U.S. patent application number 12/185994 was filed with the patent office on 2009-06-25 for subtilase variants.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Peter Bauditz, Jan Klugkist, Peter Markvardsen, Laurens Nicolaas Sierkstra, Claus Von der Osten.
Application Number | 20090163400 12/185994 |
Document ID | / |
Family ID | 30119367 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090163400 |
Kind Code |
A1 |
Sierkstra; Laurens Nicolaas ;
et al. |
June 25, 2009 |
Subtilase Variants
Abstract
The present invention relates to enzymes produced by mutating
the genes for a number of subtilases and expressing the mutated
genes in suitable hosts are presented. The enzymes exhibit improved
stability and/or improved wash performance in any detergent in
comparison to their wild type parent enzymes. The enzymes are
well-suited for use in any detergent and for some in especially
liquid or solid shaped detergent compositions.
Inventors: |
Sierkstra; Laurens Nicolaas;
(Delft, NL) ; Klugkist; Jan; (Vlaardingen, NL)
; Markvardsen; Peter; (Bagsvaerd, DK) ; Von der
Osten; Claus; (Lyngby, DK) ; Bauditz; Peter;
(Kobenhavn OE, 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: |
30119367 |
Appl. No.: |
12/185994 |
Filed: |
August 5, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10618934 |
Jul 14, 2003 |
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12185994 |
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09587747 |
Jun 5, 2000 |
6682924 |
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10618934 |
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09120577 |
Jul 22, 1998 |
6190900 |
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09587747 |
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08642987 |
May 6, 1996 |
5837517 |
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09120577 |
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Current U.S.
Class: |
510/392 ;
435/221; 435/222 |
Current CPC
Class: |
C12N 9/54 20130101; C11D
3/386 20130101 |
Class at
Publication: |
510/392 ;
435/222; 435/221 |
International
Class: |
C11D 3/386 20060101
C11D003/386; C12N 9/54 20060101 C12N009/54; C12N 9/56 20060101
C12N009/56; C12S 11/00 20060101 C12S011/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 5, 1995 |
DK |
0519/95 |
May 5, 1995 |
EP |
95201161 |
Apr 12, 1996 |
DK |
0421/96 |
Claims
1-87. (canceled)
88. A modified subtilase comprising one or more of the following
substitutions: (a) a substitution of the amino acid residue at
position 167 with Met, Pro, or Trp, (b) a substitution of the amino
acid residue at position 170 with Ile, Met, or Val, (c) a
substitution of the amino acid residue at position 171 with Met,
Pro or Trp, and (d) a substitution of the amino acid residue at
position 194 with Ile, Met, Phe, Trp, or Val, wherein each position
corresponds to a position of the amino acid sequence of subtilisin
BPN' (SEQ ID NO: 7).
89. The modified subtilase of claim 88, which comprises a
substitution of the amino acid residue at position 167 with Met,
Pro, or Trp.
90. The modified subtilase of claim 88, which comprises a
substitution of the amino acid residue at position 170 with Ile,
Met, or Val.
91. The modified subtilase of claim 88, which comprises a
substitution of the amino acid residue at position 171 with Met,
Pro or Trp.
92. The modified subtilase of claim 88, which comprises a
substitution of the amino acid residue at position 194 with Ile,
Met, Phe, Trp, or Val.
93. The modified subtilase of claim 88, further comprising at least
one further mutation at one or more of positions: 27, 36, 57, 76,
97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and 274.
94. The modified subtilase of claim 93, wherein at least one
further mutation is selected from the group consisting of K27R,
*36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S,
Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.
95. A detergent composition comprising a modified subtilase of
claim 88 and a surfactant.
96. A modified subtilisin 309 comprising one or both of the
following substitutions: (a) a substitution of Glu at position 136
with Ala, Asn, Cys, Gly, His, Ser, Thr, or Tyr, and (b) a
substitution at Y171A, Y171C, Y171G, Y171H, Y171I, Y171L, Y171M,
Y171N, Y171P, Y171Q, Y171S, or Y171W, wherein each position
corresponds to a position of the amino acid sequence of subtilisin
BPN' (SEQ ID NO: 7).
97. The modified subtilisin 309 of claim 96, which comprises a
substitution of Glu at position 136 with Ala, Asn, Cys, Gly, His,
Ser, Thr, or Tyr.
98. The modified subtilisin 309 of claim 96, which comprises Y171A,
Y171C, Y171G, Y171H, Y171I, Y171L, Y171M, Y171N, Y171P, Y171Q,
Y171S, or Y171W.
99. The modified subtilisin 309 of claim 96, further comprising at
least one further mutation at one or more of positions: 27, 36, 57,
76, 97, 101, 104, 120, 123, 194, 206, 218, 222, 224, 235 and
274.
100. The modified subtilisin 309 of claim 99, wherein at least one
further mutation is selected from the group consisting of K27R,
*36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S,
A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.
101. A detergent composition comprising a modified subtilisin 309
of claim 96 and a surfactant.
102. A modified subtilisin 147 comprising one or both of the
following substitutions: (a) a substitution of Glu at position 136
with Ala, Asn, Cys, Gly, His, Ser, Thr, or Tyr, (b) a substitution
at Y171A, Y171C, Y171G, Y171H, Y171I, Y171L, Y171M, Y171N, Y171P,
Y171Q, Y171S, or Y171W, wherein each position corresponds to a
position of the amino acid sequence of subtilisin BPN' (SEQ ID NO:
7).
103. The modified subtilisin 147 of claim 102, further comprising
at least one further mutation at one or more of positions: 27, 36,
57, 76, 97, 101, 104, 120, 123, 194, 206, 218, 222, 224, 235 and
274.
104. The modified subtilisin 147 of claim 103, wherein at least one
further mutation is selected from the group consisting of K27R,
*36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S,
A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.
105. A detergent composition comprising a modified subtilisin 147
of claim 102 and a surfactant.
106. A modified Bacillus protease PB92 comprising a substitution
Y171A, Y171C, Y171G, Y171H, Y171I, Y171L, Y171M, Y171N, Y171P,
Y171Q, Y171S, or Y171W, wherein each position corresponds to a
position of the amino acid sequence of subtilisin BPN' (SEQ ID NO:
7).
107. The modified Bacillus protease PB92 of claim 106, further
comprising at least one further mutation at one or more of
positions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 194, 206, 218,
222, 224, 235 and 274.
108. The modified Bacillus protease PB92 of claim 107, wherein at
least one further mutation is selected from the group consisting of
K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D,
N123S, A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and
T274A.
109. A detergent composition comprising a modified Bacillus
protease PB92 of claim 106 and a surfactant.
110. A modified subtilisin BPN' comprising a substitution of Lys at
position 136 with Ala, Asn, Cys, Gly, His, Ser, Thr, or Tyr,
wherein each position corresponds to a position of the amino acid
sequence of subtilisin BPN' (SEQ ID NO: 7).
111. The modified subtilisin BPN' of claim 110, further comprising
at least one further mutation at one or more of positions: 27, 36,
57, 76, 97, 101, 104, 120, 123, 194, 206, 218, 222, 224, 235 and
274.
112. The modified subtilisin BPN' of claim 111, wherein at least
one further mutation is selected from the group consisting of K27R,
*36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S,
A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.
113. A detergent composition comprising a modified subtilisin BPN'
of claim 111 and a surfactant.
114. A modified subtilisin Carlsberg comprising a substitution of
Lys at position 136 with Ala, Asn, Cys, Gly, His, Ser, Thr, or Tyr,
wherein the position corresponds to a position of the amino acid
sequence of subtilisin BPN' (SEQ ID NO: 7).
115. The modified subtilisin Carlsberg of claim 114, further
comprising at least one further mutation at one or more of
positions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 194, 206, 218,
222, 224, 235 and 274.
116. The modified subtilisin Carlsberg of claim 115, wherein at
least one further mutation is selected from the group consisting of
K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D,
N123S, A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and
T274A.
117. A detergent composition comprising a modified subtilisin
Carlsberg of claim 114 and a surfactant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/618,934 filed Jul. 14, 2003, which is a continuation of
application Ser. No. 09/587,747 filed Jun. 5, 2000, now U.S. Pat.
No. 6,682,924, which is a continuation of application Ser. No.
09/120,577 filed Jul. 22, 1998, now U.S. Pat. No. 6,190,900, which
is a continuation of application Ser. No. 08/642,987 filed May 6,
1996, now U.S. Pat. No. 5,837,517, which claims priority under 35
U.S.C. 119 of Danish application nos. 0519/95 and 0421/96 filed May
5, 1995 and Apr. 12, 1996, respectively, and European application
no. 95201161 filed May 5, 1995, the contents of which are fully
incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a paper copy and computer readable
form of a Sequence Listing, which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to novel mutant enzymes or enzyme
variants useful in formulating detergent compositions and
exhibiting improved storage stability while retaining or improving
their wash performance; cleaning and detergent compositions
containing said enzymes; mutated genes coding for the expression of
said enzymes when inserted into a suitable host cell or organism;
and such host cells transformed therewith and capable of expressing
said enzyme variants.
[0005] 2. Description of Related Art
[0006] 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 most important are proteases.
[0007] Although proteases have been used in the detergent industry
for more than 30 years, much remains unknown as to details of how
these enzymes interact with substrates and/other substances present
in e.g. detergent compositions. Some factors related to specific
residues and influencing certain properties, such as oxidative and
thermal stability in general have been elucidated, but much remains
to be found out. Also, it is still not exactly known which physical
or chemical characteristics are responsible for a good washing
performance or stability of a protease in a specific detergent
composition.
[0008] The currently used proteases have for the most part been
found by isolating proteases from nature and testing them in
detergent formulations.
[0009] An increasing number of commercially used protease are
protein engineered variants of the corresponding naturally
occurring wild type protease, e.g. DURAZYM.RTM. (Novo Nordisk A/S),
RELASE.RTM. (Novo Nordisk A/S), MAXAPEM.RTM. (Gist-Brocades N.V.),
PURAFECT.RTM. (Genencor International, Inc.).
[0010] Therefore, an object of the present invention is to provide
improved protein engineered protease variants, especially for use
in the detergent industry.
Proteases
[0011] 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). Bacteria of the Bacillus
species secrete two extracellular species of protease, a neutral,
or metalloprotease, and an alkaline protease which is functionally
a serine endopeptidase and usually referred to as subtilisin.
Secretion of these proteases has been linked to the bacterial
growth cycle, with greatest expression of protease during the
stationary phase, when sporulation also occurs. Joliffe et al.
(1980) J. Bacteriol 141 1199-1208, have suggested that Bacillus
proteases function in cell wall turnover.
Subtilases
[0012] 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).
[0013] The bacterial serine proteases have molecular weights in the
20,000 to 45,000 Daltons 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).
[0014] A sub-group of the serine proteases tentatively designated
subtilases has been proposed by Siezen et al., Protein Engng. 4
(1991) 719-737. They are defined by homology analysis of more than
40 amino acid sequences of serine proteases previously referred to
as subtilisin-like proteases. A subtilisin was previously 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 subtilisins have been identified, and
the amino acid sequence of a number of subtilisins has been
determined. These include more than six subtilisins from Bacillus
strains, namely, subtilisin 168, subtilisin BPN', subtilisin
Carlsberg, subtilisin Y, subtilisin amylosacchariticus, and
mesentericopeptidase (Kurihara et al. (1972) J. Biol. Chem. 247
5629-5631; Wells et al. (1983) Nucleic Acids Res. 11 7911-7925;
Stahl and Ferrari (1984) J. Bacteriol. 159 811-819, Jacobs et al.
(1985) Nucl. Acids Res. 13 8913-8926; Nedkov et al. (1985) Biol.
Chem. Hoppe-Seyler 366 421-430, Svendsen et al. (1986) FEBS Lett.
196 228232), one subtilisin from an actinomycetales, thermitase
from Thermoactinomyces vulgaris (Meloun et al., (1985) FEBS Lett.
198 195-200), and one fungal subtilisin, proteinase K from
Tritirachium album (Jany and Mayer (1985) Biol. Chem. Hoppe-Seyler
366 584-492). for further reference Table I from Siezen et al., has
been reproduced below.
[0015] Subtilisins are well-characterized physically and
chemically. In addition to knowledge of the primary structure
(amino acid sequence) of these enzymes, over 50 high resolution
X-ray structures of subtilisins have been determined which
delineate the binding of substrate, transition state, products, at
least three different protease inhibitors, and define the
structural consequences for natural variation (Kraut (1977) Ann.
Rev. Biochem. 46 331-358).
[0016] In the context of this application substrate should be
interpreted in its broadest form as comprising a compound
containing at least one peptide bond susceptible to hydrolysis by a
subtilisin protease.
[0017] Also the expression "product" should in the context of this
invention be interpreted to include the products of a hydrolysis
reaction involving a subtilisin protease. A product may be the
substrate in a subsequent hydrolysis reaction.
[0018] One subgroup of the subtilases, I-S1, comprises the
"classical" subtilisins, such as subtilisin 168, subtilisin BPN',
subtilisin Carlsberg (ALCALASE.RTM., Novo Nordisk A/S), and
subtilisin DY.
[0019] A further subgroup of the subtilases I-S2 is recognised by
Siezen et al., (supra). Sub-group I-S2 proteases are described as
highly alkaline subtilisins and comprise enzymes such as subtilisin
PB92 (MAXACAL.RTM., Gist-Brocades NV), subtilisin 309
(SAVINASE.RTM., Novo Nordisk A/S), subtilisin 147 (ESPERASE.RTM.,
Novo Nordisk A/S), and alkaline elastase YaB.
[0020] In the context of this invention, a 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
subtilisin protease is produced when expressed in a suitable
host.
[0021] Random and site-directed mutations of the subtilase gene
have both arisen from knowledge of the physical and chemical
properties of the enzyme and contributed information relating to
subtilase's catalytic activity, substrate specificity, tertiary
structure, etc. (Wells et al. (1987) Proc. Natl. Acad. Sci. U.S.A.
84; 1219-1223; Wells et al. (1986) Phil. Trans. R. Soc. Lond. A.
317 415-423; Hwang and Warshel (1987) Biochem. 26 26692673; Rao et
al., (1987) Nature 328 551-554.
[0022] More recent publications covering this area are Carter et
al., (1989) Proteins 6 240-248 relating to design of variants that
cleave a specific target sequence in a substrate (positions 24 and
64); Graycar et al., (1992) Annals of the New York Academy of
Sciences 672 71-79 discussing a number of previously published
results; and Takagi (1993) Int. J. Biochem. 25 307-312 also
reviewing previous results.
[0023] Especially site-directed mutagenesis of the subtilisin genes
has attracted much attention, and various mutations are described
in the following patent applications and patents:
[0024] EP 130 756 (Genentech) (corresponding to US Reissue Pat. No.
34,606 (Genencor)) relating to site specific or randomly generated
mutations in "carbonyl hydrolases" and subsequent screening of the
mutated enzymes for various properties, such as k.sub.cat/K.sub.m
ratio, pH-activity profile, and oxidation stability. This
publication reveals that site-specific mutation is feasible, and
that mutation of subtilisin BPN' in certain specified positions,
i.e. .sup.-1Tyr, .sup.32Asp, .sup.155Asn, .sup.104Tyr, .sup.222Met,
.sup.166Gly, .sup.64His, .sup.169Gly, .sup.189Phe, .sup.33Ser,
.sup.221Ser, .sup.27Tyr, .sup.156Glu or .sup.152Ala, provide for
enzymes exhibiting altered properties. Since these positions all
except position -1 were known to be involved in the functioning of
the enzyme prior to the filing of the application, and therefore
evident to select, this application does not contribute much to
solving the problem of deciding where to introduce mutations in
order to obtain enzymes with desired properties.
[0025] EP 214 435 (Henkel) relates to cloning and expression of
subtilisin Carlsberg and two mutants thereof. In this application
no reason for mutation of .sup.158Asp to .sup.158Ser and
.sup.161Ser to .sup.161Asp is provided.
[0026] International patent publication No. WO 87/04461 (Amgen)
proposes to reduce the number of Asn-Gly sequences present in the
parent enzyme in order to obtain mutated enzymes exhibiting
improved pH and heat stabilities, in the application emphasis is
put on removing, mutating, or modifying the .sup.109Asn and the
.sup.218Asn residues in subtilisin BPN'. No examples are provided
for any deletions or for modifying the Gly-residues.
[0027] International patent publication No. WO 87/05050 (Genex)
discloses random mutation and subsequent screening of a large
number of mutants of subtilisin BPN' for improved properties. In
the application mutations are described in positions .sup.218Asn,
.sup.131Gly, .sup.254Thr, .sup.166Gly, .sup.116Ala, .sup.188Ser,
.sup.126Leu, and .sup.53Ser.
[0028] EP 251 446 (Genencor) describes how homology considerations
at both primary and tertiary structural levels may be applied to
identify equivalent amino acid residues whether conserved or not.
This information together with the inventors knowledge of the
tertiary structure of subtilisin BPN' lead the inventors to select
a number of positions susceptible to mutation with an expectation
of obtaining mutants with altered properties. The positions so
identified are: .sup.124Met, .sup.222Met, .sup.104Tyr, .sup.152Ala,
.sup.156Glu, .sup.166Gly, .sup.169Gly, .sup.189Phe, .sup.217Tyr.
Also .sup.155Asn, .sup.21Tyr, .sup.22Thr, .sup.24Ser, .sup.32Asp,
.sup.33Ser, .sup.36Asp, .sup.46Gly, .sup.48Ala, .sup.49Ser,
.sup.50Met, .sup.77Asn, .sup.87Ser, .sup.94Lys, .sup.95Val,
.sup.96Leu, .sup.107Ile, .sup.110Gly, .sup.170Lys, .sup.171Tyr,
.sup.172Pro, .sup.197Asp, .sup.199Met, .sup.204Ser, .sup.213Lys,
and .sup.221Ser, which positions are identified as being expected
to influence various properties of the enzyme. Also, a number of
mutations are exemplified to support these suggestions. In addition
to single mutations in these positions the inventors also performed
a number of multiple mutations. Further the inventors identify
.sup.215Gly, .sup.67His, .sup.126Leu, .sup.135Leu, and amino acid
residues within the segments 97-103, 126-129, 213-215, and 152-172
as having interest, but mutations in any of these positions are not
exemplified.
[0029] Especially of interest for the purpose of the present
invention the inventors of EP 251 446 suggest to substitute
.sup.170Lys (in subtilisin BPN', type I-S1), specifically they
suggest to introduce Glu or Arg for the original Lys. It appears
that the Glu variant was produced and it was found that it was
highly susceptible to autolytic degradation (cf. pages 48, 121, 123
(Table XXI includes an obvious error, but indicates a reduction in
autolysis half-time from 86 to 13 minutes) and FIG. 32).
[0030] EP 260 105 (Genencor) describes modification of certain
properties in enzymes containing a catalytic triad by selecting an
amino acid residue within about 15 Angstroms from the catalytic
triad and replace the selected amino acid residue with another
residue. Enzymes of the subtilase type described in the present
specification are specifically mentioned as belonging to the class
of enzymes containing a catalytic triad. In subtilisins positions
222 and 217 are indicated as preferred positions for
replacement.
[0031] Also, Thomas, Russell, and Fersht (1985) Nature 318 375-376
shows that exchange of .sup.99Asp into .sup.99Ser in subtilisin
BPN' changes the pH dependency of the enzyme.
[0032] In a subsequent article (1987) J. Mol. Biol. 193 803-813,
the same authors also discuss the substitution of .sup.156Ser in
place of .sup.156Glu.
[0033] Both these mutations are within a distance of about 15
Angstroms from the active .sup.64His.
[0034] In Nature 328 496-500 (1987) Russel and Fersht discuss the
results of their experiments and present rules for changing
pH-activity profiles by mutating an enzyme to obtain changes in
surface charge.
[0035] WO 88/08028 (Genex) and WO 88/08033 (Amgen) relate to
modifications of amino acid residues in the calcium binding sites
of subtilisin BPN'. The enzyme is said to be stabilized by
substituting more negatively charged residues for the original
ones.
[0036] In WO 89/06279 (Novo Nordisk A/S) position 170 is indicated
as interesting and it is suggested to replace the existing residue
with Tyr. However, no data are given in respect of such a variant.
In WO 91/00345 (Novo Nordisk A/S) the same suggestion is made, and
it is shown that the Tyr variant of position 170 in subtilisin 309
(type I-S2) exhibits an improved wash performance in detergents at
a pH of about 8 (variant S003 in Tables III, IV, V, VI, VII, X).
The same substitution in combination with other substitutions in
other positions also indicates an improved wash performance (S004,
S011-S014, S022-S024, S019, S020, S203, S225, S227 in the same
Table and Table VII) all in accordance with the generic concept of
said application.
[0037] In EP 525 610 (Solvay) it is suggested to improve the
stability of the enzyme (a type 1-S2 subtilase closely related to
subtilisin PB92) towards ionic tensides by decreasing the
hydrophobicity in certain surface regions thereof. It is
consequently suggested to substitute Gln for the Arg in position
164 (170 if using BPN' numbering). No variants comprising this
substitution are disclosed in the application.
[0038] In WO 94/02618 (Gist-Brocades N.V.) a number of position 164
(170 if using BPN' numbering) variants of the I-S2 type subtilisin
PB92 are described. Examples are provided showing substitution of
Met, Val, Tyr, Ile, for the original Arg. Wash performance testing
in powder detergents of the variants indicates a slight
improvement. Especially for the Ile variant wash performance tests
on cacao an improvement of about 20-30% is indicated. No stability
data are provided.
[0039] WO 95/30011, WO 95/30010, and WO 95/29979 (Procter &
Gamble Company) describe 6 regions, especially position 199-220
(BPN' numbering), in subtilisin BPN' and subtilisin 309, which are
designed to change (i.e. decrease) the adsorption of the enzyme to
surface-bound soils. It is suggested that decreased adsorption by
an enzyme to a substrate results in better detergent cleaning
performance. No specific detergent wash performance data are
provided for the suggested variants.
[0040] WO 95/27049 (Solvay S.A.) describes a subtilisin 309 type
protease with following mutations: N43R+N116R+N117R (BPN'
numbering). Data indicate the corresponding variant is having
improved stability, compared to wild-type.
Industrial Applications of Subtilases
[0041] Proteases such as subtilisins have found much utility in
industry, particularly in detergent formulations, as they are
useful for removing proteinaceous stains.
[0042] At present at least the following proteases are known to be
commercially available and many of them are marketed in large
quantities in many countries of the world.
[0043] Subtilisin BPN' or Novo, available from e.g. Sigma, St.
Louis, U.S.A.
[0044] Subtilisin Carlsberg, marketed by Novo Nordisk A/S (Denmark)
as ALCALASE.RTM. and by Gist-Brocades N.V. (Holland) as
MAXATASE.RTM..
[0045] Both of these belong to subtilase subgroup I-S1
[0046] Among the subtilase sub-group l-S2 the following are known
to be marketed.
[0047] A Bacillus lentus subtilisin, subtilisin 309, marketed by
Novo Nordisk A/S (Denmark) as SAVINASE.RTM.. A protein engineered
variant of this enzyme is marketed as DURAZYM.RTM..
[0048] Enzymes closely resembling SAVINASE.RTM., such as subtilisin
PB92, MAXACAL.RTM. marketed by Gist-Brocades N.V. (a protein
engineered variant of this enzyme is marketed as MAXAPEM.RTM.),
OPTICLEAN.RTM. marketed by Solvay et Cie. and PURAFECT.RTM.
marketed by Genencor International.
[0049] A Bacillus lentus subtilisin, subtilisin 147, marketed by
Novo Nordisk A/S (Denmark) as ESPERASE.RTM.;
[0050] To be effective, however, such enzymes must not only exhibit
activity under washing conditions, but must also be compatible with
other detergent components during detergent production and
storage.
[0051] For example, subtilisins may be used in combination with
other enzymes active against other substrates, and the selected
subtilisin should possess stability towards such enzymes, and also
the selected subtilisin preferably should not catalyse degradation
of the other enzymes. Also, the chosen subtilisin should be
resistant to the action from other components in the detergent
formulation, such as bleaching agents, oxidizing agents, etc., in
particular an enzyme to be used in a detergent formulation should
be stable with respect to the oxidizing power, calcium binding
properties, and pH conditions rendered by the non-enzymatic
components in the detergent during storage and in the wash liquor
during wash.
[0052] The ability of an enzyme to catalyze the degradation of
various naturally occurring substrates present on the objects to be
cleaned during e.g. wash is often referred to as its washing
ability, washability, detergency, or wash performance. Throughout
this application the term wash performance will be used to
encompass this property.
[0053] The ability of an enzyme to remain active in the presence of
other components of a detergent composition prior to being put to
use (normally by adding water in the washing process) is usually
referred to as storage stability or shelf life. It is often
measured as half-life, t.sub.1/2. We will use the expression
storage stability for this property throughout this application to
encompass this property.
[0054] Naturally occurring subtilisins have been found to possess
properties which are highly variable in relation to their washing
power or ability under variations in parameters such as pH. Several
of the above marketed detergent proteases, indeed, have a better
performance than those marketed about 20 years ago, but for optimal
performance each enzyme has its own specific conditions regarding
formulation and wash conditions, e.g. pH, temperature, ionic
strength (.dbd.I), active system (tensides, surfactants, bleaching
agent, etc.), builders, etc.
[0055] As a consequence it is found that an enzyme possessing
desirable properties at low pH and low I may be less attractive at
more alkaline conditions and high 1, or an enzyme exhibiting fine
properties at high pH and high I may be less attractive at low pH,
low I conditions.
[0056] Also, it has been found that the storage stability differs
between the enzymes, but it has further been found that a specific
enzyme exhibits large variations in storage stability in respect of
different detergent formulations, dependent upon a number of
parameters, such as pH, pI, bleach system, tensides, etc., and upon
the physical state of the detergent compositions, which may be in
powder, dust, or liquid form. Furthermore it may be concentrated or
dilute.
[0057] The advent and development of recombinant DNA techniques has
had a profound influence in the field of protein chemistry.
[0058] Through the application of this technology it is possible
now to construct enzymes having desired amino acid sequences, and
as indicated above a fair amount of research has been devoted to
designing subtilisins with altered properties.
[0059] Among the proposals the technique of producing and screening
a large number of mutated enzymes as described in EP 130 756
(Genentech) (US Reissue Pat. No. 34,606 (Genencor)) and
International patent publ. no. WO 87/05050 (Genex) correspond to a
large extend to the classical method of isolating native enzymes,
submit them to classical mutagenesis programs (using radiation or
chemical mutagens) and screen them for their properties. The
difference Iles in that these methods are more efficient through
the knowledge of the presence of a large number of variant enzymes
substituted in a specific position.
[0060] A subtilisin enzyme typically comprises about 275 amino acid
residues. Each residue is capable of being 1 out of 20 possible
naturally occurring amino acids.
[0061] Therefore one very serious draw-back in that procedure is
the very large number of mutations generated that have to be
submitted to a number of preliminary screenings to determine their
properties.
[0062] A procedure as outlined in these patent applications will
consequently only be slightly better than the traditional random
mutation procedures which have been known for years.
[0063] The other known techniques relate to changing specific
properties, such as oxidation stability, thermal stability,
Ca-stability, transesterification and hydrolysis rate (EP 260 105
(Genencor)), pH-activity profile (Thomas, Russell, and Fersht,
supra), and substrate specificity (International patent publ. no.
WO 88/07578 (Genentech)). None of these publications relates to
changing either the wash performance of enzymes or their storage
stability.
[0064] International Patent Application no. PCT/DK88/00002 (Novo
Nordisk A/S) proposes to use the concept of homology comparison to
determine which amino acid positions should be selected for
mutation and which amino acids should be substituted in these
positions in order to obtain a desired change in wash
performance.
[0065] By using such a procedure the task of screening is reduced
drastically, since the number of mutants generated is much smaller,
but with that procedure it is only foreseen that enzymes exhibiting
the combined useful properties of the parent enzyme and the enzyme
used in the comparison may be obtained.
[0066] Thus, as indicated above no relationship has yet been
identified between well defined properties of an enzyme such as
those mentioned above and the wash performance and storage
stability of an enzyme in various detergent compositions.
[0067] The problem seems to be that although much research has been
directed at revealing the mechanism of enzyme activity, still only
little is known about the factors in structure and amino acid
residue combination that determine the properties, such as storage
stability in detergents, of enzymes in relation to most of their
characteristics, especially when the enzymes are present in complex
mixtures.
[0068] Consequently there still exists a need for further
improvement and tailoring of enzymes to detergent systems, as well
as a better understanding of the mechanism of protease action and
degradation in the practical use of cleaning or detergent
compositions. Such an understanding could result in rules which may
be applied for selecting mutations that with a reasonable degree of
certainty will result in an enzyme exhibiting improved storage
stability under specified conditions in a detergent
composition.
SUMMARY OF THE INVENTION
[0069] It has now surprisingly been found that a subtilase variant
having improved storage stability and/or improved performance in
detergents, can be obtained by substituting one or more amino acid
residues situated in, or in the vicinity of a hydrophobic domain of
the parent subtilase for an amino acid residue more hydrophobic
than the original residue, said hydrophobic domain comprising the
residues corresponding to residues P129, P131, I165, Y167, Y171 of
BLS309 (in BASBPN numbering), and said residues in the vicinity
thereof comprises residues corresponding to the residues E136,
G159, S164, R170, A194, and G195 of BLS309 (in BASBPN numbering),
with the exception of the R170M, R170I and R170V variants of
BABP92.
[0070] The present invention relates consequently in its first
aspect to enzyme variants exhibiting improved stability and/or
improved wash performance in detergent.
[0071] In its second aspect the invention relates to DNA constructs
capable of expressing the enzymes of the first aspect, when
inserted in a suitable manner into a host cell that subsequently is
brought to express the subtilisin enzyme(s) of the first
aspect.
[0072] In a third aspect the invention relates to the production of
the subtilisin enzymes of the invention by inserting a DNA
construct according to the second aspect into a suitable host,
cultivating the host to express the desired subtilase enzyme, and
recovering the enzyme product.
[0073] The invention relates, in part, but is not limited to,
mutants of the genes expressing the subtilase sub-group I-S2
enzymes and the ensuing enzyme variants, as indicated above.
[0074] Other subtilase gene variants encompassed by the invention
are such as those of the subtilase subgroup I-S1, e.g. subtilisin
BPN', and subtilisin Carisberg genes and ensuing variant subtilisin
BPN', Proteinase K, and subtilisin Carlsberg enzymes, which exhibit
improved stability in concentrated liquid detergents.
[0075] Still further subtilase gene variants encompassed by the
invention are such as Proteinase K and other genes and ensuing
variant Proteinase K, and other subtilase enzymes, which exhibit
improved stability in concentrated liquid detergents.
[0076] Other examples of parent subtilase enzymes that can be
modified in accordance with the invention are listed in Table
1.
[0077] Further the invention relates to the use of the mutant
enzymes in cleaning compositions and cleaning compositions
comprising the mutant enzymes, especially detergent compositions
comprising the mutant subtilisin enzymes. Specifically the
invention relates to concentrated liquid detergent compositions
comprising such enzyme variants.
Abbreviations
Amino Acids
A=Ala=Alanine
V=Val=Valine
L=Leu=Leucine
I=Ile=Isoleucine
P=Pro=Proline
F=Phe=Phenylalanine
W=Trp=Tryptophan
M=Met=Methionine
G=Gly=Glycine
S=Ser=Serine
T=Thr=Threonine
C=Cys=Cysteine
Y=Tyr=Tyrosine
N=Asn=Asparagine
Q=Gln=Glutamine
D=Asp=Aspartic Acid
E=Glu=Glutamic Acid
K=Lys=Lysine
R=Arg=Arginine
H=His=Histidine
[0078] x=Xaa=Any amino acid
Nucleic Acid Bases
A=Adenine
G=Guanine
C=Cytosine
[0079] T=Thymine (only in DNA) U=Uracil (only in RNA)
Variants
[0080] In describing the various enzyme variants produced or
contemplated according to the invention, the following
nomenclatures have been adapted for ease of reference: [0081]
Original amino acid(s) position(s) substituted amino acid(s)
[0082] According to this the substitution of Glutamic acid for
glycine in position 195 is designated as: [0083] Gly 195 Glu or
G195E a deletion of glycine in the same position is: [0084] Gly
195* or G195* and insertion of an additional amino acid residue
such as lysine is: [0085] Gly 195 GlyLys or G195GK
[0086] Where a deletion in comparison with the sequence used for
the numbering is indicated, an insertion in such a position is
indicated as: [0087] *36 Asp or *36D for insertion of an aspartic
acid in position 36
[0088] Multiple mutations are separated by pluses, i.e.: [0089] Arg
170 Tyr+Gly 195 Glu or R170Y+G195E representing mutations in
positions 170 and 195 substituting tyrosine and glutamic acid for
arginine and glycine, respectively.
Positions
[0090] In describing the variants in this application and in the
appended claims use is made of the alignment of various subtilases
in Siezen et al., supra. In other publications relating to
subtilases other alignments or the numbering of specific enzymes
have been used. It is a routine matter for the skilled person to
establish the position of a specific residue in the numbering used
here. Reference is also made to FIG. 1 showing an alignment of
residues relevant for the present invention from a large number of
subtilases. Reference is also made to Table I of WO 91/00345
showing an alignment of residues relevant for the present invention
from a number of subtilases.
TABLE-US-00001 TABLE I Presently established Subtilases (from
Siezen et al., supra) cDNA, Organism gene enzyme acronym
PROKARYOTES Bacteria: Gram-positive Bacillus subtilis 168 apr A
subtilisin I168, apr ABSS168 Bacillus amyloliquefaciens apr
subtilisin BASBPN BPN'(NOVO) Bacillus subtilis DY - subtilisin DY
BSSDY Bacillus licheniformis + subtilisin Carlsberg BLSCAR Bacillus
lentus + subtilisin 147 BLS147 Bacillus alcalophilus PB92 +
subtilisin PB92 BAPB92 Bacillus sp. DSM 4828 - alkaline protease
BDSM48 Bacillus YaB ale alkaline elastase BYSYAB YaB Bacillus
subtilis 168 epr min. extracell. prot. BSEPR Bacillus subtilis bpf
bacillopeptidase F BSBPF Bacillus subtilis IFO3013 ispl
intracell.ser. prot.1 BSISP1 Bacillus subtilis A50 - intracell.ser.
prot. BSIA50 Bacillus thuringiensis - extracell. ser. prot. BTFINI
Bacillus cereus - extracell. ser. prot. BCESPR Nocardiopsis
dassonvillei - alkaline ser. prot. NDAPII Thermoactinomyces
vulgaris - thermitase TVTHER Enterococcus faecalis cylA cytolysin
EFCYLA component A Staphylococcus epidermidis epiP epidermin lead.
SEEPIP prot. Streptococcus pyrogenes scpA C5a peptidase SPSCPA
Lactococcus lactis SK11 prtP SK11 cell wall prot. LLSK11 Bacteria:
Gram-negative Dichelobacter nodosus + basic protease DNEBPR
Xanthomonas campestris + extracellular prot. XCEXPR Serratia
marcescens + extracell. ser. prot. SMEXSP Thermus aquaticus YT-1
pstl aqualysin I TAAQUA Thermus rT41A + T41A protease TRT41A Vibrio
alginolyticus proA protease A VAPROA Streptomyces rutgersensis -
proteinase D SRESPD Archaea - halophil extra. prot. ARB172
halophilic strain 172P1 Cyanobacteria prcA Ca-dependent AVPRCA
Anabaena variabilis protease LOWER EUKARYOTES Fungi Tritirachium
album Limber + proteinase K TAPROK Tritirachium album + proteinase
R TAPROR Tritirachium album proT proteinase T TAPROT Aspergillus
oryzae + alkaline protease AOALPR Malbranchea pulchella -
thermomycolin MPTHMY Acremonium chrysogenum alp alkaline protease
ACALPR Yeasts Kluyveromyces-lactis kex1 Kex1 ser. proteinase KLKEX1
Saccharomyces cerevisiae kex2 Kex2 ser. proteinase SCKEX2
Saccharomyces cerevisiae prb1 protease B SCPRB1 Yarrowia lipolytica
xpr2 alk. extracell. prot. YLXPR2 HIGHER EUKARYOTES Worms
Caenorhabditis elegans bli4 cuticle protease CEBLI4 Insects
Drosophila (fruit fly) fur1 furin 1 DMFUR1 Drosophila (fruit fly)
fur2 furin 2 DMFUR2 Plants Cucumis melo (melon) - cucumisin CMCUCU
Mammals Human (also rat, mouse) fur furin HSFURI Human (also mouse)
+ insulinoma PC2 HSIPC2 prot. Mouse + pituitary PC3 prot. MMPPC3
Human + tripeptidyl peptid.II HSTPP
REFERENCES USED FOR TABLE I
[0091] References to amino acid sequences (GenBank.RTM./EMBL Data
Bank accession numbers are shown in brackets): [0092] ARB172
Kamekura and Seno, (1990) Biochem. Cell Biol. 68, 352-359 (amino
acid sequencing of mature protease residues 1-35; residue 14 not
determined). [0093] BSS168 Stahl and Ferrari, (1984) J. Bacteriol.
158, 411-418 (K01988). Yoshimoto, Oyama et al. (1488) J. Biochem.
103, 1060-1065 (the mature subtilisin from B. subtilis var.
amylosacchariticus differs in having T130S and T162S). Svendsen, et
al. (1986) FEBS Lett. 196, 228-232 (PIR A23624; amino acid
sequencing; the mature alkaline mesentericopeptidase From B.
mesentericus differs in having S85A, A88S, S89A, S183A and N259S).
[0094] BASBPN Wells, et al. (1983) Nucl. Acids Res. 11, 7911-7925
(X00165). Vasantha et al., (1984) J. Bacterol. 159, 811-814
(K02496). [0095] BSSDY Nedkov et al. (1983) Hoppe-Seyler's Z.
Physiol. Chem. 364, 1537-1540 (PIR A00969; amino acid sequencing).
[0096] BLSCAR Jacobs et al. (1985) Nucleic Acids Res. 13, 8913-8926
(X03341). Smith et al., (1968) J. Biol. Chem. 243, 21842191 (PIR
A00968; amino acid sequencing; mature protease sequence differs in
having T103S, P129A, S158N, N161S and S212N). [0097] BLS147 Hastrup
et al. (1989) PCT Patent Appl. WO 89/06279. Pub. Jul. 13, 1989.
(Esperase.RTM. from B. lentus). Takami et al. (1990) Appl.
Microbiol. Biotechnol., 33, 519-523 (amino acid sequencing of
mature alkaline protease residues 1-20 from Bacillus sp. no.
AH-101; this sequence differs from BLS147 in having N11S). [0098]
BABP92 van der Laan et al. (1991) Appl. Environ. Microbiol. 57
901-909. (Maxacal.RTM.). Hastrup et al. (1989) PCT Patent Appl. WO
89/06279. Pub. 13 Jul. 1989. (subtilisin 309). Savinase.RTM., from
B. lentus differs only in having N87S). Godette et al. (1991)
Abstracts 5th Protein Society Symposium, June 6, Baltimore:
abstract M8 (a high-alkaline protease from B. lentus differs in
having N87S, S99D, S101R, S103A, V104I and G159S). [0099] BDSM48
Rettenmaier et al. (1990) PCT Patent Appl. WO 90/04022. Publ. Apr.
19, 1990. [0100] BYSYAB Kaneko et al. (1989) J. Bacteriol. 171,
5232-5236 (M28537). [0101] BSEPR Sloma et al., (1988) J. Bacteriol.
170, 5557-5563 (M22407). Bruckner (1990) Mol. Gen. Genet. 221,
486-490 (X53307). [0102] BSBPF Sloma et al., (1990) J. Bacteriol.
172, 1470-1477 (M29035; corrected). Wu et al. (1990) J. Biol. Chem.
265, 6845-6850 (J05400; this sequence differs in having A169V and
586 less C-terminal residues due to a frameshift). [0103] BSISP1
Koide et al. (1986) J. Bacteriol. 167, 110-116 (M13760). [0104]
BSIA50 Strongin et al. (1978) J. Bacteriol. 133, 1401-1411 (amino
acid sequencing of mature protease residues 1-54; residues 3, 39,
40, 45, 46, 49 and 50 not determined). [0105] BTFINI Chestukhina et
al. (1985) Biokhimiya 50, 1724-1730 (amino acid sequencing of
mature protease residues 1-14 from B. thuringiensis variety
israeliensis, and residues I-16 and 223-243 from variety
finitimus). Kunitate et al. (1989) Agric. Biol. Chem. 53, 3251-3256
(amino acid sequencing of mature protease residues 6-20 from
variety kurstaki. BTKURS). [0106] BCESPR Chestukhina et al., (1985)
Biokhimiya 50, 1724-1730 (amino acid sequencing of mature residues
I-16 and 223-243). [0107] NDAPII Tsujibo et al., (1990) Agric.
Biol. Chem. 54, 2177-2179 (amino acid sequencing of mature residues
1-26). [0108] TVTHER Meloun et al. (1985) FEBS Lett. 183, 195-200
(PIR A00973; amino acid sequencing of mature protease residues
1-274). [0109] EFCYLA Segarra et al. (1991) Infect. Immun. 59,
1239-1246. [0110] SEEPIP Schnell et al. (1991) personal
communication (Siezen et al. (supra)). [0111] SPSCPA Chen et al.
(1990) J. Biol. Chem. 265, 3161-3167 (J05224). [0112] DNEBPR Kortt
et al. (1991) Abstracts 5th Protein Society Symposium, June 22-26,
Baltimore, abstract S76. [0113] LLSK11 Vos et al., (1989) J. Biol.
Chem. 264, 13579-13585 (J04962). Kok et al., (1988) Appl. Environ.
Microbiol. 54, 231-238 (M24767; the sequence from strain Wg2
differs in 44 positions, including 18 differences in the protease
domain, and a deletion of residues 1617-1676). Kiwaki et al. (1989)
Mol. Microbiol. 3, 359-369 (X14130; the sequence from strain
NCD0763 differs in 46 positions, including 22 in the protease
domain, and a deletion of residues 1617-1676). [0114] XCEXPR Liu et
al. (1990) Mol. Gen. Genet. 220, 433-440. [0115] SMEXSP Yanagida et
al. (1986) J. Bacteriol. 166, 937-994 (M13469). [0116] TAAQUA
Terada et al. (1990) J. Biol. Chem. 265, 6576-6581 (J05414). [0117]
TRT41A McHale et al. (1990) Abstracts 5th Eur. Congr. Biotechn.
Christiansen, Munck and Villadsen (eds), Munksgaard Int.
Publishers, Copenhagen. [0118] VAPROA Deane et al. (1989) Gene 76
281-288 (M25499). [0119] SRESPD Lavrenova et al. (1984)
Biochemistry USSR. 49, 447-454 (amino acid sequencing of residues
1-23; residues 13, 18 and 19 not determined). [0120] AVPRCA
Maldener et al. (1991) Mol. Gen. Genet. 225, 113-120 (the published
sequence has 28 uncertain residues near position 200-210 due to a
frameshift reading error). [0121] TAPROK Gunkel and Gassen (1989)
Eur. J. Biochem. 179, 185-194 (X14688/XI4689). Jany et al. (1986)
J. Biol. Chem. Hoppe-Seyler 367 87(PIR A24541; amino acid
sequencing; mature protease differs in having S745G,
SILST204-208DSL and VNLL264-267FNL). [0122] TAPROR Samal et al.
(1990) Mol. Microbiol. 4, 1789-1792 (X56116). [0123] TAPROT Samal
et al., (1989) Gene 85, 329-333. [0124] AOALPR Tatsumi et al.
(1989) Mol. Gen. Genet. 219, 33-38. Cheevadhanarah et al. (1991)
EMBL Data Library (X54726). [0125] MPTHMY Gaucher and Stevenson
(1976) Methods Enzymol. 45, 415-433 (amino acid sequencing of
residues 1-28, and hexapeptide LSGTSM with active site serine).
[0126] ACALPR Isogai et al. (1991) Agric. Biol. Chem. 55, 471-477.
Stepanov et al. (1986) Int. J. Biochem. 18, 369375 (amino acid
sequencing of residues 1-27: the mature protease differs in having
H13[1]Q, R13[2]N and S13[6]A). [0127] KLKEX1 Tanguy-Rougeau,
Wesolowski-Louvel and Fukuhara (1988) FEBS Lett. 234, 464-470
(X07038). [0128] SCKEX2 Mizuno et al. (1988) Biochem. Biophys. Res.
Commun. 156, 246-254(M24201). [0129] SCPRB1 Moehle et al. (1987)
Mol. Cell. Biol. 7, 43904399 (M18097). [0130] YLXYPR2Davidow et
al., (1987) J. Bacteriol. 169, 4621-4629 (M17741). Matoba et al.,
(1988) Mol. Cell. Biol. 8, 4904-4916 (M23353). [0131] CEBL14 Peters
and Rose (1991) The Worm Breeder's Gazette 11, 28. [0132]
DMFUR1Roebroek et al. (1991) FEBS Lett. 289, 133-137 (X59384).
[0133] DMFUR2Roebroek et al. (1992) 267, 17208-17215. [0134] CMCUCU
Kaneda et al. (1984) J. Biochem. 95, 825-829 (amino acid sequencing
of octapeptide NIISGTSM with active site serine). [0135] HSFURI van
den Ouweland et al. (1990) Nucl. Acids Res. 18,664 (X04329) (the
sequence of mouse furin differs in 51 positions, including five in
the catalytic domain: A15E, Y21F, S223F, A232V and N258[2]D).
Misumi et al.(1990) Nucl. Acids Res. 18,6719(X55660: the sequence
of rat furin differs in 49 positions, including three in the
catalytic domain: A15E, Y21F, H24R). [0136] HSIPC2 Smeekens and
Steiner (1990) J. Biol. Chem. 265, 2997-3000 (J05252). Seidah et
al. (1990) DNA Cell Biol. 9, 415-424 (the sequence of mouse
pituitary PC2 protease differs in 23 positions, including seven in
the protease domain: 14F, S42[2]Y, E45D, N76S, D133E, V134L and
G239[1]D). [0137] MMPPC3 Smeekens et al., (1991) Proc. Natl. Acad.
Sci. USA 88, 340-344 (M58507). Seidah et al., (1990) DNA Cell Biol.
9, 415424 (M55668/M55669; partial sequence). [0138] HSTPP Tomkinson
and Jonsson (1991) Biochemistry 30, 168-174 (J05299).
BRIEF DESCRIPTION OF THE FIGURES
[0139] FIGS. 1A and 1B show an alignment of a number of the
subtilases (SEQ ID NOs: 7-41). For optimal alignments, dashes are
shown within the sequences. Further, the numbers on the extreme
left designate the respective amino acid sequences as follows:
TABLE-US-00002 1: (BASBPN) SEQ ID NO: 7 2: (ABASS168) SEQ ID NO: 8
3: (BSSDY) SEQ ID NO: 9 4: (BLSCAR) SEQ ID NO: 10 5. (BAPB92) SEQ
ID NO: 11 6. (BYSYAB) SEQ ID NO: 12 7. (BLS147) SEQ ID NO: 13 8.
(BSEPR) SEQ ID NO: 14 9. (BSISP1) SEQ ID NO: 15 10. (TVTHER) SEQ ID
NO: 16 11. (DNEBPR) SEQ ID NO: 17 12. (XCEXPR) SEQ ID NO: 18 13.
(BSBPF) SEQ ID NO: 19 14. (EFCYLA) SEQ ID NO: 20 15. (SEEPIP) SEQ
ID NO: 21 16. (SPSCPA) SEQ ID NO: 22 17. (LLSK11) SEQ ID NO: 23 18.
(SMEXSP) SEQ ID NO: 24 19. (AVPRCA) SEQ ID NO: 25 20. (MMPPC3) SEQ
ID NO: 26 21. (HSIPC2) SEQ ID NO: 27 22. (HSFURI) SEQ ID NO: 28 23.
(DMFUR1) SEQ ID NO: 29 24. (KLKEX1) SEQ ID NO: 30 25. (SCKEX2) SEQ
ID NO: 31 26. (VAPROA). SEQ ID NO: 32 27. (TRT41A) SEQ ID NO: 33
28. (TAAQUA) SEQ ID NO: 34 29. (TAPROK) SEQ ID NO: 35 30. (TAPROR)
SEQ ID NO: 36 31. (TAPROT) SEQ ID NO: 37 32. (ACALPR) SEQ ID NO: 38
33. (AOALPR) SEQ ID NO: 39 34. (SCPRB1) SEQ ID NO: 40 35. (YLXPR2).
SEQ ID NO: 41
[0140] FIG. 2 is a 3 dimensional representation of subtilisin 309
showing the location of the hydrophobic domain and some of the
amino acid residues in the vicinity thereof to be substituted
according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0141] It has surprisingly been found that the storage stability
and/or improved performance in detergents of subtilases generally
is improved when amino acid residues situated in, or in the
vicinity of a hydrophobic domain comprising the residues P129,
P131, I165, Y167, Y171 of subtilisin 309 are substituted for a more
hydrophobic residue. The residues in question are especially E136,
G159, S164, R170, A194, and G195.
[0142] Further, said variant exhibits a particularly high improved
stability in liquid detergents and in detergents in a shaped solid
form.
[0143] FIG. 2 shows the hydrophobic domain in subtilisin 309 and
residues in the vicinity thereof a number of which are to be
substituted in order to increase the hydrophobicity of the domain.
This may be achieved by substituting hydrophobic residues for
non-hydrophobic residues and/or by substituting residues to become
even more hydrophobic than in the parent enzyme.
[0144] The same principle applies to the corresponding domain in
other subtilases, the identification of which is within the skills
of the average person working in this technical field. Graphic
representations like the one in FIG. 2 can be produced for other
subtilases to determine the target residues to be substituted
according to the invention.
[0145] A number hereof is indicated in Table II below:
TABLE-US-00003 TABLE II residues in hydrophobic domain and the
vicinity thereof Enz. Pos BASBPN BLSCAR BLS309 BLS147 TVTHER domain
129 P A P T T 131 G G P G G 165 V I I V P 167 Y Y Y Y Y 171 Y Y Y Y
Y Vicinity 136 K K E E Q 159 S S G G T 164 T T S G A 170 K K R R Y
194 P A A P S 195 E E G E V
[0146] Table II was constructed using the alignment shown in FIG.
2. It is obvious that similar or larger tables covering other
subtilases may easily be produced by the skilled person.
[0147] Consequently the invention relates to subtilase variants in
which the amino acid sequence has been changed through mutating the
gene of the subtilisin enzyme, which it is desired to modify (the
parent enzyme or gene), in the codon responsible for the expression
of the amino acid residue in positions 129, 131, 165, 167, 171,
136, 159, 164, 170, 194, and 195, which residues are more
hydrophobic than the residue(s) in the parent enzyme, especially
such hydrophobic residues that comprise a relatively long
hydrophobic side chain, such as Ile, Leu, and Val, whereby, when
the mutated gene is expressed, the amino acid residue is
substituted by a more hydrophobic residue, which increases the
hydrophobicity of the domain as such.
[0148] Hydrophobic amino acid residues are generally the
following:
Val (V), Ile (I), Leu (L), Met (M), Phe (F), Pro (P) and Trp (W).
Among these Val, Ile and Leu are preferred.
[0149] By looking at Table II and applying the principle of the
invention a number of candidates for substitution becomes
clear.
[0150] For both BASBPN and BLSCAR it seems appropriate to make
substitutions in positions 129, 131, 136, 159, 164, 167, 170, 171
and 195. In BLS309 positions 136, 164, 167, and 170, 171 would be
the first choices, and positions 159 and 195 also would be a second
choice. In BLS147 positions 129, 131, 136, 167, 170, 171 and 195
are the first choice, while positions 159 and 164 are second.
Finally, in TVTHER positions 129, 131, 136, 167, 171 and 194 are
the first choices, with 164 as a second one.
[0151] According to the invention it would entail an advantage to
substitute the Gly residues in the hydrophobic domain to bulkier
and more hydrophobic residues.
[0152] Such considerations apply for any hydrophilic or hydrophobic
residue that may occupy any of the above mentioned position,
meaning that any increase in hydrophobicity seems to be
advantageous. This means that e.g. a very hydrophilic residue such
as the charged residues Arg (R), Asp (D), Glu (E) or Lys (K) may be
substituted by any residue that is less hydrophilic. Such less
hydrophilic residues comprises the residues Gly (G), Cys (C), Ser
(S), Ala (A), Thr (T), Tyr (Y), Gin (O), His (H) or Asn (N). It
also means that a Tyr(Y) may be substituted by a more hydrophobic
residue such as Phe(F), Leu(L), or Ile(I).
[0153] Similar considerations can be applied to other subtilases
having a hydrophobic domain in this part of the surface of the
enzyme.
[0154] In the context of this invention a subtilase is defined in
accordance with Siezen et al., supra. In a more narrow sense,
applicable to many embodiments of the invention, the subtilases of
interest are those belonging to the subgroups I-S1 and I-S2. In a
more specific sense, many of the embodiments of the invention
relate to serine proteases of gram-positive bacteria which can be
brought into substantially unambiguous homology in their primary
structure, with the subtilases listed in Table I above.
[0155] The present invention also comprises any one or more
substitutions in the above mentioned positions in combination with
any other substitution, deletion or addition to the amino acid
sequence of the parent enzyme. Especially combinations with other
substitutions known to provide improved properties to the enzyme
are envisaged.
[0156] Such combinations comprise the positions: 222 (improve
oxidation stability), 218 (improves thermal stability),
substitutions in the Ca-binding sites stabilising the enzyme, e.g.
position 76, and many other apparent from the prior art.
[0157] Furthermore combinations with the variants mentioned in EP
405 901 are also contemplated specifically.
Variants
A: Single Variants:
Subtilisin BPN', Subtilisin Carlsberg, Subtilisin 168, and
Subtilisin DY Variants:
[0158] A129V, A129I, A129L, A129M, A129F,
[0159] G131V, G131I, G131L, G131M, G131F,
[0160] K136V, K136I, K136L, K136M, K136F,
[0161] S159V, S159I, S159L, S159M, S159F,
[0162] T164V, T164I, T164L, T164M, T164F,
[0163] Y167V, Y167I, Y167L, Y167M, Y167F,
[0164] K170V, K170I, K170L, K170M, K170F,
[0165] Y171V, Y171I, Y171L, Y171M, Y171F,
[0166] A194V, A194I, A194L, A194M, A194F,
[0167] E195V, E195I, E195L, E195M, E195F,
Thermitase Variants:
[0168] A129V, A129I, A129L, A129M, A129F,
[0169] G131V, G131I, G131L, G131M, G131F,
[0170] Q136V, Q136I, Q136L, Q136M, Q136F,
[0171] T159V, T159I, T159L, T159M, T159F,
[0172] A164V, A164I, A164L, A164M, A164F,
[0173] Y167V, Y167I, Y167L, Y167M, Y167F,
[0174] Y171V, Y171I, Y171L, Y171M, Y171F,
[0175] Y170V, Y170I, Y170L, Y170M, Y170F,
[0176] S194V, S194I, S194L, S194M, S194F,
Subtilisin 309, Subtilisin 147, and Bacillus PB92 Protease
Variants:
[0177] T129V, T129I, T129L, T129M, T129F,
[0178] G131V, G131I, G131L, G131M, G131F,
[0179] E136V, E136I, E136L, E136M, E136F,
[0180] G159V, G159I, G159L, G159M, G159F,
[0181] G164V, G164I, G164L, G164M, G164F, (BLS147)
[0182] S164V, S164I, S164L, S164M, S164F, (BLS309 AND BAPB92)
[0183] Y167A, Y167H, Y167N, Y167P, Y167C, Y167W, Y167Q, Y167S,
Y167T, Y167G, Y167V, Y167I, Y167L, Y167M, Y167F
[0184] R170W, R170A, R170H, R170N, R170P, R170Q, R170S, R170T,
R170Y (disclaimed for BLS309), R170V (disclaimed for BAPB92), R170I
(disclaimed for BAPB92),
[0185] R170L, R170M (disclaimed for BAPB92), R170F, R170G,
R170C,
[0186] Y171A, Y171H, Y171N, Y171P, Y171C, Y171W, Y171Q, Y171S,
Y171T, Y171G, Y171V, Y171I, Y171L, Y171M, Y171F,
[0187] A194V, A194I, A194L, A194M, A194F, (BLS309 AND BAPB92)
[0188] P194V, P194I, P194L, P194M, P194F, (BLS147)
[0189] E195V, E195I, E195L, E195M, E195F, (BLS147)
[0190] G195V, G195I, G195L, G195M, G195F, (BLS309 AND BAPB92
B: Combination Variants:
[0191] Any of the above variants are contemplated to prove
advantageous if combined with other variants in any of the
positions:
27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and
274.
[0192] Specifically the following BLS309 and BAPB92 variants are
considered appropriate for combination: K27R, *36D, S57P, N76D,
G97N, S101G, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E,
N218S, M222A, M222S, T224S, K235L and T274A.
[0193] Also such variants comprising any one or two of the
substitutions X167F, X167I, X167L, X167M, X167V, X170F, X170I,
X170L, X170M, and/or X170V, in combination with any one or more of
the other substitutions, deletions and/or insertions mentioned
above are advantageous.
[0194] Furthermore variants comprising any of the variants
V104N+S101G, K27R+V104Y+N123S+T274A, or N76D+V104A or other
combinations of these mutations (V104N, S101G, K27R, V104Y, N123S,
T274A, N76D, V104A), in combination with any one or more of the
substitutions, deletions and/or insertions mentioned above are
deemed to exhibit improved properties.
[0195] Specific combinations to be mentioned are:
a) S57P+R170L
[0196] a') S57P+R170I
b) R170L+N218S
[0197] b') R170I+N218S
c) S57P+R170L+N218S
[0198] c') S57P+R170I+N218S c'') S57P+V104Y+R170L+N218S c'')
S57P+V104Y+R170I+N218S
d) R170L+N218S+M222A
[0199] d') R170I+N218S+M222S d'') R170L+N218S+M222A d''')
R170I+N218S+M222S
e) S57P+R170L+S188P+A194P
[0200] e') S57P+R170I+S188P+A194P
f) Y167L+R170L
f) Y167L+R170I
g) Y167I+R170L
[0201] g') Y167I+R170I
h) N76D+R170L+N218S
[0202] h') N76D+R170I+N218S
i) S57P+N76D+R170L+N218S
[0203] i') S57P+N76D+R170I+N218S
j) N76D+R170L+N218S+M222A
[0204] j') N76D+R170I+N218S+M222S j'') N76D+R1 70L+N218S+M222A
j''') N76D+R170L+N218S+M222S
k) S57P+R170I+S188P+A194P+N218S
[0205] k') S57P+R170I+S188P+A194P+N218S
l) *36D+N76D+H120D+R170L+G195E+K235L
[0206] l') *36D+N76D+H120D+R170I+G195E+K235L l'')
*36D+N76D+H120D+Y167I+R170L+G195E+K235L l''')
*36D+N76D+H120D+Y167I+R170I+G195E+K235L
m) N76D+H120D+R170L+G195E+K235L
[0207] m') N76D+H120D+R170I+G195E+K235L m'')
N76D+H120D+Y167I+R170L+G195E+K235L m')
N76D+H120D+Y167I+R170I+G195E+K235L
n) *36D+G97N+V104Y+H120D+R170L+A194P+G195E+K235L
[0208] n') *36D+G97N+V104Y+H120D+R170I+A194P+G195E+K235L
o) S57P+R170L+Q206E
[0209] o') S57P+R170I+Q206E
p) R170L+Q206E
[0210] p') R170I+Q206E
q) Y167I+R170L+Q206E
[0211] q') Y167I+R170I+Q206E
r) Y167F+R170L
[0212] r') Y167F+R170I
t) Y167I+R170L+A194P
[0213] t') Y167I+R170I+A194P t'') Y167L+R170L+A194P t''')
Y167L+R170I+A194P
u) Y167I+R170L+N218S
[0214] u') Y167I+R170I+N218S u'') Y167L+R170L+N218S u''')
Y167L+R170I+N218S
v) Y167I+R170L+A194P+N218S
[0215] v') Y167I+R170I+A194P+N218S v'') Y167L+R170L+A194P+N218S
v''') Y167L+R170I+A194P+N218S
x) R170L+P131V
[0216] x') R170I+P131V
y) *36D+Y167I+R170L
[0217] y') *36D+Y167I+R170I
z) Y167I+Y171I
aa) Y167V+R170L
[0218] aa') Y167V+R170I
bb) R170L+Y171I
[0219] bb') R170I+Y171L bb'') R170L+Y171L bb''') R170I+Y171I
cc) Y167I+Y171L+N218S
[0220] cc') Y167I+Y171I+N218S
Detergent Compositions Comprising the Mutant Enzymes
[0221] The present invention also comprises the use of the mutant
enzymes of the invention in cleaning and detergent compositions and
such compositions comprising the mutant subtilisin enzymes. Such
cleaning and detergent compositions can in principle have any
physical form, but the subtilase variants are preferably
incorporated in liquid detergent compositions or in detergent
compositions in the form of bars, tablets, sticks and the like for
direct application, wherein they exhibit improved enzyme stability
or performance.
[0222] Among the liquid compositions of the present invention are
aqueous liquid detergents. having for example a homogeneous
physical character, e.g. they can consist of a micellar solution of
surfactants in a continuous aqueous phase, so-called isotropic
liquids.
[0223] Alternatively, they can have a heterogeneous physical phase
and they can be structured, for example they can consist of a
dispersion of lamellar droplets in a continuous aqueous phase, for
example comprising a deflocculating polymer having a hydrophilic
backbone and at least one hydrophobic side chain, as described in
EP-A-346 995 (Unilever) (incorporated herein by reference). These
latter liquids are heterogeneous and may contain suspended solid
particles such as particles of builder materials e.g. of the kinds
mentioned below.
[0224] Concerning powder detergent compositions such compositions
comprise in addition to any one or more of the subtilisin enzyme
variants in accordance to any of the preceding aspects of the
invention alone or in combination any of the usual components
included in such compositions which are well-known to the person
skilled in the art.
[0225] Such components comprise builders, such as phosphate or
zeolite builders, surfactants, such as anionic, cationic, non-ionic
or zwitterionic type surfactants, polymers, such as acrylic or
equivalent polymers, bleach systems, such as perborate- or
amino-containing bleach precursors or activators, structurants,
such as silicate structurants, alkali or acid to adjust pH,
humectants, and/or neutral inorganic salts.
[0226] Furthermore, a number of other ingredients are normally
present in the compositions of the invention, such as
Cosurfactants, Tartrate Succinate Builder, Neutralization System,
Suds Suppressor, Other Enzymes and Other Optional Components.
[0227] The weight ratio of anionic surfactant to nonionic
surfactant is preferably from 1:1 to 5:1. The compositions have a
pH in a 10% by weight solution in water at 20.degree. C. of from
7.0 to 9.0, a Critical Micelle Concentration of less than or equal
to 200 ppm, and an air/water Interfacial Tension at the Critical
Micelle Concentration of less than or equal to 32 dynes/cm at
35.degree. C. in distilled water. The compositions are preferably
clear, homogeneous and phase stable, and have good cleaning
performance and enzyme stability.
Various Components:
1. Anionic Surfactant
[0228] The compositions of the present invention contain from about
10% to about 50%, preferably from about 15% to about 50%, more
preferably from about 20% to 40%, and most preferably from 20% to
about 30%, by weight of a natural or synthetic anionic surfactant.
Suitable natural or synthetic anionic surfactants are e.g. soaps
and such as disclosed in U.S. Pat. Nos. 4,285,841 and
3,929,678.
[0229] Useful anionic surfactants include the water-soluble salts,
particularly the alkali metal, ammonium and alkylolammonium (e.g.,
monoethanolammonium or triethanolammonium) salts, of organic
sulfuric reaction products having in their molecular structure an
alkyl group containing from about 10 to about 20 carbon atoms and a
sulfonic acid or sulfuric acid ester group. (Included in the term
"alkyl" is the alkyl portion of aryl groups.) Examples of this
group of synthetic surfactants are the alkyl sulfates, especially
those obtained by sulfating the higher alcohols (C.sub.8-C.sub.18
carbon atoms) such as those produced by reducing the glycerides of
tallow or coconut oil; and the alkylbenzene sulfonates in which the
alkyl group contains from about 9 to about 15 carbon atoms, in
straight chain or branched chain configuration, e.g., those of the
type described in U.S. Pat. Nos. 2,220,099 and 2,477,383.
Especially valuable are linear straight chain alkylbenzene
sulfonates in which the average number of carbon atoms in the alkyl
group is from about 11 to 14.
[0230] Other anionic surfactants herein are the water-soluble salts
of: paraffin sulfonates containing from 8 to about 24 (preferably
about 12 to 18) carbon atoms; alkyl glyceryl ether sulfonates,
especially those ethers of C.sub.8-C.sub.18 alcohols (e.g., those
derived from tallow and coconut oil); alkyl phenol ethylene oxide
ether sulfates containing from 1 to about 4 units of ethylene oxide
per molecule and from 8 to 12 carbon atoms in the alkyl group; and
alkyl ethylene oxide ether sulfates containing 1 to 4 units of
ethylene oxide per molecule and from 10 to 20 carbon atoms in the
alkyl group.
[0231] Other useful anionic surfactants include the water-soluble
salts of esters of alpha-sulfonated fatty.acids containing from 6
to 20 carbon atoms in the fatty acid group and from 1 to 10 carbon
atoms in the ester group; water-soluble salts of
2-acyloxy-alkane-1-sulfonic acids containing from 2 to 9 carbon
atoms in the acyl group and from 9 to 23 carbon atoms in the alkane
moiety; water-soluble salts of olefin sulfonates containing from 12
to 24 carbon atoms; and beta-alkyloxy alkane sulfonates containing
from 1 to 3 carbon atoms in the alkyl group and from 8 to 20 carbon
atoms in the alkane moiety.
[0232] Preferred anionic surfactants are soaps, the
C.sub.10-C.sub.18 alkyl sulfates and alkyl ethoxy sulfates
containing an average of up to 4 ethylene oxide units per mole of
alkyl sulfate, C.sub.11-C.sub.13 linear alkyl benzene sulfonates,
and mixtures thereof.
2. Nonionic Surfactant
[0233] Another optional ingredient is from 2% to 14% preferably
from 2% to 8%, most preferably from 3% to 5% by weight, of an
optionally ethoxylated nonionic surfactant. The weight ratio of
natural or synthetic anionic surfactant (on an acid basis) to
nonionic surfactant is from 1:1 to 5:1 preferably from 2:1 to 5:1,
most preferably from 3:1 to 4:1. This is to ensure the formation
and adsorption of sufficient hardness surfactants at the air/water
interface to provide good greasy/oily soil removal.
[0234] The optionally ethoxylated nonionic surfactant is of the
formula R.sup.1(OC.sub.2H.sub.4).sub.nOH, wherein R.sup.1 is a
C.sub.10-C.sub.16 alkyl group or a C.sub.8-C.sub.12 alkyl phenyl
group, n is from 3 to 9, and said nonionic surfactant has an HLB
(Hydrophilic-Lipophilic Balance) of from 6 to 14, preferably from
10 to 13. These surfactants are more fully described in U.S. Pat.
Nos. 4,285,841 and 4,284,532. Particularly preferred are
condensation products of C.sub.12-C.sub.15 alcohols with from 3 to
8 moles of ethylene oxide per mole of alcohol, e.g.,
C.sub.12-C.sub.13 alcohol condensed with about 6.5 moles of
ethylene oxide per mole of alcohol. Other nonionic surfactants to
be mentioned are APG, EGE, and glucamide surfactants.
3. Detergency Builder
[0235] Among the usual detergent ingredients which may be present
in usual amounts in the detergent compositions of this invention
are the following: The compositions may be built or unbuilt, and
may be of the zero-P type (i.e. not containing any phosphorus
containing builders). Thus, the composition may contain in the
aggregate for example from 1-50%, e.g. at least about 5% and often
up to about 35-40% by weight, of one or more organic and/or
inorganic builders. Typical examples of builders include those
already mentioned above, and more broadly include alkali metal
ortho, pyro, and tripolyphosphates, alkali metal carbonates, either
alone or in admixture with calcite, alkali metal citrates, alkali
metal nitrilotriacetates, carboxymethyloxysuccinates, zeolites,
polyacetalcarboxylates, and so on.
[0236] More specifically the compositions herein contain from 5% to
20%, preferably from 10% to 15%, by weight of a detergency builder
which can be a fatty acid containing from 10 to 18 carbon atoms
and/or a polycarboxylate, zeolite, polyphoshonate and/or
polyphosphate a builder. Preferred are from 0 to 10% (more
preferably from 3% to 10%) by weight of saturated fatty acids
containing from 12 to 14 carbon atoms, along with from 0 to 10%,
more preferably from 2% to 8%, most preferably from 2% to 5%, by
weight of a polycarboxylate builder, most preferably citric acid,
in a weight ratio of from I:I to 3:1.
[0237] Since the proteolytic enzymes herein appear to provide
optimum storage stability benefits versus other enzymes when the
builder to water hardness ratio is close to one, the compositions
preferably contain sufficient builder to sequester from 2 to 10,
preferably from 3 to 8, grains per gallon of hardness.
[0238] Suitable saturated fatty acids can be obtained from natural
sources such as plant or animal esters (e.g., palm kernel oil, palm
oil and coconut oil) or synthetically prepared (e.g., via the
oxidation of petroleum or by hydrogenation of carbon monoxide via
the Fisher-Tropsch process). Examples of suitable saturated fatty
acids for use in the compositions of this invention include capric,
lauric, myristic, coconut and palm kernel fatty acid. Preferred are
saturated coconut fatty acids; from 5:1 to I:I (preferably about
3:1) weight ratio mixtures of lauric and myristic acid; mixtures of
the above with minor amounts (e.g., 1%-30% of total fatty acid) of
oleic acid; and palm kernel fatty acid.
[0239] The compositions herein preferably also contain the
polycarboxylate, polyphosphonate and polyphosphate builders
described in U.S. Pat. No. 4,284,532, Water-soluble polycarboxylate
builders, particularly citrates, are preferred of this group.
Suitable polycarboxylate builders include the various
aminopolycarboxylates, cycloalkane polycarboxylates, ether
polycarboxylates, alkyl polycarboxylates, epoxy polycarboxylates,
tetrahydrofuran polycarboxylates, benzene polycarboxylates, and
polyacetal polycarboxylates.
[0240] Examples of such polycarboxylate builders are sodium and
potassium ethylenediaminetetraacetate; sodium and potassium
nitrilotriacetate; the water-soluble salts of phyfic acid, e.g.,
sodium and potassium phytates, disclosed in U.S. Pat. No.
1,739,942, the polycarboxylate materials described in U.S. Pat. No.
3,364,103; and the water-soluble salts of polycarboxylate polymers
and copolymers described in U.S. Pat. No. 3,308,067.
[0241] Other useful detergency builders include the water-soluble
salts of polymeric aliphatic polycarboxylic acids having the
following structural and physical characteristics: (a) a minimum
molecular weight of about 350 calculated as to the acid form; (b)
an equivalent weight of 50 to 80 calculated as to acid form; (3) at
least 45 mole percent of the monomeric species having at least two
carboxyl radicals separated from each other by not more than two
carbon atoms: (d) the site of attachment of the polymer chain of
any carboxyl-containing radical being separated by not more than
three carbon atoms along the polymer chain from the site of
attachment of the next carboxyl-containing radical. Specific
examples of such builders are the polymers and copolymers of
itaconic acid, aconitic acid, maleic acid, mesaconic acid, fumaric
acid, methylene malonic acid, and citraconic acid.
[0242] Other suitable polycarboxylate builders include the
water-soluble salts, especially the sodium and potassium salts, of
mellitic acid, citric acid, pyromellitic acid, benzene
pentacarboxylic acid, oxydiacetic acid, carboxymethyloxy-succinic
acid, carboxymethyloxymalonic acid, cis-cyclohexanehexacarboxylic
acid, cis-cyclopentanetetracarboxylic acid and oxydisuccinic
acid.
[0243] Other polycarboxylates are the polyacetal carboxylates
described in U.S. Pat. Nos. 4,144,226, and 4,146,495.
[0244] Other detergency builders include the zeolites, such as the
aluminosilicate ion exchange material described in U.S. Pat. No.
4,405,483.
[0245] Other preferred builders are those of the general formula
R--CH(COOH)CH.sub.2(COOH), i.e. derivatives of succinic acid,
wherein R is C.sub.10-C.sub.20 alkyl or alkenyl, preferably
C.sub.12-C.sub.16, or wherein R may be substituted with hydroxyl,
sulfo, sulfoxy or sulfone substituents. These succinate builders
are preferably used in the form of their water soluble salts,
including the sodium, potassium and alkanolammonium salts. Specific
examples of succinate builders include: lauryl succinate, myristyl
succinate, palmityl succinate, 2-dodecenyl succinate, and the
like.
4. Proteolytic Enzyme
[0246] The enzymes of the invention can be used in well-known
standard amounts in detergent compositions. The amounts may range
very widely, e.g. about 0.0002-0.1, e.g. about 0.005-0.05, Anson
units per gram of the detergent composition. Expressed in
alternative units, the protease can be included in the compositions
in amounts in the order of from about 0.1 to 100 GU/mg (e.g. 1-50,
especially 5-20 GU/mg) of the detergent formulation, or any amount
in a wide range centering at about 0.01-4, e.g. 0.1-0.4 KNPU per g
of detergent formulation.
[0247] It may for example be suitable to use the present enzymes at
the rate of about 0.25 mg of enzyme protein per liter of wash
liquor, corresponding to an enzyme activity of the order of 0.08
KNPU per liter. Corresponding detergent formulations can contain
the enzymes in for example an amount of the order of 0.1-0.4
KNPU/g.
[0248] Expressed differently the compositions of the present
invention contain from about 0.01% to about 5%, preferably from
about 0.1% to about 2%, by weight of the proteolytic enzymes of the
invention.
[0249] The described proteolytic enzyme is preferably included in
an amount sufficient to provide an activity of from 0.05 to about
1.0, more preferably from about 0.1 to 0.75, most preferably from
about 0.125 to about 0.5 mg of active enzyme per gram of
composition.
[0250] The enzyme component may be added to the other components in
any convenient form, such as in the form of a solution, slurry, LDP
slurry, or crystals.
5. Enzyme Stabilization System
[0251] The liquid detergents according to the present invention may
comprise an enzyme stabilization system, comprising calcium ion,
boric acid, propylene glycol and/or short chain carboxylic acids.
The enzyme stabilization system comprises from about 0.5% to about
15% by weight of the composition.
[0252] The composition preferably contains from about 0.01 to about
50, preferably from about 0.1 to about 30, more preferably from
about 1 to 20 millimoles of calcium ion per liter. The level of
calcium ion should be selected so that there is always some minimum
level available for the enzyme, after allowing for complexation
with builders etc. in the composition. Any water-soluble calcium
salt can be used as the source of calcium ion, including calcium
chloride, calcium formate, and calcium acetate. A small amount of
calcium ion, generally from about 0.05 to 0.4 millimoles per liter,
is often also present in the composition due to calcium in the
enzyme slurry and formula water. From about 0.03% to about 0.6% of
calcium formate is preferred.
[0253] A second preferred enzyme stabilizer is polyols containing
only carbon, hydrogen and oxygen atoms. They preferably contain
from 2 to 6 carbon atoms and from 2 to 6 hydroxy groups. Examples
include propylene glycol (especially 1,2-propanediol, which is
preferred), ethylene glycol, glycerol, sorbitol, mannitol, and
glucose. The polyol generally represents from about 0.5% to 15%,
preferably from about 1.5% to about 8%, by weight of the
composition. Preferably, the weight ratio of polyol to any boric
acid added is at least 1, more preferably at least 1.3.
[0254] The compositions preferably also contain the water-soluble,
short chain carboxylates described in U.S. Pat. No. 4,318,818. The
formates are preferred and can be used at levels of from about
0.05% to about 5%, preferably from about 0.2% to about 2%, most
preferably from 0.4% to 1.5%, by weight of the composition. Sodium
formate is preferred.
[0255] The compositions herein also optionally contain from about
0.25% to about 5%, most preferably from about 0.5% to about 3%, by
weight of boric acid. The boric acid may be, but is preferably not,
formed by a compound capable of forming boric acid in the
composition. Boric acid is preferred, although other compounds such
as boric oxide, borax and other alkali metal borates (e.g., sodium
ortho-, meta- and pyroborate, and sodium pentaborate) are suitable.
[Substituted boric acids (e.g., phenylboronic acid, butane boronic
acid, and p-bromo phenylboronic acid) can also be used in place of
boric acid.
6. Water
[0256] The liquid compositions of the present invention may be
aqueous liquids or non-aqueous liquids. When the are aqueous
liquids, they contain from about 15% to about 60%, preferably from
about 25% to about 45%, by weight of water.
Further Optional Components
A. Cosurfactants
[0257] Optional cosurfactants for use with the above nonionic
surfactants include amides of the formula
##STR00001##
wherein R.sup.1 is an alkyl, hydroxyalkyl or alkenyl radical
containing from 8 to 20 carbon atoms, and R.sup.2 and R.sup.3 are
selected from the group consisting of hydrogen, methyl, ethyl,
propyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl,
3-hydroxypropyl, and said radicals additionally containing up to 5
ethylene oxide units, provided at least one of R.sup.2 and R.sup.3
contains a hydroxyl group.
[0258] Preferred amides are the C.sub.8-C.sub.20 fatty acid alkylol
amides in which each alkylol group contains from 1 to 3 carbon
atoms, and additionally can contain up to 2 ethylene oxide units.
Particularly preferred are the C.sub.12-C.sub.16 fatty acid
monoethanol and diethanol amides.
[0259] If used, amides are preferably present at a level such that
the above ethoxylated nonionic surfactant and amide surfactant is
in a weight ratio of from 4:1 to 1:4, preferably from 3:1 to
1:3.
[0260] Preferred and optional cosurfactants, used at a level of
from 0.15% to 1%, are the quaternary ammonium, amine and amine
oxide surfactants described in U.S. Pat. No. 4,507,219.
[0261] Of the above, the C.sub.10-C.sub.14 alkyl trimethylammonium
salts are preferred, e.g., decyl trimethylammonium methylsulfate,
lauryl trimethylammonium chloride, myristyl trimethylammonium
bromide, and coconut trimethylammonium chloride and methylsulfate.
From 0.2% to 0.8% of monoalkyl trimethylammonium chloride is
preferred.
B. Tartrate Succinate Builder
[0262] The compositions herein preferably contain from 0 to about
10%, preferably from 0 to about 6%, by weight on an acid basis, of
a tartrate succinate builder material selected from the group
consisting of:
##STR00002##
wherein X is a salt-forming cation;
##STR00003##
wherein X is a salt-forming cation; and iii) mixtures thereof.
[0263] The tartrate succinate compounds used herein are described
in U.S. Pat. No. 4,663,071.
C. Neutralization System
[0264] The present compositions can also optionally contain from
about 0 to about 0.04 moles, preferably from about 0.01 to 0.035
moles, more preferably from about 0.015 to about 0.03 moles, per
100 grams of composition of an alkanolamine selected from the group
consisting of monoethanolamine, diethanolamine, triethanolamine,
and mixtures thereof. Low levels of the alkanolamines, particularly
monoethanolamine, are preferred to enhance product stability,
detergency performance, and odour. However, the amount of
alkanolamine should be minimized for best chlorine bleach
compatibility.
[0265] In addition, the compositions contain sodium ions, and
preferably potassium ions, at a level sufficient to neutralize the
anionic species and provide the desired product pH.
D. Suds Suppressor
[0266] Another optional component for use in the liquid detergents
herein is from 0 to about 1.5%, preferably from about 0.5% to about
1.0%, by weight of silicone based suds suppressor agent.
[0267] Silicones are widely known and taught for use as highly
effective suds controlling agents. For example, U.S. Pat. No.
3,455,839 relates to compositions and processes for defoaming
aqueous solutions by incorporating therein small amounts of
polydimethylsiloxane fluids.
[0268] Useful suds controlling silicones are mixtures of silicone
and silanated silica as described, for instance, in German Patent
Application DOS 2,124,526.
[0269] Silicone defoamers and suds controlling agents have been
successfully incorporated into granular detergent compositions by
protecting them from detergent surfactants as in U.S. Pat. Nos.
3,933,672 and 4,652,392.
[0270] A preferred silicone based suds suppressor for use herein is
a suds suppressing amount of a suds controlling agent consisting
essentially of:
[0271] (i) polydimethylsiloxane fluid having a viscosity of from
about 20 cs. to about 1500 cs. at 25.degree. C.;
[0272] (ii) from about 5 to about 50 parts per 100 parts by weight
of (i) of siloxane resin composed of (CH.sub.3).sub.3SiO.sub.1/2
units and SiO.sub.2 units in a ratio of from
(CH.sub.3).sub.3SiO.sub.1/2 units and to SiO.sub.2 units of from
about 0.6:1 to about 1.2:1; and
[0273] (iii) from about 1 to about 20 parts per 100 parts by weight
of (i) of a solid silica gel.
[0274] By "suds suppressing amount" is meant that the formulator of
the composition can select an amount of this suds controlling agent
that will control the suds to the extent desired. The amount of
suds control will vary with the detergent surfactant selected. For
example, with high sudsing surfactants, relatively more of the suds
controlling agent is used to achieve the desired suds control than
with low foaming surfactants.
E. Other Enzymes
[0275] The detergent compositions of the invention may also contain
further enzymes. For example, lipase can usefully be added in the
form of a solution or a slurry of lipolytic enzyme with carrier
material (e.g. as in EP 258 068 (Novo Nordisk A/S)).
[0276] The added amount of lipase can be chosen within wide limits,
for example 50 to 30,000 LU/g per gram of the surfactant system or
of the detergent composition, e.g. often at least 100 LU/g, very
usefully at least 500 LU/g, sometimes preferably above 1000, above
2000 LU/g or above 4000 LU/g or more, thus very often within the
range of 50-4000 LU/g, and possibly within the range of 200-1000
LU/g. In this specification, lipase units are defined as they are
in EP 258 068.
[0277] The lipolytic enzyme can be chosen among a wide range of
lipases. In particular, the lipases described in for example the
following patent specifications: EP 214 761 (Novo Nordisk A/S), 258
068, and especially lipases showing immunological cross reactivity
with antisera raised against lipase from Thermomyces lanuginosus
ATCC 22070, EP 205 208 and 206 390, and especially lipases showing
immunological cross-reactivity with antisera raised against lipase
from Chromobacter viscosum var lipolyticum NRRL B-3673, or against
lipase from Alcaligenes PL-679, ATCC 31371 and FERM-P 3783, also
the lipases described in WO 87/00859 (Gist-Brocades) and EP 204 284
(Sapporo Breweries). Suitable, in particular, are for example the
following commercially available lipase preparations: Lipolase.RTM.
Novo Nordisk A/S, Amano lipases CE, P, B, AP, M-AP, AML, and CES,
and Meito lipases MY-30, OF, and PL, also Esterase.RTM. MM (Novo
Nordisk A/S), Lipozym, SP225, SP285, (all Novo Nordisk A/S) Saiken
lipase, Enzeco lipase, Toyo Jozo lipase and Diosynth lipase (Trade
Marks), Lumafast.RTM. (Genencor Inc.), Lipomax.RTM. (Gist-Brocades
N.V.), and lipases as described in WO 94/03578 (Unilever).
[0278] Amylase can for example be used when desired, in an amount
in the range of about 1 to about 100 MU (maltose units) per gram of
detergent composition (or 0.014-1.4, e.g. 0.07-0.7, KNU/g (Novo
units)). Amylases suitable are for example Termamyl.RTM., and BAN
(Novo Nordisk A/S). Cellulase can for example be used when desired,
in an amount in the range of about 0.3 to about 35 CEVU units per
gram of the detergent composition. Suitable cellulases are for
example Celluzyme.RTM., and Carezyme.RTM. (Novo Nordisk A/S).
[0279] Other enzymes contemplated to be used in the present
invention are oxidases and peroxidases
F. Other Optional Components
[0280] Other optional components for use in the liquid detergents
herein include soil removal agents, soil release polymers,
antiredeposition agents such as tetraethylene pentamine ethoxylate
(from about 0.5% to 3%, preferably from about 1% to about 3%, by
weight), suds regulants, poly vinyl pyrolidone, carboxy methyl
cellulose, clays, and hydrotropes such as sodium cumene sulfonate,
opacifiers, antioxidants, bactericides, dyes, perfumes, and
brighteners known in the art. Such optional components generally
represent less than about 15%, preferably from about 0.5% to 10%,
more preferably from about 1% to about 10%, by weight of the
composition.
[0281] The compositions may contain from 0% to about 8%, preferably
from 0% to about 5%, by weight of a C.sub.12C.sub.14 alkenyl
succinic acid or salt thereof. These materials are of the general
formula R--CH(COOX)CH.sub.2(COOX), wherein R is a C.sub.12-C.sub.14
alkenyl group and each X is H or a suitable cation, such as sodium,
potassium, ammonium or alkanolammonium (e.g., mono-, di-, or
tri-ethanolammonium).
[0282] Specific examples are 2-dodecenyl succinate (preferred) and
2-tetradecenyl succinate.
[0283] The compositions herein optionally contain from about 0.1%
to about 1%, preferably from about 0.2% to about 0.6%, by weight of
water-soluble salts of ethylenediamine tetramethylenephosphonic
acid, diethylenetriamine pentamethylenephosphonic acid,
ethylenediamine tetraacetic add (preferred), or diethylenetriamine
pentaacetic acid (most preferred) to enhance cleaning performance
when pretreating fabrics.
[0284] Furthermore, the detergent compositions may contain from
1-35% of a bleaching agent or a bleach precursor or a system
comprising bleaching agent and/or precursor with activator
therefor.
[0285] Further optional ingredients are lather boosters,
anti-corrosion agents, soil-suspending agents, sequestering agents,
anti-soil redeposition agents, and so on.
[0286] The compositions herein preferably contain up to about 10%
of ethanol.
G. Other Properties
[0287] The instant composition usually has a pH, in a 10% by weight
solution in water at 20.degree. C., of from about 7.0 to 9.0,
preferably from about 8.0 to about 8.5.
[0288] The instant compositions can also have a Critical Micelle
Concentration (CMC) of less than or equal to 200 parts per million
(ppm), and an air/water Interfacial Tension above the CMC of less
than or equal to 32, preferably less than or equal to about 30,
dynes per centimeter at 35.degree. C. in distilled water. These
measurements are described in "Measurement of Interfacial Tension
and Surface Tension--General Review for Practical Man" C. Weser,
GIT Fachzeitschrift fur das Laboratorium, 24 (1980) 642-648 and
734-742, FIT Verlag Ernst Giebeler, Darmstadt, and "Interfacial
Phenomena--Equilibrium and Dynamic Effects", C. A. Miller and P.
Neogi, Chapter 1, pp. 29-36 (1985), Marcel Dekker, Inc. New
York.
[0289] The compositions of the invention can be used for the
washing of textile materials, especially, but without limitation
cotton and polyester based textiles and mixtures thereof. For
example washing processes carried out at temperatures of about
60-65.degree. C. or lower, e.g. about 30-35.degree. C. or lower,
are particularly suitable. It can be very suitable to use the
compositions at a rate sufficient to provide about e.g. 0.4-0.8 g/l
of surfactant in the wash liquor, although it is of course possible
to use lower or higher concentrations, if desired. Without
limitation it can for example be stated that a use-rate from about
1 to 10 g/l, e.g. from about 3-6 g/l, of the detergent formulation
is suitable for use in the case when the formulations are
substantially as in the Examples.
[0290] In this aspect the invention is especially related to:
a) A detergent composition formulated as an aqueous detergent
liquid comprising anionic surfactant, nonionic surfactant,
humectant, organic acid, caustic alkali, with a pH adjusted to a
value between 9 and 10. b) A detergent composition formulated as a
non-aqueous detergent liquid comprising a liquid nonionic
surfactant consisting essentially of linear alkoxylated primary
alcohol, triacetin, sodium triphosphate, caustic alkali, perborate
monohydrate bleach precursor, and tertiary amine bleach activator,
with a pH adjusted to a value between about 9 and 10. c) An
enzymatic liquid detergent composition formulated to give a wash
liquor pH of 9 or less when used at a rate corresponding to 0.4-0.8
g/l surfactant. d) An enzymatic liquid detergent composition
formulated to give a wash liquor pH of 8.5 or more when used at a
rate corresponding to 0.4-0.8 .mu.l surfactant. e) An enzymatic
liquid detergent composition formulated to give a wash liquor ionic
strength of 0.03 or less, e.g. 0.02 or less, when used at a rate
corresponding to 0.4-0.8 g/l surfactant. f) An enzymatic liquid
detergent composition formulated to give a wash liquor ionic
strength of 0.01 or more, e.g. 0.02 or more, when used at a rate
corresponding to 0.4-0.8 g/l surfactant.
[0291] It was found that the subtilase variants of the present
invention can also be usefully incorporated in detergent
composition in the form of bars, tablets, sticks and the like for
direct application to fabrics, hard surfaces or any other surface.
In particular, they can be incorporated into soap or soap/synthetic
compositions in bar form, wherein they exhibit a remarkable enzyme
stability. Detergent composition in the form of bars, tablets,
sticks and the like for direct application, are for example
described in South African Patent 93/7274, incorporated herein by
reference.
[0292] Accordingly, the preferred bars in accordance with this
invention comprise, in addition to the subtilase variant:
[0293] i) 25 to 80%, most preferably 25 to 70%, by weight of
detergent active which is soap or a mixture of soap and synthetic
detergent active, reckoned as anhydrous;
[0294] ii) 0 to 50% and, most preferably, 10 to 30% by weight of
water;
[0295] iii) 0 to 35% and, most preferably, 0.1 to 30% by weight
filler.
[0296] In general, the amount of subtilase variant to be included
in such compositions of the invention is such that it corresponds
with a proteolytic activity of 0.1 to 100 GU/mg based on the
composition, preferably 0.5 to 20GU/mg, most preferably 1.0 to 10
GU/mg, where GU/mg is glycine unit per milligram.
Method for Producing Mutations in Subtilase Genes
[0297] Many methods for introducing mutations into genes are well
known in the art. After a brief discussion of cloning subtilase
genes, methods for generating mutations in both random sites, and
specific sites, within the subtilase gene will be discussed.
Cloning Subtilase Genes
[0298] The gene encoding a subtilase may be cloned from any of the
organisms indicated in Table I, especially gram-positive bacteria
or fungus, by various methods, well known in the art. First a
genomic, and/or cDNA library of DNA must be constructed using
chromosomal DNA or messenger RNA from the organism that produces
the subtilase to be studied. Then, if the amino-acid sequence of
the subtilase is known, homologous, labelled oligonucleotide probes
may be synthesized and used to identify subtilisin-encoding clones
from a genomic library of bacterial DNA, or from a cDNA library.
Alternatively, a labelled oligonucleotide probe containing
sequences homologous to subtilase from another strain of bacteria
or organism could be used as a probe to identify subtilase-encoding
clones, using hybridization and washing conditions of lower
stringency.
[0299] Yet another method for identifying subtilase-producing
clones would involve inserting fragments of genomic DNA into an
expression vector, such as a plasmid, transforming
protease-negative bacteria with the resulting genomic DNA library,
and then plating the transformed bacteria onto agar containing a
substrate for subtilase, such as skim milk. Those bacteria
containing subtilase-bearing plasmid will produce colonies
surrounded by a halo of clear agar, due to digestion of the skim
milk by excreted subtilase.
Generation of Random Mutations in the Subtilase Gene
[0300] Once the subtilase gene has been cloned into a suitable
vector, such as a plasmid, several methods can be used to introduce
random mutations into the gene.
[0301] One method would be to incorporate the cloned subtilase
gene, as part of a retrievable vector, into a mutator strain of
Escherichia coli.
[0302] Another method would involve generating a single stranded
form of the subtilase gene, and then annealing the fragment of DNA
containing the subtilase gene with another DNA fragment such that a
portion of the subtilase gene remained single stranded. This
discrete, single stranded region could then be exposed to any of a
number of mutagenizing agents, including, but not limited to,
sodium bisulfite, hydroxylamine, nitrous acid, formic acid, or
hydralazine. A specific example of this method for generating
random mutations is described by Shortle and Nathans (1978, Proc.
Natl. Acad. Sci. U.S.A., 75, 2170-2174). According to the Shortle
and Nathans method, the plasmid bearing the subtilase gene would be
nicked by a restriction enzyme that cleaves within the gene. This
nick would be widened into a gap using the exonuclease action of
DNA polymerase I. The resulting single-stranded gap could then be
mutagenized using any one of the above mentioned mutagenizing
agents.
[0303] Alternatively, the subtilisin gene from a Bacillus species
including the natural promoter and other control sequences could be
cloned into a plasmid vector containing replicons for both E. coli
and B. subtilis, a selectable phenotypic marker and the M13 origin
of replication for production of single-stranded plasmid DNA upon
superinfection with helper phage IR1. Single-stranded plasmid DNA
containing the cloned subtilisin gene is isolated and annealed with
a DNA fragment containing vector sequences but not the coding
region of subtilisin, resulting in a gapped duplex molecule.
Mutations are introduced into the subtilisin gene either with
sodium bisulfite, nitrous acid or formic acid or by replication in
a mutator strain of E. coli as described above. Since sodium
bisulfite reacts exclusively with cytosine in a single-stranded
DNA, the mutations created with this mutagen are restricted only to
the coding regions. Reaction time and bisulfite concentration are
varied in different experiments such that from one to five
mutations are created per subtilisin gene on average. Incubation of
10 micrograms of gapped duplex DNA in 4 M Na-bisulfite, pH. 6.0,
for 9 minutes at 37.degree. C. in a reaction volume of 400
microliters, deaminates about 1% of cytosines in the
single-stranded region. The coding region of mature subtilisin
contains about 200 cytosines, depending on the DNA strand.
Advantageously, the reaction time is varied from about 4 minutes
(to produce a mutation frequency of about one in 200) to about 20
minutes (about 5 in 200).
[0304] After mutagenesis the gapped molecules are treated in vitro
with DNA polymerase I (Klenow fragment) to make fully
double-stranded molecules and fix the mutations. Competent E. coli
are then transformed with the mutagenized DNA to produce an
amplified library of mutant subtilisins. Amplified mutant libraries
can also be made by growing the plasmid DNA in a Mut D strain of E.
coli which increases the range of mutations due to its error prone
DNA polymerase.
[0305] The mutagens nitrous acid and formic acid may also be used
to produce mutant libraries. Because these chemicals are not as
specific for single-stranded DNA as sodium bisulfite, the
mutagenesis reactions are performed according to the following
procedure. The coding portion of the subtilisin gene is cloned in
M13 phage by standard methods and single stranded phage DNA
prepared. The single-stranded DNA is then reacted with 1 M nitrous
acid pH. 4.3 for 15-60 minutes at 23.degree. C. or 2.4 M formic
acid for 1-5 minutes at 23.degree. C. These ranges of reaction
times produce a mutation frequency of from 1 in 1000 to 5 in 1000.
After mutagenesis, a universal primer is annealed to the M13 DNA
and duplex DNA is synthesized using the mutagenized single-stranded
DNA as a template so that the coding portion of the subtilisin gene
becomes fully double-stranded. At this point the coding region can
be cut out of the M13 vector with restriction enzymes and ligated
into an un-mutagenized expression vector so that mutations occur
only in the restriction fragment. (Myers et al., Science 229,
242-257 (1985)).
Generation of Site Directed Mutations in the Subtilase Gene
[0306] Once the subtilase gene has been cloned, and desirable sites
for mutation identified and the residue to substitute for the
original ones have been decided, these mutations can be introduced
using synthetic oligonucleotides. These oligonucleotides contain
nucleotide sequences flanking the desired mutation sites; mutant
nucleotides are inserted during oligonucleotide synthesis. In a
preferred method, a single stranded gap of DNA, bridging the
subtilase gene, is created in a vector bearing the subtilase gene.
Then the synthetic nucleotide, bearing the desired mutation, is
annealed to a homologous portion of the single-stranded DNA. The
remaining gap is then filled in by DNA polymerase I (Klenow
fragment) and the construct is ligated using T4 ligase. A specific
example of this method is described in Morinaga et al., (1984,
Biotechnology 2, 646-639). According to Morinaga et al., a fragment
within the gene is removed using restriction endonuclease. The
vector/gene, now containing a gap, is then denatured and hybridized
to a vector/gene which, instead of containing a gap, has been
cleaved with another restriction endonuclease at a site outside the
area involved in the gap. A single-stranded region of the gene is
then available for hybridization with mutated oligonucleotides, the
remaining gap is filled in by the Klenow fragment of DNA polymerase
1, the insertions are ligated with T4 DNA ligase, and, after one
cycle of replication, a double-stranded plasmid bearing the desired
mutation is produced. The Morinaga method obviates the additional
manipulation of constructing new restriction sites, and therefore
facilitates the generation of mutations at multiple sites. U.S.
Reissue Pat. No. 34,606 by Estell et al., issued May 10, 1994, is
able to introduce oligonucleotides bearing multiple mutations by
performing minor alterations of the cassette, however, an even
greater variety of mutations can be introduced at any one time by
the Morinaga method, because a multitude of oligonucleotides, of
various lengths, can be introduced.
Expression of Subtilase Mutants
[0307] According to the invention, a mutated subtilase gene
produced by methods described above, or any alternative methods
known in the art, can be expressed, in enzyme form, using an
expression vector. An expression vector generally falls under the
definition of a cloning vector, since an expression vector usually
includes the components of a typical cloning vector, namely, an
element that permits autonomous replication of the vector in a
microorganism independent of the genome of the microorganism, and
one or more phenotypic markers for selection purposes. An
expression vector includes control sequences encoding a promoter,
operator, ribosome binding site, translation initiation signal,
and, optionally, a repressor gene or various activator genes. To
permit the secretion of the expressed protein, nucleotides encoding
a "signal sequence" may be inserted prior to the coding sequence of
the gene. For expression under the direction of control sequences,
a target gene to be treated according to the invention is operably
linked to the control sequences in the proper reading frame.
Promoter sequences that can be incorporated into plasmid vectors,
and which can support the transcription of the mutant subtilase
gene, include but are not limited to the prokaryotic beta-lactamase
promoter (Villa-Kamaroff, et al. (1978) Proc. Natl. Acad. Sci.
U.S.A. 75, 3727-3731) and the tac promoter (DeBoer, et al. (1983)
Proc. Natl. Acad. Sci. U.S.A. 80, 21-25). Further references can
also be found in "Useful proteins from recombinant bacteria" in
Scientific American (1980) 242, 74-94.
[0308] According to one embodiment B. subtilis is transformed by an
expression vector carrying the mutated DNA. If expression is to
take place in a secreting microorganism such as B. subtilis a
signal sequence may follow the translation initiation signal and
precede the DNA sequence of interest. The signal sequence acts to
transport the expression product to the cell wall where it is
cleaved from the product upon secretion. The term "control
sequences" as defined above is intended to include a signal
sequence, when it is present.
[0309] Other host systems known to the skilled person are also
contemplated for the expression and production of the protease
variants of the invention. Such host systems comprise fungi,
including filamentous fungi, plant, avian and mammalian cells, as
well as others.
Materials and Methods
Strains:
[0310] B. subtilis 309 and 147 are variants of Bacillus lentus,
deposited with the NCIB and accorded the accession numbers NCIB
10309 and 10147, and described in U.S. Pat. No. 3,723,250
incorporated by reference herein.
[0311] E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J.
Mol. Biol. 138 179-207), was made r.sup.-,m.sup.+ by conventional
methods and is also described in U.S. Patent Application Ser. No.
039,298.
Proteolytic Activity
[0312] In the context of this invention proteolytic activity is
expressed in Kilo NOVO Protease Units (KNPU). The activity is
determined relatively to an enzyme standard (SAVINASE.TM.), and the
determination is based on the digestion of a dimethyl casein (DMC)
solution by the proteolytic enzyme at standard conditions, i.e.
50.degree. C., pH 8.3, 9 min. reaction time, 3 min. measuring time.
A folder AF 220/1 is available upon request to Novo Nordisk A/S,
Denmark, which folder is hereby included by reference.
[0313] A GU is a Glycine Unit, defined as the proteolytic enzyme
activity which, under standard conditions, during a 15 minutes'
incubation at 40.degree. C., with N-acetyl casein as substrate,
produces an amount of NH.sub.2-group equivalent to 1 .mu.mole of
glycine.
[0314] Enzyme activity can also be measured using the PNA assay,
according to reaction with the soluble substrate
succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol,
which is described in the Journal of American Oil Chemists Society,
Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., and Smith, L.
A., (1988).
EXAMPLES
[0315] For the generation of enzyme variants according to the
invention the same materials and methods as described in i.a. WO
89/06279 (Novo Nordisk A/S), EP 130,756 (Genentech), EP 479,870
(Novo Nordisk A/S), EP 214,435 (Henkel), WO 87/04461 (Amgen), WO
87/05050 (Genex), EP application no. 87303761 (Genentech), EP
260,105 (Genencor), WO 88/06624 (Gist-Brocades NV), WO 88/07578
(Genentech), WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 88/08164
(Genex), Thomas et al. (1985) Nature, 318 375-376; Thomas et al.
(1987) J. Mol. Biol., 193, 803-813; Russel and Fersht (1987) Nature
328 496-500. Other methods well established in the art may also be
used.
Example 1
Construction and Expression of Enzyme Variants
[0316] A vector suited to a synthetic gene coding for subtilase 309
and its mutants was constructed. It is essentially a pUC19 plasmid
[Yanish-Perron and Messing (1985) Gene; 33 103-119], in which the
multiple cloning site has been replaced by a linker containing the
restriction sites used to separate five sub-fragments constituting
the gene. The new linker was inserted into EcoRI-HindIII cut pUC19
thereby destroying these sites. The details of this construction
are described in WO 92/19729 on pages 25-26 and in FIG. 1 (sheets
1/7-7/7) thereof, the content of which is hereby included by
reference.
[0317] Each subfragment was made from 6 to 12 oligonucleotides. The
oligonucleotides were synthesized on an automatic DNA synthesizer
using phosphoramidite chemistry on a controlled glass support
[Beaucage and Carruthers (1981); Tetrahedron Letters 22
1859-1869].
[0318] The five subfragments were isolated on a 2% agarose gel and
inserted into pSX191. The sequence was verified by
dideoxynucleotide sequencing. Fragments A-E were isolated and
ligated together with KpnI-BamHI cut pSX191. The ligation mixtures
were used to transform competent E. coli MC1000 r.sup.-,m.sup.+
selecting for ampicillin resistance. The 850 bp KpnI-BamHI fragment
that constitutes the part of the subtilisin 309 gene coding for the
mature part of the enzyme was then used to replace the wild type
gene on pSX212 giving rise to pSX222, which was then transformed
into a competent B. subtilis strain. After fermentation of the
transformed strain and purification of the enzyme it was shown that
the product was indistinguishable from the wild type product.
[0319] Protease variants derived from the synthetic gene are made
by using oligonucleotides with altered sequence at the place(s)
where mutation is wanted (e.g. with sequences as given below) and
mixing them with the rest of the oligonucleotides appropriate to
the synthetic gene. Assembly of the variant gene is carried out
with the variant materials in a manner otherwise analogous to that
described above. Further information on synthetic genes generally
is available in Agarval et al. (1970); Nature; 227, 27-34.
[0320] A KpnI site was introduced into the beginning of the
subtilase 309 synthetic gene encoding the mature part of the
enzyme. The method used is called oligonucleotide directed
double-strand break repair mutagenesis and is described by Mandecki
(1986) Proc. Nat. Acad. Sci. USA 83 7177-7181. pSX172 is opened
with NcoI at the beginning of the mature part of the subtilase 309
gene and is mixed with the oligonucleotide NOR 789 (see WO
92/19729), heated to 100.degree. C., cooled to 0.degree. C., and
transformed into E. coli. After retransformation, the recombinants
can be screened by colony hybridisation using 32-P-labelled NOR
789. The recombinants that turned out to be positive during the
screening had the KpnI site introduced right in front of NcoI by
changing two bases without changing the amino acid sequence. pSX172
is described in EP 405 901. The KpnI site so created is inserted
into pSX120 on a 400-bp PvuI-NheI fragment, giving rise to pSX212.
pSX120 is also described in EP 405 901.
[0321] The synthetic gene is inserted between KpnI and BamHI on
pSX212, giving rise to pSX222.
[0322] Examples of mutations and corresponding sequences of
oligonucleotides are-as follows:
##STR00004##
[0323] These oligonucleoties were combined with the rest of the
oligonucleotides from the synthetic gene that was not changed.
Example 2
Purification of Enzyme Variants
[0324] This procedure relates to purification of a 10 liter scale
fermentation of subtilisin 147, subtilisin 309 or mutants
thereof.
[0325] Approximately 8 liters of fermentation broth were
centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The
supernatants were adjusted to pH 6.5 using 10% acetic acid and
filtered on Seitz Supra S100 filter plates.
[0326] The filtrates were concentrated to approximately 400 ml
using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF
cartridge. The UF concentrate was centrifuged and filtered prior to
absorption at room temperature on a Bacitracin affinity column at
pH 7. The protease was eluted from the Bacitracin column at room
temperature using 25% 2-propanol and 1 M sodium chloride in a
buffer solution with 0.01 dimethylglutaric acid, 0.1 M boric acid
and 0.002 M calcium chloride adjusted to pH 7.
[0327] The fractions with protease activity from the Bacitracin
purification step were combined and applied to a 750 ml Sephadex
G25 column (5 cm dia.) equilibrated with a buffer containing 0.01
dimethylglutaric acid, 0.2 M boric acid and 0.002 M calcium
chloride adjusted to pH 6.5.
[0328] Fractions with proteolytic activity from the Sephadex G25
column were combined and applied to a 150 ml CM Sepharose CL 6B
cation exchange column (5 cm dia.) equilibrated with a buffer
containing 0.01 M dimethylglutaric acid, 0.2 M boric acid, and
0.002 M calcium chloride adjusted to pH 6.5.
[0329] The protease was eluted using a linear gradient of 0-0.1 M
sodium chloride in 2 liters of the same buffer (0-0.2 M sodium
chloride in case of subtilisin 147).
[0330] In a final purification step protease containing fractions
from the CM Sepharose column were combined and concentrated in an
Amicon ultrafiltration cell equipped with a GR81PP membrane (from
the Danish Sugar Factories Inc.).
[0331] By using the techniques of Example 1 for the construction
and the above isolation procedure the following subtilisin 309
variants were produced and isolated:
A: G159I
B: S164I
C: Y167I
D: R170I
E: R170L
F: R170M
G: R170F
H: G195F
I: S57P+R170L
J: R170L+N218S
K: S57P+R170L+N218S
L: R170L+N218S+M222A
M: S57P+R170L+S188P+A194P
N: Y167I+R170L
O: S57P+R170L+Q206E
P: R170L+Q206E
Q: Y167I+R170L+Q206E
R: Y167I+R170L+A194P
S: Y167I+R170L+N218S
T: Y167I+R170L+A194P+N218S
U: Y167I+Y171I
V: R170G
W: R170C
X: Y171I
Y: Y167I+R170L+N218S
Example 3
Stability in Detergent Compositions Comprising Enzyme Variants
Example D1
[0332] An (isotropic) aqueous detergent liquid according to an
embodiment of the invention is formulated to contain:
TABLE-US-00004 Ingredient % NaLAS 8.0 Neodol 25-9 8.0 AES 25-3S
14.0 NaCitrate.cndot.2H.sub.2O 5.0 Propylene Glycol 5.0 Sorbitol
4.5 F-dye Tinopal UNPA-GX 0.15 Lytron 614 Opacifier 0.03 Kathon
Preservative 0.0003 Acid Blue 80 0.00117 Acid Violet 48 0.0033
SAVINASE .RTM. 16L 0.25 LIPOLASE .RTM. 100L 0.70 Fragrance 0.15
Water ad 100.0
[0333] The pH is adjusted to 7.1.
TABLE-US-00005 TABLE III Residual enzyme activity (in percentage or
original activity) after storage at 37.degree. C. for Example D1
comprising the BLS309 variant S57P + R170L + N218S. Storage time
(days) Wild-type S57P + R170L + N218S 0 100 100 3 44 74 7 11 50 10
5 36 14 7 27
[0334] From Table III it is evident that the variant
S57P+R170L+N218S exhibits a remarkably improved stability in this
type of detergent. Moreover, the variant S57P+R170L+N218S possesses
excellent compatibility towards lipase.
TABLE-US-00006 TABLE IV Residual lipase activity (in percentage of
original activity) after storage at 37.degree. C. for Example D1
comprising the BLS309 variant S57P + R170L + N218S and LIPOLASE
.RTM.. Storage time (days) LIPOLASE .RTM. plus: Wild-type S57P +
R170L + N218S 0 100 100 3 38 67 7 24 44 10 22 33 14 21 27
[0335] From Table IV it is apparent that, in addition to the
stability of the protease, the compatibility of the protease is
also improved.
Example D2
[0336] A non-aqueous detergent liquid according to an embodiment of
the invention is formulated using 38.5% C13-C15 linear primary
alcohol alkoxylated with 4.9 mol/mol ethylene oxide and 2.7 mol/mol
propylene oxide, 5% triacetin, 30% sodium triphosphate, 4% soda
ash, 15.5% sodium perborate monohydrate containing a minor
proportion of oxoborate, 4% TAED, 0.25% EDTA of which 0.1% as
phosphonic acid, Aerosil 0.6%, SCMC 1%, and 0.6% protease. The pH
is adjusted to a value between 9 and 10, e.g. about 9.8.
Example D3
[0337] Structured liquid detergents can for example contain, in
addition to a protease as described herein, 2-15% nonionic
surfactant, 5-40% total surfactant, comprising nonionic and
optionally anionic surfactant, 5-35% phosphate-containing or
non-phosphate containing builder, 0.2-0.8% polymeric thickener,
e.g. cross-linked acrylic polymer with m.w. over 10.sup.6, at least
10% sodium silicate, e.g. as neutral waterglass, alkali (e.g.
potassium-containing alkali) to adjust to desired pH, preferably in
the range 9-10 or upwards, e.g. above pH 11, with a ratio sodium
cation:silicate anion (as free silica) (by weight) less than 0.7:1,
and viscosity of 0.3-30 Pas (at 20.degree. C. and 20
s-.sup.-1).
[0338] Suitable examples contain about 5% nonionic surfactant
C13-15 alcohol alkoxylated with about 5 EO groups per mole and with
about 2.7 PO groups per mole, 15-23% neutral waterglass with 3.5
weight ratio between silica and sodium oxide, 13-19% KOH, 8-23%
STPP, 0-11% sodium carbonate, 0.5% Carbopol 941 (TM).
[0339] Protease may be incorporated at for example 0.5%.
Example D4
TABLE-US-00007 [0340] (Decoupling polymer liquid) Priolene 6907 4.5
KOH 10 Ethoxylated Alcohol.cndot.7EO (Synperonic A7) 4.5
Ethoxylated Alcohol.cndot.3EO (Synperonic A3) 4.5 Zeolite 4A 15
Fluorescer Tinopal CBS-X 0.08 Narlex DC1 1 Citric acid 8.23
Antifoam silicone DB100 0.3 LAS acid 16.5 Perfume 0.5 Water to
100
TABLE-US-00008 TABLE V Residual enzyme activity (in percentage of
original activity) after storage at 37.degree. C. for Example D4
comprising the R170L variant of BLS309. Storage time (days) R170L
Wild-type 0 100 100 2 98 73 4 96 66 10 94 46 33 87 8 81 78 2.1 101
71 0
[0341] From Table V it is evident that the R170L variant exhibits a
remarkably improved stability in this type of detergent.
TABLE-US-00009 TABLE VI Enzyme Storage Y167I + R170L + (days) WT
R170M S57P + R170L + Q206E N218S 0 100 100 100 100 0.1 90.2 78 97
94 1 58 53 95 68 2 40 34 87 55 5 16 27 75 29 6 12 22 73 24 8 8 19
77 17 14 2 11 52 4
[0342] From Table VI it can be seen that the variants tested
exhibit improved stability in comparison to the wild type enzyme in
this type of detergent
Example D5
TABLE-US-00010 [0343] (Decoupling polymer liquid) Priolene 6907 4.5
KOH 10 Ethoxylated Alcohol.cndot.7EO (Synperonic A7) 4.5
Ethoxylated Alcohol.cndot.3EO (Synperonic A3) 4.5 Zeolite 4A 15
Fluorescer Tinopal CBS-X 0.08 Narlex DC1 1 Citric acid 8.23
Antifoam silicone DB100 0.3 LAS acid 16.5 Lipolase .RTM. 100L 0.6
Perfume 0.5 Water to 100
TABLE-US-00011 TABLE VII Residual enzyme activity (in percentage of
original activity) after storage at 37.degree. C. for Example D5
comprising the BLS309 variant S57P + R170L + N218S. Residual
protease Residual lipase Storage activity activity time S57P +
R170L + S57P + R170L + (days) N218S Wild-type R170L N218S 0 100 100
100 100 2 -- 27 41 94 5 97 9 15 76 8 87 4 7 71 12 91 2.4 12 78 28
100 2.4 12 70
[0344] From Table VII it is evident that the variant
S57P+R170L+N218S exhibits a remarkably improved stability in this
type of detergent. Moreover the variant S57P+R170L+N218S possesses
excellent compatibility towards lipase.
Example D6
[0345] Soap bars were produced containing 49.7 wt. 80/20
tallow/coconut soap, 49.0% water, 20% sodium citrate, 1.0% citric
acid and 0.031% protease. After preparation of the soap bars they
were stored at ambient temperature and after specific time
intervals samples were taken and measured for protease activity.
The stability data are given below:
TABLE-US-00012 TABLE VIII Enzyme Storage R170L + N218S + (days) WT
R170L S57P R170L + Y167I 0 100 100 100 100 1 50 100 97 94 2 25 91
100 83 3 -- 100 94 80 6 -- 98 89 90 10 0 100 94 71 17 -- 93 80 73
27 -- 95 86 70
[0346] From Table VIII it is evident that the subtilase variants
R170L, R170L+N218S+S57P and R170L+Y167I exhibit a remarkably
improved stability in this type of detergent.
Example D7
[0347] Soap bars were produced containing 63.88% 80/20
tallow/coconut soap, 1% coconut fatty acid, 25.1% water, 10% sodium
citrate and 0.021% protease. The laundry soap bars were stored at
37.degree. C. and after specific time intervals samples were taken
and measured for protease activity.
TABLE-US-00013 TABLE IX Stability data: Enzyme Storage (days) WT
R170L + N218S + S57P 0 100 100 10 10 90.1 14 -- 81.5 20 0 91.4 31
-- 72.8 35 -- 79 45 -- 78
[0348] From Table IX it is evident that the subtilase variant
R170L+N218S+S57P exhibits a remarkably improved stability in this
type of detergent.
Example 4
Wash Performance of Detergent Compositions Comprising Enzyme
Variants
[0349] The following examples provide results from a number of
washing tests that were conducted under the conditions
indicated.
Experimental Conditions
TABLE-US-00014 [0350] TABLE X Experimental conditions for
evaluation of Subtilisin 309 variants. Detergent Protease model
detergent '95 Detergent dose 3 g/l pH 9.5 Wash time 15 min.
Temperature 15.degree. C. Water hardness 9.degree. dH ~1.61 mM
Ca.sup.2+/Mg.sup.2+ Enzymes Subtilisin 309 variants as listed below
Enzyme conc. 0; 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0 mg/l Test
system 150 ml beakers with a stirring rod. Cloth/volume 5 cloths (O
2.5 cm)/50 ml Detergent solution. Cloth Cotton soiled with grass
juice
[0351] Subsequent to washing the cloths were flushed in tap water
and air-dried.
[0352] The above model detergent is a simple detergent formulation.
The most characteristic features are that STP is used as builder
and the content of anionic tenside (LAS) is quite high. Further the
pH is adjusted to 9.5, which is low for a powder detergent.
Table XI
[0353] The composition of the model detergent is as follows:
25% STP (Na.sub.5P.sub.3O.sub.10)
25% Na.sub.2SO.sub.4
10% Na.sub.2CO.sub.3
20% LAS (Nansa 80S)
5% NI (Dobanol 25-7)
5% Na.sub.2Si.sub.2O.sub.5
0.5% Carboxymethylcellulose (CMC)
[0354] 9.5% water dose: 3 g/l pH is adjusted to 9.5
[0355] Measurement of remission (R) on the test material has been
done at 460 nm using an Elrepho 2000 photometer (without UV). The
measured values have been fitted to the expression:
.DELTA.R=(a.DELTA.R.sub.maxc)/(.DELTA.R.sub.max+ac)
[0356] The improvement factor is calculated by use of the initial
slope of the curve: IF=a/a.sub.ref.
.DELTA.R is the wash effect of the enzyme in remission units. a is
the initial slope of the fitted curve (c.fwdarw.0). a.sub.ref. is
the initial slope for the reference enzyme. c is the enzyme
concentration in mg/l .DELTA.R.sub.max is the theoretical maximum
wash effect of the enzyme in remission units
(c.fwdarw..infin.).
TABLE-US-00015 TABLE XII Variants and improvement factors for
subtilisin 309. Designation Variant IF S003* R170Y 2.8 S004* R170Y
+ G195E 2.6 S012* R170Y + G195E + K251E 1.6 G R170F 3.3 E R170L 3.8
F R170M 2.4 D R170I 4.1 I S57P + R170L 3.9 J R170L + N218S 1.6 K
S57P + R170L + N218S 2.3 N Y167I + R170L 6.2 P R170L + Q206E 2.6 V
R170G 2.0 W R170C 3.4 O S57P + R170L + Q206E 2.9 Q Y167I + R170L +
Q206E 2.4 R Y167I + R170L + A194P 5.1 X Y171I 1.2 Y Y167I + R170L +
N218S 4.0 T Y167I + R170I + A194P + N218S 3.6 *Described in WO
91/00345
[0357] As it can be seen from Table XII all the subtilisin 309
variants of the invention exhibits an improvement in wash
performance.
TABLE-US-00016 TABLE XIII Variants and improvement factors for
subtilisin 309 in a detergent as described in Example D4
Designation Variant IF S003* R170Y 1.5 F R170M 1.2 O S57P + R170L +
Q206E 5.0 X Y171I 4.2 R Y167I + R170L + A194P 1.2 T Y167I + R170L +
A194P + N218S 2.0 Y Y167I + R170L + N218S 2.3 *Described in WO
91/00345
[0358] As it can be seen from Table XIII all the Subtilisin 309
variants of the invention exhibits an improvement in wash
performance.
Sequence CWU 1
1
41139DNAArtificial SequenceSynthetic Construct 1aattcaggtg
caggctcaat cagctatccg gcgctctat 39241DNAArtificial
SequenceSynthetic Construct 2gtccacgtcc gagttagtcg ataggccgcg
agatacgctt g 41339DNAArtificial SequenceSynthetic Construct
3aattcaggtg caggctcaat cagctatccg gcgatctat 39441DNAArtificial
SequenceSynthetic Construct 4gtccacgtcc gagttagtcg ataggccgct
agatacgctt g 41536DNAArtificial SequenceSynthetic Construct
5agctttgtac caggggaacc gccgactcaa gatggg 36638DNAArtificial
SequenceSynthetic Construct 6aacatggtcc ccttggcggc tgagttctac
ccttaccc 387275PRTBacillus amyloliquefaciens 7Ala Gln Ser Val Pro
Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu1 5 10 15His Ser Gln Gly
Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp20 25 30Ser Gly Ile
Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala35 40 45Ser Met
Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His50 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 Leu85
90 95Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile
Glu100 105 110Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser
Leu Gly Gly115 120 125Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val
Asp Lys Ala Val Ala130 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 Ala165 170 175Val Gly Ala Val
Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val180 185 190Gly Pro
Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr195 200
205Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala
Ser210 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 Lys245 250 255Leu Gly Asp Ser Phe Tyr Tyr Gly
Lys Gly Leu Ile Asn Val Gln Ala260 265 270Ala Ala
Gln275849PRTBacillus subtilis 8Gly Gly Pro Thr Gly Ser Thr Ala Leu
Lys Thr Val Val Asp Lys Ala1 5 10 15Val Ser Ser Glu Gly Ser Ser Gly
Ser Thr Ser Thr Val Gly Tyr Pro20 25 30Ala Lys Tyr Pro Phe Ser Ser
Ala Gly Ser Glu Leu Asp Val Met Ala35 40 45Pro949PRTBacillus
subtilis 9Gly Gly Pro Ser Gly Ser Thr Ala Leu Lys Gln Ala Val Asp
Lys Ala1 5 10 15Tyr Ala Ser Ser Cys Ser Ser Gly Ser Gln Asn Thr Ile
Gly Tyr Pro20 25 30Ala Lys Tyr Asp Phe Ser Ser Val Gly Ala Glu Leu
Glu Val Met Ala35 40 45Pro1049PRTBacillus licheniformis 10Gly Gly
Pro Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala1 5 10 15Tyr
Ala Arg Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro20 25
30Ala Lys Tyr Asp Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala35
40 45Pro1145PRTBacillus alcalophilus 11Gly Ser Pro Ser Pro Ser Ala
Thr Leu Glu Gln Ala Val Asn Ser Ala1 5 10 15Thr Ser Arg Ser Gly Ala
Gly Ser Ile Ser Tyr Pro Ala Arg Tyr Ala20 25 30Phe Ser Gln Tyr Gly
Ala Gly Leu Asp Ile Val Ala Pro35 40 451245PRTUnknownBacillus YaB
12Gly Ser Ser Ala Gly Ser Ala Thr Met Glu Gln Ala Val Asn Gln Ala1
5 10 15Thr Ala Ser Ser Cys Ala Gly Asn Val Gly Phe Pro Ala Arg Tyr
Lys20 25 30Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val Ala Pro35 40
451345PRTBacillus lentus 13Gly Ser Thr Ser Gly Ser Ser Thr Leu Glu
Leu Ala Val Asn Arg Ala1 5 10 15Asn Asn Ala Thr Gly Arg Gln Gly Val
Asn Tyr Pro Ala Arg Tyr Ser20 25 30Phe Ser Thr Tyr Gly Pro Glu Ile
Glu Ile Ser Ala Pro35 40 451446PRTBacillus subtilis 14Gly Thr Thr
Ser Asp Ser Lys Ile Leu His Asp Ala Val Asn Lys Ala1 5 10 15Tyr Glu
Gln Asp Gly Asn Gly Lys Pro Val Asn Tyr Pro Ala Ala Tyr20 25 30Ser
Phe Ser Thr Thr Gly Asp Glu Val Glu Phe Ser Ala Pro35 40
451550PRTBacillus subtilis 15Gly Gly Pro Ser Asp Val Pro Glu Leu
Glu Glu Ala Val Lys Asn Ala1 5 10 15Val Lys Asn Glu Gly Asp Gly Asp
Glu Arg Thr Glu Glu Leu Ser Tyr20 25 30Pro Lys Ala Tyr Asn Phe Ser
Asn Ala Asn Lys Glu Ile Asp Leu Val35 40 45Ala
Pro501645PRTThermoactinomyces vulgaris 16Gly Gly Thr Val Gly Asn
Ser Gly Leu Gln Gln Ala Val Asn Tyr Ala1 5 10 15Trp Asn Lys Ala Gly
Asn Thr Ala Pro Asn Tyr Pro Ala Tyr Tyr Ser20 25 30Phe Ser Thr Tyr
Gly Ser Trp Val Asp Val Ala Ala Pro35 40 451747PRTDichelobacter
nodosus 17Gly Gly Gly Gly Gly Cys Ser Gln Asn Ser Gln Arg Met Ile
Asp Lys1 5 10 15Thr Thr Asn Leu Glu Asn Gln Asp Ala Ser Arg Thr Trp
Pro Ser Ser20 25 30Cys Asn Phe Ser Asn Tyr Gly Ala Arg Val His Leu
Ala Ala Pro35 40 451847PRTXanthomonas campestris 18Gly Gly Gly Gly
Ser Cys Ser Thr Thr Met Gln Asn Ala Ile Asn Gly1 5 10 15Ala Val Ser
Arg Asp Ala Ser Asn Val Ser Gly Ser Leu Pro Ala Asn20 25 30Cys Ala
Tyr Ser Asn Phe Gly Thr Gly Ile Asp Val Ser Ala Pro35 40
451958PRTBacillus subtilis 19Gly Gly Gly Ser Gly Leu Asp Glu Trp
Tyr Arg Asp Met Val Asn Ala1 5 10 15Trp Arg Ala Ala Thr Asp Leu Phe
Ile Pro Gly Gly Pro Gly Ser Ile20 25 30Ala Asn Pro Ala Asn Tyr Pro
Phe Ser Leu Gln Gly Pro Ser Pro Tyr35 40 45Asp Glu Ile Lys Pro Glu
Ile Ser Ala Pro50 552059PRTEnterococcus faecalis 20Gly Ser Tyr Lys
Asn Met Glu Ile Asp Asp Glu Arg Phe Thr Val Glu1 5 10 15Ala Phe Arg
Lys Val Val Asn Tyr Ala Arg Lys Asn Glu Ser Arg Asp20 25 30Ile Ser
Thr Gly Asn Glu Lys His Ile Pro Gly Gly Leu Glu Tyr Ser35 40 45Asn
Tyr Gly Ser Asn Val Ser Ile Tyr Gly Pro50 552168PRTStaphylococcus
epidermidis 21Gly Asn Tyr Leu Ile Arg Asp Asp Glu Lys Val Asp Tyr
Asp Ala Leu1 5 10 15Gln Lys Ala Ile Asn Tyr Ala Gln Lys Lys Asp Gly
Ile Asn Val Lys20 25 30Lys Val Lys Glu Ile Asn Lys Lys Arg Thr Ser
Lys Lys Val Tyr Asp35 40 45Ser Pro Ala Asn Leu Asn Phe Ser Asn Tyr
Gly Asn Asn Phe Ile Asp50 55 60Leu Met Thr
Ile652271PRTStreptococcus pyogenes 22Gly Asn Ala Ala Leu Ala Tyr
Ala Asn Leu Pro Asp Glu Thr Lys Lys1 5 10 15Ala Phe Asp Tyr Ala Lys
Ser Lys Asp Ser Ser Phe Gly Gly Lys Thr20 25 30Arg Leu Pro Leu Ala
Asp His Pro Asp Tyr Gly Val Val Gly Thr Pro35 40 45Ala Ala Ala Asp
Phe Ser Ser Trp Gly Leu Thr Ala Asp Gly Asn Ile50 55 60Lys Pro Asp
Ile Ala Ala Pro65 702373PRTLactococcus lactis 23Gly Ser Asn Ser Gly
Asn Gln Thr Leu Glu Asp Pro Glu Leu Ala Ala1 5 10 15Val Gln Asn Ala
Asn Glu Ser Ser Gly Thr Ser Gly Ser Ala Thr Glu20 25 30Gly Val Asn
Lys Asp Tyr Tyr Gly Leu Gln Asp Asn Glu Met Val Gly35 40 45Ser Pro
Gly Thr Ser Arg Phe Thr Ser Tyr Gly Pro Val Ser Asn Leu50 55 60Ser
Phe Lys Pro Asp Ile Thr Ala Pro65 702463PRTSerratia marcescens
24Gly Ile Ala Pro Asp Gln Pro Val Pro Thr Gly Gly His Ser Ala Met1
5 10 15Ser Thr Leu Leu Arg Ala Ala Arg His Tyr Asn Asn Tyr Asn Ile
Pro20 25 30Glu Ala Gln Lys Ser Leu Pro Tyr Ala Phe Pro Asp Val Leu
Asn Ser35 40 45Ser Thr Ser Cys Gly Gln Thr Ala Ser Tyr Cys Val Ser
Ala Pro50 55 602554PRTAnabaena variabilis 25Gly Pro Pro Asp Gly Lys
Gln Lys Val Pro Leu Pro Asp Ser Thr Arg1 5 10 15Leu Ala Met Asp Tyr
Ala Ile Asn Lys Gly Gly Asn Glu Ser Val Asp20 25 30Asn Asp Gly Tyr
Ala Ser Tyr Glu Lys Tyr Ser Asp Phe Gly Thr Ala35 40 45Val Trp Cys
Ala Phe Pro502658PRTMouse 26Gly Pro Asn Asp Asp Gly Lys Thr Val Glu
Gly Pro Gly Arg Leu Ala1 5 10 15Gln Lys Ala Phe Glu Tyr Gly Val Lys
Gln Gly Gly Gly Arg Gln Gly20 25 30Asp Asn Cys Asp Cys Asp Gly Tyr
Thr Asp Ser Ile Tyr Tyr Ala Glu35 40 45Lys Cys Ser Ser Thr Leu Ala
Thr Ser Tyr50 552757PRTHuman 27Gly Pro Thr Asp Asn Gly Lys Thr Val
Asp Gly Pro Arg Asp Val Thr1 5 10 15Leu Gln Ala Met Ala Asp Gly Val
Asn Lys Gly Gly Gly Ser Tyr Asp20 25 30Asp Cys Asn Cys Asp Gly Tyr
Ala Ser Ser Met Trp Tyr Asp Glu Ser35 40 45Cys Ser Ser Thr Leu Ala
Ser Thr Phe50 552858PRTHuman 28Gly Pro Glu Asp Asp Gly Lys Thr Val
Asp Gly Pro Ala Arg Leu Ala1 5 10 15Glu Glu Ala Phe Phe Arg Gly Val
Ser Gln Gly Gly Gly Arg Glu His20 25 30Asp Ser Cys Asn Cys Asp Gly
Tyr Thr Asn Ser Ile Tyr Tyr Ser Glu35 40 45Ala Cys Ser Ser Thr Leu
Ala Thr Thr Tyr50 552958PRTUnknownDrosophila 29Gly Pro Asp Asp Asp
Gly Lys Thr Val Asp Gly Pro Gly Glu Leu Ala1 5 10 15Ser Arg Ala Phe
Ile Glu Gly Thr Thr Lys Gly Gly Gly Arg Glu Gln20 25 30Asp Asn Cys
Asn Cys Asp Gly Tyr Thr Asn Ser Ile Trp Tyr Ser Glu35 40 45Lys Cys
Ser Ser Thr Leu Ala Thr Thr Tyr50 553058PRTKluyveromyces lactis
30Gly Pro Ser Asp Asp Gly Lys Thr Met Gln Ala Pro Asp Thr Leu Val1
5 10 15Lys Lys Ala Ile Ile Lys Gly Val Thr Glu Gly Gly Gly Met Phe
Gly20 25 30Asp Ser Cys Asn Phe Asp Gly Tyr Thr Asn Ser Ile Phe Tyr
Ser Glu35 40 45Ser Cys Ser Ala Val Met Val Val Thr Tyr50
553158PRTSaccharomyces cerevisiae 31Gly Pro Ala Asp Asp Gly Arg His
Leu Gln Gly Pro Ser Asp Leu Val1 5 10 15Lys Lys Ala Leu Val Lys Gly
Val Thr Glu Gly Gly Gly Thr Arg Gly20 25 30Asp Asn Cys Asn Tyr Asp
Gly Tyr Thr Asn Ser Ile Tyr Tyr Ser Glu35 40 45Gly Cys Ser Ala Val
Met Ala Val Thr Tyr50 553245PRTVibrio alginolyticus 32Gly Gly Gly
Gln Ser Val Ala Leu Asp Ser Ala Val Gln Ser Ala Val1 5 10 15Gln Ser
Ser Asn Ala Asp Ala Cys Asn Tyr Ser Pro Ala Arg Val Ala20 25 30Phe
Ser Asn Trp Gly Ser Cys Val Asp Val Phe Ala Pro35 40
453345PRTUnknownThermus 33Gly Gly Gly Ala Ser Thr Ala Leu Asp Thr
Ala Val Met Asn Ala Ile1 5 10 15Asn Ala Asp Asn Arg Asp Ala Cys Phe
Tyr Ser Pro Ala Arg Val Thr20 25 30Phe Ser Asn Tyr Gly Arg Cys Leu
Asp Leu Phe Ala Pro35 40 453445PRTThermus aquaticus 34Gly Gly Gly
Val Ser Thr Ala Leu Asp Asn Ala Val Lys Asn Ser Ile1 5 10 15Ala Ala
Asp Asn Ala Asn Ala Cys Asn Tyr Ser Pro Ala Arg Val Ala20 25 30Phe
Ser Asn Tyr Gly Ser Cys Val Asp Leu Phe Ala Pro35 40
453545PRTTritirachium album 35Gly Gly Gly Tyr Ser Ser Ser Val Asn
Ser Ala Ala Ala Arg Leu Gln1 5 10 15Ser Ser Asn Asn Ala Asp Ala Arg
Asn Tyr Ser Pro Ala Ser Glu Pro20 25 30Phe Ser Asn Tyr Gly Ser Val
Leu Asp Ile Phe Gly Pro35 40 453645PRTTritirachium album 36Gly Gly
Gly Tyr Ser Ser Ser Val Asn Ser Ala Ala Ala Asn Leu Gln1 5 10 15Gln
Ser Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Ser20 25
30Phe Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Ala Pro35 40
453745PRTTritirachium album 37Gly Gly Pro Ser Ser Ser Ala Val Asn
Arg Ala Ala Ala Glu Ile Thr1 5 10 15Ser Ala Glu Ala Thr Asp Ala Ser
Ser Ser Ser Pro Ala Ser Glu Glu20 25 30Tyr Ser Asn Phe Gly Ser Val
Val Asp Leu Leu Ala Pro35 40 453845PRTAcremonium chrysogenum 38Gly
Gly Gly Tyr Ser Ser Ala Phe Asn Asn Ala Val Asn Thr Ala Tyr1 5 10
15Ser Arg Asp Asn Gln Asn Ala Ala Asn Tyr Ser Pro Ala Ser Ala Ala20
25 30Phe Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Ala Pro35 40
453945PRTAspergillus oryzae 39Gly Gly Gly Tyr Ser Lys Ala Phe Asn
Asp Ala Val Glu Asn Ala Phe1 5 10 15Glu Gln Glu Asn Ser Asp Ala Gly
Gln Thr Ser Pro Ala Ser Ala Pro20 25 30Phe Ser Asn Phe Gly Lys Val
Val Asp Val Phe Ala Pro35 40 454045PRTSaccharomyces cerevisiae
40Gly Gly Gly Lys Ser Pro Ala Leu Asp Leu Ala Val Asn Ala Ala Val1
5 10 15Glu Val Glu Asn Gln Asp Ala Cys Asn Thr Ser Pro Ala Ser Ala
Asp20 25 30Phe Ser Asn Trp Gly Lys Cys Val Asp Val Phe Ala Pro35 40
454151PRTYarrowia lipolytica 41Gly Gly Pro Lys Ser Ala Ser Gln Asp
Ala Leu Trp Ser Arg Ala Thr1 5 10 15Gln Glu Asp Ala Val Asp Ala Cys
Asn Asp Ser Pro Gly Asn Ile Gly20 25 30Gly Trp Ser Gly Gly Gln Gly
Ser Asn Tyr Gly Thr Cys Val Asp Val35 40 45Phe Ala Pro50
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