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AU721693B2 - A recombinant enzyme with mutanase activity - Google Patents

A recombinant enzyme with mutanase activity Download PDF

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AU721693B2
AU721693B2 AU32544/97A AU3254497A AU721693B2 AU 721693 B2 AU721693 B2 AU 721693B2 AU 32544/97 A AU32544/97 A AU 32544/97A AU 3254497 A AU3254497 A AU 3254497A AU 721693 B2 AU721693 B2 AU 721693B2
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mutanase
pro
ser
sequence
ala
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Claus Crone Fuglsang
Torben Halkier
Mogens Trier Hansen
Charlotte Johansen
Jill Angela Wahleithner
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Novozymes AS
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Novo Nordisk AS
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Nutrition Science (AREA)
  • Animal Husbandry (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Fodder In General (AREA)
  • Cosmetics (AREA)

Description

WO 98/00528 DrT/'KmnTt nAoI Title: A recombinant enzyme with mutanase activity FIELD OF THE INVENTION The present invention relates to a method for constructing an 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 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.
BACKGROUND OF THE INVENTION Mutanases are a-1,3-glucanases (also known as a-1,3glucanohydrolases) which degrade the a-1,3-glycosidic linkages in mutan. Mutanases have been described from two species of Trichoderma (Hasegawa et al., (1969), Journal of Biological Chemistry 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.
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 antiplaque agent in dental applications and personal care products, 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 removal using mutanase produced by Trichoderma harzianum CBS 243.71 to degrade mutan synthesized by cultivating Streptococcus 2 muilans 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 pMT1 815, Figure 3 shows an outline of the construction of the A. oryzac recombinant mutanase expression vector pMT 1802, 1i 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 7' harzianum CBS 243.71 mutanase at pH 7, Figure 6 shows the temperature stability of recombinant and wild-type T harzianun CBS 243.71 mutanase at pH 7, Figure 7 shows the indirect Malthus standard curve for a mix culture of S. nmuans, A.
viscosus and F. nuclealum grown in BHI at 37 0
C.
Summary of the Invention The object of the invention is to provide a recombinant mutanase derived from a 0. 2 fi'lamentous 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.
According to a first embodiment the invention provides a method for constructing an 5 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- M) 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.
I:\DAY LI B\libzz\ 15548.docsak 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 kex2like site between the DNA sequences encoding said (pre)pro-peptide and the mature region of the mutanasc.
According to a further embodiment the invention provides 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 in region of the mutanse.
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.
Accordingly in a further embodiment the invention provides a filamentous host cell is comprising a heterologous mutanase gene derived from a filamentous fungus being from the genus Trichoderma or the genus Aspergillus or the genus Fusarium.
In a further embodiment, the invention provides 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 21 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 recombinant mutanase 5 frliom 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.
o 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.
In a further embodiment the invention provides the use of the recombinant mutanase of the invention or the substantially purified mutanase of the invention or composition or I:\[)AYLIB\Iibzz\1 5548.docsak 4 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 li lamentous fungus by heterologous expression.
The present inventors have as the first been able to express the mutanase gene of a lilamentous fingus 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 il filamentous fungi, such as from filamentous fungi of the genus Trichodernma, such a strain of iTrichoderma harzianum, especially Trichoderna harzianuium CBS 243.71, and the genera 'Streplonyces, Cladosporium or Aspergillus.
Previously it has not been possible to produce mutanases of lilamentous fungi heterologously. Consequently, according to prior art mutanases are produced homnologously and comprise a mixture of other enzyme activities besides the mutanase with undesired contaminants).
An example of this is Trichodernia harzianuni CBS 243.71 which are known to prodLIuce a mutanase as also described above. The mutanase derived from Trichodernia hcrziaCnoin CBS 243.71 has before the successful findings of the present invention only been 2) 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 a we .e .e a a a I:\DAYI I1\Ibzz\1 5548.docsak WO 98/00528 PCT/DK9700283 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 GLA 6 amino acids) to relatively long peptides PEPA 49 amino acids).
The pro-peptide can perform a number of different functions.
Firstly, pro-peptides might contribute to the efficiency of cotranslational translocation of the protein across the ER-membrane. Secondly, pro-peptides might contribute to co-translational 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 dibasic cleavage is thought to occur in the Golgi, the internal di-basic peptide sequences in cytoplasmic proteins will not be attacked by this processing.
WO 98/00528 PCT/DK97/00283 6 Kex2 sites Kex2 sites (see e.g. Methods in Enzymology Vol 185, ed. D.
Goeddel, Academic Press Inc. (1990), San Diego, CA, "Gene Expression Technology") and kex2-like sites are di-basic recognition sites cleavage sites) found between the propeptide 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 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.
369-376, found that insertion of a kex2 site in the secretion signal of chymosin signal peptide and pro-peptide), which encoded the glucoamylase signal peptide and pro-peptide fused to prochymosin, did not increase the secretion level of recombinant chymosin expressed in a Aspergillus nidulans host 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) 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.
The term "homologous" production means expression of the wildtype 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: a) isolating a DNA sequence encoding a mutanase from a filamentous fungus known to produce a mutanase, WO 98/00528 PCT/DK97/00283 7 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 T. 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 TAKAamylase (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 -4 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 oligonucleotide 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.
WO 98/00528 PCT7DK97/00283 8 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.
is 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 WO 98/00528 PCT/DK97/00283 9 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 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 Trichoderma, 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 Fusarium, 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 roseum f. sp. cerealis), or Fusarium sulphureum (in 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 e.g. the strain deposited as Fusarium graminearum ATCC 20334. The strain ATCC WO 98/00528 PCT/DK97/00283 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.
WO 98/00528 PCT/DK97/00283 11 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 centrifugation 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 molecular 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 The temperature optimum of both the recombinant and wild-type mutanase was found to be around 45 0 C at pH 7 and around 550C at pH 5.5 (see Figure Further, the residual activity starts to decline at 4000C at pH 7, while the enzyme is more stable at pH 5.5, where the residual activity starts to decline at 550C.
The inventors have also provided a substantially pure wildtype 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 WO 98/00528 PCT/DK97/00283 12 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/ml 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 the acidic proteases described in WO 95/02044 (Novo Nordisk endoglucosidases, lipases, amylases, 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 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.
WO 98/00528 PCT/DK97/00283 13 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, humectant, 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 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.
WO 98/00528 PCT/DK97/00283 14 Humectants are in general present in from 0% to 80%, preferably 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, amphoteric 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 monoglycerides 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 WO 98/00528 PCT/DK97/00283 Whitening/bleaching agents Whitening/bleaching agents include H 2 0 2 and may be added in amounts less that preferably from 0.25 to 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 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 WO 98/00528 PCT/DK97/00283 16 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 Humectant 0 to Thickener 0.1 to Binder 0.01 to Sweetener 0.1% to Foaming agent 0 to Whitener 0 to Enzymes 0.0001% to In a specific embodiment of the invention the oral care product is toothpaste having a pH in the range from 6.0 to about 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.
WO 98/00528 PCT/DK97/00283 17 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 Enzymes 0-20% Ethanol 0-2% Other ingredients 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 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.
WO 98/00528 PCT/DK97/00283 18 Turner; Blackie Academic and Professional), using the A. oryzae pyrG gene as marker.
E. coli Plasmids and Vectors: pMT1796 (Figure 1 and Figure 2) pMT1802 (Figure 2) pMT1815 (Figure 2) pHD414: Aspergillus expression vector is a derivative of the plasmid p775 (described in EP 238.023). The construction of the pHD414 is further described in WO 93/11249. pHD414 contains the A. niger glucoamylase terminator and the A. oryzae TAKA amylase promoter.
pHD414+mut (Figure 3) pHan37 containing the TAKA:TPI promoter Linkers: Linker #1: GATCCTCACA ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GG GAGTGT TAC AAC CCG CAA CAG GCT GCA GAT CCG GAT CCG C Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Linker #2: C CAA TAC TGT TAG T GT ACG GTT ATG ACA ATC AGATC Ala Cys Gin Tyr Cys Primers: Primer 1: 5' GGGGGGATCCACCATGAG 3' (SEQ ID No. 3) Primer 2: 5' ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC 3' (SEQ ID No. 4) Primer 3: 5' GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT 3' (SEQ ID No. Primer 4: 5' CCACGGTCACCCAAAATAC 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. WO 98/00528 PCT/K97/00283 19 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: 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 fermenter 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 standard techniques (Sambrook, Fritsch, E. 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 0 C (kept constant), and with an aeration rate of rpm in a medium comprised of the following components: WO 98/00528 PCT/DK97/00283 NZ-Case 6.5 g/litre Yeast Extract 6 g/litre
(NH
4 )2SO 4 20 g/litre
K
2 P0 4 3 g/litre Glucose 50 g/litre Pluronic PE6100 0.1% After 35 hours, sucrose is added to a final concentration of 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 with acetic acid) and the solution is stirred overnight at 37 0 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 gmol reducing sugar (calculated as glucose) per minute. Reducing sugars were measured with alkaline
K
3 Fe(CN) 6 Standard Conditions mutan Reaction minutes 0
C
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 and the suspension is homogenised for 15 minutes in an Ultra Turrax T25 homogenizer at 4 0 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 WO 98/00528 PCT/DK97/00283 21 analyzed for mutanase activity is applied in sample wells punched in the agarose, and the plate is incubated overnight at 37 0
C,
whereafter clearing zones are formed around mutanase containing samples.
Western hybridization Western hybridizations are performed using the ECL western blotting system (Amersham International, plc, 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 (acyano-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 0
C
for 4 hours and finally hydrated with sterile deionized water.
Plaque coating of Hydroxyapatite disks (HA) Hydroxyapatite disks (HA) were dry sterilised (121 0 C, 2 bar, minutes) and coated with filter sterilised saliva for 18 hours at 37 0 C. The HA disks were placed in a sterile rack in a beaker, Brain Heart Infusion broth (BHI) containing 0.2% sucrose was poured into the beaker covering the disks. Sterile Na 2 S (pH was added immediately before inoculation given the final concen- WO 98/00528 PCT/DK97/00283 22 tration of 5 g/litre. A mixture 1:1:1 of Streptococcus mutans, Actinomyces viscosus and Fusobacterium nucleatum grown anaerobically (BHI, 37 0 C, 24 h) was used as inoculum in the concentration of approximately 106 cfu/ml. The disks were incubated anaerobic at 37 0 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 0
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 acetate, 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 kDa cut-off membrane and followed by a buffer change to 10 mM sodium acetate, pH 4.7. The concentrate was filtrated through a WO 98/00528 PCT/DK97/00283 23 Whatmann 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 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 performed 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 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 NH 4
HCO
3 for 16 hours at 37°C. The WO 98/00528 PCT/DK97/00283 24 resulting peptides were separated by reversed phase HPLC using a Vydac Ci1 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 1 i 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-PPh-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, pHD414+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, NarI and XhoI. From this digestion a 3499 nt (nucleotide) vector fragment and a 610 nt NarI/XhoI fragment were isolated. These two fragments were then ligated with linker #1 (see above) and a 618 nt EcoRI/BamHI fragment from pHan37 containing the TAKA:TPI promoter, giving plasmid pJW99.
HD414+mut was next digested with XhoI and SphI, and a 1790 nt WO 98/00528 PCT/DK97/00283 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 XbaI and XhoI. 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 XhoI/KpnI 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.
WO 98/00528 PCT/DK97/00283 26 Example 4 Expression of recombinant Mutanase in Aspergillus oryzae The strain A. oryzae JaL125 was transformed using a PEGmediated protocol (see EP 238 023) and a DNA mixture containing 0.5 pg of a plasmid encoding the gene that confers resistance to the herbicide Basta and 8.0 gg 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 maltose, 1% bactopeptone and 0.5% yeast extract) was inoculated with spores and grown for 5 days with shaking at 30 0 C. The supernatant was harvested after 5 days growth.
highest mutanase number of Construct level detected transformants tested pMT1802, mutanase <0.001 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 Purification of recombinant mutanase 700 ml fermentation broth was filtered and concentrated. The pH was adjusted to 4.7 (conductivity around 300 pS/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 WO 98/00528 PCT/DK97/00283 27 mutanase was eluted in a linear NaC1 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 uS/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 (16/60) column (Pharmacia) in sodium acetate pH 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 400C 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 gl filtrate in Eppendorf vials were added 750 il ferricyanide reagent (0.4 g/l
K
3 Fe(CN) 6 20 g/l Na 2
CO
3 and incubated 15 minutes at 85 0
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 WO 98/00528 PCT/DK97/00283 28 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 ml 0.4 M NaOH and the samples were filtered on Munktell filters. 100 gl filtrate in Eppendorf vials were added 750 l ferricyanide reagent (0.4 g/l K 3 Fe(CN) 6 20 g/l Na 2
CO
3 and incubated 15 minutes at 850C. 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 OC. The temperature optimum at pH 5.5 was above 550 (See Figure 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 OC at pH 7, while the enzyme is more stable at pH 5.5, where the residual activity starts to decline at 55 0 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 WO 98/00528 PCT/DK97/00283 29 to the enzyme.
Example 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), Actinomyces viscosus (DSM 43329) and Fusobacterium nucleatum subsp. polymorphum (DSM 20482).
HA disks with plaque were transferred to acetate buffer (pH 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 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 (200C). The DAPI stained cells were examined with the x 100 oil immersion fluorescence objective on an Olympus model 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.
WO 98/00528 PCT/DK97/00283 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 Loglo reduction Removal of No. of (MU/ml) (cfu/cm 2 plaque observations 0 0 0 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.
WO 98/00528 PCT/DK97/00283 SEQUENCE LISTING GENERAL INFORMATION:
APPLICANT:
NAME: Novo Nordisk A/S STREET: Novo Alle CITY: Bagsvaerd COUNTRY: Denmark POSTAL CODE (ZIP): DK-2880 TELEPHONE: +45 4444 8888 TELEFAX: +45 4449 3256 (ii) TITLE OF INVENTION: A recombinant enzyme with mutanase activity (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1905 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: STRAIN: Trichoderma harzianum CBS 243.71 (ix) FEATURE: NAME/KEY: CDS LOCATION:1..1905 NAME/KEY: sig peptide LOCATION:1..120 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ATG
Met 1 TTG GGC GTT Leu Gly Val GTC CGC CGT CTA GGC CTA GGC GCC CTT GCT GCC GCA Val Arg Arg Leu Gly Leu Gly Ala Leu Ala Ala Ala 5 10 GCT CTG TCT Ala Leu Ser TCT CTC GAG Ser Leu Glu CTC GGC AGT GCC GCT CCC GCC AAT GTT Leu Gly Ser Ala Ala Pro Ala Asn Val GCT ATT CGG Ala Ile Arg TTC TGT CAC Phe Cys His GAA CGT GCT TCT Glu Arg Ala Ser GCT GAC CGT CTC Ala Asp Arg Leu
GTA
Val TTC ATG Phe Met ATT GGT ATT GTT Ile Gly Ile Val GAC CGT GGC AGC Asp Arg Gly Ser
TCA
Ser GCA GAC TAT GAT Ala Asp Tyr Asp GAC ATG CAA CGT Asp Met Gln Arg GCC AAA GCC GCT GGC ATT GAC GCA TTC GCT CTG Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu 75 AAC ATC GGC Asn Ile Gly GAC TCT GCC Asp Ser Ala AAC TGG TGG Asn Trp Trp 115
GTT
Val GGC TAT ACC GAC Gly Tyr Thr Asp CAA CTC GGG TAT Gln Leu Gly Tyr GCC TAT Ala Tyr GAC CGT AAT GGC ATG AAA GTC TTC ATT TCA Asp Arg Asn Gly Met Lys Val Phe Ile Ser 100 105 TTC GAT TTC Phe Asp Phe 110 AAG ATT GCG Lys Ile Ala AGC CCC GGT AAT Ser Pro Gly Asn GTT GGT GTT GGC Val Gly Val Gly
CAG
Gln 125 WO 98/00528 WO 9800528PCT/DK97/00283 CAG TAT Gin Tyr 130 GCC AGC CGT CCC Ala Ser Arg Pro CAG CTG TAT GTT Gin Leu Tyr Val
GAC
Asp 140 AAC CGG CCA TTC Asn Arg Pro Phe
GCC
Ala 145 TCT TCC TTC GCT Ser Ser Phe Ala GAC GGT TTG GAT Asp Gly Leu Asp AAT GCG TTG CGC Asn Ala Leu Arg
TCT
Ser 160 GCT GCA GGC TCC Ala Ala Gly Ser
AAC
Asn 165 GTT TAC TTT GTG Val Tyr Phe Val AAC TTC CAC CCT Asn Phe His Pro GGT CAA Gly Gin 175 432 480 528 576 624 TCT TCC CCC Ser Ser Pro AAT GAT GGA Asn Asp Gly 195 AAC ATT GAT GGC Asn Ile Asp Gly CTC AAC TGG ATG Leu Asn Trp Met GCC TGG GAT Ala Trp Asp 190 GTC ACG GTG Val Thr Val AAC AAC AAG GCA Asn Asn Lys Ala AAG CCG GGC CAG Lys Pro Gly Gln
ACT
Thr 205 GCA GAC Ala Asp 210 GGT GAC AAC GCT Gly Asp Asn Ala AAG AAT TGG TTG Lys Asn Trp, Leu
GGT
Gly 220 GGC AAG CCT TAC Gly Lys Pro Tyr
CTA
Leu 225 GCG CCT GTC TCC Ala Pro Val Ser CCT TGG TTT TTC ACC CAT TTT GGC CCT GAA GTT Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val 230 235 240 TCA TAT TCC AAG Ser Tyr Ser Lys TGG GTC TTC CCA Trp Val Phe Pro GGT CCT CTG ATC Gly Pro Leu Ile TAT AAC Tyr Aen 255 CGG TGG CAA Arg Trp Gin GTC TTG CAG CAG Val Leu Gin Gin TTC CCC ATG GTT Phe Pro Met Val GAG ATT GTT Glu Ile Val 270 ACC TGG AAT GAC TAC GGC GAG TCT CAC TAC GTC GGT CCT CTG AAG TCT Thr Trp Asn Asp Tyr Gly Giu Ser His Tyr Val Giy Pro Leu Lys Ser 275 280 285 768 816 864 912 960 AAG CAT Lys His 290 TTC GAT GAT GGC Phe Asp Asp Gly TCC AAA TGG GTC Ser Lys Trp Val
AAT
Asn 300 GAT ATG CCC CAT Asp Met Pro His
GAT
Asp 305 GGA TTC TTG GAT Gly Phe Leu Asp TCA AAG CCG TTT Ser Lys Pro Phe OCT GCA TAT AAG Ala Ala Tyr Lys
AAC
Aen 320 AGG GAT ACT GAT ATA TCT AAG, TAT GTT CAA Arg Asp Thr Asp Ile Ser Lys Tyr Val Gin 325 330 MAT GAG CAG CTT Asn Oiu Gin Leu GTT TAC Val Tyr 335 TGG TAC CGC Trp Tyr Arg ACC TCT MAC Thr Ser Asn 355 MAC TTG MAG GCA Asn Leu Lys Ala GAC TGC GAC GCC Asp CyB Asp Ala ACC GAC ACC Thr Asp Thr 350 TTT ATG GGA Phe Met Gly CGC CCG GCT MAT Arg Pro Ala Asn GGA AGT GGC AAT Gly Ser Gly Asn
TAC
Tyr 365 1008 1056 1104 1152 1200 CGC CCT Arg Pro 370 GAT GOT TGO CAA Asp Gly Trp Gin ATG CAT GAT ACC Met Asp Asp Thr
OTT
Val 380 TAT OTT 0CC GCA Tyr Val Ala Ala CTT CTC MAG ACC GCC GOT AGC GTC ACG GTC ACG TOT GOC GOC ACC ACT Leu Leu Lye Thr Ala Gly Ser Val Thr Val Thr Ser Gly Gly Thr Thr WO 98/00528 PCTIDK97/00283 395 400 CAA ACG TTO CAG Gin Thr Phe Gin
GCC
Ala 405 AAC GCC GGA GCC Asn Ala Gly Ala CTC TTC CAA ATO Leu Phe Gin Ile CCT GCC Pro Ala 415 AGO ATC GGO Ser Ile Gly TTT AGC GGA Phe Ser Gly 435
CAG
Gin 420 CAA AAG TTT GCT Gin Lys Phe Ala
CTA
Leu 425 ACT CGC AAC GGT Thr Arg Asn Gly CAG ACC GTC Gin Thr Val 430 TCT TGC GGT Ser Cys Gly ACC TCA TTG Thr Ser Leu ATG GAT Met Asp 440 ATO ACC AAC GTT Ile Thr Asn Val
TGO
Cys 445 ATO TAO Ile Tyr 450 AAT TTC AAC CCA Asn Phe Asn Pro GTT GGC ACC ATT Val Gly Thr Ile
CCT
Pro 460 GCC GOC TTT GAO Ala Gly Phe Asp
GAO
Asp 465 OCT OTT CAG GCT Pro Leu Gin Ala GAO GOT OTT TTO TOT TTG ACC ATC GGA TTG OAT Asp Gly Leu Phe Ser Leu Thr Ile Gly Leu His 470 475 480 GTC ACG ACT TGT Val Thr Thr Cys
CAG
Gin 485 GCC AAG OCA TCT Ala Lys Pro Ser
OTT
Leu 490 GGA ACC AAC CCT Gly Thr Asn Pro CCT GTC Pro Val 495 ACT TOT GGC Thr Ser Gly TCC TOG CCT Ser Ser Pro 515
OCT
Pro 500 GTG TCC TOG CTG Val Ser Ser Leu
OCA
Pro 505 GCT TCC TCC ACC Ala Ser Ser Thr ACC CGC GCA Thr Arg Ala 510 CCT GTC TCT Pro Val Ser OCT GTT TOT TCA Pro Vai Ser Ser CGT GTC TOT TCT Arg Val Ser Ser
CCC
Pro 525 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 .1824 1872 1905 TCC CCT Ser Pro 530 OCA GTT TOT CGC Pro Vai Ser Arg
ACC
Thr 535 TCT TOT CCC CCT Ser Ser Pro Pro CCT COG GOC AGO Pro Pro Ala Ser
AGO
Ser 545 ACG COG OCA TCG Thr Pro Pro Ser CAG GTT TGC GTT Gin Val Cys Val
GCC
Ala 555 GGC ACC GTT GCT Gly Thr Val Ala
GAO
Asp 560 GGC GAG TCC GGC AAC TAO ATO GGC CTG TGC Gly Giu Ser Gly Asn Tyr Ile Gly Leu Cys 565 570 CAA TTO AGO TGO Gin Phe Ser Cys AAO TAO Aen Tyr 575 GGT TAO TGT Gly Tyr Cys ATO TOG OCA Ile Ser Pro 595
CCA
Pro 580 CCG GGA COG TGT Pro Gly Pro Cys
AAG
Lys 585 TGO ACC GCC TTT Cys Thr Ala Phe GGT GOT CCC Gly Ala Pro 590 OTA COG GGA Leu Pro Gly CCG GCA AGO AAT Pro Ala Ser Asn
GGG
Gly 600 CGC AAC GGC TGC Arg Asn Gly Cys
OCT
Pro 605 OAA GGC Glu Gly 610 OAT GGT TAT CTG Asp Gly Tyr Leu
GGC
Gly 615 OTG TGC AGT TTO Leu Cys Ser Phe TGT AAO CAT AAT Cys Asn His Asn TAO TGC COG OCA ACG OCA TGC CAA TAO TOT TAG Tyr Cys Pro Pro Thr Ala Cys Gin Tyr Cys 625 630 635 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 635 amino acids TYPE: amino acid TOPOLOGY: linear WO 98/00528 WO 9800528PCT/DK97/00283 34 (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 Ala Ser Phe Asp Asn Asp Asn Gln Ala 145 Ala Ser Asn Ala Leu 225 Ser Arg Thr Lys Asp 305 Arg Trp Leu Leu Met Asp Ile Ser Trp Tyr 130 Ser Ala Ser Asp Asp 210 Ala Tyr Trp Trp His 290 Gly Asp Tyr Ser Giu I le Met Gly Ala Trp 115 Ala Ser Gly Pro Gly 195 Gly Pro Ser Gin Asn 275 Phe Phe Thr Arg Ser Glu Gly Gin Val Asp 100 Ser Ser Phe Ser Ser 180 Asn Asp Val Lys Gin 260 Asp Asp Leu Asp Arg Leu Arg Ile Arg Asp Arg Pro Arg Ala Asn 165 Asn Asn Asn Ser Asn 245 Val Tyr Asp Asp Ile 325 Asn Gly Ala Val Ala 70 Gly Asn Gly Pro Gly 150 Val Ile Lys Ala Pro 230 Trp Leu Gly Gly Leu 310 Ser Leu Ser Ser Giy 55 Lys Tyr Gly Asn Ala 135 Asp Tyr Asp Ala Tyr 215 Trp Val Gin Giu Asn 295 Ser Lys Lys Ala Ser 40 Asp Ala Thr Met Ala 120 Gin Gly Phe Gly Pro 200 Lys Phe Phe Gin Ser 280 Ser Lys Tyr Ala Ala 25 Ala Arg Ala Asp Lys 105 Val Leu Leu Val Ala 185 Lys Asn Phe Pro Gly 265 His Lys Pro Val Leu Pro Asp Gly Gly Gin 90 Vai Gly Tyr Asp Pro 170 Leu Pro Trp Thr Gly 250 Phe Tyr Trp Phe Gin 330 Asp Ala Arg Ser Ile 75 Gin Phe Val Val Val 155 Asn Asn Gly Leu His 235 Gly Pro Val Val Ile 315 Asn Cys Asn Leu Ser Asp Leu Ile Gly Asp 140 Asn Phe Trp Gin Gly 220 Phe Pro Met Gly Asn 300 Ala Giu Asp Val Val Ala Ala Gly Ser Gin 125 Asn Ala His Met Thr 205 Gly Gly Leu Val Pro 285 Asp Ala Gin Ala Ala Phe Asp Phe Tyr Phe 110 Lys Arg Leu Pro Ala 190 Val Lys Pro Ile Glu 270 Leu Met Tyr Leu Thr Ile Cys Tyr Ala Ala Asp Ilie Pro Arg Gly 175 Trp Thr Pro Giu Tyr 255 Ile Lys Pro Lys Val 335 Asp Arg His Asp Leu Tyr Phe Ala Phe Ser 160 Gin Asp Val Tyr Val 240 Asn Val Ser His Asn 320 Tyr Thr WO 98/00528 WO 9800528PCTIDK97/00283 Thr Arg Leu 385 Gin Ser Phe Ile Asp 465 Vai Thr Ser Ser Ser 545 Gly Giy I le Giu Tyr 625 Ser Pro 370 Leu Thr Ile Ser Tyr 450 Pro Thr Ser Ser Pro 530 Thr Giu Tyr Ser Gly 610 Cys Asn 355 Asp Lys Phe Gly Gly 435 Asn Leu Thr Giy Pro 515 Pro Pro Ser Cys Pro 595 Asp 340 Arg Giy Thr Gin Gin 420 Thr Phe Gin Cys Pro 500 Pro Vai Pro Gly Pro 580 Pro Giy Pro Trp Ala Ala 405 Gin Ser Asn Ala Gin 485 Val Val Ser Ser Asn 565 Pro Ala Tyr Ala Gin Gly 390 Asn Lys Leu Pro Asp 470 Ala Ser Ser Arg Gly 550 Tyr Gly Ser Leu Asn Thr 375 Ser Ala Phe Met Tyr 455 Gly Lys Ser Ser Thr 535 Gin Ile Pro Asn Gly 615 Cys 345 Gly Asp Thr Aia Leu 425 Ile Gly Phe Ser Pro 505 Arg Ser Cys Leu Lys 585 Arg Cys Asn Vai 380 Ser Phe Asn Val Pro 460 Thr Thr Ser Ser Pro 540 Giy Phe Aia Cys Ser 620 Tyr 365 Tyr Gly Gin Giy Cys 445 Aia Ile Asn Thr Pro 525 Pro Thr Ser Phe Pro 605 Cys 350 Phe Val Gly Ile Gin 430 Ser Giy Gly Pro Thr 510 Pro Pro Val Cys Gly 590 Leu Asn Pro Pro Thr Ala Gin Tyr Cys INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucieic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucieic acid DESCRIPTION: /desc "Primer I', WO 98/00528 PCT/DK97/00283 36 CAGCGTCCAC ATCACGAGC 19 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer 2" GAAGAAGCAC GTTTCTGCAG AGACCG 26 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer 3" CGGTCTCTCG AGAAACGTGC TTCTTC 26 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer 4" GCCACTTCCG TTATTAGCC 19 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer GGGGGGATCC ACCATGAG 18 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer 6" ACGGTCAGCA GAAGAAGCTC GACGAATAGG ACTGGC 36 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 98/00528 WO 9800528PCT/DK97/00283 37 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer 7"1 GCCAGTCCTA TTCGTCGAGC TTCTTCTGCT GACCGT INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer 8"1 CCACGGTCAC CAACAATAC INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 6032 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: STRAIN: Trichoderma harzianum CBS 243.71 (ix) FEATURE: NAME/KEY: CDS LOCATION:3188. .5092 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GACGAAAGGG
CTTAGACGTC
TCTAAATACA
AATATTGAAA
TTGCGGCATT
CTGAAGATCA
TCCTTGAGAG
TATGTGGCGC
ACTATTCTCA
GCATGACAGT
ACTTACTTCT
GGGATCATGT
ACGAGCGTGA
GCGAACTACT
TTGCAGGACC
GAGCCGGTGA.
CCCGTATCGT
AGATCGCTGA
CATATATACT
TCCTTTTTGA
CAGACCCCGT
GCTGCTTGCA
TACCAACTCT
TTCTAGTGTA
TCGCTCTGCT
GGTTGGACTC
CGTGCACACA
AGCATTGAGA
CCTCGTGATA
AGGTGGCACT
TTCAAATATG
AAGGAAGAGT
TTGCCTTCCT
GTTGGGTGCA
TTTTCGCCCC
GGTATTATCC
GAATGACTTG
AAGAGAATTA
GACAACGATC
AACTCGCCTT
CACCACGATG
TACTCTAGCT
ACTTCTGCGC
GCGTGGGTCT
AGTTATCTAC
GATAGGTGCC
TTAGATTGAT
TAATCTCATG
AGAAAAGATC
AACAAAAAAA
TTTTCCGAAG
GCCGTAGTTA
AATCCTGTTA
AAGACGATAG
GCCCAGCTTG
AAGCGCCACG
CGCCTATTTT
TTTCGGGGAA
TATCCGCTCA
ATGAGTATTC
GTTTTTGCTC
CGAGTGGGTT
GAAGAACGTT
CGTATTGACG
GTTGAGTACT
TGCAGTGCTG
GGAGGACCGA
GATCGTTGGG
CCTGTAGCAA
TCCCGGCAAC
TCGGCCCTTC
CGCGGTATCA
ACGACGGGGA
TCACTGATTA
TTAAAACTTC
ACCAAAATCC
AAAGGATCTT
CCACCGCTAC
GTAACTGGCT
GGCCACCACT
CCAGTGGCTG
TTACCGGATA
GAGCGAACGA
CTTCCCGAAG
TATAGGTTAA
ATGTGCGCGG
TGAGACAATA
AACATTTCCC
ACCCAGAAAC
ACATCGAACT
TTCCAATGAT
CCGGGCAAGA
CACCAGTCAC
CCATAACCAT
AGGAGCTAAC
AACCGGAGCT
TGGCAACAAC
AATTAATAGA
CGGCTGGCTG
TTGCAGCACT
GTCAGGCAAC
AGCATTGGTA
ATTTTTAATT
CTTAACGTGA
CTTGAGATCC
CAGCGGTGGT
TCAGCAGAGC
TCAAGAACTC
CTGCCAGTGG
AGGCGCAGCG
CCTACACCGA
GGAGAAAJGGC
TGTCATGATA
AACCCCTATT
ACCCTGATAA
TGTCGCCCTT
GCTGGTGAAA
GGATCTCAAC
GAGCACTTTT
GCAACTCGGT
AGAAAAGCAT
GAGTGATAAC
CGCTTTTTTG
GAATGAAGCC
GTTGCGCAAA
CTGGATGGAG
GTTTATTGCT
GGGGCCAGAT
TATGGATGAA
ACTGTCAGAC
TAAAAGGATC
GTTTTCGTTC
TTTTTTTCTG
TTGTTTGCCG
GCAGATACCA
TGTAGCACCG
CGATAAGTCG
GTCGGGCTGA
ACTGAGATAC
GGACAGGTAT
ATAATGGTTT
TGTTTATTTT
ATGCTTCAAT
ATTCCCTTTT
GTAAAAGATG
AGCGGTAAGA
AAAGTTCTGC
CGCCGCATAC
CTTACGGATG
ACTGCGGCCA
CACAACATGG
ATACCAAACG
CTATTARCTG
'GCGGATAAAG
GATAAATCTG
GGTAAGCCCT
CGAAATAGAC
CAAGTTTACT
TAGGTGAAGA
CACTGAGCGT
CGCGTAATCT
GATCAAGAGC
AATACTGTCC
CCTACATACC
TGTCTTACCG
ACGGGGGGTT
CTACAGCGTG
CCGGTA.AGCG
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 WO 98/00528 WO 9800528PCT/DK97/00283
GCAGGGTC
ATAGTCCT
GGGGGCGG
GCTGGCC I
TTACCGCC
CAGTGAGC
CGATTCAT
CCGAGCC
TACGCTT~
TTTAAATC
TTAATTAG
TAAACAGA
CCGAAATC
TAGGCGCG
CCCGAATC
TTTTAAAM
ATTACGT I
AACCCCGG
ATCACGAG
CGAAACAG
GCGATCC1
ACTCAACC
CTGCGAAT
TGCGATG I
AATTTACC
CCTCACA
GCC GCA Ala Ala ~GG AACAGGAGAG C( 'GT CGGGTTTCGC Cl ~AG CCTATGGAAA Al TT TGCTCACATG T TT TGAGTGAGCT GI GA GGAAGCGGAA GI 'TA ATGCAGCCTG AJ LGT TCAGCGCCTA A LAA AAGCTACTTA A AA CTGATTAAAG G~ ~AG CAATATCAGG C( LTT ACTTTTGAAA A :AG GCAGATAAAG C( ICT CCATCTAAAT G' :GA TAGAACTACT Cl 'TT TTATATGGCG G( 'AG GGCTGATATT TI ~AA GTCAACAGCA T( ICG AAGGACCACC T( ICC CAAGAAAAAG G~ AC CAACACCCTC C] AC AAATCACAGT C( 'CG CTTGGATTCC C( 'AT CACAACATAT A TC TATCCACACT T( ATG TTG GGC GTT Met Leu Gly Val 1 GCT CTG TCT TCT Ala Leu Ser Ser 20 TCT CTC GAG GAA Ser Leu Giu Glu TTC ATG ATT GGT Phe Met Ile Gly
;CACGAGGG
kCCTCTGAC iCGCCAGCA rCTTTCCTG 1
.TACCGCTC
kGCGCCCAA rTAATGATT kACGCCTTA
V.ATCGATC
PGCCGAACG
,GCGCACGA
kGGCACATC
'ATACAGGC
ErTCTGGCTG k.TTTTTATA
;TGGTGGGC
CGTGAAAA
XCAAGCCCA
TAGGCATC
LVCGGCCCGT
k.GAGTGACT
;TCCCCGGT
'GCCCCTAG
kATACTAGC
=TCTTCCTT
AGCTTCCAGG
TTGAGCGTCG
ACGCGGCCTT
CGTTATCCCC
GCCGCAGCCG
TACGCAAACC
ACATACGCCT
TACAATTAAG
TCGCAGTCCC
AGCTATAAAT
AAGGCAACTT
AGTATTTAAA
AGATAGACOT
TGGTGTACAG
TAGAAGTCAG
AACTCGCTTG
TCGTCAAOGGG
AGTCCTTCAC
GGACGCACCA
CGGCCTTTTC
AGGGGCGGAA
ATTGTCCTGC
TCGTAGAGCT
AAGGGATGCC
CCTCAATCCT
GGGAAACGCC
ATTTTTGTGA
TTTACGGTTC
TGATTCTGTG
AACGACCGAG
GCCTCTCCCC
C!CGGGTAGTA
CAGTTAAAGA
GATTCGCCTA
GATATAACAA
AAAAAGCGAA
GCCCGAATCC
CTACCTATTA
GGGCATAAAA
AATTCATAGT
CGCGGGCAAC
ATGCAAGACC
GGAGAAACCC
TCCAATTAGA
TGCAACGCTG
ATTTAAAGGG
AGAATGCAAT
TAAAGTATGT
ATGCTTGGAG
CTATATACAC
TGGTATCTTT
TGCTCGTCAG
CTGGCCTTTT
GATAACCGTA
CGCAGCGAGT
GCGCGTTGGC
GACCGAGCAG
AGTTAGAATC
TCAAAACCAG
TATTAAAGCA
AGCGCTCTAC
TTATTAAGCG
AATCGGCTTC
TTACGCACTA
GTTTTGATCA
TCGCTTACCG
AAAGTAGTAA
CAGCGTCCAC
AGCAGCAAAG
ATCACGGGCA
ATTAATTTCC
TTAAACTCTT
CCCTTGTCGA
TTTCCAACTC
AACTGGGGAT
GTC
Val 5
CGC
Arg
ATT
Ile
TGT
CYB
TAT
Tyr
GCT
Ala
GCC
Ala
GAT
Asp
ATT
Ile CTC GGC Leu Gly CGT OCT Arg Ala COT CTA GGC Arg Leu Gly AGT GCC GCT Ser Ala Ala 25 TCT TCT OCT Ser Ser Ala
CTA
Leu
C.CC
Pro GCC AAT OTT Ala Asn Val
OCT
Ala GGC GCC CTT OCT Gly Ala Leu Ala
CGG
Arg
CAC
His
GAT
Asp
CTG
Leu s0
TAT
Tyr GAC CGT CTC Asp Arg Leu GTA TTC Val Phe 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3229 3277 3325 3373 3421 3469 3517 3565 3613 3661- 3709 3757 3805 3853
ATT
Ile OAT GAC Asp Asp AAC ATC Asn Ile ATG CAA CGT Met Gin Arg GGC OTT GAC Gly Val Asp 85 GCC GAC CGT Ala Asp Arg 100 TGG AGC CCC Trp Ser Pro GTT GOT Val Gly 55 GCC AAA Ala Lys 70 GGC TAT Oly Tyr AAT GGC Asn Gly GGT AAT Gly Asn 40
GAC
Asp
GCC
Al a
CGT
Arg
GCT
Ala AGC TCA GCA GAC Ser Ser Ala Asp ATT GAC GCA TTC Ile Asp Ala Phe
GAC
Asp TTC AAC Phe Asn
TCT
Ser
TG
Trp ACC GAC CAG Thr Asp Gin ATG AAA GTC Met Lys Val 105 GCA OTT GOT Ala Val Gly 120 CAG CTG TAT Gin Leu Tyr GOT TTG GAT Gly Leu Asp GCG CAG TAT Ala Gin Tyr 130 115
GCC
Ala AGC CGT CCC Ser Arg Pro CAA CTC GGG Gin Leu Oly TTC ATT TCA Phe Ile Ser OTT 000 CAG Val Gly Gin 125 OTT GAC AAC Val Asp Asn 140 GTA AAT GCG Val Asn Ala 155 AAC TTC CAC Asn Phe His AAC TGG ATG Asn Trp Met
TAT
Tyr
TTC
Phe 110
AAG
Lys
CG
Arg
TTG
Leu
CCT
Pro CCA TTC GCC Pro Phe Ala 145 CGC TCT OCT Arg Ser Ala
TCT
Ser TCC TTC GCT Ser Phe Ala
GGT
Gly 150
OTT
Val GCA GGC TCC Ala Gly Ser TAC TTT GTG Tyr Phe Val
GGT
Gly 175
TOO
Trp 160
CAA
Gin
CCC
Pro 170
CTC
Leu TOT TCC CCC Ser Ser Pro GAT AAT OAT Asp Asn Asp
GGA
Gly 195
GGT
Gly TCO AAC Ser Aen 180 AAC AAC Asn Asn GAO AAC Asp Asn ATT OAT GOC Ile Asp Gly AAG GCA CCC Lys Ala Pro 200 OCT TAO AAG Ala Tyr Lys 215 CCO GOC CAG Pro Gly Gin ACT GTC Thr Val 205 GGC AAG Gly Lys ACG GTG GCA Thr Val Ala
GAO
Asp 210 AAT TGG TTO Asn Trp Leu
GOT
Gly 220 WO 98100528 WO 9800528PCT/DK97/00283 CCT TAC Pro Tyr GAA GTT Giu Val 240 CTA GCG Leu Ala 225 TCA TAT Ser Tyr
CCT
Pro GTC TCC Val. Ser
TAT
Tyr 255
ATT
Ile
AAG
Lys
CCC
Pro
AAG
Lys
GTT
Val 335
GAC
Asp AAC CCC Asn Arg GTT ACC Val Thr TCT AAG Ser Lys CAT GAT His Asp 305 AAC AGG Asn Arg 320 TAC TGG Tyr Trp ACC ACC Thr Thr
TGG
Trp
TGG
Trp
CAT
His 290
GGA
Gly
GAT
Asp
TAC
Tyr
TCT
Ser
CCT
Pro 370
CTC
Leu TCC AAG Ser Lys CAA CAG Gin Gin 260 AAT GAC Asn Asp
AAC
Asn 245
GTC
Val
CCT
Pro 230
TGG
Trp
TTG
Leu TTT TTC ACC Phe Phe Thr TTC CCA GGT Phe Pro Gly 250 CAG GGC TTC Gin Gly Phe
CAT
His 235
GGT
Gly TTT GGC CCT Phe Gly Pro CCT CTG ATC Pro Leu Ile
CAG
Gin CCC ATG GTT Pro Met Val
GAG
Giu 270 265 TAC GGC Tyr Gly
GAG
C iu 275
TTC
Phe GAT GAT GGC Asp Asp Ciy
AAC
Asn 295
TCA
Ser TTC TTG GAT Phe Leu Asp ACT CAT ATA Thr Asp Ile 325 CGC CGC AAC Arg Arg Asn
CTT
Leu 310
TCT
Ser 280
TCC
Ser
AAG
Lys
TAT
Tyr
GCA
Ala
AAC
Asn 360
TGG
Trp
TTT
Phe GTC AAT Val Asn 300 ATT GCT Ile Ala
CAC
His TAC GTC GGT Tyr Val Gly CCT CTG Pro Leu 285 CAT ATG Asp Met GCA TAT Ala Tyr TCT AAG Ser Lys TTG AAG Leu Lys GCT AAT Ala Asn CTT CAA Val Gin 330 TTG GAC Leu Asp 315
AAT
Asn GAG CAG CTT Ciu Gin Leu TGC GAO CC Cys Asp Ala
ACC
Thr 350 ATG CGA Met Giy GCC GCA Ala Ala ACC ACT Thr Thr 400 CCT GCC Pro Ala 340 AAC CGC Asn Arg 355 GAT COT Asp Giy AAC ACC Lys Thr
CCG
Pro AGT GCC Ser Giy
AAT
Asn TGG CAA ACT ATG GAT Trp Gin Thr Met Asp 375 CCC GGT AGC GTC ACG Ala Gly Ser Val Thr ACG TTC CAG Thr Phe Gin
GCC
Aia 405
CAA
Gin 390
AAC
Asn CCC GGA CC Ala Gly Ala AGC ATC GGC Ser Ile Giy AAG TTT GCT Lys Phe Ala 415
ACC
Thr
CTA
Leu 425
ATC
Ile GAT ACC GTT Asp Thr Val 380 GTC ACG TCT Val Thr Ser 395 AAC CTC TTC Asn Leu Phe 410 ACT CGC AAC Thr Arg Asn ACC AAC GTT Thr Aen Vai ACC ATT CCT Thr Ile Pro 460 TCT TTG ACC Ser Leu Thr 475 CTT GCA ACC Leu Cly Thr TAC TTT Tyr Phe 365 TAT GTT Tyr Val CCC GGC Gly Cly CAA ATC Gin Ile GTC TTT AC Val Phe Ser
GGA
Cly 435
AAT
Asn 3901 3949 3997 4045 4093 4141 4189 4237 4285 4333 4381 4429 4477 4525 4573 4621 4669 4717 4765 4813 4861 4909 4957 TCA TTG ATC Ser Leu Met
CAT
Asp 440
GGT
Cly
TGC
Cys 445
CAG
Gin 430
TCT
Ser TGC GGT ATC Cys Gly Ile
TAC
Tyr 450
CCT
Pro TTC AAC CCA Phe Asn Pro
TAT
Tyr 455
GGT
Giy GTT CGC Val Gly CTT TTC Leu Phe TTT GAC Phe Asp TTG CAT Leu His 480 CCT GTC Pro Val
GAC
Asp 465
GTC
Val OTT CAG GCT Leu Gin Ala
CAC
Asp 470
CC
Ala GCC GC Ala Cly ATC GGA Ile Cly AAC CCT Asn Pro ACG ACT TGT Thr Thr Cys
CAG
Gin 485 AAC CCA TOT Lys Pro Ser 495
CC
Arg
GTC
Val1
GCA
Ala
TOT
Ser ACT TCT CCC CCT CTG Thr Ser Cly Pro Val 500 TCC TCG CCT CCT OTT Ser Ser Pro Pro Vai 515 TOO CCT CCA GTT TCT Ser Pro Pro Vai Ser 530 ACC ACG CCG CCA TOG Ser Thr Pro Pro Ser 545 GGC GAG TCC CCC AAC Cly Giu Ser Gly Aen TOC TOG CTG Ser Ser Leu
CCA
Pro 505
COT
Arg 490
GCT
Ala TCC TCC ACC Ser Ser Thr
ACC
Thr 510 TCT TCA Ser Ser
ACT
Thr 520
TCT
Ser CTC TCT TOT Val Ser Ser CCC CCT Pro Pro 525 CCC AGC Ala Ser OCT GAC Ala Asp 560 AAC TAC
CC
Arg
OCT
Gly 550
TAC
Tyr
ACC
Thr 535
CAG
Gin TOT CCC CCT Ser Pro Pro GTT TCC GTT Val Cys Val
CC
Ala 555
CAA
Gin CCC CCT CCC Pro Pro Pro 540 CCC ACC CTT Gly Thr Val TTO AGC TC Phe Ser Cys ATC CCC CTG Ile Gly Leu
CCT
TGC
Cys 570
TGC
TAC TGT CCA CCA CCC TGT AAG ACC CCC TTT GCT WO 98/00528 WO 9800528PCT/DK97/00283 Asn 575
GCT
Ala Tyr
CCC
Pro Tyr
TCG,
Ser Cys
CCA
Pro 595
GAT
ABP
Pro Gly GCA AGC Ala Ser Pro Cys AAT GGG Asn Gly 600 Lys 585 CGc Arg Thr Ala Phe Gly 590 CCG GGA Pro Gly CAT AAT His Asn GAA GGC Glu Gly 610 TAC TGC Tyr Cys 625 GGT TAT Gly Tyr CCG CCA ACG Pro Pro Thr
GACTGACACC
GGCAATTGGT
CAAGTCATGT
GAAAGCCATG
ACAAAGCACT
ATTCAATGCA
CACAGTGGAG
TCAAGAGTAT
AAGAGGGTCC
TTAGGCAGTA
TGCCATCTGC
GCGCGGCGTC
ATCTGTGCGG
CATAGTTAAG
TGCTCCCGGC
GGTTTTCACC
TGGCGGTAGA
TATATGATCA
GATTGTAATC
GTCTTTCCTT
AGAAAATTAG
TAGCCATGAG
CAGCAACATT
ATCTCTAC!CG,
CCATCCATCA
TTGCTGGAAT
CACTAAATCC
CAGGTTCAAC
TATTTCACAC
CCAGCCCCGA
ATCCGCTTAC
GTCATCACCG
CAATCP
TGTATC
GACCGP
CGTGTm
CATTCC
CTCATC
CCCCAI
TCCAAI
AACCC
GTCGGC
GATCAI
TCTCTC
CGCATP.
CACCCG
AGACAP
AAACGC
CTG GGC CTG TGC Leu Gly Leu Cys 615 GCA TGC CAA TAC Ala Cys.Gin Tyr 630 ATCC ATTTCGCTAT ~TAGT GGGTGTGCAT LCGGA ATTGAGGATA LGAAG ACCAGACAGA ~ATCC TTCTCTGCTT ~TTAG ATCCAAGCAC CATT GCTTTCCCCA AGAT CGTCTTCGCT LGTTC AATAATAGCC ~GCCA GTTGGCCGGG 'TGAT CCACCGCCCA ~CTCT AGCGCCTGAT LTGGT GCACTCTCAG ;CCAA CACCCGCTGA GCTG TGACCGTCTC
GCGA
AAC GGC TGC CCT CTA Asn Gly Cys Pro Leu 605 AGT TTC AGT TGT AAC Ser Phe Ser Cys Asn 620 TGT TAG TCTAGAGGGT Cys 635
AGTTAAAGGA
AATAGTAGTG
TCCGGAAATA
CAGTCCCTGA
GCTCTGCTGA
GTAATTCCAT
GGGGCCTCCC
TCAAAATCTT
GAGATGCATG
TGGTCATTGG
CGAGGGCGTC
GCGGTATTTT
TACAATCTGC
CGCGCCCTGA
CGGGAGCTGC
TGGGGATGAG
AAATGGAAGC
CAGACACCGT
TTTACCCTGC
TATCACTGTC
AGCCGAGGTC
AACGACTAAA
TGACAATTCC
GTGGAGTCAA
CCGCCTGTGA
TTTGCTTTTT
CTCCTTACGC
TCTGATGCCG
CGGGCTTGTC
ATGTGTCAGA
5005 5053 5102 5162 5222 5282 5342 5402 5462 5522 5582 5642 5702 5762 5822 5882 5942 6002 6032

Claims (24)

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.
3. The method according to claim 2, wherein the mutanase is obtained from 15 a strain of the species T. harzianum.
4. The method according to claim 3, wherein the mutanase is obtained from the strain T. harzianum CBS 243.71. The method according to any one of claims 2 to 4, in which the mutanase DNA sequence is isolated from or produced on the basis of a nucleic acid library of Trichoderma harzianurn CBS 243.71.
6. The method according to any one of claims 1 to 5, wherein the mutanase (pre)pro-sequence is replaced by the Lipolase® (pre)pro-sequence or the TAKA- amylase (pre)pro-sequence.
7. A method for constructing an expression vector comprising a mutanase gene obtained from a filamentous fungus suitable for heterologous production, substantially as hereinbefore described with reference to any one of the examples.
8. An expression vector comprising a mutanase gene obtained from a filamentous fungus suitable for heterologous production, produced by the method according to any one of claims 1 to 7.
9. 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. The expression vector according to claim 9, further comprising an operably linked promoter sequence and/or a prepro-sequence.
11. The expression vector according to claim 9 or claim 10, wherein the prepro-sequence comprises 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 s kex2-like site, or the Lipolase® pro-sequence, or the TAKA pro-sequence. C00144
12. The expression vector according to claim 11, wherein the promoter is the TAKA promoter or TAKA:TPI promoter.
13. The expression vector according to any of claims 9 to 12, being the vector pMT1796.
14. An expression vector, substantially as hereinbefore described with reference to any one of the examples. A lilamentous host cell comprising a heterologous mutanase gene derived from. a filamentous fungus being from the genus Trichodernma or the genus Aspergillus or the genus Fusarium. in 16. The host cell according to claim 15, derived from a strain of T. harzianum.
17. The host cell according to claim 15, derived from a strain of A. oryzae or A. niger.
18. The host cell according to claim 15, derived from a strain of Fusarium ovxysvporiiun, Fusarium graminearum, Fusarium sulphureum or Fusarium cerealis. i 19. The host cell according to any one of claims 15 to 18, wherein the host cell is a protease deficient or protease minus strain. The host cell according to claim 19, wherein the host cell is the protease deficient strain Aspergillus oryzae JaL125 having the alkaline protease gene named "alp" S deleted. 2 21. A filamentous host cell, substantially as hereinbefore described with reference to any one of the examples.
22. A process for producing a recombinant mutanase in a host cell, comprising the S steps: a) transforming an expression vector comprising a mutanase gene with a kex2 site 5 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 recombinant mutanase flom the culture medium.
23. The process according to claim 22, wherein the recombinant expression vector is prepared according to the method of any one of claims 1 to 7.
24. The process according to claim 22 or claim 23, wherein the filamentous host is a host cell according to any one of claims 15 to 21. I:\DAYLIB\libzz\1 5548.docsak 43 A process for producing a recombinant mutanase in a host cell, substantially as hereinbefore described with reference to any one of the examples.
26. An isolated recombinant mutanase produced according to the process according to any one of claims 22 to
27. A composition comprising a recombinant mutanase according to claim 26 and further other ingredients conventionally used in food, feed and/or pet food products.
28. An oral care composition comprising a recombinant mutanase according to claim 26. further comprising a dextranase, oxidase, peroxidase, haloperoxidase, laccase, protease, endoglucosidase, lipase or amylase or mixtures thereof. I 29. An oral care product comprising a recombinant mutanase according to claim 26 or an oral care composition according to claim 28 and further comprising ingredients conventionally used in oral care products. The oral care product according to claim 29, being a dentifrice.
31. The oral care product according to claim 30 being a toothpaste, tooth powder or a mouth wash.
32. Use of the recombinant mutanase according to claim 26 or an oral care composition of claim 28 or oral care product according to any one of claims 29 to 31 for preventing the formation of dental plaque or removing dental plaque.
33. The use of the recombinant mutanase according to claim 26 or an oral care 20 composition of claim 28 or oral care product according to any one of claims 29 to 31 in oral i care products for humans and/or animals.
34. Use of the composition according to claim 27, in food, feed and/or pet food products. Dated 13 April, 2000 9. Novo Nordisk A/S Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 9 9 I:\I)ALIB\Iizz\ I 5548.docsak
AU32544/97A 1996-06-28 1997-06-30 A recombinant enzyme with mutanase activity Ceased AU721693B2 (en)

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CN1226282A (en) 1999-08-18

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