Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-03-23
2004-08-17
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S221000, C435S252300, C435S032000, C435S471000, C536S023200, C510S350000
Reexamination Certificate
active
06777218
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. 119 of Danish application PA 2000 00405 filed on Mar. 14, 2000, the contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to novel subtilases having an improved wash performance on egg stains. These subtilases are useful exhibiting excellent or improved wash performance on egg stains when used in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dishwash compositions, including automatic dishwash compositions.
The present invention also relates to isolated nucleic acid sequences encoding the subtilases, nucleic acid constructs, recombinant expression vectors, host cells comprising the nucleic acid construct, and methods for producing and using the subtilases of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the subtilase enzymes of the invention as well as to use of such enzymes in detergent compositions and for removal of egg stains.
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. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM® (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 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 (1985)
Nature
318 375-376; Thomas, Russell, and Fersht (1987)
J. Mol. 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); 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 Al (SOLVAY); and WO 94/02618 (GIST-BROCADES N.V.).
However, even though a number of useful proteases and protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses.
In particular, the problem of removing egg stains from e.g. laundry or hard surfaces has been pronounced due to the fact that many serine proteases are inhibited by substances present in the egg white.
Therefore, an object of the present invention, is to provide improved subtilase enzymes, which are suitable for removal of egg stains from, for example, laundry and/or hard surfaces.
SUMMARY OF THE INVENTION
Thus, in a first aspect the present invention relates to a subtilase enzyme having improved wash performance on egg stains, the subtilase being selected from the group consisting of
(a) a subtilase having an amino acid sequence which has at least 88% identity with the amino acid sequence shown as amino acids 1 to 269 of SEQ ID NO:2;
(b) a subtilase which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with
(i) a complementary strand of the nucleic acid sequence shown as nucleotides 1 to 807 of SEQ ID NO:1, or
(ii) a subsequence of (i) of at least 100 nucleotides; and
(c) a subtilase encoded by the subtilase encoding part of the nucleic acid sequence cloned into a plasmid fragment present in
Escherichia coli
MT173 DSM 13306, or a variant thereof having at least 88% identity to said subtilase.
In a second aspect the present invention relates to an isolated nucleic acid sequence comprising a nucleic acid sequence which encodes for the subtilases according to the invention.
In a third aspect the present invention relates to an isolated nucleic acid sequence encoding a subtilase, selected from the group consisting of
(a) a nucleic acid sequence having at least 88% identity with the nucleic acid sequence shown as nucleotides 1 to 807 SEQ ID NO:1; and
(b) a subtilase which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with
(i) a complementary strand of the nucleic acid sequence shown as nucleotides 1 to 807 of SEQ ID NO:1, or
(ii) a subsequence of (i) of at least 100 nucleotides; and
(c) the subtilase encoding part of the nucleic acid sequence which has been cloned into a plasmid present in
Escherichia coli
MT173 DSM 13306, or a variant thereof having at least 88% identity to said nucleic acid sequence.
In a fourth aspect the present invention relates to a nucleic acid construct comprising the nucleic acid sequence according to the invention operably linked to one or more control sequences capable of directing the expression of the subtilase in a suitable host.
In a fifth aspect the present invention relates to a recombinant expression vector comprising the nucleic acid construct according to the invention, a promoter, and transcriptional and translational stop signals.
In a sixth aspect the present invention relates to a recombinant host cell comprising the nucleic acid construct of the invention.
In a seventh aspect the present invention relates to a method for producing the subtilase according to the invention, the method comprising:
(a) cultivating a recombinant host cell according to the invention under conditions conducive to the production of the subtilase; and
(b) recovering the subtilase.
In an eight aspect the present invention relates to a cleaning or detergent composition, preferably a laundry or dishwash composition, comprising the subtilase according to the invention.
Further aspects of the present invention relate to use of the subtilases according to the invention in a cleaning or detergent composition; use of the subtilases or the compositions according to the invention for removal of egg stains; a method for cleaning or washing, including a method for removal of egg stains from, a hard surface or laundry comprising contacting the hard surface or the laundry with the composition of the invention.
Concerning alignment and numbering reference is made to
FIG. 1
which shows alignments between subtilisin BPN′ (a) (BASBPN) and the novel subtilase of the invention (b).
These alignments are in this patent application used as a reference for numbering the residues.
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 various subtilase enzyme variants produced or contemplated according to the invention, the following nomenclatures and conventions have been adapted for ease of reference:
A frame of reference is first defined by aligning the isolated or parent enzyme with subtilisin BPN′ (BASBPN).
The alignment can be obtained by the GAP routine of the GCG package v
Borchert Torben V.
Cherry Joel
Fanø Tina Sejersgård
Giver Lorraine J.
Mikkelsen Frank
Achutamurthy Ponnathapu
Lambiris Elias J.
Moore William W.
Novozymes A/S
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