Cleaning compositions for solid surfaces – auxiliary compositions – Cleaning compositions or processes of preparing – For cleaning a specific substrate or removing a specific...
Reexamination Certificate
2000-08-01
2003-09-23
Saidha, Tekchand (Department: 1652)
Cleaning compositions for solid surfaces, auxiliary compositions
Cleaning compositions or processes of preparing
For cleaning a specific substrate or removing a specific...
C435S196000, C435S198000, C435S252300, C435S320100, C435S069100, C510S305000, C510S392000, C530S350000, C536S023200, C536S023700
Reexamination Certificate
active
06624129
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to lipase variants suited for use in detergent compositions, particularly detergents with a high content of anionic surfactant. More particularly, the invention relates to variants of the wild-type lipase from
Humicola lanuginosa
strain DSM 4109.
BACKGROUND OF THE INVENTION
For a number of years, lipases have been used as detergent enzymes to remove lipid or fatty stains from clothes and other textiles, particularly a lipase derived from
Humicola lanuginosa
(EP 258 068 and EP 305 216) sold under the tradename Lipolase® (product of Novo Nordisk A/S).
WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079 and WO 97/07202 disclose variants of the
H. lanuginosa
lipase having improved properties for detergent purposes. Thus, WO 97/04079 discloses variants having a peptide addition (extension) at the N-terminal. WO 97/07202 discloses lipase variants with “first wash performance” which are capable of removing substantial amounts of lard from a lard stained swatch in a one-cycle wash.
There is an ever existing need for providing novel lipases with improved properties, in particular improved washing properties in commercial detergents, including detergents with a high content of anionic surfactants. The present invention relates to such novel lipases.
SUMMARY OF THE INVENTION
The inventors have found that variants of Lipolase (wild-type
Humicola lanuginosa
lipase) with a certain distribution of electrically charged amino acids have a particularly good first-wash performance in a detergent solution with a high ratio of anionic to non-ionic surfactant.
The inventors found that the effect is achieved by attaching a positively charged peptide extension at the N-terminal and by imposing certain restrictions on the charge distribution in the region corresponding to amino acid positions 90-101 and at position 210. The inventors further devised a method of developing variants with such performance from Lipolase by attaching a peptide extension at the N-terminal and substituting amino acids in the region 90-101 or in the immediate surroundings in the three-dimensional structure. The lipases may further provide additional benefits, such as whiteness maintenance and dingy cleanup.
Accordingly, the invention provides a lipase which is a polypeptide having an amino acid sequence which:
a) has at least 90% identity with the wild-type lipase derived from
Humicola lanuginosa
strain DSM 4109;
b) compared to said wild-type lipase, comprises a positively charged peptide extension attached to the N-terminal;
c) comprises a negative amino acid in position E210 of said wild-type lipase.
Further, the amino acid sequence may:
d) comprise a negatively charged amino acid in the region corresponding to positions 90-101 of said wild-type lipase; and
e) comprise a neutral or negative amino acid at a position corresponding to N94 of said wild-type lipase and/or has a negative or neutral net electric charge in the region corresponding to positions 90-101 of said wild-type lipase.
Alternatively, the amino acid sequence may:
d) comprise amino acids with negative or unchanged electric charge in at least two of positions N94, D96 and E99 of said wild-type lipase.
The invention also provides a detergent composition comprising the lipase, a DNA sequence encoding the lipase, an expression vector harboring the DNA sequence, a transformed host cell containing said DNA sequence or said expression vector, and a method of producing the lipase by culturing the transformed host cell.
Further, the invention provides a method of producing a variant lipase, which method comprises:
a) selecting a parent lipolytic enzyme having an amino acid sequence which has at least 90% identity with the wild-type lipase derived from Humicola lanuginosa strain DSM 4109;
b) modifying the sequence of a nucleic acid encoding the parent lipase to produce a nucleic acid encoding a lipase which comprises a peptide extension at the N-terminal and an amino acid substitution at as location:
i) in the region corresponding to positions 90-101 of said wild-type lipase, or
ii) at the surface of the three-dimensional structure within 6 Å of any of the positions 90-101,
c) expressing the modified nucleic acid in a host cell to produce the variant lipase,
d) testing the first-wash effect of the variant lipase in a detergent solution comprising anionic surfactant in an amount of more than 70% by weight of the total surfactant,
e) optionally repeating steps b-d, and
f) selecting a variant having improved first-wash effect.
REFERENCES:
patent: 0 258 068 (1988-03-01), None
patent: 0 305 216 (1989-03-01), None
patent: WO 92/05249 (1992-04-01), None
patent: WO 94/25577 (1994-11-01), None
patent: WO 95/22615 (1995-08-01), None
patent: WO 96/00292 (1996-01-01), None
patent: WO 97/04079 (1997-02-01), None
patent: WO 97/07202 (1997-02-01), None
Anant Patkar Shamkant
Bojsen Kirsten
Borch Kim
Halkier Dorte Aaby
Svendsen Allan
Garbell Jason I.
Lambrias Elias J.
Novozymes A/S
Saidha Tekchand
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