Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-12-13
2004-04-27
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S221000, C435S222000, C435S252300, C435S320100, C435S471000, C536S023200, C510S392000
Reexamination Certificate
active
06727085
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the use of subtilase variants for removal of egg stains from laundry or from hard surfaces. In particular the present invention relates to the use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, where the subtilase variant comprises at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering, vide infra). These subtilase variants 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 composition, including automatic dishwash compositions. The present invention also relates to novel subtilase variants, to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention.
BACKGROUND OF THE INVENTION
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. A thorough list of prior art protease variants is given in WO 99/27082.
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.
In particular, the problem of removing egg stains from e.g. laundry or hard surfaces has been pronounced due to the fact that many proteases are inhibited by substances present in the egg white. Examples of such substances include trypsin inhibitor type IV-0 (Ovo-inhibitor) and trypsin inhibitor type III-0 (Ovomucoid).
Therefore, an object of the present invention, is to provide improved subtilase variants, which are not, or which are only to a limited extent, inhibited by such substances. A further object of the present invention is to provide improved subtilase variants, 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 the use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, the subtilase variant comprising at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering).
In a second aspect the present invention relates to a subtilase variant selected from the group consisting of
a variant comprising at least one additional amino acid residue in the active site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 98 and 99 and further comprising at least one additional modification (BASBPN numbering), and
a variant comprising at least one additional amino acid residue in the active site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising at least one additional modification (BASBPN numbering),
where the variant—when tested in the “Ovo-inhibition Assay” disclosed in Example 4 herein—has a Residual Activity of at least 10%.
In a third aspect the present invention relates to a subtilase variant selected from the group consisting of
a variant comprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further comprising a substitution in positions 133 and 143,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising a substitution in position 99,
a variant comprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further comprising substitutions in positions 167, 170 and 194,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 216 and 217,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 217 and 218,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 42 and 43, and
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 129 and 130.
In a fourth aspect the present invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a fifth aspect the present invention relates to an expression vector comprising the isolated DNA sequence of the invention.
In a sixth aspect the present invention relates to a microbial host cell transformed with the expression vector of the invention.
In a seventh aspect the present invention relates to a method for producing a subtilase variant according to the invention, wherein a host according to the invention is cultured under conditions conducive to the expression and secretion of said variant, and the variant is recovered.
In an eight aspect the present invention relates to a cleaning or detergent composition, preferably a laundry or dishwash composition, comprising the variant of the invention.
In a ninth aspect the present invention relates to a method for removal of egg stains from a hard surface or from laundry, the method comprising contacting the egg stain-containing hard surface or the egg stain-containing laundry with a cleaning or detergent composition, preferably a laundry or dishwash composition, containing a subtilase variant comprising at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering).
Still other aspect of the present invention will be apparent from the below description and from the appended claims.
Concerning alignment and numbering reference is made to
FIG. 1
which shows an alignments between subtilisin BPN' (a) (BASBPN) and subtilisin 309 (BLSAVI) (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 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 version 9.1 to number the variants using the following parameters: gap creation penalty=8 and gap extension penalty=8 and all other parameters kept at their default values.
Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance.
Thereby a number of deletions and insertions will be defined in relation to BASBPN. In
FIG. 1
, subtilisin 309 (Savinase®) has 6 deletions in positions 36, 58, 158, 162, 163, and 164 in comparison to BASBPN. These deletions are in
FIG. 1
ind
Fanø Tina Sejersgård
Mikkelsen Frank
Lambiris Elias J.
Moore William W.
Nashed Nashaat T.
LandOfFree
Subtilase variants having an improved wash performance on... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Subtilase variants having an improved wash performance on..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Subtilase variants having an improved wash performance on... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3209005