Cutinase variants

Details Cutinase variants Cutinase variants

2001-05-31

2004-11-09

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S071100, C435S320100, C435S252330, C435S325000, C435S041000, C435S009000, C435S455000, C435S471000, C536S023200, C510S300000

Reexamination Certificate

active

06815190

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a cutinase variant, more particularly to a cutinase variant having improved thermostability. The invention also relates to a DNA sequence encoding the variant, a vector comprising the DNA sequence, a transformed host cell harboring the DNA sequence or the vector, to a method of producing the variant, and to use of the variant.
BACKGROUND OF THE INVENTION
Cutinases are lipolytic enzymes capable of hydrolyzing the substrate cutin. Cutinases are known from various fungi (P. E. Kolattukudy in “Lipases”, Ed. B. Borgström and H. L. Brockman, Elsevier 1984, 471-504). The amino acid sequence and the crystal structure of a cutinase of
Fusarium solani pisi
have been described (S. Longhi et al., Journal of Molecular Biology, 268 (4), 779-799 (1997)). The amino acid sequence of a cutinase from
Humicola insolens
has also been published (U.S. Pat. No. 5,827,719).
A number of variants of the cutinase of
Fusarium solani pisi
have been published WO 94/14963; WO 94/14964; Appl. Environm. Microbiol. 64, 2794-2799, 1998; Proteins: Structure, Function and Genetics 26, 442-458, 1996; J. of Computational Chemistry 17, 1783-1803, 1996; Protein Engineering 6, 157-165, 1993; Proteins: Structure, Function, and Genetics 33, 253-264, 1998; J. of Biotechnology 66, 11-26, 1998; Biochemistry 35, 398-410, 1996.
Fungal cutinases may be used in the enzymatic hydrolysis of cyclic oligomers of poly(ethylene terephthalate), e.g. in the finishing of yarn or fabric from poly(ethylene terephthalate) fibers (WO 97/27237). However, it is desirable to improve the thermostability of known fungal cutinases to allow a higher process temperature.
SUMMARY OF THE INVENTION
The inventors have found certain variants of fungal cutinases having improved thermostability.
Accordingly, the invention provides a variant of a parent fungal cutinase comprising substitution of one or, more amino acid residues which is located:
a) within 17 Å from the location of the N-terminal amino acid (as calculated from amino acid residues in a crystal structure), and/or
b) within 20 positions from the N-terminal amino acid.
The invention also provides a DNA sequence encoding the variant, an expression vector comprising the DNA sequence, a transformed host cell harboring the DNA sequence or the expression vector, a method of producing the variant, processes using the variant and a detergent composition comprising the variant.


REFERENCES:
patent: 5827719 (1998-10-01), Sandal
patent: 6254645 (2001-07-01), Kellis, Jr.
patent: WO 94/14964 (1994-07-01), None
Nicolas,A. and Cambillau,C. (1995) Fusarium Solani Cutinase Mutant With Thr 45 Replaced By Ala (gi|1827561|pdb|1XZG| [1827561]) and Fusarium Solani Cutinase Mutant With Thr 119 Replaced By His (gi|1827559|pdb|1XZI| [1827559]).*
Longhi,S. and Cambillau,C. (1995) Fusarium Solani Cutinase Mutant With Thr 38 Replaced By Phe gi|1827558|pdb|pdb|1XZJ|, Cutinase, R196e Mutant. (gi|1633261|pdb|1CUH| [1633261]), and Cutinase, A199c Mutant (gi|1633258|pdb|1CUU| [1633258]).*
WF Fett, HC Gerard, RA Moreau, SF Osman and LE Jones Screening of Nonfilamentous Bacteria for Production of Cutin-Degrading Enzymes Appl. Environ. Microbiol., Jul. 1992, 2123-2130, vol. 58, No. 7 (Abstract).*
Alignment of SEQ ID No.: 2 from 08/827,997 with Accession# Q99174 by BLOSUM62.*
Ausbel, FM (1995) Enzymatic manipulation of DNA and RNA In: Current Protocols in Molecular Biology Unit 3.16.

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