Classifications

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

Definitions

• compositions useful in oral care products for humans and animals are also the object of the invention to provide compositions useful in oral care products for humans and animals.
• Figure 2 shows plasmid construction of plasmids pMT1796, pMT1802, and pMT1815
• Figure 3 shows an outline of the construction of the A. oryzae recombinant mutanase expression vector pMT1802
• Figure 6 shows the temperature stability of recombinant and wild- type ⁇ . harzianum CBS 243.71 mutanase at pH 7,
• the mutanase is obtained from a strain within the genus Trichoderma .
• the invention relates to a process for producing a recombinant mutanase in a host cell, comprising the steps: a) transforming an expression vector comprising a mutanase gene with a kex2 site or kex2-like site between the DNA sequences encoding the pro-peptide and the mature region of the mutanase into a suitable filamentous fungus host cell, b) cultivating the host cell in a suitable culture medium under conditions permitting expression and secretion of an active mutanase, c) recovering and optionally purifying the secreted active re- combinant mutanase from the culture medium.
• the expression vector may be prepared according to the above described method of the invention.
• a recombinant mutanase may according to the invention be produced according to the process of the invention.
• a substantially pure wild-type mutanase obtained from Trichoderma harzianum CBS 243.71 essentially free of any contaminants is also part of the invention.
• the invention also relates to a composition comprising a recombinant mutanase of the invention or a substantially pure mutanase of the invention useful in oral care products and food, feed and/or pet food products.
• mutanases are produced homologously and comprise a mixture of other enzyme activities besides the mutanase (i.e. with undesired contaminants) .
• An example of this is Trichoderma harzianum CBS 243.71 which are known to produce a mutanase as also described above.
• the mutanase derived from Trichoderma harzianum CBS 243.71 has before the successful findings of the present invention only been produced homologously. It is advantageous to be able to produce the mutanase heterologously, as it is then possible to provide a single component mutanase free of undesired contaminants. Further, it facilitates providing an isolated and purified enzyme of the invention in industrial scale.
• Removal of the pro-peptide from the mature protein occurs in general by processing by a specific endopeptidase, usually after the two positively charged amino acid residues Arg-Arg, Arg-Lys or Lys-Arg.
• other amino acid combinations containing at least one basic amino acid, have been found to be processed.
• Kex2 sites see e . g. Methods in Enzymology Vol 185, ed. D. Goeddel, Academic Press Inc. (1990) , San Diego, CA, "Gene 5 Expression Technology"
• kex2-like sites are di-basic recognition sites (i.e. cleavage sites) found between the pro- peptide encoding region and the mature region of some proteins. Insertion of a kex2 site or a kex2-like site have in certain cases been shown to improve correct endopeptidase processing at
• primers are designed from the knowledge to at least a part of SEQ ID No. 2. Fragments of mutanase gene are then PCR amplified by the use of these primers. These fragments are used as probes for cloning the complete gene.
• the DNA sequence encoding a mutanase may be isolated by a general method involving
• the promoter may be the TAKA promoter or the TAKA:TPI promoter.
• the expression vector is the pMT1796 used to illustrate the concept of the invention in Example 3 below.
• the host cell is a protease deficient or protease minus strain.
• the pH optimum of the recombinant mutanase was found to lie in the range from 3.5 to 5.5 which equals the pH optimum of the wild-type mutanase (see Figure 4) .
• the temperature optimum of both the recombinant and wild-type mutanase was found to be around 45°C at pH 7 and around 55°C at pH 5.5 (see Figure 5). Further, the residual activity starts to decline at 40°C at pH 7, while the enzyme is more stable at pH 5.5, where the residual activity starts to decline at 55°C.
• the abrasive product may be present in from 0 to 70% by weight, preferably from 1% to 70%.
• the abrasive material content typically lies in the range of from 10% to 70% by weight of the final toothpaste product.
• Humectants are employed to prevent loss of water from e .g. toothpastes.
• Suitable humectants for use in oral care products according to the invention include the following compounds and mixtures thereof: glycerol, polyol, sorbitol, polyethylene glycols (PEG), propylene glycol, 1,3-propanediol, 1,4-butanediol, hydrogenated partially hydrolysed polysaccharides and the like.
• Humectants are in general present in from 0% to 80%, preferably 5 to 70% by weight in toothpaste.
• foaming agent soap an-ionic, cat-ionic, non-ionic, a pho- teric and/or zwitterionic surfactants can be used. These may be present at levels of from 0% to 15%, preferably from 0.1 to 13%, more preferably from 0.25 to 10% by weight of the final product.
• the oral care composition and products of the present invention can be made using methods which are common in the oral product area.
• Primer 1 5' GGGGGGATCCACCATGAG 3' (SEQ ID No. 3)
• Primer 2 5' ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC 3' (SEQ ID No. 4)
• Primer 3 5' GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT 3 1 (SEQ ID No. 5)
• Primer 4 5' CCACGGTCACCAACAATAC 3' (SEQ ID No. 6)
• Primer 5 GGGGGGATCCACCATGAG (SEQ ID No. 7)
• Primer 6 ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC (SEQ ID No.
• Phenyl-sepharose FF (high sub) column (Pharmacia)
• a 5% mutan suspension is made in 50 mM sodium acetate, pH 5.5 and the suspension is homogenised for 15 minutes in an Ultra
• the sample to be analyzed for mutanase activity is applied in sample wells punched in the agarose, and the plate is incubated overnight at 37 °C, whereafter clearing zones are formed around mutanase containing samples .
• Mass spectrometry of purified wild-type mutanase is done using matrix assisted laser desorption ionization time-of-flight mass spectrometry in a VG Analytical TofSpec.
• 2 ml of sample is mixed with 2 ml saturated matrix solution (a- cyano-4-hydroxycinnamic acid in 0.1% TFA:acetonitrile (70:30)) and 2 ml of the mixture spotted onto the target plate. Before introduction into the mass spectrometer the solvent is removed by evaporation. Samples are desorbed and ionized by 4 ns laser pulses (337 nm) at threshold laser power and accelerated into the field-free flight tube by an accelerating voltage of 25 kV. Ions are detected by a microchannel plate set at 1850 V.
• Hydroxyapatite tablets are prepared by compressing 250 mg of hydroxyapatite in a tablet die at about 5,900 kg (13,000 lbs) of pressure for 5 minutes. The tablets are then sintered at 600°C for 4 hours and finally hydrated with sterile deionized water.
• the Malthus-method is based on the methods described in Johnston et al., (1995), Journal of Microbiological Methods 21, p. 15-26 and Johansem et al. (1995), Journal of Applied Bacteriology 78, p. 297-303.
• the combined mutanase pool was concentrated in a Filtron concentrator equipped with a 10 kDa cut-off membrane and followed by a buffer change to 10 mM Tris-HCl, pH 8.0.
• the final volume of the concentrate was 870 ml.
• Biosystems 473A protein sequencer To generate peptides reduced and S-carboxymethylated mutanase
• Vydac Ci ⁇ column eluted with a linear gradient of 80% 2-propanol containing 0.08% TFA in 0.1% aqueous TFA.
• Peptides were repurified by reversed-phase-HPLC using a Vydac C ⁇ column eluted with linear gradients of 80% acetonitrile containing 0.08% TFA in
• the disks were either DAPI stained or transferred to Malthus cells, as indirect impedance measurements were used when enumerating living adherent cells (Malthus Flexi M2060, Malthus Instrument Limited) .
• the removal of plaque from the disks was also determined by fluorescent microscopy, after mutanase treatment disks were stained with DAPI (3 mM) and incubated in the dark for 5 minutes (20°C) .
• the DAPI stained cells were examined with the x 100 oil immersion fluorescence objective on an Olympus model BX50 microscope equipped with a 200 W mercury lamp and an UV- filter. The result was compared with the quantitative data obtained by the impedance measurements.
• the number of living cells on the saliva treated HA-surface after enzyme treatment was determined by the Malthus method and shown in Table 1. However, by the Malthus method it is not possible to distinguish between a bactericidal activity of mutanase or an enzymatic removal of the plaque. Therefore a decrease in living bacteria on the surface has to be compared with the simultaneously removal of plaque from the surface which is estimated by the DAPI staining.
• mutanase reduced the amount of adhering cells.
• the activity was observed as a removal of plaque and not as a bactericidal activity against cells in plaque.
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• AGCATTGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG 1680 GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT 1740 ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG 1800 GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT 1860 GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA 1920 TTACCGCCTT TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT 1980 CAGTGAGCGA GGAAGCGGAA GAGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC 2040 CGATTCATTA ATGC
• GCT CCC ATC TCG CCA CCG GCA AGC AAT GGG CGC AAC GGC TGC CCT CTA 5005

Landscapes

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

Applications Claiming Priority (5)

Publications (1)

Family

ID=26064514

Family Applications (1)

Country Status (6)

Families Citing this family (19)

Family Cites Families (5)

• 1997
  • 1997-06-30 CA CA002258291A patent/CA2258291A1/en not_active Abandoned
  • 1997-06-30 EP EP97928131A patent/EP0954570A1/en not_active Withdrawn
  • 1997-06-30 CN CN97196831A patent/CN1226282A/zh active Pending
  • 1997-06-30 AU AU32544/97A patent/AU721693B2/en not_active Ceased
  • 1997-06-30 WO PCT/DK1997/000283 patent/WO1998000528A1/en not_active Application Discontinuation
  • 1997-06-30 JP JP10503763A patent/JP2000514288A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events