatgg acattgcaaa
cttaagctta ggtagctcag cacctagcgc gacactcgag 720caagcagtgg
atgaagcaac cgcaaatggt gtcctcgttg ttgccgcttc tgggaactct
780ggtgcaagtt ccattggtta tccagctcgc tatgataatg ctatggccgt
tggcgccacc 840gaccagtcag atggcctagc tagcttttct cagtacggtg
atggcttaga catcgttgct 900ccaggtgttg gcatcgatag tacctatcct
ggtagctcat acgatagctt aagtggaaca 960tcaatggcaa cacctcatgt
tgctggtgcc gcagcattgg tgaaagaaaa gaatccactt 1020tggtcaaatg
aacaaattcg cgctcattta aacgaaactg caactgacct tggcgatatg
1080tatcgttttg gtaatggact tttaaacgca catgccgctg ttgaa
11252375PRTunknownBacillus sp. DSM 8714 2Met Asn Arg Lys Pro Val
Lys Leu Ile Ala Gly Thr Ala Leu Val Met 1 5 10 15 Gly Phe Val Ile
Ser Ser Ser Ser Ile Ser Thr Ala Glu Glu Thr Lys 20 25 30 Lys Thr
Tyr Leu Ile Gly Phe Asp Ala Gln Glu Glu Val Glu Thr Phe 35 40 45
Thr Asn Met Val Asp Ser Glu Ile Gly Ala Leu Ser Glu Glu Glu Ile 50
55 60 Asp Ile Thr Tyr Glu Phe Lys Glu Ile Pro Val Val Ser Ala Glu
Met 65 70 75 80 Ser Glu Glu Glu Tyr Ala Ala Leu Leu Glu Asp Pro Ser
Ile Ser Tyr 85 90 95 Ile Glu Glu Asp Ile Glu Val Thr Thr Met Ala
Gln Ala Ile Pro Trp 100 105 110 Gly Ile Ser Gln Ile Ser Ala Pro Glu
Ala Gln Ile Ala Gly Phe Thr 115 120 125 Gly Glu Gly Val Asn Val Ala
Val Leu Asp Thr Gly Ile Glu Asp His 130 135 140 Pro Asp Leu Asn Val
Gln Gly Gly Val Ser Phe Val Gln Gly Glu Pro 145 150 155 160 Asp Tyr
Gln Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Ile 165 170 175
Ala Ala Leu Asp Asn Asp Glu Gly Val Ile Gly Val Ala Pro Asn Ala 180
185 190 Asp Leu Tyr Ala Val Lys Val Leu Gly Ala Asn Gly Ser Gly Ser
Val 195 200 205 Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly Glu Asn
Gly Met Asp 210 215 220 Ile Ala Asn Leu Ser Leu Gly Ser Ser Ala Pro
Ser Ala Thr Leu Glu 225 230 235 240 Gln Ala Val Asp Glu Ala Thr Ala
Asn Gly Val Leu Val Val Ala Ala 245 250 255 Ser Gly Asn Ser Gly Ala
Ser Ser Ile Gly Tyr Pro Ala Arg Tyr Asp 260 265 270 Asn Ala Met Ala
Val Gly Ala Thr Asp Gln Ser Asp Gly Leu Ala Ser 275 280 285 Phe Ser
Gln Tyr Gly Asp Gly Leu Asp Ile Val Ala Pro Gly Val Gly 290 295 300
Ile Asp Ser Thr Tyr Pro Gly Ser Ser Tyr Asp Ser Leu Ser Gly Thr 305
310 315 320 Ser Met Ala Thr Pro His Val Ala Gly Ala Ala Ala Leu Val
Lys Glu 325 330 335 Lys Asn Pro Leu Trp Ser Asn Glu Gln Ile Arg Ala
His Leu Asn Glu 340 345 350 Thr Ala Thr Asp Leu Gly Asp Met Tyr Arg
Phe Gly Asn Gly Leu Leu 355 360 365 Asn Ala His Ala Ala Val Glu 370
375 3269PRTunknownBacillus sp. DSM 8714 3Ala Gln Ala Ile Pro Trp
Gly Ile Ser Gln Ile Ser Ala Pro Glu Ala 1 5 10 15 Gln Ile Ala Gly
Phe Thr Gly Glu Gly Val Asn Val Ala Val Leu Asp 20 25 30 Thr Gly
Ile Glu Asp His Pro Asp Leu Asn Val Gln Gly Gly Val Ser 35 40 45
Phe Val Gln Gly Glu Pro Asp Tyr Gln Asp Gly Asn Gly His Gly Thr 50
55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Glu Gly Val
Ile 65 70 75 80 Gly Val Ala Pro Asn Ala Asp Leu Tyr Ala Val Lys Val
Leu Gly Ala 85 90 95 Asn Gly Ser Gly Ser Val Ser Ser Ile Ala Gln
Gly Leu Glu Trp Ala 100 105 110 Gly Glu Asn Gly Met Asp Ile Ala Asn
Leu Ser Leu Gly Ser Ser Ala 115 120 125 Pro Ser Ala Thr Leu Glu Gln
Ala Val Asp Glu Ala Thr Ala Asn Gly 130 135 140 Val Leu Val Val Ala
Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Gly 145 150 155 160 Tyr Pro
Ala Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175
Ser Asp Gly Leu Ala Ser Phe Ser Gln Tyr Gly Asp Gly Leu Asp Ile 180
185 190 Val Ala Pro Gly Val Gly Ile Asp Ser Thr Tyr Pro Gly Ser Ser
Tyr 195 200 205 Asp Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val
Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Glu Lys Asn Pro Leu Trp
Ser Asn Glu Gln Ile 225 230 235 240 Arg Ala His Leu Asn Glu Thr Ala
Thr Asp Leu Gly Asp Met Tyr Arg 245 250 255 Phe Gly Asn Gly Leu Leu
Asn Ala His Ala Ala Val Glu 260 265 41125DNAunknownBacillus sp. DSM
8714 4atgaagaaaa gatcaaacgt tttaatcgca ggaacagcga tcgcaaccat
tgctttaata 60ggaacaccat ccatttcaga agctgcagag gaaaaaaaat cttatttaat
tggctttgat 120gaacctcaag aagttgagca atttacaaca aatttggaag
aagagattcg tacacaagca 180gatgatgcta ttgatgtaac gtacgagttt
aaagatattc ctgttcttgc cgtagatatg 240acggaagaag aaatgactga
actcaaaaat gaagagagta tttcctatat tgaagaagat 300caagaagtga
caacgatggc gcaaagcatt ccatggggaa ttgaaagaat tggcacgcca
360gcagcacacg catcaggatt cacaggtagc ggtgtaagtg tcgcggtcct
tgatacaggg 420attgatccac attctgactt aaatgtacaa gggggggtta
gttttgtacc aggcgaaagt 480ggagcagatg atggaaatgg acacggtact
catgtagcag gaacgattgc agcgttagat 540aatgatgaag gcgttttagg
cgttgctcca gaggttgatc tctttgcagt aaaagtttta 600agtgcatctg
gatcaggatc aattagttcg attgcgcaag gtttagagtg gacagctgaa
660aacaacattg atgtggctaa tttaagctta ggcagtccct ctcctagtca
gacgctagaa 720caagcggtta atgacgccac agatagtggt gtgcttgtag
tagcagcagc agggaattct 780ggaacaagct cattaggtta tccagctcgt
tatgataatg caatggctgt tggcgctacc 840gaccaatccg atagcctggc
tagcttctca cagtatggcg agggtcttga cttagtcgct 900cctggtgttg
gtgtagaaag cacgtaccca ggtggaggtt atgacagctt aagcggcaca
960tctatggctg ctccacatgt tgcaggtgca gcagcactcg ttaaacaaaa
aaatccaggc 1020tggacaaacg aacaaatacg aagccattta aacgatacag
ccaatgatct tggcgattcg 1080ttccgcttcg gtagtggctt attgaatgcc
gaaaatgccg ttcaa 11255375PRTunknownBacillus sp. DSM 8714 5Met Lys
Lys Arg Ser Asn Val Leu Ile Ala Gly Thr Ala Ile Ala Thr 1 5 10 15
Ile Ala Leu Ile Gly Thr Pro Ser Ile Ser Glu Ala Ala Glu Glu Lys 20
25 30 Lys Ser Tyr Leu Ile Gly Phe Asp Glu Pro Gln Glu Val Glu Gln
Phe 35 40 45 Thr Thr Asn Leu Glu Glu Glu Ile Arg Thr Gln Ala Asp
Asp Ala Ile 50 55 60 Asp Val Thr Tyr Glu Phe Lys Asp Ile Pro Val
Leu Ala Val Asp Met 65 70 75 80 Thr Glu Glu Glu Met Thr Glu Leu Lys
Asn Glu Glu Ser Ile Ser Tyr 85 90 95 Ile Glu Glu Asp Gln Glu Val
Thr Thr Met Ala Gln Ser Ile Pro Trp 100 105 110 Gly Ile Glu Arg Ile
Gly Thr Pro Ala Ala His Ala Ser Gly Phe Thr 115 120 125 Gly Ser Gly
Val Ser Val Ala Val Leu Asp Thr Gly Ile Asp Pro His 130 135 140 Ser
Asp Leu Asn Val Gln Gly Gly Val Ser Phe Val Pro Gly Glu Ser 145 150
155 160 Gly Ala Asp Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr
Ile 165 170 175 Ala Ala Leu Asp Asn Asp Glu Gly Val Leu Gly Val Ala
Pro Glu Val 180 185 190 Asp Leu Phe Ala Val Lys Val Leu Ser Ala Ser
Gly Ser Gly Ser Ile 195 200 205 Ser Ser Ile Ala Gln Gly Leu Glu Trp
Thr Ala Glu Asn Asn Ile Asp 210 215 220 Val Ala Asn Leu Ser Leu Gly
Ser Pro Ser Pro Ser Gln Thr Leu Glu 225 230 235 240 Gln Ala Val Asn
Asp Ala Thr Asp Ser Gly Val Leu Val Val Ala Ala 245 250 255 Ala Gly
Asn Ser Gly Thr Ser Ser Leu Gly Tyr Pro Ala Arg Tyr Asp 260 265 270
Asn Ala Met Ala Val Gly Ala Thr Asp Gln Ser Asp Ser Leu Ala Ser 275
280 285 Phe Ser Gln Tyr Gly Glu Gly Leu Asp Leu Val Ala Pro Gly Val
Gly 290 295 300 Val Glu Ser Thr Tyr Pro Gly Gly Gly Tyr Asp Ser Leu
Ser Gly Thr 305 310 315 320 Ser Met Ala Ala Pro His Val Ala Gly Ala
Ala Ala Leu Val Lys Gln 325 330 335 Lys Asn Pro Gly Trp Thr Asn Glu
Gln Ile Arg Ser His Leu Asn Asp 340 345 350 Thr Ala Asn Asp Leu Gly
Asp Ser Phe Arg Phe Gly Ser Gly Leu Leu 355 360 365 Asn Ala Glu Asn
Ala Val Gln 370 375 6269PRTunknownBacillus sp. DSM 8714 6Ala Gln
Ser Ile Pro Trp Gly Ile Glu Arg Ile Gly Thr Pro Ala Ala 1 5 10 15
His Ala Ser Gly Phe Thr Gly Ser Gly Val Ser Val Ala Val Leu Asp 20
25 30 Thr Gly Ile Asp Pro His Ser Asp Leu Asn Val Gln Gly Gly Val
Ser 35 40 45 Phe Val Pro Gly Glu Ser Gly Ala Asp Asp Gly Asn Gly
His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn
Asp Glu Gly Val Leu 65 70 75 80 Gly Val Ala Pro Glu Val Asp Leu Phe
Ala Val Lys Val Leu Ser Ala 85 90 95 Ser Gly Ser Gly Ser Ile Ser
Ser Ile Ala Gln Gly Leu Glu Trp Thr 100 105 110 Ala Glu Asn Asn Ile
Asp Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Gln
Thr Leu Glu Gln Ala Val Asn Asp Ala Thr Asp Ser Gly 130 135 140 Val
Leu Val Val Ala Ala Ala Gly Asn Ser Gly Thr Ser Ser Leu Gly 145 150
155 160 Tyr Pro Ala Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp
Gln 165 170 175 Ser Asp Ser Leu Ala Ser Phe Ser Gln Tyr Gly Glu Gly
Leu Asp Leu 180 185 190 Val Ala Pro Gly Val Gly Val Glu Ser Thr Tyr
Pro Gly Gly Gly Tyr 195 200 205 Asp Ser Leu Ser Gly Thr Ser Met Ala
Ala Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys
Asn Pro Gly Trp Thr Asn Glu Gln Ile 225 230 235 240 Arg Ser His Leu
Asn Asp Thr Ala Asn Asp Leu Gly Asp Ser Phe Arg 245 250 255 Phe Gly
Ser Gly Leu Leu Asn Ala Glu Asn Ala Val Gln 260 265
71149DNABacillus pseudalcaliphilus DSM 8725 7gtgaatcaag gatggaaaaa
acttctcaca atgacagcgg ttgttttatt attttcatta 60acaagtatga cagtattggc
agatgaagag aaaaagacct atttaatcgg gttccataat 120cagctagatg
tcaacgaatt tattgaggag gatgtaacga atacaaatgg cgtgcaatta
180tatacgtcag aggataagtc tgcacaggta caattagagg tcttacatga
atttgagcaa 240atcccagttg ttgctgttga gctgagtcca gctgatatca
aggcattaga ggcagagtca 300ggtattgcct atattgaaga agactttgac
gttacgattg cgaaccaaac cgtaccgtgg 360ggaatcgctc aggtacaagc
tccacaagcg catgaattag gccacagtgg gtcaggaaca 420aaagtagcgg
tacttgatac tggtattgct gagcatgctg atttattcat tcatggagga
480gcaagctttg ttgcaggtga gccagattat catgatttaa atgggcacgg
aactcacgta 540gcaggaacaa tcgctgcact taatgatgga gccggagtaa
tcggtgttgc accagacgca 600gaattatatg cggtcaaagt attaggggca
agtggtagtg gttcggtaag ttcaattgca 660caaggtttag aatgggctgg
tgataatggt atggacgtag ccaatctaag cttaggtagc 720ccggttggta
gtgatacgtt agagcaagca gttaattacg caacggattc aggggttctt
780gttgtggctg cttctggtaa tagtgggtca gggactgttt cttacccagc
tcgatatgat 840aacgcatttg ctgttggtgc aacagaccaa gtgaataacc
gtgcaagctt ttcacaatat 900ggaacggggt tagatattgt cgcacctggt
gttgaagttg aaagtacgta cttaaatggt 960gagtatgcga gcttgagtgg
tacttccatg gcgacaccac atgtcgcggg ggtcgcggcg 1020ttaataaaag
ctaaaaatcc aatgttatct aatgaagaga ttcgtcagca attagttcag
1080acagctacac cgttaggaag tgctgatatg tatggaagtg gtttagttaa
tgcagaggtg 1140gctgtacaa 11498383PRTBacillus pseudalcaliphilus DSM
8725 8Val Asn Gln Gly Trp Lys Lys Leu Leu Thr Met Thr Ala Val Val
Leu 1 5 10 15 Leu Phe Ser Leu Thr Ser Met Thr Val Leu Ala Asp Glu
Glu Lys Lys 20 25 30 Thr Tyr Leu Ile Gly Phe His Asn Gln Leu Asp
Val Asn Glu Phe Ile 35 40 45 Glu Glu Asp Val Thr Asn Thr Asn Gly
Val Gln Leu Tyr Thr Ser Glu 50 55 60 Asp Lys Ser Ala Gln Val Gln
Leu Glu Val Leu His Glu Phe Glu Gln 65 70 75 80 Ile Pro Val Val Ala
Val Glu Leu Ser Pro Ala Asp Ile Lys Ala Leu 85 90 95 Glu Ala Glu
Ser Gly Ile Ala Tyr Ile Glu Glu Asp Phe Asp Val Thr 100 105 110 Ile
Ala Asn Gln Thr Val Pro Trp Gly Ile Ala Gln Val Gln Ala Pro 115 120
125 Gln Ala His Glu Leu Gly His Ser Gly Ser Gly Thr Lys Val Ala Val
130 135 140 Leu Asp Thr Gly Ile Ala Glu His Ala Asp Leu Phe Ile His
Gly Gly 145 150 155 160 Ala Ser Phe Val Ala Gly Glu Pro Asp Tyr His
Asp Leu Asn Gly His 165 170 175 Gly Thr His Val Ala Gly Thr Ile Ala
Ala Leu Asn Asp Gly Ala Gly 180 185 190 Val Ile Gly Val Ala Pro Asp
Ala Glu Leu Tyr Ala Val Lys Val Leu 195 200 205 Gly Ala Ser Gly Ser
Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu 210 215 220 Trp Ala Gly
Asp Asn Gly Met Asp Val Ala Asn Leu Ser Leu Gly Ser 225 230 235 240
Pro Val Gly Ser Asp Thr Leu Glu Gln Ala Val Asn Tyr Ala Thr Asp 245
250 255 Ser Gly Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ser Gly
Thr 260 265 270 Val Ser Tyr Pro Ala Arg Tyr Asp Asn Ala Phe Ala Val
Gly Ala Thr 275 280 285 Asp Gln Val Asn Asn Arg Ala Ser Phe Ser Gln
Tyr Gly Thr Gly Leu 290 295 300 Asp Ile Val Ala Pro Gly Val Glu Val
Glu Ser Thr Tyr Leu Asn Gly 305 310 315 320 Glu Tyr Ala Ser Leu Ser
Gly Thr Ser Met Ala Thr Pro His Val Ala 325 330 335 Gly Val Ala Ala
Leu Ile Lys Ala Lys Asn Pro Met Leu Ser Asn Glu 340 345 350 Glu Ile
Arg Gln Gln Leu Val Gln Thr Ala Thr Pro Leu Gly Ser Ala 355 360 365
Asp Met Tyr Gly Ser Gly Leu Val Asn Ala Glu Val Ala Val Gln 370 375
380 9269PRTBacillus pseudalcaliphilus DSM 8725 9Asn Gln Thr Val Pro
Trp Gly Ile Ala Gln Val Gln Ala Pro Gln Ala 1 5 10 15 His Glu Leu
Gly His Ser Gly Ser Gly Thr Lys Val Ala Val Leu Asp 20 25 30 Thr
Gly Ile Ala Glu His Ala Asp Leu Phe Ile His Gly Gly Ala Ser 35 40
45 Phe
Val Ala Gly Glu Pro Asp Tyr His Asp Leu Asn Gly His Gly Thr 50 55
60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asp Gly Ala Gly Val Ile
65 70 75 80 Gly Val Ala Pro Asp Ala Glu Leu Tyr Ala Val Lys Val Leu
Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly
Leu Glu Trp Ala 100 105 110 Gly Asp Asn Gly Met Asp Val Ala Asn Leu
Ser Leu Gly Ser Pro Val 115 120 125 Gly Ser Asp Thr Leu Glu Gln Ala
Val Asn Tyr Ala Thr Asp Ser Gly 130 135 140 Val Leu Val Val Ala Ala
Ser Gly Asn Ser Gly Ser Gly Thr Val Ser 145 150 155 160 Tyr Pro Ala
Arg Tyr Asp Asn Ala Phe Ala Val Gly Ala Thr Asp Gln 165 170 175 Val
Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Thr Gly Leu Asp Ile 180 185
190 Val Ala Pro Gly Val Glu Val Glu Ser Thr Tyr Leu Asn Gly Glu Tyr
195 200 205 Ala Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala
Gly Val 210 215 220 Ala Ala Leu Ile Lys Ala Lys Asn Pro Met Leu Ser
Asn Glu Glu Ile 225 230 235 240 Arg Gln Gln Leu Val Gln Thr Ala Thr
Pro Leu Gly Ser Ala Asp Met 245 250 255 Tyr Gly Ser Gly Leu Val Asn
Ala Glu Val Ala Val Gln 260 265 101125DNABacillus oshimensis
10atgaagaaaa gaacacacgt attaattgca ggaacagcag tcgcaaccat tgctttaata
60ggaacaccat ccatttcaga agcagcagag gaaaaaaaat cttatttaat tggctttgat
120gaacctcagg aagtggagca atttacaaca aatttagcag aagagattcg
cacacaagca 180gatgatgcga ttgatgtaac gtacgaattt aaggagattc
ctgttcttgc agtagaaatg 240acagaagaag agatggctga actcaaaaat
gaagagagta tttcctatat tgaagaggat 300caagaagtga caacgatggc
acaaagcatt ccatggggaa tcgaaagaat tggcacgcca 360gctgcacagg
cctcaggatt tacaggcagt ggtgtaagtg tagcagtcct tgatacagga
420attgatccac actctgactt aaatatacaa ggtggcgtta gttttgtacc
aggcgaaagt 480gggtcagatg atggaaatgg acacggtact catgtagcag
gtacgattgc agcgttagat 540aatgatcaag gggtattggg tgttgcgcca
gacgttgatc tttttgcagt aaaagtctta 600agtgcttctg gatcaggatc
gattagttcg attgcgcaag ggttagagtg gacagcagaa 660aacaatattg
atgtagccaa tctaagttta ggaagcccct ctcctagtca gacattagag
720caagcggtta atgatgccac agatagcggt gtgcttgtag tagcagcagc
agggaattct 780gggacaagtt cattaggata tccagctcgt tatgatcatg
caatggctgt tggcgctacc 840gatgagtcgg atagtctcgc tagcttctca
cagtatggag agggactcga tttagtcgca 900cctggcgttg gtgtagaaag
tacgtaccca ggtggaggtt atgacagctt aagcggaaca 960tctatggctg
ctccacatgt tgcaggtgcc gcagcactcg ttaagcaaaa aaatccaagc
1020tggacaaacg aacaaatacg aggccattta aacgatacag ccaatgatct
tggcgattcg 1080ttccgctttg gtagtggctt actgaatgtt gaaaatgccg ttcaa
112511375PRTBacillus oshimensis 11Met Lys Lys Arg Thr His Val Leu
Ile Ala Gly Thr Ala Val Ala Thr 1 5 10 15 Ile Ala Leu Ile Gly Thr
Pro Ser Ile Ser Glu Ala Ala Glu Glu Lys 20 25 30 Lys Ser Tyr Leu
Ile Gly Phe Asp Glu Pro Gln Glu Val Glu Gln Phe 35 40 45 Thr Thr
Asn Leu Ala Glu Glu Ile Arg Thr Gln Ala Asp Asp Ala Ile 50 55 60
Asp Val Thr Tyr Glu Phe Lys Glu Ile Pro Val Leu Ala Val Glu Met 65
70 75 80 Thr Glu Glu Glu Met Ala Glu Leu Lys Asn Glu Glu Ser Ile
Ser Tyr 85 90 95 Ile Glu Glu Asp Gln Glu Val Thr Thr Met Ala Gln
Ser Ile Pro Trp 100 105 110 Gly Ile Glu Arg Ile Gly Thr Pro Ala Ala
Gln Ala Ser Gly Phe Thr 115 120 125 Gly Ser Gly Val Ser Val Ala Val
Leu Asp Thr Gly Ile Asp Pro His 130 135 140 Ser Asp Leu Asn Ile Gln
Gly Gly Val Ser Phe Val Pro Gly Glu Ser 145 150 155 160 Gly Ser Asp
Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Ile 165 170 175 Ala
Ala Leu Asp Asn Asp Gln Gly Val Leu Gly Val Ala Pro Asp Val 180 185
190 Asp Leu Phe Ala Val Lys Val Leu Ser Ala Ser Gly Ser Gly Ser Ile
195 200 205 Ser Ser Ile Ala Gln Gly Leu Glu Trp Thr Ala Glu Asn Asn
Ile Asp 210 215 220 Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro Ser
Gln Thr Leu Glu 225 230 235 240 Gln Ala Val Asn Asp Ala Thr Asp Ser
Gly Val Leu Val Val Ala Ala 245 250 255 Ala Gly Asn Ser Gly Thr Ser
Ser Leu Gly Tyr Pro Ala Arg Tyr Asp 260 265 270 His Ala Met Ala Val
Gly Ala Thr Asp Glu Ser Asp Ser Leu Ala Ser 275 280 285 Phe Ser Gln
Tyr Gly Glu Gly Leu Asp Leu Val Ala Pro Gly Val Gly 290 295 300 Val
Glu Ser Thr Tyr Pro Gly Gly Gly Tyr Asp Ser Leu Ser Gly Thr 305 310
315 320 Ser Met Ala Ala Pro His Val Ala Gly Ala Ala Ala Leu Val Lys
Gln 325 330 335 Lys Asn Pro Ser Trp Thr Asn Glu Gln Ile Arg Gly His
Leu Asn Asp 340 345 350 Thr Ala Asn Asp Leu Gly Asp Ser Phe Arg Phe
Gly Ser Gly Leu Leu 355 360 365 Asn Val Glu Asn Ala Val Gln 370 375
12269PRTBacillus oshimensis 12Ala Gln Ser Ile Pro Trp Gly Ile Glu
Arg Ile Gly Thr Pro Ala Ala 1 5 10 15 Gln Ala Ser Gly Phe Thr Gly
Ser Gly Val Ser Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Asp Pro
His Ser Asp Leu Asn Ile Gln Gly Gly Val Ser 35 40 45 Phe Val Pro
Gly Glu Ser Gly Ser Asp Asp Gly Asn Gly His Gly Thr 50 55 60 His
Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Gln Gly Val Leu 65 70
75 80 Gly Val Ala Pro Asp Val Asp Leu Phe Ala Val Lys Val Leu Ser
Ala 85 90 95 Ser Gly Ser Gly Ser Ile Ser Ser Ile Ala Gln Gly Leu
Glu Trp Thr 100 105 110 Ala Glu Asn Asn Ile Asp Val Ala Asn Leu Ser
Leu Gly Ser Pro Ser 115 120 125 Pro Ser Gln Thr Leu Glu Gln Ala Val
Asn Asp Ala Thr Asp Ser Gly 130 135 140 Val Leu Val Val Ala Ala Ala
Gly Asn Ser Gly Thr Ser Ser Leu Gly 145 150 155 160 Tyr Pro Ala Arg
Tyr Asp His Ala Met Ala Val Gly Ala Thr Asp Glu 165 170 175 Ser Asp
Ser Leu Ala Ser Phe Ser Gln Tyr Gly Glu Gly Leu Asp Leu 180 185 190
Val Ala Pro Gly Val Gly Val Glu Ser Thr Tyr Pro Gly Gly Gly Tyr 195
200 205 Asp Ser Leu Ser Gly Thr Ser Met Ala Ala Pro His Val Ala Gly
Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Thr Asn
Glu Gln Ile 225 230 235 240 Arg Gly His Leu Asn Asp Thr Ala Asn Asp
Leu Gly Asp Ser Phe Arg 245 250 255 Phe Gly Ser Gly Leu Leu Asn Val
Glu Asn Ala Val Gln 260 265 131125DNABacillus patagoniensis
13atgaatcgaa aaccagttaa actaatcgca ggaacagttc ttgttatggg ctttgtcatc
60agttcatcat ccatatcaac tgccgaggaa acaaaaaaga cttatcttat tggttttgac
120gctcaggaag aagtcgaaac attcacgaat atcgttgatt ctgagatagg
ggctttatct 180gaagaagata ttgacattac ctacgaattt aaagacatac
cggtcgtctc tgctgaaatg 240agtgatgagg agtatgcagc attactagaa
gacccatcga tatcatatat tgaagaagac 300atcgaagtaa caacaatggc
ccaaaccatt ccatggggca ttagtcaaat tagtgctcct 360gaagcacaaa
tcgctggatt tactggtgag ggcgtaaacg tcgcggtgct ggatactgga
420atagaagatc accccgactt aaacgttcaa ggcggtgtta gctttgttca
aggagagccg 480gattatcagg atggaaatgg acacggaacc catgtcgccg
gtacaatcgc tgcccttgat 540aacgacgaag gcgtaattgg agtcgcacca
aatgcagatc tttatgcagt caaagttctt 600ggtgcaaatg gttcaggctc
ggtcagctca attgctcaag ggcttgaatg ggcaggagaa 660aatgggatgg
acattgcaaa cttaagccta ggtagctctg cacctagcgc gacactcgag
720caagcagtgg atgaagcaac cgcaaatggc gtcctcgttg tagccgcttc
tgggaactcg 780ggtgcaagtt ctattggtta tccggctcgc tatgataacg
ctatggccgt tggcgccacc 840gaccagtcag acagcctagc taacttttct
caatatggcg aaggcttaga cattgtagct 900ccaggtgttg gcatcgatag
tacctatact ggcagctcat acgacagctt aagtggaaca 960tcaatggcca
cccctcatgt tgctggatcc gcagcattgg tgaaagaaaa gaatccactt
1020tggtcaaatg aacaaattcg tgctcattta aacgaaactg caactgacct
tggagatacg 1080tatcgttttg gtaatgggct tttaaacgca catgccgctg ttgaa
112514375PRTBacillus patagoniensis 14Met Asn Arg Lys Pro Val Lys
Leu Ile Ala Gly Thr Val Leu Val Met 1 5 10 15 Gly Phe Val Ile Ser
Ser Ser Ser Ile Ser Thr Ala Glu Glu Thr Lys 20 25 30 Lys Thr Tyr
Leu Ile Gly Phe Asp Ala Gln Glu Glu Val Glu Thr Phe 35 40 45 Thr
Asn Ile Val Asp Ser Glu Ile Gly Ala Leu Ser Glu Glu Asp Ile 50 55
60 Asp Ile Thr Tyr Glu Phe Lys Asp Ile Pro Val Val Ser Ala Glu Met
65 70 75 80 Ser Asp Glu Glu Tyr Ala Ala Leu Leu Glu Asp Pro Ser Ile
Ser Tyr 85 90 95 Ile Glu Glu Asp Ile Glu Val Thr Thr Met Ala Gln
Thr Ile Pro Trp 100 105 110 Gly Ile Ser Gln Ile Ser Ala Pro Glu Ala
Gln Ile Ala Gly Phe Thr 115 120 125 Gly Glu Gly Val Asn Val Ala Val
Leu Asp Thr Gly Ile Glu Asp His 130 135 140 Pro Asp Leu Asn Val Gln
Gly Gly Val Ser Phe Val Gln Gly Glu Pro 145 150 155 160 Asp Tyr Gln
Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Ile 165 170 175 Ala
Ala Leu Asp Asn Asp Glu Gly Val Ile Gly Val Ala Pro Asn Ala 180 185
190 Asp Leu Tyr Ala Val Lys Val Leu Gly Ala Asn Gly Ser Gly Ser Val
195 200 205 Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly Glu Asn Gly
Met Asp 210 215 220 Ile Ala Asn Leu Ser Leu Gly Ser Ser Ala Pro Ser
Ala Thr Leu Glu 225 230 235 240 Gln Ala Val Asp Glu Ala Thr Ala Asn
Gly Val Leu Val Val Ala Ala 245 250 255 Ser Gly Asn Ser Gly Ala Ser
Ser Ile Gly Tyr Pro Ala Arg Tyr Asp 260 265 270 Asn Ala Met Ala Val
Gly Ala Thr Asp Gln Ser Asp Ser Leu Ala Asn 275 280 285 Phe Ser Gln
Tyr Gly Glu Gly Leu Asp Ile Val Ala Pro Gly Val Gly 290 295 300 Ile
Asp Ser Thr Tyr Thr Gly Ser Ser Tyr Asp Ser Leu Ser Gly Thr 305 310
315 320 Ser Met Ala Thr Pro His Val Ala Gly Ser Ala Ala Leu Val Lys
Glu 325 330 335 Lys Asn Pro Leu Trp Ser Asn Glu Gln Ile Arg Ala His
Leu Asn Glu 340 345 350 Thr Ala Thr Asp Leu Gly Asp Thr Tyr Arg Phe
Gly Asn Gly Leu Leu 355 360 365 Asn Ala His Ala Ala Val Glu 370 375
15269PRTBacillus patagoniensis 15Ala Gln Thr Ile Pro Trp Gly Ile
Ser Gln Ile Ser Ala Pro Glu Ala 1 5 10 15 Gln Ile Ala Gly Phe Thr
Gly Glu Gly Val Asn Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Glu
Asp His Pro Asp Leu Asn Val Gln Gly Gly Val Ser 35 40 45 Phe Val
Gln Gly Glu Pro Asp Tyr Gln Asp Gly Asn Gly His Gly Thr 50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Glu Gly Val Ile 65
70 75 80 Gly Val Ala Pro Asn Ala Asp Leu Tyr Ala Val Lys Val Leu
Gly Ala 85 90 95 Asn Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly
Leu Glu Trp Ala 100 105 110 Gly Glu Asn Gly Met Asp Ile Ala Asn Leu
Ser Leu Gly Ser Ser Ala 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala
Val Asp Glu Ala Thr Ala Asn Gly 130 135 140 Val Leu Val Val Ala Ala
Ser Gly Asn Ser Gly Ala Ser Ser Ile Gly 145 150 155 160 Tyr Pro Ala
Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Ser
Asp Ser Leu Ala Asn Phe Ser Gln Tyr Gly Glu Gly Leu Asp Ile 180 185
190 Val Ala Pro Gly Val Gly Ile Asp Ser Thr Tyr Thr Gly Ser Ser Tyr
195 200 205 Asp Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala
Gly Ser 210 215 220 Ala Ala Leu Val Lys Glu Lys Asn Pro Leu Trp Ser
Asn Glu Gln Ile 225 230 235 240 Arg Ala His Leu Asn Glu Thr Ala Thr
Asp Leu Gly Asp Thr Tyr Arg 245 250 255 Phe Gly Asn Gly Leu Leu Asn
Ala His Ala Ala Val Glu 260 265 161041DNAartificial
sequenceSynthetic Construct 16gaggaaacaa aaaagactta tcttattggc
tttgatgctc aggaagaagt cgaaacattc 60acgaatatgg tcgattctga gataggggct
ctatctgaag aagaaattga tattacctac 120gaatttaaag aaataccggt
cgtctctgct gaaatgagtg aagaagaata tgcagcatta 180ctagaagacc
catcgatatc atatattgaa gaagacatcg aagtaacaac aatggcccaa
240gccattccat ggggaattag tcaaattagt gcccctgaag cgcaaattgc
tggatttact 300ggtgagggtg taaatgttgc ggtgctggat actggaatag
aggatcaccc cgatttaaac 360gttcaaggcg gtgttagctt tgttcaagga
gagccggatt atcaggatgg aaatggacac 420ggaacccatg tcgccggtac
aatcgctgcc cttgataacg acgaaggcgt aattggagtc 480gcaccaaatg
cagatcttta tgcagtcaaa gttctgggtg caaatggttc tggctcagtc
540agctcaattg ctcaagggct tgaatgggca ggagaaaacg gaatggacat
tgcaaactta 600tcattaggta gctcagcacc tagcgcgaca ctggaacaag
cagtggatga agcaaccgca 660aatggtgtcc tcgttgttgc cgcttctggg
aactctggtg caagttccat tggttatcca 720gctcgctatg ataatgctat
ggccgttggc gccaccgacc agtcagatgg cctagcatca 780ttttctcagt
acggtgatgg cttagacatc gttgctccag gtgttggcat cgatagtacc
840tatcctggta gctcatacga tagcttaagt ggaacatcaa tggcaacacc
tcatgttgct 900ggtgccgcag cattggtgaa agaaaagaat ccactttggt
caaatgaaca aattcgcgct 960catttaaacg aaactgcaac tgaccttggc
gatatgtatc gttttggtaa tggactttta 1020aacgcacatg ccgctgttga a
104117347PRTartificial sequenceProtein expressed from synthetic
construct 17Glu Glu Thr Lys Lys Thr Tyr Leu Ile Gly Phe Asp Ala Gln
Glu Glu 1 5 10 15 Val Glu Thr Phe Thr Asn Met Val Asp Ser Glu Ile
Gly Ala Leu Ser 20 25 30 Glu Glu Glu Ile Asp Ile Thr Tyr Glu Phe
Lys Glu Ile Pro Val Val 35 40 45 Ser Ala Glu Met Ser Glu Glu Glu
Tyr Ala Ala Leu Leu Glu Asp Pro 50 55 60 Ser Ile Ser Tyr Ile Glu
Glu Asp Ile Glu Val Thr Thr Met Ala Gln 65 70 75 80 Ala Ile Pro Trp
Gly Ile Ser Gln Ile Ser Ala Pro Glu Ala Gln Ile 85 90 95 Ala Gly
Phe Thr Gly Glu Gly Val Asn Val Ala Val Leu Asp Thr Gly 100 105 110
Ile Glu Asp His Pro Asp Leu Asn Val Gln Gly Gly Val Ser Phe Val 115
120 125 Gln Gly Glu Pro Asp Tyr Gln Asp Gly Asn Gly His Gly Thr His
Val 130 135 140 Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Glu Gly Val
Ile Gly Val 145 150 155 160 Ala Pro Asn Ala Asp Leu Tyr Ala Val Lys
Val Leu Gly Ala Asn Gly 165 170 175 Ser Gly Ser Val Ser Ser Ile Ala
Gln Gly Leu Glu Trp Ala Gly Glu 180 185 190 Asn Gly Met Asp Ile Ala
Asn Leu Ser Leu Gly Ser Ser Ala Pro Ser 195 200 205 Ala Thr Leu Glu
Gln Ala Val Asp Glu Ala Thr Ala Asn Gly Val Leu 210 215 220 Val Val
Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Gly Tyr Pro 225 230
235
240 Ala Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp Gln Ser Asp
245 250 255 Gly Leu Ala Ser Phe Ser Gln Tyr Gly Asp Gly Leu Asp Ile
Val Ala 260 265 270 Pro Gly Val Gly Ile Asp Ser Thr Tyr Pro Gly Ser
Ser Tyr Asp Ser 275 280 285 Leu Ser Gly Thr Ser Met Ala Thr Pro His
Val Ala Gly Ala Ala Ala 290 295 300 Leu Val Lys Glu Lys Asn Pro Leu
Trp Ser Asn Glu Gln Ile Arg Ala 305 310 315 320 His Leu Asn Glu Thr
Ala Thr Asp Leu Gly Asp Met Tyr Arg Phe Gly 325 330 335 Asn Gly Leu
Leu Asn Ala His Ala Ala Val Glu 340 345 181041DNAartificial
sequenceSynthetic Construct 18gcagaggaaa aaaaatctta tttaattggc
tttgatgaac ctcaagaagt tgagcaattt 60acaacaaatt tggaagaaga gattcgtaca
caagcagatg atgctattga tgtaacgtac 120gagtttaaag atattcctgt
tcttgccgta gatatgacgg aagaagaaat gactgaactc 180aaaaatgaag
agagtatttc ctatattgaa gaagatcaag aagtgacaac gatggcgcaa
240agcattccat ggggaattga aagaattggc acgccagcag cacacgcatc
aggattcaca 300ggtagcggtg taagtgtcgc ggtccttgat acagggattg
atccacattc tgacttaaat 360gttcaagggg gggttagttt tgtaccaggc
gaaagtggag cagatgatgg aaatggacac 420ggtactcatg tagcaggaac
gattgcagcg ttagataatg atgaaggcgt tttaggcgtt 480gctccagagg
ttgatctctt tgcagtaaaa gttttaagtg catctggatc aggatcaatt
540agttcgattg cgcaaggttt agagtggaca gctgaaaaca acattgatgt
ggctaattta 600tctttaggca gtccctctcc tagtcagacg ctagaacaag
cggttaatga cgccacagat 660agtggtgtgc ttgtagtagc agcagcaggg
aactctggaa caagctcatt aggttatcca 720gctcgttatg ataatgcaat
ggctgttggc gctaccgacc aatccgatag cctggcatca 780ttctcacagt
atggcgaggg tcttgactta gtcgctcctg gtgttggtgt agaaagcacg
840tacccaggtg gaggttatga cagcttaagc ggcacatcta tggctgctcc
acatgttgca 900ggtgcagcag cactcgttaa acaaaaaaat ccaggctgga
caaacgaaca aatacgaagc 960catttaaacg atacagccaa tgatcttggc
gattcgttcc gcttcggtag tggcttattg 1020aatgccgaaa atgccgttca a
104119347PRTartificial sequenceProtein expressed from synthetic
construct 19Ala Glu Glu Lys Lys Ser Tyr Leu Ile Gly Phe Asp Glu Pro
Gln Glu 1 5 10 15 Val Glu Gln Phe Thr Thr Asn Leu Glu Glu Glu Ile
Arg Thr Gln Ala 20 25 30 Asp Asp Ala Ile Asp Val Thr Tyr Glu Phe
Lys Asp Ile Pro Val Leu 35 40 45 Ala Val Asp Met Thr Glu Glu Glu
Met Thr Glu Leu Lys Asn Glu Glu 50 55 60 Ser Ile Ser Tyr Ile Glu
Glu Asp Gln Glu Val Thr Thr Met Ala Gln 65 70 75 80 Ser Ile Pro Trp
Gly Ile Glu Arg Ile Gly Thr Pro Ala Ala His Ala 85 90 95 Ser Gly
Phe Thr Gly Ser Gly Val Ser Val Ala Val Leu Asp Thr Gly 100 105 110
Ile Asp Pro His Ser Asp Leu Asn Val Gln Gly Gly Val Ser Phe Val 115
120 125 Pro Gly Glu Ser Gly Ala Asp Asp Gly Asn Gly His Gly Thr His
Val 130 135 140 Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Glu Gly Val
Leu Gly Val 145 150 155 160 Ala Pro Glu Val Asp Leu Phe Ala Val Lys
Val Leu Ser Ala Ser Gly 165 170 175 Ser Gly Ser Ile Ser Ser Ile Ala
Gln Gly Leu Glu Trp Thr Ala Glu 180 185 190 Asn Asn Ile Asp Val Ala
Asn Leu Ser Leu Gly Ser Pro Ser Pro Ser 195 200 205 Gln Thr Leu Glu
Gln Ala Val Asn Asp Ala Thr Asp Ser Gly Val Leu 210 215 220 Val Val
Ala Ala Ala Gly Asn Ser Gly Thr Ser Ser Leu Gly Tyr Pro 225 230 235
240 Ala Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp Gln Ser Asp
245 250 255 Ser Leu Ala Ser Phe Ser Gln Tyr Gly Glu Gly Leu Asp Leu
Val Ala 260 265 270 Pro Gly Val Gly Val Glu Ser Thr Tyr Pro Gly Gly
Gly Tyr Asp Ser 275 280 285 Leu Ser Gly Thr Ser Met Ala Ala Pro His
Val Ala Gly Ala Ala Ala 290 295 300 Leu Val Lys Gln Lys Asn Pro Gly
Trp Thr Asn Glu Gln Ile Arg Ser 305 310 315 320 His Leu Asn Asp Thr
Ala Asn Asp Leu Gly Asp Ser Phe Arg Phe Gly 325 330 335 Ser Gly Leu
Leu Asn Ala Glu Asn Ala Val Gln 340 345 201068DNAartificial
sequenceSynthetic Construct 20gatgaagaga aaaagaccta tttaatcggg
ttccataatc agctagatgt caacgaattt 60attgaggagg atgtaacgaa tacaaatggc
gtgcaattat atacgtcaga ggataagtct 120gcacaggtac aattagaggt
cttacatgaa tttgagcaaa tcccagttgt tgctgttgag 180ctgagtccag
ctgatatcaa ggcattagag gcagagtcag gtattgccta tattgaagaa
240gactttgacg ttacgattgc gaaccaaacc gtaccgtggg gaatcgctca
ggtacaagct 300ccacaagcgc atgaattagg ccacagtggg tcaggaacaa
aagtagcggt acttgatact 360ggtattgctg agcatgctga tttattcatt
catggaggag catcatttgt tgcaggtgag 420ccagattatc atgatttaaa
tgggcacgga actcacgtag caggaacaat cgctgcactt 480aatgatggag
ccggagtaat cggtgttgca ccagacgcag aattatatgc ggtcaaagta
540ttaggggcaa gtggtagtgg ttcggtaagt tcaattgcac aaggtttaga
atgggctggt 600gataatggta tggacgtagc caatctatca ttaggtagcc
cggttggtag tgatacgtta 660gagcaagcag ttaattacgc aacggattca
ggggttcttg ttgtggctgc ttctggtaat 720agtgggtcag ggactgtttc
ttacccagct cgatatgata acgcatttgc tgttggtgca 780acagaccaag
tgaataaccg tgcatcattt tcacaatatg gaacggggtt agatattgtc
840gcacctggtg ttgaagttga aagtacgtac ttaaatggtg agtatgcgag
cttgagtggt 900acttccatgg cgacaccaca tgtcgcgggg gtcgcggcgt
taataaaagc taaaaatcca 960atgttatcta atgaagagat tcgtcagcaa
ttagttcaga cagctacacc gttaggaagt 1020gctgatatgt atggaagtgg
tttagttaat gcagaggtgg ctgttcaa 106821356PRTartificial
sequenceProtein expressed from synthetic construct 21Asp Glu Glu
Lys Lys Thr Tyr Leu Ile Gly Phe His Asn Gln Leu Asp 1 5 10 15 Val
Asn Glu Phe Ile Glu Glu Asp Val Thr Asn Thr Asn Gly Val Gln 20 25
30 Leu Tyr Thr Ser Glu Asp Lys Ser Ala Gln Val Gln Leu Glu Val Leu
35 40 45 His Glu Phe Glu Gln Ile Pro Val Val Ala Val Glu Leu Ser
Pro Ala 50 55 60 Asp Ile Lys Ala Leu Glu Ala Glu Ser Gly Ile Ala
Tyr Ile Glu Glu 65 70 75 80 Asp Phe Asp Val Thr Ile Ala Asn Gln Thr
Val Pro Trp Gly Ile Ala 85 90 95 Gln Val Gln Ala Pro Gln Ala His
Glu Leu Gly His Ser Gly Ser Gly 100 105 110 Thr Lys Val Ala Val Leu
Asp Thr Gly Ile Ala Glu His Ala Asp Leu 115 120 125 Phe Ile His Gly
Gly Ala Ser Phe Val Ala Gly Glu Pro Asp Tyr His 130 135 140 Asp Leu
Asn Gly His Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu 145 150 155
160 Asn Asp Gly Ala Gly Val Ile Gly Val Ala Pro Asp Ala Glu Leu Tyr
165 170 175 Ala Val Lys Val Leu Gly Ala Ser Gly Ser Gly Ser Val Ser
Ser Ile 180 185 190 Ala Gln Gly Leu Glu Trp Ala Gly Asp Asn Gly Met
Asp Val Ala Asn 195 200 205 Leu Ser Leu Gly Ser Pro Val Gly Ser Asp
Thr Leu Glu Gln Ala Val 210 215 220 Asn Tyr Ala Thr Asp Ser Gly Val
Leu Val Val Ala Ala Ser Gly Asn 225 230 235 240 Ser Gly Ser Gly Thr
Val Ser Tyr Pro Ala Arg Tyr Asp Asn Ala Phe 245 250 255 Ala Val Gly
Ala Thr Asp Gln Val Asn Asn Arg Ala Ser Phe Ser Gln 260 265 270 Tyr
Gly Thr Gly Leu Asp Ile Val Ala Pro Gly Val Glu Val Glu Ser 275 280
285 Thr Tyr Leu Asn Gly Glu Tyr Ala Ser Leu Ser Gly Thr Ser Met Ala
290 295 300 Thr Pro His Val Ala Gly Val Ala Ala Leu Ile Lys Ala Lys
Asn Pro 305 310 315 320 Met Leu Ser Asn Glu Glu Ile Arg Gln Gln Leu
Val Gln Thr Ala Thr 325 330 335 Pro Leu Gly Ser Ala Asp Met Tyr Gly
Ser Gly Leu Val Asn Ala Glu 340 345 350 Val Ala Val Gln 355
221041DNAartificial sequenceSynthetic Construct 22gcagaggaaa
aaaaatctta tttaattggc tttgatgaac ctcaggaagt ggagcaattt 60acaacaaatt
tagcagaaga gattcgcaca caagcagatg atgcgattga tgtaacgtac
120gaatttaagg agattcctgt tcttgcagta gaaatgacag aagaagagat
ggctgaactc 180aaaaatgaag agagtatttc ctatattgaa gaggatcaag
aagtgacaac gatggcacaa 240agcattccat ggggaatcga aagaattggc
acgccagctg cacaggcctc aggatttaca 300ggcagtggtg taagtgtagc
agtccttgat acaggaattg atccacactc tgacttaaat 360atacaaggtg
gcgttagttt tgtaccaggc gaaagtgggt cagatgatgg aaatggacac
420ggtactcatg tagcaggtac gattgcagcg ttagataatg atcaaggggt
attgggtgtt 480gcgccagacg ttgatctttt tgcagtaaaa gtcttaagtg
cttctggatc aggatcgatt 540agttcgattg cgcaagggtt agagtggaca
gcagaaaaca atattgatgt agccaatcta 600agtttaggaa gcccctctcc
tagtcagaca ttagagcaag cggttaatga tgccacagat 660agcggtgtgc
ttgtagtagc agcagcaggg aactctggga caagttcatt aggatatcca
720gctcgttatg atcatgcaat ggctgttggc gctaccgatg agtcggatag
tctcgcatca 780ttctcacagt atggagaggg actcgattta gtcgcacctg
gcgttggtgt agaaagtacg 840tacccaggtg gaggttatga cagcttaagc
ggaacatcta tggctgctcc acatgttgca 900ggtgccgcag cactcgttaa
gcaaaaaaat ccaagctgga caaacgaaca aatacgaggc 960catttaaacg
atacagccaa tgatcttggc gattcgttcc gctttggtag tggcttactg
1020aatgttgaaa atgccgttca a 104123347PRTartificial sequenceProtein
expressed from synthetic construct 23Ala Glu Glu Lys Lys Ser Tyr
Leu Ile Gly Phe Asp Glu Pro Gln Glu 1 5 10 15 Val Glu Gln Phe Thr
Thr Asn Leu Ala Glu Glu Ile Arg Thr Gln Ala 20 25 30 Asp Asp Ala
Ile Asp Val Thr Tyr Glu Phe Lys Glu Ile Pro Val Leu 35 40 45 Ala
Val Glu Met Thr Glu Glu Glu Met Ala Glu Leu Lys Asn Glu Glu 50 55
60 Ser Ile Ser Tyr Ile Glu Glu Asp Gln Glu Val Thr Thr Met Ala Gln
65 70 75 80 Ser Ile Pro Trp Gly Ile Glu Arg Ile Gly Thr Pro Ala Ala
Gln Ala 85 90 95 Ser Gly Phe Thr Gly Ser Gly Val Ser Val Ala Val
Leu Asp Thr Gly 100 105 110 Ile Asp Pro His Ser Asp Leu Asn Ile Gln
Gly Gly Val Ser Phe Val 115 120 125 Pro Gly Glu Ser Gly Ser Asp Asp
Gly Asn Gly His Gly Thr His Val 130 135 140 Ala Gly Thr Ile Ala Ala
Leu Asp Asn Asp Gln Gly Val Leu Gly Val 145 150 155 160 Ala Pro Asp
Val Asp Leu Phe Ala Val Lys Val Leu Ser Ala Ser Gly 165 170 175 Ser
Gly Ser Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Thr Ala Glu 180 185
190 Asn Asn Ile Asp Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro Ser
195 200 205 Gln Thr Leu Glu Gln Ala Val Asn Asp Ala Thr Asp Ser Gly
Val Leu 210 215 220 Val Val Ala Ala Ala Gly Asn Ser Gly Thr Ser Ser
Leu Gly Tyr Pro 225 230 235 240 Ala Arg Tyr Asp His Ala Met Ala Val
Gly Ala Thr Asp Glu Ser Asp 245 250 255 Ser Leu Ala Ser Phe Ser Gln
Tyr Gly Glu Gly Leu Asp Leu Val Ala 260 265 270 Pro Gly Val Gly Val
Glu Ser Thr Tyr Pro Gly Gly Gly Tyr Asp Ser 275 280 285 Leu Ser Gly
Thr Ser Met Ala Ala Pro His Val Ala Gly Ala Ala Ala 290 295 300 Leu
Val Lys Gln Lys Asn Pro Ser Trp Thr Asn Glu Gln Ile Arg Gly 305 310
315 320 His Leu Asn Asp Thr Ala Asn Asp Leu Gly Asp Ser Phe Arg Phe
Gly 325 330 335 Ser Gly Leu Leu Asn Val Glu Asn Ala Val Gln 340 345
241044DNAartificial sequenceSynthetic Construct 24gaggaaacaa
aaaagactta tcttattggt tttgacgctc aggaagaagt cgaaacattc 60acgaatatcg
ttgattctga gataggggct ttatctgaag aagatattga cattacctac
120gaatttaaag acataccggt cgtctctgct gaaatgagtg atgaggagta
tgcagcatta 180ctagaagacc catcgatatc atatattgaa gaagacatcg
aagtaacaac aatggcccaa 240accattccat ggggcattag tcaaattagt
gctcctgaag cacaaatcgc tggatttact 300ggtgagggcg taaacgtcgc
ggtgctggat actggaatag aagatcaccc cgacttaaac 360gttcaaggcg
gtgttagctt tgttcaagga gagccggatt atcaggatgg aaatggacac
420ggaacccatg tcgccggtac aatcgctgcc cttgataacg acgaaggcgt
aattggagtc 480gcaccaaatg cagatcttta tgcagtcaaa gttcttggtg
caaatggttc aggctcggtc 540agctcaattg ctcaagggct tgaatgggca
ggagaaaatg ggatggacat tgcaaactta 600agcctaggta gctctgcacc
tagcgcgaca ctggaacaag cagtggatga agcaaccgca 660aatggcgtcc
tcgttgtagc cgcttctggg aactcgggtg caagttctat tggttatccg
720gctcgctatg ataacgctat ggccgttggc gccaccgacc agtcagacag
cctagctaac 780ttttctcaat atggcgaagg cttagacatt gtagctccag
gtgttggcat cgatagtacc 840tatactggca gctcatacga cagcttaagt
ggaacatcaa tggccacccc tcatgttgct 900ggctcagcag cattggtgaa
agaaaagaat ccactttggt caaatgaaca aattcgtgct 960catttaaacg
aaactgcaac tgaccttgga gatacgtatc gttttggtaa tgggctttta
1020aacgcacatg ccgctgttga ataa 104425333PRTartificial
sequenceProtein expressed from synthetic construct 25Glu Glu Thr
Lys Lys Thr Tyr Leu Ile Gly Phe Asp Ala Gln Glu Glu 1 5 10 15 Val
Glu Thr Phe Thr Asn Ile Val Asp Ser Glu Ile Gly Ala Leu Ser 20 25
30 Glu Glu Asp Ile Asp Ile Thr Tyr Glu Phe Lys Asp Ile Pro Val Val
35 40 45 Ser Ala Glu Met Ser Asp Glu Glu Tyr Ala Ala Leu Leu Glu
Asp Pro 50 55 60 Ser Ile Ser Tyr Ile Glu Glu Asp Ile Glu Val Thr
Thr Met Ala Gln 65 70 75 80 Thr Ile Pro Trp Gly Ile Ser Gln Ile Ser
Ala Pro Glu Ala Gln Ile 85 90 95 Ala Gly Phe Thr Gly Glu Gly Val
Asn Val Ala Val Leu Asp Thr Gly 100 105 110 Ile Glu Asp His Pro Asp
Leu Asn Val Gln Gly Gly Val Ser Phe Val 115 120 125 Gln Gly Glu Pro
Asp Tyr Gln Asp Gly Asn Gly His Gly Thr His Val 130 135 140 Ala Gly
Thr Ile Ala Ala Leu Asp Asn Asp Glu Gly Val Ile Gly Val 145 150 155
160 Ala Pro Asn Ala Asp Leu Tyr Ala Val Lys Val Leu Gly Ala Asn Gly
165 170 175 Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala
Gly Glu 180 185 190 Asn Gly Met Asp Ile Ala Asn Leu Ser Leu Gly Ser
Ser Ala Pro Ser 195 200 205 Ala Thr Leu Glu Gln Ala Val Asp Glu Ala
Thr Ala Asn Gly Val Leu 210 215 220 Val Val Ala Ala Ser Gly Asn Ser
Gly Ala Ser Ser Ile Gly Tyr Pro 225 230 235 240 Ala Arg Tyr Asp Asn
Ala Met Ala Val Gly Ala Thr Asp Gln Ser Asp 245 250 255 Ser Leu Ala
Asn Phe Ser Gln Tyr Gly Glu Gly Leu Asp Ile Val Ala 260 265 270 Pro
Gly Val Gly Ile Asp Ser Thr Tyr Thr Gly Ser Ser Tyr Asp Ser 275 280
285 Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ser Ala Ala
290 295 300 Leu Val Lys Glu Lys Asn Pro Leu Trp Ser Asn Glu Gln Ile
Arg Ala 305 310 315 320 His Leu Asn Glu Thr Ala Thr Asp Leu Gly Asp
Thr Tyr 325 330 2620PRTunknownwherein X is any amino acid and Xa is
N or S or Xa is N or Xa is SMISC_FEATURE(5)..(6)X is any amino
acidMISC_FEATURE(10)..(10)X is N or S or X is N or X is
SMISC_FEATURE(14)..(17)X is any amino acidMISC_FEATURE(20)..(20)X
is any amino acid 26Asp Leu Gly Asp Xaa Xaa Arg Phe Gly Xaa Gly Leu
Leu Xaa Xaa Xaa 1 5 10 15 Xaa Ala Val Xaa 20
27269PRTunknownBacillus sp. DSM 8714 27Ala Gln Ala Ile Pro Trp Gly
Ile Ser Gln Ile Ser Ala Pro Glu Ala 1 5 10 15 Gln Ile Ala Gly Phe
Thr Gly Glu Gly Val Asn Val Ala Val Leu Asp 20
25 30 Thr Gly Ile Glu Asp His Pro Asp Leu Asn Val Gln Gly Gly Val
Ser 35 40 45 Phe Val Gln Gly Glu Pro Asp Tyr Gln Asp Gly Asn Gly
His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn
Asp Glu Gly Val Ile 65 70 75 80 Gly Val Ala Pro Asn Ala Asp Leu Tyr
Ala Val Lys Val Leu Gly Ala 85 90 95 Asn Gly Ser Gly Ser Val Ser
Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Glu Asn Gly Met
Asp Ile Ala Asn Leu Ser Leu Gly Ser Ser Ala 115 120 125 Pro Ser Ala
Thr Leu Glu Gln Ala Val Asp Glu Ala Thr Ala Asn Gly 130 135 140 Val
Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Gly 145 150
155 160 Tyr Pro Ala Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp
Gln 165 170 175 Ser Asp Gly Leu Ala Ser Phe Ser Gln Tyr Gly Asp Gly
Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Gly Ile Asp Ser Thr Tyr
Pro Gly Ser Ser Tyr 195 200 205 Asp Ser Leu Ser Gly Thr Ser Met Ala
Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Glu Lys
Asn Pro Leu Trp Ser Asn Glu Gln Ile 225 230 235 240 Arg Ala His Leu
Asn Glu Thr Ala Thr Asp Leu Gly Asp Met Tyr Arg 245 250 255 Phe Gly
Asn Gly Leu Leu Asn Ala His Ala Ala Val Glu 260 265
28269PRTBacillus patagoniensis 28Ala Gln Thr Ile Pro Trp Gly Ile
Ser Gln Ile Ser Ala Pro Glu Ala 1 5 10 15 Gln Ile Ala Gly Phe Thr
Gly Glu Gly Val Asn Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Glu
Asp His Pro Asp Leu Asn Val Gln Gly Gly Val Ser 35 40 45 Phe Val
Gln Gly Glu Pro Asp Tyr Gln Asp Gly Asn Gly His Gly Thr 50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Glu Gly Val Ile 65
70 75 80 Gly Val Ala Pro Asn Ala Asp Leu Tyr Ala Val Lys Val Leu
Gly Ala 85 90 95 Asn Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly
Leu Glu Trp Ala 100 105 110 Gly Glu Asn Gly Met Asp Ile Ala Asn Leu
Ser Leu Gly Ser Ser Ala 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala
Val Asp Glu Ala Thr Ala Asn Gly 130 135 140 Val Leu Val Val Ala Ala
Ser Gly Asn Ser Gly Ala Ser Ser Ile Gly 145 150 155 160 Tyr Pro Ala
Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Ser
Asp Ser Leu Ala Asn Phe Ser Gln Tyr Gly Glu Gly Leu Asp Ile 180 185
190 Val Ala Pro Gly Val Gly Ile Asp Ser Thr Tyr Thr Gly Ser Ser Tyr
195 200 205 Asp Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala
Gly Ser 210 215 220 Ala Ala Leu Val Lys Glu Lys Asn Pro Leu Trp Ser
Asn Glu Gln Ile 225 230 235 240 Arg Ala His Leu Asn Glu Thr Ala Thr
Asp Leu Gly Asp Thr Tyr Arg 245 250 255 Phe Gly Asn Gly Leu Leu Asn
Ala His Ala Ala Val Glu 260 265 29269PRTunknownBacillus sp. DSM
8717 29Ala Gln Ser Ile Pro Trp Gly Ile Glu Arg Ile Gly Thr Pro Ala
Ala 1 5 10 15 His Ala Ser Gly Phe Thr Gly Ser Gly Val Ser Val Ala
Val Leu Asp 20 25 30 Thr Gly Ile Asp Pro His Ser Asp Leu Asn Val
Gln Gly Gly Val Ser 35 40 45 Phe Val Pro Gly Glu Ser Gly Ala Asp
Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala
Ala Leu Asp Asn Asp Glu Gly Val Leu 65 70 75 80 Gly Val Ala Pro Glu
Val Asp Leu Phe Ala Val Lys Val Leu Ser Ala 85 90 95 Ser Gly Ser
Gly Ser Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Thr 100 105 110 Ala
Glu Asn Asn Ile Asp Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120
125 Pro Ser Gln Thr Leu Glu Gln Ala Val Asn Asp Ala Thr Asp Ser Gly
130 135 140 Val Leu Val Val Ala Ala Ala Gly Asn Ser Gly Thr Ser Ser
Leu Gly 145 150 155 160 Tyr Pro Ala Arg Tyr Asp Asn Ala Met Ala Val
Gly Ala Thr Asp Gln 165 170 175 Ser Asp Ser Leu Ala Ser Phe Ser Gln
Tyr Gly Glu Gly Leu Asp Leu 180 185 190 Val Ala Pro Gly Val Gly Val
Glu Ser Thr Tyr Pro Gly Gly Gly Tyr 195 200 205 Asp Ser Leu Ser Gly
Thr Ser Met Ala Ala Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu
Val Lys Gln Lys Asn Pro Gly Trp Thr Asn Glu Gln Ile 225 230 235 240
Arg Ser His Leu Asn Asp Thr Ala Asn Asp Leu Gly Asp Ser Phe Arg 245
250 255 Phe Gly Ser Gly Leu Leu Asn Ala Glu Asn Ala Val Gln 260 265
30269PRTBacillus oshimensis 30Ala Gln Ser Ile Pro Trp Gly Ile Glu
Arg Ile Gly Thr Pro Ala Ala 1 5 10 15 Gln Ala Ser Gly Phe Thr Gly
Ser Gly Val Ser Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Asp Pro
His Ser Asp Leu Asn Ile Gln Gly Gly Val Ser 35 40 45 Phe Val Pro
Gly Glu Ser Gly Ser Asp Asp Gly Asn Gly His Gly Thr 50 55 60 His
Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Gln Gly Val Leu 65 70
75 80 Gly Val Ala Pro Asp Val Asp Leu Phe Ala Val Lys Val Leu Ser
Ala 85 90 95 Ser Gly Ser Gly Ser Ile Ser Ser Ile Ala Gln Gly Leu
Glu Trp Thr 100 105 110 Ala Glu Asn Asn Ile Asp Val Ala Asn Leu Ser
Leu Gly Ser Pro Ser 115 120 125 Pro Ser Gln Thr Leu Glu Gln Ala Val
Asn Asp Ala Thr Asp Ser Gly 130 135 140 Val Leu Val Val Ala Ala Ala
Gly Asn Ser Gly Thr Ser Ser Leu Gly 145 150 155 160 Tyr Pro Ala Arg
Tyr Asp His Ala Met Ala Val Gly Ala Thr Asp Glu 165 170 175 Ser Asp
Ser Leu Ala Ser Phe Ser Gln Tyr Gly Glu Gly Leu Asp Leu 180 185 190
Val Ala Pro Gly Val Gly Val Glu Ser Thr Tyr Pro Gly Gly Gly Tyr 195
200 205 Asp Ser Leu Ser Gly Thr Ser Met Ala Ala Pro His Val Ala Gly
Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Thr Asn
Glu Gln Ile 225 230 235 240 Arg Gly His Leu Asn Asp Thr Ala Asn Asp
Leu Gly Asp Ser Phe Arg 245 250 255 Phe Gly Ser Gly Leu Leu Asn Val
Glu Asn Ala Val Gln 260 265 31269PRTunknownBacillus sp. B001 31Ala
Gln Ser Ile Pro Trp Gly Ile Glu Arg Ile Gly Thr Pro Ala Ala 1 5 10
15 Gln Ala Ser Gly Phe Thr Gly Ser Gly Val Ser Val Ala Val Leu Asp
20 25 30 Thr Gly Ile Asp Pro His Ser Asp Leu Asn Val Gln Gly Gly
Val Ser 35 40 45 Phe Val Pro Gly Glu Ser Gly Ala Asp Asp Gly Asn
Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asp
Asn Asp Glu Gly Val Leu 65 70 75 80 Gly Val Ala Pro Glu Val Asp Leu
Phe Ala Val Lys Val Leu Ser Ala 85 90 95 Ser Gly Ser Gly Ser Ile
Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Ala Glu Asn Asn
Ile Asp Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser
Gln Thr Leu Glu Gln Ala Val Asn Asp Ala Thr Asp Ser Gly 130 135 140
Val Leu Val Val Ala Ala Ala Gly Asn Ser Gly Thr Ser Ser Leu Gly 145
150 155 160 Tyr Pro Ala Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr
Asp Gln 165 170 175 Ser Asp Ser Leu Ala Ser Phe Ser Gln Tyr Gly Glu
Gly Leu Asp Leu 180 185 190 Val Ala Pro Gly Val Gly Val Glu Ser Thr
Tyr Pro Gly Gly Gly Tyr 195 200 205 Asp Ser Leu Ser Gly Thr Ser Met
Ala Ala Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln
Lys Asn Pro Gly Trp Thr Asn Glu Gln Ile 225 230 235 240 Arg Ser His
Leu Asn Asp Thr Ala Asn Asp Leu Gly Asp Ser Phe Arg 245 250 255 Phe
Gly Ser Gly Leu Leu Asn Ala Glu Asn Ala Val Gln 260 265
32269PRTunknownGeomicrobium sp. JCM 19038 32Ser Gln Thr Ile Pro Trp
Gly Ile Asp Arg Val Asn Ala Pro Ala Ala 1 5 10 15 Asn Ala Ser Gly
Val Thr Gly Gly Gly Val Ser Val Ala Ile Leu Asp 20 25 30 Thr Gly
Ile Ser Thr His Glu Asp Leu Asn Ile Gln Gly Gly Glu Ser 35 40 45
Phe Val Pro Gly Glu Pro Gly Ile Asp Asp Gly Asn Gly His Gly Thr 50
55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Asp Leu Gly Val
Leu 65 70 75 80 Gly Val Ser Pro Asp Val Asp Leu Tyr Ala Val Lys Val
Leu Gly Ser 85 90 95 Asp Gly Ser Gly Asn Ile Ser Ser Ile Ala Glu
Gly Leu Glu Trp Ala 100 105 110 Gly Glu Asn Gly Met Asp Val Ala Asn
Met Ser Leu Gly Ser Pro Leu 115 120 125 Pro Ser Pro Thr Leu Glu Gln
Ala Val Asp Glu Ala Thr Asp Arg Gly 130 135 140 Val Leu Val Val Ala
Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Gly 145 150 155 160 Tyr Pro
Ala Ala Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Thr Gln 165 170 175
Asn Asp Thr Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180
185 190 Val Ala Pro Gly Val Gly Val Glu Ser Thr Tyr Pro Gly Gly Gly
Tyr 195 200 205 Arg Ser Leu Asp Gly Thr Ser Met Ala Ala Pro His Val
Ala Gly Val 210 215 220 Ala Ala Leu Val Leu Glu Gln Asn Pro Ser Trp
Ser Pro Gln Gln Val 225 230 235 240 Arg Asn His Leu Asn Asp Thr Ala
Thr Asp Leu Gly Asp Ser Asn Gln 245 250 255 Tyr Gly Ser Gly Leu Val
Asp Ala Val Ser Ala Thr Glu 260 265 33269PRTunknownGeomicrobium sp.
JCM 19055 33Ser Gln Thr Val Pro Trp Gly Ile Asp Arg Val Asn Ala Pro
Ala Ala 1 5 10 15 Asn Ala Ser Gly Val Thr Gly Gly Gly Val Ser Val
Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Glu Asp Leu Asn
Ile Gln Gly Gly Glu Ser 35 40 45 Phe Val Pro Gly Glu Pro Gly Ile
Asp Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile
Ala Ala Leu Asp Asn Asp Thr Gly Val Val 65 70 75 80 Gly Val Ser Pro
Asp Ala Asp Leu Tyr Ala Val Lys Val Leu Gly Ser 85 90 95 Asp Gly
Ser Gly Asn Ile Ser Ser Ile Ala Gln Gly Leu Gln Trp Ala 100 105 110
Gly Glu Asn Gly Met Asp Val Ala Asn Met Ser Leu Gly Ser Pro Leu 115
120 125 Pro Ser Pro Thr Leu Glu Gln Ala Val Asp Glu Ala Thr Asp Arg
Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser
Ser Leu Ser 145 150 155 160 Tyr Pro Ala Ala Tyr Asp Asn Ala Met Ala
Val Gly Ala Thr Thr Gln 165 170 175 Ser Asp Ala Arg Ala Ser Phe Ser
Gln Tyr Gly Ala Gly Leu Asp Leu 180 185 190 Val Ala Pro Gly Val Gly
Val Glu Ser Thr Tyr Pro Gly Gly Gly Tyr 195 200 205 Arg Ser Leu Asp
Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val 210 215 220 Ala Ala
Leu Val Leu Glu Gln Asn Pro Ser Trp Ser Pro Gln Gln Val 225 230 235
240 Arg Ser His Val Asn Asp Thr Ala Thr Asp Leu Gly Asp Thr Asn Gln
245 250 255 Phe Gly Ser Gly Leu Val Asp Ala Glu Ser Ala Thr Asp 260
265 34269PRTunknownGeomicrobium sp. JCM 19037 34Ser Gln Thr Ile Pro
Trp Gly Ile Asp Arg Val Gln Ala Thr Ala Ala 1 5 10 15 His Asn Arg
Gly Ile Thr Gly Asn Gly Val Arg Val Ala Val Leu Asp 20 25 30 Thr
Gly Ile Ser Asn His Pro Asp Leu Asn Ile Gln Gly Gly Thr Ser 35 40
45 Phe Val Pro Gly Glu Pro Gly Ile Ala Asp Gly Asn Gly His Gly Thr
50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Asn Val Gly
Val Leu 65 70 75 80 Gly Val Ala Pro Asp Val Asp Leu Phe Ala Val Lys
Val Leu Gly Arg 85 90 95 Ser Gly Ser Gly Ser Ile Ser Gly Ile Ala
Gln Gly Leu Gln Trp Ser 100 105 110 Ser Asn Asn Asn Met Asp Val Ala
Asn Met Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Pro Thr Leu Glu
Arg Ala Val Asn Gln Ala Thr Asn Ser Gly 130 135 140 Val Leu Val Val
Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Gly 145 150 155 160 Tyr
Pro Ala Arg Tyr Gln Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170
175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Phe Gly Thr Gly Leu Asp Ile
180 185 190 Met Ala Pro Gly Val Gly Val Gln Ser Thr Tyr Pro Gly Asn
Gly Tyr 195 200 205 Arg Ser Leu Ser Gly Thr Ser Met Ala Ala Pro His
Val Ala Gly Val 210 215 220 Ala Ala Leu Val Met Ser Asn Asn Pro Ser
Trp Ser Pro Ala Gln Val 225 230 235 240 Arg Ser His Leu Asn Gln Thr
Ala Thr Pro Ile Gly Ala Ser Asn Gln 245 250 255 Tyr Gly Asn Gly Leu
Val Asn Ala Asn Ala Ala Thr Gln 260 265 35269PRTBacillus okhensis
35Asn Gln Thr Ile Pro Trp Gly Ile Thr Arg Val Gln Ala Pro Ala Ala 1
5 10 15 Ile Asn Arg Gly Phe Thr Gly Ala Gly Val Arg Val Ala Val Leu
Asp 20 25 30 Thr Gly Ile Ser Asn His Pro Asp Leu Asn Ile Arg Gly
Gly Val Ser 35 40 45 Phe Val Pro Gly Glu Ser Thr Tyr Gln Asp Gly
Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu
Asn Asn Ser Ile Gly Val Val 65 70 75 80 Gly Val Ala Pro Asn Thr Glu
Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Asn Gly Ser Gly Ser
Ile Ser Ser Ile Ala Gln Gly Leu Gln Trp Thr 100 105 110 Ala Gln Asn
Asn Ile His Val Ala Asn Leu Ser Leu Gly Ser Pro Thr 115 120 125 Gly
Ser Gln Thr Leu Glu Leu Ala Val Asn Gln Ala Thr Ser Ala Gly 130
135
140 Val Leu Val Val Ala Ala Ser Gly Asn Asn Gly Ser Gly Thr Ile Ser
145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Leu Ala Val Gly Ala
Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly
Thr Gly Leu Asn Ile 180 185 190 Val Ala Pro Gly Val Gly Val Gln Ser
Thr Tyr Pro Gly Asn Arg Tyr 195 200 205 Ala Ser Leu Ser Gly Thr Ser
Met Ala Thr Pro His Val Ala Gly Val 210 215 220 Ala Ala Leu Val Lys
Gln Lys Asn Pro Gly Trp Ser Asn Thr Gln Ile 225 230 235 240 Arg Gln
His Leu Leu Asn Thr Ala Thr Pro Leu Gly Ser Ser Asn Gln 245 250 255
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265
36269PRTBacillus gibsonii 36Gln Gln Thr Val Pro Trp Gly Ile Thr Arg
Val Gln Ala Pro Ala Val 1 5 10 15 His Asn Arg Gly Ile Thr Gly Ser
Gly Val Arg Val Ala Ile Leu Asp 20 25 30 Ser Gly Ile Ser Ala His
Ser Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly
Glu Pro Thr Thr Ala Asp Leu Asn Gly His Gly Thr 50 55 60 His Val
Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Ile 65 70 75 80
Gly Val Ala Pro Asn Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85
90 95 Asn Gly Ser Gly Ser Val Ser Gly Ile Ala Gln Gly Leu Glu Trp
Ala 100 105 110 Ala Thr Asn Asn Met His Ile Ala Asn Met Ser Leu Gly
Ser Asp Phe 115 120 125 Pro Ser Ser Thr Leu Glu Arg Ala Val Asn Tyr
Ala Thr Ser Arg Asp 130 135 140 Val Leu Val Ile Ala Ala Thr Gly Asn
Asn Gly Ser Gly Ser Val Gly 145 150 155 160 Tyr Pro Ala Arg Tyr Ala
Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Arg Arg
Ala Asn Phe Ser Gln Tyr Gly Thr Gly Ile Asp Ile 180 185 190 Val Ala
Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Asn Arg Tyr 195 200 205
Val Ser Met Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210
215 220 Ala Ala Leu Val Lys Gln Arg Tyr Pro Ser Trp Asn Ala Thr Gln
Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Asn Leu Gly
Asn Ser Ser Gln 245 250 255 Phe Gly Ser Gly Leu Val Asn Ala Glu Ala
Ala Thr Arg 260 265 37269PRTBacillus lentus 37Ala Gln Ser Val Pro
Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg
Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr
Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40
45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly
Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys
Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala
Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala
Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu
Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val
Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr
Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170
175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile
180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser
Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His
Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser
Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr
Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu
Val Asn Ala Glu Ala Ala Thr Arg 260 265 38269PRTunknownBacillus
lentus; DSM 5483 Synthetic 38Ala Gln Ser Ile Pro Trp Gly Ile Ser
Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly
Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr
His Glu Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro
Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His
Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu 65 70
75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly
Ala 85 90 95 Asp Gly Glu Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu
Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser
Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val
Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser
Gly Asn Ser Gly Ala Ser Ser Ile Gly 145 150 155 160 Tyr Pro Ala Arg
Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn
Asn Arg Ala Ser Phe Ser Arg Tyr Gly Ala Gly Leu Asp Ile 180 185 190
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195
200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly
Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn
Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser
Leu Gly Asp Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala
Glu Ala Ala Thr Arg 260 265 39269PRTBacillus pseudalcaliphilus
39Asn Gln Thr Val Pro Trp Gly Ile Ala Gln Val Gln Ala Pro Gln Ala 1
5 10 15 His Glu Leu Gly His Ser Gly Ser Gly Thr Lys Val Ala Val Leu
Asp 20 25 30 Thr Gly Ile Ala Glu His Ala Asp Leu Phe Ile His Gly
Gly Ala Ser 35 40 45 Phe Val Ala Gly Glu Pro Asp Tyr His Asp Leu
Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu
Asn Asp Gly Ala Gly Val Ile 65 70 75 80 Gly Val Ala Pro Asp Ala Glu
Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser
Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asp Asn
Gly Met Asp Val Ala Asn Leu Ser Leu Gly Ser Pro Val 115 120 125 Gly
Ser Asp Thr Leu Glu Gln Ala Val Asn Tyr Ala Thr Asp Ser Gly 130 135
140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ser Gly Thr Val Ser
145 150 155 160 Tyr Pro Ala Arg Tyr Asp Asn Ala Phe Ala Val Gly Ala
Thr Asp Gln 165 170 175 Val Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly
Thr Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Glu Val Glu Ser
Thr Tyr Leu Asn Gly Glu Tyr 195 200 205 Ala Ser Leu Ser Gly Thr Ser
Met Ala Thr Pro His Val Ala Gly Val 210 215 220 Ala Ala Leu Ile Lys
Ala Lys Asn Pro Met Leu Ser Asn Glu Glu Ile 225 230 235 240 Arg Gln
Gln Leu Val Gln Thr Ala Thr Pro Leu Gly Ser Ala Asp Met 245 250 255
Tyr Gly Ser Gly Leu Val Asn Ala Glu Val Ala Val Gln 260 265
40273PRTBacillus pseudofirmus 40Ala Gln Thr Val Pro Trp Gly Ile Pro
Tyr Ile Tyr Ser Asp Val Val 1 5 10 15 His Arg Gln Gly Tyr Phe Gly
Asn Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Val Ala Pro
His Pro Asp Leu His Ile Arg Gly Gly Val Ser 35 40 45 Phe Ile Ser
Thr Glu Asn Thr Tyr Val Asp Tyr Asn Gly His Gly Thr 50 55 60 His
Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Tyr Gly Val Leu 65 70
75 80 Gly Val Ala Pro Gly Ala Glu Leu Tyr Ala Val Lys Val Leu Asp
Arg 85 90 95 Asn Gly Ser Gly Ser His Ala Ser Ile Ala Gln Gly Ile
Glu Trp Ala 100 105 110 Met Asn Asn Gly Met Asp Ile Ala Asn Met Ser
Leu Gly Ser Pro Ser 115 120 125 Gly Ser Thr Thr Leu Gln Leu Ala Ala
Asp Arg Ala Arg Asn Ala Gly 130 135 140 Val Leu Leu Ile Gly Ala Ala
Gly Asn Ser Gly Gln Gln Gly Gly Ser 145 150 155 160 Asn Asn Met Gly
Tyr Pro Ala Arg Tyr Ala Ser Val Met Ala Val Gly 165 170 175 Ala Val
Asp Gln Asn Gly Asn Arg Ala Asn Phe Ser Ser Tyr Gly Ser 180 185 190
Glu Leu Glu Ile Met Ala Pro Gly Val Asn Ile Asn Ser Thr Tyr Leu 195
200 205 Asn Asn Gly Tyr Arg Ser Leu Asn Gly Thr Ser Met Ala Ser Pro
His 210 215 220 Val Ala Gly Val Ala Ala Leu Val Lys Gln Lys His Pro
His Leu Thr 225 230 235 240 Ala Ala Gln Ile Arg Asn Arg Met Asn Gln
Thr Ala Ile Pro Leu Gly 245 250 255 Asn Ser Thr Tyr Tyr Gly Asn Gly
Leu Val Asp Ala Glu Tyr Ala Ala 260 265 270 Gln 41274PRTBacillus
licheniformis 41Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala
Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys
Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His Pro Asp
Leu Asn Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala
Tyr Asn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His Val Ala Gly
Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 65 70 75 80 Leu Gly Val
Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn 85 90 95 Ser
Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp 100 105
110 Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala
115 120 125 Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr
Ala Arg 130 135 140 Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly
Ser Ser Gly Asn 145 150 155 160 Thr Asn Thr Ile Gly Tyr Pro Ala Lys
Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser Asn Ser
Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu Leu Glu Val
Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200 205 Pro Thr Asn
Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His
Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu 225 230
235 240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr
Leu 245 250 255 Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val
Glu Ala Ala 260 265 270 Ala Gln 42276PRTunknownBacillus sp. Strain
LG12 42Ala Gln Thr Val Pro Tyr Gly Val Pro His Ile Lys Ala Asp Val
Ala 1 5 10 15 His Ala Gln Asn Val Thr Gly Ser Gly Val Lys Val Ala
Val Leu Asp 20 25 30 Thr Gly Ile Asp Ala Ser His Glu Asp Leu Arg
Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ser Glu Glu Pro Asp Ala
Leu Thr Asp Gly Asn Gly His 50 55 60 Gly Thr His Val Ala Gly Thr
Ile Ala Ala Leu Asn Asn Asn Val Gly 65 70 75 80 Val Leu Gly Val Ser
Tyr Asp Val Asp Leu Tyr Ala Val Lys Val Leu 85 90 95 Ser Ala Gly
Gly Ser Gly Thr Leu Ala Gly Ile Ala Gln Gly Ile Glu 100 105 110 Trp
Ala Ile Asp Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120
125 Ser Thr Gly Ser Thr Thr Leu Lys Gln Ala Ser Asp Asn Ala Tyr Asn
130 135 140 Ser Gly Ile Val Val Ile Ala Ala Ala Gly Asn Ser Gly Ser
Val Leu 145 150 155 160 Gly Leu Val Asn Thr Ile Gly Tyr Pro Ala Arg
Tyr Asp Ser Val Ile 165 170 175 Ala Val Gly Ala Val Asp Ser Asn Asn
Asn Arg Ala Ser Phe Ser Ser 180 185 190 Val Gly Ser Gln Leu Glu Val
Met Ala Pro Gly Val Ala Ile Asn Ser 195 200 205 Thr Leu Pro Gly Asn
Gln Tyr Gly Glu Leu Asn Gly Thr Ser Met Ala 210 215 220 Ser Pro His
Val Ala Gly Ala Ala Ala Leu Leu Leu Ala Gln Asn Pro 225 230 235 240
Asn Leu Thr Asn Val Gln Val Arg Glu Arg Leu Arg Asp Thr Ala Thr 245
250 255 Asn Leu Gly Ser Ala Phe Asn Tyr Gly His Gly Val Ile Asn Leu
Glu 260 265 270 Arg Ala Leu Gln 275 43275PRTunknownBacillus sp.
KSM-LD1 43Ala Gln Thr Thr Pro Trp Gly Val Thr His Ile Asn Ala His
Arg Ala 1 5 10 15 His Ser Ser Gly Val Thr Gly Ser Gly Val Lys Val
Ala Ile Leu Asp 20 25 30 Thr Gly Ile His Ala Ser His Pro Asp Leu
Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Ile Ser Gly Glu Ser Asn
Pro Tyr Ile Asp Ser Asn Gly His 50 55 60 Gly Thr His Val Ala Gly
Thr Val Ala Ala Leu Asn Asn Thr Val Gly 65 70 75 80 Val Leu Gly Val
Ala Tyr Asn Ala Glu Leu Tyr Ala Val Lys Val Leu 85 90 95 Ser Ala
Ser Gly Ser Gly Thr Leu Ser Gly Ile Ala Gln Gly Val Glu 100 105 110
Trp Ser Ile Ala Asn Lys Met Asp Val Ile Asn Met Ser Leu Gly Gly 115
120 125 Ser Ser Gly Ser Thr Ala Leu Gln Arg Ala Val Asp Asn Ala Tyr
Arg 130 135 140 Asn Asn Ile Val Val Val Ala Ala Ala Gly Asn Ser Gly
Ala Gln Gly 145 150 155 160 Asn Arg Asn Thr Ile Gly Tyr Pro Ala Arg
Tyr Ser Ser Val Ile Ala 165 170 175 Val Gly Ala Val Asp Ser Asn Asn
Asn Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Ser Glu Leu Glu Val
Met Ala Pro Gly Val Ser Ile Leu Ser Thr 195 200 205 Val Pro Gly Ser
Ser Tyr Ala Ser Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His
Val Ala Gly Ala Ala Ala Leu Leu Lys Ala Lys Tyr Pro Asn 225 230 235
240 Trp Ser Ala Ala Gln
Ile Arg Asn Lys Leu Asn Ser Thr Thr Thr Tyr 245 250 255 Leu Gly Ser
Ser Phe Tyr Tyr Gly Asn Gly Val Ile Asn Val Glu Arg 260 265 270 Ala
Leu Gln 275 44275PRTunknownBacillus sp. Strain LG12 44Ala Gln Thr
Val Pro Trp Gly Ile Pro His Ile Lys Ala Asp Lys Ala 1 5 10 15 His
Ala Ala Gly Val Thr Gly Ser Gly Val Lys Val Ala Ile Leu Asp 20 25
30 Thr Gly Ile Asp Ala Asn His Ala Asp Leu Asn Val Lys Gly Gly Ala
35 40 45 Ser Phe Val Ser Gly Glu Pro Asn Ala Leu Gln Asp Gly Asn
Gly His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn
Asn Thr Thr Gly 65 70 75 80 Val Leu Gly Val Ala Tyr Asn Ala Asp Leu
Tyr Ala Val Lys Val Leu 85 90 95 Ser Ala Ser Gly Ser Gly Thr Leu
Ser Gly Ile Ala Gln Gly Ile Glu 100 105 110 Trp Ser Ile Ser Asn Gly
Met Asn Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Ser Ser Gly Ser
Thr Ala Leu Gln Gln Ala Cys Asn Asn Ala Tyr Asn 130 135 140 Arg Gly
Ile Val Val Ile Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly 145 150 155
160 Asn Arg Asn Thr Met Gly Tyr Pro Ala Arg Tyr Ser Ser Val Ile Ala
165 170 175 Val Gly Ala Val Ser Ser Asn Asn Thr Arg Ala Ser Phe Ser
Ser Val 180 185 190 Gly Ser Glu Leu Glu Val Met Ala Pro Gly Val Asn
Ile Leu Ser Thr 195 200 205 Thr Pro Gly Asn Asn Tyr Ala Ser Phe Asn
Gly Thr Ser Met Ala Ala 210 215 220 Pro His Val Ala Gly Ala Ala Ala
Leu Ile Lys Ala Lys Tyr Pro Ser 225 230 235 240 Met Thr Asn Val Gln
Ile Arg Glu Arg Leu Lys Asn Thr Ala Thr Asn 245 250 255 Leu Gly Asp
Pro Phe Phe Tyr Gly Lys Gly Val Ile Asn Val Glu Ser 260 265 270 Ala
Leu Gln 275 45275PRTBacillus amyloliquefaciens 45Ala Gln Ser Val
Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser
Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30
Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35
40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser
His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn
Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr
Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser
Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met
Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala
Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val
Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160
Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165
170 175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser
Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile
Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly
Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu
Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val
Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser
Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala
Gln 275 46275PRTBacillus atrophaeus 46Ala Gln Ser Val Pro Tyr Gly
Ile Ser Gln Ile Lys Ala Pro Ala Val 1 5 10 15 His Ser Gln Gly Tyr
Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile
Asp Ser Ser His Pro Asp Leu Lys Val Ser Gly Gly Ala 35 40 45 Ser
Phe Val Pro Ser Glu Pro Asn Pro Phe Gln Asp Gly Asn Ser His 50 55
60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Val Gly
65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys
Val Leu 85 90 95 Ser Ser Ser Gly Ser Gly Asp Tyr Ser Trp Ile Ile
Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile
Asn Met Ser Leu Gly Gly 115 120 125 Pro Gln Gly Ser Thr Ala Leu Lys
Ala Val Val Asp Lys Ala Val Ser 130 135 140 Gln Gly Ile Val Val Val
Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser
Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val
Gly Ala Val Asp Ser Asn Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185
190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr
195 200 205 Leu Pro Gly Ser Ser Tyr Gly Ser Tyr Asn Gly Thr Ser Met
Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Val Leu Ser
Lys His Pro Asn 225 230 235 240 Trp Thr Asn Ser Gln Val Arg Asn Ser
Leu Glu Ser Thr Ala Thr Asn 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr
Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275
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