Laccase mutants

Details Laccase mutants Laccase mutants

2000-03-06

2001-04-17

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S189000, C435S320100, C435S254300, C536S023200, C530S350000

Reexamination Certificate

active

06218170

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to laccase mutants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O
2
/OH
−
pathway.
BACKGROUND OF THE INVENTION
Laccase is a polyphenol oxidase (EC 1.10.3.2) which catalyses the oxidation of a variety of inorganic and aromatic compounds, particularly phenols, with the concomitant reduction of molecular oxygen to water.
Laccase belongs to a family of blue copper-containing oxidases which includes ascorbate oxidase and the mammalian plasma protein ceruloplasmin. All these enzymes are multi-copper-containing proteins.
Because laccases are able to catalyze the oxidation of a variety of inorganic and aromatic compounds, laccases have been suggested in many potential industrial applications such as lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair colouring, and waste water treatment.
The various applications ask for laccases with specific properties. It is the purpose of the present application to create laccase variants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O
2
/OH
−
-pathway.
BRIEF DISCLOSURE OF THE INVENTION
The present invention relates to laccase variants, in particular to
A variant of a parent laccase, which variant has laccase activity, and increased oxidation potential and comprises a mutation in a position corresponding to at least one of the following positions:
G511A,V,P,L,I,F,Y,W;
T428A,V,P,L,I,F,Y,W;
S510A,V,P,L,I,F,Y,W;
D106A,V,P,L,I,F,Y,W;
N109A,V,P,L,I,F,Y,W,Q;
L500I,F,Y,W;
A108V,P,L,I,F,Y,W;
G514A,V,P,L,I,F,Y,W;
 wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1;
A variant of a parent laccase, which variant has laccase activity and an altered pH optimum and comprises a mutation in a position corresponding to at least one of the following positions:
192-193;
234-236;
269;
293-294;
364-365;
372-373;
426-433;
503-513;
 wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1;
A variant of a parent laccase, which variant has laccase activity and an altered mediator efficiency and comprises a mutation in a position corresponding to at least one of the following positions:
185-194;
235;
293-294;
365-373;
427-429;
505;
507-508;
510-511;
 wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1; and
A variant of a parent laccase, which variant has laccase activity and an altered O
2
/OH
−
-pathway and comprises a mutation in a position corresponding to at least one of the following positions:
A506E;
N109D;
H93E;
H95E;
M433E;
M480E;
 wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1.
In still further aspects the invention relates to DNA encoding such variants and methods of preparing the variants.
Finally, the invention relates to the use of the variants for various industrial purposes.


REFERENCES:
patent: 5770419 (1998-06-01), Feng et al.
patent: 5856164 (1999-01-01), Outtrup et al.
patent: WO 95/11296 (1995-04-01), None
patent: WO 95/33836 (1995-12-01), None
patent: WO 97/09431 (1997-03-01), None
patent: WO 95/33837 (1997-12-01), None
Germann et al., (1988) The Journal of Biological Chemistry, 2:885-896.
Xu et al., (1996) Biochimica et Biophysica Acta 1292: 303-311.
Kojima et al., (1990) The Journal of Biological Chemistry, 265(25):15224-15230.

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