EP0954570A1 - Rekombinantes Enzym mit Mutanaseaktivität - Google Patents

Rekombinantes Enzym mit Mutanaseaktivität

Info

Publication number
EP0954570A1
EP0954570A1 EP97928131A EP97928131A EP0954570A1 EP 0954570 A1 EP0954570 A1 EP 0954570A1 EP 97928131 A EP97928131 A EP 97928131A EP 97928131 A EP97928131 A EP 97928131A EP 0954570 A1 EP0954570 A1 EP 0954570A1
Authority
EP
European Patent Office
Prior art keywords
mutanase
pro
gly
ser
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97928131A
Other languages
English (en)
French (fr)
Inventor
Jill Angela Wahleithner
Claus Crone Fuglsang
Torben Halkier
Charlotte Johansen
Mogens Trier Hansen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0954570A1 publication Critical patent/EP0954570A1/de
Withdrawn legal-status Critical Current

Links

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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

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)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Nutrition Science (AREA)
  • Epidemiology (AREA)
  • Birds (AREA)
  • Animal Husbandry (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Cosmetics (AREA)
  • Fodder In General (AREA)
EP97928131A 1996-06-28 1997-06-30 Rekombinantes Enzym mit Mutanaseaktivität Withdrawn EP0954570A1 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DK71596 1996-06-28
DK71596 1996-06-28
DK100496 1996-09-17
DK100496 1996-09-17
PCT/DK1997/000283 WO1998000528A1 (en) 1996-06-28 1997-06-30 A recombinant enzyme with mutanase activity

Publications (1)

Publication Number Publication Date
EP0954570A1 true EP0954570A1 (de) 1999-11-10

Family

ID=26064514

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97928131A Withdrawn EP0954570A1 (de) 1996-06-28 1997-06-30 Rekombinantes Enzym mit Mutanaseaktivität

Country Status (6)

Country Link
EP (1) EP0954570A1 (de)
JP (1) JP2000514288A (de)
CN (1) CN1226282A (de)
AU (1) AU721693B2 (de)
CA (1) CA2258291A1 (de)
WO (1) WO1998000528A1 (de)

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Publication number Priority date Publication date Assignee Title
AU716989B2 (en) * 1996-04-16 2000-03-16 Novozymes A/S Compositions for the removal of dental plaque
AU4550997A (en) * 1996-10-11 1998-05-11 Novo Nordisk A/S Cellulose binding domains (cbds) for oral care products
ES2163988B1 (es) * 1999-07-31 2003-04-01 Newbiotechnic Sa Proteina con actividad antifungica.
AU782624B2 (en) * 1999-12-30 2005-08-18 Board Of Trustees Of Miami University Methods and compositions for inhibiting adhesion by microorganisms
JP4495904B2 (ja) * 2002-03-01 2010-07-07 天野エンザイム株式会社 改変プロモーター
JP2007501218A (ja) 2003-08-04 2007-01-25 ファイザー・プロダクツ・インク 非晶質薬物の吸着物および親油性ミクロ相形成物質の医薬組成物
US8809392B2 (en) 2008-03-28 2014-08-19 Ecolab Usa Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
US8871807B2 (en) 2008-03-28 2014-10-28 Ecolab Usa Inc. Detergents capable of cleaning, bleaching, sanitizing and/or disinfecting textiles including sulfoperoxycarboxylic acids
WO2009118714A2 (en) 2008-03-28 2009-10-01 Ecolab Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
US12203056B2 (en) 2008-03-28 2025-01-21 Ecolab Usa Inc. Sulfoperoxycarboxylic acids, their preparation and methods of use as bleaching and antimicrobial agents
DK2280686T3 (en) * 2008-04-11 2015-01-26 Danisco Us Inc Alpha-glucanase and oral care composition containing the same
CN102277374B (zh) * 2011-07-27 2013-04-17 浙江诺倍威生物技术有限公司 一种鸡传染性法氏囊病基因工程亚单位疫苗的制备和应用
US9321664B2 (en) 2011-12-20 2016-04-26 Ecolab Usa Inc. Stable percarboxylic acid compositions and uses thereof
CA2867565C (en) 2012-03-30 2021-01-19 Victor KEASLER Use of peracetic acid/hydrogen peroxide and peroxide-reducing agents for treatment of drilling fluids, frac fluids, flowback water and disposal water
US20140256811A1 (en) 2013-03-05 2014-09-11 Ecolab Usa Inc. Efficient stabilizer in controlling self accelerated decomposition temperature of peroxycarboxylic acid compositions with mineral acids
US8822719B1 (en) 2013-03-05 2014-09-02 Ecolab Usa Inc. Peroxycarboxylic acid compositions suitable for inline optical or conductivity monitoring
US10165774B2 (en) 2013-03-05 2019-01-01 Ecolab Usa Inc. Defoamer useful in a peracid composition with anionic surfactants
MX2021014340A (es) 2019-05-31 2022-01-06 Ecolab Usa Inc Metodo de monitoreo de las concentraciones de peracidos mediante mediciones de conductividad y composiciones de peracidos.
US12096768B2 (en) 2019-08-07 2024-09-24 Ecolab Usa Inc. Polymeric and solid-supported chelators for stabilization of peracid-containing compositions
WO2021202625A1 (en) 2020-03-31 2021-10-07 Ecolab Usa Inc. Method for quenching peroxycarboxylic acid runaway reactions
CN113151330B (zh) * 2021-03-30 2023-09-08 云南师范大学 一种酸性蛋白酶突变体及其制备方法和应用
CN113684194B (zh) * 2021-08-31 2023-06-13 湖南大地同年生物科技有限公司 一种突变的马达蛋白及其应用、试剂盒
CN115975956A (zh) * 2022-02-23 2023-04-18 杨挥 一种重组酶突变体及包含该突变体的核酸等温扩增反应体系

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Also Published As

Publication number Publication date
JP2000514288A (ja) 2000-10-31
CN1226282A (zh) 1999-08-18
WO1998000528A1 (en) 1998-01-08
AU3254497A (en) 1998-01-21
AU721693B2 (en) 2000-07-13
CA2258291A1 (en) 1998-01-08

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