WO2002061068A2 - Oxidase free of catalase side activities - Google Patents

Abstract

The invention is directed to a method for inactivating catalase side activities in a composition comprising oxidase and catalase by subjecting the composition to a sulphur-containing reducing agent. By this method an oxidase composition is obtained with substantially no catalase side activity. The composition may be used for bleaching purposes, e.g. for bleaching teeth and textile.

Description

OXIDASE FREE OF CATALASE SIDE ACTIVITIES

FIELD OF INVENTION

The present invention relates to oxidase compositions free of catalase side activities, to a method for its production and to its use in e.g. bleaching compositions, as well as in baking or anti-microbial compositions..

BACKGROUND OF THE INVENTION

Oxidases are enzymes, which catalyse reactions wherein oxygen acts as electron acceptor. The reaction products include hydrogen peroxide. The overall reaction mechanism is:

Substrate + O₂ → Oxidized substrate + H₂O₂

An example of this kind of enzymes is glucose oxidase. Glucose oxidases are enzymes that catalyze the oxidation of glucose with oxygen whereby hydrogen peroxide is formed. Such enzymes are known from microbial, plant and animal origins, e.g., glucose oxidase from Aspergillus, Penicillium and Talaromyces. Glucose oxidase has been described as useful for various purposes, e.g., for bleaching purposes and in the baking industry, useful for strengthening the dough.

An example of a commercial glucose oxidase is Gluzyme™, an Aspergillus niqer glucose oxidase, available from Novozymes A/S, Denmark. US A 5,094,951 describes the cloning and expressing of this enzyme in a recombinant nucleic acid system. After fermentation the glucose oxidase may be recovered from the fermentation medium by conventional procedures including, centrifugation, filtration, spray-drying, evaporation or precipitation. The enzyme may then be further purified by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like.

Despite these procedures catalase side activities are still present in the purified fermentation broth. Catalase breaks down hydrogen peroxide into water and oxygen. Therefore residual catalase activity is unwanted for many industrial applications. When e.g. glucose oxidase is used in connection with bleaching, e.g. tooth or textile bleaching and as an anti-microbial agent it would be desirable that no catalase activity is present in the product.

US A 5,919,684 discloses a method for reducing catalase activity in alcohol oxidase compositions which comprises aging the composition obtained by the fermentation of Pichia pastohs cells at a temperature and for a time period sufficient to accomplish inactivating said catalase while maintaining alcohol oxidase activity. In particular conditions for the aging are a temperature in the range off about 1 to about 15°C for at least 20 days.

It is an object of the present invention to provide a composition comprising an oxidase with substantially no catalase activity.

SUMMARY OF THE INVENTION

The present inventors have found a method for preparing an oxidase composition which is substantially free from catalase activity, thereby providing an oxidase composition which can be used for different purposes, e.g. bleaching, antimicrobial or baking purposes.

Consequently, in the first aspect the present relates to a method for inactivation catalase activity in a composition comprising oxidase and catalase, wherein the composition is treated with a sulphur-containing reducing agent. In the second aspect the invention relates to a composition comprising an oxidase substantially free from catalase activity.

In a third aspect the invention relates to the use of the composition for bleaching purposes, disinfecting purposes, baking purposes etc.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there is provided a method for inactivation of the catalase activity of a composition comprising an oxidase and a catalase by treating the composition with a sulphur-containing reducing agent. Before the methods of the invention are described, it is to be understood that this invention is not limited to the particular methods described. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "oxidase" includes mixtures of such oxidases, reference to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the particular methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the material for which the reference was cited in connection with.

The term "bleaching" is here defined as a whitening of a surface, e.g. a fiber, fabric, garment, yarn or teeth, and can be measured by using the change in the colour space coordinates L^*a^*b^* (CIELAB-system): L^* gives the change in white/black at a scale of from 0 to 100. A decrease in L^* means an increase in black colour (decrease of white colour), an increase in L^* means an increase in white colour (a decrease in black colour). Bleaching may also be measured using Stensby units (W = L + 3a - 3b). In the context of the present invention the term whitening refers to the same as the term bleaching. The term "inactivating" as used herein describes a process whereby enzyme activity is irreversibly decreased.

The term "reducing agent" as used herein describes a compound or composition which has reducing properties.

Oxidases

Oxidases which are contemplated include e.g. glucose oxidase (E.C. 1.1.3.4), hexose oxidase (E.C. 1.1.3.5), L-amino-acid oxidase (E.C. 1.4.3.2), xylitol oxidase, galactose oxidase (E.C. 1.1.3.9), pyranose oxidase (E.C. 1.1.3.10), alcohol oxidase (E.C. 1.1.3.13), xanthine oxidase (E.C. 1.1 .3.22), aldose oxidase, and cellobiose oxidase.

A suitable glucose oxidase may originate from Aspergillus sp., such as a strain of Aspergillus niger, such as the glucose oxidase from A.niger described in US 5,094,951 or US 5,783,414, or from a strain of Cladosporium sp. in particular Cladosporium oxysporum, especially CI. oxysporum CBS 163 described in WO 95/29996 (from Novozymes A/S). Another example is an alkaline glucose oxidase which may be derived from a strain of Cladosporium, e.g. C. oxysporum, in particular C. oxysporum CBS 163.94, such as the glucose oxidase described in WO 98/20136. Hexose oxidases from the red sea-weed Chondrus crispus (commonly known as Irish moss)(Sullivan and Ikawa, (1973), Biochim. Biophys. Acts, 309, p. 11- 22; Ikawa, (1982), Meth. in Enzymol. 89, carbohydrate metabolism part D, 145-149) oxidises a broad spectrum of carbohydrates, such as D-glucose, D-galactose, maltose, cellobiose, lactose, D-glucose 6-phosphate, D-mannose, 2-deoxy-D-glucose, 2-deoxy-D-galactose, D-fucose, D-glucuronic acid, and D-xylose.

Also the red sea-weed Iridophycus flaccidum produces easily extractable hexose oxidases, which oxidise several different mono- and disaccharides (Bean and Hassid, (1956), J. Biol. Chem, 218, p. 425; Rand et al. (1972, J. of Food Science 37, p. 698-710). The broad substrate spectrum of hexose oxidase is advantageous in the connection with tooth bleaching or teeth whitening as the total amount of usable substrate (i.e. carbohydrate) present in the mouth is significantly greater than for related enzymes having more specific catalytic properties.

If a L-amino acid oxidase is used it may be a bacterial L-amino acid oxidase, such as the Corynebacteπum L-amino acid oxidase described by Koyama H., Agric. Biol. Chem., 1988, 52(3), 743-78 or the Cryptococcus laurentii L-amino acid oxidase which is obtainable from the deposited strain DSM 2762 or it may be a fungal L-amino acid oxidase, such as a L-amino oxidase derived from a strain of Neurospora, e.g. N. crassa such as the enzyme described by Niedermann et al., J. Biol. Chem., 1990, 265(28), 17240-17251 or from Trichoderma sp. such as Thchoderma harzianum, such as the L-amino acid oxidase described in WO 94/25574 (from Novozymes A/S), or Trichoderma viride.

Another relevant group of tooth bleaching or teeth whitening enzymes is xylitol oxidases (see e.g. JP 80892242) which oxidises xylitol, D-sorbitol, D-galactitol, D-mannitol and D-arabinitol in the presence of oxygen. A xylitol oxidase can be obtained from strains of Streptomyces sp. (e.g. Streptomyces IKD472, FERM P- 14339) having a pH optimum at 7.5, is stable at pH 5.5 to 10.5 and at temperatures up to 65°C; properties very well suited for oral care compositions and products.

A galactose oxidase may be derived from a strain of Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma or Fusaruium, e.g. F. venenatum, such as the F. venenatum galactose oxidase described in WO 00/50606. The aldose oxidase (also called carbohydrate oxidase) may be derived from, Microdochium, e.g. M. nivale such as the carbohydrate oxidase described in WO 99/31990.

If a pyranose oxidase is used it may be derived, e.g., from a microbial source, such as a fungus, e.g., a filamentous fungus or a yeast, in particular a Basidomycete fungus or the pyranose oxidase may be derived from genera belonging to Agaricales, such as Oudemansiella or Mycena, or to Aphyllophorales, such as Trametes, e.g. T. hirsuta, T. versicolor, T. gibbosa, T suaveolens, T. ochracea, T. pubescens, or to Phanerochaete, e.g P. chrysosporium, Lenzites or Peniophora, e.g. P. gigantea (Huwig et al., 1994, Journal of Biotechnology 32, 309-315; Huwig et el., 1992, Med. Fac. Landbouww, Univ. Gent, 57/4a, 1749-1753; Danneel et al., 1993, Eur. J. Biochem. 214, 795-802), or Polyporus, e.g. P. pinsitus (Ruelius et al., 1968, Biochim. Biophys. Acta, 167, 493-500), or Bierkandera, e.g. B adusta or Phlebiopsis, e.g. P. gigantea (Huwig et al., 1992, op. cit). It may also be derived from a bacterial source.

Another example of an oxidase is a lysyl oxidase, i.e. an enzyme capable of oxidative deamination of lysyl.

The oxidase may also be a benzylamine oxidase, such as a benzylamine oxidase derived from the genus Pichia, such as Pichia pastoris, or it may be the enzyme described by Tur, S.S. and Lerch, K. in FEBS letters, vol. 238, No. 1 , September 1988, pp. 74-76. It may be advantageous to use enzyme(s) which can act on substrates which are not cariogenic (i.e. substrates which are not or is not immediately degraded into cariogenic substrates such as sucrose, glucose, fructose, maltose etc.).

Examples of such a substrate include amino acids, alcohol, sugar alcohol, such as xylitol, sorbitol etc.

Sulphur containing reducing agents

The sulphur containing reducing agents according to the present invention may be sulphurous acid (H₂SO₃) and salts thereof, e.g. sodium sulphite, sodium hydrogen sulphite, thiosulphuric acid (H₂S₂O₃) and salts thereof, dithiorous acid (H₂S₂O₄) salts, pyrosulphurous acid (H₂S₂O ) salts and dithionic acid (H₂S₂Oβ) and salts thereof. However any sulphur containing reducing agent which is able to carry out the process is within the scope of the present invention. In a particular embodiment, the sulphur containing reducing agent is Na₂SO₃. In carrying out the method of the present invention, there is employed a composition comprising the oxidase and catalase. The composition may be obtained in accordance with the following production method.

Production of Oxidases

Oxidases of the invention may be produced by aerobic cultivation of an appropriate microbial strain on a nutrient medium containing suitable carbon and nitrogen sources, such media being known in the art. The strains may be any microbial stain which is able to express the oxidase in question. Examples of strains includes Aspergillus, Penicillium, Trichoderma, Microdochium and Talaromyces.

A temperature in the range of from 20°C to 50°C is suitable for growth and oxidase production.

Alternatively, oxidases of the invention can be produced by aerobic cultivation of a transformed host organism containing the appropriate genetic information from the above mentioned strains. Such transformants can be prepared and cultivated by methods known in the art:

Cloning a DNA Seguence Encoding an Oxidase

The DNA sequence encoding an oxidase of the invention may be isolated from any cell or microorganism producing the oxidase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the oxidase to be studied. Then, if the amino acid sequence of the oxidase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify oxidase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known oxidase gene could be used as a probe to identify oxidase-encoding clones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying oxidase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming glucose oxidase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for oxidase thereby allowing clones expressing the oxidase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers in Tetrahedron Letters 22, 1981 , pp. 1859-1869 or the method described by Matthes et al. in The EMBQ J. 3, 1984, pp. 801-805. In the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or R.K. Saiki et al. in Science 239, 1988, pp. 487-491.

Expression of Oxidase

According to the invention, a oxidase-encoding DNA sequence produced by methods described above, or by any alternative methods known in the art, can be expressed in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding an oxidase of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transchptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding a oxidase of the invention, especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus Amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizo- mucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae those phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the oxidase of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and plJ702.

The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the host cell, such as the dal genes from β. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicil- lin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co- transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. Procedures suitable for constructing vectors of the invention encoding an oxidase and containing the promoter, terminator and other elements, respectively, are well known to persons skilled in the art (cf., for instance, Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an oxidase of the invention. The cell may be transformed with the DNA construct of the invention encoding the oxidase conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells. The cell may be of a higher organism such as a mammal or an insect, but is particularly a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are grampositive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermo- philus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuhngiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E.coli.

The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known perse.

The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Fusarium, e.g. Fusarium oxysporum or Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the oxidase of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

The oxidase secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Inactivating catalase activity

In accordance with the present invention a further or alternative purification step is employed in which the catalase activity is substantially decreased by contacting the composition comprising the oxidase and catalase with a sulphur containing reducing agent. The addition of the sulphur containing reducing agent may also constitute an alternative step to the chromatographic procedures mention above and thereby improve the process economy. The term "substantially free from" or "substantially decreased" is here defined as a residual catalase activity of below 800 ClU/ml, particularly below 500 ClU/ml, more particularly below 100 ClU/ml and even more particularly below 50 ClU/ml. CIU is the amount of catalase which decomposes 1 μmole of H₂O₂ per minute at pH 7.0 and 25°C. The degradation of hydrogen peroxide can be followed spectrophotometrically at 240 nm, and the time consumption for a specified decrease in absorbance at a specified H₂O₂ concentration is a measure of the catalase activity. A folder AF 250/1 describing this analytical method is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

The treatment with the sulphur containing reducing agent may be improved by including a diafiltration and/or ultra filtration step prior to, during and/or after the treatment with the reducing agent.

During the treatment the pH of the oxidase containing composition is maintained in the acidic range, in particular in the range of pH about 3.0 to 7.0, more particular about pH 4.0 to 6.0 or in the range of pH 3.0 to 5.0. The pH may be adjusted with an acid or a base depending on the initial pH. Examples of acids are acetic acid and phosphoric acid. In particular it may be an acid with a pK_a-value below 4, such as phosphoric acid, oxalic acid, sulphuric acid and hydrochloric acid. It appears that acids with a pKg-value below 4 result in a fast inactivation of catalase with optimal oxidase activity. In particular the pK_a-value may be between -20 and 4, more particularly between -10 and 4, more particularly between -5 and 4, more particularly between 0 and 4, more particularly between 1 and 4, even more particularly between 2 and 4. The temperature of the treatment should be in the range of from 1°C to 60°C. In particular, the temperature should be in the range of from 4°C to 25°C.

In particular, inactivation of catalase activities is carried out by adjusting the pH to about 4 and adding the sulphur containing reducing agent (final concentration of 20-200 mM) to the composition (up to five times) without further adjustment of the pH, and leave it either with or without stirring until the catalase activity is reduced to the level wanted. Consecutive additions of the sulphur containing reducing agent (up to five times) may be needed for complete inactivation. Addition of a cationic polymer (flocculent) may improve the inactivation process.

Examples of such cationic flocculants are cationic polyamine or polyacrylamide flocculants, including SUPERFLOC®, such as SUPERFLOC®C521 , C581 , C591 and HE28000 (Cytec Industries Inc.). The method can be carried out on compositions comprising oxidase regardless of the purity of the composition. As mentioned above, the method is particular carried out on a composition, which have been subjected to one or more of the following procedures, including separating the cells from the medium by cent fugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like. In accordance with the second and third aspects of the invention, the invention provides a composition comprising an oxidase substantially free from catalase activity and the use of said composition for different purposes, e.g. for bleaching, baking etc.

Industrial Applications

The oxidase of the invention possesses valuable properties allowing for various industrial applications. In particular the enzyme finds potential application in bleaching compositions as a hydrogen peroxide source, used alone or used together with oxidizable substrate. The oxidase may also be used in combination with a peroxidase, including a haloperoxidase and a peroxidase substrate.

Peroxidases Within the group of peroxidases classified under the Enzyme Classification number E.C. 1.11 (Acting on peroxide as acceptor), E.C. 1.11.1 (Peroxidases), peroxidase (E.C.1.11.1.7) are especially contemplated.

Particularly, the peroxidase employed in the composition of the invention is producible by plants (e.g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria.

Some particular fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, e.g., Fusahum, Humicola, Trichoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusahum oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucaha (IFO 6113), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alii or Dreschlera halodes.

Other particular fungi include strains belonging to the subdivision

Basidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete, Coriolus or

Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371 ), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus), e.g., T. versicolor(e.g. PR4 28-A).

Further particular fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particular Mucor hiemalis. Some particular bacteria include strains of the order Actinomycetales, e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.

Other particular bacteria include Bacillus pumilus (ATCC 12905), Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11 ).

Further particular bacteria include strains belonging to Myxococcus, e.g., M. virescens.

The peroxidase may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant DNA vector which carries a DNA sequence encoding said peroxidase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the peroxidase, in a culture medium under conditions permitting the expression of the peroxidase and recovering the peroxidase from the culture. Particularly, a recombinantly produced peroxidase is a peroxidase derived from a Coprinus sp., in particular C. macrorhizus or C. cinereus according to WO 92/16634.

Examples of peroxidase substrates include the compounds described in WO 94/12621 and WO 95/01426, which is hereby incorporated by reference, and represented by the general formula I:

Specifically contemplated compounds within the above formula I include the following: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS); 6-hydroxy-2-naphtoic acid; 7-methoxy-2-naphtol; 7-amino-2-naphthalene sulphonic acid; 5-amino-2-naphthalene sulphonic acid; 1 ,5-diaminonaphthalene; 7-hydroxy-1 ,2-naphthimidazole; 10- methylphenothiazine; 10-phenothiazine-propionic acid (PPT); N-hydroxysuccinimide- 10-phenothiazine-propionate; benzidine; 3,3'-dimethylbenzidine; 3,3'-dimethoxy- benzidine; 3,3',5,5'-tetramethylbenzidine; 4'-hydroxy-4-biphenylcarboxylic acid; 4- amino-4'-methoxystilbene; 4,4'-diaminostilbene-2,2'-disulfonic acid; 4,4'-diaminodiphe- nylamine; 2,7-diaminofluorene; 4,4'-dihydroxy-biphenylene; triphenylamine; 10-ethyl- 4-phenothiazinecarboxylic acid; 10-ethylphenothiazine; 10-propylphenothiazine; 10- isopropylphenothiazine; methyl-10-phenothiazinepropionate; 10-phenylphenothiazine; 10-allylphenothiazine; 10-phenoxazinepropionic acid (POP); 10-(3-(4-methyl-1- piperazinyl)propyl)phenothiazine; 10-(2-pyrrolidinoethyl)phenothiazine; 10-methyl- phenoxazine; iminostilbene; 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid; N-benzylidene-4-biphenylamine; 5-amino-2-naphthalenesulfonic acid; 7-methoxy-2- naphtol; 4,4'-dihydroxybenzophenone; N-(4-(dimethylamino)benzylidene)-p-anisidine; 3-methyl-2-benzothiazolinone(4-(dimethylamino)benzylidene)hydrazone; 2-acethyl-10- methylphenothiazine; 10-(2-hydroxyethyl)phenothiazine; 10-(2-hydroxyethyl)phenoxa- zine; 10-(3-hydroxypropyl)phenothiazine; 4,4'-dimethoxy-N-methyl-diphenylamine, and vanillin azine.

Other substrates contemplated include 4-hydroxybenzoic acid, L-tyrosine, sy ngate acids, ferulic acid, sinapic acid, chlorogenic acid, caffeic acid and esters thereof.

Still further examples include organic compounds described in WO 96/10079, which is hereby incorporated by reference, and represented by the general formula II:

Specific compounds covered by the above formula II are acetosyringone, syringaldehyde, methylsyringate, syringic acid, ethylsyringate, propylsyringate, butylsyringate, hexylsyringate, octylsyringate and ethyl 3-(4- hydroxy-3,5-dimethoxyphenyl)acrylate.

The enzyme may be useful in the bleaching of fabrics and fibres, in bleaching of dyes in solution and in anti-microbial compositions.

The enzyme may also be very useful in the baking industry due to its excellent ability for improving the properties of doughs/breads.

The enzyme also has many potential applications in the personal care area, for example in personal care products such as tooth paste, mouthwash, denture cleaner, liquid soap, skin care creams and lotions, hair care and body care formulations, and solutions for cleaning contact lenses. In particular the oxidase of the invention may be very useful in tooth paste, alone or together with other enzymes, particularly together with mutanase and/or dextranase, and/or an amyloglucosidase and a lactoperoxidase as such a combination of enzymes forms a very efficient antibacterial system:

Polysacchahdes from plaques ~>(Amyloglucosidase) Glucose -> (Glucose oxidase of the invention)

Gluconic acid + H₂O₂ ; the formed hydrogen peroxide may react with thiocyanate in the following way: H₂O₂ + SCN^" -> (Lactoperoxidase)

OSCN^", in which OSCN^" is a bactehostatic agent.

The oxidase may also be used as a source for hydrogen peroxide in combination with e.g. a haloperoxidase, and a halide source.

Haloperoxidases

Haloperoxidases form a class of enzymes which are able to oxidize halides (CI-, Br-, I-) in the presence of hydrogen peroxide or a hydrogen peroxide generating system to the corresponding hypohalous acids according to:

H₂O₂ + X^" + H⁺ -> H₂O + HOX

If a convenient nucleophilic acceptor is present, a reaction will occur with HOX and a halogenated compound will be formed.

There are three types of haloperoxidases, classified according to their specificity for halide ions: Chloroperoxidases (E.C. 1.11.1.10) which catalyse the chlorination, bromination and ionidation of compounds; Bromoperoxidases which show specificity for bromide and iodide ions; and iodoperoxidases (E.C. 1.11.1.8) which solely catalyze the oxidation of iodide ions.

Haloperoxidases have been isolated from various organisms: mammals, marine animals, plants, algae, fungi and bacteria (for reference see Biochimica et

Biophysica Acta 1161 , 1993, pp. 249-256). It is generally accepted that haloperoxidases are the enzymes responsible for the formation of halogenated compounds in nature, although other enzymes may be involved.

Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaha, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis (see US Patent No. 4,937,192). According to an aspect of the present invention a haloperoxidase obtainable from Curvularia, in particular C. verruculosa is particularly such as C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70. Curvulaha haloperoxidase and recombinant production hereof is described in WO 97/04102.

Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia (for reference see The Journal of Biological Chemistry 263, 1988, pp. 13725-13732) and Streptomyces, e.g., S. aureofaciens (for reference see Structural Biology 1 , 1994, pp. 532-537).

Bromide peroxidase has been isolated from algae (see US Patent No. 4,937,192).

In use, the concentration of the haloperoxidase may be varied in order to achieve the desired effect within the desired time frame. However, according to the invention the haloperoxidase will normally be added in a concentration of 0.001-10 mg enzyme protein per litre, particularly in a concentration of 0.001-5 mg enzyme protein per litre, more particularly in a concentration of 0.001-1 mg enzyme protein per litre.

In a particular embodiment the haloperoxidase is derivable from Curvularia sp., in particular C. verruculosa and C. inaequalis.

Halide Sources

According to the invention, the halide source for the reaction with haloperoxidase may be achieved in many different ways: The halide source may be sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide, or potassium iodide. The concentration of the halide source will typically correspond to 0.01-1000 mM, particularly in the range of from 0.1-100 mM.

Industrial Compsitions

Anti-microbial compositions

The present invention provides an antimicrobial composition, comprising one or more enzymatic component(s) and one or more non-enzymatic biocides. The enzymatic component and the non-enzymatic biocides of the composition may be selected so that the number of living cells of E. coli (DSM1576), when incubated 10 min at 20°C in an aqueous solution containing 50% w/w (particularly 25% w/w, more particularly 10% w/w, most particularly 5% w/w) of the biocide and 0.5 ppm (particularly 0.1 ppm) of the enzymatic component, are reduced at least 5% (particularly 10%) more than compared to what is obtained by adding the results of separate incubations with the biocides and the enzymatic component alone, i.e. a simple additive effect.

The enzymatic component and the non-enzymatic biocides of the composition may also be selected so that the outgrowth of E. coli (DSM1576) at 25°C in a microbial growth substrate containing 500 ppm (particularly250 ppm, more particularlylOO ppm, most particularly 50 ppm) of the biocide and 0.5 ppm (particularly 0.1 ppm) of the enzymatic component, are inhibited at least 5% longer time than compared to what is obtained by adding the results of separate incubations with the biocides and the enzymatic component alone, i.e. a simple additive effect. The composition may be formulated as a solid, liquid, gel or paste.

When formulated as a solid all components may be mixed together, e.g., as a powder, a granulate or a gelled product.

When other than dry form compositions are used and even in that case, it is preferred to use a two-part formulation system having the enzyme(s) separate from the rest of the composition.

The composition of the invention may further comprise auxiliary agents such as wetting agents, thickening agents, buffer, stabilisers, perfume, colourants, fillers and the like.

Useful wetting agents are surfactants, i.e., non-ionic, anionic, amphoteric or zwittehonic surfactants.

The composition of the invention may be a concentrated product or a ready-to- use product. In use, the concentrated product is typically diluted with water to provide a medium having an effective antimicrobial activity, applied to the object to be disinfected or preserved, and allowed to react with the microorganisms present. The pH of an aqueous solution of the composition is in the range of from pH 2 to 11 , particularly in the range of from pH 4 to 10, more particularly in the range of from pH 5 to 9, and most particularly in the range of from pH 6 to 8.

The amount of oxidase and other enzymes, such as peroxidase or haloperoxidase in the composition may be in the range of from 0.001 to 5 mg enzymeprotein/l, particular from 0.01 to 2 mg enzymeprotein/l, more particular 0.01 to 1 mg enzymeprotein/l.

Bleaching Compositions

The bleaching compositions of the invention may be used for bleaching textile, for bleaching/whitening teeth or for bleaching paper/pulp.

Bleaching textiles

The process of bleaching textiles may be carried out in the presence of conventional fabric, garment, or yarn finishing agents, including wetting agents, polymeric agents, dispersing agents, etc.

A conventional wetting agent may be used to improve the contact between the substrate and the enzyme used in the process. The wetting agent may be a non-ionic surfactant, e.g. an ethoxylated fatty alcohol. A very useful wetting agent is an ethoxylated and propoxylated fatty acid ester such as Berol 087 (product of Akzo Nobel, Sweden).

Examples of suitable polymers include proteins (e.g., bovine serum albumin, whey, casein or legume proteins), protein hydrolysates (e.g., whey, casein or soy protein hydrolysate), polypeptides, lignosulfonates, polysaccharides and derivatives thereof, polyethylene glycol, polypropylene glycol, polyvinyl pyrrolidone, ethylene diamine condensed with ethylene or propylene oxide, ethoxylated polyamines, or ethoxylated amine polymers.

The dispersing agent may suitably be selected from nonionic, anionic, cationic, ampholytic or zwitterionic surfactants. More specifically, the dispersing agent may be selected from carboxymethylcellulose, hydroxypropylcellulose, alkyl aryl sulphonates, long-chain alcohol sulphates (primary and secondary alkyl sulphates), sulphonated olefins, sulphated monoglycerides, sulphated ethers, sulphosuccinates, sulphonated methyl ethers, alkane sulphonates, phosphate esters, alkyl isothionates, acylsarcosides, alkyltauhdes, fluorosurfactants, fatty alcohol and alkylphenol condensates, fatty acid condensates, condensates of ethylene oxide with an amine, condensates of ethylene oxide with an amide, sucrose esters, sorbitan esters, alkyloamides, fatty amine oxides, ethoxylated monoamines, ethoxylated diamines, alcohol ethoxylate and mixtures thereof. A very useful dispersing agent is an alcohol ethoxylate such as Berol 08 (product of Akzo Nobel, Sweden).

The bleaching processing may be performed in any machinery known in the art.

Oral compositions

When the oxidase according to the invention is to be applied in oral compositions it is advantageous to use enzymes being substantially active at pHs prevailing in the mouth, i.e. between pH 5.0 to 9.0, particularly between pH 6.0 to 8.5, especially between pH 6,0 to 7.5.

The term "substantially active" enzyme means in this context that the enzyme(s) has(have) an relative activity (pH-optimum defines 100% at the same conditions) higher than 30%, better 50%, even better more than 70%, such as 80%, and in the best case up to about 100% of the activity at the pH optimum.

In a particular embodiment the oral composition or oral care product of the invention comprises also a substrate for the oxidase.

The amount of oxidase(s) needed in an oral composition of the invention to obtain tooth bleaching depends on the particular compound employed, but ranges generally from 0.0001 % to 5%, particularly from about 0.001 % to about 1 %, and most particularly from about 0.001% to about 0.1% by weight enzyme protein of the weight of the final composition.

The oral composition or oral care product of the invention may comprise at least one other enzyme activity, which includes the activity of a protease, and/or plaque degrading enzymes such as mutanase and/or dextranase and/or lipase and/or amylase and/or anti-microbial enzyme system.

In a particular embodiment of the invention the oral composition or oral care product comprise an oxidase and a dextranase and/or a mutanase.

An oral composition of the invention may advantageously be used in conventional oral care products having any suitable physical form (i.e. powder, paste, gel, liquid, ointment, tablet, lozenges etc.).

An "oral care product" of the invention is defined as a product which can be used for maintaining and/or improving oral hygiene and/or appearance in the mouth of humans and animals, and/or preventing or treating dental diseases. Examples of such oral care products include toothpaste, dental cream, gel or tooth powder, odontic, mouth washes, denture cleaning agents, pre- or post brushing rinse formulations, chewing gum, lozenges, and candy.

Toothpastes and tooth gels typically include abrasive polishing materials, foaming agents, flavouring agents, humectants, binders, thickeners, sweetening agents, and water.

Mouth washes, including plaque removing liquids, typically comprise a water/alcohol solution, flavour, humectants, sweetener, foaming agent, and colorant.

According to the invention said abrasive polishing material includes alumina and hydrates thereof, such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, sodium bicarbonate ("Baking soda"), kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum silicate, calcium carbonate, zirconium silicate, and also powdered plastics, such as polyvinyl chloride, polyamides, polymethyl methacrylate, polystyrene, phenol-formaldehyde resins, melamine- formaldehyde resins, urea-formaldehyde resins, epoxy resins, powdered polyethylene, silica xerogels, hydrogels and aerogels and the like. Also suitable as abrasive agents are calcium pyrophosphate, water-insoluble alkali metaphosphates, dicalcium phosphate and/or its dihydrate, dicalcium orthophosphate, tricalcium phosphate, particulate hydroxyapatite and the like. It is also possible to employ mixtures of these substances. Dependent on the oral care product the abrasive product may be present in from 0 to 70% by weight, particularly from 1% to 70%. For toothpastes the abrasive material content typically lies in the range from 10% to 70% by weight of the final toothpaste product.

Humectants are employed to prevent loss of water from e.g. toothpastes. Suitable humectants for use in oral care products according to the invention include the following compounds and mixtures thereof: glycerol, polyol, sorbitol, polyethylene glycols (PEG), propylene glycol, 1 ,3-propanediol, 1 ,4-butanediol, hydrogenated partially hydrolysed polysaccharides and the like. Humectants are in general present from 0% to 80%, particularly 5 to 70% by weight in toothpaste.

Silica, starch, xanthan gum, extracts of Irish moss, alginates, pectin, cellulose derivatives, such as hydroxyethyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl cellulose, polyacrylic acid and its salts, polyvinylpyrrolidone, can be mentioned as examples of suitable thickeners and binders, which help stabilizing the dentifrice product. Thickeners may be present in toothpaste creams and gels in an amount of from 0.1 to 20% by weight, and binders to the extent from 0.01 to 10% by weight of the final product.

As foaming agent soap, anionic, cationic, non-ionic, amphoteric and/or zwitterionic surfactants can be used. These may be present at levels from 0% to 15%, particularly from 0.1 to 13%, more particularly from 0.25 to 10% by weight of the final product. Surfactants are only suitable to the extent that they do not exert an inactivation effect on the present enzymes. Surfactants include fatty alcohol sulphates, salts of sulphonated mono-glycehdes or fatty acids having 10 to 20 carbon atoms, fatty acid-albumen condensation products, salts of fatty acids amides and tauhnes and/or salts of fatty acid esters of isethionic acid. Suitable sweeteners include saccharin and/or other appropriate sweeteners.

Flavours, such as spearmint and peppermint, are usually present in low amounts, such as from 0.01 % to about 5% by weight, especially from 0.1 % to 5%.

Water is usually added in an amount giving e.g. toothpaste a flowable form, i.e. between 40% to 70% by weight of the final product.

Further water-soluble anti-bacterial agents, such as chlorhexidine digluconate, hexetidine, alexidine, quaternary ammonium anti-bacterial compounds and water-soluble sources of certain metal ions such as zinc, copper, silver and stannous (e.g. zinc, copper and stannous chloride, and silver nitrate) may also be included. Also contemplated according to the invention is the addition of anti- calculus agents, anti-plaque agents, compounds which can be used as fluoride source, dyes/colorants, preservatives, vitamins, pH-adjusting agents, anti-caries agents, desensitizing agents etc.

A toothpaste produced from an oral composition of the invention (in weight % of the final toothpaste composition) may e.g. comprise the following ingredients:

Abrasive material 10 to 70%

Humectant 0 to 80%

Thickener 0.1 to 20% Binder 0.01 to 10%

Sweetener 0.1 % to 5%

Foaming agent 0 to 15%

Bleaching enzyme 0.0001 % to 20%

Other enzymes 0 to 20% Enzyme substrate 0 to 5%

A mouth wash produced from an oral care composition of the invention (in weight % of the final mouth wash composition) may typically comprise the following ingredients: 0-20% Humectant

0-2% Surfactant 0-5% Enzymes 0-20% Ethanol

0-2% Other ingredients (e.g. flavour, sweetener active ingredients such as florides).

0-70% Water The mouth wash composition may be buffered with an appropriate buffer e.g. sodium citrate or phosphate in the pH-range 6-7.5.

The mouth wash may be in none-diluted form (i.e. must be diluted before use).

Uses

The invention also encompasses various uses of a composition comprising an enzymatic component and one or more non-enzymatic biocides. Said composition is typically useful at any locus subject to contamination by bacteria, fungi, yeast or algae. Typically, loci are in aqueous systems such as cooling water systems, laundry rinse water, oil systems such as cutting oils, lubricants, oil fields and the like, where microorganisms need to be killed or where their growth needs to be controlled. However, the present invention may also be used in all applications for which known antimicrobial compositions are useful, such as protection of wood, latex, adhesive, glue, paper, cardboard, textile, leather, plastics, caulking, and feed.

Other uses include preservation of foods, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouth wash, contact lens products, enzyme formulations, or food ingredients.

Thus, the composition used in the method of the invention may by useful as a disinfectant, e.g., in the treatment of acne, infections in the eye or the mouth, skin infections; in antiperspirants or deodorants; in foot bath salts; for cleaning and disinfection of contact lenses, hard surfaces, teeth (oral care), wounds, bruises and the like.

In general it is contemplated that the composition of the present invention is useful for cleaning, disinfecting or inhibiting microbial growth on any hard surface. Examples of surfaces, which may advantageously be contacted with the composition of the invention are surfaces of process equipment used e.g. dairies, chemical or pharmaceutical process plants, water sanitation systems, oil processing plants, paper pulp processing plants, water treatment plants, and cooling towers. The composition of the invention should be used in an amount, which is effective for cleaning, disinfecting or inhibiting microbial growth on the surface in question.

Further, it is contemplated that the composition of the invention can advantageously be used in a cleaning-in-place (C.I.P.) system for cleaning of process equipment of any kind.

The composition of the invention may additionally be used for cleaning surfaces and cooking utensils in food processing plants and in any area in which food is prepared or served such as hospitals, nursing homes, restaurants, especially fast food restaurants, delicatessens and the like. It may also be used as an antimicrobial in food products and would be especially useful as a surface antimicrobial in cheeses, fruits and vegetables and food on salad bars.

It may also be used as a preservation agent or a disinfection agent in water based paints. The composition of the present invention is also useful for microbial control of water lines, and for disinfection of water, in particular for disinfection of industrial water.

Furthermore, the composition may be used as a biosensor or an analytical reagent. In another aspect of the invention the enzyme may be used for enzymatic deoxygenation (oxygenscavenger) of food items, including beverage. The reduction of the amount of dissolved oxygen in juice has been described in Journal of Food Processing and Preservation 14, 1990, pp. 253-256.

Baking

It is also contemplated that oxidase compositions free of catalase activity obtained according to the present invention can be used within the baking industry. It is well-known within the art of baking that addition of oxidases improves the properties of dough and the quality of bread see for example US 4,990,343, EP 0 321 811 , WO 97/21351 , WO 97/22257 and EP 0 468 731. Dough generally comprises wheat meal or wheat flour and/or other types of meal, flour or starch such as corn flour, corn starch, rye meal, rye flour, oat flour, oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch. The dough may be fresh, frozen or pre-baked. The dough is typically leavened e.g. by adding chemical leavening agents or yeast, usually Saccharomyces cerevisiae (baker's yeast). The dough may be a laminated dough.

The dough may also comprise other conventional dough ingredients, e.g.: proteins, such as milk powder, gluten, and soy; eggs (either whole eggs, egg yolks or egg whites); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino acid such as L- cysteine; a sugar; a salt such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate. The dough may comprise fat (triglyceride) such as granulated fat or shortening. The dough may further comprise an emulsifier such as a monoglyceride. The oxidase composition free of catalase activity obtained according to the present invention may be used for any kind of baked product prepared from dough, either of a soft or a crisp character, either of a white, light or dark type. Examples are bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, pie crusts, crisp bread, steamed bread, pizza and the like.

A baking composition comprises the oxidase composition free of catalase activity and optionally other ingredients. The baking composition may be in the form of a granulate or agglomerated powder. It may have a narrow particle size distribution with more than 95 % (by weight) of the particles in the range from 25 to 500 mm. Granulates and agglomerated powders may be prepared by conventional methods, e.g. by spraying the amylase onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g. a salt (such as NaCI or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.

The baking composition may, in addition to enzymes, comprise other baking ingredients, particularly flour. Thus, the composition may be a dough or a flour pre- mix.

Optionally, an additional enzyme may be used together with the oxidase. The additional enzyme may be an amylase, a cyclodextrin glucanotransferase, a peptidase, in particular an exopeptidase, a transglutaminase, a lipase, a phospholipase, a cellulase, a hemicellulase, a protease, a glycosyltransferase, a branching enzyme (1 ,4-alpha-glucan branching enzyme) or a second oxidoreductase (in addition to the hydrogen peroxide-forming oxidase). The additional enzyme may be of any origin, including mammalian and plant, and particularly of microbial (bacterial, yeast or fungal) origin.

The amylase may be fungal or bacterial, e.g. a maltogenic alpha-amylase from β. stearothermophilus or an alpha-amylase from Bacillus, e.g. B. licheniformis or β. amyloliquefaciens, a beta-amylase, e.g. from plant (e.g. soy bean) or from microbial sources (e.g. Bacillus), a glucoamylase, e.g. from A. niger, or a fungal alpha- amylase, e.g. from A. oryzae.

The hemicellulase may be a pentosanase, e.g. a xylanase which may be of microbial origin, e.g. derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of A. aculeatus, A. niger, A. awamoh, or A. tubigensis, from a strain of Trichoderma, e.g. T. reesei, or from a strain of Humicola, e.g. H. insolens. The protease may be from Bacillus, e.g. B. amyloliquefaciens.

The lipase may be derived from a strain of Thermomyces (Humicola), Rhizomucor, Candida, Aspergillus, Rhizopus, or Pseudomonas, in particular from T. lanuginosus (H. lanuginosa), Rhizomucor miehei, C. antarctica, A. niger, Rhizopus delemar, Rhizopus arrhizus or P. cepacia. The phospholipase may have phospholipase A1 or A2 or lysophospholipase activity; it may or may not have lipase activity. It may be of animal origin, e.g. from pancreas, snake venom or bee venom, or it may be of microbial origin, e.g. from filamentous fungi, yeast or bacteria, such as Aspergillus or Fusahum, e.g. A. niger, A. oryzae or F. oxysporum. Also, the variants described in WO 00/32758 may be used. The second oxidoreductase may be a peroxidase, a laccase or a lipoxygenase. MATERIALS AND METHODS

Materials

Enzymes:

Coprinus cinereus peroxidase (available from Novozymes A/S, Denmark)

Aspergillus niger glucose oxidase obtained by recombinant expression in Aspergillus oryzae, Gluzyme™ (available from Novozymes A/S, Denmark)

Flocculants:

SUPERFLOC®: C521 , C581 , C591 all available from Cytec Industries Inc.

Methods

Micro plate reader Vmax from Molecular Devices

Catalase assay in micro scale:

100μl Substrate (12 mM H₂O₂ in 25 mM phosphate pH 7) is mixed with 25 μl catalase containing solution and incubated at ambient temperature. After 5-7 minutes 375 μl stop reagent (50 mM citrate pH 3.5) is added and mixed by gentle agitation. 25 μl mixture is withdrawn and added to 225 μl ABTS/POD-reagent (2 mM ABTS, 50 μg/ml Coprinus cinereus peroxidase in 50 mM citrate pH 4) in a micro-well plate. After 3-5 minutes absorbance at 405 nm is measured on a micro plate reader. Catalase will reduce formation of the green ABTS radical colour. For quantification of the catalase content in the samples, dilutions of a known catalase standard are assayed in the same plate.

Assay for Glucose Oxidase Activity (GODU)

Glucose oxidase activity is determined in the following way: Glucose oxidase oxidizes D-glucose in the presence of oxygen producing hydrogen peroxide. The hydrogen peroxide formed, in the presence of peroxidase, oxidizes ABTS (2,2'- azinobis(3-ethylbenzothiazoline-6-sulfonate)). The greenish-blue colour resulting after a fixed reaction time measured as the absorbance at 418 nm is a function of the amount of hydrogen peroxide. In the assay the following concentrations, pH, temperature and reaction time are used: D-glucose: 100 mM; ABTS: 0.4 mM; phosphate buffer: 100 mM; pH: 7.0; temperature: 30°C; reaction time: 20 min. The activity of glucose oxidase is given in UNITS (1 UNIT is the amount of glucose oxidase which under the above standard conditions forms 1 μmole of hydrogen peroxide per minute).

Assay for catalase activity (CIU)

One CIU decomposes one μmol of H₂0₂ per minute at pH 7.0 and 25°C. The degradation of hydrogen peroxide can be followed spectrophotometrically at 240 nm, and the time consumption for a specified decrease in absorbance at a specified H₂O₂ concentration is a measure of the catalase activity. A folder AF 250/1 describing this analytical method is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

EXAMPLES

The present invention is further illustrated in the following examples which are not intended to be in any way limiting to the scope of the invention as claimed.

Example 1

Samples of recombinant A.niger glucose oxidase concentrate expressed in Aspergillus oryzae containing catalase (approximately 25000 ClU/ml) are diluted 10 times in a 20 mM citrate buffer at pH 4.0, 4.5, 5.0, 5.5 and 6.0, respectively. Samples from each pH are added sodium sulphite (100 mM stock solution) to a concentration of 20 mM. Reference samples are added water instead of sodium sulphite. Alle samples are incubated at 25°C for 3 days and residual catalase activity (ClU/ml) was measured by a micro plate assay relative to a catalase standard. The results are shown in the table:

Example 2

Samples of recombinant A.niger glucose oxidase concentrate expressed in Aspergillus oryzae containing catalase (approximately 25000 ClU/ml) are diluted 10 times in:

A) 20 mM citrate buffer pH 4.5

B) 20 mM citrate buffer pH 4.5 containing 1 mM sulphite

1 ml of each diluted sample is dialysed against 1 L dilution medium for 2 days at 4°C, and residual catalase activity (ClU/ml) was measured by a micro assay relative to a catalase standard.

The results are shown in the table:

Example 3

100 ml recombinant A.niger glucose oxidase concentrate expressed in Aspergillus oryzae containing catalase (approximately 25000 ClU/ml) was dialysed against 3 x 5 I water. Dialysed samples are added:

A) Nothing (reference)

B) 20 mM sodium sulphite

C) 0.5% C521 + 20 mM sodium sulphite

D) 0,5% C581 + 20 mM sodium sulphite

E) 0.5% C591 + 20 mM sodium sulphite

Samples are stored 2 days at 4°C, and residual catalase activity was measured by a micro plate assay relative to a catalase standard. The results are shown in the table below:

The results show that the catalase inactivation is enhanced by addition of cationic flocculants.

Example 4

A sample of concentrated recombinant A. niger glucose oxidase expressed in A. oryzae has been washed with water by ultrafiltration (Danish Separation System A/S with a membrane cut-off of 10 kDa) to a dry matter content in the permeate of 0.7%. After wash the sample showed a glucose oxidase activity of 3484 GODU/ml and contained catalase of 22300 ClU/ml.

50 ml of this enzyme was adjusted to pH 4.0 and added 158 mg sodium sulphite powder to a concentration of 50 mM sulphite. The sample was stored at 5°C. After 1 , 2 and 5 days, respectively, the sample was further treated with 158 mg sodium sulphite and residual catalase activity (ClU/ml) was measured by a micro plate assay relative to a catalase standard. Further, glucose oxidase activity as well as pH was measured in the sample.

The results are shown in the table below:

The catalase activity has been reduced to approximately 0.1 % of the original activity without reducing the glucose oxidase activity.

Example 5

A sample of concentrated recombinant A. niger glucose oxidase expressed in A. oryzae was washed with water by ultrafiltration (Danish Separation System A/S with a membrane cut-off of 10 kDa) to a dry matter content in the concentrate of 10%. After wash the sample showed a glucose oxidase activity of 17243 GODU/ml and contained catalase of 89521 ClU/ml.

A sample of this enzyme was adjusted to pH 4.0 with either acetic acid or with phosphoric acid and sodium sulphite powder was added to a final concentration of 50 mM sulphite. The sample was stored at 10°C. After 1 day further sodium sulphite powder was added to a final concentration of 50 mM. The sample was then stored at 10°C for another 4 days. Residual catalase activity (ClU/ml) was measured by a micro plate assay relative to a catalase standard. Further, glucose oxidase activity was measured in the sample.

The results are shown in the table below:

Phosphoric acid is very effective for the pH adjustment for inactivation of the catalase activity.

Example 6

A concentrate of recombinant A. niger glucose oxidase expressed in A. oryzae (39345 ClU/ml, 7658 GODU/ml) was adjusted to pH 4.0 at 10°C with respectively:

Acetic acid pKa = 4,76 Phosphoric acid pKa = 2,15 Oxalic acid pKa = 1,19 Sulphuric acid pKa = -2 Hydrochloric acid pKa = -6

Na₂SO₃ (as powder) was added to the sample(s) with low stirring to a final concentration of 50 mM Na₂SO₃. The samples were kept at 5-10°C with slight stirring and Catalase and Glucose Oxidase activity was measured after 2 hour, 1 day, and up to 5 days - depending on the results after day 1. Another 50 mM Na₂SO₃ was added, until the catalase activity was below 50-100 ClU/ml. When the catalase was inactivated (<50-100 ClU/ml), it was investigated if the inactivation of catalase was reversible, i.e. whether it was possible to reactivate the catalase with oxygen. Thus atm. air was bubbled through the solutions for at least 1 hour before reanalysing the level of catalase.

The catalase (CIU) and glucose oxidase (GODU) activity was measure as described under "Methods".The results of the catalase and glucose oxidase activities are shown in the table below:

* 3 times 24h treatment with 50 mM Na₂SO₃ addition every 24h

As shown in the table the catalase did not reactivate, when the solutions were bubbled with atm. air for 1 hour.

Furthermore, table 1 shows that acids which have a pKa-value lower than 4 are able to inactive catalase fast (addition of Na₂SO₃ once is enough) while for acids with a pKa-value higher than 4 it is necessary to add Na₂SO₃ several times to obtain sufficient inactivation of the catalase. It is also shown, that phosphoric acid gives the lowest catalase activity with the highest residual glucose oxidase activity.

Claims

1. A method for inactivating catalase activity in a composition comprising oxidase and catalase, wherein the composition is treated with a sulphur-containing reducing agent.

2. The method according to claim 1 , wherein the treatment is carried out prior to, during and/or after a diafiltration and/or ultrafiltration step.

3. The method according to any of claims 1-2, wherein the composition is of microbial origin.

4. The method according to any of claims 1-3, wherein the oxidase is a glucose oxidase, particular a glucose oxidase derived from Aspergillus niger.

5. The method according to any of claims 1-4, wherein the sulphur-containing compounds are selected from the group consisting of sulphurous acid (H₂SO₃) and salts thereof, thiosulphuric acid (H₂S₂O₃) and salts thereof, dithiorous acid (H₂S₂O₄) salts, pyrosulphurous acid (H₂S₂O₅) salts and dithionic acid (H₂S₂Oβ) and salts thereof.

6. The method according to claim 5, wherein the sulphur-containing compound is Na₂SO₃.

7. The method according to any of claims 1-6, wherein the method is carried out at a pH in the range of from about 3.0 to 7.0, particularly at a pH about 4.0 to 6.0.

8. The method according to claim 7, wherein the pH is adjusted with an acid having a pKa-value below 4.

9. The method according to claim 8, wherein the acid is phosphoric acid.

10. The method according to any of claims 1-9, wherein the method is carried out at a temperature in the range of from 1 to 60°C.

11. The method according to any of claims 1-10, wherein the composition is added a cationic flocculent.

12. A composition obtained according to any of the preceding claims.

13. A composition comprising an oxidase substantially free from catalase activity.

14. The composition according to any of claims 12-13, wherein the oxidase is a glucose oxidase, particularly a glucose oxidase derived from Aspergillus niger.

15. The composition according to any of claims 12-14, wherein the composition further comprising a substrate for the oxidase.

16. The composition according to any of claims 12-15, wherein the composition further comprises a peroxidase and a substrate for the peroxidase.

17. The composition according to claim 16, wherein the peroxidase is a haloperoxidase and the composition further comprises a halide source.

18. A composition according to any of claims 12-15, wherein the composition is a baking composition.

19. The composition according to any of claims 12-17, wherein the composition is an oral care composition.

20. The composition according to any of claims 12-17, wherein the composition is an anti-microbial composition.

21. The composition according to any of claims 12-17, wherein the composition is a bleaching composition.

22. Use of a composition according to any of claims 12-15 for baking purposes.

23. Use of a composition according to any of claims 12-17 for bleaching purposes.

24. The use according to claim 23 for bleaching teeth.

25. The use according to claim 23 for bleaching textile.

26. Use of a composition according to any of claims 12-17 as an anti-microbial agent.

27. Use of a composition according to any of claims 12-17 in a biosensor or as an analytical reagent.

28. Use of a composition according to any of claims 21-17 as a dye transfer inhibitor.