Cleaning compositions for solid surfaces – auxiliary compositions – Cleaning compositions or processes of preparing – With oxygen or halogen containing chemical bleach or oxidant...
Reexamination Certificate
1998-10-06
2001-07-10
Prouty, Rebecca E. (Department: 1652)
Cleaning compositions for solid surfaces, auxiliary compositions
Cleaning compositions or processes of preparing
With oxygen or halogen containing chemical bleach or oxidant...
C435S192000, C435S440000, C435S471000, C510S392000, C536S023200
Reexamination Certificate
active
06258769
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a novel variant of peroxidase, and a bleaching agent 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.
One drawback of these commonly used bleaching agents is that they are not particularly efficient at the lower temperatures at which coloured fabrics are usually washed. Their efficiency may be enhanced by the use of activators (e.g. organic acid anhydrides, esters or imides) which give rise to the formation of peracids.
Apart from being employed for bleaching stains on fabric, such conventional bleaching agents have also been suggested for preventing surplus dyes from coloured fabrics which leach from the fabrics when these are washed from being deposited on other fabrics present in the same wash (this phenomenon is commonly known as dye transfer). The problem of dye transfer, of course, is most noticeable when white or light-coloured fabrics are washed together with fabrics of a darker colour from which dye is leached during washing.
It has been found that peroxidases utilizing hydrogen peroxide as their substrate are able to enhance the bleaching effect of hydrogen peroxide during washing. The use of peroxidase 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. The use of peroxidase for inhibiting the transfer of dye from a dyed fabric to another fabric during washing 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 may be prepared by recombinant DNA techniques.
Accordingly, the present invention relates to a peroxidase variant with improved hydrogen peroxide stability, characterized by insertion, deletion or substitution of one or more amino acid residues located in or near the substrate channel of the parent peroxidase, near the heme group of the parent peroxidase or at or near the active site of the parent peroxidase.
Information about the three-dimensional structure of the parent peroxidase was obtained 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 press). The sequence alignment showed that the parent peroxidase was homologous to yeast cytochrome c peroxidase (CCP) which has a known structure (J. Wang et al. , Biochemistry 29, 1990, p. 7160), and the overall structure of the parent peroxidase could therefore be inferred from the CCP structure. An example of how structural information for a homologous peroxidase can be inferred from the crystal structure of CCP is given in P. Du et al., Protein Engineering 5, 1992, pp. 679-691.
In the present context, the term “substrate channel” is intended to indicate a passage in the peroxidase molecule through which the substrate passes before oxidation at the heme group of the peroxidase. The term “active site” is intended to indicate the substrate-binding site of the peroxidase (cf. Eur. J. Biochem. 213, 1993, pp 605-611). The term “near” should be taken to indicate a distance from the substrate channel, heme group or active site, respectively, of not more than 15 Å, more preferably not more than 10 Å, most preferably not more than 5Å, in any direction. The term “stability” is intended to indicate that the enzyme is active in the presence of hydrogen peroxide at a concentration of up to 20 mM.
In another aspect, the present invention relates to a bleaching agent comprising a peroxidase variant according to the invention, optionally in the form of a non-dusting granulate, a liquid, in particular a stabilised liquid, or a protected enzyme.
REFERENCES:
patent: 4732973 (1988-03-01), Barr et al.
patent: 4752585 (1988-06-01), Koths et al.
patent: 5286638 (1994-02-01), Tanaka et al.
patent: 5346823 (1994-09-01), Estell et al.
patent: 179486 (1986-04-01), None
patent: 505311 (1991-09-01), None
patent: 486067 (1992-05-01), None
patent: WO 91/05858 (1991-02-01), None
patent: 91/05839 (1991-05-01), None
M. Kjalke et al. “Comparison of Structure and Activities of Peroxidases fromCoprinus cinereus, Coprinus macrorhizus,andArthromyces ramosus”, Biochem. Biophys. Acta 1120: 248-256, Apr. 1992.*
K. Welinder et al. “Structure of Plant and Fungal Peroxidases”, Biochem. Soc. Trans. 20: 337-340, May 1992.
Andersen Morten Birket
Welinder Karen Gjesing
Lambiris Esq. Elias
Novozymes A/S Patents
Prouty Rebecca E.
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