A RECOMBINANT ENZYME WITH MUTANASE ACTIVITY

Abstract: The present invention relates to method for constructing an expression vector comprising a mutanase gene obtained from a filamentous fungus suitable for heterologous production comprising the steps of a) isolating a DNA sequence encoding a mutanase from a filamentous fungus, b) introducing a kex2-site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase, c) cloning the DNA sequence obtained in step b) into a suitable expression vector. The invention also relates to a recombinant expression vector comprising said mutanase gene sequence and a kex2 cleavage site between the DNA sequence encoding the pro-peptide and the region encoding the mature mutanase, a filamentous fungus host cell, a process for producing recombinant mutanase and a recombinant mutanase. It is also the object of the invention to provide compositions useful in oral care products for humans and animals.

Description: Title: A recoir-binant enzyme with mutanase activity FIELD OF THE INVENTION The present invention relates to a method for constructing an 5 expression vector comprising a mutanase gene obtained from a filamentous fungus suitable for heterologous production, a recombinant expression vector comprising said mutanase gene sequence and a kex2 cleavage site between the DNA sequence encoding the pro-peptide and the DNA sequence encoding the mature 10 mutanase, a filamentous fungus host cell, a process of producing recombinant mutanase, and said recombinant mutanase. It is also the object of the invention to provide compositions useful in oral care products for humans and animals. 15 BACKGROUND OF THE INVENTION Mutanases are α-1, 3-glucanases (also known as α-1,3- glucanohydrolases) which degrade the α-l,3-glycosidic linkages in mutan. Mutanases have been described from two species of Trichoderma (Hasegawa et al. , (1969), Journal of Biological Chemistry 20 244, p. 5460-5470; Guggenheim and Haller, (1972), Journal of Dental Research 51, p. 394-402) and from a strain of Streptomyces (Takehara et al., (1981), Journal of Bacteriology 145, p. 729- 735) , Cladosporium resinae (Hare et al. (1978), Carbohydrate Research 66, p. 245-264), Pseudomonas sp. (US patent no. 25 4,438,093), Flavobacterium sp. (JP 77038113), Bacillus circulanse (JP 63301788) and Aspergillus sp.. A mutanase gene from Trichoderma harzianum has been cloned and sequenced (Japanese Patent No. 4-58889-A from Nissin Shokuhin Kaisha LDT) . Although mutanases have commercial potential for use as an 30 antiplaque agent in dental applications and personal care products, e . g. , toothpaste, chewing gum, or other oral and dental care products, the art has been unable to produce mutanases in significant quantities to be commercial useful. US patent no. 4,353,891 (Guggenheim et al.) concerns plaque 35 removal using mutanase produced by Trichoderma harzianum CBS 243.71 to degrade mutan synthesized by cultivating Streptococcus mutans strain CBS 350.71 identifiable as OMZ 176. It is an object of the present invention to provide a recombinant mutanase from Trichoderma harzianum which can be produced in commercially useful quantities. BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows plasmid pMT1796 Figure 2 shows plasmid construction of plasmids pMT1796, pMT1802, and pMT1815, Figure 3 shows an outline of the construction of the A. oryzae recombinant mutanase expression vector pMT1802 , Figure 4 shows the pH-profile of recombinant and wild- type T. harzianum CBS 243.71 mutanase Figure 5 shows the temperature profile of recombinant and wild- type T. harzianum CBS 243.71 mutanase at pH 7, Figure 6 shows the temperature stability of recombinant and wild- type Γ. harzianum CBS 243.71 mutanase at pH 7, Figure 7 shows the indirect Malthus standard curve for a mix culture of S . mutans , A . viscosus and F . nucleatu grown in BHI at 37°C. SUMMARY OF THE INVENTION The object of the invention is to provide a recombinant mutanase derived from a filamentous fungus by heterologous expression. The present inventors have as the first been able to express the mutanase gene of a filamentous fungus heterologously and thus cleared the way for providing a single component, recombinant mutanase essentially free of any contaminants . In the first aspect the invention relates to a method for constructing an expression vector comprising a mutanase gene obtained from a filamentous fungus suitable for heterologous production comprising the steps of: a) isolating a DNA sequence encoding a mutanase from a filamentous fungus, b) introducing a kex2 site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase, or replacing the mutanase (pre) pro-sequence with a (pre) pro-sequence comprising a kex2 or kex2-like site of another fungal enzyme, c) cloning the DNA sequence obtained in step b) into a suitable expression vector. In a preferred embodiment the mutanase is obtained from a strain within the genus Trichoderma . In step b) the mutanase (pre) pro-sequence may for instance be replaced with the Lipolase® (pre) pro-sequence or the TAKA- amylase (pre) pro-sequence. It is also an object of the invention to provide an expression vector comprising a mutanase gene and a DNA sequence encoding a (pre) pro-peptide with a kex2 site or kex2-like site between the DNA sequences encoding said (pre) pro-peptide and the mature region of the mutanase. The invention also relates to a filamentous host cell for production of recombinant mutanase derived from a filamentous fungus. Preferred host cells include filamentous fungi of the genera Trichoderma , Aspergillus, and Fusarium . Further, the invention relates to a process for producing a recombinant mutanase in a host cell, comprising the steps: a) transforming an expression vector comprising a mutanase gene with a kex2 site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase into a suitable filamentous fungus host cell, b) cultivating the host cell in a suitable culture medium under conditions permitting expression and secretion of an active mutanase, c) recovering and optionally purifying the secreted active re- combinant mutanase from the culture medium. The expression vector may be prepared according to the above described method of the invention. A recombinant mutanase may according to the invention be produced according to the process of the invention. A substantially pure wild-type mutanase obtained from Trichoderma harzianum CBS 243.71 essentially free of any contaminants is also part of the invention. The invention also relates to a composition comprising a recombinant mutanase of the invention or a substantially pure mutanase of the invention useful in oral care products and food, feed and/or pet food products. Finally the invention relates to the use of the recombinant mutanase of the invention or the substantially purified mutanase of the invention or composition or product of the invention preventing the formation of human or animal dental plaque or removing dental plaque and for the use in food, feed and/or pet food products. DETAILED DESCRIPTION OF THE INVENTION The object of the invention is to provide a recombinant mutanase derived from a filamentous fungus by heterologous expression. The present inventors have as the first been able to express the mutanase gene of a filamentous fungus heterologously and thus cleared the way for providing a single component recombinant mutanase essentially free of any contaminants. The principle of the invention can be used for all mutanases derivable from filamentous fungi, such as from filamentous fungi of the genus Trichoderma, such a strain of Trichoderma harzianum, especially Trichoderma harzianum CBS 243.71, and the genera Streptomyceε , Cladosporium or Aspergillus . Previously it has not been possible to produce mutanases of filamentous fungi heterologously. Consequently, according to prior art mutanases are produced homologously and comprise a mixture of other enzyme activities besides the mutanase (i.e. with undesired contaminants) . An example of this is Trichoderma harzianum CBS 243.71 which are known to produce a mutanase as also described above. The mutanase derived from Trichoderma harzianum CBS 243.71 has before the successful findings of the present invention only been produced homologously. It is advantageous to be able to produce the mutanase heterologously, as it is then possible to provide a single component mutanase free of undesired contaminants. Further, it facilitates providing an isolated and purified enzyme of the invention in industrial scale. According to the invention it is possible to express mutanases derived from filamentous fungi in a suitable host cell by introducing a kex2 cleavage site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature mutanase, or replacing the mutanase (pre) pro-sequence with a (pre) pro-sequence comprising a kex2 site or kex2-like site of another fungal enzyme. The (pre) pro-sequence have for instance be the Lipolase® (pre) pro-sequence or the TAKA-amylase (pre) pro-sequence. Pro-peptides A large number of mature proteins are initially synthesised with a N-terminal extension, the pro-peptide, varying from very small peptides (e . g . GLA 6 amino acids) to relatively long pep- tides (e.g. PEPA 49 amino acids). The pro-peptide can perform a number of different functions. Firstly, pro-peptides might contribute to the efficiency of co- translational translocation of the protein across the ER-mem- brane. Secondly, pro-peptides might contribute to co-transla- tional proteolytic processing of the polypeptide. Thirdly, they might act as intracellular targeting signal for routing to specific cellular compartments. Fourthly, in some pro-proteins the pro-peptide keeps the protein inactive until it reaches its site of action. Removal of the pro-peptide from the mature protein occurs in general by processing by a specific endopeptidase, usually after the two positively charged amino acid residues Arg-Arg, Arg-Lys or Lys-Arg. However, also other amino acid combinations, containing at least one basic amino acid, have been found to be processed. The absence of these doublets in mature, endogenous secreted proteins might protect them from proteolytic cleavage. As di- basic cleavage is thought to occur in the Golgi, the internal di-basic peptide sequences in cytoplasmic proteins will not be attacked by this processing. Kex2 sites Kex2 sites (see e . g. Methods in Enzymology Vol 185, ed. D. Goeddel, Academic Press Inc. (1990) , San Diego, CA, "Gene 5 Expression Technology") and kex2-like sites are di-basic recognition sites (i.e. cleavage sites) found between the pro- peptide encoding region and the mature region of some proteins. Insertion of a kex2 site or a kex2-like site have in certain cases been shown to improve correct endopeptidase processing at 10 the pro-peptide cleavage site resulting in increased protein secretion levels. However, in a number of other cases insertion of a Kex2 cleavage site did not increase the secretion level. For instance, Cullen et al., (1987), Bio/Technology, vol. 5, p. 15 369-376, found that insertion of a kex2 site in the secretion signal of chymosin (i.e. signal peptide and pro-peptide), which encoded the glucoamylase signal peptide and pro-peptide fused to prochy osin, did not increase the secretion level of recombinant chymosin expressed in a Aspergillus nidulans host 20 cell. Other examples of references showing that insertion of a kex2 site or a kex2-like site do not always increase the secretion level include Valverde et al., (1995), J. of Biolog. Chem, p. 15821-15826) 25 In the context of the present invention the term "heterologous" production means expression of a recombinant enzyme in an host organism different from the original donor organism or expression of a recombinant enzyme by the donor organism. 30 The term "homologous" production means expression of the wild- type enzyme by the original organism. In the first aspect the invention relates to a method for construction of an expression vector comprising a mutanase gene suitable for heterologous production comprising the steps of: 35 a) isolating a DNA sequence encoding a mutanase from a filamentous fungus known to produce a mutanase, b) introducing a kex2 site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase, or replacing the mutanase (pre) pro-sequence with a (pre) pro-sequence comprising a kex2 or kex2-like site of another fungal enzyme, c) cloning the mutanase gene with the kex2 site or kex2-like site obtained in step b) into a suitable expression vector. In a preferred embodiment of the mutanase gene is obtained from the genus Trichoderma , preferably a strain of the species cr. harzianum , especially the strain T. harzianum CBS 243.71. The complete mutanase gene DNA sequence derived from Trichoderma harzianum CBS 243.71 is shown in SEQ ID No. 1 In step b) the mutanase (pre) pro-sequence may for instance be replaced with the Lipolase® (pre) pro-sequence or the TAKA- amylase (pre) pro-sequence. In the examples below illustrating the present invention a kex2-site is inserted into the Trichoderma harzianum mutanase gene presented in SEQ ID No. 1 as the site specific mutation E36 → K36. Isolation of the mutanase gene The DNA sequence encoding a mutanase may, in accordance with well-known procedures, conveniently be isolated from DNA from a suitable source, such as any of the above mentioned organisms known to comprise a mutanase gene, by use of synthetic oligo- nucleotide probes prepared on the basis of the DNA sequence disclosed herein. For instance, a suitable oligonucleotide probe may be prepared on the basis of the nucleotide sequences shown in SEQ ID no. 1 or the amino acid sequence shown in SEQ ID no. 2 or any suitable sub-sequence thereof. According to this method primers are designed from the knowledge to at least a part of SEQ ID No. 2. Fragments of mutanase gene are then PCR amplified by the use of these primers. These fragments are used as probes for cloning the complete gene. Alternatively, the DNA sequence encoding a mutanase may be isolated by a general method involving - cloning, in suitable vectors, a DNA or cDNA library from a strain of genus Trichoderma , - transforming suitable host cells with said vectors, - culturing the host cells under suitable conditions to express any enzyme of interest encoded by a clone in the DNA library, - screening for positive clones by determining any mutanase activity of the enzyme produced by such clones, and - isolating the DNA coding an enzyme from such clones. The general method is further disclosed in WO 93/11249 the contents of which are hereby incorporated by reference. Expression vector In another aspect the invention relates to an expression vector comprising a mutanase gene and a DNA sequence encoding a pro-peptide with a kex2 site or kex2-like site inserted between the DNA sequences encoding said pro-peptide and the mature region of the mutanase. In preferred embodiments of the invention the expression vector comprises besides the kex2 site or kex2-like site an operably linked DNA sequence encoding a prepro-peptide (i.e. signal peptide and a pro-peptide) . The prepro-sequence may advantageously be the original mutanase signal-sequence or the Lipolase® signal-sequence or the TAKA signal-sequence and the original mutanase pro-sequence or the Lipolase® pro-sequence or the TAKA pro-sequence. The promoter may be the TAKA promoter or the TAKA:TPI promoter. In a specific embodiment of the invention the expression vector is the pMT1796 used to illustrate the concept of the invention in Example 3 below. 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. 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 encoding the mutanase should also be operably connected to a suitable promoter and terminator sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The procedures used to ligate the DNA sequences coding for the mutanase, a prepro-sequence including the kex2 site or kex2-like site, the promoter and the terminator, respectively, and to insert them into suitable vectors are well known to persons skilled in the art (cf., for instance, Sambrook et al., (1989) , Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY). Host Cell A third aspect of the invention relates to a filamentous fungi host cell for production of recombinant mutanase derived from a filamentous fungus of the genus Tric o--.er-7ia, such as a strain of T . harzianum , especially T. harzianum CBS 243.71, or the genus Aspergillus , such as a strain of A . oryzae or A . niger, or a strain of the genus Fuεarium , such as a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gribberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella roseu f . sp. cerealis) , or Fusarium sulphureum fin the prefect state named Gibberella puricaris, synonym with Fusarium trichothecioides , Fusarium bactridioides, Fusarium sambucium, Fusarium roseum, and Fusarium roseum var. graminearum) , Fusarium cerealis (synonym with Fusarium crokkwellnse) or Fusarium venenatum . The host cell may advantageously be a F. graminearum described in WO 96/00787 (from Novo Nordisk A/S), e.g. the strain deposited as Fusarium graminearum ATCC 20334. The strain ATCC 20334 was previously wrongly classified as Fusarium graminearum (Yoder, W. and Christianson, L. 1997) . RAPD-based and classical taxonomic analyses have now revealed that the true identity of the Quorn fungus, ATCC 20334, is Fusarium venenatum Nirenburg sp. nov. In a preferred embodiment of the invention the host cell is a protease deficient or protease minus strain. This may for instance be the protease deficient strain Aspergillus oryzae JaL125 having the alkaline protease gene named "alp" deleted. This strain is described in PCT/DK97/00135 (from Novo Nordisk A/S) . Filamentous fungi 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. The use of Aspergillus as a host microorganism is described in EP 238 023 (Novo Nordisk A/S) , the contents of which are hereby incorporated by reference. According to a further aspect the invention relates to a process for producing a recombinant mutanase in a host cell. Said process comprises the following steps: a) transforming an expression vector encoding a mutanase gene with a kex2 site or a kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase into a suitable filamentous fungus host cell, b) cultivating the host cell in a suitable culture medium under conditions permitting the expression of the expression vector, c) recovering the secreted recombinant mutanase from the culture medium, d) and optionally purifying the recombinant mutanase. The recombinant expression vector may advantageously be any of the above described. Further, the filamentous fungi host cells to be used for production of the recombinant mutanase of the invention according to the process of the invention may be any of the above mentioned host cell, especially of the genera Aspergillus, Fusarium or Trichoderma . The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed mutanase is secreted into the culture medium and may be recovered from there by well-known procedures including separating the cells from the medium by centrif gation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like. It is also an important object of the invention to provide a recombinant mutanase produced according to the process of the invention. The isolated recombinant mutanase has essentially an amino acid sequence as shown in SEQ ID no. 2. From SDS-PAGE a mole- cular weight around 80 kDa was found. The pH optimum of the recombinant mutanase was found to lie in the range from 3.5 to 5.5 which equals the pH optimum of the wild-type mutanase (see Figure 4) . The temperature optimum of both the recombinant and wild-type mutanase was found to be around 45°C at pH 7 and around 55°C at pH 5.5 (see Figure 5). Further, the residual activity starts to decline at 40°C at pH 7, while the enzyme is more stable at pH 5.5, where the residual activity starts to decline at 55°C. The inventors have also provided a substantially pure wild- type mutanase obtained from Trichoderma harzianum CBS 243.71 essentially free of any active contaminants, such as other enzyme activities. Composition It is also an object of the invention to provide a composition comprising the recombinant mutanase of the invention or the purified wild-type mutanase essentially free of any active contaminants of the invention. Oral care composition In a still further aspect, the present invention relates to an oral care composition useful as an ingredient in oral care products. An oral care composition of the invention may suitably comprise an amount of the recombinant Trichoderma harzianum mutanase equivalent to an enzyme activity, calculated as enzyme activity units in the final oral care product, in the range from 0.001 MU to 1000 MU/ml, preferably from 0.01 MU/ l to 500 MU/ml, such as from 0.1 MU/ml to 100 MU/ml, especially 0.05 MU/ml to 100 MU/ml. It is also contemplated according to the invention to include other enzyme activities than mutanase activity in the oral care composition. Contemplated enzyme activities include activities from the group of enzymes comprising dextranases, oxidases, such as glucose oxidase, L-amino acid oxidase, peroxidases, such as e .g. the Coprinus sp. peroxidases described in WO 95/10602 (from Novo Nordisk A/S) or lactoperoxidaseor, haloperoxidases, laccases, proteases, such as papain, acidic protease (e .g. the acidic proteases described in WO 95/02044 (Novo Nordisk A/S)), endoglucosidases, lipases, amylaεes, including amyloglucosidases, such as AMG (from Novo Nordisk A/S) , and mixtures thereof. Oral care products The oral care product may have any suitable physical form (i.e. powder, paste, gel, liquid, ointment, tablet etc.). An "oral care product" can be defined as a product which can be used for maintaining or improving the oral hygiene in the mouth of humans and animals, by preventing dental caries, preventing the formation of dental plaque and tartar, removing dental plaque and tartar, preventing and/or treating dental diseases etc. At least in the context of the present invention oral care products do also encompass products for cleaning dentures, artificial teeth and the like. Examples of such oral care products include toothpaste, dental cream, gel or tooth powder, odontic, mouth washes, 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, whitening/bleaching/ stain removing agents, water, and optionally enzymes. Mouth washes, including plaque removing liquids, typically comprise a water/alcohol solution, flavour, hu ectant, sweetener, foaming agent, colorant, and optionally enzymes. Abrasives Abrasive polishing material might also be incorporated into the dentifrice product of the invention. According to the invention said abrasive polishing material includes alumina and hydrates thereof, such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, 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, preferably from 1% to 70%. For toothpastes the abrasive material content typically lies in the range of 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 in from 0% to 80%, preferably 5 to 70% by weight in toothpaste. Silica, starch, tragacanth gum, 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 helps 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 of from 0.01 to 10% by weight of the final product. Foaming agents As foaming agent soap, an-ionic, cat-ionic, non-ionic, a pho- teric and/or zwitterionic surfactants can be used. These may be present at levels of from 0% to 15%, preferably from 0.1 to 13%, more preferably from 0.25 to 10% by weight of the final product. Surfactants 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- glycerides or fatty acids having 10 to 20 carbon atoms, fatty acid-albumen condensation products, salts of fatty acids amides and taurines and/or salts of fatty acid esters of isethionic acid. Sweetening agents Suitable sweeteners include saccharin. Flavouring agents Flavours, such as spearmint, are usually present in low amounts, such as from 0.01% to about 5% by weight, especially from 0.1% to 5%. Whitening/bleaching agents Whitening/bleaching agents include H 2 O 2 and may be added in amounts less that 5%, preferably from 0.25 to 4%, calculated on the basis of the weight of the final product. The whitening/bleaching agents may be an enzyme, such as an oxidoreductase. Examples of suitable teeth bleaching enzymes are described in WO 97/06775 (from Novo Nordisk A/S) . Water Water is usually added in an amount giving e .g. toothpaste a flowable form. Additional agents 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 compounds which can be used as fluoride source, dyes/colorants, preservatives, vitamins, pH-adjusting agents, anti-caries agents, desensitizing agents etc. Enzymes Other essential components used in oral care products and in oral care products of the invention are enzymes. Enzymes are biological catalysts of chemical reactions in living systems. Enzymes combine with the substrates on which they act forming an intermediate enzyme-substrate complex. This complex is then converted to a reaction product and a liberated enzyme which continue its specific enzymatic function. Enzymes provide several benefits when used for cleansing of the oral cavity. Proteases break down salivary proteins, which are adsorbed onto the tooth surface and form the pellicle, the first layer of resulting plaque. Proteases along with lipases destroy bacteria by lysing proteins and lipids which form the structural components of bacterial cell walls and membranes. Dextranase breaks down the organic skeletal structure produced by bacteria that forms a matrix for bacterial adhesion. Proteases and amylases, not only prevents plaque formation, but also prevents the development of calculus by breaking-up the carbohydrate-protein complex that binds calcium, preventing mineralization. Toothpaste A toothpaste produced from an oral care composition of the invention (in weight % of the final toothpaste composition) may typically 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% Whitener 0 to 5% Enzymes 0.0001% to 20% In a specific embodiment of the invention the oral care product is toothpaste having a pH in the range from 6.0 to about 8.0 comprising a) 10% to 70% Abrasive material b) 0 to 80% Humectant c) 0.1 to 20% Thickener d) 0.01 to 10% Binder e) 0.1% to 5% Sweetener f) 0 to 15% Foaming agent g) 0 to 5% Whitener i) 0.0001% to 20% Enzymes. Said enzymes referred to under i) include the recombinant mutanase of the invention, and optionally other types of enzymes mentioned above known to be used in toothpastes and the like. Mouth wash 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 fluorides) . 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) . Method of Manufacture The oral care composition and products of the present invention can be made using methods which are common in the oral product area. According to the present invention the recombinant mutanase and/or the substantially purified mutanase free of active contaminants can be use in food, feed and/or pet food products. MATERIALS AND METHODS Materials Micro-organisms Trichoderma harzianum CBS 243.71 A. oryzae JaL 125: Aspergillus oryzae IFO 4177 available from Institute for Fermentation, Osaka; 17-25 Juso Hammachi 2-Chome Yodogawa-ku, Osaka, Japan, having the alkaline protease gene named "alp" (described by Murakami K et al., (1991), Agric. Biol. Chem. 55, p. 2807-2811) deleted by a one step gene replacement method (described by G. May in "Applied Molecular Genetics of Filamentous Fungi" (1992) , p. 1-25. Eds. J. R. Kinghorn and G. Turner; Blackie Academic and Professional) , using the A. oryzae pyrG gene as marker. E. coli DH50C Plasmids and Vectors: pMT1796 (Figure 1 and Figure 2) PMT1802 (Figure 2) PMT1815 (Figure 2) pHD414: Aspergillus expression vector is a derivative of the plasmid p775 (described in EP 238.023). The construction of the pHD414 is further described in WO 93/11249. pHD414 contains the A . niger glucoamylase terminator and the A . oryzae TAKA amylase promoter. pHD414+mut (Figure 3) pHan37 containing the TAKA:TPI promoter Linkers : Linker #1: GATCCTCACA ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GG GAGTGT TAC AAC CCG CAA CAG GCT GCA GAT CCG GAT CCG C Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Linker #2 : C CAA TAC TGT TAG T GT ACG GTT ATG ACA ATC AGATC Ala Cys Gin Tyr Cys *** Primers Primer 1: 5' GGGGGGATCCACCATGAG 3' (SEQ ID No. 3) Primer 2: 5' ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC 3' (SEQ ID No. 4) Primer 3: 5' GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT 3 1 (SEQ ID No. 5) Primer 4: 5' CCACGGTCACCAACAATAC 3' (SEQ ID No. 6) Primer 5: GGGGGGATCCACCATGAG (SEQ ID No. 7), Primer 6: ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC (SEQ ID No. 8) Primer 7: GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT (SEQ ID NO. 9), Primer 8: CCACGGTCACCAACAATAC (SEQ ID No. 10). Enzymes : lysyl-specific protease from Achromobacter Trichoderma harzianum CBS 243.71 fermentation broth (Batch no. PPM 3897) Media- Substrates and Solutions: YPM: 2% maltose, 1% bactopeptone and 0.5% yeast extract) DAPI: 4 • , 6-diamidino-2-phenylindole (Sigma D-9542) Britton-Robinson Buffer BHI: Brain Heart Infusion broth Equipment: 10 kDa cut-off ultra-filtration cassette (Alpha Minisette from Filtron) . Phenyl-sepharose FF (high sub) column (Pharmacia) Seitz EK1 filter plate Q-sepharose FF column (Pharmacia) Applied Biosystems 473A protein sequencer 2 litre Kieler fer enter Olympus model BX50 microscope Malthus Flexi M2060 (Malthus Instrument Limited) Methods: Molecular biology procedures All molecular biology procedures including restriction digests, DNA ligations, E. coli transformations, DNA isolations, Southern hybridizations, PCR amplifications, and library constructions and screenings were completed using stan- dard techniques (Sambrook, J. , Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning: A laboratory manual/E.F. Cold Spring Harbor Laboratory Press, Plainview, NY) . Preparation of Mutan Mutan is prepared by growing Streptococcus mutans CBS 350.71 at pH 6.5, 37°C (kept constant), and with an aeration rate of 75 rpm in a medium comprised of the following components: NZ-Case 6.5 g/litre Yeast Extract 6 g/litre (NH 4 ) 2 S0 4 20 g/litre K 2 P0 4 3 g/litre Glucose 50 g/litre Pluronic PE6100 0.1% After 35 hours, sucrose is added to a final concentration of 60 g/litre to induce glucosyltransferase. The total fermentation time is 75 hours. The supernatant from the fermentation is centrifuged and filtered (sterile) . Sucrose is then added to the supernatant to a final concentration of 5% (pH is adjusted to pH 7.0 with acetic acid) and the solution is stirred overnight at 37 °C. The solution is filtered and the insoluble mutan is harvested on propex and washed extensively with deionized water containing 1% sodium benzoate, pH 5 (adjusted with acetic acid) . Finally, the insoluble mutan is lyophilized and ground. Determination of mutanase activity (MU) One Mutanase Unit (MU) is the amount of enzyme which under standard conditions liberates 1 μmol reducing sugar (calculated as glucose) per minute. Reducing sugars were measured with alkaline K 3 Fe(CN) 6 . Standard Conditions Substrate 1.5% mutan Reaction time 15 minutes Temperature 40°C pH 5.5 A detailed description of Novo Nordisk' s analytical method (AF 180/1-GB) is available from Novo Nordisk A/S on request. Mutanase Plate Assay A 5% mutan suspension is made in 50 mM sodium acetate, pH 5.5 and the suspension is homogenised for 15 minutes in an Ultra Turrax T25 homogenizer at 4°C. 1% agarose in 50 mM sodium acetate, pH 5.5 is made 0.2% with respect to mutan and 12.5 ml agarose is casted in each petri dish (d=10 cm) . The sample to be analyzed for mutanase activity is applied in sample wells punched in the agarose, and the plate is incubated overnight at 37 °C, whereafter clearing zones are formed around mutanase containing samples . Western hybridization Western hybridizations are performed using the ECL western blotting system (Amersham International, pic, Buckinghamshire, England) and a primary antibody solution containing polyclonal rabbit-anti-mutanase. The limit of detection is 0.001 MU/ml. Mass spectrometry Mass spectrometry of purified wild-type mutanase is done using matrix assisted laser desorption ionization time-of-flight mass spectrometry in a VG Analytical TofSpec. For mass spectrometry 2 ml of sample is mixed with 2 ml saturated matrix solution (a- cyano-4-hydroxycinnamic acid in 0.1% TFA:acetonitrile (70:30)) and 2 ml of the mixture spotted onto the target plate. Before introduction into the mass spectrometer the solvent is removed by evaporation. Samples are desorbed and ionized by 4 ns laser pulses (337 nm) at threshold laser power and accelerated into the field-free flight tube by an accelerating voltage of 25 kV. Ions are detected by a microchannel plate set at 1850 V. Preparation of Hydroxyapatite disks (HA) Hydroxyapatite tablets are prepared by compressing 250 mg of hydroxyapatite in a tablet die at about 5,900 kg (13,000 lbs) of pressure for 5 minutes. The tablets are then sintered at 600°C for 4 hours and finally hydrated with sterile deionized water. Plaque coating of Hydroxyapatite disks (HA) Hydroxyapatite disks (HA) were dry sterilised (121°C, 2 bar, 20 minutes) and coated with filter sterilised saliva for 18 hours at 37 °C. The HA disks were placed in a sterile rack in a beaker, Brain Heart Infusion broth (BHI) containing 0.2% sucrose was poured into the beaker covering the disks. Sterile Na 2 S (pH 7.0) was added immediately before inoculation given the final concen- tration of 5 g/litre. A mixture 1:1:1 of Streptococcus mutans, Actinomyceε viscosus and Fusobacterium nucleatum grown anaero- bically (BHI, 37 °C, 24 h) was used as inoculum in the concentration of approximately 10 cfu/ml. The disks were incubated anaerobic at 37°C for 4 days with slight stirring. Malthus-method for plaque The Malthus-method is based on the methods described in Johnston et al., (1995), Journal of Microbiological Methods 21, p. 15-26 and Johansem et al. (1995), Journal of Applied Bacteriology 78, p. 297-303. EXAMPLES Example 1 Purification of wild-type Mutanase 100 g fermentation broth of Trichoderma harzianum CBS 243.71 (Batch no. PPM 3897) were dissolved in 1 litre 10 mM sodium acetate, pH 5.2 overnight at 4°C. 65 g DEAE-Sephadex A-50 were swelled in 3 litre 10 mM sodium acetate, pH 5.2. Excess buffer was removed after swelling. DEAE- Sephadex was mixed with the crude mutanase preparation for 1 hour and unbound material was collected by filtration through Propex cloth. The gel was further washed with 2.5 1 of 10 mM sodium ace- tate, pH 5.2. A pool containing the unbound material was made; volume 4 litre. Remaining DEAE-Sephadex particles were removed by filtration through a Whatman GF/F filter. 350 ml S-Sepharose was equilibrated in 10 mM sodium acetate, pH 5.2 and mixed with 600 ml of the pool from the DEAE-Sephadex for 10 minutes. Unbound material was collected by filtration through Propex cloth and the gel was washed with 500 ml 10 mM sodium acetate buffer, pH 5.2. Bound material was eluted with the same buffer containing 1 M NaCl. The procedure was repeated 7 times. The combined pool containing the unbound material (7 litre) was concentrated on a Filtron concentrator equipped with a 10 kDa cut-off membrane and followed by a buffer change to 10 mM sodium acetate, pH 4.7. The concentrate was filtrated through a What ann GF/F filter. The final volume of the concentrate was 600 ml. An S-Sepharose column (180 ml, 2.6 x 33 cm) was equilibrated with 10 mM sodium acetate, pH 4.7. The pH adjusted concentrate from the S-Sepharose batch ion exchange was applied onto the column in 50 ml portions with a flow of 10 ml/min. The mutanase was eluted with a linear gradient from 0 to 20 mM NaCl in 3 column volumes. The residual protein was eluted with the same buffer containing 1 M NaCl. Fractions were analyzed for mutanase activity (plate assay) and fractions with high activity were pooled. The procedure was repeated 12 times. The combined mutanase pool was concentrated in a Filtron concentrator equipped with a 10 kDa cut-off membrane and followed by a buffer change to 10 mM Tris-HCl, pH 8.0. The final volume of the concentrate was 870 ml. The concentrated pool from the S-Sepharose column was further purified on a HiLoad Q-Sepharose column (50 ml, 2.6 x 10 cm) equilibrated with 10 mM Tris-HCl, pH 8.0. Portions of 130 ml was applied with a flow of 8 ml/min. Elution of the mutanase was per- formed with a linear gradient from 0 to 50 mM NaCl in 12 column volumes. Fractions with high mutanase activity (plate assay) were pooled, concentrated in an Amicon cell equipped with a 10 kDa cut-off membrane. Finally, the mutanase preparation was dialyzed extensively against 10 mM sodium phosphate, pH 7.0 and filtrated through a 0.45 mm filter. The yield of the mutanase in the purification described above was 300 mg. The purity of the HiLoad-Q preparation was analyzed by SDS-PAGE and N-terminal sequencing and judged by both methods the purity was around 95%. Example 2 N-terminal sequencing of wild-type Mutanase N-terminal amino acid sequencing was carried out in an Applied Biosystems 473A protein sequencer. To generate peptides reduced and S-carboxymethylated mutanase (» 450 mg) was digested with the lysyl-specific protease from Achromobacter (10 mg) in 20 mM NH4HCO 3 for 16 hours at 37°C. The resulting peptides were separated by reversed phase HPLC using a Vydac Ciβ column eluted with a linear gradient of 80% 2-propanol containing 0.08% TFA in 0.1% aqueous TFA. Peptides were repurified by reversed-phase-HPLC using a Vydac C^ column eluted with linear gradients of 80% acetonitrile containing 0.08% TFA in 0.1% aqueous TFA before being subjected to N-terminal amino acid sequencing. The amino acid sequences determined are given below. N-terminal: Ala-Ser-Ser-Ala-Asp-Arg-Leu-Val-Phe-Cys-His-Phe-Met-Ile-Gly-Ile- Val-Gly-Asp-Arg-Gly-Ser-Ser-Ala-Asp-Tyr-Asp-Asp-Asp- Peptide 1: Val Phe-Ile-Ser-Phe-Asp-Phe-Asn-Trp-Trp-Ser-Pro-Gly-Asn-Ala-Val- Gly-Val-Gly-Gln-Lys Peptide 2: Pro-Tyr-Leu-Ala-Pro-Val-Ser-Pro-Trp-Phe-Phe-Thr-His-Phe-Gly-Pro- Glu-Val-Ser-Tyr-Ser- Peptide 3: Trp-Val-Asn-Asp-Met-Pro-His-Asp-Gly-Phe-Leu-Asp-Leu-Ser-Lys Example 3 Construction of the mutanase expression vectors / pMT1796, pMT1802 and pMT1815 A cDNA clone encoding mutanase was identified in a Trichoderma harzianum CBS 243.71 library by hybridization with a fragment of the gene amplified by PCR using primers based on the mutanase sequence shown in SEQ ID NO. 1. DNA sequence analysis of the isolated clone, pHD4l4+mut, showed that it indeed encoded the mutanase gene, and that the 5' end of the construct contained a long leader sequence. To remove this leader, pHD414+mut was restricted with the enzymes EcoRI , Narl and Xhol . From this digestion a 3499 nt (nucleotide) vector fragment and a 610 nt Narl/Xhol fragment were isolated. These two fragments were then ligated with linker #1 (see above) and a 618 nt -ScσRI/BaroHI fragment from pHan37 containing the TAKA:TPI promoter, giving plasmid pJW99. HD414+rout was next digested with Xhol and SphI , and a 1790 nt fragment encoding amino acids 35-598 of the mutanase gene was isolated. This fragment was ligated with linker #2 (see above) and pJW99 that had been linearized with the restriction enzymes Xjal and Xhol. The resulting plasmid, pMT1802, contains the T. harzianum mutanase gene under the control of the TAKA:TPI promoter. Plasmid pMT1796 is identical to pMT1802 except that E36 of the mutanase protein has been changed to K36 by replacing the Xhol/Kpnl fragment of pMT1802 with a PCR amplified fragment containing the desired mutation. This PCR fragment was created in a two step procedure as reported in Ho, et al. (1989), Gene, 77, p. 51-59, using the following primers: Primer 1 ( nt 2751 5 ' CAGCGTCCACATCACGAGC nt 2769) and Primer 2 ( nt 3306 5 ' GAAGAAGCACGTTTCTCGAGAGACCG nt 3281). Primer 3 (nt 3281 5' CGGTCTCTGAGAAACGTGCTTCTTC nt 3306) and Primer 4 ( nt 4266 5 ' GCCACTTCCGTTATTAGCC nt 4248). nucleotide numbers refer to the pMT1802 plasmid (See SEQ ID No. 11) . To create pMT1815, a 127 nt DNA fragment was PCR amplified using again a two step procedure and the primers: Primer 5: GGGGGGATCCACCATGAG; Primer 6: ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC; Primer 7: GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT; Primer 8: CCACGGTCACCAACAATAC, and the plasmids pHan37 and pMT1802 as templates in the first round of amplification. This fragment contains a BamHI restriction enzyme site followed by the Lipolase® prepro-sequence in frame with residues 38-54 of the mutanase protein and ending with a BstEII site. The fragment was digested with the restriction enzymes BstEII and BamHI and inserted into pMT1802 that had been linearized with the same pair of enzymes. Changes in constructs were confirmed and the integrity of the resulting coding regions were checked by nucleotide sequencing. Example 4 Expression of recombinant Mutanase in Aspergillus oryzae The strain A . oryzae JaL125 was transformed using a PEG- mediated protocol (see EP 238 023) and a DNA mixture containing 0.5 μg of a plasmid encoding the gene that confers resistance to the herbicide Basta and 8.0 μg of one of the three mutanase expression plasmids. Transformants were selected on minimal plates containing 0.5% basta and 50 mM urea as a nitrogen source. Shake flask cultures Transformed colonies were spore purified twice on selection media and spores were harvested. A 20 ml universal container (Nunc, cat #364211) containing 10 ml YPM (2% maltose, 1% bactopeptone and 0.5% yeast extract) was inoculated with spores and grown for 5 days with shaking at 30°C. The supernatant was harvested after 5 days growth. highest mutanase number of Construct level detected transformants tested pMT1802, mutanase <0.001 10 prepro + mutanase pMT1796, mutanase 3.8 4 prepro + KEX2 + mutanase pMT1815, Lipolase® 0.16 22 prepro + mutanase Table 1 Comparison of mutanase expression from the three different expression constructs. The limit of detection was 0.001 MU/ml The presence of mutanase in culture supernatants was examined by western hybridizations. SDS-PAGE and protein transfers were performed using standard protocols. Example 5 Purification of recombinant mutanase 700 ml fermentation broth was filtered and concentrated. The pH was adjusted to 4.7 (conductivity around 300 μS/cm) and the broth was loaded onto an S-Sepharose column (XK 50/22) (Pharmacia) equilibrated in 10 mM sodium acetate pH 4.7. The mutanase was eluted in a linear NaCl gradient. The major part of the mutanase appeared in the unbound fractions. These fractions were pooled and concentrated. Then the concentrate was loaded onto a HiLoad Q-Sepharose column (Pharmacia) equilibrated in 10 mM Tris-HCl, pH 8.0 (around 600 μS/cm) . The mutanase was eluted in a linear gradient of NaCl and the mutanase containing-fractions were pooled according to purity and activity. The pooled fractions were concentrated and a fraction was further purified by gelfiltration on a Superdex 75 (16/60) column (Pharmacia) in sodium acetate pH 6.0. The purified mutanase has a specific activity around 19 MU pr. absorption unit at 280 nm. From SDS-PAGE (Novex 4-20 % ; run according to the manufacturer's instructions) a molecular weight around 80 kDa is found. The N-terminal amino acid sequence was confirmed to be identical to the N-terminal amino acid sequence of the wt mutanase (Ala-Ser-Ser-Ala-) (see Example 2) Example 6 pH-profile of mutanase 500 ml 5 % mutan in 50 mM Britton-Robinson buffer at varying pH was added 2 ml enzyme sample (diluted in MilliQ-filtered water) in large vials (to ensure sufficient agitation) and incubated for 15 minutes at 40°C while shaking vigorously. The reaction was terminated by adding 0.5 ml 0.4 M NaOH and the samples were filtered on Munktell filters. 100 μl filtrate in Eppendorf vials were added 750 μl ferricyanide reagent (0.4 g/1 K 3 Fe(CN) 6 , 20 g/1 Na 2 C0 3 ) and incubated 15 minutes at 85°C. After allowing the samples to cool, the decrease in absorption at 420 nm was measured. A dilution series of glucose was included as a standard. Substrate and enzyme blanks were always included. Samples were run in duplicate. The pH-optimum for both wild-type and recombinant enzyme is around pH 3.5-5.5 (see Figure 4) . Example 7 Temperature profile of mutanase: 500 ml 5 % mutan in 100 mM sodium acetate, pH 5.5 or in 100 mM sodium phosphate, pH 7 was added 2 ml enzyme sample (diluted in MilliQ-filtered water) in large vials (to ensure sufficient agitation) and incubated for 15 minutes at various temperatures while shaking vigorously. The reaction was terminated by adding 0.5 ml 0.4 M NaOH and the samples were filtered on Munktell filters. 100 μl filtrate in Eppendorf vials were added 750 μl ferricyanide reagent (0.4 g/1 K 3 Fe(CN) 6 , 20 g/1 Na 2 C0 3 ) and in- cubated 15 minutes at 85°C. After allowing the samples to cool, the drop in absorption at 420 nm was measured. A dilution series of glucose was included as a standard. Substrate and enzyme blanks were always included. Samples were run in duplicate. The temperature profiles for the recombinant and wt mutanase were identical. The temperature optimum at pH 7 was around 45 °C. The temperature optimum at pH 5.5 was above 55° (See Figure 5) . Example 8 Temperature stability of mutanase: The temperature stability was investigated by pre-incubating enzyme samples for 30 minutes at various temperatures in 0.1 M sodium acetate, pH 5.5 or in 0.1 M sodium phosphate, pH 7 before assaying the residual activity. Both recombinant and wt mutanase have similar temperature stability profiles. The residual activity starts to decline at 40 °C at pH 1 , while the enzyme is more stable at pH 5.5, where the residual activity starts to decline at 55°C (See Figure 6) . Example 9 Molecular weight of purified wild-type Mutanase The mass spectrometry, performed as described above, of the mutanase revealed an average mass around 75 kDa. In addition, it was clear from the spectra that the glycosylation of the mutanase is heterogeneous. The peptide mass of the mutanase is more than 64 kDa meaning that more than 11 kDa of carbohydrate is attached to the enzyme. Example 10 Activity of mutanase against Dental Plaque A plaque biofilm was grown anaerobic on saliva coated hydroxyapatite disks as described in the Material and Methods Section above. The plaque was a mixed culture of Streptococcus mutans (SFAG, CBS 350.71), Actinomyceε viscosus (DSM 43329) and Fuso- bacterium nucleatum subsp. polymorphum (DSM 20482) . HA disks with plaque were transferred to acetate buffer (pH 5.5) containing recombinant Trichoderma mutanase 1 MU/ml and whirled for 2 minutes (sterile buffer was used as control) . After enzyme treatment, the disks were either DAPI stained or transferred to Malthus cells, as indirect impedance measurements were used when enumerating living adherent cells (Malthus Flexi M2060, Malthus Instrument Limited) . For the impedance measurements 3 ml of BHI were transferred to the outer chamber of the indirect Malthus cells, and 0.5 ml of sterile KOH (0.1 M) was transferred to the inner chamber. After mutanase treatment the disks with plaque were slightly rinsed with phosphate buffer and transferred to the outer chamber. The detection times (dt) in Malthus were converted to colony counts by use of a calibration curve relating cfu/ml to dt (Figure 7) . The calibration curve was constructed by a series of 10-fold dilution rate prepared from the mixed culture. Conductance dt of each dilution step was determined in BHI and a calibration curve relating cfu/ml of the 10 fold dilutions to dt in BHI was constructed for the mixed culture (Figure 7) . The removal of plaque from the disks was also determined by fluorescent microscopy, after mutanase treatment disks were stained with DAPI (3 mM) and incubated in the dark for 5 minutes (20°C) . The DAPI stained cells were examined with the x 100 oil immersion fluorescence objective on an Olympus model BX50 microscope equipped with a 200 W mercury lamp and an UV- filter. The result was compared with the quantitative data obtained by the impedance measurements. The number of living cells on the saliva treated HA-surface after enzyme treatment was determined by the Malthus method and shown in Table 1. However, by the Malthus method it is not possible to distinguish between a bactericidal activity of mutanase or an enzymatic removal of the plaque. Therefore a decrease in living bacteria on the surface has to be compared with the simultaneously removal of plaque from the surface which is estimated by the DAPI staining. Mutanase Logio reduction Removal of No. of (MU/ml) (cfu/cm ) plaque (%) observations 0 0 0 10 1 1.4 96 6 Table 2: Enzymatic plaque removal (pH 5.5, 2 minutes) from saliva treated hydroxyapatite determined by impedance measurements. A significant removal of plaque was determined by fluorescent microscopy after treatment with mutanase. Thus mutanase reduced the amount of adhering cells. However, the activity was observed as a removal of plaque and not as a bactericidal activity against cells in plaque. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Novo Nordisk A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: Denmark (F) POSTAL CODE (ZIP): DK-2880 (G) TELEPHONE: +45 4444 8888 (H) TELEFAX: +45 4449 3256 (ii) TITLE OF INVENTION: A recombinant enzyme with mutanase activity (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1905 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (B) STRAIN: Trichoderma harzianum CBS 243.71 ( ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1905 (A) NAME/KEY: sig_peptide (B) LOCATION: 1..120 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GGC GCC CTT GCT GCC GCA 48 Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Ala Leu Ala Ala Ala 1 5 10 15 GCT CTG TCT TCT CTC GGC AGT GCC GCT CCC GCC AAT GTT GCT ATT CGG 96 Ala Leu Ser Ser Leu Gly Ser Ala Ala Pro Ala Asn Val Ala He Arg 20 25 30 TCT CTC GAG GAA CGT GCT TCT TCT GCT GAC CGT CTC GTA TTC TGT CAC 144 Ser Leu Glu Glu Arg Ala Ser Ser Ala Asp Arg Leu Val Phe Cys His 35 40 45 TTC ATG ATT GGT ATT GTT GGT GAC CGT GGC AGC TCA GCA GAC TAT GAT 192 Phe Met He Gly He Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp 50 55 60 GAT GAC ATG CAA CGT GCC AAA GCC GCT GGC ATT GAC GCA TTC GCT CTG 240 Asp Asp Met Gin Arg Ala Lys Ala Ala Gly He Asp Ala Phe Ala Leu 65 70 75 80 AAC ATC GGC GTT GAC GGC TAT ACC GAC CAG CAA CTC GGG TAT GCC TAT 288 Asn He Gly Val Asp Gly Tyr Thr Asp Gin Gin Leu Gly Tyr Ala Tyr 85 90 95 GAC TCT GCC GAC CGT AAT GGC ATG AAA GTC TTC ATT TCA TTC GAT TTC 336 Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe He Ser Phe Asp Phe 100 105 110 AAC TGG TGG AGC CCC GGT AAT GCA GTT GGT GTT GGC CAG AAG ATT GCG 384 Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gin Lys He Ala 115 120 125 CAG TAT GCC AGC CGT CCC GCC CAG CTG TAT GTT GAC AAC CGG CCA TTC 432 Gin Tyr Ala Ser Arg Pro Ala Gin Leu Tyr Val Asp Asn Arg Pro Phe 130 135 140 GCC TCT TCC TTC GCT GGT GAC GGT TTG GAT GTA AAT GCG TTG CGC TCT 480 Ala Ser Ser Phe Ala Gly Asp Gly Leu Asp Val Asn Ala Leu Arg Ser 145 150 155 160 GCT GCA GGC TCC AAC GTT TAC TTT GTG CCC AAC TTC CAC CCT GGT CAA 528 Ala Ala Gly Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro Gly Gin 165 170 175 TCT TCC CCC TCC AAC ATT GAT GGC GCC CTC AAC TGG ATG GCC TGG GAT 576 Ser Ser Pro Ser Asn lie Asp Gly Ala Leu Asn Trp Met Ala Trp Asp 180 185 190 AAT GAT GGA AAC AAC AAG GCA CCC AAG CCG GGC CAG ACT GTC ACG GTG 624 Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gin Thr Val Thr Val 195 200 205 GCA GAC GGT GAC AAC GCT TAC AAG AAT TGG TTG GGT GGC AAG CCT TAC 672 Ala Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys Pro Tyr 210 215 220 CTA GCG CCT GTC TCC CCT TGG TTT TTC ACC CAT TTT GGC CCT GAA GTT 720 Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val 225 230 235 240 TCA TAT TCC AAG AAC TGG GTC TTC CCA GGT GGT CCT CTG ATC TAT AAC 768 Ser Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Pro Leu lie Tyr Asn 245 250 255 CGG TGG CAA CAG GTC TTG CAG CAG GGC TTC CCC ATG GTT GAG ATT GTT 816 Arg Trp Gin Gin Val Leu Gin Gin Gly Phe Pro Met Val Glu lie Val 260 265 270 ACC TGG AAT GAC TAC GGC GAG TCT CAC TAC GTC GGT CCT CTG AAG TCT 864 Thr Trp Asn Asp Tyr Gly Glu Ser His Tyr Val Gly Pro Leu Lys Ser 275 280 285 AAG CAT TTC GAT GAT GGC AAC TCC AAA TGG GTC AAT GAT ATG CCC CAT 912 Lys His Phe Asp Asp Gly Asn Ser Lys Trp Val Asn Asp Met Pro His 290 295 300 GAT GGA TTC TTG GAT CTT TCA AAG CCG TTT ATT GCT GCA TAT AAG AAC 960 Asp Gly Phe Leu Asp Leu Ser Lys Pro Phe lie Ala Ala Tyr Lys Asn 305 310 315 320 AGG GAT ACT GAT ATA TCT AAG TAT GTT CAA AAT GAG CAG CTT GTT TAC 1008 Arg Asp Thr Asp lie Ser Lys Tyr Val Gin Asn Glu Gin Leu Val Tyr 325 330 335 TGG TAC CGC CGC AAC TTG AAG GCA TTG GAC TGC GAC GCC ACC GAC ACC 1056 Trp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala Thr Asp Thr 340 345 350 ACC TCT AAC CGC CCG GCT AAT AAC GGA AGT GGC AAT TAC TTT ATG GGA 1104 Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe Met Gly 355 360 365 CGC CCT GAT GGT TGG CAA ACT ATG GAT GAT ACC GTT TAT GTT GCC GCA 1152 Arg Pro Asp Gly Trp Gin Thr Met Asp Asp Thr Val Tyr Val Ala Ala 370 375 380 CTT CTC AAG ACC GCC GGT AGC GTC ACG GTC ACG TCT GGC GGC ACC ACT 1200 Leu Leu Lys Thr Ala Gly Ser Val Thr Val Thr Ser Gly Gly Thr Thr 385 390 395 400 CAA ACG TTC CAG GCC AAC GCC GGA GCC AAC CTC TTC CAA ATC CCT GCC 1248 Gin Thr Phe Gin Ala Asn Ala Gly Ala Asn Leu Phe Gin He Pro Ala 405 410 415 AGC ATC GGC CAG CAA AAG TTT GCT CTA ACT CGC AAC GGT CAG ACC GTC 1296 Ser He Gly Gin Gin Lys Phe Ala Leu Thr Arg Asn Gly Gin Thr Val 420 425 430 TTT AGC GGA ACC TCA TTG ATG GAT ATC ACC AAC GTT TGC TCT TGC GGT 1344 Phe Ser Gly Thr Ser Leu Met Asp He Thr Asn Val Cys Ser Cys Gly 435 440 445 ATC TAC AAT TTC AAC CCA TAT GTT GGC ACC ATT CCT GCC GGC TTT GAC 1392 He Tyr Asn Phe Asn Pro Tyr Val Gly Thr He Pro Ala Gly Phe Asp 450 455 460 GAC CCT CTT CAG GCT GAC GGT CTT TTC TCT TTG ACC ATC GGA TTG CAT 1440 Asp Pro Leu Gin Ala Asp Gly Leu Phe Ser Leu Thr He Gly Leu His 465 470 475 480 GTC ACG ACT TGT CAG GCC AAG CCA TCT CTT GGA ACC AAC CCT CCT GTC 1488 Val Thr Thr Cys Gin Ala Lys Pro Ser Leu Gly Thr Asn Pro Pro Val 485 490 495 ACT TCT GGC CCT GTG TCC TCG CTG CCA GCT TCC TCC ACC ACC CGC GCA 1536 Thr Ser Gly Pro Val Ser Ser Leu Pro Ala Ser Ser Thr Thr Arg Ala 500 505 510 TCC TCG CCT CCT GTT TCT TCA ACT CGT GTC TCT TCT CCC CCT GTC TCT 1584 Ser Ser Pro Pro Val Ser Ser Thr Arg Val Ser Ser Pro Pro Val Ser 515 520 525 TCC CCT CCA GTT TCT CGC ACC TCT TCT CCC CCT CCC CCT CCG GCC AGC 1632 Ser Pro Pro Val Ser Arg Thr Ser Ser Pro Pro Pro Pro Pro Ala Ser 530 535 540 AGC ACG CCG CCA TCG GGT CAG GTT TGC GTT GCC GGC ACC GTT GCT GAC 1680 Ser Thr Pro Pro Ser Gly Gin Val Cys Val Ala Gly Thr Val Ala Asp 545 550 555 560 GGC GAG TCC GGC AAC TAC ATC GGC CTG TGC CAA TTC AGC TGC AAC TAC 1728 Gly Glu Ser Gly Asn Tyr He Gly Leu Cys Gin Phe Ser Cys Asn Tyr 565 570 575 GGT TAC TGT CCA CCG GGA CCG TGT AAG TGC ACC GCC TTT GGT GCT CCC 1776 Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly Ala Pro 580 585 590 ATC TCG CCA CCG GCA AGC AAT GGG CGC AAC GGC TGC CCT CTA CCG GGA 1824 He Ser Pro Pro Ala Ser Asn Gly Arg Asn Gly Cys Pro Leu Pro Gly 595 600 605 GAA GGC GAT GGT TAT CTG GGC CTG TGC AGT TTC AGT TGT AAC CAT AAT 1872 Glu Gly Asp Gly Tyr Leu Gly Leu Cys Ser Phe Ser Cys Asn His Asn 610 615 620 TAC TGC CCG CCA ACG GCA TGC CAA TAC TGT TAG 1905 Tyr Cys Pro Pro Thr Ala Cys Gin Tyr Cys * 625 630 635 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 635 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Ala Leu Ala Ala Ala 1 5 10 15 Ala Leu Ser Ser Leu Gly Ser Ala Ala Pro Ala Asn Val Ala He Arg 20 25 30 Ser Leu Glu Glu Arg Ala Ser Ser Ala Asp Arg Leu Val Phe Cys His 35 40 45 Phe Met He Gly He Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp 50 55 60 Asp Asp Met Gin Arg Ala Lys Ala Ala Gly He Asp Ala Phe Ala Leu 65 70 75 80 Asn He Gly Val Asp Gly Tyr Thr Asp Gin Gin Leu Gly Tyr Ala Tyr 85 90 95 Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe He Ser Phe Asp Phe 100 105 110 Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gin Lys He Ala 115 120 125 Gin Tyr Ala Ser Arg Pro Ala Gin Leu Tyr Val Asp Asn Arg Pro Phe 130 135 140 Ala Ser Ser Phe Ala Gly Asp Gly Leu Asp Val Asn Ala Leu Arg Ser 145 150 155 160 Ala Ala Gly Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro Gly Gin 165 170 175 Ser Ser Pro Ser Asn He Asp Gly Ala Leu Asn Trp Met Ala Trp Asp 180 185 190 Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gin Thr Val Thr Val 195 200 205 Ala Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys Pro Tyr 210 215 220 Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val 225 230 235 240 Ser Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Pro Leu He Tyr Asn 245 250 255 Arg Trp Gin Gin Val Leu Gin Gin Gly Phe Pro Met Val Glu He Val 260 265 270 Thr Trp Asn Asp Tyr Gly Glu Ser His Tyr Val Gly Pro Leu Lys Ser 275 280 285 Lys His Phe Asp Asp Gly Asn Ser Lys Trp Val Asn Asp Met Pro His 290 295 300 Asp Gly Phe Leu Asp Leu Ser Lys Pro Phe He Ala Ala Tyr Lys Asn 305 310 315 320 Arg Asp Thr Asp He Ser Lys Tyr Val Gin Asn Glu Gin Leu Val Tyr 325 330 335 Trp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala Thr Asp Thr 340 345 350 Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe Met Gly 355 360 365 Arg Pro Asp Gly Trp Gin Thr Met Asp Asp Thr Val Tyr Val Ala Ala 370 375 380 Leu Leu Lys Thr Ala Gly Ser Val Thr Val Thr Ser Gly Gly Thr Thr 385 390 395 400 Gin Thr Phe Gin Ala Asn Ala Gly Ala Asn Leu Phe Gin He Pro Ala 405 410 415 Ser He Gly Gin Gin Lys Phe Ala Leu Thr Arg Asn Gly Gin Thr Val 420 425 430 Phe Ser Gly Thr Ser Leu Met Asp He Thr Asn Val Cys Ser Cys Gly 435 440 445 He Tyr Asn Phe Asn Pro Tyr Val Gly Thr He Pro Ala Gly Phe Asp 450 455 460 Asp Pro Leu Gin Ala Asp Gly Leu Phe Ser Leu Thr He Gly Leu His 465 470 475 480 Val Thr Thr Cys Gin Ala Lys Pro Ser Leu Gly Thr Asn Pro Pro Val 485 490 495 Thr Ser Gly Pro Val Ser Ser Leu Pro Ala Ser Ser Thr Thr Arg Ala 500 505 510 Ser Ser Pro Pro Val Ser Ser Thr Arg Val Ser Ser Pro Pro Val Ser 515 520 525 Ser Pro Pro Val Ser Arg Thr Ser Ser Pro Pro Pro Pro Pro Ala Ser 530 535 540 Ser Thr Pro Pro Ser Gly Gin Val Cys Val Ala Gly Thr Val Ala Asp 545 550 555 560 Gly Glu Ser Gly Asn Tyr He Gly Leu Cys Gin Phe Ser Cys Asn Tyr 565 570 575 Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly Ala Pro 580 585 590 He Ser Pro Pro Ala Ser Asn Gly Arg Asn Gly Cys Pro Leu Pro Gly 595 600 605 Glu Gly Asp Gly Tyr Leu Gly Leu Cys Ser Phe Ser Cys Asn His Asn 610 615 620 Tyr Cys Pro Pro Thr Ala Cys Gin Tyr Cys * 625 630 635 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer 1" CAGCGTCCAC ATCACGAGC 19 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer 2" GAAGAAGCAC GTTTCTGCAG AGACCG 26 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer 3" CGGTCTCTCG AGAAACGTGC TTCTTC 26 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer 4" GCCACTTCCG TTATTAGCC 19 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer 5" GGGGGGATCC ACCATGAG 18 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer 6" ACGGTCAGCA GAAGAAGCTC GACGAATAGG ACTGGC 36 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer 7" GCCAGTCCTA TTCGTCGAGC TTCTTCTGCT GACCGT 36 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE : other nucleic acid (A) DESCRIPTION: /desc = "Primer 8" CCACGGTCAC CAACAATAC 19 ( 2 ) INFORMATION FOR SEQ ID NO : 11 : ( i ) SEQUENCE CHARACTERISTICS : ( A ) LENGTH : 6032 base pairs ( B ) TYPE : nucleic acid ( C ) STRANDEDNESS : single ( D ) TOPOLOGY : l inear ( ii ) MOLECULE TYPE : DNA ( genomic ) ( vi ) ORIGINAL SOURCE : ( B ) STRAIN : Trichoderma harzianum CBS 243 . 71 ( ix ) FEATURE : (A) NAME/KEY: CDS (B) LOCATION: 3188..5092 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA ATAATGGTTT 60 CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT 120 TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT 180 AATATTGAAA AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT 240 TTGCGGCATT TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG 300 CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA 360 TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC 420 TATGTGGCGC GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC 480 ACTATTCTCA GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG 540 GCATGACAGT AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA 600 ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG 660 GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG 720 ACGAGCGTGA CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG 780 GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG 840 TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG 900 GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT 960 CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC 1020 AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT 1080 CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA 1140 TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT 1200 CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT 1260 GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC 1320 TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTCC 1380 TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC 1440 TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG 1500 GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT 1560 CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG 1620 AGCATTGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG 1680 GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT 1740 ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG 1800 GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT 1860 GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA 1920 TTACCGCCTT TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT 1980 CAGTGAGCGA GGAAGCGGAA GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC 2040 CGATTCATTA ATGCAGCCTG ATTAATGATT ACATACGCCT CCGGGTAGTA GACCGAGCAG 2100 CCGAGCCAGT TCAGCGCCTA AAACGCCTTA TACAATTAAG CAGTTAAAGA AGTTAGAATC 2160 TACGCTTAAA AAGCTACTTA AAAATCGATC TCGCAGTCCC GATTCGCCTA TCAAAACCAG 2220 TTTAAATCAA CTGATTAAAG GTGCCGAACG AGCTATAAAT GATATAACAA TATTAAAGCA 2280 TTAATTAGAG CAATATCAGG CCGCGCACGA AAGGCAACTT AAAAAGCGAA AGCGCTCTAC 2340 TAAACAGATT ACTTTTGAAA AAGGCACATC AGTATTTAAA GCCCGAATCC TTATTAAGCG 2400 CCGAAATCAG GCAGATAAAG CCATACAGGC AGATAGACCT CTACCTATTA AATCGGCTTC 2460 TAGGCGCGCT CCATCTAAAT GTTCTGGCTG TGGTGTACAG GGGCATAAAA TTACGCACTA 2520 CCCGAATCGA TAGAACTACT CATTTTTATA TAGAAGTCAG AATTCATAGT GTTTTGATCA 2580 TTTTAAATTT TTATATGGCG GGTGGTGGGC AACTCGCTTG CGCGGGCAAC TCGCTTACCG 2640 ATTACGTTAG GGCTGATATT TACGTGAAAA TCGTCAAGGG ATGCAAGACC AAAGTAGTAA 2700 AACCCCGGAA GTCAACAGCA TCCAAGCCCA AGTCCTTCAC GGAGAAACCC CAGCGTCCAC 2760 ATCACGAGCG AAGGACCACC TCTAGGCATC GGACGCACCA TCCAATTAGA AGCAGCAAAG 2820 CGAAACAGCC CAAGAAAAAG GTCGGCCCGT CGGCCTTTTC TGCAACGCTG ATCACGGGCA 2880 GCGATCCAAC CAACACCCTC CAGAGTGACT AGGGGCGGAA ATTTAAAGGG ATTAATTTCC 2940 ACTCAACCAC AAATCACAGT CGTCCCCGGT ATTGTCCTGC AGAATGCAAT TTAAACTCTT 3000 CTGCGAATCG CTTGGATTCC CCGCCCCTAG TCGTAGAGCT TAAAGTATGT CCCTTGTCGA 3060 TGCGATGTAT CACAACATAT AAATACTAGC AAGGGATGCC ATGCTTGGAG TTTCCAACTC 3120 AATTTACCTC TATCCACACT TCTCTTCCTT CCTCAATCCT CTATATACAC AACTGGGGAT 3180 CCTCACA ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GGC GCC CTT GCT 3229 Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Ala Leu Ala 1 5 10 GCC GCA GCT CTG TCT TCT CTC GGC AGT GCC GCT CCC GCC AAT GTT GCT 3277 Ala Ala Ala Leu Ser Ser Leu Gly Ser Ala Ala Pro Ala Asn Val Ala 15 20 25 30 ATT CGG TCT CTC GAG GAA CGT GCT TCT TCT GCT GAC CGT CTC GTA TTC 3325 lie Arg Ser Leu Glu Glu Arg Ala Ser Ser Ala Asp Arg Leu Val Phe 35 40 45 TGT CAC TTC ATG ATT GGT ATT GTT GGT GAC CGT GGC AGC TCA GCA GAC 3373 Cys His Phe Met lie Gly lie Val Gly Asp Arg Gly Ser Ser Ala Asp 50 55 60 TAT GAT GAT GAC ATG CAA CGT GCC AAA GCC GCT GGC ATT GAC GCA TTC 3421 Tyr Asp Asp Asp Met Gin Arg Ala Lys Ala Ala Gly lie Asp Ala Phe 65 70 75 GCT CTG AAC ATC GGC GTT GAC GGC TAT ACC GAC CAG CAA CTC GGG TAT 3469 Ala Leu Asn lie Gly Val Asp Gly Tyr Thr Asp Gin Gin Leu Gly Tyr 80 85 90 GCC TAT GAC TCT GCC GAC CGT AAT GGC ATG AAA GTC TTC ATT TCA TTC 3517 Ala Tyr Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe lie Ser Phe 95 100 105 110 GAT TTC AAC TGG TGG AGC CCC GGT AAT GCA GTT GGT GTT GGC CAG AAG 3565 Asp Phe Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gin Lys 115 120 125 ATT GCG CAG TAT GCC AGC CGT CCC GCC CAG CTG TAT GTT GAC AAC CGG 3613 lie Ala Gin Tyr Ala Ser Arg Pro Ala Gin Leu Tyr Val Asp Asn Arg 130 135 140 CCA TTC GCC TCT TCC TTC GCT GGT GAC GGT TTG GAT GTA AAT GCG TTG 3661 Pro Phe Ala Ser Ser Phe Ala Gly Asp Gly Leu Asp Val Asn Ala Leu 145 150 155 CGC TCT GCT GCA GGC TCC AAC GTT TAC TTT GTG CCC AAC TTC CAC CCT 3709 Arg Ser Ala Ala Gly Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro 160 165 170 GGT CAA TCT TCC CCC TCC AAC ATT GAT GGC GCC CTC AAC TGG ATG GCC 3757 Gly Gin Ser Ser Pro Ser Asn lie Asp Gly Ala Leu Asn Trp Met Ala 175 180 185 190 TGG GAT AAT GAT GGA AAC AAC AAG GCA CCC AAG CCG GGC CAG ACT GTC 3805 Trp Asp Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gin Thr Val 195 200 205 ACG GTG GCA GAC GGT GAC AAC GCT TAC AAG AAT TGG TTG GGT GGC AAG 3853 Thr Val Ala Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys 210 215 220 CCT TAC CTA GCG CCT GTC TCC CCT TGG TTT TTC ACC CAT TTT GGC CCT 3901 Pro Tyr Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro 225 230 235 GAA GTT TCA TAT TCC AAG AAC TGG GTC TTC CCA GGT GGT CCT CTG ATC 3949 Glu Val Ser Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Pro Leu lie 240 245 250 TAT AAC CGG TGG CAA CAG GTC TTG CAG CAG GGC TTC CCC ATG GTT GAG 3997 Tyr Asn Arg Trp Gin Gin Val Leu Gin Gin Gly Phe Pro Met Val Glu 255 260 265 270 ATT GTT ACC TGG AAT GAC TAC GGC GAG TCT CAC TAC GTC GGT CCT CTG 4045 lie Val Thr Trp Asn Asp Tyr Gly Glu Ser His Tyr Val Gly Pro Leu 275 280 285 AAG TCT AAG CAT TTC GAT GAT GGC AAC TCC AAA TGG GTC AAT GAT ATG 4093 Lys Ser Lys His Phe Asp Asp Gly Asn Ser Lys Trp Val Asn Asp Met 290 295 300 CCC CAT GAT GGA TTC TTG GAT CTT TCA AAG CCG TTT ATT GCT GCA TAT 4141 Pro His Asp Gly Phe Leu Asp Leu Ser Lys Pro Phe lie Ala Ala Tyr 305 310 315 AAG AAC AGG GAT ACT GAT ATA TCT AAG TAT GTT CAA AAT GAG CAG CTT 4189 Lys Asn Arg Asp Thr Asp lie Ser Lys Tyr Val Gin Asn Glu Gin Leu 320 325 330 GTT TAC TGG TAC CGC CGC AAC TTG AAG GCA TTG GAC TGC GAC GCC ACC 4237 Val Tyr Trp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala Thr 335 340 345 350 GAC ACC ACC TCT AAC CGC CCG GCT AAT AAC GGA AGT GGC AAT TAC TTT 4285 Asp Thr Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe 355 360 365 ATG GGA CGC CCT GAT GGT TGG CAA ACT ATG GAT GAT ACC GTT TAT GTT 4333 Met Gly Arg Pro Asp Gly Trp Gin Thr Met Asp Asp Thr Val Tyr Val 370 375 380 GCC GCA CTT CTC AAG ACC GCC GGT AGC GTC ACG GTC ACG TCT GGC GGC 4381 Ala Ala Leu Leu Lys Thr Ala Gly Ser Val Thr Val Thr Ser Gly Gly 385 390 395 ACC ACT CAA ACG TTC CAG GCC AAC GCC GGA GCC AAC CTC TTC CAA ATC 4429 Thr Thr Gin Thr Phe Gin Ala Asn Ala Gly Ala Asn Leu Phe Gin lie 400 405 410 CCT GCC AGC ATC GGC CAG CAA AAG TTT GCT CTA ACT CGC AAC GGT CAG 4477 Pro Ala Ser lie Gly Gin Gin Lys Phe Ala Leu Thr Arg Asn Gly Gin 415 420 425 430 ACC GTC TTT AGC GGA ACC TCA TTG ATG GAT ATC ACC AAC GTT TGC TCT 4525 Thr Val Phe Ser Gly Thr Ser Leu Met Asp lie Thr Asn Val Cys Ser 435 440 445 TGC GGT ATC TAC AAT TTC AAC CCA TAT GTT GGC ACC ATT CCT GCC GGC 4573 Cys Gly lie Tyr Asn Phe Asn Pro Tyr Val Gly Thr lie Pro Ala Gly 450 455 460 TTT GAC GAC CCT CTT CAG GCT GAC GGT CTT TTC TCT TTG ACC ATC GGA 4621 Phe Asp Asp Pro Leu Gin Ala Asp Gly Leu Phe Ser Leu Thr lie Gly 465 470 475 TTG CAT GTC ACG ACT TGT CAG GCC AAG CCA TCT CTT GGA ACC AAC CCT 4669 Leu His Val Thr Thr Cys Gin Ala Lys Pro Ser Leu Gly Thr Asn Pro 480 485 490 CCT GTC ACT TCT GGC CCT GTG TCC TCG CTG CCA GCT TCC TCC ACC ACC 4717 Pro Val Thr Ser Gly Pro Val Ser Ser Leu Pro Ala Ser Ser Thr Thr 495 500 505 510 CGC GCA TCC TCG CCT CCT GTT TCT TCA ACT CGT GTC TCT TCT CCC CCT 4765 Arg Ala Ser Ser Pro Pro Val Ser Ser Thr Arg Val Ser Ser Pro Pro 515 520 525 GTC TCT TCC CCT CCA GTT TCT CGC ACC TCT TCT CCC CCT CCC CCT CCG 4813 Val Ser Ser Pro Pro Val Ser Arg Thr Ser Ser Pro Pro Pro Pro Pro 530 535 540 GCC AGC AGC ACG CCG CCA TCG GGT CAG GTT TGC GTT GCC GGC ACC GTT 4861 Ala Ser Ser Thr Pro Pro Ser Gly Gin Val Cys Val Ala Gly Thr Val 545 550 555 GCT GAC GGC GAG TCC GGC AAC TAC ATC GGC CTG TGC CAA TTC AGC TGC 4909 Ala Asp Gly Glu Ser Gly Asn Tyr lie Gly Leu Cys Gin Phe Ser Cys 560 565 570 AAC TAC GGT TAC TGT CCA CCG GGA CCG TGT AAG TGC ACC GCC TTT GGT 4957 Asn Tyr Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly 575 580 585 590 GCT CCC ATC TCG CCA CCG GCA AGC AAT GGG CGC AAC GGC TGC CCT CTA 5005 Ala Pro lie Ser Pro Pro Ala Ser Asn Gly Arg Asn Gly Cys Pro Leu 595 600 605 CCG GGA GAA GGC GAT GGT TAT CTG GGC CTG TGC AGT TTC AGT TGT AAC 5053 Pro Gly Glu Gly Asp Gly Tyr Leu Gly Leu Cys Ser Phe Ser Cys Asn 610 615 620 CAT AAT TAC TGC CCG CCA ACG GCA TGC CAA TAC TGT TAG TCTAGAGGGT 5102 His Asn Tyr Cys Pro Pro Thr Ala Cys Gin Tyr Cys * 625 630 635 GACTGACACC TGGCGGTAGA CAATCAATCC ATTTCGCTAT AGTTAAAGGA TGGGGATGAG 5162 GGCAATTGGT TATATGATCA TGTATGTAGT GGGTGTGCAT AATAGTAGTG AAATGGAAGC 5222 CAAGTCATGT GATTGTAATC GACCGACGGA ATTGAGGATA TCCGGAAATA CAGACACCGT 5282 GAAAGCCATG GTCTTTCCTT CGTGTAGAAG ACCAGACAGA CAGTCCCTGA TTTACCCTGC 5342 ACAAAGCACT AGAAAATTAG CATTCCATCC TTCTCTGCTT GCTCTGCTGA TATCACTGTC 5402 ATTCAATGCA TAGCCATGAG CTCATCTTAG ATCCAAGCAC GTAATTCCAT AGCCGAGGTC 5462 CACAGTGGAG CAGCAACATT CCCCATCATT GCTTTCCCCA GGGGCCTCCC AACGACTAAA 5522 TCAAGAGTAT ATCTCTACCG TCCAATAGAT CGTCTTCGCT TCAAAATCTT TGACAATTCC 5582 AAGAGGGTCC CCATCCATCA AACCCAGTTC AATAATAGCC GAGATGCATG GTGGAGTCAA 5642 TTAGGCAGTA TTGCTGGAAT GTCGGGGCCA GTTGGCCGGG TGGTCATTGG CCGCCTGTGA 5702 TGCCATCTGC CACTAAATCC GATCATTGAT CCACCGCCCA CGAGGGCGTC TTTGCTTTTT 5762 GCGCGGCGTC CAGGTTCAAC TCTCTCCTCT AGCGCCTGAT GCGGTATTTT CTCCTTACGC 5822 ATCTGTGCGG TATTTCACAC CGCATATGGT GCACTCTCAG TACAATCTGC TCTGATGCCG 5882 CATAGTTAAG CCAGCCCCGA CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC 5942 TGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA 6002 GGTTTTCACC GTCATCACCG AAACGCGCGA 6032

Claims: PATENT CLAIMS 1. A method for constructing an expression vector comprising a mutanase gene obtained from a filamentous fungus suitable for heterologous production comprising the steps of: a) isolating a DNA sequence encoding a mutanase from a filamentous fungus, b) introducing a kex2 site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase, or replacing the mutanase (pre) pro-sequence with a (pre) pro-sequence comprising a kex2 or kex2-like site of another fungal enzyme, c) cloning the DNA sequence obtained in step b) into a suitable expression vector. 2. The method according to claim 1, wherein the mutanase is obtained from the genus Trichoderma , preferably a strain of the species T. harzianum , especially the strain T. harzianum CBS 243.71. 3. The method according to claim 2, in which the mutanase DNA sequence is isolated from or produced on the basis of a nucleic acid library of Trichoderma harzianum CBS 243.71. 4. The method according to any of claims 1 to 3 , wherein the mutanase (pre) pro-sequence is replaced by the Lipolase® (pre) pro-sequence or the TAKA-amylase (pre) pro-sequence. 5. An expression vector comprising a mutanase gene and a DNA sequence encoding a pro-peptide with a kex2 site or kex2-like site between the DNA sequences encoding said pro-peptide and the mature region of the mutanase. 6. The expression vector according to claim 5, further comprising an operably linked promoter sequence and/or a prepro- sequence. 7. The expression vector according to claims 5 and 6, wherein the prepro-sequence comprise the original mutanase signal sequence, or the Lipolase® signal-sequence, or the TAKA pro- sequence and the original mutanase pro-sequence with a kex2 or 5 kx2-like site, or the Lipolase® pro-sequence, or the TAKA pro- sequence. 8. The expression vector according to claim 7, wherein the promoter is the TAKA promoter or TAKA.TPI promoter. 10 9. The expression vector according to any claims 5 to 8, being the vector pMT1796. 10. A filamentous host cell for production of recombinant 15 mutanase derived from a filamentous fungus being from the genus Trichoderma , such as a strain of T. harzianum , or the genus Aspergillus , such as a strain of A . oryzae or A . niger, or a strain of the genus Fusarium, such as a strain of Fusarium oxy- sporium , Fusarium graminearum, Fusarium sulphureum, Fusarium 20 cerealis . 11. The host cell according to claim 10 wherein the host cell is a protease deficient of protease minus strain. 25 12. The host cell according to claim 11, wherein the host cell is the protease deficient strain Aspergillus oryzae JaL125 having the alkaline protease gene named "alp" deleted. 13. A process for producing a recombinant mutanase in a host 30 cell, comprising the steps: a) transforming an expression vector comprising a mutanase gene with a kex2 site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase into a suitable filamentous fungus host cell, 35 b) cultivating the host cell in a suitable culture medium under conditions permitting expression and secretion of an active mutanase, c) recovering and optionally purifying the secreted active recombinant mutanase from the culture medium. 14. The process according to claim 13 wherein the recombinant 5 expression vector is prepared according to the method of claim 1 to 4. 15. The process according to claim 13 and 14, wherein the filamentous host is a host cell according to any of claims 7 to 10 9. 16. An isolated recombinant mutanase produced according to the process according to any of claims 13 to 15. 15 17. A substantially pure wild-type mutanase obtained from Trichoderma harzianum CBS 243.71 essentially free of any contaminants . 18. A composition comprising a recombinant mutanase according 20 to claim 16 or a substantially pure wild-type mutanase according to claim 17 and further other ingredients conventionally used in food, feed and/or pet food products. 19. An oral care composition comprising a recombinant mutanase 25 according to claim 16 or a substantially pure wild-type mutanase according to claim 17, further comprising an enzyme selected from the group of dextranases, oxidases, peroxidases, haloperoxidases , laccases, proteases, endoglucosidases. Upases, amylases, and mixtures thereof. 30 20. An oral care product comprising a recombinant mutanase according to claim 16 or a substantially purified mutanase according to claim 17 or an oral care composition according to claim 19 and further comprising ingredients conventionally used 35 in oral care products. 21. The oral care product according to claim 20, being a dentifrice, such as a toothpaste, tooth powder or a mouth wash. 22. Use of the recombinant mutanase according to claim 16 or the 5 substantially purified mutanase according to claim 17 or an oral care composition of claim 19 or oral care product according to claims 20 and 21 for preventing the formation of dental plaque or removing dental plaque. 10 23. The use of the recombinant mutanase according to claims 16 or the substantially purified mutanase according to claim 17 or a oral care composition of claim 19 or oral care product according to claims 18 and 20 in oral care products for humans and/or animals. 15 24. Use of the composition according to claim 18, in food, feed and/or pet food products.

Inventors: FUGLSANG CLAUS CRONE, HALKIER TORBEN, HANSEN MOGENS TRIER, JOHANSEN CHARLOTTE, WAHLEITHNER JILL ANGELA, FUGLSANG, CLAUS, CRONE, HALKIER, TORBEN, HANSEN, MOGENS, TRIER, JOHANSEN, CHARLOTTE, WAHLEITHNER, JILL, ANGELA
Applicants: FUGLSANG CLAUS CRONE, HALKIER TORBEN, HANSEN MOGENS TRIER, JOHANSEN CHARLOTTE, NOVONORDISK AS, WAHLEITHNER JILL ANGELA, FUGLSANG, CLAUS, CRONE, HALKIER, TORBEN, HANSEN, MOGENS, TRIER, JOHANSEN, CHARLOTTE, NOVO NORDISK A/S, WAHLEITHNER, JILL, ANGELA
Application year: 1997
Main classification: C12N9
IPC codes: A23K1, A23L1, A61K8, A61K38, A61Q11, C12N1, C12N9, C12N15, C12R1