U.S. patent application number 11/588435 was filed with the patent office on 2008-11-06 for multiply-substituted protease variants with altered net charge for use in detergents.
Invention is credited to Robert M. Caldwell, Paech Christian, Katherine D. Collier, James T. Kellis, Joanne Nadherny, Donald P. Naki, Ayrookaran J. Poulose, Volker Schellenberger.
Application Number | 20080274938 11/588435 |
Document ID | / |
Family ID | 39939969 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274938 |
Kind Code |
A1 |
Poulose; Ayrookaran J. ; et
al. |
November 6, 2008 |
Multiply-substituted protease variants with altered net charge for
use in detergents
Abstract
Novel protease variants derived from the DNA sequences of
naturally-occurring or recombinant non-human proteases are
disclosed. The variant proteases, in general, are obtained by in
vitro modification of a precursor DNA sequence encoding the
naturally-occurring or recombinant protease to generate the
substitution of a plurality of amino acid residues in the amino
acid sequence of a precursor protease. Protease variants are
provided that contain substitutions of the amino acids at one or
more residue positions so that the substitution alters the charge
at that position to make the charge more negative or less positive
compared to a precursor protease and thus the protease variant is
more effective in a low detergent concentration system than a
precursor protease. Also provided are protease variants containing
substitutions of the amino acids at one or more residue positions
so that the substitution alters the charge at that position to make
the charge more positive or less negative compared to a precursor
protease and thus the protease variant is more effective in a high
detergent concentration system than a precursor protease. Protease
variants are provided that contain substitutions of the amino acids
at one or more residue positions so that the substitution alters
the charge at that position to make the charge more negative or
less positive compared to a precursor protease and thus the
protease variant is more effective in a medium detergent
concentration system than a precursor protease. Also provided are
protease variants containing substitutions of the amino acids at
one or more residue positions so that the substitution alters the
charge at that position to make the charge more positive or less
negative compared to a precursor protease and thus the protease
variant is more effective in a medium detergent concentration
system than a precursor protease. Further provided is a method of
producing a protease variant that is more effective in a low
detergent concentration system, medium detergent concentration
system and high detergent concentration system than a precursor
protease.
Inventors: |
Poulose; Ayrookaran J.;
(Belmont, CA) ; Schellenberger; Volker; (Palo
Alto, CA) ; Kellis; James T.; (Portola Valley,
CA) ; Christian; Paech; (Dale City, CA) ;
Nadherny; Joanne; (San Francisco, CA) ; Naki; Donald
P.; (San Francisco, CA) ; Collier; Katherine D.;
(Redwood City, CA) ; Caldwell; Robert M.;
(Belmont, CA) |
Correspondence
Address: |
GENENCOR INTERNATIONAL, INC.;ATTENTION: LEGAL DEPARTMENT
925 PAGE MILL ROAD
PALO ALTO
CA
94304
US
|
Family ID: |
39939969 |
Appl. No.: |
11/588435 |
Filed: |
October 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10423649 |
Apr 25, 2003 |
7129076 |
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11588435 |
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08956323 |
Oct 23, 1997 |
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10423649 |
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Current U.S.
Class: |
510/392 ;
435/222 |
Current CPC
Class: |
A61K 8/66 20130101; C11D
3/386 20130101; A23G 4/123 20130101; A61K 2800/86 20130101; C12Y
304/21062 20130101; A61Q 11/00 20130101; C12N 9/54 20130101 |
Class at
Publication: |
510/392 ;
435/222 |
International
Class: |
C11D 7/42 20060101
C11D007/42; C12N 9/56 20060101 C12N009/56 |
Claims
1-2. (canceled)
3. A Bacillus subtilisin variant comprising a substitution of an
amino acid at one or more residue positions of a precursor Bacillus
subtilisin, wherein said substitution alters the overall charge of
the precursor subtilisin resulting in a subtilisin variant that is
less positive or more negative relative to the precursor
subtilisin, wherein said substitution makes the variant more
effective than the precursor in a low detergent concentration
system having less than about 800 ppm detergent components present
in the wash water.
4. The subtilisin variant according to claim 3 wherein the
precursor subtilisin is a Bacillus lentus subtilisin.
5-7. (canceled)
8. A cleaning composition comprising the subtilisin variant of
claim 3.
9-26. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part
application of U.S. patent application Ser. No. 08/956,323, filed
Oct. 23, 1998, U.S. patent application Ser. No. 08/956,564, filed
Oct. 23, 1998, and U.S. patent application Ser. No. 08/956,324
filed Oct. 23, 1998, all of which are hereby incorporated herein in
their entirety.
BACKGROUND OF THE INVENTION
[0002] Serine proteases are a subgroup of carbonyl hydrolases. They
comprise a diverse class of enzymes having a wide range of
specificities and biological functions. Stroud, R. Sci. Amer.,
131:74-88. Despite their functional diversity, the catalytic
machinery of serine proteases has been approached by at least two
genetically distinct families of enzymes: 1) the subtilisins and 2)
the mammalian chymotrypsin-related and homologous bacterial serine
proteases (e.g., trypsin and S. gresius trypsin). These two
families of serine proteases show remarkably similar mechanisms of
catalysis. Kraut, J. (1977), Annu. Rev. Biochem., 46:331-358.
Furthermore, although the primary structure is unrelated, the
tertiary structure of these two enzyme families bring together a
conserved catalytic triad of amino acids consisting of serine,
histidine and aspartate.
[0003] Subtilisins are serine proteases (approx. MW 27,500) which
are secreted in large amounts from a wide variety of Bacillus
species and other microorganisms. The protein sequence of
subtilisin has been determined from at least nine different species
of Bacillus. Markland, F. S., et al. (1983), Hoppe-Seyler's Z.
Physiol. Chem., 364:1537-1540. The three-dimensional
crystallographic structure of subtilisins from Bacillus
amyloliquefaciens, Bacillus licheniformis and several natural
variants of B. lentus have been reported. These studies indicate
that although subtilisin is genetically unrelated to the mammalian
serine proteases, it has a similar active site structure. The x-ray
crystal structures of subtilisin containing covalently bound
peptide inhibitors (Robertus, J. D., et al. (1972), Biochemistry,
11:2439-2449) or product complexes (Robertus, J. D., et al. (1976),
J. Biol. Chem., 251:1097-1103) have also provided information
regarding the active site and putative substrate binding cleft of
subtilisin. In addition, a large number of kinetic and chemical
modification studies have been reported for subtilisin; Svendsen,
B. (1976), Carlsberg Res. Commun., 41:237-291; Markland, F. S. Id.)
as well as at least one report wherein the side chain of methionine
at residue 222 of subtilisin was converted by hydrogen peroxide to
methionine-sulfoxide (Stauffer, D. C., et al. (1965), J. Biol.
Chem., 244:5333-5338) and extensive site-specific mutagenesis has
been carried out (Wells and Estell (1988) TIBS 13:291-297)
[0004] A common issue in the development of a protease variant for
use in a detergent formulation is the variety of wash conditions
including varying detergent formulations that a protease variant
might be used in. For example, detergent formulations used in
different areas have different concentrations of their relevant
components present in the wash water. For example, a European
detergent system typically has about 4500-5000 ppm of detergent
components in the wash water while a Japanese detergent system
typically has approximately 667 ppm of detergent components in the
wash water. In North America, particularly the United States, a
detergent system typically has about 975 ppm of detergent
components present in the wash water. Surprisingly, a method for
the rational design of a protease variant for use in a low
detergent concentration system, a high detergent concentration
system, and/or a medium detergent concentration system as well as
for use in all three types of detergent concentration systems has
been developed.
SUMMARY OF THE INVENTION
[0005] It is an object herein to provide protease variants
containing substitutions of the amino acids at one or more residue
positions so that the substitution alters the charge at that
position to make the charge more negative or less positive compared
to a precursor protease and thus the protease variant is more
effective in a low detergent concentration system than a precursor
protease. A low detergent concentration system is a wash system
that has less than about 800 ppm of detergent components present in
the wash water.
[0006] It is another object herein to provide protease variants
containing substitutions of the amino acids at one or more residue
positions so that the substitution alters the charge at that
position to make the charge more positive or less negative compared
to a precursor protease and thus the protease variant is more
effective in a high detergent concentration system than a precursor
protease. A high detergent concentration system is a wash system
that has greater than about 2000 ppm of detergent components
present in the wash water.
[0007] It is another object herein to provide protease variants
containing substitutions of the amino acids at one or more residue
positions so that the substitution alters the charge at that
position to make the charge more positive or less negative compared
to a precursor protease and thus the protease variant is more
effective in a medium detergent concentration system than a
precursor protease. A medium detergent concentration system is a
system that has between about 800 ppm and about 2000 ppm of
detergent components present in the wash water.
[0008] It is another object herein to provide protease variants
containing substitutions of the amino acids at one or more residue
positions so that the substitution alters the charge at that
position to make the charge more negative or less positive compared
to a precursor protease and thus the protease variant is more
effective in a medium detergent concentration system than a
precursor protease. A medium detergent concentration system is a
wash system that has between about 800 ppm to about 2000 ppm of
detergent components present in the wash water.
[0009] It is a further object to provide DNA sequences encoding
such protease variants, as well as expression vectors containing
such variant DNA sequences.
[0010] Still further, another object of the invention is to provide
host cells transformed with such vectors, as well as host cells
which are capable of expressing such DNA to produce protease
variants either intracellularly or extracellularly.
[0011] There is further provided a cleaning composition comprising
a protease variant of the present invention.
[0012] Additionally, there is provided an animal feed comprising a
protease variant of the present invention.
[0013] Also provided is a composition for the treatment of a
textile comprising a protease variant of the present invention.
[0014] There is further provided a method of producing a protease
variant that is more effective in a low, medium and high detergent
concentration system than a precursor protease including: [0015] a)
substituting an amino acid at one or more residue positions wherein
the substitution alters the charge at that position to make the
charge more positive or less negative compared to the precursor
protease; [0016] b) substituting an amino acid at one or more
residue positions wherein the substitution alters the charge at
that position to make the charge more negative or less positive
compared to the precursor protease; [0017] c) testing the variant
to determine its effectiveness in a high, medium and low detergent
concentration system compared to the precursor protease; and [0018]
d) repeating steps a)-c) as necessary to produce a protease variant
that is more effective in a low, medium and high detergent
concentration system than a precursor protease wherein steps a) and
[0019] b) can be done in any order.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-C depict the DNA and amino acid sequence for
Bacillus amyloliquefaciens subtilisin and a partial restriction map
of this gene.
[0021] FIG. 2 depicts the conserved amino acid residues among
subtilisins from Bacillus amyloliquefaciens (BPN)' and Bacillus
lentus (wild-type).
[0022] FIGS. 3A and 3B depict the amino acid sequence of four
subtilisins. The top line represents the amino acid sequence of
subtilisin from Bacillus amyloliquefaciens subtilisin (also
sometimes referred to as subtilisin BPN'). The second line depicts
the amino acid sequence of subtilisin from Bacillus subtilis. The
third line depicts the amino acid sequence of subtilisin from B.
licheniformis. The fourth line depicts the amino acid sequence of
subtilisin from Bacillus lentus (also referred to as subtilisin 309
in PCT WO89/06276). The symbol denotes the absence of specific
amino acid residues as compared to subtilisin BPN'.
DETAILED DESCRIPTION OF THE INVENTION
[0023] As noted above, certain geographies have certain wash
conditions and, as such, use different types of detergents. For
example, Japan uses a low detergent concentration system while
Europe uses a high detergent concentration system. As discussed
previously, the United States uses a medium detergent concentration
system. We have found that different protease variants perform
optimally in these different detergent formulations. However, as a
result of these observations, one would expect that it would be
impossible to find a protease that would work well in all three
types of detergents. Surprisingly, this is not the case. A method
of rationally designing a protease variant to be used in either a
low detergent concentration system or a high detergent
concentration system or even a medium detergent concentration
system as well as one that works in all three detergent
concentration systems has been developed.
[0024] We have found that in order to produce a protease variant
that is more efficacious in a low detergent concentration system,
it is necessary to replace positively charged residue(s) either
with negatively charged residue(s) or neutral residue(s) and/or
neutral residue(s) with negatively charged residue(s). In contrast,
we note that in order to produce a protease variant that is more
efficacious in a high detergent concentration system, it is
necessary to replace negatively charged residue(s) either with
positively charged residue(s) or neutral residue(s) and/or neutral
residue(s) with positively charged residue(s). Further, we have
found that many of the protease variants useful in the low
detergent concentration system and/or the high detergent
concentration system also are effective in a medium detergent
concentration system. By balancing these changes, it is possible to
produce a protease variant that works well in low detergent
concentration systems, low and medium detergent concentration
systems, medium and high detergent concentration systems, high
detergent concentration systems, or all three detergent
concentration systems.
[0025] The electrostatic charge of any ionizable amino acid side
chain with an acidic or basic function assumes in aqueous solution
is a function of the pH. The acidic residues Glu and Asp, in an
equilibrium process, lose a proton by dissociation between pH 3 and
6 thereby acquiring a negative charge. In a similar fashion, His,
Lys, and Arg gradually deprotonate between pH 5 and 8, pH 8.5 and
11.5, and pH 11 and 14, respectively, thereby losing a positive
charge. The proton of Tyr OH increasingly dissociates between pH
8.5 and 11.5, whereby Tyr acquires a negative charge. The
dissociation range for the carboxy terminus is pH 1 to 4, yielding
a negative charge, and for the amino terminus it is pH 8 to 11,
accompanied by the loss of a positive charge. The dissociation
range for amino acid side chains given here are average values for
many proteins but they are known to be affected by unusual
structural configurations in some proteins.
[0026] The cumulative effect of all charges determines whether a
protein has a net positive or net negative charge at a given pH.
The pH at which positive and negative charges are equally effective
and convey an electrostatically neutral state to a protein is
called the isoelectric point (pI). A protein will lose or gain
charge when the pH is shifted or when an amino acid with an
ionizable side chain residue is added or removed. An increase in
net positive charge can be achieved either by replacing a residue
that at a given pH is negatively charged with an uncharged or a
positively charged residue, leading to a formal charge change of +1
and +2, respectively. By replacing an uncharged side chain residue
with one that is protonated at the given pH the formal charge
change would be +1. Similarly, net negative charge can be increased
by replacing positively and uncharged side chains with negatively
charged side chains at the pH of observation and gain a formal in
crease of negative charge by -1 and -2, respectively.
[0027] A low detergent concentration system includes detergents
where less than about 800 ppm of detergent components are present
in the wash water. Japanese detergents are typically considered low
detergent concentration system as they have approximately 667 ppm
of detergent components present in the wash water.
[0028] A medium detergent concentration includes detergents where
between about 800 ppm and about 2000 ppm of detergent components
are present in the wash water. North American detergents are
generally considered to be medium detergent concentration systems
as they have approximately 975 ppm of detergent components present
in the wash water. Brazil typically has approximately 1500 ppm of
detergent components present in the wash water.
[0029] A high detergent concentration system includes detergents
where greater than about 2000 ppm of detergent components are
present in the wash water. European detergents are generally
considered to be high detergent concentration systems as they have
approximately 4500-5000 ppm of detergent components in the wash
water.
[0030] Latin American detergents are generally high suds phosphate
builder detergents and the range of detergents used in Latin
America can fall in both the medium and high detergent
concentrations as they range from 1500 ppm to 6000 ppm of detergent
components in the wash water. As mentioned above, Brazil typically
has approximately 1500 ppm of detergent components present in the
wash water. However, other high suds phosphate builder detergent
geographies, not limited to other Latin American countries, may
have high detergent concentration systems up to about 6000 ppm of
detergent components present in the wash water.
[0031] In light of the foregoing, it is evident that concentrations
of detergent compositions in typical wash solutions throughout the
world varies from less than about 800 ppm of detergent composition
("low detergent concentration geographies"), for example about 667
ppm in Japan, to between about 800 ppm to about 2000 ppm ("medium
detergent concentration geographies"), for example about 975 ppm in
U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm
("high detergent concentration geographies"), for example about
4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high
suds phosphate builder geographies.
[0032] The concentrations of the typical wash solutions are
determined empirically. For example, in the U.S., a typical washing
machine holds a volume of about 64.4 L of wash solution.
Accordingly, in order to obtain a concentration of about 975 ppm of
detergent within the wash solution about 62.79 g of detergent
composition must be added to the 64.4 L of wash solution. This
amount is the typical amount measured into the wash water by the
consumer using the measuring cup provided with the detergent.
[0033] Proteases are carbonyl hydrolases which generally act to
cleave peptide bonds of proteins or peptides. As used herein,
"protease" means a naturally-occurring protease or a recombinant
protease. Naturally-occurring proteases include
.alpha.-aminoacylpeptide hydrolase, peptidylamino acid hydrolase,
acylamino hydrolase, serine carboxypeptidase,
metallocarboxypeptidase, thiol proteinase, carboxylproteinase and
metalloproteinase. Serine, metallo, thiol and acid proteases are
included, as well as endo and exo-proteases.
[0034] The present invention includes protease enzymes which are
non-naturally occurring carbonyl hydrolase variants (protease
variants) having a different proteolytic activity, stability,
substrate specificity, pH profile and/or performance characteristic
as compared to the precursor carbonyl hydrolase from which the
amino acid sequence of the variant is derived. Specifically, such
protease variants have an amino acid sequence not found in nature,
which is derived by substitution of a plurality of amino acid
residues of a precursor protease with different amino acids. The
precursor protease may be a naturally-occurring protease or
recombinant protease.
[0035] The protease variants useful herein encompass the
substitution of any of the nineteen naturally occurring L-amino
acids at the designated amino acid residue positions. Such
substitutions can be made in any precursor subtilisin (procaryotic,
eucaryotic, mammalian, etc.). Throughout this application reference
is made to various amino acids by way of common one- and
three-letter codes. Such codes are identified in Dale, M. W.
(1989), Molecular Genetics of Bacteria, John Wiley & Sons,
Ltd., Appendix B.
[0036] The protease variants useful herein are preferably derived
from a Bacillus subtilisin. More preferably, the protease variants
are derived from Bacillus lentus subtilisin and/or subtilisin
309.
[0037] Subtilisins are bacterial or fungal proteases which
generally act to cleave peptide bonds of proteins or peptides. As
used herein, "subtilisin" means a naturally-occurring subtilisin or
a recombinant subtilisin. A series of naturally-occurring
subtilisins is known to be produced and often secreted by various
microbial species. Amino acid sequences of the members of this
series are not entirely homologous. However, the subtilisins in
this series exhibit the same or similar type of proteolytic
activity. This class of serine proteases shares a common amino acid
sequence defining a catalytic triad which distinguishes them from
the chymotrypsin related class of serine proteases. The subtilisins
and chymotrypsin related serine proteases both have a catalytic
triad comprising aspartate, histidine and serine. In the subtilisin
related proteases the relative order of these amino acids, reading
from the amino to carboxy terminus, is aspartate-histidine-serine.
In the chymotrypsin related proteases, the relative order, however,
is histidine-aspartate-serine. Thus, subtilisin herein refers to a
serine protease having the catalytic triad of subtilisin related
proteases. Examples include but are not limited to the subtilisins
identified in FIG. 3 herein. Generally and for purposes of the
present invention, numbering of the amino acids in proteases
corresponds to the numbers assigned to the mature Bacillus
amyloliquefaciens subtilisin sequence presented in FIG. 1.
[0038] "Recombinant subtilisin" or "recombinant protease" refer to
a subtilisin or protease in which the DNA sequence encoding the
subtilisin or protease is modified to produce a variant (or mutant)
DNA sequence which encodes the substitution, deletion or insertion
of one or more amino acids in the naturally-occurring amino acid
sequence. Suitable methods to produce such modification, and which
may be combined with those disclosed herein, include those
disclosed in U.S. Pat. RE 34,606, U.S. Pat. No. 5,204,015 and U.S.
Pat. No. 5,185,258, U.S. Pat. No. 5,700,676, U.S. Pat. No.
5,801,038, and U.S. Pat. No. 5,763,257.
[0039] "Non-human subtilisins" and the DNA encoding them may be
obtained from many procaryotic and eucaryotic organisms. Suitable
examples of procaryotic organisms include gram negative organisms
such as E. coli or Pseudomonas and gram positive bacteria such as
Micrococcus or Bacillus. Examples of eucaryotic organisms from
which subtilisin and their genes may be obtained include yeast such
as Saccharomyces cerevisiae, fungi such as Aspergillus sp.
[0040] A "protease variant" has an amino acid sequence which is
derived from the amino acid sequence of a "precursor protease". The
precursor proteases include naturally-occurring proteases and
recombinant proteases. The amino acid sequence of the protease
variant is "derived" from the precursor protease amino acid
sequence by the substitution, deletion or insertion of one or more
amino acids of the precursor amino acid sequence. Such modification
is of the "precursor DNA sequence" which encodes the amino acid
sequence of the precursor protease rather than manipulation of the
precursor protease enzyme per se. Suitable methods for such
manipulation of the precursor DNA sequence include methods
disclosed herein, as well as methods known to those skilled in the
art (see, for example, EP 0 328299, WO89/06279 and the US patents
and applications already referenced herein).
[0041] These amino acid position numbers refer to those assigned to
the mature Bacillus amyloliquefaciens subtilisin sequence presented
in FIG. 1. The invention, however, is not limited to the mutation
of this particular subtilisin but extends to precursor proteases
containing amino acid residues at positions which are "equivalent"
to the particular identified residues in Bacillus amyloliquefaciens
subtilisin. In a preferred embodiment of the present invention, the
precursor protease is Bacillus lentus subtilisin and the
substitutions are made at the equivalent amino acid residue
positions in B. lentus corresponding to those listed above.
[0042] A residue (amino acid) position of a precursor protease is
equivalent to a residue of Bacillus amyloliquefaciens subtilisin if
it is either homologous (i.e., corresponding in position in either
primary or tertiary structure) or analogous to a specific residue
or portion of that residue in Bacillus amyloliquefaciens subtilisin
(i.e., having the same or similar functional capacity to combine,
react, or interact chemically).
[0043] In order to establish homology to primary structure, the
amino acid sequence of a precursor protease is directly compared to
the Bacillus amyloliquefaciens subtilisin primary sequence and
particularly to a set of residues known to be invariant in
subtilisins for which sequence is known. For example, FIG. 2 herein
shows the conserved residues as between B. amyloliquefaciens
subtilisin and B. lentus subtilisin. After aligning the conserved
residues, allowing for necessary insertions and deletions in order
to maintain alignment (i.e., avoiding the elimination of conserved
residues through arbitrary deletion and insertion), the residues
equivalent to particular amino acids in the primary sequence of
Bacillus amyloliquefaciens subtilisin are defined. Alignment of
conserved residues preferably should conserve 100% of such
residues. However, alignment of greater than 75% or as little as
50% of conserved residues is also adequate to define equivalent
residues. Conservation of the catalytic triad, Asp32/His64/Ser221
should be maintained. Siezen et al. (1991) Protein Eng.
4(7):719-737 shows the alignment of a large number of serine
proteases. Siezen et al. refer to the grouping as subtilases or
subtilisin-like serine proteases.
[0044] For example, in FIG. 3, the amino acid sequence of
subtilisin from Bacillus amyloliquefaciens, Bacillus subtilis,
Bacillus licheniformis (carlsbergensis) and Bacillus lentus are
aligned to provide the maximum amount of homology between amino
acid sequences. A comparison of these sequences shows that there
are a number of conserved residues contained in each sequence.
These conserved residues (as between BPN' and B. lentus) are
identified in FIG. 2.
[0045] These conserved residues, thus, may be used to define the
corresponding equivalent amino acid residues of Bacillus
amyloliquefaciens subtilisin in other subtilisins such as
subtilisin from Bacillus lentus (PCT Publication No. WO89/06279
published Jul. 13, 1989), the preferred protease precursor enzyme
herein, or the subtilisin referred to as PB92 (EP 0 328 299), which
is highly homologous to the preferred Bacillus lentus subtilisin.
The amino acid sequences of certain of these subtilisins are
aligned in FIGS. 3A and 3B with the sequence of Bacillus
amyloliquefaciens subtilisin to produce the maximum homology of
conserved residues. As can be seen, there are a number of deletions
in the sequence of Bacillus lentus as compared to Bacillus
amyloliquefaciens subtilisin. Thus, for example, the equivalent
amino acid for Val165 in Bacillus amyloliquefaciens subtilisin in
the other subtilisins is isoleucine for B. lentus and B.
licheniformis.
[0046] "Equivalent residues" may also be defined by determining
homology at the level of tertiary structure for a precursor
protease whose tertiary structure has been determined by x-ray
crystallography. Equivalent residues are defined as those for which
the atomic coordinates of two or more of the main chain atoms of a
particular amino acid residue of the precursor protease and
Bacillus amyloliquefaciens subtilisin (N on N, CA on CA, C on C and
O on O) are within 0.13 nm and preferably 0.1 nm after alignment.
Alignment is achieved after the best model has been oriented and
positioned to give the maximum overlap of atomic coordinates of
non-hydrogen protein atoms of the protease in question to the
Bacillus amyloliquefaciens subtilisin. The best model is the
crystallographic model giving the lowest R factor for experimental
diffraction data at the highest resolution available.
R factor = h Fo ( h ) - Fc ( h ) h Fo ( h ) ##EQU00001##
[0047] Equivalent residues which are functionally analogous to a
specific residue of Bacillus amyloliquefaciens subtilisin are
defined as those amino acids of the precursor protease which may
adopt a conformation such that they either alter, modify or
contribute to protein structure, substrate binding or catalysis in
a manner defined and attributed to a specific residue of the
Bacillus amyloliquefaciens subtilisin. Further, they are those
residues of the precursor protease (for which a tertiary structure
has been obtained by x-ray crystallography) which occupy an
analogous position to the extent that, although the main chain
atoms of the given residue may not satisfy the criteria of
equivalence on the basis of occupying a homologous position, the
atomic coordinates of at least two of the side chain atoms of the
residue lie with 0.13 nm of the corresponding side chain atoms of
Bacillus amyloliquefaciens subtilisin. The coordinates of the three
dimensional structure of Bacillus amyloliquefaciens subtilisin are
set forth in EPO Publication No. 0 251 446 (equivalent to U.S. Pat.
No. 5,182,204, the disclosure of which is incorporated herein by
reference) and can be used as outlined above to determine
equivalent residues on the level of tertiary structure.
[0048] Some of the residues identified for substitution are
conserved residues whereas others are not. In the case of residues
which are not conserved, the substitution of one or more amino
acids is limited to substitutions which produce a variant which has
an amino acid sequence that does not correspond to one found in
nature. In the case of conserved residues, such substitutions
should not result in a naturally-occurring sequence. The protease
variants of the present invention include the mature forms of
protease variants, as well as the pro- and prepro-forms of such
protease variants. The prepro-forms are the preferred construction
since this facilitates the expression, secretion and maturation of
the protease variants.
[0049] "Prosequence" refers to a sequence of amino acids bound to
the N-terminal portion of the mature form of a protease which when
removed results in the appearance of the "mature" form of the
protease. Many proteolytic enzymes are found in nature as
translational proenzyme products and, in the absence of
post-translational processing, are expressed in this fashion. A
preferred prosequence for producing protease variants is the
putative prosequence of Bacillus amyloliquefaciens subtilisin,
although other protease prosequences may be used.
[0050] A "signal sequence" or "presequence" refers to any sequence
of amino acids bound to the N-terminal portion of a protease or to
the N-terminal portion of a proprotease which may participate in
the secretion of the mature or pro forms of the protease. This
definition of signal sequence is a functional one, meant to include
all those amino acid sequences encoded by the N-terminal portion of
the protease gene which participate in the effectuation of the
secretion of protease under native conditions. The present
invention utilizes such sequences to effect the secretion of the
protease variants as defined herein. One possible signal sequence
comprises the first seven amino acid residues of the signal
sequence from Bacillus subtilis subtilisin fused to the remainder
of the signal sequence of the subtilisin from Bacillus lentus (ATCC
21536).
[0051] A "prepro" form of a protease variant consists of the mature
form of the protease having a prosequence operably linked to the
amino terminus of the protease and a "pre" or "signal" sequence
operably linked to the amino terminus of the prosequence.
[0052] "Expression vector" refers to a DNA construct containing a
DNA sequence which is operably linked to a suitable control
sequence capable of effecting the expression of said DNA in a
suitable host. Such control sequences include a promoter to effect
transcription, an optional operator sequence to control such
transcription, a sequence encoding suitable mRNA ribosome binding
sites and sequences which control termination of transcription and
translation. The vector may be a plasmid, a phage particle, or
simply a potential genomic insert. Once transformed into a suitable
host, the vector may replicate and function independently of the
host genome, or may, in some instances, integrate into the genome
itself. In the present specification, "plasmid" and "vector" are
sometimes used interchangeably as the plasmid is the most commonly
used form of vector at present. However, the invention is intended
to include such other forms of expression vectors which serve
equivalent functions and which are, or become, known in the
art.
[0053] The "host cells" used in the present invention generally are
procaryotic or eucaryotic hosts which preferably have been
manipulated by the methods disclosed in U.S. Pat. RE 34,606 to
render them incapable of secreting enzymatically active
endoprotease. A preferred host cell for expressing protease is the
Bacillus strain BG2036 which is deficient in enzymatically active
neutral protease and alkaline protease (subtilisin). The
construction of strain BG2036 is described in detail in U.S. Pat.
No. 5,264,366. Other host cells for expressing protease include
Bacillus subtilis 1168 (also described in U.S. Pat. RE 34,606 and
U.S. Pat. No. 5,264,366, the disclosure of which are incorporated
herein by reference), as well as any suitable Bacillus strain such
as B. licheniformis, B. lentus, etc.
[0054] Host cells are transformed or transfected with vectors
constructed using recombinant DNA techniques. Such transformed host
cells are capable of either replicating vectors encoding the
protease variants or expressing the desired protease variant. In
the case of vectors which encode the pre- or prepro-form of the
protease variant, such variants, when expressed, are typically
secreted from the host cell into the host cell medium.
[0055] "Operably linked," when describing the relationship between
two DNA regions, simply means that they are functionally related to
each other. For example, a presequence is operably linked to a
peptide if it functions as a signal sequence, participating in the
secretion of the mature form of the protein most probably involving
cleavage of the signal sequence. A promoter is operably linked to a
coding sequence if it controls the transcription of the sequence; a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to permit translation.
[0056] The genes encoding the naturally-occurring precursor
protease may be obtained in accord with the general methods known
to those skilled in the art. The methods generally comprise
synthesizing labeled probes having putative sequences encoding
regions of the protease of interest, preparing genomic libraries
from organisms expressing the protease, and screening the libraries
for the gene of interest by hybridization to the probes. Positively
hybridizing clones are then mapped and sequenced.
[0057] The cloned protease is then used to transform a host cell in
order to express the protease. The protease gene is then ligated
into a high copy number plasmid. This plasmid replicates in hosts
in the sense that it contains the well-known elements necessary for
plasmid replication: a promoter operably linked to the gene in
question (which may be supplied as the gene's own homologous
promoter if it is recognized, i.e., transcribed, by the host), a
transcription termination and polyadenylation region (necessary for
stability of the mRNA transcribed by the host from the protease
gene in certain eucaryotic host cells) which is exogenous or is
supplied by the endogenous terminator region of the protease gene
and, desirably, a selection gene such as an antibiotic resistance
gene that enables continuous cultural maintenance of
plasmid-infected host cells by growth in antibiotic-containing
media. High copy number plasmids also contain an origin of
replication for the host, thereby enabling large numbers of
plasmids to be generated in the cytoplasm without chromosomal
limitations. However, it is within the scope herein to integrate
multiple copies of the protease gene into host genome. This is
facilitated by procaryotic and eucaryotic organisms which are
particularly susceptible to homologous recombination.
[0058] The gene can be a natural B. lentus gene. Alternatively, a
synthetic gene encoding a naturally-occurring or mutant precursor
protease may be produced. In such an approach, the DNA and/or amino
acid sequence of the precursor protease is determined. Multiple,
overlapping synthetic single-stranded DNA fragments are thereafter
synthesized, which upon hybridization and ligation produce a
synthetic DNA encoding the precursor protease. An example of
synthetic gene construction is set forth in Example 3 of U.S. Pat.
No. 5,204,015, the disclosure of which is incorporated herein by
reference.
[0059] Once the naturally-occurring or synthetic precursor protease
gene has been cloned, a number of modifications are undertaken to
enhance the use of the gene beyond synthesis of the
naturally-occurring precursor protease. Such modifications include
the production of recombinant proteases as disclosed in U.S. Pat.
RE 34,606 and EPO Publication No. 0 251 446 and the production of
protease variants described herein.
[0060] The following cassette mutagenesis method may be used to
facilitate the construction of the protease variants of the present
invention, although other methods may be used. First, the
naturally-occurring gene encoding the protease is obtained and
sequenced in whole or in part. Then the sequence is scanned for a
point at which it is desired to make a mutation (deletion,
insertion or substitution) of one or more amino acids in the
encoded enzyme. The sequences flanking this point are evaluated for
the presence of restriction sites for replacing a short segment of
the gene with an oligonucleotide pool which when expressed will
encode various mutants. Such restriction sites are preferably
unique sites within the protease gene so as to facilitate the
replacement of the gene segment. However, any convenient
restriction site which is not overly redundant in the protease gene
may be used, provided the gene fragments generated by restriction
digestion can be reassembled in proper sequence. If restriction
sites are not present at locations within a convenient distance
from the selected point (from 10 to 15 nucleotides), such sites are
generated by substituting nucleotides in the gene in such a fashion
that neither the reading frame nor the amino acids encoded are
changed in the final construction. Mutation of the gene in order to
change its sequence to conform to the desired sequence is
accomplished by M13 primer extension in accord with generally known
methods. The task of locating suitable flanking regions and
evaluating the needed changes to arrive at two convenient
restriction site sequences is made routine by the redundancy of the
genetic code, a restriction enzyme map of the gene and the large
number of different restriction enzymes. Note that if a convenient
flanking restriction site is available, the above method need be
used only in connection with the flanking region which does not
contain a site.
[0061] Once the naturally-occurring DNA or synthetic DNA is cloned,
the restriction sites flanking the positions to be mutated are
digested with the cognate restriction enzymes and a plurality of
end termini-complementary oligonucleotide cassettes are ligated
into the gene. The mutagenesis is simplified by this method because
all of the oligonucleotides can be synthesized so as to have the
same restriction sites, and no synthetic linkers are necessary to
create the restriction sites.
[0062] As used herein, proteolytic activity is defined as the rate
of hydrolysis of peptide bonds per milligram of active enzyme. Many
well known procedures exist for measuring proteolytic activity (K.
M. Kalisz, "Microbial Proteinases," Advances in Biochemical
Engineering/Biotechnology, A. Fiechter ed., 1988). In addition to
or as an alternative to modified proteolytic activity, the variant
enzymes of the present invention may have other modified properties
such as K.sub.m, k.sub.cat, k.sub.cat/K.sub.m ratio and/or modified
substrate specificity and/or modified pH activity profile. These
enzymes can be tailored for the particular substrate which is
anticipated to be present, for example, in the preparation of
peptides or for hydrolytic processes such as laundry uses.
[0063] In one aspect of the invention, the objective is to secure a
variant protease having altered proteolytic activity as compared to
the precursor protease, since increasing such activity (numerically
larger) enables the use of the enzyme to more efficiently act on a
target substrate. Also of interest are variant enzymes having
altered thermal stability and/or altered substrate specificity as
compared to the precursor. In some instances, lower proteolytic
activity may be desirable, for example a decrease in proteolytic
activity would be useful where the synthetic activity of the
proteases is desired (as for synthesizing peptides). One may wish
to decrease this proteolytic activity, which is capable of
destroying the product of such synthesis. Conversely, in some
instances it may be desirable to increase the proteolytic activity
of the variant enzyme versus its precursor. Additionally, increases
or decreases (alteration) of the stability of the variant, whether
alkaline or thermal stability, may be desirable. Increases or
decreases in k.sub.cat, K.sub.m or k.sub.cat/K.sub.m are specific
to the substrate used to determine these kinetic parameters.
[0064] In another aspect of the invention, it has been found that
protease variants containing substitutions of the amino acids at
one or more residue positions so that the substitution alters the
charge at that position to make the charge more negative or less
positive compared to a precursor protease are more effective in a
low detergent concentration than a precursor protease.
[0065] In a further aspect of the invention, it has been found that
protease variants containing substitutions of the amino acids at
one or more residue positions so that the substitution alters the
charge at that position to make the charge more positive or less
negative compared to a precursor protease are more effective in a
high detergent concentration than a precursor protease.
[0066] Further, we have found that many of the protease variants
useful in the low detergent concentration system and/or the high
detergent concentration system also are effective in a medium
detergent concentration system.
[0067] These substitutions are preferably made in Bacillus lentus
(recombinant or native-type) subtilisin, although the substitutions
may be made in any Bacillus protease, preferably Bacillus
subtilisins.
[0068] Based on the screening results obtained with the variant
proteases, the noted mutations in Bacillus amyloliquefaciens
subtilisin are important to the proteolytic activity, performance
and/or stability of these enzymes and the cleaning or wash
performance of such variant enzymes.
[0069] Many of the protease variants of the invention are useful in
formulating various detergent compositions or personal care
formulations such as shampoos or lotions. A number of known
compounds are suitable surfactants useful in compositions
comprising the protease mutants of the invention. These include
nonionic, anionic, cationic or zwitterionic detergents, as
disclosed in U.S. Pat. No. 4,404,128 to Barry J. Anderson and U.S.
Pat. No. 4,261,868 to Jiri Flora, et al. A suitable detergent
formulation is that described in Example 7 of U.S. Pat. No.
5,204,015 (previously incorporated by reference). The art is
familiar with the different formulations which can be used as
cleaning compositions. In addition to typical cleaning
compositions, it is readily understood that the protease variants
of the present invention may be used for any purpose that native or
wild-type proteases are used. Thus, these variants can be used, for
example, in bar or liquid soap applications, dishcare formulations,
contact lens cleaning solutions or products, peptide hydrolysis,
waste treatment, textile applications, as fusion-cleavage enzymes
in protein production, etc. The variants of the present invention
may comprise enhanced performance in a detergent composition (as
compared to the precursor). As used herein, enhanced performance in
a detergent is defined as increasing cleaning of certain enzyme
sensitive stains such as grass or blood, as determined by usual
evaluation after a standard wash cycle.
[0070] Proteases of the invention can be formulated into known
powdered and liquid detergents having pH between 6.5 and 12.0 at
levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by
weight. These detergent cleaning compositions can also include
other enzymes such as known proteases, amylases, cellulases,
lipases or endoglycosidases, as well as builders and
stabilizers.
[0071] The addition of proteases of the invention to conventional
cleaning compositions does not create any special use limitation.
In other words, any temperature and pH suitable for the detergent
is also suitable for the present compositions as long as the pH is
within the above range, and the temperature is below the described
protease's denaturing temperature. In addition, proteases of the
invention can be used in a cleaning composition without detergents,
again either alone or in combination with builders and
stabilizers.
[0072] The present invention also relates to cleaning compositions
containing the protease variants of the invention. The cleaning
compositions may additionally contain additives which are commonly
used in cleaning compositions. These can be selected from, but not
limited to, bleaches, surfactants, builders, enzymes and bleach
catalysts. It would be readily apparent to one of ordinary skill in
the art what additives are suitable for inclusion into the
compositions. The list provided herein is by no means exhaustive
and should be only taken as examples of suitable additives. It will
also be readily apparent to one of ordinary skill in the art to
only use those additives which are compatible with the enzymes and
other components in the composition, for example, surfactant.
[0073] When present, the amount of additive present in the cleaning
composition is from about 0.01% to about 99.9%, preferably about 1%
to about 95%, more preferably about 1% to about 80%.
[0074] The variant proteases of the present invention can be
included in animal feed such as part of animal feed additives as
described in, for example, U.S. Pat. No. 5,612,055; U.S. Pat. No.
5,314,692; and U.S. Pat. No. 5,147,642.
[0075] One aspect of the invention is a composition for the
treatment of a textile that includes variant proteases of the
present invention. The composition can be used to treat for example
silk or wool as described in publications such as RD 216,034; EP
134,267; U.S. Pat. No. 4,533,359; and EP 344,259.
[0076] The following is presented by way of example and is not to
be construed as a limitation to the scope of the claims.
[0077] All publications and patents referenced herein are hereby
incorporated by reference in their entirety.
EXAMPLE 1
[0078] A large number of protease variants were produced and
purified using methods well known in the art. All mutations were
made in Bacillus lentus GG36 subtilisin.
[0079] The protease variants produced were tested for performance
in two types of detergent and wash conditions using a microswatch
assay described in "An improved method of assaying for a preferred
enzyme and/or preferred detergent composition", U.S. Ser. No.
60/068,796.
[0080] Tables 1-13 list the variant proteases assayed and the
results of testing in two different detergents. All values are
given as comparison to the first protease shown in the table (i.e.,
a value of 1.32 indicates an ability to release 132% of the stain
as opposed to the 100% of the first variant in the table).
[0081] Column A shows the charge difference of a variant. For
column B, the detergent was 0.67 g/l filtered Ariel Ultra (Procter
& Gamble, Cincinnati, Ohio, USA), in a solution containing 3
grains per gallon mixed Ca.sup.2+/Mg.sup.2+ hardness, and 0.3 ppm
enzyme was used in each well at 25.degree. C. (low concentration
detergent system). For column C, the detergent was 3.38 g/l
filtered Ariel Futur (Procter & Gamble, Cincinnati, Ohio, USA),
in a solution containing 15 grains per gallon mixed
Ca.sup.2+/Mg.sup.2+ hardness, and 0.3 ppm enzyme was used in each
well at 40.degree. C. (high concentration detergent system).
TABLE-US-00001 TABLE 1 A B C N76D S103A V104I Q109R 1.00 1.00 N76D
S103A V104I Q109R Q245R +1 0.48 1.41
TABLE-US-00002 TABLE 2 A B C V68A N76D S103A V104I G159D Q236H
Q245R 1.00 1.00 V68A N76D S103A V104I G159D N204D Q236H Q245R -1
1.11 0.03
TABLE-US-00003 TABLE 3 A B C V68A N76D S103A V104I 1.00 1.00 T22K
V68A N76D S103A V104I +1 0.74 1.85
TABLE-US-00004 TABLE 4 A B C N76D S103A V104I M222S 1.00 1.00 N76D
S103A V104I N173R M222S 0 0.66 1.84 Q12R N76D S103A V104I M222S
Q245R +1 0.41 5.84
TABLE-US-00005 TABLE 5 A B C Q12R N76D S103A I104T S130T M222S
Q245R 1.00 1.00 Q12R N76D S103A I104T S130T M222S Q245R N261D -1
1.79 0.81 Q12R N76D S103A I104T S130T R170S N185D M222S N243D Q245R
-3 2.87 0.02
TABLE-US-00006 TABLE 6 A B C V68A N76D S103A V104I G159D Q236H 1.00
1.00 V68A N76D S103A V104I G159D Q236H Q245R +1 0.94 6.80 V68A
S103A V104I G159D A232V Q236H Q245R N252K +3 0.44 20.60
TABLE-US-00007 TABLE 7 A B C V68A N76D S103A V104I G159D A232V
Q236H Q245R 1.00 1.00 V68A N76D S103A V104I G159D P210R A232V Q236H
Q245R +1 0.44 2.66
TABLE-US-00008 TABLE 8 A B C V68A S103A V104I G159D A232V Q236H
Q245R N252K 1.00 1.00 V68A S103A V104I G159D A232V Q236H Q245R
N248D N252K -1 1.96 0.65
TABLE-US-00009 TABLE 9 A B C V68A S103A V104I G159D A232V Q236H
Q245R 1.00 1.00 V68A S103A V104I G159D A232V Q236H K237E Q245R -2
1.27 0.12
TABLE-US-00010 TABLE 10 A B C V68A S103A V104I G159D A232V Q236H
Q245R L257V 1.00 1.00 V68A N76D S103A V104I G159D A232V Q236H Q245R
L257V -1 1.56 0.48
TABLE-US-00011 TABLE 11 A B C S103A V104I G159D A232V Q236H Q245R
N248D N252K 1.00 1.00 S103A V104I G159D L217E A232V Q236H Q245R
N248D N252K -1 1.90 0.15
TABLE-US-00012 TABLE 12 A B C S103A V104I S101G G159D A232V Q236H
Q245R N248D N252K 1.00 1.00 N76D S103A V104I S101G G159D A232V
Q236H Q245R N248D N252K -1 1.28 0.39
TABLE-US-00013 TABLE 13 A B C N62D S103A V104I G159D T213R A232V
Q236H Q245R N248D N252K 1.00 1.00 N62D S103A V104I Q109R G159D
T213R A232V Q236H Q245R N248D N252K +1 0.40 1.74
EXAMPLE 2
[0082] The following protease variants were made and tested as
noted in Example 1.
[0083] The variants in Table 14 are protease variants which have
both types of substitutions: those which alter the charge at a
position to make the charge more negative or less positive and
those which alter the charge at a position to make the charge more
positive or less negative compared to B. lentus GG36 as well as
neutral substitutions that do not affect the charge at a given
residue position. This produces protease variants that perform
better than a standard in both low detergent concentration systems
(column A; 0.67 g/l filtered Ariel Ultra (Procter & Gamble,
Cincinnati, Ohio, USA), in a solution containing 3 grains per
gallon mixed Ca.sup.2+/Mg.sup.2+ hardness, and 0.3 ppm enzyme was
used in each well at 25.degree. C.) and high detergent
concentration systems (column B; 3.38 g/l filtered Ariel Futur
(Procter & Gamble, Cincinnati, Ohio, USA), in a solution
containing 15 grains per gallon mixed Ca.sup.2+/Mg.sup.2+ hardness,
and 0.3 ppm enzyme was used in each well at 40.degree. C.).
TABLE-US-00014 TABLE 14 A B N76D S103A V104I 1.00 1.00 V68A S103A
V104I G159D A232V Q236H Q245R N252K 1.41 1.85 V68A N76D S103A V104I
G159D T213R A232V Q236H Q245R T260A 1.30 1.73 V68A S103A V104I
G159D A232V Q236H Q245R N248D N252K 2.77 1.20 V68A S103A V104I
N140D G159D A232V Q236H Q245R N252K 2.96 1.42 N43K V68A S103A V104I
G159D A232V Q236H Q245R 2.05 1.78 N43D V68A S103A V104I G159D A232V
Q236H Q245R N252K 2.00 1.34 V68A N76D S103A V104I G159D A215R A232V
Q236H Q245R 1.67 1.45 Q12R V68A N76D S103A V104I G159D A232V Q236H
Q245R 2.16 1.72 N76D S103A V104I V147I G159D A232V Q236H Q245R
N248S K251R 1.35 1.29 V68A N76D S103A V104I G159D A232V Q236H Q245R
S256R 2.01 1.72 V68A N76D S103A V104I G159D Q206R A232V Q236H Q245R
2.09 1.62 S103A V104I G159D A232V Q236H Q245R N248D N252K 1.44 1.41
G20R V68A S103A V104I G159D A232V Q236H Q245R N248D N252K 1.81 1.72
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K L257R 1.51
1.41 V68A S103A V104I A232V Q236H Q245R N248D N252K 1.04 1.50 N76D
S103A V104I G159D A232V Q236H Q245R L257V 1.92 1.09
Sequence CWU 1
1
611497DNAB. amyloliquefaciensCDS(96)...(1245) 1ggtctactaa
aatattattc catactatac aattaataca cagaataatc tgtctattgg 60ttattctgca
aatgaaaaaa aggagaggat aaaga gtg aga ggc aaa aaa gta 113 Met Arg Gly
Lys Lys Val 1 5tgg atc agt ttg ctg ttt gct tta gcg tta atc ttt acg
atg gcg ttc 161Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu Ile Phe Thr
Met Ala Phe 10 15 20ggc agc aca tcc tct gcc cag gcg gca ggg aaa tca
aac ggg gaa aag 209Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly Lys Ser
Asn Gly Glu Lys 25 30 35aaa tat att gtc ggg ttt aaa cag aca atg agc
acg atg agc gcc gct 257Lys Tyr Ile Val Gly Phe Lys Gln Thr Met Ser
Thr Met Ser Ala Ala 40 45 50aag aag aaa gat gtc att tct gaa aaa ggc
ggg aaa gtg caa aag caa 305Lys Lys Lys Asp Val Ile Ser Glu Lys Gly
Gly Lys Val Gln Lys Gln 55 60 65 70ttc aaa tat gta gac gca gct tca
gtc aca tta aac gaa aaa gct gta 353Phe Lys Tyr Val Asp Ala Ala Ser
Val Thr Leu Asn Glu Lys Ala Val 75 80 85aaa gaa ttg aaa aaa gac ccg
agc gtc gct tac gtt gaa gaa gat cac 401Lys Glu Leu Lys Lys Asp Pro
Ser Val Ala Tyr Val Glu Glu Asp His 90 95 100gta gca cat gcg tac
gcg cag tcc gtg cct tac ggc gta tca caa att 449Val Ala His Ala Tyr
Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile 105 110 115aaa gcc cct
gct ctg cac tct caa ggc tac act gga tca aat gtt aaa 497Lys Ala Pro
Ala Leu His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys 120 125 130gta
gcg gtt atc gac agc ggt atc gat tct tct cat cct gat tta aag 545Val
Ala Val Ile Asp Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys135 140
145 150gta gca agc gga gcc agc atg gtt cct tct gaa aca aat cct ttc
caa 593Val Ala Ser Gly Ala Ser Met Val Pro Ser Glu Thr Asn Pro Phe
Gln 155 160 165gac aac aac tct cac gga act cac gtt gcc ggc aca gtt
gcg gct ctt 641Asp Asn Asn Ser His Gly Thr His Val Ala Gly Thr Val
Ala Ala Leu 170 175 180aat aac tca atc ggt gta tta ggc gtt gcg cca
agc gca tca ctt tac 689Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pro
Ser Ala Ser Leu Tyr 185 190 195gct gta aaa gtt ctc ggt gct gac ggt
tcc ggc caa tac agc tgg atc 737Ala Val Lys Val Leu Gly Ala Asp Gly
Ser Gly Gln Tyr Ser Trp Ile 200 205 210att aac gga atc gag tgg gcg
atc gca aac aat atg gac gtt att aac 785Ile Asn Gly Ile Glu Trp Ala
Ile Ala Asn Asn Met Asp Val Ile Asn215 220 225 230atg agc ctc ggc
gga cct tct ggt tct gct gct tta aaa gcg gca gtt 833Met Ser Leu Gly
Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val 235 240 245gat aaa
gcc gtt gca tcc ggc gtc gta gtc gtt gcg gca gcc ggt aac 881Asp Lys
Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala Gly Asn 250 255
260gaa ggc act tcc ggc agc tca agc aca gtg ggc tac cct ggt aaa tac
929Glu Gly Thr Ser Gly Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr
265 270 275cct tct gtc att gca gta ggc gct gtt gac agc agc aac caa
aga gca 977Pro Ser Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln
Arg Ala 280 285 290tct ttc tca agc gta gga cct gag ctt gat gtc atg
gca cct ggc gta 1025Ser Phe Ser Ser Val Gly Pro Glu Leu Asp Val Met
Ala Pro Gly Val295 300 305 310tct atc caa agc acg ctt cct gga aac
aaa tac ggg gcg tac aac ggt 1073Ser Ile Gln Ser Thr Leu Pro Gly Asn
Lys Tyr Gly Ala Tyr Asn Gly 315 320 325acg tca atg gca tct ccg cac
gtt gcc gga gcg gct gct ttg att ctt 1121Thr Ser Met Ala Ser Pro His
Val Ala Gly Ala Ala Ala Leu Ile Leu 330 335 340tct aag cac ccg aac
tgg aca aac act caa gtc cgc agc agt tta gaa 1169Ser Lys His Pro Asn
Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu 345 350 355aac acc act
aca aaa ctt ggt gat tct ttg tac tat gga aaa ggg ctg 1217Asn Thr Thr
Thr Lys Leu Gly Asp Ser Leu Tyr Tyr Gly Lys Gly Leu 360 365 370atc
aac gta caa gcg gca gct cag taa a acataaaaaa ccggccttgg 1265Ile Asn
Val Gln Ala Ala Ala Gln *375 380ccccgccggt tttttattat ttttcttcct
ccgcatgttc aatccgctcc ataatcgacg 1325gatggctccc tctgaaaatt
ttaacgagaa acggcgggtt gacccggctc agtcccgtaa 1385cggccaactc
ctgaaacgtc tcaatcgccg cttcccggtt tccggtcagc tcaatgccat
1445aacggtcggc ggcgttttcc tgataccggg agacggcatt cgtaatcgga tc
14972382PRTB. amyloliquefaciens 2Met Arg Gly Lys Lys Val Trp Ile
Ser Leu Leu Phe Ala Leu Ala Leu1 5 10 15Ile Phe Thr Met Ala Phe Gly
Ser Thr Ser Ser Ala Gln Ala Ala Gly 20 25 30Lys Ser Asn Gly Glu Lys
Lys Tyr Ile Val Gly Phe Lys Gln Thr Met 35 40 45Ser Thr Met Ser Ala
Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly 50 55 60Gly Lys Val Gln
Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Val Thr65 70 75 80Leu Asn
Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala 85 90 95Tyr
Val Glu Glu Asp His Val Ala His Ala Tyr Ala Gln Ser Val Pro 100 105
110Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr
115 120 125Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile
Asp Ser 130 135 140Ser His Pro Asp Leu Lys Val Ala Ser Gly Ala Ser
Met Val Pro Ser145 150 155 160Glu Thr Asn Pro Phe Gln Asp Asn Asn
Ser His Gly Thr His Val Ala 165 170 175Gly Thr Val Ala Ala Leu Asn
Asn Ser Ile Gly Val Leu Gly Val Ala 180 185 190Pro Ser Ala Ser Leu
Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser 195 200 205Gly Gln Tyr
Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn 210 215 220Asn
Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala225 230
235 240Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val
Val 245 250 255Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser
Ser Thr Val 260 265 270Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala
Val Gly Ala Val Asp 275 280 285Ser Ser Asn Gln Arg Ala Ser Phe Ser
Ser Val Gly Pro Glu Leu Asp 290 295 300Val Met Ala Pro Gly Val Ser
Ile Gln Ser Thr Leu Pro Gly Asn Lys305 310 315 320Tyr Gly Ala Tyr
Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly 325 330 335Ala Ala
Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln 340 345
350Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp Ser Leu
355 360 365Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln
370 375 3803275PRTB. amyloliquefaciens 3Ala Gln Ser Val Pro Tyr Gly
Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15His Ser Gln Gly Tyr
Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30Ser Gly Ile Asp
Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45Ser Met Val
Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60Gly Thr
His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly65 70 75
80Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu
85 90 95Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile
Glu 100 105 110Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser
Leu Gly Gly 115 120 125Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val
Asp Lys Ala Val Ala 130 135 140Ser Gly Val Val Val Val Ala Ala Ala
Gly Asn Glu Gly Thr Ser Gly145 150 155 160Ser Ser Ser Thr Val Gly
Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175Val Gly Ala Val
Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190Gly Pro
Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200
205Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser
210 215 220Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His
Pro Asn225 230 235 240Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu
Asn Thr Thr Thr Lys 245 250 255Leu Gly Asp Ser Phe Tyr Tyr Gly Lys
Gly Leu Ile Asn Val Gln Ala 260 265 270Ala Ala Gln 2754275PRTB.
subtilis 4Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro
Ala Leu 1 5 10 15His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val
Ala Val Ile Asp 20 25 30Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn
Val Arg Gly Gly Ala 35 40 45Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr
Gln Asp Gly Ser Ser His 50 55 60Gly Thr His Val Ala Gly Thr Ile Ala
Ala Leu Asn Asn Ser Ile Gly65 70 75 80Val Leu Gly Val Ser Pro Ser
Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95Asp Ser Thr Gly Ser Gly
Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110Trp Ala Ile Ser
Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125Pro Thr
Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135
140Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser
Gly145 150 155 160Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro
Ser Thr Ile Ala 165 170 175Val Gly Ala Val Asn Ser Ser Asn Gln Arg
Ala Ser Phe Ser Ser Ala 180 185 190Gly Ser Glu Leu Asp Val Met Ala
Pro Gly Val Ser Ile Gln Ser Thr 195 200 205Leu Pro Gly Gly Thr Tyr
Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220Pro His Val Ala
Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr225 230 235 240Trp
Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250
255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala
260 265 270Ala Ala Gln 2755274PRTB. licheniformis 5Ala Gln Thr Val
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val 1 5 10 15Gln Ala
Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp 20 25 30Thr
Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala 35 40
45Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly
50 55 60Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly
Val65 70 75 80Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys
Val Leu Asn 85 90 95Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser
Gly Ile Glu Trp 100 105 110Ala Thr Thr Asn Gly Met Asp Val Ile Asn
Met Ser Leu Gly Gly Ala 115 120 125Ser Gly Ser Thr Ala Met Lys Gln
Ala Val Asp Asn Ala Tyr Ala Arg 130 135 140Gly Val Val Val Val Ala
Ala Ala Gly Asn Ser Gly Asn Ser Gly Ser145 150 155 160Thr Asn Thr
Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175Gly
Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185
190Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr
195 200 205Pro Thr Asn Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala
Ser Pro 210 215 220His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys
His Pro Asn Leu225 230 235 240Ser Ala Ser Gln Val Arg Asn Arg Leu
Ser Ser Thr Ala Thr Tyr Leu 245 250 255Gly Ser Ser Phe Tyr Tyr Gly
Lys Gly Leu Ile Asn Val Glu Ala Ala 260 265 270Ala Gln6269PRTB.
lentus 6Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala
Ala 1 5 10 15His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala
Val Leu Asp 20 25 30Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg
Gly Gly Ala Ser 35 40 45Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly
Asn Gly His Gly Thr 50 55 60His Val Ala Gly Thr Ile Ala Ala Leu Asn
Asn Ser Ile Gly Val Leu65 70 75 80Gly Val Ala Pro Ser Ala Glu Leu
Tyr Ala Val Lys Val Leu Gly Ala 85 90 95Ser Gly Ser Gly Ser Val Ser
Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110Gly Asn Asn Gly Met
His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125Pro Ser Ala
Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140Val
Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser145 150
155 160Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp
Gln 165 170 175Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly
Leu Asp Ile 180 185 190Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr
Pro Gly Ser Thr Tyr 195 200 205Ala Ser Leu Asn Gly Thr Ser Met Ala
Thr Pro His Val Ala Gly Ala 210 215 220Ala Ala Leu Val Lys Gln Lys
Asn Pro Ser Trp Ser Asn Val Gln Ile225 230 235 240Arg Asn His Leu
Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255Tyr Gly
Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265
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