SUBTILASE ENZYMES OF THE I-S1 AND I-S2 SUB-GROUPS HAVING AN ADDITIONAL AMINO ACID RESIDUE IN AN ACTIVE SITE LOOP REGION

ENZYMES SUBTILASES DES SOUS-GROUPES I-S1 ET I-S2 AYANT UN RESIDU D'ACIDE AMINE ADDITIONNEL DANS UNE REGION BOUCLE DE SITE ACTIF

English Abstract

Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino
acid residue in position 100 of the active site loop (b) region from position
95 to 103. Variant subtilases exhibit improved wash performance in a detergent
in comparison to its parent enzyme.

French Abstract

Cette invention se rapporte à des enzymes subtilases des sous-groupes I-S1 et I-S2 comportant un résidu d'acide aminé additionnel dans la position 100 de la région boucle de site actif (b) entre la position 95 et 103. Des variants de ces substilases possèdent des performances de lavage améliorées dans un détergent par rapport à leur enzyme parente.

Claims

51

CLAIMS:


1. An isolated subtilase enzyme of the I-S1 or I-S2 sub-groups
having one or two additional amino acid residues in position 100
of the active site loop (b) region from position 95 to 103,
whereby said one or two amino acid residues is/are inserted
between positions 100 and 101, which amino acid residues are
selected from the group consisting of T, S, C, N, Q, A, G, V, P,
I, F, L, M, W and Y, and wherein the positions are numbered
according to the amino acid sequence of mature subtilisin BPN.

2. The isolated subtilase enzyme of claim 1, wherein said
subtilase enzyme is a constructed variant.


3. The isolated subtilase enzyme of claim 1 or 2, wherein said
insertion(s) between positions 100 and 101 are combined with one
or more further modification(s) in any other position(s).


4. The isolated subtilase enzyme of claim 3, wherein said
further modification(s) are in one or more of the positions 27,
36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222,
224, 235 and 274.


5. The isolated subtilase enzyme of claim 3 or 4 wherein said
modification(s) is/are combined with modification(s) in one or
more of the positions 129, 131, 133 and 194.


6. The isolated subtilase enzyme of any one of the claims 1-5,
wherein the subtilase belongs to the sub-group I-S1.


7. The isolated subtilase enzyme of claim 6, which is chosen
from the group consisting of BSS168, BASBPN, BSSDY and BLSCAR.

8. The isolated subtilase enzyme according to any one of claims
1-5, wherein the subtilase belongs to the sub-group I-S2.




52

9. The isolated subtilase enzyme of claim 8, which is chosen
from the group consisting of BLS147, BLS309, BAALKP and BYSYAB.

10. The isolated subtilase enzyme of claim 8 or 9, wherein said
further modification(s) are chosen from the group consisting of
K27R, *36D, S57P, N76D, S87N, G97N, S101G, V104A, V104N, V104Y,
H120D, N123S, Y167X, R170X, Q206E, N218S, M222S, M222A, T224S,
K235L and T274A.


11. The isolated subtilase enzyme of claim 8 or 9, wherein said
further modification(s) are chosen from the group consisting of
S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A,
N76D+S103A+V104I 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 in any one
of claims 1 to 7.


12. The isolated subtilase enzyme of claim 8 or 9, wherein said
modification(s) are chosen from the group consisting of P129K,
P131H, A133P, A133D and A194P.


13. The isolated subtilase enzyme according to any one of claims
1-12 comprising the modification A98G+G100GA+S101A+S103T or
S99G+G100GGT+S101T.


14. The isolated subtilase enzyme of claim 1, which has the amino
acid sequence:

A-Q-T-V-P-Y-G-I-P-L-I-K-A-D-K-V-Q-A-Q-G-F-K-G-A-N-V-K-V-A-V

L-D-T-G-I-Q-A-S-H-P-D-L-N-V-V-G-G-A-S-F-V-A-G-E-A-*-Y-N-T-D

G-N-G-H-G-T-H-V-A-G-T-V-A-A-L-D-N-T-T-G-V-L-G-V-A-P-S-V-S-L

Y-A-V-K-V-L-N-S-S-G-X-S-G-T-Y-S-G-I-V-S-G-I-E-W-A-T-T-N-G-M-D



53

V-I-N-M-S-L-G-G-P-S-G-S-T-A-M-K-Q-A-V-D-N-A-Y-A-R-G-V-V-V-V

A-A-A-G-N-S-G-S-S-G-N-T-N-T-I-G-Y-P-A-K-Y-D-S-V-I-A-V-G-A-V

D-S-N-S-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A-P-G-A-G-V-Y-S-T-Y-P

T-S-T-Y-A-T-L-N-G-T-S-M-A-S-P-H-V-A-G-A-A-A-L-I-L-S-K-H-P-N

L-S-A-S-Q-V-R-N-R-L-S-S-T-A-T-Y-L-G-S-S-F-Y-Y-G-K-G-L-I-N-V

E-A-A-A-Q
or an isolated subtilase enzyme having an amino acid sequence comprising a
position 100a amino acid residue and exhibiting an identity of more than
70%, 75%, 80%, 85%, 90%, or 95% therewith.


15. The subtilase of claim 1, which has the amino acid sequence:

A-Q-S-V-P-W-G-I-S-R-V-Q-A-P-A-A-H-N-R-G-L-T-G-S-G-V-K-V-A-V-

L-D-T-G-I-*-S-T-H-P-D-L-N-I-R-G-G-A-S-F-V-P-G-E-P-*-S-T-Q-D-

G-N-G-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-I-G-V-L-G-V-A-P-S-A-E-L-

Y-A-V-K-V-L-G-A-S-G-X-S-G-S-V-S-S-I-A-Q-G-L-E-W-A-G-N-N-G-M-H-

V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S-R-G-V-L-V-V-

A-A-S-G-N-S-G-A-*-G-S-I-S-*-*-*-Y-P-A-R-Y-A-N-A-M-A-V-G-A-T-

D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A-P-G-V-N-V-Q-S-T-Y-P-

G-S-T-Y-A-S-L-N-G-T-S-M-A-T-P-H-V-A-G-A-A-A-L-V-K-Q-K-N-P-S-

W-S-N-V-Q-I-R-N-H-L-K-N-T-A-T-S-L-G-S-T-N-L-Y-G-S-G-L-V-N-A-





54

E-A-A-T-R

or an isolated subtilase enzyme having an amino acid sequence comprising a
position 100a amino acid residue and exhibiting an identity of more than 70%,
75%, 80%, 85%, 90%, or 95% therewith.


16. An isolated DNA sequence encoding an isolated subtilase
enzyme according to any one of claims 1 to 15.


17. An expression vector comprising an isolated DNA sequence of
claim 16.


18. A microbial host cell transformed with an expression vector
of claim 17.


19. The microbial host of claim 18, which is a bacterium.


20. The microbial host of claim 19, wherein said bacterium is a
Bacillus.


21. The microbial host of claim 20, wherein said Bacillus is B.
lentus.


22. The microbial host of claim 19, which is a fungus or yeast.

23. The microbial host of claim 22, wherein said fungus is a
filamentous fungus.


24. The microbial host of claim 23, wherein said filamentous
fungus is an Aspergillus.


25. A method for producing an isolated subtilase enzyme according
to any one of claims 1 to 15, wherein a host of any of claims
18-24 is cultured under conditions conducive to the expression
and secretion of said enzyme, and the enzyme is recovered.




55

26. A composition comprising an isolated subtilase enzyme
according to any one of claims 1 to 15 and a carrier.


27. The composition according to claim 26, which additionally
comprises a cellulase, lipase, cutinase, oxidoreductase, another
protease, or an amylase.


28. The composition according to claim 26 or 27, wherein the
composition is a detergent composition.


29. Use of an isolated subtilase enzyme according to any one of
claims 1 to 15 or an enzyme composition according to claims 26
or 27 in a laundry and/or a dishwash detergent.

Description

CA 02355579 2009-01-21
I

Title: Subtilase enzymes of the I-Si and I-S2 sub-groups having
an additional amino acid residue in an active site loop region.
TECHNICAL FIELD

This invention relates to novel subtilase enzymes c the
I-S1 and I-S2 sub-groups having at least one additional amino
acid residue in position 100 of the active site loop (b) region
from position 95 to 103. These proteases are useful exhibiting
excellent or improved wash performance when used in detergents;

io cleaning and, detergent compositions. The invention further
relates to 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, and methods for producing the novel enzymes.

BACKGROUND OF THE INVENTION

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 enzymes are proteases.
An increasing number of commercially used proteases are
protein engineered variants of naturally occurring wild type

proteases, e.g. DURAZXM' (Novo Nordisk A/S), RELASE (Novo
Nordisk A/S), MAXAPEMV (Gist-Brocades N.V.), PURAFECT (Genencor
International, Inc.).

Further a number of protease variants are described in
the art, such as in EP 130756 (GENENTECH)(corresponding to US
3o Reissue Patent No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO

87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR);
Thomas, Russell, and Fersht (1985) Nature 318 375-376; Thomas,


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WO Q&/37624 2 PCTIDK99/00715
Russell, and Fersht (1987) J. Mot. Biol. 193 803-813; Russel and
Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO
88/08033 (Amgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011
(PROCTER & GAMBLE COMPANY); WO 95/30010 (PROCTER & GAMBLE

COMPANY); WO 95/29979 (PROCTER & GAMBLE COMPANY); US 5.543.302
(SOLVAY S.A.); EP 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK
A/S); WO 91/00345 (NOVO NORDISK A/S); EP 525 610 Al (SOLVAY);
and WO 94/02618 (GIST-BROCADES N.V.).

However, even though a number of useful protease variants
to have been described, there is still a need for new improved
proteases or protease variants for a number of industrial uses.

Therefore, an object of the present invention, is to
provide improved proteases or protein engineered protease
variants, especially for use in the detergent industry.


SUMMARY OF THE INVENTION

The present inventors have found that subtilisins wherein
at least one of the active site loops are longer than those
presently known, exhibit improved wash performance properties in

detergent compositions. The identification thereof was done in
constructing subtilisin variants, especially of the subtilisin
309 (BLSAVI or Savinase(D), exhibiting improved wash performance
properties in detergent compositions relative to the parent wild
type enzyme. This has been described in our earlier application
DK1332/97.

It has now been found that certain subtilases or variants
thereof of the I-S1 (true " subtilisins" ) and I-S2 (high
alkaline subtilisins) sub-groups having at least one additional
amino acid residue in position 100 (or rather between positions

100 and 101) of the active site loop (b) region from position 95
to 103, exhibit surprisingly improved wash performance in


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WO 00137624 3 PCT/DK99/00715
comparison to those presently known and those described in said
application.

The improved proteases according to the invention may be
obtained by isolation from natural resources or by the

introduction of at least one further amino acid residue (an
insertion) in the active site loop (b) between positions 100 and
101 in a wild type subtilase (for a definition of the active
site loops and the numbering of positions see below).

Although this finding was done in subtilisin 309 it is
io predicted that it will be possible to produce or isolate similar
advantageous subtilases or subtilase variants.

Furthermore it will be possible to specifically screen
natural isolates to identify novel wild type subtilases
comprising an active site loop (b) which is longer than the

corresponding active site loop in known wild type subtilases,
such as subtilisin 309, which subtilases can be considered to
have an inserted amino acid residue between positions 100 and
101, and exhibiting excellent wash performance in a detergent,
in comparison to their closest related known subtilisin, such as
20. subtilisin 309.

Concerning alignment and numbering reference is made to
Figs. 1, la, 2 and 2a below showing alignments between
subtilisin BPN' (BASBPN)(a) and subtilisin 309 (BLSAVI)(b), and
alignments between subtilisin BPN'(a) (BASBPN) and subtilisin

Carlsberg (g) (BLSCAR). In Figs. 1 and 2 the alignments were
established by the use of the GAP routine of the GCG package as
indicated below, whereas the alignments of Figs. la and 2a are
the same as shown in WO 91/00345. These alignments are in this
patent application used as a reference for numbering the

3o residues.


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The seven active site loops (a) to (g) (including both
the end amino acid residues indicated) are here defined to
encompass the amino acid residues in the segments given below

s (a) the region between amino acid residue 33 and 43;
(b) the region between amino acid residue 95 and 103;
(c) the region between amino acid residue 125 and 132;
(d) the region between amino acid residue 153 and 173;
(e) the region between amino acid residue 181 and 195;

(f) the region between amino acid residue 202 and 204;
(g) the region between amino acid residue 218 and 219.
Accordingly, in a first aspect the invention relates to

an isolated (i.e. greater than 10 % pure) subtilase enzymes of
is the I-S1 and I-S2 sub-groups having at least one additional
amino acid residue in position 100 of the active site loop (b)
region from position 95 to 103, whereby said additional amino
acid residue(s) correspond to the insertion of at least one
amino acid residue between positions 100 and 101.

In a second aspect the invention relates to an isolated
DNA sequence encoding a subtilase variant of the invention.

In a third aspect the invention relates to an expression
vector comprising an isolated DNA sequence encoding a subtilase
variant of the invention.

In a fourth aspect the invention relates to a microbial
host cell transformed with an expression vector according to the
fourth aspect.

In a further aspect the invention relates to the produc-
tion of the subtilisin enzymes of the invention.

The enzymes of the invention can generally be produced by
either cultivation of a microbial strain from which the enzyme
was isolated and recovering the enzyme in substantially pure


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WO 00737624 5 PCT/DK99/00715
form; or by inserting an expression vector according to the
fourth aspect of the invention into a suitable microbial host,
cultivating the host to express the desired subtilase enzyme,
and recovering the enzyme product.

Further the invention relates to a composition comprising
a subtilase or subtilase variant of the invention.

Even further the invention relates to the use of the
enzymes of the invention for a number of industrial relevant
uses, in particular for use in cleaning compositions and

io cleaning compositions comprising the mutant enzymes, especially
detergent compositions comprising the mutant subtilisin enzymes.
DEFINITONS

Prior to discussing this invention in further detail, the
following terms and conventions will first be defined.


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W0.00737624 6 PCT/DK99/00715
NOMENCLATURE OF AMINO ACIDS

A = Ala = Alanine
V = Val = Valine
L = Leu = Leucine
s I = Ile = Isoleucine

P = Pro = Proline

F = Phe = Phenylalanine
W = Trp = Tryptophan
M = Met = Methionine

to G = Gly = Glycine
S = Ser = Serine
T = Thr = Threonine
C = Cys = Cysteine
Y = Tyr = Tyrosine

1s N = Asn = Asparagine
Q = Gln = Glutamine
D = Asp = Aspartic Acid
E = Glu = Glutamic Acid
K = Lys = Lysine

20 R = Arg = Arginine
H = His = Histidine

X = Xaa = Any amino acid
NOMENCLATURE OF NUCLEIC ACIDS

25 A = Adenine
G = Guanine
C = Cytosine

T = Thymine (only in DNA)
U = Uracil (only in RNA)

NOMENCLATURE AND CONVENTIONS FOR DESIGNATION OF VARIANTS


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WO OU/37624 7 PCT/DK99/00715
In describing the various enzyme variants produced or
contemplated according to the invention, the following nomen-
clatures and conventions have been adapted for ease of

reference:
s A frame of reference is first defined by aligning the
isolated or parent wild type enzyme with subtilisin BPN'
(BASBPN).
The alignment can be obtained by the GAP routine of the
GCG package version 9.1 to number the variants using the

to following parameters: gap creation penalty = 8 and gap extension
penalty = 8 and all other parameters kept at their default
values.
Another method is to use known recognised alignments
between subtilases, such as the alignment indicated in WO

15 91/00345. In most cases the differences will not be of any
importance.

Such alignments between subtilisin BPN' (BASBPN) and
subtilisin 309 (BLSAVI) and subtilisin Carlsberg (BLSCAR),
respectively are indicated in Figs. 1, la, 2, and 2a. They

20 define a number of deletions and insertions in relation to
BASBPN. In Fig. 1 subtilisin 309 has 6 deletions in positions
36, 58, 158, 162, 163, and 164 in comparison to BASBPN, whereas
in Fig. la subtilisin 309 has the same deletions in positions
36, 56, 159, 164, 165, and 166 in comparison to BASBPN. In Fig.

25 2 subtilisin Carlsberg has one deletion in position 58 in
comparison to BASBPN, whereas in Fig. 2a subtilisin Carlsberg
has the one deletion in position 56 in comparison to BASBPN.
These deletions are in Figs. 1, la, 2, and 2a indicated by
asterixes (*).
30 The various modifications performed in a wild type enzyme
is indicated in general using three elements as follows:


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WO 9@/37624 8 PCT/DK99/00715
Original amino acid position substituted amino acid

The notation G195E thus means a substitution of a glycine
in position 195 with a glutamic acid.

In the case when the original amino acid residue may be
any amino acid residue, a short hand notation may at times be
used indicating only the position and substituted amino acid,
Position substituted amino acid

Such a notation is particular relevant in connection with
modification(s) in homologous subtilases (vide infra).

Similarly when the identity of the substituting amino
acid residue(s) is immaterial,


Original amino acid position

When both the original amino acid(s) and substituted
amino acid(s) may comprise any amino acid, then only the

position is indicated, e.g.: 170.
When the original amino acid(s) and/or substituted amino
acid(s) may comprise more than one, but not all amino acid(s),
then the selected amino acids are indicated inside brackets { },

Original amino acid position {substituted amino acid,, . ,
substituted amino acidn}

For specific variants the specific three or one letter
codes are used, including the codes Xaa and X to indicate any
3o amino acid residue.

SUBSTITUTIONS:


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The substitution of Glutamic acid for glycine in position
195 is designated as:

Gly195Glu or G195E

or the substitution of any amino acid residue acid for glycine
in position 195 is designated as:

G1y195Xaa or G195X
or

G1y195 or G195

The substitution of serine for any amino acid residue in
position 170 would thus be designated

Xaa170Ser or X170S.
or

170Ser or 170S

Such a notation is particular relevant in connection with
modification(s) in homologous subtilases (vide infra). 170Ser is
thus meant to comprise e.g. both a Lys170Ser modification in

BASBPN and Arg170Ser modification in BLSAVI (cf. Fig. 1).
For a modification where the original amino acid(s)
and/or substituted amino acid(s) may comprise more than one, but
not all amino acid(s), the substitution of glycine, alanine,
serine or threonine for arginine in position 170 would be
indicated by

Arg170{Gly,Ala,Ser,Thr} or R170{G,A,S,T}
to indicate the variants

R170G, R170A, R170S, and R170T.
DELETIONS:

A deletion of glycine in position 195 will be indicated
by:

Gly195* or G195*


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WO OO/37624 10 PCT/DK99/00715
Correspondingly the deletion of more than one amino acid
residue, such as the deletion of glycine and leucine in
positions 195 and 196 will be designated

Glyl95*+Leu196* or G195*+L196*

INSERTIONS:
The insertion of an additional amino acid residue such as
e.g. a lysine after G195 is :

Gly195GlyLys or G195GK; or

to when more than one amino acid residue is inserted, such as e.g.
a Lys, Ala and Ser after G195 this is :

Gly195GlyLysAlaSer or G195GKAS

In such cases the inserted amino acid residue(s) are
numbered by the addition of lower case letters to the position
number of the amino acid residue preceding the inserted amino
acid residue(s). In the above example the sequences 194 to 196
would thus be:

194 195 196
BLSAVI A - G - L

194 195 195a 195b 195c 196
Variant A - G - K - A - S - L

In cases where an amino acid residue identical to the
existing amino acid residue is inserted it is clear that a
degeneracy in the nomenclature arises. If for example a glycine
is inserted after the glycine in the above example this would be
indicated by G195GG. The same actual change could just as well

3o be indicated as A194AG for the change from
194 195 196

BLSAVI A - G - L


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WO 00137624 11 PCTIDK99/00715
to

194 195 195a 196
Variant A - G - G - L
194 194a 195 196

Such instances will be apparent to the skilled person,
and the indication G195GG and corresponding indications for this
type of insertions are thus meant to comprise such equivalent
degenerate indications.


FILLING A GAP:

Where a deletion in an enzyme exists in the reference
comparison with the subtilisin BPN' sequence used for the
numbering, an insertion in such a position is indicated as:
*36Asp or *36D

for the insertion of an aspartic acid in position 36
MULTIPLE MODIFICATIONS

Variants comprising multiple modifications are separated
by pluses, e.g. :

Argl70Tyr+Glyl95Glu or R170Y+G195E

representing modifications in positions 170 and 195 substituting
tyrosine and glutamic acid for arginine and glycine, respec-
tively.

or e.g. Tyrl67{Gly,Ala,Ser,Thr}+Argl70{Gly,Ala,Ser,Thr}
designates the variants

Tyrl67Gly+Argl7OGly, Tyrl67Gly+Argl7OAla,
Tyrl67Gly+Argl7OSer, Tyrl67Gly+Argl7OThr,
Tyrl67Ala+Argl7OGly, Tyrl67Ala+Argl7OAla,

Tyrl67Ala+Argl7OSer, Tyrl67Ala+Argl7OThr,
Tyrl67Ser+Argl7OGly, Tyrl67Ser+Argl7OAla,
Tyrl67Ser+Argl7OSer, Tyrl67Ser+Argl7OThr,


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WO 00137624 12 PCT/DK99/00715
Tyrl67Thr+Argl7OGly, Tyrl67Thr+Argl7OAla,
Tyrl67Thr+Argl7OSer, and Tyrl67Thr+Argl7OThr.

This nomenclature is particular relevant relating to
modifications aimed at substituting, replacing, inserting or

s deleting amino acid residues having specific common properties,
such as residues of positive charge (K, R, H), negative charge
(D, E), or conservative amino acid modification(s) of e.g.
Tyrl67{Gly,Ala,Ser,Thr}+Argl70{Gly,Ala,Ser,Thr}, which signifies
substituting a small amino acid for another small amino acid.

to See section "Detailed description of the invention" for further
details.

Proteases
Enzymes cleaving the amide linkages in protein substrates
is are classified as proteases, or (interchangeably) peptidases

(see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman
and Company, San Francisco, Chapter 3).

Numbering of amino acid positions/residues

20 If nothing else is mentioned the amino acid numbering
used herein correspond to that of the subtilase BPN' (BASBPN)
sequence. For further description of the BPN' sequence see Figs.
1 and 2, or Siezen et al., Protein Engng. 4 (1991) 719-737.

25 Serine proteases

A serine protease is an enzyme which catalyzes the
hydrolysis of peptide bonds, and in which there is an essential
serine residue at the active site (White, Handler and Smith,
1973 "Principles of Biochemistry," Fifth Edition, McGraw-Hill
3o Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in
the 20,000 to 45,000 Dalton range. They are inhibited by


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WO.00/37624 13 PCT/DK99/00715
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

s the serine proteases, from pH 9.0 to 11.0 (for review, see
Priest (1977) Bacteriological Rev. 41 711-753).
Subtilases
A sub-group of the serine proteases tentatively designated

to subtilases has been proposed by Siezen et al., Protein Engng. 4
(1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-
523. They are defined by homology analysis of more than 170
amino acid sequences of serine proteases previously referred to
as subtilisin-like proteases. A subtilisin was previously often

is defined as a serine protease produced by Gram-positive bacteria
or fungi, and according to Siezen et al. now is a subgroup of
the subtilases. A wide variety of subtilases have been
identified, and the amino acid sequence of a number of
subtilases has been determined. For a more detailed description

20 of such subtilases and their amino acid sequences reference is
made to Siezen et al.(1997).

One subgroup of the subtilases, I-S1 or " true#
subtilisins, comprises the "classical" subtilisins, such as
subtilisin 168 (BSS168), subtilisin BPN', subtilisin Carlsberg

25 (ALCALASE!", NOVO NORDISK A/S), and subtilisin DY (BSSDY).
A further subgroup of the subtilases, I-S2 or high
alkaline subtilisins, is recognised by Siezen et al. (supra).
Sub-group I-S2 proteases are described as highly alkaline
subtilisins and comprises enzymes such as subtilisin PB92

30 (BAALKP) (MAXACAL", Gist-Brocades NV), subtilisin 309 (SAVINASET,
NOVO NORDISK A/S), subtilisin 147 (BLS147) (ESPERASE', NOVO
NORDISK A/S), and alkaline elastase YaB (BSEYAB).


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WO 90/37624 14 PCT/DK99/00715
List of acronyms for subtilases:

I-Si
Subtilisin 168, BSS168 (BSSAS (Subtilisin amylosacchariticus),
BSAPRJ (Subtilisin J), BSAPRN (Subtilisin NAT), BMSAMP (Mes-
entericopeptidase),

Subtilisin BPN', BASBPN,
Subtilisin DY, BSSDY,

Subtilisin Carlsberg, BLSCAR (BLKERA (Keratinase), BLSCA1,
io BLSCA2, BLSCA3),

BSSPRC, Serine protease C
BSSPRD, Serine protease D
I-S2

Subtilisin Sendai, BSAPRS
Subtilisin ALP 1, BSAPRQ,

Subtilisin 147, Esperase BLS147 (BSAPRM (SubtilisinAprM),
BAH101),

Subtilisin 309, Savinase , BLS309/BLSAVI (BSKSMK (M-protease),
BAALKP(Subtilisin PB92, Bacillus alkalophilic alkaline prote-
ase), BLSUBL (Subtilisin BL)),

Alkaline elastase YaB, BYSYAB,
"SAVINASE "

SAVINASE is marketed by NOVO NORDISK A/S. It is
subtilisin 309 from B. Lentus and differs from BAALKP only in
one position (N87S, see Fig. 1 herein). SAVINASE has the amino
acid sequence designated b) in Fig. 1.


Parent subtilase


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The term ''parent subtilase " describes a subtilase
defined according to Siezen et al. (1991 and 1997). For further
details see description of " SUBTILASES " immediately above. A
parent subtilase may also be a subtilase isolated from a natural

s source, wherein subsequent modification have been made while
retaining the characteristic of a subtilase. Alternatively the
term "parent subtilase'' may be termed "wild type subtilase " .
Modification(s) of a subtilase variant

The term "modification(s)" used herein is defined to
include chemical modification of a subtilase as well as genetic
manipulation of the DNA encoding a subtilase. The
modification(s) can be replacement(s) of the amino acid side
chain(s), substitution(s), deletion(s) and/or insertions in or

is at the amino acid(s) of interest.
Subtilase variant
In the context of this invention, the term subtilase
variant or mutated subtilase means a subtilase that has been
produced by an organism which is expressing a mutant gene

derived from a parent microorganism which possessed an original
or parent gene and which produced a corresponding parent enzyme,
the parent gene having been mutated in order to produce the
mutant gene from which said mutated subtilase protease is

produced when expressed in a suitable host.
Homologous subtilase sequences

Specific active site loop regions, and amino acid
insertions in said loops of SAVINASE subtilase are identified
for modification herein to obtain a subtilase variant of the
invention.


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W0.00137624 16 PCTIDK99/00715
However, the invention is not limited to modifications of
this particular subtilase, but extend to other parent (wild-
type) subtilases, which have a homologous primary structure to
that of SAVINASEO. The homology between two amino acid sequences

is in this context described by the parameter "identity" .

In order to determine the degree of identity between two
subtilases the GAP routine of the GCG package version 9.1 can be
applied (infra) using the same settings. The output from the
routine is besides the amino acid alignment the calculation of

to the "Percent Identity" between the two sequences.

Based on this description it is routine for a person
skilled in the art to identify suitable homologous subtilases
and corresponding homologous active site loop regions, which can
be modified according to the invention.


Wash performance

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 or hard surface cleaning is often

referred to as its washing ability, wash-ability, detergency, or
wash performance. Throughout this application the term wash
performance will be used to encompass this property.

Isolated DNA sequence

The term "isolated", when applied to a DNA sequence
molecule, denotes that the DNA sequence has been removed from
its natural genetic milieu and is thus free of other extraneous
or unwanted coding sequences, and is in a form suitable for use
within genetically engineered protein production systems. Such

isolated molecules are those that are separated from their
natural environment and include cDNA and genomic clones.
Isolated DNA molecules of the present invention are free of


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other genes with which they are ordinarily associated, but may
include naturally occurring 5' and 3' untranslated regions such
as promoters and terminators. The identification of associated
regions will be evident to one of ordinary skill in the art (see

s for example, Dynan and Tijan, Nature 316:774-78, 1985). The term
"an isolated DNA sequence" may alternatively be termed "a
cloned DNA sequence".

Isolated protein

When applied to a protein, the term "isolated" indicates
that the protein has been removed from its native environment.
In a preferred form, the isolated protein is

substantially free of other proteins, particularly other
homologous proteins (i.e. "homologous impurities" (see below)).
An isolated protein is greater than 10 % pure, preferably

greater than 20 % pure, more preferably greater than 30 % pure,
as determined by SDS-PAGE. Further it is preferred to provide
the protein in a highly purified form, i.e., greater than 40%
pure, greater than 60% pure, greater than 80% pure, more

preferably greater than 95% pure, and even more preferably
greater than 99% pure, as determined by SDS-PAGE.

The term "isolated protein" may alternatively be termed
"purified protein''.

Homologous impurities

The term "homologous impurities" means any impurity
(e.g. another polypeptide than the polypeptide of the invention)
which originate from the homologous cell where the polypeptide of
the invention is originally obtained from.



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Obtained from

The term "obtained from" as used herein in connection
with a specific microbial source, means that the polynucleotide
and/or polypeptide produced by the specific source, or by a cell

s in which a gene from the source has been inserted.
Substrate

The term "Substrate" used in connection with a
substrate for a protease is should be interpreted in its

io broadest form as comprising a compound containing at least one
peptide bond susceptible to hydrolysis by a subtilisin protease.
Product
The term "product" used in connection with a product

is derived from a protease enzymatic reaction should in the context
of this invention be interpreted to include the products of a
hydrolysis reaction involving a subtilase protease. A product
may be the substrate in a subsequent hydrolysis reaction.

20 BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 shows an alignment between subtilisin BPN' (a) and
Savinase (b) using the GAP routine mentioned above.

Fig. la shows the alignment between subtilisin BPN' and
Savinase A as taken from WO 91/00345.

25 Fig. 2 shows an alignment between subtilisin BPN' and
subtilisin Carlsberg using the GAP routine mentioned above.
Fig. 2a shows the alignment between subtilisin BPN' and

subtilisin Carlsberg as taken from WO 91/00345.

Fig. 3 shows the three dimensional structure of Savinase
30 (Protein data bank (PDB) entry 1SVN) . In the Figure the active
site loop (b) is indicated.


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DETAILED DESCRIPTION OF THE INVENTION

The subtilases of the invention in a first aspect relates
to an isolated (i.e. greater than 10 o pure) subtilase enzyme of
the I-S1 and I-S2 sub-groups having at least one additional

s amino acid residue in position 100 of the active site loop (b)
region from position 95 to 103, whereby said additional amino
acid residue(s) correspond to the insertion of at least one
amino acid residue between positions 100 and 101.

In other words the subtilases of the invention are

io characterized by comprising an active site loop (b) region of
more than 9 amino acid residue and wherein the additional amino
acid residue is or can be considered as being inserted between
positions 100 and 101 as compared to the parent or a known wild
type subtilase.
15 A subtilase of the first aspect of the invention may be a
parent or wildtype subtilase identified and isolated from
nature.
Such a parent wildtype subtilase may be specifically
screened for by standard techniques known in the art.

20 One preferred way of doing this may be by specifically
PCR amplify DNA regions known to encode active site loops in
subtilases from numerous different microorganism, preferably
different Bacillus strains.
Subtilases are a group of conserved enzymes, in the sense
25 that their DNA and amino acid sequences are homologous.
Accordingly it is possible to construct relatively specific
primers flanking active site loops.

One way of doing this is by investigating an alignment of
different subtilases (see e.g. Siezen et al. Protein Science 6

30 (1997) 501-523). It is from this routine work for a person
skilled in the art to construct PCR primers flanking the active
site loop corresponding to the active site loop (b) between


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WO Q0t37624 20 PCT/DK99/00715
amino acid residue 95 to 103 in any of the group I-S1 or I-S2
groups, such as from BLSAVI. Using such PCR primers to amplify
DNA from a number of different microorganism, preferably
different Bacillus strains, followed by DNA sequencing of said

amplified PCR fragments, it will be possible to identify strains
which produce subtilases of these groups comprising a longer, as
compared to e.g. BLSAVI, active site region corresponding to the
active site loop region from positions 95 to 103, and where an
insertion can be considered to exist between positions 100 and

l0 101. Having identified the strain and a partial DNA sequence of
such a subtilase of interest, it is routine work for a person
skilled in the art to complete cloning, expression and
purification of such a subtilase of the invention.

However, it is envisaged that a subtilase enzyme of the
invention predominantly is a variant of a parent subtilase.
Accordingly, in one embodiment the invention relates to

an isolated subtilase enzyme according to the first aspect of
the invention, wherein said subtilase enzyme is a constructed
variant having a longer active site loop (b) than its parent

enzyme by having at least one amino acid insertion between amino
acid residues 100 and 101.

The subtilases of the invention exhibit excellent wash
performance in a detergent, and if the enzyme is a constructed
variant an improved wash performance in a detergent in

comparison to its closest related subtilase, such as subtilisin
309.

Different subtilase products will exhibit a different
wash performance in different types of detergent compositions. A
subtilase of the invention has improved wash performance, as

compared to its closest relative in a majority of such different
types of detergent compositions.


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WO 80/37624 21 PCT/DK99/00715
Preferably a subtilase enzyme of the invention has
improved wash performance, as compared to its closest relative
in the detergent composition shown in Example 3 herein (vide
infra).

s In order to determine if a given subtilase amino acid
sequence (irrelevant whether said subtilase sequence is a parent
wildtype subtilase sequence or a subtilase variant sequence
produced by any other method than by site directed mutagenesis)
is within the scope of the invention, the following procedure

io may be used:

i) Align said subtilase sequence to the amino acid sequence
of subtilisin BPN' (see section "Definitions" herein
(vide supra);

ii) Based on the alignment performed in step i) identify the
15 active site loop (b), in said subtilase sequence
corresponding to the active site loop (b) region of
subtilisin BPN' comprising the region (both of the end
amino acids included) between amino acid residue from 95
to 103;

20 iii) Determine if the active site loop (b) in said subtilase
sequence, identified in step ii) is longer than the
corresponding active site loop in BLSAVI and if said
prolongation corresponds to the insertion of at least one
amino acid residue between positions 100 and 101.

25 If this is the case the subtilase investigated is a
subtilase within the scope of the present invention.

The alignment performed in step i) above is performed as
described above by using the GAP routine.

Based on this description it is routine for a person
30 skilled in the art to identify the active site loop (b) in a
subtilase and determine if the subtilase in question is within
the scope of the invention. If a variant is constructed by site


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WO 00/37624 22 PCT/DK99/00715
directed mutagenesis, it is of course known beforehand if the
subtilase variant is within the scope of the invention.

A subtilase variant of the invention may be constructed
by standard techniques known in the art such as by site-

s directed/random mutagenesis or by DNA shuffling-of different
subtilase sequences. See section "PRODUCING A SUBTILASE
VARIANT'' and Material and methods herein (vide infra) for
further details.
In further embodiments the invention relates to

l0 1.an isolated subtilase enzyme according to the invention,
wherein said at least one inserted amino acid residue is chosen
from the group comprising: T,G,A, and S;

2.an isolated subtilase enzyme according to the invention,
wherein said at least one inserted amino acid residue is chosen
15 from the group of charged amino acid residues comprising:

D,E,H,K, and R, more preferably D,E,K and R;

3.an isolated subtilase enzyme according to the invention,
wherein said at least one inserted amino acid residue is chosen
from the group of hydrophilic amino acid residues comprising:

20 C,N,Q,S and T, more preferably N,Q,S and T;

4.an isolated subtilase enzyme according to the invention,
wherein said at least one inserted amino acid residue is chosen
from the group of small hydrophobic amino acid residues
comprising: A,G and V; or

25 5.an isolated subtilase enzyme according to the invention,
wherein said at least one inserted amino acid residue is chosen
from the group of large hydrophilic amino acid residues
comprising: F,I,L,M,P,W and Y, more preferably F,I,L,M, and Y.

In a further embodiment, the invention relates to an
30 isolated subtilase enzyme according to the invention, wherein
said insertion between positions 100 and 101 comprises at least


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WO QO/37624 23 PCT/DK99/00715
two amino acids, as compared to the corresponding active site
loop in BLSAVI.
In further embodiments the invention relates to an
isolated subtilase enzyme comprising at least one insertion,
s chosen from the group comprising (in BASBPN numbering):
Xl00X{T,G,A,S}

X100X{D, E,K,R}
X10OX{H, V,C,N,Q}

X100X{F,I,L,M,P,W,Y}

or more specific for subtilisin 309 and closely related
subtilases, such as BAALKP, BLSUBL, and BSKSMK

G100GA
is G100GT
Gl00GG
G100GS
G10OGD

G100GE
G100GK
G100GR
Gl00GH

G100GV
G100GC
Gl00GN
G100GQ

3o G100GF
G10OGI
G100GL


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WO 00137624 24 PCT/DK99/00715
G100GM

G100GP
G100OW
G100GY
or any of the following combinations
A98G+G100GA+SlOlA+S103T
S99G+G100GGT+SlO1T

It is well known in the art that a so-called conservative
substitution of one amino acid residue to a similar amino acid
residue is expected to produce only a minor change in the
characteristic of the enzyme.
Table III below list groups of conservative amino acid
substitutions.


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W0.00/37624 25 PCT/DK99/00715
Table III

Conservative amino acid substitutions
Common Property Amino Acid

Basic (positive charge) K = lysine

H = histidine
Acidic (negative charge) E = glutamic acid
D = aspartic acid
Polar Q = glutamine

N = asparagine
Hydrophobic L = leucine

I = isoleucine
V = valine

M = methionine
Aromatic F = phenylalanine
W = tryptophan

Y = tyrosine
Small G = glycine
A = alanine
S = serine

T = threonine

According to this principle subtilase variants comprising
conservative substitutions, such as G97A+A98AS+S99G,

s G97S+A98AT+S99A are expected to exhibit characteristics that are
not drastically different from each other.

Based on the disclosed and/or exemplified subtilase
variants herein, it is routine work for a person skilled in the
art to identify suitable conservative modification(s) to these

io variants in order to obtain other subtilase variants exhibiting
similarly improved wash-performance.

According to the invention it the subtilases of the
invention belong to the subgroups I-S1 and I-S2, especially


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WO 00/37624 26 PCT/DK99/00715
subgroup I-S2, both for isolating novel enzymes of the invention
from nature or from the artificial creation of diversity, and
for designing and producing variants from a parent subtilase.

In relation to variants from subgroup I-Si, it is

preferred to choose a parent subtilase from the group comprising
BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR
(BLKERA, BLSCA1, BLSCA2, BLSCA3), BSSPRC, and BSSPRD, or
functional variants thereof having retained the characteristic
of sub-group I-S1.

io In relation to variants from subgroup I-S2 it is
preferred to choose a parent subtilase from the group comprising
BSAPRQ, BLS147 (BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP,
BLSUBL), BYSYAB, and BSAPRS, or functional variants thereof
having retained the characteristic of sub-group I-S2.

is In particular said parent subtilase is BLSAVI (SAVINASE
NOVO NORDISK A/S), and a preferred subtilase variant of the in-
vention is accordingly a variant of SAVINASE .

The present invention also comprises any of the above
mentioned subtilases of the invention in combination with any

20 other modification to the amino acid sequence thereof. Especial-
ly combinations with other modifications known in the art to
provide improved properties to the enzyme are envisaged. The art
describe a number of subtilase variants with different improved
properties and a number of those are mentioned in the "Back-

25 ground of the invention" section herein (vide supra). Those ref-
erences are disclosed here as references to identify a subtilase
variant, which advantageously can be combined with a subtilase
variant of the invention.

Such combinations comprise the positions: 222 (improve
30 oxidation stability), 218 (improves thermal stability), substi-
tutions in the Ca-binding sites stabilizing the enzyme, e.g. po-
sition 76, and many other apparent from the prior art.


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W0.00/37624 2 7 PCT/DK99/00715
In further embodiments a subtilase variant of the inven-
tion may advantageously be combined with one or more modifica-
tion(s) in any of the positions:

27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206, 218,
222, 224, 235 and 274.

Specifically the following BLSAVI, BLSUBL, BSKSMK, and
BAALKP variants are considered appropriate for combination:
K27R, *36D, S57P, N76D, S87N, G97N, S101G, S103A, V104A, V1041,
V104N, V104Y, H120D, N123S, Y167, R170, Q206E, N218S, M222S,

io M222A, T224S, K235L and T274A.

Furthermore variants comprising any of the variants
S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A,
N76D+S103A+V104I or N76D+V104A or other combinations of these
mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A)

i5 in combination with any one or more of the modification(s) men-
tioned above exhibit improved properties.

Even further subtilase variants of the main aspect(s) of
the invention are preferably combined with one or more modifica-
tion(s) in any of the positions 129, 131, 133 and 194, prefera-

2o bly as 129K, 131H, 133P, 133D and 194P modifications, and most
preferably as P129K, P131H, A133P, A133D and A194P modifica-
tions. Any of those modification(s) are expected to provide a
higher expression level of a subtilase variant of the invention
in the production thereof.

25 Accordingly, an even further embodiment of the invention
relates to a variant according to the invention, wherein said
modification is chosen from the group comprising:

PRODUCING A SUBTILASE VARIANT

30 Many methods for cloning a subtilase of the invention and
for introducing insertions into genes (e.g. subtilase genes) are


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well known in the art, cf. the references cited in the
"BACKGROUND OF THE INVENTION" section.

In general standard procedures for cloning of genes and
introducing insertions (random and/or site directed) into said
s genes may be used in order to obtain a subtilase variant of the

invention. For further description of suitable techniques refer-
ence is made to Examples herein (vide infra) and (Sambrook et
al. (1989) Molecular cloning: A laboratory manual, Cold Spring
Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al.

to (eds.) "Current protocols in Molecular Biology". John Wiley and
Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular
Biological Methods for Bacillus". John Wiley and Sons, 1990);
and WO 96/34946.

Further a subtilase variant of the invention may be
15 constructed by standard techniques for artificial creation of
diversity, such as by DNA shuffling of different subtilase genes
(WO 95/22625; Stemmer WPC, Nature 370:389-91 (1994)). DNA
shuffling of e.g. the gene encoding Savinase with one or more
partial subtilase sequences identified in nature to comprise an

20 active site (b) loop regions longer than the active site (b)
loop of Savinase , will after subsequent screening for improved
wash performance variants, provide subtilase variants according
to the invention.

25 EXPRESSION VECTORS

A recombinant expression vector comprising a DNA
construct encoding the enzyme of the invention may be any vector
which may conveniently be subjected to recombinant DNA pro-
cedures.
30 The choice of vector will often depend on the host cell
into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e. a vector which exists as


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WO 00/37624 2 9 PCT/DK99/00715
an extrachromosomal entity, the replication of which is indepen-
dent of chromosomal replication, e.g. a plasmid. Alternatively,
the vector may be one that on introduction into a host cell is
integrated into the host cell genome in part or in its entirety

and replicated together with the chromosome(s) into which it has
been integrated.
The vector is preferably an expression vector in which
the DNA sequence encoding the enzyme of the invention is
operably linked to additional segments required for

1o transcription of the DNA. In general, the expression vector is
derived from plasmid or viral DNA, or may contain elements of
both. The term, "operably linked" indicates that the segments
are arranged so that they function in concert for their intended
purposes, e.g. transcription initiates in a promoter and

1s proceeds through the DNA sequence coding for the enzyme.
The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell.
20 Examples of suitable promoters for use in bacterial host
cells include the promoter of the Bacillus stearothermophilus
maltogenic amylase gene, the Bacillus licheniformis alpha-
amylase gene, the Bacillus amyloliquefaciens alpha-amylase gene,
the Bacillus subtilis alkaline protease gen, or the Bacillus

25 pumilus xylosidase gene, or the phage Lambda PR or PL promoters
or the E. coli lac, trp or tac promoters.
The DNA sequence encoding the enzyme of the invention may
also, if necessary, be operably connected to a suitable
terminator.
30 The recombinant vector of the invention may further
comprise a DNA sequence enabling the vector to replicate in the
host cell in question.


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WO QW37624 30 PCT/DK99/00715
The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell,
or a gene encoding resistance to e.g. antibiotics like

kanamycin, chloramphenicol, erythromycin, tetracycline,

spectinomycine, or the like, or resistance to heavy metals or
herbicides.
To direct an enzyme of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(also known as a leader sequence, prepro sequence or pre

to sequence) may be provided in the recombinant vector. The
secretory signal sequence is joined to the DNA sequence encoding
the enzyme in the correct reading frame. Secretory signal
sequences are commonly positioned 5' to the DNA sequence
encoding the enzyme. The secretory signal sequence may be that

is normally associated with the enzyme or may be from a gene
encoding another secreted protein.
The procedures used to ligate the DNA sequences coding
for the present enzyme, the promoter and optionally the termin-
ator and/or secretory signal sequence, respectively, or to

20 assemble these sequences by suitable PCR amplification schemes,
and to insert them into suitable vectors containing the
information necessary for replication or integration, are well
known to persons skilled in the art (cf., for instance, Sambrook
et al., op.cit.).


HOST CELL
The DNA sequence encoding the present enzyme introduced into the
host cell may be either homologous or heterologous to the host
in question. If homologous to the host cell, i.e. produced by

the host cell in nature, it will typically be operably connected
to another promoter sequence or, if applicable, another
secretory signal sequence and/or terminator sequence than in its


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WO 00/37624 31 PCT/DK99/00715
natural environment. The term "homologous" is intended to
include a DNA sequence encoding an enzyme native to the host
organism in question. The term "heterologous" is intended to
include a DNA sequence not expressed by the host cell in nature.

s Thus, the DNA sequence may be from another organism, or it may
be a synthetic sequence.
The host cell into which the DNA construct or the
recombinant vector of the invention is introduced may be any
cell which is capable of producing the present enzyme and

io includes bacteria, yeast, fungi and higher eukaryotic cells
including plants.
Examples of bacterial host cells which, on cultivation,
are capable of producing the enzyme of the invention are gram-
positive bacteria such as strains of Bacillus, such as strains
is of B. subtilis, B. licheniformis, B. lentus, B. brevis, B.

stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.
coagulans, B. circulans, B. lautus, B. megatherium or B.
thuringiensis, or strains of Streptomyces, such as S. lividans
or S. murinus, or gram-negative bacteria such as Echerichia

20 coli .
The transformation of the bacteria may be effected by
protoplast transformation, electroporation, conjugation, or by
using competent cells in a manner known per se (cf. Sambrook et
al., supra).
25 When expressing the enzyme in bacteria such as E. coli,
the enzyme may be retained in the cytoplasm, typically as insol-
uble granules (known as inclusion bodies), or may be directed to
the periplasmic space by a bacterial secretion sequence. In the
former case, the cells are lysed and the granules are recovered

30 and denatured after which the enzyme is refolded by diluting the
denaturing agent. In the latter, case, the enzyme may be
recovered from the periplasmic space by disrupting the cells,


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- WO 00/37624 32 PCT/DK99/00715
e.g. by sonication or osmotic shock, to release the contents of
the periplasmic space and recovering the enzyme.

When expressing the enzyme in gram-positive bacteria such
as Bacillus or Streptomyces strains, the enzyme may be retained

s in the cytoplasm, or may be directed to the extracellular medium
by a bacterial secretion sequence. In the latter case, the
enzyme may be recovered from the medium as described below.
METHOD OF PRODUCING SUBTILASE

The present invention provides a method of producing an
isolated enzyme according to the invention, wherein a suitable
host cell, which has been transformed with a DNA sequence

encoding the enzyme, is cultured under conditions permitting the
production of the enzyme, and the resulting enzyme is recovered
from the culture.

When an expression vector comprising a DNA sequence
encoding the enzyme is transformed into a heterologous host cell
it is possible to enable heterologous recombinant production of
the enzyme of the invention.

Thereby it is possible to make a highly purified
subtilase composition, characterized in being free from
homologous impurities.

In this context homologous impurities mean any impurities
(e.g. other polypeptides than the enzyme of the invention) which
originate from the homologous cell where the enzyme of the

invention is originally obtained from.

The medium used to culture the transformed host cells may
be any conventional medium suitable for growing the host cells

in question. The expressed subtilase may conveniently be

secreted into the culture medium and may be recovered therefrom
by well-known procedures including separating the cells from the
medium by centrifugation or filtration, precipitating


CA 02355579 2001-06-18

WO 00/37624 33 PCT/DK99/00715
proteinaceous components of the medium by means of a salt such
as ammonium sulfate, followed by chromatographic procedures such
as ion exchange chromatography, affinity chromatography, or the
like.


USE OF A SUBTILASE VARIANT OF THE INVENTION

A subtilase protease variant of the invention may be used
for a number of industrial applications, in particular within
the detergent industry.

Further the invention relates to an enzyme composition,
which comprise a subtilase variant of the invention.

An summary of preferred industrial applications and
corresponding preferred enzyme compositions are described below.
This summary is not in any way intended to be a complete
is list of suitable applications of a subtilase variant of the

invention. A subtilase variants of the invention may be used in
other industrial applications known in the art to include use of
a protease, in particular a subtilase.

DETERGENT COMPOSITIONS COMPRISING THE MUTANT ENZYMES

The present invention 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 are well described in

the art and reference is made to WO 96/34946; WO 97/07202; WO
95/30011 for further description of suitable cleaning and
detergent compositions.

Furthermore the example(s) below demonstrate the
improvements in wash performance for a number of subtilase
variants of the invention.

Detergent Compositions


CA 02355579 2001-06-18

WO Q0137624 34 PCT/DK99/00715
The enzyme of the invention may be added to and thus be-
come a component of a detergent composition.

The detergent composition of the invention may for exam-
ple be formulated as a hand or machine laundry detergent compo-
s sition including a laundry additive composition suitable for

pre-treatment of stained fabrics and a rinse added fabric sof-
tener composition, or be formulated as a detergent composition
for use in general household hard surface cleaning operations,
or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent
additive comprising the enzyme of the invention. The detergent
additive as well as the detergent composition may comprise one
or more other enzymes such as a protease, a lipase, a cutinase,
an amylase, a carbohydrase, a cellulase, a pectinase, a man-

es nanase, an arabinase, a galactanase, a xylanase, an oxidase,
e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should
be compatible with the selected detergent, (i.e. pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredi-

2o ents, etc.), and the enzyme(s) should be present in effective
amounts.

Proteases: Suitable proteases include those of animal, vegetable
or microbial origin. Microbial origin is preferred. Chemically
modified or protein engineered mutants are included. The

25 protease may be a serine protease or a metallo protease,
preferably an alkaline microbial protease or a trypsin-like
protease. Examples of alkaline proteases are subtilisins,
especially those derived from Bacillus, e.g., subtilisin Novo,
subtilisin Carlsberg, subtilisin 309, subtilisin 147 and

30 subtilisin 168 (described in WO 89/06279) . Examples of trypsin-
like proteases are trypsin (e.g. of porcine or bovine origin)


CA 02355579 2001-06-18

WO 00137624 35 PCT/DK99/00715
and the Fusarium protease described in WO 89/06270 and WO
94/25583.

Examples of useful proteases are the variants described in
WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946,
especially the variants with substitutions in one or more of the

following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123,
167, 170, 194, 206, 218, 222, 224, 235 and 274.

Preferred commercially available protease enzymes include
AlcalaseTM, SavinaseTM, PrimaseTM, DuralaseTM, EsperaseTM, and
to KannaseTM (Novo Nordisk A/S) , MaxataseTM, MaxacalTM, MaxaperTM,

ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, and FN3TM (Genencor
International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal
origin. Chemically modified or protein engineered mutants are
included. Examples of useful lipases include lipases from

Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T.
lanuginosus) as described in EP 258 068 and EP 305 216 or from
H. insolens as described in WO 96/13580, a Pseudomonas lipase,
e.g. from P. alcaligenes or P. pseudoal cal i genes (EP 218 272),

P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.
fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO
96/27002) , P. wisconsinensis (WO 96/12012), a Bacillus lipase,
e.g. from B. subtilis (Dartois et al. (1993), Biochemica et
Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP
64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described
in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO
95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO
95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include
LipolaseTM and Lipolase Ultra'' (Novo Nordisk A/S).


CA 02355579 2001-06-18

W0.00137624 36 PCT/DK99/00715
Amylases: Suitable amylases (a and/or (3) include those of bac-
terial or fungal origin. Chemically modified or protein
engineered mutants are included. Amylases include, for example,
a-amylases obtained from Bacillus, e.g. a special strain of B.
s licheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in
WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424,
especially the variants with substitutions in one or more of the
following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156,

io 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408,
and 444.

Commercially available amylases are DuramylTm, TermamylTM,
FungamylTM and BANTM (Novo Nordisk A/S), RapidaseT" and Purastar",
(from Genencor International Inc.).

15 Cellulases: Suitable cellulases include those of bacterial or
fungal origin. Chemically modified or protein engineered mutants
are included. Suitable cellulases include cellulases from the
genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
Acremonium, e.g. the fungal cellulases produced from Humicola

20 insolens, Myceliophthora thermophila and Fusarium oxysporum dis-
closed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757
and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral
cellulases having colour care benefits. Examples of such cellu-
25 lases are cellulases described in EP 0 495 257, EP 0 531 372, WO

96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants such as those described in WO 94/07998, EP 0 531 315,
US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO
98/12307 and PCT/DK98/00299.

30 Commercially available cellulases include CelluzymeTM, and
CarezymeTM (Novo Nordisk A/S), ClazinaseTM, and Puradax HATM


CA 02355579 2001-06-18

- WO 00{37624 37 PCT/DK99/00715
(Genencor International Inc.), and KAC-500(B)TM (Kao
Corporation).
Peroxidases/Oxidases: Suitable peroxidases/oxidases include
those of plant, bacterial or fungal origin. Chemically modified
or protein engineered mutants are included. Examples of useful

peroxidases include peroxidases from Coprinus, e.g. from C.
cinereus, and variants thereof as those described in WO
93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GuardzymeTM
to (Novo Nordisk A/S) .
The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of
these enzymes. A detergent additive of the invention, i.e. a

separate additive or a combined additive, can be formulated e.g.
as a granulate, a liquid, a slurry, etc. Preferred detergent ad-
ditive formulations are granulates, in particular non-dusting
granulates, liquids, in particular stabilized liquids, or slur-
ries.
Non-dusting granulates may be produced, e.g., as dis-
closed in US 4,106,991 and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating ma-
terials are poly(ethylene oxide) products (polyethylene glycol,
PEG) with mean molar weights of 1000 to 20000; ethoxylated non-

ylphenols having from 16 to 50 ethylene oxide units; ethoxylated
fatty alcohols in which the alcohol contains from 12 to 20 car-
bon atoms and in which there are 15 to 80 ethylene oxide units;
fatty alcohols; fatty acids; and mono- and di- and triglycerides
of fatty acids. Examples of film-forming coating materials suit-

3o able for application by fluid bed techniques are given in GB
1483591. Liquid enzyme preparations may, for instance, be stabi-
lized by adding a polyol such as propylene glycol, a sugar or


CA 02355579 2001-06-18

WO 0x/37624 3 8 PCT/DK99/00715
sugar alcohol, lactic acid or boric acid according to estab-
lished methods. Protected enzymes may be prepared according to
the method disclosed in EP 238,216.

The detergent composition of the invention may be in any
s convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste or a liquid. A liquid detergent may be aqueous, typically
containing up to 70 % water and 0-30 % organic solvent, or non-
aqueous.

The detergent composition comprises one or more surfac-
lo tants, which may be non-ionic including semi-polar and/or ani-
onic and/or cationic and/or zwitterionic. The surfactants are
typically present at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain
from about 1% to about 40% of an anionic surfactant such as lin-
15 ear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate

(fatty alcohol sulfate), alcohol ethoxysulfate, secondary al-
kanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or
alkenylsuccinic acid or soap.

When included therein the detergent will usually contain
20 from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanol-
amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid
amide, or N-acyl N-alkyl derivatives of glucosamine
25 ( "glucamides" ) .
The detergent may contain 0-65 % of a detergent builder
or complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, carbonate, citrate, nitrilotriacetic acid, ethyl-
enediaminetetraacetic acid, diethylenetriaminepentaacetic acid,

3o alkyl- or alkenylsuccinic acid, soluble silicates or layered
silicates (e.g. SKS-6 from Hoechst).


CA 02355579 2001-06-18

- WO 00737624 39 PCTIDK99/00715
The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethyl-
ene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates,

s maleic/acrylic acid copolymers and lauryl methacrylate/acrylic
acid copolymers.

The detergent may contain a bleaching system that may
comprise a H202 source such as perborate or percarbonate, which
may be combined with a peracid-forming bleach activator such as

to tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Al-
ternatively, the bleaching system may comprise peroxyacids of
e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the inven-
tion may be stabilized using conventional stabilizing agents,
15 e.g., a polyol such as propylene glycol or glycerol, a sugar or

sugar alcohol, lactic acid, boric acid, or a boric acid deriva-
tive, e.g., an aromatic borate ester, or a phenyl boronic acid
derivative such as 4-formylphenyl boronic acid, and the com-
position may be formulated as described in e.g. WO 92/19709 and
20 WO 92/19708.
The detergent may also contain other conventional deter-
gent ingredients such as e.g. fabric conditioners including
clays, foam boosters, suds suppressors, anti-corrosion agents,
soil-suspending agents, anti-soil redeposition agents, dyes,

2s bactericides, optical brighteners, hydrotropes, tarnish inhibi-
tors, or perfumes.
It is at present contemplated that in the detergent com-
positions any enzyme, in particular the enzyme of the invention,
may be added in an amount corresponding to 0.01-100 mg of enzyme

30 protein per litre of wash liquor, preferably 0.05-5 mg of enzyme
protein per litre of wash liquor, in particular 0.1-1 mg of en-
zyme protein per litre of wash liquor.


CA 02355579 2009-01-21

The enzyme of the invention may additionally be ircorne-
rated in the detergent formulations disclosed in WO 971107202.
s LEATHER INDUSTRY APPLICATIONS

A subtilase of the invention may be used in the leather
industry, in particular for use in depilation of skins.

In said application a subtilase variant of the invention
is preferably used in an enzyme composition, which further

io comprises another protease.

For a more detailed description of suitable other
proteases see section relating to suitable enzymes for use in a
detergent composition (vide supra).

is WOOL INDUSTRY APPLICATIONS

A subtilase of the invention may be used in the wool
industry, in particular for use in cleaning of clothes
comprising wool.

In said application a subtilase variant of the invention
20 is preferably used in an enzyme composition, which further
comprises another protease.

For a more detailed description of suitable other
proteases see section relating to suitable enzymes for use in a
detergent composition (vide supra).
25 The invention is described in further detail in the
following examples, which are not in any way intended to limit
the scope of the invention as claimed.

MATERIALS AND METHODS

STRAINS:

B. subtilis DN1885 (Diderichsen et al., 1990).


CA 02355579 2009-01-21
41

B. lentus 309 and 147 are specific strains of Bacillus
lentus, deposited with the NCIB and accorded the accession
numbers NCIB 10309 and 10147, and described in US Patent No.
3,723,250,

s E. coli MC 1000 (M.J_ Casadaban and S.N. Cohen (1980); J.
Mot. Biol. 138 179-207), was made r-,m+ by conventional methods
and is also described in US Patent No. 5,036,002.

PLASMIDS:
pJS3: E. coli - B. subtilis shuttle vector containing a
synthetic gene encoding for subtilase 309. (Described by Jacob
SchiOdt et al. in Protein and Peptide letters 3:39-44 (1996)).

1s pSX222: B. subtilis expression vector (Described in WO
96/34946).

GENERAL MOLECULAR BIOLOGY METHODS:

Unless otherwise mentioned the DNA manipulations and
20 transformations were performed using standard methods of
molecular biology (Sambrook et al. (1989) Molecular cloning: A
laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor,
NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular
Biology". John Wiley and Sons, 1995; Harwood, C. R., and

2s Cutting, S. M. (eds.) "Molecular Biological Methods for
Bacillus". John Wiley and Sons, 1990).

Enzymes for DNA manipulations were used according to the
specifications of the suppliers.

3o ENZYMES FOR DNA MANIPULATIONS
Unless otherwise mentioned all enzymes for DNA manipula-
tions, such as e.g. restiction endonucleases, ligases etc., are


CA 02355579 2009-01-21
42

obtained from New England Biolabs, Inc.
PROTEOLYTIC ACTIVITY
In the context of this invention proteolytic activity is
s expressed in Kilo NOVO Protease Units (KNPU). The activity is
determined relatively to an enzyme standard (SAVINASE ), and the
determination is based on the digestion of a dimethyl casein
(DMC) solution by the proteolytic enzyme at standard conditions,
i.e. 50 C, pH 8.3, 9 min. reaction time, 3 min. measuring time.

1o A folder AF 220/1 is available upon request to Novo Nordisk A/S,
Denmark.

A GU is a Glycine Unit, defined as the proteolytic enzyme
activity that, under standard conditions, during a 15 minutes'
incubation at 40 C, with N-acetyl casein as substrate, produces

15 an amount of NH2-group equivalent to 1 mmole of glycine.

Enzyme activity can also be measured using the PNA assay,
according to reaction with the soluble substrate succinyl-
alanine-alanine-proline-phenyl-alanine-para-nitro-phenol, which
is described in the Journal of American Oil Chemists Society,

20 Rothgeb, T.M., Goodlander, B.D., Garrison, P.H., and Smith,
L.A., (1988).

FERMENTATION:
Fermentations for the production of subtilase enzymes

25 were performed at 30 C on a rotary shaking table (300 r.p.m.) in
500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium
for 5 days.
Consequently in order to make an e.g. 2 litre broth 20
Erlenmeyer flasks were fermented simultaneously.


MEDIA:
BPX Medium Composition (per litre)


CA 02355579 2001-06-18

WO 90737624 43 PCT/DK99/00715
Potato starch 100 g

Ground barley 50 g
Soybean flour 20 g
Na2HPO4 x 12 H2O 9 g
Pluronic 0.1 g
Sodium caseinate 10 g

The starch in the medium is liquefied with a-amylase and
the medium is sterilized by heating at 120 C for 45 minutes.
After sterilization the pH of the medium is adjusted to 9 by

addition of NaHCO3 to 0.1 M.
EXAMPLE 1

CONSTRUCTION AND EXPRESSION OF ENZYME VARIANTS:
SITE-DIRECTED MUTAGENESIS:

Subtilase 309 site-directed variants of the invention
comprising specific insertions in the active site loop (b)
between positions 100 and 101 were made by traditional cloning
of DNA fragments (Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced

by PCR of oligos containing the desired insertions (see below).
The template plasmid DNA was pJS3, or an analogue of this
containing a variant of Subtilase 309._

Insertions were introduced by oligo directed mutagenesis
to the construction of G100GX insertion variants (X = any amino
acid residue inserted between positions 100 and 101) resulting
in G100GX subtilase 309 variants.

The Subtilase 309 variants were transformed into E. coli.
DNA purified from a over night culture of these transformants
were transformed into B. subtilis by restriction endonuclease

digestion, purification of DNA fragments, ligation,
transformation of B. subtilis. Transformation of B. subtilis was


CA 02355579 2001-06-18

- W0.00137624 44 PCT/DK99/00715
performed as described by Dubnau et al., 1971, J. Mol. Biol. 56,
pp. 209-221.

LOCALIZED RANDOM MUTAGENESIS IN ORDER TO INSERT RANDOM
s INSERTIONS IN A LOCALIZED REGION:

The overall strategy to used to perform localized random
mutagenesis was:

a mutagenic primer (oligonucleotide) was synthesized
corresponding to the DNA sequence flanking the site of

io insertion, separated by the DNA base pairs defining the
insertion.

Subsequently, the resulting mutagenic primer was used in
a PCR reaction with a suitable opposite primer. The resulting
PCR fragment was purified and extended in a second PCR-reaction,

is before being digested by endonucleases and cloned into the E.
coli - B. subtilis shuttle vector (see below).

Alternatively, and if necessary, the resulting PCR
fragment is used in a second PCR reaction as a primer with a
second suitable opposite primer to allow digestion and cloning

20 of the mutagenized region into the shuttle vector. The PCR
reactions are performed under normal conditions.

Following this strategy a localized random library was
constructed in SAVINASE wherein insertions were introduced in
the active site loop region between positions 100 and 101.

25 The mutations were introduced by mutagenic primers (see
below), so that all 20 amino acids are represented (N = 25% of
A, T, C, and G; whereas S = 50% C and G. The produced PCR
fragment were extended towards the N-terminal of Savinase by
another round of PCR by combination of a overlapping sequence

30 with a PCR-fragment produced by PCR-amplification with primers;
5' CTA AAT ATT CGT GGTGGC GC 3' (sense) and 5' GAC TTT AAC AGC
GTA TAG CTC AGC 3' (antisense). The extended DNA-fragments were


CA 02355579 2001-06-18

W0.00/37624 45 PCT/DK99/00715
cloned into the Hind III- and Mlu I- sites of the modified
plasmid pJS3 (see above), and ten randomly chosen E. coli
colonies were sequenced to confirm the mutations designed._

The mutagenic primer (5' GTT AAA GTC CTA GGG GCG AGC GGT
s NNS TCA GGT TCG GTC AGC TCG ATT G3'(sense)) were used in a PCR
reaction with a suitable anti-sense opposite primer, situated
downstream of the Mlu I site in pJS3 (e.g. 5'- CCC TTT AAC CGC
ACA GCG TTT -3' (anti-sense)) and the plasmid pJS3 as template.
This resulting PCR product was cloned into the pJS3 shuttle

io vector by using the restriction enzymes Hind III and Mlu I.

The random library was transformed into E. coli by well
known techniques.

The library prepared contained approximately 100,000
individual clones/library.

15 Ten randomly chosen colonies were sequenced to confirm
the mutations designed.

In order to purify a subtilase variant of the invention,
the B. subtilis pJS3 expression plasmid comprising a variant of
the invention was transformed into a competent B. subtilis

20 strain and was fermented as described above in a medium
containing 10 g/ml Chloramphenicol (CAM).

EXAMPLE 2

PURIFICATION OF ENZYME VARIANTS:

25 This procedure relates to purification of a 2 liter scale
fermentation for the production of the subtilases of the
invention in a Bacillus host cell.

Approximately 1.6 litres of fermentation broth were
centrifuged at 5000 rpm for 35 minutes in 1 litre beakers. The
30 supernatants were adjusted to pH 6.5 using 10% acetic acid and
filtered on Seitz Supra S100 filter plates.


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WO Q0/37624 4 6 PCTIDK99/00715
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.

The fractions with protease activity from the Bacitracin
to 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.
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.

The protease was eluted using a linear gradient of 0-0.1
M sodium chloride in 2 litres of the same buffer (0-0.2 M sodium
chloride in case of Subtilisin 147).

In a final purification step protease containing
fractions from the CM Sepharose column were combined and con-
centrated in an Amicon ultrafiltration cell equipped with a

GR81PP membrane (from the Danish Sugar Factories Inc.).

By using the techniques of Example 1 for the construction
and fermentation, and the above isolation procedure the
following subtilisin 309 variants were produced and isolated:
G100GT
G100GA
Gl00GS
Gl00GD


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WO QO/37624 47 PCT/DK99/00715
G100GE
G100GP
G100GG
G100GH
G100GI
G100GT+Y167A
S99G+G100GT+S101T
A98G+G100GA+S101A+S103T
These variants exhibited better wash performance than Savinase
in a preliminary assay.

s EXAMPLE 3

WASH PERFORMANCE OF DETERGENT COMPOSITIONS COMPRISING ENZYME
VARIANTS

The following examples provide results from a number of
washing tests that were conducted under the conditions indicated


CA 02355579 2001-06-18

WO Q&/37624 48 PCT/DK99/00715
MINI WASH

WASH CONDITIONS:

Europe Detergent 95 US
Detergent 4 g/l 3 g/l 1 g/l
Dosage

Wash Temp 30 C 15 C 25 C
Wash Time 30 min 15 min. 10 min

Water 18 dH (Ca '/Mg ' 6 dH 6 dH (Ca2+/Mg2+ _
hardness =5:1) 2:1)

pH Not adjusted 10.5 Not adjusted
Enzyme conc. 1, 2, 5, 10, 30 1, 2, 5, 10, 30
nM nM

Test system 150 ml glass 10 nm 150 ml glass
beakers with a beakers with a
stirring rod stirring rod

Textile/volu 5 textile 5 textile 5 textile pieces
me pieces (0 2.5 pieces (0 2.5 (0 2.5 cm) in 50
cm) in 50 ml cm) in 50 ml ml detergent
detergent detergent

Test EMPA116 EMPA117 EMPA117
Material

DETERGENTS:
The detergents used were either a model detergent, named
Detergent 95 or obtained from supermarkets in Denmark (OMO,
datasheet ED-9745105) and the USA (Wisk, datasheet ED-9711893),
respectively. Prior to use all enzymatic activity in the
detergents was inactivated by micro wave treatment.

Detergent 95 is a simple model formulation. pH is
adjusted to 10.5 which is within the normal range for a powder
detergent. The composition of model detergent 95 is as follows:


CA 02355579 2001-06-18

WO 40/37624 49 PCT/DK99/00715
25% STP (Na5P3O_ )

2 5 % Na2SO4
100-0 Na2CO3

20% LAS (Nansa 80S)

s 5.0% Non-ionic tenside (Dobanol 25-7)
5.001 Na2Si2O5
0.5% Carboxymethylcellulose (CMC)
9.5% Water

SWATCHES ;
The swatches used were EMPA116 and EMPA117, obtained from
EMPA Testmaterialen, Movenstrasse 12, CH-9015 St. Gall,
Switzerland.

1s REFLECTANCE
Measurement of reflectance (R) on the test material was
done at 460 nm using a Macbeth ColorEye 7000 photometer. The
measurements were done according to the manufacturers protocol.
EVALUATION
The evaluation of the wash performance of a subtilase is
determined by either the improvement factor or the performance
factor for the subtilase investigated.

The improvement factor, IFDose/response is defined as
the ratio between the slopes of the wash performance curves for
a detergent containing the subtilase investigated and the same
detergent containing a reference subtilase at the asymptotic
concentration of the subtilase goes to zero

IFDose/response= a/aref


CA 02355579 2001-06-18

WO 00137624 5 0 PCT/DK99/00715
The wash performance is calculated according to the
formula I:

a=ORmax =c
R = RO + DR +a c (I);
max
wherein
R is the wash performance in reflectance units; Ro is the
intercept of the fitted curve with y-axis (blind); a is the
slope of the fitted curve as c -a 0; c is the enzyme
concentration; and LRmax is the theoretical maximal wash effect
as c --* oo.


The performance factor, P, is calculated according to the
formula II

RVariant -RBlank (II)
RSavinase -RBlank
where
Rvariant is the reflectance of test material washed with lOnM
variant; Rsavinase is the reflectance of test material washed
with lOnM Savinase; Rblank is the reflectance of test material
washed with no enzyme

US (detergent: US Wisk, Swatch: EMPA117)
Variants P
G100GA 1,2
S99G+G100GGT+SlO1T 1,6
* P calculated at [E] = 5nM

The subtilases of the inventions are thus seen to exhibit
improved wash performance in comparison to Savinase .



CA 02355579 2001-06-18
50a

SEQUENCE LISTING
<110> NOVOZYMES A/S

<120> Subtilase enzymes of the I-S1 and I-S2 sub-groups
having an additional amino acid residue in an active
site loop region.

<130> 15194-33CA
<150> PCT/DK99/00715
<151> 1999-12-20
<150> PA 1998 01673
<151> 1998-12-18
<160> 2

<170> Patentln Ver. 2.1
<210> 1
<211> 275
<212> PRT
<213> Bacillus licheniformis
<400> 1
Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val
1 5 10 15
Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala
35 40 45

Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly
50 55 60
Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val
65 70 75 80
Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn
85 90 95

Ser Ser Gly Xaa Ser Gly Thr Tyr Ser Gly Ile Val Ser Gly Ile Glu
100 105 110
Trp Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly
115 120 125
Pro Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala
130 135 140

Arg Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly
145 150 155 160


CA 02355579 2001-06-18
50b

Asn Thr Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala
165 170 175
Val Gly Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val
180 185 190
Gly Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr
195 200 205

Tyr Pro Thr Ser Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser
210 215 220
Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn
225 230 235 240
Leu Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr
245 250 255
Leu Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala
260 265 270
Ala Ala Gln
275
<210> 2
<211> 270
<212> PRT
<213> Bacillus lentus
<400> 2
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala
1 5 10 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu
65 70 75 80
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95

Ser Gly Xaa Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp
100 105 110
Ala Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro
115 120 125
Ser Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg
130 135 140


CA 02355579 2001-06-18
50c

Gly Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile
145 150 155 160
Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp
165 170 175

Gln Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp
180 185 190
Ile Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr
195 200 205
Tyr Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly
210 215 220

Ala Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln
225 230 235 240
Ile Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn
245 250 255

Leu Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
260 265 270