U.S. patent application number 11/482424 was filed with the patent office on 2007-07-12 for subtilase variants.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Henriette Draborg, Stefan Minning, Vibeke Skovgaard Nielsen.
Application Number | 20070161531 11/482424 |
Document ID | / |
Family ID | 38233421 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070161531 |
Kind Code |
A1 |
Draborg; Henriette ; et
al. |
July 12, 2007 |
Subtilase variants
Abstract
The present invention relates to novel subtilase variants
exhibiting improvements relative to the parent subtilase in one or
more properties including: wash performance, thermal stability,
storage stability or catalytic activity. The variants of the
invention are suitable for use in e.g., cleaning or detergent
compositions, such as laundry detergent compositions and dish wash
compositions, including automatic dish wash compositions.
Inventors: |
Draborg; Henriette;
(Allerod, DK) ; Nielsen; Vibeke Skovgaard;
(Bagsvaerd, DK) ; Minning; Stefan; (Ballerup,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
38233421 |
Appl. No.: |
11/482424 |
Filed: |
July 7, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60698254 |
Jul 11, 2005 |
|
|
|
Current U.S.
Class: |
510/320 ;
435/222; 435/252.3; 435/254.2; 435/471; 435/483; 435/69.1;
536/23.2 |
Current CPC
Class: |
C12N 15/52 20130101;
C11D 3/38636 20130101; C12N 15/74 20130101; C12N 9/54 20130101 |
Class at
Publication: |
510/320 ;
435/069.1; 435/252.3; 435/254.2; 435/471; 435/483; 435/222;
536/023.2 |
International
Class: |
C11D 3/386 20060101
C11D003/386; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 9/56 20060101 C12N009/56; C12N 1/21 20060101
C12N001/21; C12N 1/18 20060101 C12N001/18; C12N 15/74 20060101
C12N015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2005 |
DK |
2005 01007 |
Claims
1. A subtilase variant comprising one or more sets of modifications
selected from the group consisting of T143K, Y167A, R170S, A194P;
Y167A, R170S, A194P, K251 R; Y167A, R170S, A194P, S265K; Y167A,
R170S, A194P, V244R; S141E, Y167A, R170S, A194P; Y167A, R170S,
M1751; Y167A, R170S, A172T; Y167A, R170S, A174V, M175F; Y167A,
R170S, A172V, A174V; Y167A, R170S, A172E; Y167A, R170S, M175L;
Y167A, R170S, A174T; Y167A, R170S, A174T, M175L; G53C, G61E; A98S,
S99D, G100S; S9R, T22A, V68A, S99A, *99aD; S9R, P14H, R19L, N62D;
G61P, *99aS; N43S, N62D; *96aG, P131S, V203A, A228T; N62D, A232C,
Q236L, Q245N; *96aA, A98T, R247K; S99D, S101R, S103A, V104I, G160S,
A194P, L217D; *61aD; N62D, S106A; V68A, S106M, N184D; S9R, A15T,
*97aV, H120N; A15M, A16P, *99aD; *99aE, G160S, S163T, G195S, G211S,
K237R, G258A, T260L; G23S, *99aD, A194P, S242T, Q245R; G100S,
N173D; Y167A, R170S, A172E; A98T, Q137L, Y167A, R170S, M175L;
*98aA, S99D; S99A, *99aD, V203A; N62D, K237R; V11M, N76D, L126F,
K251R; S9F, A15L, A16P, T22I, *98aA, S99D, R170H; *96aA, *130aG,
P131H; E54D, N62D; *98aA, *98bS, S99G, S101T; S9R, A15T, V68A,
I79T, G102S, P131H, Q137H; *100aA, *100bG, *100cS, *100dG; V68A,
L111I; *98aA, R170H, Q245R; I35V, N62D, N183D, T224S; *97aG, P131S,
V203A, A228T; S9R, R10K, P14Q, T22A, Y167A, R170S; S9R, *22aL,
S57A, G61E, *98aA, V139L, N173S; P14T, N18K, Y167A, R170S; S9R,
Q12E, P14Q, K27R, Y167A, R170S; N62D, R170L; N62D, R170S, Q245R;
Y167A, R170S, A194P, K251R, S265K; P14T, N18K, Y167A, R170S, A194P;
N62D, A151G, K237R; N62D, A151G, Q245R; N62D, A151G, K237R, Q245R;
S103A, V104I, G159D, A232V, Q236H, Q245R; S9R, A15T, T22A, V139L;
S9R, A15T, G61E, A85T, E89Q, P239L, Q245C; S9R, A15T, V68A, H120N,
Q245R; N248R; S9R, A15T, *22aL, V139L, N204D, Q245L; N218S; S9R,
A15T, V68A, Q245R, N252K; S9R, A15T, V68A, Q245R, H120N; V68A,
S106A, H120N; V68A, S106A, N252K; A15T, V68A, S99G, Q245R, N261D;
S9R, V68A, S99G, Q245R, N261D; V68A, S99G, Q245R, N261D; S9R, A15T,
V68A, S99G, N261D; S9R, A15T, V68A, Q245R, N261D; S9R, A15T, *22aL,
V139L, S163G, N204D, Q245L; Q245R, N252H; S9R, *22aL, G61 E, *97aA,
M119I, Q137H, N173S; V68A, S106A, T213A; S9R, A15T, V68A,
H.sub.120N, P131S, Q137H, Q245M; S9R, A15T, V68A, 172F, S99G,
Q245R, N261D; S9R, A15T, V68A, S99D, Q245R, N261D; S9R, A15T, V68A,
S99G, A194P, Q245R, N261D; S9R, A15T, V68A, N76I, S99G, Q245R,
N261D and S9R, A15T, V68A, S99G, A228V, Q245R, N261D.
2. A subtilase variant according to claim 1, wherein said variant
further comprises one or more of the modifications K27R, *36D,
S56P, N62D, V68A, N76D, S87N, G97N, S99SE, S101G, S101R, S103A,
V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D,
Y167A, R170S, R170L, A194P, N204D, V2051, Q206E, L217D, N218S,
N218D, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D,
N252K, T274A, S101G+V104N, S87N+S100G+V104N,
K27R+V104Y+N123S+T274A, N76D+S103A+V104I,
S99D+S101R+S103A+V104I+G160S,
S3T+V4I+S99D+S101R+S103A+V104I+G160S+V199M+V205I+L217D,
S3T+V4I+S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217D,
S3T+V4I+S99D+S101R+S103A+V104I+G160S+V205I and N76D+V104A.
3. A subtilase variant according to claim 1 comprising the
following substitutions: S101
G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.
4. The variant according to claim 1, wherein the parent subtilase
belongs to the sub-group I-S1.
5. The variant according to claim 1, wherein the parent subtilase
belongs to the sub-group I-S2, and wherein the parent preferably is
BLSAVI.
6. A cleaning or detergent composition, comprising a variant of
claim 1 and a surfactant.
7. A composition of claim 6, which additionally comprises a
cellulase, a lipase, an amylase, a cutinase, a protease, a
hemicellulase, an esterase, a lactase, a glycoamylase, a
polygalacturonase, a beta-galactosidase, a ligninase, or a mixture
thereof.
8. An isolated DNA sequence encoding a subtilase variant of claim
1.
9. An expression vector comprising the isolated DNA sequence of
claim 8.
10. A microbial host cell transformed with the expression vector of
claim 9.
11. A microbial host cell of claim 10, which is a bacterium.
12. A microbial host cell of claim 10, which is a fungus or
yeast.
13. A method for producing a subtilase variant, comprising (a)
culturing a host of claim 1 under conditions conducive to the
expression and secretion of the variant, and (b) recovering the
variant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority or the benefit under 35
U.S.C. 119 of Danish application no. PA 2005 01007 filed Jul. 8,
2005 and U.S. provisional application No. 60/698,254 filed Jul. 11,
2005, the contents of which are fully incorporated herein by
reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The present invention relates to novel subtilase variants
exhibiting alterations relative to the parent subtilase in one or
more properties including: wash performance, thermal stability,
storage stability and catalytic activity. The variants of the
invention are suitable for use in e.g., cleaning or detergent
compositions, such as laundry detergent compositions and dish wash
compositions, including automatic dish wash compositions. The
present invention also relates to isolated DNA sequences encoding
the variants, expression vectors, host cells, and methods for
producing and using the variants of the invention. Further, the
present invention relates to cleaning and detergent compositions
comprising the variants of the invention.
BACKGROUND OF THE INVENTION
[0004] In the detergent industry enzymes have for more than 30
years been implemented in washing formulations. Enzymes used in
such formulations comprise proteases, lipases, amylases,
cellulases, as well as other enzymes, or mixtures thereof.
Commercially the most important enzymes are proteases.
[0005] An increasing number of commercially used proteases are
protein engineered variants or naturally occurring wild type
proteases, e.g., RELASE.RTM., ALCALASE.RTM., SAVINASE.RTM.,
PRIMASE.RTM., EVERLASE.RTM., ESPERASE.RTM., OVOZYME.RTM.,
CORONOASE, POLARZYME.RTM. and KANNASE.RTM. (Novozymes A/S),
MAXATASE.TM., MAXACAL.TM., MAXAPEM.TM., PROPERASE.TM.,
PURAFECT.TM., PURAFECT OXP.TM., FN2.TM., FN3.TM., FN4.TM. and
PURAFECT PRIME.TM. (Genencor International, Inc.), BLAP X and BLAP
S (Henkel). Further, a number of protease variants are described in
the art. A list of prior art protease variants is given in WO
99/27082.
[0006] However, even though a large number of useful protease
variants have been described, there is still a need for new
improved proteases or protease variants for a number of industrial
uses such as laundry or hard surface cleaning. Therefore, an object
of the present invention is to provide improved subtilase variants
for such purposes.
SUMMARY OF THE INVENTION
[0007] Thus, in a first aspect the present invention relates to a
subtilase variant comprising one or more of the modifications
listed in Table 1. TABLE-US-00001 TABLE 1 Modifications in
subtilase variants. T143K, Y167A, R170S, A194P Y167A, R170S, A194P,
K251R Y167A, R170S, A194P, S265K Y167A, R170S, A194P, V244R S141E,
Y167A, R170S, A194P Y167A, R170S, M1751 Y167A, R170S, A172T Y167A,
R170S, A174V, M175F Y167A, R170S, A172V, A174V Y167A, R170S, A172E
Y167A, R170S, M175L Y167A, R170S, A174T Y167A, R170S, A174T, M175L
G53C, G61E A98S, S99D, G100S S9R, T22A, V68A, S99A, *99Ad S9R,
P14H, R19L, N62D G61P, *99aS N43S, N62D *96aG, P131S, V203A, A228T
N62D, A232C, Q236L, Q245N *96aA, A98T, R247K S99D, S101R, S103A,
V104I, G160S, A194P, L217D *61aD N62D, S106A V68A, S106M, N184D
S9R, A15T, *97aV, H120N A15M, A16P, *99aD *99aE, G160S, S163T,
G195S, G211S, K237R, G258A, T260L G23S, *99aD, A194P, S242T, Q245R
G100S, N173D Y167A, R170S, A172E A98T, Q137L, Y167A, R170S, M175L
*98aA, S99D S99A, *99aD, V203A N62D, K237R V11M, N76D, L126F, K251R
S9F, A15L, A16P, T22I, *98aA, S99D, R170H *96aA, *130aG, P131H
E54D, N62D *98aA, *98bS, S99G, S101T S9R, A15T, V68A, I79T, G102S,
P131H, Q137H *100aA, *100bG, *100cS, *100dG V68A, L111I *98aA,
R170H, Q245R I35V, N62D, N183D, T224S *97aG, P131S, V203A, A228T
S9R, R10K, P14Q, T22A, Y167A, R170S S9R, *22aL, S57A, G61E, *98aA,
V139L, N173S P14T, N18K, Y167A, R170S S9R, Q12E, P14Q, K27R, Y167A,
R170S N62D, R170L N62D, R170S, Q245R Y167A, R170S, A194P, K251R,
S265K P14T, N18K, Y167A, R170S, A194P N62D, A151G, K237R N62D,
A151G, Q245R N62D, A151G, K237R, Q245R S103A, V104I, G159D, A232V,
Q236H, Q245R S9R, A15T, T22A, V139L S9R, A15T, G61E, A85T, E89Q,
P239L, Q245C S9R, A15T, V68A, H120N, Q245R N248R S9R, A15T, *22AI,
V139L, N204D, Q245L N218S S9R, A15T, V68A, Q245R, N252K S9R, A15T,
V68A, Q245R, H120N V68A, S106A, H120N V68A, S106A, N252K A15T,
V68A, S99G, Q245R, N261D S9R, V68A, S99G, Q245R, N261D V68A, S99G,
Q245R, N261D S9R, A15T, V68A, S99G, N261D S9R, A15T, V68A, Q245R,
N261D S9R, A15T, *22aL, V139L, S163G, N204D, Q245L Q245R, N252H
S9R, *22aL, G61E, *97aA, M119I, Q137H, N173S V68A, S106A, T213A
S9R, A15T, V68A, H120N, P131S, Q137H, Q245M S9R, A15T, V68A, I72F,
S99G, Q245R, N261D S9R, A15T, V68A, S99D, Q245R, N261D S9R, A15T,
V68A, S99G, A194P, Q245R, N261D S9R, A15T, V68A, N76I, S99G, Q245R,
N261D S9R, A15T, V68A, S99G, A228V, Q245R, N261D
[0008] The variants listed in Table 1 exhibit protease activity.
Each position corresponds to a position of the amino acid sequence
of subtilisin BPN' set forth in FIG. 1 and SEQ ID NO: 1.
[0009] In a second aspect the present invention relates to an
isolated polynucleotide encoding a subtilase variant of the
invention.
[0010] In a third aspect the present invention relates to an
expression vector comprising the isolated polynucleotide of the
invention.
[0011] In a fourth aspect the present invention relates to a
microbial host cell transformed with the expression vector of the
invention.
[0012] In a fifth aspect the present invention relates to a method
for producing a subtilase variant according to the invention,
comprising culturing a host according to the invention under
conditions conducive to the expression and secretion of the
variant, and recovering the variant.
[0013] In a sixth aspect the present invention relates to a
cleaning or detergent composition, preferably a laundry or dish
wash composition, comprising the variant of the invention.
[0014] Concerning alignment and numbering, reference is made to
FIG. 1 which shows an alignment between subtilisin BPN' (a)
(BASBPN) and subtilisin 309 (b) (BLSAVI). This alignment is in this
patent application used as a reference for numbering the
residues.
Definitions
[0015] Prior to discussing this invention in further detail, the
following terms and conventions will first be defined. For a
detailed description of the nomenclature of amino acids and nucleic
acids, we refer to WO 00/71691 beginning at page 5, which is herein
incorporated by reference.
Nomenclature and Conventions for Designation of Variants
[0016] In describing the various subtilase enzyme variants produced
or contemplated according to the invention, the following
nomenclatures and conventions have been adapted for ease of
reference: A frame of reference is first defined by aligning the
isolated or parent enzyme with subtilisin BPN' (BASBPN).
[0017] The alignment can be obtained by the GAP routine of the GCG
package version 9.1 to number the variants using the following
parameters: gap creation penalty=8 and gap extension penalty=8 and
all other parameters kept at their default values.
[0018] Another method is to use known recognized alignments between
subtilases, such as the alignment indicated in WO 91/00345. In most
cases the differences will not be of any importance.
[0019] Thereby a number of deletions and insertions will be defined
in relation to BASBPN (SEQ ID NO: 1). In FIG. 1, subtilisin 309
(SEQ ID NO: 2) has 6 deletions in positions 36, 58, 158, 162, 163,
and 164 in comparison to BASBPN. These deletions are in FIG. 1
indicated by asterixes (*). For a detailed description of the
nomenclature of modifications introduced in a polypeptide by
genetic manipulation we refer to WO 00/71691 page 7-12, which is
herein incorporated by reference.
[0020] Proteases Enzymes cleaving the amide linkages in protein
substrates are classified as proteases, or (interchangeably)
peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H.
Freeman and Company, San Francisco, Chapter 3).
[0021] Numbering of amino acid positions/residues If nothing else
is mentioned the amino acid numbering used herein correspond to
that of the subtilase BPN' (BASBPN) sequence. For further
description of the BPN' sequence, see FIG. 1, SEQ ID NO: 1 or
Siezen et al., 1991, Protein Engng. 4:719-737.
[0022] Serine proteases A serine protease is an enzyme which
catalyzes the hydrolysis of peptide bonds, and in which there is an
essential serine residue at the active site (White, Handler and
Smith, 1973 "Principles of Biochemistry," Fifth Edition,
McGraw-Hill Book Company, NY, pp. 271-272). The bacterial serine
proteases have molecular weights in the 20,000 to 45,000 Dalton
range. They are inhibited by diisopropylfluorophosphate. They
hydrolyze simple terminal esters and are similar in activity to
eukaryotic chymotrypsin, also a serine protease. A more narrow
term, alkaline protease, covering a sub-group, reflects the high pH
optimum of some of the serine proteases, from pH 9.0 to 11.0 (for
review, see Priest, 1977, Bacteriological Rev. 41:711-753).
[0023] Subtilases A sub-group of the serine proteases tentatively
designated subtilases has been proposed by Siezen et al., 1991,
Protein Engng. 4:719-737 and Siezen et al., 1997, Protein Science
6:501-523. They are defined by homology analysis of more than 170
amino acid sequences of serine proteases previously referred to as
subtilisin-like proteases. A subtilisin was previously often
defined as a serine protease produced by Gram-positive bacteria or
fungi, and according to Siezen et al., now is a subgroup of the
subtilases. A wide variety of subtilases have been identified, and
the amino acid sequence of a number of subtilases has been
determined. For a more detailed description of such subtilases and
their amino acid sequences reference is made to Siezen et al.
(1997).
[0024] One subgroup of the subtilases, I-S1 or "true" subtilisins,
comprises the "classical" subtilisins, such as subtilisin 168
(BSS168), subtilisin BPN', subtilisin Carlsberg (ALCALASE.RTM.,
Novozymes A/S), and subtilisin DY (BSSDY).
[0025] A further subgroup of the subtilases, I-S2 or high alkaline
subtilisins, is recognized by Siezen et al. (supra). Sub-group I-S2
proteases are described as highly alkaline subtilisins and
comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL.RTM.,
Genencor International Inc.), subtilisin 309 (SAVINASE.RTM.,
Novozymes A/S), subtilisin 147 (BLS147) (ESPERASE.RTM., Novozymes
A/S), and alkaline elastase YaB (BSEYAB).
[0026] "SAVINASE.RTM.". SAVINASE.RTM. which is marketed by
Novozymes A/S is subtilisin 309 from B. lentus and differs from
BMLKP only in one position (N87S). SAVINASE.RTM. has the amino acid
sequence designated b) in FIG. 1 and in SEQ ID NO: 2.
[0027] Parent subtilase. The term "parent subtilase" describes a
subtilase defined according to Siezen et al. (1991and 1997). For
further details see description of "Subtilases" above. A parent
subtilase may also be a subtilase isolated from a natural source,
wherein subsequent modifications have been made while retaining the
characteristic of a subtilase. Furthermore, a parent subtilase may
be a subtilase which has been prepared by the DNA shuffling
technique, such as described by J. E. Ness et al., 1999, Nature
Biotechnology 17:893-896. Alternatively the term "parent subtilase"
may be termed "wild type subtilase".
[0028] Modification(s) of a subtilase variant. The term
"modification(s)" used herein is defined to include chemical
modification of a subtilase as well as genetic manipulation of the
DNA encoding a subtilase. The modification(s) can be replacement(s)
of the amino acid side chain(s), substitution(s), deletion(s)
and/or insertions in or at the amino acid(s) of interest.
[0029] Subtilase variant. In the context of this invention, the
term subtilase variant or mutated subtilase means a subtilase that
has been produced by an organism which is expressing a mutant gene
derived from a parent microorganism which possessed an original or
parent gene and which produced a corresponding parent enzyme, the
parent gene having been mutated in order to produce the mutant gene
from which said mutated subtilase protease is produced when
expressed in a suitable host.
[0030] Homologous subtilase sequences. The homology between two
amino acid sequences is in this context described by the parameter
"identity". In order to determine the degree of identity between
two subtilases the GAP routine of the GCG package version 9.1 can
be applied (infra) using the same settings. The output from the
routine is besides the amino acid alignment the calculation of the
"Percent Identity" between the two sequences. Based on this
description it is routine for a person skilled in the art to
identify suitable homologous subtilases, which can be modified
according to the invention.
[0031] Isolated polynucleotide. The term "isolated", when applied
to a polynucleotide, denotes that the polynucleotide has been
removed from its natural genetic milieu and is thus free of other
extraneous or unwanted coding sequences, and is in a form suitable
for use within genetically engineered protein production systems.
Such isolated molecules are those that are separated from their
natural environment and include cDNA and genomic clones. Isolated
DNA molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985). The term "an isolated
polynucleotide" may alternatively be termed "a cloned
polynucleotide".
[0032] Isolated protein. When applied to a protein, the term
"isolated" indicates that the protein has been removed from its
native environment. In a preferred form, the isolated protein is
substantially free of other proteins, particularly other homologous
proteins (i.e., "homologous impurities" (see below)). An isolated
protein is more than 10% pure, preferably more than 20% pure, more
preferably more than 30% pure, as determined by SDS-PAGE. Further
it is preferred to provide the protein in a highly purified form,
i.e., more than 40% pure, more than 60% pure, more than 80% pure,
more preferably more than 95% pure, and most preferably more than
99% pure, as determined by SDS-PAGE. The term "isolated protein"
may alternatively be termed "purified protein".
[0033] Homologous impurities. The term "homologous impurities"
means any impurity (e.g., another polypeptide than the subtilase of
the invention), which originate from the homologous cell where the
subtilase of the invention is originally obtained from.
[0034] Obtained from. The term "obtained from" as used herein in
connection with a specific microbial source, means that the
polynucleotide and/or subtilase produced by the specific source, or
by a cell in which a gene from the source has been inserted.
[0035] Substrate. The term "substrate" used in connection with a
substrate for a protease should be interpreted in its broadest form
as comprising a compound containing at least one peptide (amide)
bond susceptible to hydrolysis by a subtilisin protease.
[0036] Product. The term "product" used in connection with a
product derived from a protease enzymatic reaction should, in the
context of the present invention, be interpreted to include the
products of a hydrolysis reaction involving a subtilase protease. A
product may be the substrate in a subsequent hydrolysis
reaction.
[0037] Wash Performance. In the present context the term "wash
performance" is used as an enzyme's ability to remove proteinaceous
or organic stains present on the object to be cleaned during e.g.,
wash or hard surface cleaning. See also the wash performance test
in Example 3 herein.
BRIEF DESCRIPTION OF THE DRAWING
[0038] FIG. 1 shows an alignment between subtilisin BPN' (a) and
SAVINASE.RTM. (b) using the GAP routine mentioned above.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention relates to novel subtilase variants
exhibiting alterations relative to the parent subtilase in one or
more properties including: Wash performance, thermal stability,
storage stability or catalytic activity. Variants which are
contemplated as being part of the invention are such variants
where, when compared to the wild-type subtilase, one or more amino
acid residues have been modified by substitution, deletion or
insertion. The variants of the present invention comprise one or
more of those modifications listed in Table 1.
[0040] The variants listed in Table 1 exhibit protease activity,
and each position corresponds to a position of the amino acid
sequence of subtilisin BPN' showed in FIG. 1 and SEQ ID NO: 1.
[0041] A subtilase variant of the first aspect of the invention may
be a parent or wild-type subtilase identified and isolated from
nature. Such a parent wild-type subtilase may be specifically
screened for by standard techniques known in the art.
[0042] One preferred way of doing this may be by specifically PCR
amplify conserved DNA regions of interest from subtilases from
numerous different microorganism, preferably different Bacillus
strains.
[0043] Subtilases are a group of conserved enzymes, in the sense
that their DNA and amino acid sequences are homologous. Accordingly
it is possible to construct relatively specific primers flanking
the polynucleotide sequences of interest.
[0044] Using such PCR primers to amplify DNA from a number of
different microorganisms, preferably different Bacillus strains,
followed by DNA sequencing of said amplified PCR fragments, it will
be possible to identify strains which produce subtilase variants of
the invention. Having identified the strain and a partial DNA
sequence of such a subtilase of interest, it is routine work for a
person skilled in the art to complete cloning, expression and
purification of such a subtilase. However, it is envisaged that a
subtilase variant of the invention is predominantly a variant of a
parent subtilase.
[0045] A subtilase variant suitable for the uses described herein
may be constructed by standard techniques known in the art such as
by site-directed/random mutagenesis or by DNA shuffling of
different subtilase sequences. See the "Material and Methods"
section and Example 1 herein for further details.
[0046] As will be acknowledged by the skilled person, the variants
described herein may comprise one or more additional modifications,
in particular one or more additional substitutions or insertions.
Moreover, the variants described herein may encompass mutation at
more than just one position. For example the variant according to
the invention may contain mutations at one position, two positions,
three positions or more than three positions, such as four to eight
positions. It is preferred that the parent subtilase belongs to the
subgroups I-S1 or I-S2, especially subgroup I-S2, both for enzymes
from nature or from the artificial creation of diversity, and for
designing and producing variants from a parent subtilase.
[0047] In relation to variants from subgroup I-S1, it is preferred
to select a parent subtilase from the group consisting of BSS168
(BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA,
BLSCA1, BLSCA2, BLSCA3), BSSPRC, and BSSPRD, or functional variants
thereof having retained the characteristic of sub-group I-S1.
[0048] In relation to variants from subgroup I-S2 it is preferred
to select a parent subtilase from the group consisting of BSAPRQ,
BLS147 (BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYAB,
BAPB92, TVTHER, and BSAPRS, or functional variants thereof having
retained the characteristic of sub-group I-S2. In particular, the
parent subtilase is BLSAVI (SAVINASE.RTM., Novozymes A/S), and a
preferred subtilase variant of the invention is accordingly a
variant of SAVINASE.RTM..
[0049] The present invention also encompasses any of the above
mentioned subtilase variants in combination with any other
modification to the amino acid sequence thereof. Especially
combinations with other modifications known in the art to provide
improved properties to the enzyme are envisaged. The art describes
a number of subtilase variants with different improved properties
and a number of those are mentioned in the "Background of the
invention" section. Those references are disclosed here as
references to identify a subtilase variant, which advantageously
can be combined with a subtilase variant described herein. Such
combinations comprise the positions: 222 (improves oxidation
stability), 218 (improves thermal stability), substitutions in the
Ca.sup.2+-binding sites stabilizing the enzyme, e.g., position 76,
and many other apparent from the prior art.
[0050] In further embodiments a subtilase variant described herein
may advantageously be combined with one or more modification(s) in
any of the positions:
27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
120, 123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245,
248, 252 and 274.
[0051] Specifically, the following BLSAVI, BLSUBL, BSKSMK, and
BAALKP modifications are considered appropriate for
combination:
[0052] K27R, *36D, S56P, N62D, V68A, N76D, S87N, G97N, S99SE,
S101G, S101R, S103A, V104A, V104I, V104N, V104Y, S106A, H120D,
H120N, N123S, G159D, Y167A, R170S, R170L, A194P, N204D, V2051,
Q206E, L217D, N218S, N218D, M222S, M222A, T224S, A232V, K235L,
Q236H, Q245R, N248D, N252K and T274A.
[0053] Furthermore variants comprising any of the modifications
S101G+V104N, S87N+S100G+V104N, K27R+V104Y+N123S+T274A,
N76D+S103A+V104I, S99D+S101R+S103A+V104I+G160S,
S3T+V4I+S99D+S101R+S103A+V104I+G160S+V199M+V205I+L217D,
S3T+V4I+S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217D,
S3T+V4I+S99D+S101R+S103A+V104I+G160S+V205I or N76D+V104A, or other
combinations of the modifications K27R, *36D, S56P, N62D, V68A,
N76D, S87N, G97N, S99SE, S101G, S103A, V104A, V104I, V104N, V104Y,
S106A, H120D, H120N, N123S, G159D, Y167A, R170S, R170L, A194P,
N204D, V2051, Q206E, L217D, N218D, N218S, M222A, M222S, T224S,
A232V, K235L, Q236H, Q245R, N248D, N252K and T274A in combination
with any one or more of the modification(s) mentioned above exhibit
improved properties. A particular interesting variant is a variant,
which, in addition to a modification according to the invention,
contains the following substitutions: S101
G+S103A+V104I+G159D+A232V+Q236H+ Q245R+N248D+N252K.
[0054] Moreover, subtilase variants of the main aspect(s) of the
invention are preferably combined with one or more modification(s)
in any of the positions 129, 131and 194, preferably as 129K, 131H
and 194P modifications, and most preferably as P129K, P131H and
A194P modifications. Any of those modification(s) are expected to
provide a higher expression level of the subtilase variant in the
production thereof.
[0055] The wash performance of a selected variant of the invention
may be tested in the wash performance test disclosed in Example 3
herein. The wash performance test may be employed to assess the
ability of a variant, when incorporated in a standard or commercial
detergent composition, to remove proteinaceous stains from a
standard textile as compared to a reference system, namely the
parent subtilase or a similar subtilase exhibiting an even better
wash performance (incorporated in the same detergent system and
tested under identical conditions). The enzyme variants of the
present application were tested using the Automatic Mechanical
Stress Assay (AMSA). With the AMSA test the wash performance of a
large quantity of small volume enzyme-detergent solutions can be
examined rapidly. Using this test, the wash performance of a
selected variant can be initially investigated, the rationale being
that if a selected variant does not show a significant improvement
in the test compared to the parent subtilase, it is normally not
necessary to carry out further test experiments.
[0056] Therefore, variants which are particularly interesting for
the purposes described herein, are such variants which, when tested
in a commercial detergent composition such as a US type detergent,
an Asian type, a European type or a Latin American type detergent
as described in the wash performance test (Example 3), shows an
improved wash performance as compared to the parent subtilase
tested under identical conditions.
[0057] The improvement in the wash performance may be quantified by
calculating the so-called intensity value (Int) defined in Example
3, herein.
[0058] Evidently, it is preferred that the variant of the invention
fulfils the above criteria on at least the stated lowest level,
more preferably at the stated highest level.
Producing a Subtilase Variant
[0059] Many methods for cloning a subtilase and for introducing
substitutions, deletions or insertions into genes (e.g., subtilase
genes) are well known in the art.
[0060] In general standard procedures for cloning of genes and
introducing mutations (random and/or site directed) into said genes
may be used in order to obtain a subtilase variant of the
invention. For further description of suitable techniques reference
is made to Example 1 herein (vide infra) and (Sambrook et al.,
1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
lab., Cold Spring Harbor, N.Y.; Ausubel et al. (eds.) "Current
protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood
and Cutting (eds.) "Molecular Biological Methods for Bacillus".
John Wiley and Sons, 1990), and WO 96/34946.
[0061] Further, a subtilase variant may be constructed by standard
techniques for artificial creation of diversity, such as by DNA
shuffling of different subtilase genes (WO 95/22625; Stemmer, 1994,
Nature 370:389-91). DNA shuffling of, e.g., the gene encoding
SAVINASE.RTM. with one or more partial subtilase sequences
identified in nature, will after subsequent screening for improved
wash performance variants, provide subtilase variants suitable for
the purposes described herein.
Expression Vectors
[0062] A recombinant expression vector comprising a DNA construct
encoding the enzyme of the invention may be any vector that may
conveniently be subjected to recombinant DNA procedures. The choice
of vector will often depend on the host cell into which it is to be
introduced. Thus, the vector may be an autonomously replicating
vector, i.e., a vector that exists as an extra-chromosomal entity,
the replication of which is independent of chromosomal replication,
e.g., a plasmid.
[0063] Alternatively, the vector may be one that on introduction
into a host cell is integrated into the host cell genome in part or
in its entirety and replicated together with the chromosome(s) into
which it has been integrated.
[0064] The vector is preferably an expression vector in which the
DNA sequence encoding the enzyme of the invention is operably
linked to additional segments required for transcription of the
DNA. In general, the expression vector is derived from plasmid or
viral DNA, or may contain elements of both. The term, "operably
linked" indicates that the segments are arranged so that they
function in concert for their intended purposes, e.g.,
transcription initiates in a promoter and proceeds through the DNA
sequence coding for the enzyme.
[0065] The promoter may be any DNA sequence that shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell.
[0066] Examples of suitable promoters for use in bacterial host
cells include the promoter of the Bacillus stearothermophilus
maltogenic amylase gene, the Bacillus licheniformis alpha-amylase
gene, the Bacillus amyloliquefaciens alpha-amylase gene, the
Bacillus subtilis alkaline protease gene, or the Bacillus pumilus
xylosidase gene, or the phage Lambda P.sub.R or P.sub.L promoters
or the E. coli lac, trp or tac promoters. The DNA sequence encoding
the enzyme of the invention may also, if necessary, be operably
connected to a suitable terminator.
[0067] The recombinant vector of the invention may further comprise
a DNA sequence enabling the vector to replicate in the host cell in
question. The vector may also comprise a selectable marker, e.g., a
gene the product of which complements a defect in the host cell, or
a gene encoding resistance to e.g., antibiotics like kanamycin,
chloramphenicol, erythromycin, tetracycline, spectinomycine, or the
like, or resistance to heavy metals or herbicides.
[0068] To direct an enzyme of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(also known as a leader sequence, prepro sequence or pre sequence)
may be provided in the recombinant vector. The secretory signal
sequence is joined to the DNA sequence encoding the enzyme in the
correct reading frame. Secretory signal sequences are commonly
positioned 5' to the DNA sequence encoding the enzyme. The
secretory signal sequence may be that normally associated with the
enzyme or may be from a gene encoding another secreted protein.
[0069] The procedures used to ligate the DNA sequences coding for
the present enzyme, the promoter and optionally the terminator
and/or secretory signal sequence, respectively, or to assemble
these sequences by suitable PCR amplification schemes, and to
insert them into suitable vectors containing the information
necessary for replication or integration, are well known to persons
skilled in the art (cf., for instance, Sambrook et al., op.
cit.).
Host Cell
[0070] The DNA sequence encoding the present enzyme introduced into
the host cell may be either homologous or heterologous to the host
in question. If homologous to the host cell, i.e., produced by the
host cell in nature, it will typically be operably connected to
another promoter sequence or, if applicable, another secretory
signal sequence and/or terminator sequence than in its natural
environment. The term "homologous" is intended to include a DNA
sequence encoding an enzyme native to the host organism in
question. The term "heterologous" is intended to include a DNA
sequence not expressed by the host cell in nature. Thus, the DNA
sequence may be from another organism, or it may be a synthetic
sequence.
[0071] The host cell into which the DNA construct or the
recombinant vector of the invention is introduced may be any cell
that is capable of producing the present enzyme and includes
bacteria, yeast, fungi and higher eukaryotic cells including
plants.
[0072] Examples of bacterial host cells which, on cultivation, are
capable of producing the enzyme of the invention are gram-positive
bacteria such as strains of Bacillus, such as strains of B.
subtilis, B. licheniformis, B. lentus, B. brevis, B.
stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.
coagulans, B. circulans, B. lautus, B. megaterium or B.
thuringiensis, or strains of Streptomyces, such as S. lividans or
S. murinus, or gram-negative bacteria such as Escherichia coli.
[0073] The transformation of the bacteria may be effected by
protoplast transformation, electroporation, conjugation, or by
using competent cells in a manner known per se (cf. Sambrook et
al., supra).
[0074] When expressing the enzyme in bacteria such as E. coli, the
enzyme may be retained in the cytoplasm, typically as insoluble
granules (known as inclusion bodies), or may be directed to the
periplasmic space by a bacterial secretion sequence. In the former
case, the cells are lysed and the granules are recovered and
denatured after which the enzyme is refolded by diluting the
denaturing agent. In the latter case, the enzyme may be recovered
from the periplasmic space by disrupting the cells, e.g., by
sonication or osmotic shock, to release the contents of the
periplasmic space and recovering the enzyme.
[0075] When expressing the enzyme in gram-positive bacteria such as
Bacillus or Streptomyces strains, the enzyme may be retained in the
cytoplasm, or may be directed to the extracellular medium by a
bacterial secretion sequence. In the latter case, the enzyme may be
recovered from the medium as described below.
Method for Producing a Subtilase Variant
[0076] The present invention provides a method of producing an
isolated enzyme according to the invention, wherein a suitable host
cell, which has been transformed with a DNA sequence encoding the
enzyme, is cultured under conditions permitting the production of
the enzyme, and the resulting enzyme is recovered from the
culture.
[0077] When an expression vector comprising a DNA sequence encoding
the enzyme is trans-formed into a heterologous host cell it is
possible to enable heterologous recombinant production of the
enzyme of the invention. Thereby it is possible to make a highly
purified subtilase composition, characterized in being free from
homologous impurities.
[0078] The medium used to culture the transformed host cells may be
any conventional medium suitable for growing the host cells in
question. The expressed subtilase may conveniently be secreted into
the culture medium and may be recovered there-from by well-known
procedures including separating the cells from the medium by
centrifugation or filtration, precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulfate, followed by chromatographic procedures such as ion
exchange chromatography, affinity chromatography, or the like.
Detergent Applications
[0079] The enzyme of the invention may be added to and thus become
a component of a detergent composition. The detergent composition
of the invention may for example be formulated as a hand or machine
laundry detergent composition including a laundry additive
composition suitable for pre-treatment of stained fabrics and a
rinse added fabric softener composition, or be formulated as a
detergent composition for use in general household hard surface
cleaning operations, or be formulated for hand or machine
dishwashing operations.
[0080] In a specific aspect, the invention provides a detergent
additive comprising the enzyme of the invention. The detergent
additive as well as the detergent composition may comprise one or
more other enzymes such as a protease, a lipase, a cutinase, an
amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an
arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase,
and/or a peroxidase.
[0081] In general the properties of the chosen enzyme(s) should be
compatible with the selected detergent, (i.e., pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective
amounts.
[0082] Proteases: Suitable proteases include those of animal,
vegetable or microbial origin. Microbial origin is preferred.
Chemically modified or protein engineered mutants are included. The
protease may be a serine protease or a metallo protease, preferably
an alkaline microbial protease or a trypsin-like protease. Examples
of alkaline proteases are subtilisins, especially those derived
from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg,
subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO
89/06279). Examples of trypsin-like proteases are trypsin (e.g., of
porcine or bovine origin) and the Fusarium protease described in WO
89/06270 and WO 94/25583.
[0083] Examples of useful proteases are the variants described in
WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially
the variants with substitutions in one or more of the following
positions: 27, 36, 57, 68, 76, 87, 97, 101, 104, 106, 120, 123,
167, 170, 194, 206, 218, 222, 224, 235, 245, 252 and 274. Preferred
commercially used protease enzymes include RELASE.RTM.,
ALCALASE.RTM., SAVINASE.RTM., PRIMASE.RTM., EVERLASE.RTM.,
ESPERASE.RTM., OVOZYME.RTM., CORONASE.RTM., POLARZYME.RTM. and
KANNASE.RTM. (Novozymes A/S), MAXATASE.TM., MAXACAL.TM.,
MAXAPEM.TM., PROPERASE.TM., PURAFECT.TM., PURAFECT OXP.TM.,
FN2.TM., FN3.TM., FN4.TM. and PURAFECT PRIME.TM. (Genencor
International, Inc.), BLAP X and BLAP S (Henkel).
[0084] Lipases: Suitable lipases include those of bacterial or
fungal origin. Chemically modified or protein engineered mutants
are included. Examples of useful lipases include lipases from
Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T.
lanuginosus) as described in EP 258 068 and EP 305 216 or from H.
insolens as described in WO 96/13580, a Pseudomonas lipase, e.g.,
from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.
cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,
Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.
wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B.
subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta
1131:253-360), B. stearothermophilus (JP 64/744992) or B. pumilus
(WO 91/16422). Other examples are lipase variants such as those
described in WO 92/05249, WO 94/01541, EP 407225, EP 260105, WO
95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO
95/22615, WO 97/04079 and WO 97/07202. Preferred commercially used
lipase enzymes include LIPOLASE.RTM., LIPOLASE ULTRA.RTM. and
LIPEX.RTM. (Novozymes A/S).
[0085] Amylases: Suitable amylases (alpha and/or beta) include
those of bacterial or fungal origin.
[0086] Chemically modified or protein engineered mutants are
included. Amylases include, for example, .alpha.-amylases obtained
from Bacillus, e.g., a special strain of B. licheniformis,
described in more detail in GB 1,296,839. Examples of useful
amylases are the variants described in WO 94/02597, WO 94/18314, WO
96/23873, and WO 97/43424, especially the variants with
substitutions in one or more of the following positions: 15, 23,
105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208,
209, 243, 264, 304, 305, 391, 408, and 444. Commercially used
amylases are DURAMYL.RTM., TERMAMYL.RTM., STAINZYME.RTM.,
FUNGAMYL.RTM. and BAN.RTM. (Novozymes A/S), RAPIDASE.TM.,
PURASTAR.TM. and PURASTAR OXAM.TM. (from Genencor International
Inc.).
[0087] Cellulases: Suitable cellulases include those of bacterial
or fungal origin. Chemically modified or protein engineered mutants
are included. Suitable cellulases include cellulases from the
genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
Acremonium, e.g., the fungal cellulases produced from Humicola
insolens, Myceliophthora thermophila and Fusarium oxysporum
disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S.
Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral
cellulases having colour care and whiteness maintenance benefits.
Examples of such cellulases are cellulases described in EP 0 495
257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other
examples are cellulase variants such as those described in WO
94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.
5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and
PCT/DK98/00299. Commercially used cellulases include RENOZYME.RTM.,
CELLUZYME.RTM., and CAREZYME.RTM. (Novozymes A/S), CLAZINASE.TM.,
and PURADEX HA.TM. (Genencor Int. Inc.), and KAC-500(B).TM. (Kao
Corporation).
[0088] Peroxidases/Oxidases: Suitable peroxidases/oxidases include
those of plant, bacterial or fungal origin. Chemically modified or
protein engineered mutants are included. Examples of useful
peroxidases include peroxidases from Coprinus, e.g., from C.
cinereus, and variants thereof as those described in WO 93/24618,
WO 95/10602, and WO 98/15257. Commercially used peroxidases include
GUARDZYME.TM. (Novozymes A/S).
[0089] Hemicellulases: Suitable hemicellulases include those of
bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Suitable hemicellulases include
mannanase, lichenase, xylanase, arabinase, galactanase acetyl xylan
esterase, glucorunidase, ferulic acid esterase, coumaric acid
esterase and arabinofuranosidase as described in WO 95/35362.
Suitable mannanases are described in WO 99/64619.
[0090] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive of the invention, i.e., a separate
additive or a combined additive, can be formulated e.g., as a
granulate, a liquid, a slurry, etc. Preferred detergent additive
formulations are granulates, in particular non-dusting granulates,
liquids, in particular stabilized liquids, or slurries.
[0091] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (polyethylene glycol,
PEG) with mean molar weights of 1000 to 20000; ethoxylated
nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated
fatty alcohols in which the alcohol contains from 12 to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty
alcohols; fatty acids; and mono- and di- and triglycerides of fatty
acids. Examples of film-forming coating materials suitable for
application by fluid bed techniques are given in GB 1483591. Liquid
enzyme preparations may, for instance, be stabilized by adding a
polyol such as propylene glycol, a sugar or sugar alcohol, lactic
acid or boric acid according to established methods. Protected
enzymes may be prepared according to the method disclosed in EP
238,216.
[0092] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste, a gel or a liquid. A liquid detergent may be aqueous,
typically containing up to 70% water and 0-30% organic solvent, or
non-aqueous.
[0093] The detergent composition comprises one or more surfactants,
which may be non-ionic including semi-polar and/or anionic and/or
cationic and/or zwitterionic. The surfactants are typically present
at a level of from 0.1% to 60% by weight.
[0094] When included therein the detergent will usually contain
from about 1% to about 40% of an anionic surfactant such as linear
alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty
alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid
or soap.
[0095] When included therein the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpoly-glycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0096] The detergent may contain 0-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, carbonate, citrate, nitrilotriacetic acid,
ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic
acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered
silicates (e.g., SKS-6 from Hoechst).
[0097] The detergent may comprise one or more polymers. Examples
are carboxymethyl-cellulose, poly(vinylpyrrolidone), poly (ethylene
glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates,
maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid
copolymers.
[0098] The detergent may contain a bleaching system which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively, the bleaching system may comprise peroxyacids of
e.g., the amide, imide, or sulfone type.
[0099] The enzyme(s) of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, 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, and the composition may be
formulated as described in e.g., WO 92/19709 and WO 92/19708.
[0100] The detergent may also contain other conventional detergent
ingredients such as e.g., fabric conditioners including clays, foam
boosters, suds suppressors, anti-corrosion agents, soil-suspending
agents, anti-soil redeposition agents, dyes, bactericides, optical
brighteners, hydrotropes, tarnish inhibitors, or perfumes.
[0101] It is at present contemplated that in the detergent
compositions any enzyme, in particular the enzyme of the invention,
may be added in an amount corresponding to 0.01-100 mg of enzyme
protein per litre of wash liquor, preferably 0.05-5 mg of enzyme
protein per litre of wash liquor, in particular 0.1-1 mg of enzyme
protein per litre of wash liquor.
[0102] Variations in local and regional conditions, such as water
hardness and wash temperature calls for regional detergent
compositions. Detergent Examples 1and 2 provide ranges for the
composition of a typical Latin American detergent and a typical
European powder detergent respectively.
DETERGENT EXAMPLE 1
Typical Latin American Detergent Composition
[0103] TABLE-US-00002 Group Subname Content Surfactants 0-30%
Sulphonates 0-30% Sulphates 0-5% Soaps 0-5% Non-ionics 0-5%
Cationics 0-5% FAGA 0-5% Bleach 0-20% SPT/SPM 0-15% NOBS, TAED 0-5%
Builders 0-60% Phosphates 0-30% Zeolite 0-5% Na2OSiO2 0-10% Na2CO3
0-20% Fillers 0-40% Na2SO4 0-40% Others up to 100% Polymers Enzymes
Foam regulators Water Hydrotropes Others
DETERGENT EXAMPLE 2
Typical European powder Detergent Composition
[0104] TABLE-US-00003 Group Subname Content Surfactants 0-30%
Sulphonates 0-20% Sulphates 0-15% Soaps 0-10% Non-ionics 0-10%
Cationics 0-10% Other 0-10% Bleach 0-30% SPT/SPM 0-30% NOBS + TAED
0-10% Builders 0-60% Phosphates 0-40% Zeolite 0-40% Na2OSiO2 0-20%
Na2CO3 0-20% Fillers 0-40% Na2SO4 0-40% NaCl 0-40% Others up to
100% Polymers Enzymes Foam regulators Water Hydrotropes Others
Other Applications
[0105] The subtilase variants of the present invention may be used
in the processing of food, especially in the field of diary
products, such as milk, cream and cheese, but also in the
processing of meat and vegetables. The subtilase variants of the
present invention may also be used in the processing of feed for
cattle, poultry, and pigs and especially for pet food. Further, the
subtilase variants of the invention may be used for the treatment
of hides. The subtilase variants of the invention may also be used
in processes for decontaminating instruments, surfaces, and other
materials in hospitals, clinics, and meat processing plants, etc.
in order to decompose prions or other infectious agents.
Materials and Methods
Method for Producing a Protease Variant
[0106] The present invention provides a method of producing an
isolated enzyme according to the invention, wherein a suitable host
cell, which has been transformed with a DNA sequence encoding the
enzyme, is cultured under conditions permitting the production of
the enzyme, and the resulting enzyme is recovered from the
culture.
[0107] When an expression vector comprising a DNA sequence encoding
the enzyme is transformed into a heterologous host cell it is
possible to enable heterologous recombinant production of the
enzyme of the invention. Thereby it is possible to make a highly
purified RP-II protease composition, characterized in being free
from homologous impurities.
[0108] The medium used to culture the transformed host cells may be
any conventional medium suitable for growing the host cells in
question. The expressed subtilase variant may conveniently be
secreted into the culture medium and may be recovered there-from by
well-known procedures including separating the cells from the
medium by centrifugation or filtration, precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulfate, followed by chromatographic procedures such as ion
exchange chromatography, affinity chromatography, or the like.
Proteolytic Activity
[0109] Enzyme activity can be measured using the PNA assay using
succinyl-alanine-alanine-proline-glutamicacid-paranitroaniline as a
substrate. The principle of the PNA assay is described in the
Journal of American Oil Chemists Society, Rothgeb, T. M.,
Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).
Textiles
[0110] Standard textile pieces are obtained from EMPA St. Gallen,
Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland or CFT, Center
For Testmaterials, Vlaardingen, Netherlands. Especially important
are EMPA 116 (cotton textile stained with blood, milk and ink),
EMPA 117 (polyester/cotton textile stained with blood, milk and
ink), C-03 (cotton textile stained with chocolate milk and soot),
C-05 (cotton textile stained with blood, milk and ink) and C-10
(cotton textile stained with milk, oil and pigment).
[0111] Wash Conditions TABLE-US-00004 Latin North Asia excl. Region
America America Europe Japan Japan Temperature 20-25.degree. C.
20-32.degree. C. 30-60.degree. C. 15-30.degree. C. 15-20.degree. C.
Washing 14-16 min 12 min 20-40 min 14-20 min 15 min time Water
6-12.degree.dH 6.degree.dH 15.degree.dH 14.degree.dH 3.degree.dH
hardness* Detergent 1.5-4 g/l 1.0-1.5 g/l 4-10 g/l 1.5-2.5 g/l
0.5-0.7 g/l dosage Washing pH As it is As it is As it is As it is
As it is *.degree.dH: adjusted by adding CaCl.sub.2*2H.sub.2O,
MgCl.sub.2*6H.sub.2O and NaHCO.sub.3 to Milli-Q water.
Detergents
[0112] The enzymes of the invention may be tested in the detergent
formulations disclosed in WO 97/07202 or in the detergent examples
above. Further, tests could be done in detergents formulations
purchased from wfk testgewebe GmbH (Germany) or similar supplier,
or in commercial detergents.
[0113] List of test detergents from wfk testgewebe:
[0114] IEC 60456 Type A* Base Detergent
[0115] IEC 60456 Type B Base Detergent
[0116] IEC 60456 Type C Detergent
[0117] ECE Reference Detergent with Phosphate (1977)
[0118] ECE Reference Detergent without Phosphate (1998)
[0119] AHAM Standard Detergent
[0120] EU ECOLABEL (detergents) Light Duty Detergent
[0121] EU ECOLABEL (detergents) PVP
[0122] However, also one of the following commercial detergents may
be used in the wash assay, e.g., Ariel HDP, P&G, Mexico; Omo
Multi Acao HDP, Unilever, Brazil; Breeze HDP, Unilever Thailand;
Diao Pai, Nice, China; Tide HDL, P&G, US; Wisk HDL, Unilever,
US; TOP HDP, Lion, Japan; Attack HDP, Kao, Japan; Ariel Regular
HDP, P&G, Europe; Ariel Compact HDPC, P&G, Europe; Persil
Megaperls, Henkel, Germany and Persil, Unilever, UK.
[0123] Furthermore, a brand extension or color/compact version for
the above specified detergent could be used as well.
[0124] If the detergent contains enzymes, the detergent should be
in-activated before use in order to eliminate the enzyme activity
already present in the detergent. This is done by heating a
detergent stock solution to 85.degree. C. in 5 minutes in a micro
wave oven. The concentration of the detergent stock solution to be
inactivated in the micro wave oven is 4-20 g/l.
Automatic Mechanical Stress Assay
[0125] The Automatic Mechanical Stress Assay (AMSA) is described in
Example 3 below.
Mini Wash Assay
[0126] The milliliter scale wash performance assay is conducted
under the following conditions: TABLE-US-00005 Detergent Latin
American HDP Detergent dose 1.5-4 g/l pH As it is Wash time 14-16
min. Temperature 20-25.degree. C. Water hardness 6-12.degree.dH,
adjusted by adding CaCl.sub.2*2H.sub.2O, MgCl.sub.2*6H.sub.2O and
NaHCO.sub.3 to milli-Q water. Enzyme conc. 5 nM, 10 nM, 30 nM Test
system 125 ml glass beakers. Textile dipped in test solution.
Continuously lifted up and down into the detergent solution, 50
times per minute. Test solution volume 50 ml
[0127] After washing the textile piece is flushed in tap water and
air-dried and the remission (R) of the test material is measured at
460 nm using a Zeiss MCS 521 VIS spectrophotometer. The
measurements are done according to the manufacturer's protocol.
[0128] The performance of the new variants is compared to the
performance of Savinase by calculating the relative performance:
RP=(R.sub.variant-R.sub.BLANK)/(R.sub.SAVINASE-R.sub.BLANK)
[0129] A variant is considered to exhibit improved wash
performance, if it performs better than the reference in at least
one detergent composition.
EXAMPLE 1
Construction and Expression of Enzyme Variants:
Site-Directed Mutagenesis:
[0130] Subtilisin 309 (SAVINASE.RTM.) site-directed variants of the
invention comprising specific insertions/deletions/substitutions
are made by traditional cloning of DNA fragments (Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor, 1989) produced by PCR with oligonucleotides containing the
desired mutations.
[0131] Briefly, plasmid DNA pSX222 (E. coli/B. subtilis shuttle
vector including appropriate selection marker, origins of
replication for Bacillus and E. coli, digestion sites, etc.
disclosed in WO 96/34946) bearing the subtilisin 309 wild-type or a
subtilisin 309 variant gene is used as template in the PCR
reaction. In a first PCR an oligonucleotide containing the desired
mutation (anti-sense) and a suitable opposite oligonucleotide
(sense) is used. The resulting DNA fragment is used as a sense
oligonucleotide in a second PCR together with a suitable anti-sense
oligonucleotide. The resulting DNA fragment is digested with
suitable restriction enzymes and ligated into a suitable E.
coli/Bacillus shuttle vector (e.g., pSX222) digested with the same
enzymes.
[0132] The ligation product is transformed into competent E. coli
and plated on a solid agar containing an appropriate selection
marker. DNA purified from a single colony is sequenced to confirm
the designed mutation. Plasmid DNA is isolated from E. coli cells
bearing plasmids containing subtilisin 309 genes with the designed
mutation and is transformed into a suitable competent B. subtilis
strain, i, B. subtilis DN1885: Disclosed in WO 01/16285 (e.g., as
described by Dubnau et al., 1971, J. Mol. Biol. 56:209-221) and
plated on a solid agar containing an appropriate selection
marker.
[0133] Plasmid DNA from single Bacillus colonies showing protease
activity is isolated and sequenced to confirm the designed
mutation. Bacillus colonies bearing plasmid DNA including
subtilisin 309 genes with the desired mutations are fermented in
baffled shake flasks in a suitable media.
EXAMPLE 2
Purification and Assessment of Enzyme Concentration
[0134] After fermentation purification of subtilisin variants is
accomplished using Hydrophobic Charge Induction Chromatography
(HCIC) and subsequent vacuum filtration. To capture the enzyme, the
HCIC uses a cellulose matrix to which 4-Mercapto-Ethyl-Pyridine
(4-MEP) is bound.
[0135] Beads of the cellulose matrix sized 80-100 micro-m are mixed
with a media containing yeast extract and the transformed B.
subtilis capable of secreting the subtilisin variants and incubated
at pH 9.5 in Unifilter.RTM. microplates. As 4-MEP is hydrophobic at
pH >7 and the subtilisin variants are hydrophobic at pH 9.5 a
hydrophobic association is made between the secreted enzyme and the
4-MEP on the beads. After incubation the media and cell debris is
removed by vacuum filtration while the beads and enzyme are kept on
the filter. To elute the enzyme from the beads the pH is now
lowered by washing the filter with an elution buffer (pH 5). Hereby
the enzymes part from the beads and can be retrieved from the
buffer.
[0136] The concentration of the purified subtilisin enzyme variants
is assessed by active site titration (AST). The purified enzyme is
incubated with the high affinity inhibitor CI-2A at different
concentrations to inhibit a varying amount of the active sites. The
protease and inhibitor binds to each other at a 1:1 ratio and
accordingly the enzyme concentration can be directly related to the
concentration of inhibitor, at which all protease is inactive. To
measure the residual protease activity, a substrate (0.6 mM
Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCl buffer) is added after the
incubation with inhibitor and during the following 4 minutes the
development of the degradation product pNA (paranitrophenol) is
measured periodically at 405 nm on an Elisa Reader. Each of the
variants of the invention listed in Table 1 herein was purified
according to the above procedure and subsequently the enzyme
concentration was determined.
[0137] Known concentrations of the variants of Table 1 were tested
for wash performance in detergent as described below.
EXAMPLE 3
Wash Performance of Detergent Composition Comprising Subtilase
Variants
[0138] The enzyme variants of the present application are tested
using the Automatic Mechanical Stress Assay (AMSA). With the AMSA
test the wash performance of a large quantity of small volume
enzyme-detergent solutions can be examined. The AMSA plate has a
number of slots for test solutions and a lid firmly squeezing the
textile swatch to be washed against all the slot openings. During
the washing time, the plate, test solutions, textile and lid are
vigorously shaken to bring the test solution in contact with the
textile and apply mechanical stress. For further description see WO
02/42740 especially the paragraph "Special method embodiments" at
page 23-24. The assay is conducted under the experimental
conditions specified below. TABLE-US-00006 Detergent Latin American
type HDP Detergent dosage 2.2 g/l Test solution volume 160 micro l
pH Adjusted to pH 9.5-10.5 with NaHCO.sub.3. Wash time 14 minutes
Temperature 20.degree. C. Water hardness 9.degree.dH* Enzyme
concentration 5 nM, 10 nM and 30 nM in test solution Test material
C-10 *.degree.dH: adjusted by adding CaCl.sub.2*2H.sub.2O;
MgCl.sub.2*6H.sub.2O (Ratio Ca.sup.2+:Mg.sup.2+- = 2:1) to milli-Q
water.
[0139] The Latin American type detergent was composed according to
the provisions in Detergent Example 1 herein. After washing the
textile pieces are flushed in tap water and air-dried.
[0140] The performance of the enzyme variant is measured as the
brightness of the colour of the textile samples washed with that
specific enzyme variant. Brightness can also be expressed as the
intensity of the light reflected from the textile sample when
aluminated with white light. When the textile is stained the
intensity of the reflected light is lower, than that of a clean
textile. Therefore the intensity of the reflected light can be used
to measure wash performance of an enzyme variant.
[0141] Color measurements are made with a professional flatbed
scanner (PFU DL2400pro), which is used to capture an image of the
washed textile samples. The scans are made with a resolution of 200
dpi and with an output colour dept of 24 bits. In order to get
accurate results, the scanner is frequently calibrated with a Kodak
reflective IT8 target. To extract a value for the light intensity
from the scanned images, a special designed software application is
used (Novozymes Color Vector Analyzer). The program retrieves the
24 bit pixel values from the image and converts them into values
for red (r), green (g) and blue (b). The intensity value (Int) is
calculated by adding the (r), (g) and (b) values together as
vectors and then taking the length of the resulting vector: Int
{square root over (r.sup.2+g.sup.2+b.sup.2)}.
[0142] The wash performance (P) of a variant is defined as the
light intensity value of textile surface washed with enzyme
variant: P=Int(v)
[0143] The results are presented in Table 2 where the performance
is given as relative performance of a new variant versus the
performance of Savinase at 10 nM protease concentration: RP is
(P.sub.VARIANT-P.sub.BLANK)/(P.sub.SAVINASE-P.sub.BLANK)
TABLE-US-00007 TABLE 2 Wash performance test results with subtilase
variants relative to the performance of Savinase. Relative
Mutations in variant performance T143K, Y167A, R170S, A194P 1.8
Y167A, R170S, A194P, K251R 1.6 Y167A, R170S, A194P, S265K 2.0
Y167A, R170S, A194P, V244R 1.9 S141E, Y167A, R170S, A194P 1.1
Y167A, R170S, M175I 1.2 Y167A, R170S, A172T 1.2 Y167A, R170S,
A174V, M175F 1.4 Y167A, R170S, A172V, A174V 1.5 Y167A, R170S, A172E
1.3 Y167A, R170S, M175L 1.5 Y167A, R170S, A174T 1.2 Y167A, R170S,
A174T, M175L 1.4 G53C, G61E 1.3 A98S, S99D, G100S 1.4 S9R, T22A,
V68A, S99A, *99aD 1.7 S9R, P14H, R19L, N62D 1.8 G61P, *99aS 1.7
N43S, N62D 1.7 *96aG, P131S, V203A, A228T 1.8 N62D, A232C, Q236L,
Q245N 2.0 *96aA, A98T, R247K 2.0 S99D, S101R, S103A, V104I, G160S,
A194P, L217D 1.4 *61aD 1.3 N62D, S106A 2.2 V68A, S106M, N184D 1.6
S9R, A15T, *97aV, H120N 1.4 A15M, A16P, *99aD 1.6 *99aE, G160S,
S163T, G195S, G211S, K237R, 1.2 G258A, T260L G23S, *99aD, A194P,
S242T, Q245R 1.5 G100S, N173D 1.4 Y167A, R170S, A172E 1.1 A98T,
Q137L, Y167A, R170S, M175L 1.1 *98aA, S99D 1.8 S99A, *99aD, V203A
1.8 N62D, K237R 2.1 V11M, N76D, L126F, K251R 1.4 S9F, A15L, A16P,
T22I, *98aA, S99D, R170H 1.2 *96aA, *130aG, P131H 1.5 E54D, N62D
2.0 *98aA, *98bS, S99G, S101T 1.8 S9R, A15T, V68A, I79T, G102S,
P131H, Q137H 1.7 *100aA, *100bG, *100cS, *100dG 1.7 V68A, L111I 1.8
*98aA, R170H, Q245R 1.8 I35V, N62D, N183D, T224S 1.2 *97aG, P131S,
V203A, A228T 1.4 S9R, R10K, P14Q, T22A, Y167A, R170S 1.6 S9R,
*22aL, S57A, G61E, *98aA, V139L, N173S 1.3 P14T, N18K, Y167A, R170S
2.0 S9R, Q12E, P14Q, K27R, Y167A, R170S 1.7 N62D, R170L 1.7 N62D,
R170S, Q245R 1.4 Y167A, R170S, A194P, K251R, S265K 2.0 P14T, N18K,
Y167A, R170S, A194P 2.0 N62D, A151G, K237R 2.0 N62D, A151G, Q245R
1.9 N62D, A151G, K237R, Q245R 2.0
EXAMPLE 4
Wash Performance of Detergent Composition Comprising Subtilase
Variants
[0144] The milliliter scale wash performance assay was conducted
under the following conditions:
[0145] Mini Wash Assay TABLE-US-00008 Detergent Persil, Lever, UK,
HDP Detergent dose 6 g/l pH As it is Wash time 20 min. Temperature
30.degree. C. Water hardness 15.degree.dH, adjusted by adding
CaCl.sub.2*2H.sub.2O, MgCl.sub.2*6H.sub.2O and NaHCO.sub.3
(4:1:7.5) to milli-Q water. Enzyme conc. 2.5 nM, 5 nM, 10 nM, 30
nM, 60 nM Test system 125 ml glass beakers. Textile dipped in test
solution. Continuously lifted up and down into the detergent
solution, 50 times per minute. Swatch used: EMPA 116 (2.5 cm
.times. 7 cm) Test solution volume 50 ml
[0146] After washing the textile piece is flushed in tap water and
air-dried and the remission (R) of the test material is measured at
460 nm using a Zeiss MCS 521 VIS spectrophotometer. The
measurements are done according to the manufacturer's protocol.
[0147] The performance of the new variants is compared to the
performance of Savinase at 10 nM protease concentration by
calculating the relative performance:
RP=(R.sub.variant-R.sub.BLANK)/(R.sub.SAVINASE-R.sub.BLANK)
[0148] A variant is considered to exhibit improved wash
performance, if it performs better than the reference in at least
one detergent composition.
[0149] Scoring: A score=1 is given for variants with an improved
wash performance equal to or better than 1.1. TABLE-US-00009
Mutations Score S103A, V104I, G159D, A232V, Q236H, Q245R 1 S9R,
A15T, T22A, V139L 1 S9R, A15T, G61E, A85T, E89Q, P239L, Q245C 1
S9R, A15T, V68A, H120N, Q245R 1 N248R 1 S9R, A15T, *22aL, V139L,
N204D, Q245L 1 N218S 1 S9R, A15T, V68A, Q245R, N252K 1 S9R, A15T,
V68A, Q245R, H120N 1 V68A, S106A, H120N 1 V68A, S106A, N252K 1
A15T, V68A, S99G, Q245R, N261D 1 S9R, V68A, S99G, Q245R, N261D 1
V68A, S99G, Q245R, N261D 1 S9R, A15T, V68A, S99G, N261D 1 S9R,
A15T, V68A, Q245R, N261D 1 S9R, A15T, *22aL, V139L, S163G, N204D,
Q245L 1 Q245R, N252H 1 S9R, *22aL, G61E, *97aA, M119I, Q137H, N173S
1 V68A, S106A, T213A 1 S9R, A15T, V68A, H120N, P131S, Q137H, Q245M
1 S9R, A15T, V68A, I72F, S99G, Q245R, N261D 1 S9R, A15T, V68A,
S99D, Q245R, N261D 1 S9R, A15T, V68A, S99G, A194P, Q245R, N261D 1
S9R, A15T, V68A, N76I, S99G, Q245R, N261D 1 S9R, A15T, V68A, S99G,
A228V, Q245R, N261D 1
[0150]
Sequence CWU 1
1
2 1 275 PRT B. amyloliquefaciens (subtilisin BPN') 1 Ala 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 2 269 PRT B. lentus (subtilisin 309) 2 Ala 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
* * * * *