Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1999-09-15
2001-02-06
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
Reexamination Certificate
active
06184015
ABSTRACT:
FIELD OF THE INVENTION
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.
No three-dimensional structural information has been available for a laccase before.
We have now elucidated the three-dimensional structure of a
Coprinus cinereus
laccase. By having this three-dimensional structure we are able to create laccase variants with altered properties: 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 three-dimensional structure of a laccase has now been elucidated. On the basis of an analysis of said structure it is possible to identify structural parts or specific amino acid residues which from structural or functional considerations appear to be important for increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O
2
/OH
−
-pathway of a laccase.
Furthermore, when comparing the three-dimensional structure of the Coprinus laccase structure with known amino acid sequences of various laccases, it has been found that some similarities exist between the sequences. The present invention is based on these findings.
Accordingly, in a first aspect the invention relates to a method of constructing a variant of a parent Coprinus laccase, which variant has laccase activity and increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O
2
/OH
−
-pathway as compared to said parent laccase, which method comprises
i) analysing the three-dimensional structure of the parent Coprinus laccase to identify at least one amino acid residue or at least one structural part of the Coprinus laccase structure, which amino acid residue or structural part is believed to be of relevance for altering the oxidation potential and/or altering the pH optimum and/or altering the mediator pathway and/or altering the O
2
/OH
−
-pathway of the parent Coprinus laccase (as evaluated on the basis of structural or functional considerations),
ii) constructing a Coprinus laccase variant, which as compared to the parent Coprinus laccase, has been modified in the amino acid residue or structural part identified in i) so as to alter the oxidation potential and/or alter the pH optimum and/or alter the mediator pathway and/or alter the O
2
/OH
−
-pathway, and, optionally,
iii) testing the resulting Coprinus laccase variant with respect to oxidation potential and/or pH optimum and/or mediator pathway and/or O
2
/OH
−
-pathway.
In a second aspect the present invention relates to a method of constructing a variant of a parent Coprinus-like laccase, which variant has laccase activity and increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O
2
/OH
−
-pathway as compared to said parent laccase, which method comprises
i) comparing the three-dimensional amino acid structure of the Coprinus laccase with an amino acid sequence of a Coprinus-like laccase,
ii) identifying a part of the Coprinus-like laccase amino acid sequence which is different from the Coprinus laccase amino acid sequence and which from structural or functional considerations is contemplated to be responsible for differences in the stability of the Coprinus and Coprinus-like laccase,
iii) modifying the part of the Coprinus-like laccase identified in ii) whereby a Coprinus-like laccase variant is obtained, which has an increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O
2
/OH
−
-pathway as compared to the parent Coprinus-like laccase, and optionally,
iv) testing the resulting Coprinus-like laccase variant with respect to oxidation potential and/or pH optimum and/or mediator pathway and/or O
2
/OH
−
-pathway.
In still further aspects the invention relates to variants of a Coprinus laccase and of Coprinus-like laccases, 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: 5480801 (1996-01-01), Wahleithner et al.
patent: 5998353 (1999-12-01), Pedersen et al.
patent: WO 92/01046 (1992-01-01), None
patent: WO 95/33836 (1995-12-01), None
patent: WO 96/00290 (1996-01-01), None
patent: WO 96/06930 (1996-03-01), None
patent: WO 96/23874 (1996-08-01), None
patent: WO 97/09431 (1997-03-01), None
Branden et al. Introduction to Protein Structure. Garland Publishing, Inc. (1991) pp. 269-271.
Ducros et al. Crystallization and preliminary X-ray analysis of the laccase fromCoprinus cinereus. Acta Cryst. (1997) D53, 605-607, Sep. 1997.
Branden et al. Introduction to Protein Structure. Garland Publishing, Inc. (1991) pp. 247-268.
Xu et al., BBA, vol. 1292, pp. 303-311 (1996).
Messerschmidt et al., Chemical Abstracts, vol. 111, No. 3, p. 297 (Jul. 17, 1989).
Hanna et al., Biochem J., vol. 253, pp. 561-568 (1988).
Kojima et al., (1990) J. Biol. Chem. 265(25) :15224-15230.
Svendsen Allan
Xu Feng
Achutamurthy Ponnathapu
Green, Esq. Reza
Kerr Kathleen
Lambiris Esq. Elias J.
Novo Nordisk A S
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