WO1998000528A1 - A recombinant enzyme with mutanase activity - Google Patents

A recombinant enzyme with mutanase activity Download PDF

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Publication number
WO1998000528A1
WO1998000528A1 PCT/DK1997/000283 DK9700283W WO9800528A1 WO 1998000528 A1 WO1998000528 A1 WO 1998000528A1 DK 9700283 W DK9700283 W DK 9700283W WO 9800528 A1 WO9800528 A1 WO 9800528A1
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WO
WIPO (PCT)
Prior art keywords
mutanase
pro
gly
ser
ala
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PCT/DK1997/000283
Other languages
French (fr)
Inventor
Jill Angela Wahleithner
Claus Crone Fuglsang
Torben Halkier
Charlotte Johansen
Mogens Trier Hansen
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Novo Nordisk A/S
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Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to JP10503763A priority Critical patent/JP2000514288A/en
Priority to EP97928131A priority patent/EP0954570A1/en
Priority to AU32544/97A priority patent/AU721693B2/en
Publication of WO1998000528A1 publication Critical patent/WO1998000528A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01084—Glucan 1,3-alpha-glucosidase (3.2.1.84), i.e. mutanase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • 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.
  • compositions useful in oral care products for humans and animals are also the object of the invention to provide compositions useful in oral care products for humans and animals.
  • Mutanases are ⁇ -1, 3-glucanases (also known as ⁇ -1,3- glucanohydrolases) which degrade the ⁇ -l,3-glycosidic linkages in mutan. Mutanases have been described from two species of Trichoderma (Hasegawa et al. , (1969), Journal of Biological Chemistry
  • FIG. 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 ⁇ . 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.
  • 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 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
  • the mutanase is obtained from a strain within the genus Trichoderma .
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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 Streptomyce ⁇ , Cladosporium or Aspergillus .
  • 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 Streptomyce ⁇ , Cladosporium or Aspergillus .
  • 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.
  • 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-peptide 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).
  • 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.
  • other amino acid combinations containing at least one basic amino acid, have been found to be processed.
  • Kex2 sites see e . g. Methods in Enzymology Vol 185, ed. D. Goeddel, Academic Press Inc. (1990) , San Diego, CA, "Gene 5 Expression Technology"
  • 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
  • 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.
  • the invention relates to a method for construction of an expression vector comprising a mutanase gene suitable for heterologous production comprising the steps 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA sequence encoding a mutanase may be isolated by a general method involving
  • the invention in another aspect 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.
  • 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.
  • the expression vector is the pMT1796 used to illustrate the concept of the invention in Example 3 below.
  • vector will often depend on the host cell into which it is to be introduced.
  • 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.
  • 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.
  • 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.
  • 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 .
  • a strain of the genus Fu ⁇ arium 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.
  • 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.
  • 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.
  • Aspergillus as a host microorganism is described in EP 238 023 (Novo Nordisk A/S) , the contents of which are hereby incorporated by reference.
  • 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.
  • 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
  • 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 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, 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.
  • 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.
  • oral care products 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.
  • oral care products do also encompass products for cleaning dentures, artificial teeth and the like.
  • 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.
  • Abrasive polishing material might also be incorporated into the dentifrice product of 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.
  • alumina and hydrates thereof such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calcined aluminum
  • 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.
  • the abrasive product may be present in from 0 to 70% by weight, preferably from 1% to 70%.
  • 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 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 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.
  • Suitable sweeteners include saccharin.
  • Flavours such as spearmint
  • Whitening/bleaching agents 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 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.
  • 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.
  • 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.
  • 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:
  • the oral care product is toothpaste having a pH in the range from 6.0 to about
  • 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 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) .
  • the oral care composition and products of the present invention can be made using methods which are common in the oral product area.
  • the recombinant mutanase and/or the substantially purified mutanase free of active contaminants can be use in food, feed and/or pet food products.
  • 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.
  • Plasmids and Vectors Plasmids and Vectors: pMT1796 ( Figure 1 and 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
  • 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.
  • YPM 2% maltose, 1% bactopeptone and 0.5% yeast extract
  • DAPI 4 • , 6-diamidino-2-phenylindole (Sigma D-9542) Britton-Robinson Buffer
  • Phenyl-sepharose FF (high sub) column (Pharmacia)
  • 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
  • sucrose is added to a final concentration of
  • the insoluble mutan is lyophilized and ground.
  • MU Mutanase Unit
  • 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
  • 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 .
  • 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.
  • 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.
  • 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.
  • HA Hydroxyapatite disks
  • 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.
  • 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%.
  • Biosystems 473A protein sequencer To generate peptides reduced and S-carboxymethylated mutanase
  • Vydac Ci ⁇ 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
  • amino acid sequences determined are given below.
  • 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.
  • pHD4l4+mut 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
  • 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 -Sc ⁇ RI/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.
  • 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).
  • nucleotide numbers refer to the pMT1802 plasmid (See SEQ ID No. 11) .
  • 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 strain A . oryzae JaL125 was transformed using a PEG- mediated protocol (see EP 238 023) and a DNA mixture containing 0.5 ⁇ g of a plasmid encoding the gene that confers resistance to the herbicide Basta and 8.0 ⁇ g 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.
  • 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.
  • 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)
  • 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) .
  • the mass spectrometry, performed as described above, of the mutanase revealed an average mass around 75 kDa.
  • the glycosylation of the mutanase is heterogeneous.
  • the peptide mass of the mutanase is more than
  • 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), Actinomyce ⁇ 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) .
  • 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) .
  • 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 reduced the amount of adhering cells.
  • the activity was observed as a removal of plaque and not as a bactericidal activity against cells in plaque.
  • MOLECULE TYPE DNA (genomic)
  • ORIGINAL SOURCE
  • 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 GAGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC 2040 CGATTCATTA ATGC
  • GCT CCC ATC TCG CCA CCG GCA AGC AAT GGG CGC AAC GGC TGC CCT CTA 5005

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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 H2O2 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 31 (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 (NH4)2S04 20 g/litre K2P04 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 K3Fe(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 Na2S (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 NH4HCO3 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 K3Fe(CN)6, 20 g/1 Na2C03) 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 K3Fe(CN)6, 20 g/1 Na2C03) 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.
PCT/DK1997/000283 1996-06-28 1997-06-30 A recombinant enzyme with mutanase activity WO1998000528A1 (en)

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WO2001049255A2 (en) * 1999-12-30 2001-07-12 University Of Louisville Research Foundation, Inc. Methods and compositions for inhibiting adhesion by microorganisms
US6264925B1 (en) * 1996-10-11 2001-07-24 Novozymes A/S Cellulose binding domains (CBDs) for oral care products
ES2163988A1 (en) * 1999-07-31 2002-02-01 Newbiotechnic Sa Antifungic compositions and method for controlling fungi
AU2003211620B8 (en) * 2002-03-01 2003-10-08 Amano Enzyme Inc. Modified promoter
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US9242879B2 (en) 2012-03-30 2016-01-26 Ecolab Usa Inc. Use of peracetic acid/hydrogen peroxide and peroxide-reducing agents for treatment of drilling fluids, frac fluids, flowback water and disposal water
US9253978B2 (en) 2008-03-28 2016-02-09 Ecolab USA, Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
US9288992B2 (en) 2013-03-05 2016-03-22 Ecolab USA, Inc. Efficient stabilizer in controlling self accelerated decomposition temperature of peroxycarboxylic acid compositions with mineral acids
US9290448B2 (en) 2008-03-28 2016-03-22 Ecolab USA, Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
US9540598B2 (en) 2008-03-28 2017-01-10 Ecolab Usa Inc. Detergents capable of cleaning, bleaching, sanitizing and/or disinfecting textiles including sulfoperoxycarboxylic acids
US9585397B2 (en) 2013-03-05 2017-03-07 Ecolab Usa Inc. Peroxycarboxylic acid compositions suitable for inline optical or conductivity monitoring
US9902627B2 (en) 2011-12-20 2018-02-27 Ecolab Usa Inc. Stable percarboxylic acid compositions and uses thereof
US10165774B2 (en) 2013-03-05 2019-01-01 Ecolab Usa Inc. Defoamer useful in a peracid composition with anionic surfactants
CN113151330A (en) * 2021-03-30 2021-07-23 云南师范大学 Acid protease mutant and preparation method and application thereof
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US6254856B1 (en) * 1996-04-16 2001-07-03 Novo Nordisk, A/S Compositions for the removal of dental plaque
US6264925B1 (en) * 1996-10-11 2001-07-24 Novozymes A/S Cellulose binding domains (CBDs) for oral care products
ES2163988A1 (en) * 1999-07-31 2002-02-01 Newbiotechnic Sa Antifungic compositions and method for controlling fungi
WO2001049255A2 (en) * 1999-12-30 2001-07-12 University Of Louisville Research Foundation, Inc. Methods and compositions for inhibiting adhesion by microorganisms
WO2001049255A3 (en) * 1999-12-30 2002-02-21 Univ Louisville Res Found Methods and compositions for inhibiting adhesion by microorganisms
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US8648183B2 (en) 2002-03-01 2014-02-11 Amano Enzyme Inc. Modified promoter
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AU2003211620B8 (en) * 2002-03-01 2003-10-08 Amano Enzyme Inc. Modified promoter
USRE47033E1 (en) 2003-08-04 2018-09-11 Bend Research, Inc. Pharmaceutical compositions of adsorbates of amorphous drugs and lipophilic microphase-forming materials
US9023393B2 (en) 2003-08-04 2015-05-05 Bend Research, Inc. Pharmaceutical compositions of adsorbates of amorphous drugs and lipophilic microphase-forming materials
US11015151B2 (en) 2008-03-28 2021-05-25 Ecolab Usa Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
US10669512B2 (en) 2008-03-28 2020-06-02 Ecolab Usa Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
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US10323218B2 (en) 2008-03-28 2019-06-18 Ecolab Usa Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
US9253978B2 (en) 2008-03-28 2016-02-09 Ecolab USA, Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
US10077415B2 (en) 2008-03-28 2018-09-18 Ecolab Usa Inc. Detergents capable of cleaning, bleaching, sanitizing and/or disinfecting textiles including sulfoperoxycarboxylic acids
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US8709386B2 (en) 2008-04-11 2014-04-29 Danisco Us Inc. Method of making and using an alpha-glucanase composition to reduce or remove biofilm
US9902627B2 (en) 2011-12-20 2018-02-27 Ecolab Usa Inc. Stable percarboxylic acid compositions and uses thereof
US10017403B2 (en) 2012-03-30 2018-07-10 Ecolab Usa Inc. Use of peracetic acid/hydrogen peroxide and peroxide-reducing enzymes for treatment of drilling fluids, frac fluids, flowback water and disposal water
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US9585397B2 (en) 2013-03-05 2017-03-07 Ecolab Usa Inc. Peroxycarboxylic acid compositions suitable for inline optical or conductivity monitoring
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US10893674B2 (en) 2013-03-05 2021-01-19 Ecolab Usa Inc. Efficient stabilizer in controlling self accelerated decomposition temperature of peroxycarboxylic acid compositions with mineral acids
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AU3254497A (en) 1998-01-21
CN1226282A (en) 1999-08-18

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