Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2000-05-23
2001-08-21
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
Reexamination Certificate
active
06277611
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a me-hod of designing laccase mutants with improved stability properties, which method is based on the hitherto unknown three-dimensional structure of laccases.
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. A major problem with the use of laccases are their poor storage stability at temperatures above room temperature, especially at 40° C.
In Example 1 of the present application we have tested the stability of various laccases at 40° C., and it can be seen that after 2 weeks of storage the laccase activity is down to less than 50% of the initial value, and at low pH the laccase activity after 2 weeks is zero. For many purposes such a decrease is unacceptable, so it is the purpose of the present invention to create laccase variants with improved stability by using the information of a three-dimensional structure of a
Coprinus cinereus
laccase. No three-dimensional structural information has been available for a laccase before.
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 the stability 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 Corrinus laccase, which variant has laccase activity and improved stability 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 stability 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 stability, and, optionally,
iii) testing the resulting Coprinus laccase variant with respect to stability
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 improved stability 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 improved stability as compared to the parent Coprinus-like laccase, and optionally,
iv) testing the resulting Coprinus-like laccase variant with respect to stability.
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.
DETAILED DISCLOSURE OF THE INVENTION
The Coprinus-like laccases
A number of laccases produced by different fungi are homologous on the amino acid level. For instance, when using the homology percent obtained from UWGCG program using the GAP program with the default parameters (penalties: gap weight=3.0, length weight=0.l; WISCONSIN PACKAGE Version 8.1-UNIX, August 1995, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) the following homology was found:
Coprinus cinereus
laccase comprising the amino acid sequence shown in SEQ ID No. 1: 100%;
Polyporus pinsitus
(I) laccase comprising the amino acid sequence shown in SEQ ID No. 2: 74.4%;
Polyporus pinsitus
(II) laccase comprising the amino acid sequence shown in SEQ ID No. 3: 73.8%;
Phlebia radiata
laccase comprising the amino acid sequence shown in SEQ ID No. 4: 69.9%;
Rhizoctonia solani
(I) laccase comprising the amino acid sequence shown in SEQ ID No. 5: 64.8%;
Rhizoctonia solani
(II) laccase comprising the amino acid sequence shown in SEQ ID No. 6: 63.0%;
Rhizoctonia solani
(III) laccase comprising the amino acid sequence shown in SEQ ID No. 7: 61.0%;
Rhizoctonia solani
(IV) laccase comprising the amino acid sequence shown in SEQ ID No. 8: 59.7%;
Scytalidiurn thermophilum
laccase comprising the amino acid sequence shown in SEQ ID No. 9: 57.4%;
Myceliophthora thermophila
laccase comprising the amino acid sequence shown in SEQ ID No. 10: 56.5%.
Because of the homology found between the above mentioned laccases, they are considered to belong to the same class of laccases, namely the class of “Coprinus-like laccases”.
Accordingly, in the present context, the term “Coprinus-like laccase” is intended to indicate a laccase which, on the amino acid level, displays a homology of at least 50% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 55% and less than 100% to the Coprinus cinereus laccase SEQ ID NO 1, or at least 60% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 65% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 70% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 75% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 80% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 85% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 90% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1, or at least 95% and less than 100% to the
Coprinus cinereus
laccase SEQ ID NO 1.
In the present context, “derived from” is intended not only to indicate a laccase produced or producible by a strain of the organism in question, but also a la-case encoded by a DNA sequence isolated from such strain and produced in a host organism containing said DNA sequence. Finally, the term is intended to indicate a laccase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the laccase in question.
The three-dimensional Coprinus laccase structure
The Coprinus laccase which was used to elucidate the three-dimensional structure forming the basis for the present invention consists of the 539 amino acids derived from
Coprinus cinereus
laccase IFO 8371 as disclosed in sequence ID No. 1.
The obtained three-dimensional structure is believed to be representative for the structure of any Coprinus-like laccase.
The structure of the laccase was solved in accordance with the principle for X-ray crystallographic methods given in “X-Ray Struc
Cherry Joel
Pedersen Anders Hjelholt
Rasmussen Grethe
Schneider Palle
Svendsen Allan
Garbell Esq. Jason I.
Laubiris, Esq Elian J.
Novozymeo A/S
Slobodyansky Elizabeth
LandOfFree
Laccase mutants does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Laccase mutants, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Laccase mutants will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2500863