Subtilase enzymes

Details Subtilase enzymes Subtilase enzymes

2002-09-12

2004-08-10

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S221000, C435S222000, C435S252300, C435S320100, C435S471000, C510S350000, C536S023200

Reexamination Certificate

active

06773907

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to subtilase enzymes having an additional amino acid in the active site loop (b) region from position 95 to 103 and detergent and cleaning compositions comprising same. The invention further relates to genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and host cells transformed therewith, and methods for producing the enzymes.
2. Description of the Related Art
In the detergent industry, enzymes have been used in washing formulations for more than 30 years. Such enzymes include proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. The most important commercially are proteases.
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild-type proteases, e.g. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).
In addition, a number of protease variants have been described in the art, such as in EP 130756 (GENENTECH) (corresponding to U.S. Reissue Pat. No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht,
Nature,
318, 375-376 (1985); Thomas, Russell, and Fersht,
J. Mol. Biol.,
193, 803-813 (1987); 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); U.S. Pat. No. 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 A1 (SOLVAY); and WO 94/02618 (GIST-BROCADES N.V.).
However, even though a number of useful protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses.
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 is longer than those presently known, exhibit improved wash performance properties in detergent compositions. The identification thereof was done by constructing subtilisin variants, especially of subtilisin 309 (BLSAVI or SAVINASE®), which exhibited improved wash performance properties in detergent compositions relative to the parent wild-type enzyme. This was described in our earlier application DK 1332/97, which published as WO 99/27082.
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 the active site loop (b) region from position 95 to 103, exhibit surprisingly improved wash performance in 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) region 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 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 wild-type subtilases comprising an active site loop (b) region which is longer than the corresponding active site loop region in known wild-type subtilases, such as subtilisin 309, which subtilases can be considered to have an inserted amino acid residue in the active site loop (b) region, and exhibiting excellent wash performance in a detergent, in comparison to their closest related known subtilisin, such as subtilisin 309.
Concerning alignment and numbering reference is made to
FIGS. 1A
,
1
B,
2
A and
2
B showing alignments between subtilisin BPN′ (BASBPN) (a) and subtilisin 309 (BLSAVI) (b), and alignments between subtilisin BPN′ (a) (BASBPN) and subtilisin Carlsberg (g) (BLSCAR). These alignments are used herein as a reference for numbering the residues.
The seven active site loops (a) to (g) are herein defined as the segments of amino acid residues provided below (including the terminal amino acid residues):
(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 enzyme of the I-S1 and I-S2 sub-groups having at least one additional amino acid residue in the active site loop (b) region from position 95 to 103, whereby said additional amino acid residue(s) corresponds to the insertion of at least one amino acid residue.
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 third aspect.
In a further aspect the invention relates to the production 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 form; or by inserting an expression vector according to the third 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 and detergent compositions, comprising the subtilisin enzymes of the present invention.
Definitions
Prior to discussing this invention in further detail, the following terms and conventions will first be defined.
NOMENCLATURE OF 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
X = Xaa = Any amino acid
NOMENCLATURE OF NUCLEIC ACIDS
A = Adenine
G = Guanine
C = Cytosine
T = Thymine (only in DNA)
U = Uracil (only in RNA)
Nomenclature and Conventions for Designation of Variants
In describing the subtilases of the present invention, the following nomenclatures and conventions have been adapted for ease of reference:
A frame of reference is first defined by aligning the isolated or parent 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 following parameters: ga

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