US20020085980A1

US20020085980A1 - Oral care product comprising a mutan binding domain

Info

Publication number: US20020085980A1
Application number: US09/938,743
Authority: US
Inventors: Claus Fuglsang
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 1996-10-25
Filing date: 2001-08-24
Publication date: 2002-07-04
Also published as: WO1998018437A1; AU4616797A; US6355228B1; EP0951272A1

Abstract

Disclosed is a polypeptide hybrid containing an amino acid sequence with binding affinity for mutan, the amino acid sequence being bound to an active component useful for oral care purposes; an oral care composition comprising a mutan binding domain; an oral care product comprising such an oral care composition of the invention; and finally the use of a mutan binding polypeptide hybrid or a single unit MBD for oral care purposes, including preventing dental plaque formation and/or removal of existing dental plaque.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 09/295,744 filed Apr. 20, 1999, which is a continuation of PCT application No. PCT/DK97/00470 filed Oct. 27, 1997, and claims priority under 35 U.S.C. 119 of Danish application 1186/96 filed Oct. 25, 1996, the contents of which are fully incorporated herein by reference.[0001]

FIELD OF THE INVENTION

The present invention relates to a polypeptide with specific binding affinity for mutan, an oral care composition comprising a polypeptide hybrid of the invention, an oral care product comprising such an oral care composition, and finally the use of a polypeptide with a specific binding affinity for mutan for oral care purposes, including preventing dental plaque formation and/or removal of existing dental plaque.

BACKGROUND OF THE INVENTION

The formation of dental plaque leads to dental caries, gingival inflammation, periodontal disease, and eventually tooth loss. Dental plaque is a mixture of bacteria, epithelial cells, leukocytes, macrophages, and other oral exudate. Said bacteria produce highly branched polysaccharides which together with microorganisms from the oral cavity form an adhesive matrix for the continued proliferation of dental plaque.

As plaque continues to accumulate rock hard white or yellowish deposits arise. These deposits are called calcified plaque, calculus or tartar, and are formed in the saliva from plaque and minerals, such as in particular calcium.

Oral Polysaccharides

Oral polysaccharides mainly consist of the adhesive polysaccharides termed “fructans” and “glucans”.

Glucans are produced from carbohydrates, such as sucrose introduced into the mouth, e.g. as a food or beverage constituent, by the action of cariogenic microorganisms, such as Streptococcus sobrinus or Streptococcus sanguis, growing in the oral cavity.

The term “glucan” is a general common term covering a number of polysaccharides and includes cellulose, starch, dextran, mutan, pullulan etc.

Oral glucans comprise water-soluble dextran, having large portions of alpha-1,6-glucosidic linkage and as the major component a water-insoluble extra-cellular polysaccharide called “mutan” comprised of a backbone with alpha-1,3-glycosidic linkages and branches with alpha-1,6-glycosidic linkages.

Mutan bind to almost any surface such as the surface of teeth, (i.e. hydroxyapatite constituting the hard outer porous layer of the teeth), pellicle, the cell surface of oral microorganisms as well as to acceptor proteins on the cell of said cariogenic bacteria adhering to the teeth surface.

WO 95/31556 (Unilever) discloses the glucan binding domain of glycosyltransferase having specificity for binding to dextran (being a polysaccharide with mainly alpha-1,6-glucosidic linkages).

According to WO 95/31556 the glucan binding domain is covalently chemically bound to “material” having an activity, such as inhibitory effect against the formation of dental plaque. Said material may be an enzyme, such as galactose oxidase (see Example 6).

Polysaccharide binding domains conjugated to other proteins and peptides to aid in downstream processing of recombinant fermentation are known. For instance, researchers have made fusion or hybrid proteins containing a starch binding domain (Chen et al. (1991), Gene 991, p. 121-126), cellulose binding domain (Ong et al. (1989), TIBTech 7, p. 239-243) for the purpose of purifying the proteins on starch and cellulose resins, Polysaccharide binding fusion proteins useful as removable labels (WO 93/21331).

SUMMARY OF THE INVENTION

It is the object of the present invention to provide oral care products which efficiently prevent the formation of dental plaque and/or facilitates removal of already deposited dental plaque.

In the first aspect the present invention relates to a polypeptide hybrid comprising an amino acid sequence with binding affinity for mutan, said amino acid sequence being bound to an active component useful for oral care purposes.

In the second aspect the invention relates to an oral care composition comprising a polypeptide hybrid comprising an amino acid sequence with binding affinity for mutan bound, said amino acid sequence being bound to an active component useful for oral care purposes and further ingredients conventionally used in oral care compositions.

In the third aspect the invention relates to an oral care product comprising an oral care composition of the invention.

In the final aspect the invention relates to the use of a composition of the invention or oral care product of the invention for preventing the formation of dental plaque and/or removing dental plaque.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding isotherm of [0019] T. harzianum mutanase binding to mutan,
FIG. 2 shows lineage of the mutanase constructs pMT1796, pJW104 and pJW105. Regions of the plasmids are labelled as follows: the lipase and mutanase genes as open boxes; the mutan binding domain as “cooh terminus”; a thick-lined arc=promoter; an arrow signifies the direction of transcription; ø=mutanase signal sequence; ▪=mutanase prosequence; restriction enzyme sites are labelled. [0020]
FIG. 3 outlines the construction of the expression plasmid pJW105. Regions are marked as follows: the [0021] Humicola lanuginosa lipase gene (lipolase gene) as a striped box; the mutanase gene as an open box; primers by an arrow indicating their 5′ to 3′ orientation; the TAKA promoter by a heavy-line; restriction enzyme sites are labelled.

DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide oral care products which efficiently prevent the formation of dental plaque and/or facilitates removal of already deposited dental plaque. [0022]
The inventors of the present invention have provided polypeptide hybrids which increase the amount of active component delivered to dental plaque. This results in improved removal of dental plaque and/or in an improved dental plaque inhibiting/preventing effect when used in oral care compositions and products in comparison to the effect obtained by prior art oral care compositions and products, e.g. the composition disclosed in WO 95/31556, which delivers the active component to the dextran component of dental plaque. [0023]
The increased delivery of active component is obtained by a polypeptide (or amino acid sequence) which target(s) (ie. binds to) the major component of dental plaque, i.e. mutan. [0024]
Said polypeptide capable of binding to mutan may be bound to an active component useful for oral care purposes to form a polypeptide hybrid. [0025]
By using a polypeptide hybrid having a binding affinity for mutan an increased amount of active component (e.g. an enzyme) is brought in close contact with the substrate (i.e. the mutan component of the dental plaque), in comparison with e.g. polypeptide hybrids which target minor components, such as the dextran component, of dental plaque. [0026]
Consequently, when targeting the dental plaque using a polypeptide hybrid capable of binding to mutan more active component (e.g. enzyme) is delivered to the place of action resulting in a more efficient processing (e.g. degradation) of oral polysaccharides and hereby removal of dental plaque. In other words the mutan binding domain of the polypeptide hybrid provides access and proximity between the active component its substrate. [0027]
Further, the dental plaque formation is more efficiently prevented as the mutan binding domain acts as a sort of competitive inhibitor blocking the binding sites to which the dental plaque forming bacteria, such as [0028] Streptococcus sobrinus, and glucan binding proteins, such as glucosyltransferases (GTFs), can adhere. Consequently, a mutan binding domain may according to the present invention be a “single unit Mutan Binding Domain” as defined below.
As described in Example 12 it was found that the isolated single unit mutan binding domain binds to mutan, indicating that mutan binding domains are suitable for increasing the delivery of active component fused to the mutan binding domain to the mutan of dental plaque. Further, Example 12 also shows that single unit mutan binding domains are suitable for preventing the formation of dental plaque as the mutan binding domain when binding to mutan of dental plaque) inhibit further access of plaque forming microorganisms. [0029]
MBD [0030]
In the following “mutan binding domain” will be abbreviated as “MBD” and is meant to define all polypeptide sequences or peptide sequences having affinity for binding to mutan. [0031]
Most known MBDs today are found internally or at the N or C termini of mutanases. [0032]
As described in the Examples below, illustrating the present invention, the mutanase derived from [0033] Trichoderma harzianum CBS 243.71 comprises a mutan binding domain in the C-terminal end and a catalytic domain in the N-terminal end.
Single Unit Mutan Binding Domain (Single Uunit MBD) [0034]
The term “single unit MBD” may also be referred to as “Isolated MBD” or “Separated MBD”. [0035]
In the context of the present invention a “single unit MBD” includes up to the entire part of the amino acid sequence of a MBD-containing enzyme, e.g. a polysaccharide hydrolysing enzyme, being essentially free of the catalytic domain, but retaining the MBD(s). [0036]
Thus, in the context of the invention, the entire catalytic amino acid sequence of an enzyme (e.g. a mutanase) or other enzymes comprising one or more MBDs is not to be regarded as a single unit MBD. [0037]
Typically a single unit MBD constitutes one or more MBDs of a polysaccharide hydrolysing enzyme, one or more MBDs of a mutan binding protein or a protein designed and/or engineered to be capable of binding to mutan. [0038]
The single unit MBD is at least as large as the minimum number of amino acids in the amino acid sequence required to bind to mutan. [0039]
A single unit MBD may also be an amino acid sequence in which the binding and catalytic domain are one and the same. [0040]
Isolation of a MBD [0041]
In order to isolate the MBD of e.g. a mutanase, several genetic approaches may be used. One method uses restriction enzymes to remove a portion of the gene and then to fuse the remaining gene-vector fragment in frame to obtain a mutated gene that encodes a protein truncated for a particular gene fragment. Another method involves the use of exonucleases such as Ba131 to systematically delete nucleotides either externally from the 5′ and the 3′ ends of the DNA or internally from a restricted gap within the gene. These gene deletion methods result in a mutated gene encoding a shortened gene molecule which may then be evaluated for substrate binding ability. Appropriate substrates for evaluating the binding activity include mutan. [0042]
Once a nucleotide sequence encoding the substrate binding region has been identified, either as cDNA or chromosomal DNA, it may then be manipulated in a variety of ways to fuse it to a DNA sequence encoding the enzyme of interest. The mutan binding encoding fragment and the DNA encoding the enzyme of interest are then ligated with or without a linker. The resulting ligated DNA may then be manipulated in a variety of ways to provide for expression. Microbial hosts such as Aspergillus, e.g., [0043] A. niger and A. oryzae, Bacillus, E. coli or S. cerevisiae are preferred.
According to the invention the MBD may be bound to any active component including organic compounds, inorganic complexes, proteins, enzymes, peptides, antibodies and various ligands, which are useful for oral purposes, especially anti-plaque or anti-stain agents. [0044]
The coupling of the MBD and the active component may be performed through e.g. ester, sulfhydryl, peptide, isopeptide, amide and other types of chemical bonds. [0045]
MBD Hybrids [0046]
In the first aspect the invention relates to a polypeptide hybrid comprising an amino acid sequence with binding affinity for mutan bound to an active component useful for oral care purposes. [0047]
In an embodiment of the invention the MBD is conjugated to an enzyme to form a fusion protein or polypeptide-enzyme hybrid. [0048]
The enzyme moiety of the MBD-enzyme hybrid may be an enzyme from the following group of enzyme including oxidases, peroxidases, proteases, lipases, glucosidases, lipases, esterases, deaminases, ureases and polysaccharide hydrolases or mixtures thereof. [0049]
Examples 3 to 5 describe the construction, expression and purification of a MBD-enzyme hybrid constituted of the [0050] T. harzianum CBS 243.71 MBD fused to a Humicola lanuginosa lipase (Lipolase®). Other MBD-enzyme hybrids may be prepared in a corresponding way.
The above mentioned enzyme activities are preferred for oral care purposes as these activities are known to be suitable for oral care purposes. Some of the included enzyme activities are known to be capable of contributing to the degradation of different constituents of dental plaque. [0051]
Preferred as the enzyme moiety are glucosidases, preferably alpha-glycosidases, especially mutanases, dextranases, pullulanases and alpha-amylases, and mixtures thereof. [0052]
In the case of using a dextranase as the enzyme moiety it may for instance be derived from a strain of the genera Penicillium, Paecilomyces, Aspergillus, Fusarium, Spicaria, Verticillium, Helminthosporium and Chaetomium; bacteria of the genera Lactobacillus, Streptococcus, Celivibrio, Cytophaga, Brevibacterium, Pseudomonas, Corynebacterium, Arthrobacter and Flavobacterium, and yeasts such as [0053] Lipomyces starkeyi.
Specifically contemplated is dextranases derived from a strain of Paecilomyces or Penicillium, such as [0054] Paecilomyces lilacinum or Penicilium lilacinus.
In the case of using a mutanase as the enzyme moiety it may for instance be derived from a strain of the genera Trichoderma, Streptomyces, Cladosporium, Bacillus, Aspergillus [0055]
Specifically contemplated is mutanases derived from Trichoderna, such as [0056] Trichoderma harziaum, especially the deposited strain Trichoderma harzianum CBS 243.71.
It is preferred that the enzyme(s) is(are) substantially active at temperatures and pHs prevailing in the mouth when using the oral care product of the invention. This normally means that the enzymes should be substantially active between 20° C. and 40° C., and at pHs in the range from pH 4.0 to 8.0. [0057]
The term “substantially active” means in the context of the present invention that the enzyme in question has a relative activity above 70%, in particular above 80%, especially above 90% of the activity at the temperature optimum. [0058]
When using a MBD-enzyme hybrid of the invention a smaller amount of enzyme need to be used to obtain the desired effect and/or less time is needed to obtain the desired effect. [0059]
Preparation of MBD Hybrids [0060]
MBD hybrids of the invention may be prepared by recombinant DNA technology e.g. as described in WO 95/16782, WO 95/31556, WO 93/21331, WO 90/00609 or other documents referred to in the “Background of the Invention” section above. [0061]
More specifically MBD hybrids may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the MBD ligated, with or without a linker, to a DNA sequence encoding the polypeptide, e.g. an enzyme, of interest and growing the host cell to express the fused gene. The MBD hybrids may be described by the following formula:[0062]
MBD—MR—X,
wherein: [0063]
MBD can be either the N-terminal or the C-terminal region of an amino acid sequence corresponding to at least the MBD; [0064]
MR is the middle region (the linker), and may be a bond, or a short linking group of from about 2 to about 100 carbon atoms, in particular of from 2 to 40 carbon atoms, or typically from about 2 to about 100 amino acids, in particular of from 2 to 40 amino acids; and [0065]
X can be either the N-terminal or the C-terminal region and is the polypeptide of interest. [0066]
Other MBD Coniugates [0067]
The MBD hybrid may also be a conjugate of a MBD and another polypeptide, such as a non-enzymatic protein or peptide, or a chemical moiety. This will add an additional property to the oral care composition. [0068]
The MBD may be bound to anti-plaque agents, anti-staining agents, anti-microbial agents, antibodies, antibody fragments, histamins, lactoferins, defensins, magainins, cecropins, and other cationic anti-bacteriocins and bacteriocins. [0069]
Further, the MBD may also be chemically conjugated to e.g. microbicides including, but not limited to, triclosan, chlorhexidine, quaternary ammonium compounds, chloroxylenol, chloroxyethanol, thymol, and fluoride. [0070]
Anti-microbial cat-ions such as Zn, Sn, Cu and others can also be complexed to MBDs by forming conjugates with appropriate chelating agents such as (poly)carboxylic acids, amino acids and so on. [0071]
The user of an oral care product of the invention (which will be describe in more details below), prepared from the oral care composition of the invention also described below, will benefit from the present invention, as the direct and indirect disadvantages (e.g. yellow deposits on the teeth and prevention of dental holes and gingivitis, respectively) can be prevented more effectively than with prior art products. [0072]
Oral Care Compositions [0073]
In a second aspect the invention relates to an oral care composition comprising a MBD hybrid or single unit MBD and further ingredients conventionally used in oral care compositions. [0074]
The oral care composition of the invention may advantageously comprise MBD hybrids of the invention described above. [0075]
The enzyme moiety of the MBD-enzyme hybrids in the oral care composition may be an enzyme from the following group of enzyme including oxidases, peroxidases, proteases, lipases, glucosidases, lipases, esterases, deaminases, ureases and polysaccharide hydrolases, or mixtures thereof. [0076]
An oral care composition of the invention may suitably have incorporated an amount of 0.001-10 mg/ml MBD-hybrid calculated on the basis of final oral care product. [0077]
In a preferred embodiment the MBD hybrid is a MBD-enzyme hybrid. Preferred enzyme activities are glycosidase activities, such as an alpha-glycosidase activity, such as dextranase, mutanase, pullulanase and/or alpha-amylase activity. [0078]
In the cases of using a hybrid MBD-dextranase, MBD-mutanase, and/or MBD-pullulanase the enzyme activity should lie in the range from 0.001 KDU to 1000 KDU/ml, preferably from 0.01 KDU/ml to 500 KDU/ml, especially from 0.1 KDU/ml to 100 KDU/ml for MBD-dextranases, from 0.001 MU/ml to 1000 MU/ml, preferably from 0.01 MU/ml to 500 MU/ml, especially from 0.01 MU/ml to 100 MU/ml and from 0.01 MU/ml to 100 MU/ml, for MBD-mutanases, in the range from 0.001 KDU to 1000 KPU/ml, preferably from 0.01 KPU/ml to 500 KPU/ml, especially from 0.1 KPU/ml to 100 KPU/ml for MBD-pullulanase. [0079]
It is also contemplated according to the invention to include other enzyme activities in the oral care compositions of the invention. Contemplated enzymes, beside dextranase and mutanase, may be from the group including proteases, such as papain, endoglucosidases, lipases, amylase and mixtures thereof. [0080]
Oral Care Products [0081]
The invention also relates to oral care products comprising an oral care composition of the invention. 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 formation of dental plaque, removing dental plaque, preventing and/or treating dental diseases etc. [0082]
At least in the context of the present invention, oral care products also encompass products for cleaning dentures, artificial teeth and the like. [0083]
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. [0084]
Toothpastes and tooth gels typically include abrasive polishing materials, foaming agents, flavouring agents, humectants, binders, thickeners, sweetening agents, whitening/bleaching/stain Ad removing agents, water, and optionally enzymes. [0085]
Mouth washes, including plaque removing liquids, typically comprise a water/alcohol solution, flavor, humectant, sweetener, foaming agent, colorant, and optionally enzymes. [0086]
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 ion 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. [0087]
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. [0088]
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 hydrolyzed polysaccharides and the like. Humectants are in general present in from 0% to 80%, preferably 5 to 70% by weight in toothpaste. [0089]
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. [0090]
As foaming agent soap, anionic, cationic, 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. [0091]
Surfactants are only suitable to the extent that they do not exert an inactivation effect on the present MBD hybrids. 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. [0092]
Suitable sweeteners include saccharin. [0093]
Flavors, 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%. [0094]
Whitening/bleaching agents include H[0095] ₂O₂and 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.
Water is usually added in an amount giving e.g. toothpaste a flowable form. [0096]
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. [0097]
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. [0098]
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. [0099]
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. [0100]
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. [0101]
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. [0102]

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 to 20%
	MBD hybrid and/or single unit MBD	0.0001% to 1%

In a specific embodiment of the invention the oral care product comprises



	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
	h) 0 to 20%	Enzymes
	i) 0.0001% to 1%	MBD hybrid and/or single unit MBD

Said MBD hybrid referred to in connection with the specific toothpaste and mouth wash above may have any activity, such as enzymatic activity, suitable for oral care purposes. Preferred enzyme activities are alpha-glycosidases, especially dextranases, mutanases, pullulanases, and alpha-amylases. Said enzyme referred to include all enzyme activities suitable in oral care products. [0105]
Use of an Oral Care Composition or Product [0106]
In the third aspect the invention relates to the use of the composition of the invention or an oral care product of the invention for preventing the formation of plaque and/or for removing dental plaque. [0107]
Method of Manufacture [0108]
The oral care composition and products of the present invention can be made using methods which are common in the oral product area. [0109]

MATERIALS AND METHODS

Materials [0110]
Enzymes [0111]
Mutanase produced by [0112] Trichoderma harzianum CBS 243.71 (available from Novo Nordisk A/S) Humicola lanuginosa lipase (available from Novo Nordisk ANS as Lipolase®) and is described in EP 305 216.
Plasmids [0113]
pAHL: a [0114] Humicola lanuginosa lipase gene (sometimes referred to as lipolase gene) containing plasmid described in EP 305,216.
pMT1 796: mutanase expression plasmid prepared as described in Example 8. [0115]
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 [0116] A. niger glucoamylase terminator and the A. oryzae TAKA amylase promoter.
pHD414+mut (pHD414 comprising the [0117] T. harzianum mutanase gene)
pHan37 containing the TAKA:TPI promoter [0118]

Linkers


Linker #1:
GATCCTCACA ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GG	(SEQ ID NO: 15)

GAGTGT TAC AAC CCG CAA CAG GCT GCA GAT CCG GAT CCG C	(SEQ ID NO: 16)

Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly	(SEQ ID NO: 17)

Linker #2:
C CAA TAC TGT TAG T	(SEQ ID NO: 18)

GT ACG GTT ATG ACA ATC AGATC	(SEQ ID NO: 19)

Ala Cys Gln Tyr Cys ***	(SEQ ID NO: 20)

Microorganisms [0120]
[0121] Trichoderma harzianum CBS 243.71
[0122] Streptococcus sobnnus strain CBS 350.71 identifiable as OMZ 176
[0123] Actinomyces viscosus DSM 43329
[0124] Fusobacterium nucleatum subsp. polymorphum DSM 20482
[0125] A. oryzae JaL125: Aspergillus oryzae host strain with the alkaline protease gene named “alp” deleted. Strain JaL125 is disclosed in WO 97/35956 (Novo Nordisk A/S)
Solutions and the Like [0126]
Britton-Robinson Buffer [0127]
CAPS (3-cyclohexylamino-1-propanesulfonic acid) (Sigma) [0128]
Coomassie Brilliant Blue R250(Sigma) [0129]
Peroxidase-conjugated swine immunoglobulins (DAKO, Denmark) [0130]
Erythrosin B (Sigma) [0131]
Equipment [0132]
Shaker (Eppndorf Thermomixer, Type 5436) [0133]
473A Protein Sequencer from Applied Biosystems [0134]
SDS-PAGE 4-20% (Novex) [0135]
Tricine 16% gel (Novex) [0136]
Tris-Glycine 4-20% (Novex) [0137]
Immobolin P[0138] ^SQPVDF membrane (Millipore)
Chromameter CR-200 (Minolta) [0139]
Preparation of Hydroxyapatite Disks [0140]
Hydroxyapatite (HA) disks are prepared by compressing 250 mg of hydroxyapatite in a disk die at about 5,900 kg (13,000 lbs) of pressure for 5 minutes. The disks are then sintered at 600° C. for 4 hours and finally hydrated with sterile deionized water. [0141]
Sterilisation of Hydroxyapatite Disks [0142]
HA disks are sterilised at 180° C. for two hours. [0143]
Preparation of Mutan [0144]
Mutan is prepared by growing [0145] Streptococcus sobrinus 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/liter

Yeast Extract 6 g/liter

(NH₄)₂SO₄ 20 g/liter

K₂PO₄ 3 g/liter

Glucose 50 g/liter

Pluronic PE6100 0.1%
After 35 hours, sucrose is added to a final concentration of 60 glliter 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. [0146]
Methods [0147]
Molecular Biology Procedures [0148]
All molecular biology procedures including restriction digests, DNA ligations, [0149] E. coli transformations, DNA isolations, Southern hybridizations, PCR amplifications, and library constructions and screenings were completed using standard techniques (Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning: A laboratory manual/E. F. Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Determination of Dextranase Activity (KDU) [0150]
One Kilo Novo Dextranase Unit (1 KDU) is the amount of enzyme which breaks down dextran forming reducing sugar equivalent to 1 g maltose per hour in Novo Nordisk' method for determination of dextranase based on the following standard conditions: [0151]

Substrate Dextran 500 (Pharmacia)

Reaction time 20 minutes

Temperature 40° C.

PH 5.4
A detailed description of Novo Nordisk's analytical method (AF 120) is available on request. [0152]
Determination of Mutanase Activity (MU) [0153]
One Mutanase Unit (MU) is the amount of enzyme which under standard conditions liberates 1 mmol reducing sugar (calculated as glucose) per minute. [0154]

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. [0155]
Preparation of Mutan Adhered Glass Wall [0156]
[0157] Streptococcus sobrinus OMZ 176 (CBS 350.71) is inoculated in a glass tube (22 mm diameter×150 mm height) containing 10 ml Todd Hewitt Broth with 2% sucrose and the tube is allowed to stand overnight at 37° C. The broth is discarded and adhered mutan and Streptococcus sobrinus cells on glass wall are washed twice with 10 ml of 0.85% NaCl solution.
Langmuir Fit [0158]
A=(Amax*E)/((1/K(ads)+E) [0159]
A: Adsorbed enzyme [0160]
E: Free enzyme [0161]
Amax: Max adsorbed enzyme [0162]
K(ads): Adsorption coefficient [0163]
The Langmuir fit is described in Stuart, J. Y. & Ristoph, D. L., (1984), Biotechnol. Bioengng, 27, p 1056+. [0164]
Assessment of the Plaque Inhibition Effect [0165]
The method used for assessing the plaque removal effect is based on the method described by Kao in JP 2250816. According to the present method the hydroxyapatite disks, sterilised as described above, become coated with a biofilm by being placed overnight in the presence of three strains of oral microorganisms ([0166] Streptococcus sobrinus, Actinomyces viscosus and Fusobacterium nucleatum) and various enzymes in a Brain Heart Infusion Medium (Difco) containing 0.2% sucrose.
To test plaque inhibition effect, 0.1% Erythrosin B in PBS (phosphate buffered saline) is used to stain plaque present on the hydroxyapatite disks red. The intensity of the red colour (referred to as a*) is measured on a Chromameter CR-200. The maximum a* value is 60. Values below that indicate a less intensive red colour (i.e. less plaque present). An a* value of zero indicated no red colouring (i.e. no plaque). A plaque inhibition effect is expressed as a relative figure based on the value of a* for a non-treated biofilm being 100%. [0167]

EXAMPLES

Example 1

Binding of Purified Mutanase to Mutan [0168]
The binding of purified [0169] T. harzianum CBS 243.71 mutanase (SEQ ID NO: 2) to mutan was investigated by incubating mutanase in varying concentration with 1 mg/ml mutan in 10 mM Britton-Robinson buffer, pH 7, at 4° C. for 1 hour while stirring. The samples were then centrifuged at 15,000 g for 2.5 minutes and filtered through 0.45 micrometer filters (Millipore). The residual activity was measured in the supernatant. The binding isotherm obtained can be filted to simple Langmuir binding and an affinity constant and a max adsorption constant can be obtained. FIG. 1 displays the result of the test. As can be seen the mutanase binds to mutan.

Example 2

Construction of the Recombinant Mutanase Expression Vector pMT1796 [0170]
A cDNA clone encoding mutanase was identified in a [0171] Trichoderma harzianum CBS 243.71 library by hybridization with a fragment of the mutanase 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, Nail and Xhol. From this digestion a 3499 nt (nucleotide) vector fragment and a 610 nt NarliXhol fragment were isolated. These two fragments were then ligated with linker #1 (see above) and a 618 nt EcoRl/BamHI fragment from pHan37 containing the TAKA:TPI promoter, giving plasmid pJW99. HD414+mut was next digested with Xhol and Sphl, and a 1790 nt fragment encoding amino acids 35-598 of the mutanase gene was isolated. [0172]
This fragment was ligated with linker #2 (see above) and pJW99 that had been linearized i′ E with the restriction enzymes Xbal and Xhol. The resulting plasmid, pMT1802, contains the [0173] 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 (SEQ ID NO: 8)

(nt 2751 5′-CAGCGTCCACATCACGAGC nt 2769);

and

Primer 2 (SEQ ID NO: 9)

(nt 3306 5′-GAAGAAGCACGTTTCTCGAGAGACCG nt 3281);

Primer 3 (SEQ ID NO: 10)

(nt 3281 5′-CGGTCTCTGAGAAACGTGCTTCTTC nt 3306) and

Primer 4 (SEQ ID NO: 11)

(nt 4266 5′-GCCACTTCCGTTATTAGCC nt 4248);

nucleotide numbers refer to the pMT1802

plasmid (See SEQ ID NO: 12).

Example 3

Construction of Recombinant MBD-Lipase Hybrid [0175] A. oryzae Expression Plasmid pJW105
Construction of pJW 104 (DNA construct with a mutan binding domain) [0176]
pJW104 containing an internal deletion of the mutanase coding region encompassing amino acids 32-536 (nt 94-1608) was constructed as follows: [0177]
The mutanase expression plasmid, pMT1796 (described in Example 2), contains two unique restriction sites which were used in the construction of pJW104: a Xhol site that sits 6 [0178] nucleaotides 5′ to the first codon of the mature protein and an Xbal site that immediately follows that stop codon (see FIG. 2).
A primer, Primer C (SEQ ID NO: 3), was made. The 3′ end of this primer consists of 21 nucleotides matching the sequence of the mutanase gene corresponding to amino acids 537 to 543 in the sense direction. The 5′ end of the primer harbors a Xhol site. [0179]

Primer C:

5′ CATACTCGAGAAACGT GCC AGC AGC ACG CCG CCA TCG 3′

Xho I Ala Ser Ser Thr Pro Pro Ser

537
Another primer, Primer D (SEQ ID NO: 4), was made. This primer match a sequence of pMT1796 downstream from the Xbal site, and oriented in the opposite direction of primer C. Primer D: [0180]
5′ [0181] GATTACAATCACATGACTTGGC 3′
pMT 1796 was PCR amplified using the primers C and D. Digestion of the 433 nt amplicon with Xhol and Xbal yielded a fragment of 290 nt that was cloned into pMT1796 that had been linearized with these same two enzymes. Altered regions of pJW104 were DNA sequences to confirm the presence of the deletion and to check the integrity of the mutanase gene. [0182]
Construction of pJW105 [0183]
To create pJW105, three DNA fragments (shown in FIG. 3) were ligated: a vector fragment from pMT1796, a fragment containing the amino portion of the [0184] Humicola lanuginosa lipase gene, and a PCR amplicon containing the in-frame fusion between the COOH terminus of the Humicola lanuginosa lipase gene and the MBD of T. harzianum mutanase. pMT1796 contains two unique restriction enzyme sites, a BamHl fragment that sits at the 3′ end of the TAKA promoter just before the initiating Met and a Nael site that sits over codons 425 and 426 in the COOH terminus of mutanase gene. Digestion of the plasmid with these two enzymes gives a 4359 nt fragment that was gel purified. The Humicola lanuginosa lipase gene portion of the construct is a 727 nt BamHI/BstXl restriction fragment from the expression plasmid pAHL. The unique BamHI site within this plasmid lies immediately 5′ to the start codon of the gene, and the unique BstXI site lies over codons 214-216 of the mature Humicola lanuginosa lipase protein. The final piece of the construct was created using overlap-PCR-extension (Ho et al. (1989), Gene, 77, 51-59) with two rounds of PCR amplification and the primers E, F, G and further Primer D (see above):
Primer E (SEQ ID NO: 5): [0185]

5′ CGGAACACTCTACCGCATTACC 3′

Primer F (SEQ ID NO: 6):

5′ CGGCGTGCTGCTGGCAGGAAGACATGTCCCAATTAAC 3′,

Primer G (SEQ ID NO: 7)

5′ GTTAATTGGGACATGTCTTCCTGCCAGCAGCACGCCG 3′
Primers F and G overlap the [0186] Humicola lanuginosa lipase/mutanase fusion of the construct. First round amplification consisted of two separate reactions one of which paired primers E and F with the lipase plasmid pAHL, and the second that paired primers G and D with the mutanase plasmid pMT1796. This reaction yielded fragments of 237 and 449 nt, respectively. Second round amplification paired primers E and D with 0.25 ml of the reaction mixtures from the amplification round 1. The resulting 670 nt fragment was digested with BstXl and Nael, gel purified and ligated together with the vector fragment and the 727 nt BamHI/BstXl fragment from the lipase gene. The protein coding region of the resulting plasmid, pJW105, was DNA sequenced.
The DNA and amino acid sequence of the MBD-lipase hybrid is shown in SEQ ID NO: 13 and 14, respectively. [0187]

Example 4

Expression of Recombinant MBD-lipase Hybrid in [0188] Aspergillus oryzae
0.1 ug pJW105 was transformed into [0189] A. oryzae strain JaL125 using a PEG-mediated protocol (see EP 238,023) and a DNA mixture containing 0.5 microgram of a plasmid encoding the gene that confers resistance to the herbicide Basta. Transformants were selected on minimal plates containing 0.5% basta and 50 mM urea as a nitrogen source.
Shake Flask Cultures [0190]
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. [0191]
SDS-PAGE and protein transfers were performed using standard protocols. [0192]

Example 5

Purification of the Recombinant MBD-lipase Hybrid [0193]
Purification of the recombinant MBD-lipase hybrid from the A. oryzae JaL125 fermentation broth was performed as follows: Filtered fermentation broth was incubated with 5% mutan in 0.1% sodium acetate, pH 5.5, for 30 minutes while stirring. The sample was centrifuged for 10 minutes at 10,000 g. The precipitate was re-suspended in 1 ml 0.1 M sodium acetate, pH 5.5, and centrifuged. This step was repeated 3 times before eluting the MBD with water (MilliQ-filtered). The eluate was concentrated on an Amicon cell (YM10 membrane). The fusion protein (i.e. the MBD hybrid) was further purified by gel filtration using a Superdex75 16/60 column (Pharmacia) in 0.1 M sodium acetate, pH 6 hybrid. [0194]

Example 6

Binding of Recombinant MBD-lipase Hybrid to Mutan [0195]
The binding of purified MBD-lipase hybrid (SEQ ID NO: 14) to mutan is investigated using the procedure described in Example 1. [0196]

Example 7

Expression of Recombinant Single Unit MBD in [0197] Aspergillus oryzae
Expression of the [0198] A. oryzae expression plasmid pJW104 coding for the single unit MBD of the T. harzianum mutanase was carried out as described in Example 4.

Example 8

Purification of the Recombinant Single Unit MBD [0199]
Isolated MBD was purified by incubating 30 ml fermentation broth of [0200] A. orzyae expressing the C-terminal domain of the mutanase with 3 ml 5% mutan in 0.1 sodium acetate, pH 5.5, for minutes while stirring.
The sample was centrifuged for 10 minutes at 10,000 g. The precipitate was re-suspended in 1 ml 0.1 M sodium acetate, pH 5.5, and centrifuged. This step was repeated 3 times before eluting the MBD with water (MilliQ-filtered). Purified MBD appeared at a molecular weight around 10 kDa in westerns. [0201]

Example 9

Immuno Detection of MBD [0202]
The MBD can be detected by Mancini immuno diffusion. 12 ml of 1% agarose in 20 mM Tris-Maleate, pH 7, at 56° C. was added 100 μl of mutanase antibodies raised in rabbits. The solution was poured onto GelBond Film. 10 μl samples were added to each well and the gel was left over night at room temperature. The gel was then washed in 0.5% NaCl and 3 times in water before drying. The gel was stained in 0.5% Coomassie Brilliant Blue 45% ethanol, 10% acetic acid for 1 minute and de-stained in 25% ethanol, 10% acetic acid. [0203]

Example 10

Western Analysis of MBD, Catalytic Domain and [0204] Humicola lanuginosa Lipase-MBD Fusion Protein
SDS-PAGE was performed using a Tricine 16% gel from Novex (for the MBD) or Tris-Glycine 4-20% (Novex) (for higher M[0205] _wproteins). The proteins were blotted onto a Millipore Immobolin P^SQPVDF membrane in 10 mM glycine, 20% methanol, pH 11.8 at 175 mA for 3 hours. Standards and controls were Coomassie stained (see Example 9) while the rest of the membrane was blocked with 4.875 ml Tween20 in 245 ml washing solution (30.3 g Tris base, 43.8 g NaCl, 2.5 ml Tween20 in 5 liters water) for 3 minutes before washing with the washing solution. The membrane was then reacted with 50 μl of the detecting antibodies (mutanase antibodies raised in rabbits) diluted to 50 ml with washing solution and washed 4 times with washing solution before incubating with peroxidase-conjugated swine immunoglobulins to rabbit immunoglobulins (50 μl in 50 ml washing solution). The membrane was washed again with washing solution and once with 50 mM sodium acetate, pH 5, and stained with carbazol (2 ml 3-amino-9-ethylcarbazol in 48 ml acetate buffer added 25 μl 30% H₂O₂). The membrane was then washed in water and fixed in 12.4 g sodium thiosulfate in 1 liter of water.

Example 11

Isolation of Single Unit MBD of [0206] T. harzianum
Single unit MBD of [0207] T. harzianum CBS 243.71 was isolated by proteolytic degradation of mutanase.
Purified [0208] T. harzianum mutanase was digested with chymoptrypsin in a ratio of 1:100 (protease:mutanase) in 0.1 M Tris-HCI buffer, pH 8.5 at 30° C. for 2.5 hours. The resulting digest was investigated using SDS-PAGE (Novex 4-20%). The 42 kDa band observed was blotted onto a Millipore Immobolin p^SQPVDF membrane in 10 mM CAPS, 6% methanol at 175 mA for 3 hours. The membrane was stained with 0.1% Coomassie Brilliant Blue R250 in 60% methanol, 1% acetic acid and de-stained in 40% methanol. The 42 kDa band was subjected to N-terminal amino acid sequencing using the 473A Protein Sequencer from Applied Biosystems according to the manufacturer's description.
The N-terminal sequence obtained was Ser-Leu-Thr-Ile-Gly-Leu corresponding to a proteolytic cleavage between Phe436 and Ser437 (see SEQ ID NO. 1 or 2) in the mature mutanase. The C-terminal domain obtained by chymotrypsin digestion was shown to bind to mutan by incubating the 50 microliter chymotrypsin digest with 50 microliter 5% mutan in 0.1 M ammonium bicarbonate, pH 8.15 for 30 minutes at 25°0C. The sample was then centrifuged for 5 minutes at 15,000 g and 30 microliter sample was then loaded onto SDS-PAGE (Novex 4-20%). A control without mutan (buffer) was included. [0209]

Example 12

Binding of Purified MBD to Mutan [0210]
The single unit MBD was mixed 1:1 with 0.1 M acetate buffer, pH 5.5 with/without 5% mutan. The samples were incubated for 30 minutes at room temperature before centrifuging the [0211] samples 5 minutes at 15,000 g. Ten microliter of supernatant was analyzed by Mancini immuno diffusion. No MBD was detected in the sample pre-incubated with mutan indicating that the single unit MBD binds to mutan.

Example 13

Catalytic Domain [0212]
Fermentation broth of [0213] A. oryzae expressing the N-terminal domain of the mutanase was analyzed for. It was observed that said N-terminal domain has catalytic activity.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. [0214]
Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. [0215]
1 20 1905 base pairs nucleic acid single linear DNA (genomic) Trichoderma harzianum CBS 243.71 sig_peptide 1..120 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 Ile 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 Ile Gly Ile 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 Gln Arg Ala Lys Ala Ala Gly Ile 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 Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln 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 Ile 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 Gln Lys Ile Ala 115 120 125 CAG TAT GCC AGC CGT CCC GCC CAG CTG TAT GTT GAC AAC CGG CCA TTC 432 Gln Tyr Ala Ser Arg Pro Ala Gln 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 Gln 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 Ile 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 Gln 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 Ile Tyr Asn 245 250 255 CGG TGG CAA CAG GTC TTG CAG CAG GGC TTC CCC ATG GTT GAG ATT GTT 816 Arg Trp Gln Gln Val Leu Gln Gln Gly Phe Pro Met Val Glu Ile 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 Ile 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 Ile Ser Lys Tyr Val Gln Asn Glu Gln 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 Gln 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 Gln Thr Phe Gln Ala Asn Ala Gly Ala Asn Leu Phe Gln Ile Pro Ala 405 410 415 AGC ATC GGC CAG CAA AAG TTT GCT CTA ACT CGC AAC GGT CAG ACC GTC 1296 Ser Ile Gly Gln Gln Lys Phe Ala Leu Thr Arg Asn Gly Gln 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 Ile 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 Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Ile 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 Gln Ala Asp Gly Leu Phe Ser Leu Thr Ile 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 Gln 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 Gln 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 Ile Gly Leu Cys Gln 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 Ile 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 Gln Tyr Cys * 625 630 635 634 amino acids amino acid linear protein 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 Ile 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 Ile Gly Ile Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp 50 55 60 Asp Asp Met Gln Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu 65 70 75 80 Asn Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Gly Tyr Ala Tyr 85 90 95 Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe Ile Ser Phe Asp Phe 100 105 110 Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gln Lys Ile Ala 115 120 125 Gln Tyr Ala Ser Arg Pro Ala Gln 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 Gln 165 170 175 Ser Ser Pro Ser Asn Ile Asp Gly Ala Leu Asn Trp Met Ala Trp Asp 180 185 190 Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gln 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 Ile Tyr Asn 245 250 255 Arg Trp Gln Gln Val Leu Gln Gln Gly Phe Pro Met Val Glu Ile 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 Ile Ala Ala Tyr Lys Asn 305 310 315 320 Arg Asp Thr Asp Ile Ser Lys Tyr Val Gln Asn Glu Gln 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 Gln 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 Gln Thr Phe Gln Ala Asn Ala Gly Ala Asn Leu Phe Gln Ile Pro Ala 405 410 415 Ser Ile Gly Gln Gln Lys Phe Ala Leu Thr Arg Asn Gly Gln Thr Val 420 425 430 Phe Ser Gly Thr Ser Leu Met Asp Ile Thr Asn Val Cys Ser Cys Gly 435 440 445 Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Ile Pro Ala Gly Phe Asp 450 455 460 Asp Pro Leu Gln Ala Asp Gly Leu Phe Ser Leu Thr Ile Gly Leu His 465 470 475 480 Val Thr Thr Cys Gln 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 Gln Val Cys Val Ala Gly Thr Val Ala Asp 545 550 555 560 Gly Glu Ser Gly Asn Tyr Ile Gly Leu Cys Gln 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 Ile 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 Gln Tyr Cys 625 630 37 base pairs nucleic acid single linear other nucleic acid /desc = “Primer C” 3 CATACTCGAG AAACGTGCCA GCAGCACGCC GCCATCG 37 22 base pairs nucleic acid single linear other nucleic acid /desc = “Primer D” 4 GATTACAATC ACATGACTTG GC 22 22 base pairs nucleic acid single linear other nucleic acid /desc = “Primer e” 5 CGGAACACTC TACCGCATTA CC 22 37 base pairs nucleic acid single linear other nucleic acid /desc = “Primer F” 6 CGGCGTGCTG CTGGCAGGAA GACATGTCCC AATTAAC 37 37 base pairs nucleic acid single linear other nucleic acid /desc = “Primer G” 7 GTTAATTGGG ACATGTCTTC CTGCCAGCAG CACGCCG 37 19 base pairs nucleic acid single linear other nucleic acid /desc = “Primer 1” 8 CAGCGTCCAC ATCACGAGC 19 26 base pairs nucleic acid single linear other nucleic acid /desc = “Primer 2” 9 GAAGAAGCAC GTTTCTGCAG AGACCG 26 26 base pairs nucleic acid single linear other nucleic acid /desc = “Primer 3” 10 CGGTCTCTCG AGAAACGTGC TTCTTC 26 19 base pairs nucleic acid single linear other nucleic acid /desc = “Primer 4” 11 GCCACTTCCG TTATTAGCC 19 6032 base pairs nucleic acid single linear DNA (genomic) Trichoderma harzianum CBS 243.71 CDS 3188..5092 12 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 CGCAGCGAGR 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 Ile 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 Ile Gly Ile 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 Gln Arg Ala Lys Ala Ala Gly Ile 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 Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln 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 Ile 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 Gln Lys 115 120 125 ATT GCG CAG TAT GCC AGC CGT CCC GCC CAG CTG TAT GTT GAC AAC CGG 3613 Ile Ala Gln Tyr Ala Ser Arg Pro Ala Gln 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 Gln Ser Ser Pro Ser Asn Ile 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 Gln 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 Ile 240 245 250 TAT AAC CGG TGG CAA CAG GTC TTG CAG CAG GGC TTC CCC ATG GTT GAG 3997 Tyr Asn Arg Trp Gln Gln Val Leu Gln Gln 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 Ile 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 Ile 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 Ile Ser Lys Tyr Val Gln Asn Glu Gln 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 Gln 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 Gln Thr Phe Gln Ala Asn Ala Gly Ala Asn Leu Phe Gln Ile 400 405 410 CCT GCC AGC ATC GGC CAG CAA AAG TTT GCT CTA ACT CGC AAC GGT CAG 4477 Pro Ala Ser Ile Gly Gln Gln Lys Phe Ala Leu Thr Arg Asn Gly Gln 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 Ile 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 Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Ile 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 Gln Ala Asp Gly Leu Phe Ser Leu Thr Ile 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 Gln 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 Gln 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 Ile Gly Leu Cys Gln 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 Ile 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 Gln 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 1155 base pairs nucleic acid single linear other nucleic acid /desc = “mbd-lipase hybrid” sig_peptide 1..66 CDS 1..1155 13 ATG AGG AGC TCC CTT GTG CTG TTC TTT GTC TCT GCG TGG ACG GCC TTG 48 Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu 1 5 10 15 GCC AGT CCT ATT CGT CGA GAG GTC TCG CAG GAT CTG TTT AAC CAG TTC 96 Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe 20 25 30 AAT CTC TTT GCA CAG TAT TCT GCA GCC GCA TAC TGC GGA AAA AAC AAT 144 Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn 35 40 45 GAT GCC CCA GCT GGT ACA AAC ATT ACG TGC ACG GGA AAT GCC TGC CCC 192 Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro 50 55 60 GAG GTA GAG AAG GCG GAT GCA ACG TTT CTC TAC TCG TTT GAA GAC TCT 240 Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser 65 70 75 80 GGA GTG GGC GAT GTC ACC GGC TTC CTT GCT CTC GAC AAC ACG AAC AAA 288 Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys 85 90 95 TTG ATC GTC CTC TCT TTC CGT GGC TCT CGT TCC ATA GAG AAC TGG ATC 336 Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile 100 105 110 GGG AAT CTT AAC TTC GAC TTG AAA GAA ATA AAT GAC ATT TGC TCC GGC 384 Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly 115 120 125 TGC AGG GGA CAT GAC GGC TTC ACT TCG TCC TGG AGG TCT GTA GCC GAT 432 Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp 130 135 140 ACG TTA AGG CAG AAG GTG GAG GAT GCT GTG AGG GAG CAT CCC GAC TAT 480 Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr 145 150 155 160 CGC GTG GTG TTT ACC GGA CAT AGC TTG GGT GGT GCA TTG GCA ACT GTT 528 Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val 165 170 175 GCC GGA GCA GAC CTG CGT GGA AAT GGG TAT GAT ATC GAC GTG TTT TCA 576 Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser 180 185 190 TAT GGC GCC CCC CGA GTC GGA AAC AGG GCT TTT GCA GAA TTC CTG ACC 624 Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr 195 200 205 GTA CAG ACC GGC GGA ACA CTC TAC CGC ATT ACC CAC ACC AAT GAT ATT 672 Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile 210 215 220 GTC CCT AGA CTC CCG CCG CGC GAA TTC GGT TAC AGC CAT TCT AGC CCA 720 Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro 225 230 235 240 GAG TAC TGG ATC AAA TCT GGA ACC CTT GTC CCC GTC ACC CGA AAC GAT 768 Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp 245 250 255 ATC GTG AAG ATA GAA GGC ATC GAT GCC ACC GGC GGC AAT AAC CAG CCT 816 Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro 260 265 270 AAC ATT CCG GAT ATC CCT GCG CAC CTA TGG TAC TTC GGG TTA ATT GGG 864 Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly 275 280 285 ACA TGT CTT CCT GCC AGC AGC ACG CCG CCA TCG GGT CAG GTT TGC GTT 912 Thr Cys Leu Pro Ala Ser Ser Thr Pro Pro Ser Gly Gln Val Cys Val 290 295 300 GCC GGC ACC GTT GCT GAC GGC GAG TCC GGC AAC TAC ATC GGC CTG TGC 960 Ala Gly Thr Val Ala Asp Gly Glu Ser Gly Asn Tyr Ile Gly Leu Cys 305 310 315 320 CAA TTC AGC TGC AAC TAC GGT TAC TGT CCA CCG GGA CCG TGT AAG TGC 1008 Gln Phe Ser Cys Asn Tyr Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys 325 330 335 ACC GCC TTT GGT GCT CCC ATC TCG CCA CCG GCA AGC AAT GGG CGC AAC 1056 Thr Ala Phe Gly Ala Pro Ile Ser Pro Pro Ala Ser Asn Gly Arg Asn 340 345 350 GGC TGC CCT CTA CCG GGA GAA GGC GAT GGT TAT CTG GGC CTG TGG AGT 1104 Gly Cys Pro Leu Pro Gly Glu Gly Asp Gly Tyr Leu Gly Leu Trp Ser 355 360 365 TTC AGT TGT AAC CAT AAT TAC TGC CCG CCA ACG GCA TGC CAA TAC TGT 1152 Phe Ser Cys Asn His Asn Tyr Cys Pro Pro Thr Ala Cys Gln Tyr Cys 370 375 380 TAG 1155 * 385 384 amino acids amino acid linear protein 14 Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu 1 5 10 15 Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe 20 25 30 Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn 35 40 45 Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro 50 55 60 Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser 65 70 75 80 Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys 85 90 95 Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile 100 105 110 Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly 115 120 125 Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp 130 135 140 Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr 145 150 155 160 Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val 165 170 175 Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser 180 185 190 Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr 195 200 205 Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile 210 215 220 Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro 225 230 235 240 Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp 245 250 255 Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro 260 265 270 Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly 275 280 285 Thr Cys Leu Pro Ala Ser Ser Thr Pro Pro Ser Gly Gln Val Cys Val 290 295 300 Ala Gly Thr Val Ala Asp Gly Glu Ser Gly Asn Tyr Ile Gly Leu Cys 305 310 315 320 Gln Phe Ser Cys Asn Tyr Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys 325 330 335 Thr Ala Phe Gly Ala Pro Ile Ser Pro Pro Ala Ser Asn Gly Arg Asn 340 345 350 Gly Cys Pro Leu Pro Gly Glu Gly Asp Gly Tyr Leu Gly Leu Trp Ser 355 360 365 Phe Ser Cys Asn His Asn Tyr Cys Pro Pro Thr Ala Cys Gln Tyr Cys 370 375 380 42 base pairs nucleic acid single linear other nucleic acid <Unknown> 15 GATCCTCACA ATGTTGGGCG TTGTCCGCCG TCTAGGCCTA GG 42 40 base pairs nucleic acid single linear other nucleic acid <Unknown> 16 CGCCTAGGCC TAGACGTCGG ACAACGCCCA ACATTGTGAG 40 11 amino acids amino acid linear protein 17 Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly 5 10 14 base pairs nucleic acid single linear other nucleic acid <Unknown> 18 CCAATACTGT TAGT 14 22 base pairs nucleic acid single linear other nucleic acid <Unknown> 19 CTAGACTAAC AGTATTGGCA TG 22 5 amino acids amino acid linear protein 20 Ala Cys Gln Tyr Cys 5

Claims

What is claimed is:

1. A polypeptide hybrid comprising an amino acid sequence with binding affinity for mutan, said amino acid sequence being bound to an active component useful for oral care purposes.

2. The polypeptide hybrid of claim 1, wherein the active component is an anti-plaque, anti-stain or anti-microbial agent.

3. The polypeptide hybrid of claim 1, wherein the active component is an enzyme selected from the group consisting of oxidases, peroxidases, proteases, lipases, glucosidases, lipases, esterases, deaminases, ureases and polysaccharide hydrolases.

4. The polypeptide hybrid of claim 3, which polypeptide is bound to an enzyme with glycosidase activity.

5. An oral care composition comprising a mutan binding domain (MBD) hybrid or single unit MBD and further ingredients conventionally used in oral care compositions.

6. The oral care composition of claim 5, wherein the MBD hybrid is a polypeptide hybrid according to claim 1.

7. An oral care product comprising an oral care composition of claim 5.

8. The oral care product of claim 7, being a dentifrice.

9. A hybrid comprising:

(a) an amino acid sequence which has binding affinity for mutan and which lacks a functional catalytic domain of a mutanase, linked to

(b) an enzyme selected from the group consisting of deaminases, esterases, glucosidases, lipases, oxidases, peroxidases, polysaccharide hydrolases, proteases, and ureases.

10. The hybrid of claim 9, wherein the enzyme is a glycosidase.

11. An oral care composition comprising (a) a hybrid of claim 9 or a single unit mutan binding domain and (b) further ingredients conventionally used in oral care compositions.

12. An oral care product comprising an oral care composition of claim 11.

13. The oral care product of claim 12, which is a dentifrice.