Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1996-05-07
1998-10-06
Prouty, Rebecca E.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
4351723, 536 232, 2521861, 25218627, 25218628, 25218629, 2521863, 25218631, 25218638, 510305, 510374, C12N 908, C11D 754, C11D 742
Patent
active
058174958
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/DK94/00382 filed Oct. 13, 1994, which is incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a novel variant of peroxidase, and a bleaching or detergent composition comprising the peroxidase variant.
BACKGROUND OF THE INVENTION
The use of bleaching agents in washing procedures and as constituents of detergent compositions is well known in the art. Thus, bleaching agents are incorporated in or sold as constituents of a major part of the commercially available detergent compositions. Important conventional bleaching agents incorporated in detergent compositions are compounds which act as precursors of hydrogen peroxide formed in the course of the washing procedure. Perborates and percarbonates are the most important examples of compounds which are employed as bleaching agents and which exert a bleaching effect in this fashion. The detailed mechanism of bleaching by means of these bleaching agents is not known at present, but it is generally assumed that the hydrogen peroxide formed during washing converts coloured substances (responsible for stains on fabric) into non-coloured materials by oxidation and that some oxidation of the coloured substances may also take place due to their direct interaction with perborate or percarbonate.
It has been found that peroxidases, utilizing hydrogen peroxide as substrate, are able to enhance the bleaching effect of hydrogen peroxide during washing. The use of peroxidases for bleaching stains on fabrics is described in WO 89/09813. It was also found that coloured substances leached from dyed fabrics could be bleached by means of peroxidases together with hydrogen peroxide. The use of peroxidases for inhibiting dye transfer in this way is described in WO 91/05839.
SUMMARY OF THE INVENTION
It has surprisingly been found that peroxidase variants with an improved stability towards hydrogen peroxide at alkaline conditions may be prepared by recombinant DNA techniques.
Accordingly, the present invention relates to a peroxidase variant with improved hydrogen peroxide stability at alkaline conditions, characterized by insertion, deletion or substitution of one or more amino acid residues in the region from amino acid residue 48 to 56, 76, 109, 214, 239, 258 to 262, 264, 266 to 272 of the parent peroxidase, a Coprinus cinereus peroxidase encoded by the sequence shown in SEQ ID 1.
Information about the crystal structure of the parent peroxidase was obtained by X-ray diffraction, and also by aligning the amino acid sequence of the parent peroxidase to amino acid sequences of other known peroxidases (K. G. Welinder et al., "Structure and evolution of peroxidases" in Plant Peroxidase Biochemistry and Physiology, K. G. Welinder et al. (eds.), University of Copenhagen and Geneva 1993).
In the present context the term "improved hydrogen peroxide stability" is intended to indicate that the peroxidase variant is at least 10% more stable than the parent peroxidase in the presence of hydrogen peroxide at a concentration of up to 20 mM H.sub.2 O.sub.2. More specifically this is intended to mean that the peroxidase variant at one or more temperatures in the temperature interval of from 10.degree. C. to 60.degree. C. and at one or more H.sub.2 O.sub.2 concentrations of up to 20 mM H.sub.2 O.sub.2 has an at least 10% longer half-life than the wild type peroxidase (parent peroxidase). (Half-life is determined by fitting residual activities to 1st order decay). The term "alkaline conditions" is intended to indicate the pH-range from 7 to 11.
In other aspects, the present invention relates to a bleaching composition comprising a peroxidase variant according to the invention and a hydrogen peroxide source, optionally additionally comprising an oxidizable substrate; and to a detergent composition comprising a surfactant, a peroxidase variant according to the invention and a hydrogen peroxide source, optionally additionally comprising an oxidizable substrate.
BRIEF DESCRIPTION OF DRAWI
Cherry Joel R.
Jensen Birger Rostgaard
Lamsa Michael
Pedersen Anders Hjelholt
Schneider Palle
Gregg, Esq. Valeta
Novo Nordisk A S
Prouty Rebecca E.
Zelson Esq. Steve T.
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