WO1994005778A1 - Nouveaux produits de depolymerisation de chitosane partiellement acetyle, nouvelles enzymes et nouvelles souches de thermoactinomyces destinees a leur production - Google Patents

Nouveaux produits de depolymerisation de chitosane partiellement acetyle, nouvelles enzymes et nouvelles souches de thermoactinomyces destinees a leur production Download PDF

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Publication number
WO1994005778A1
WO1994005778A1 PCT/EP1993/002352 EP9302352W WO9405778A1 WO 1994005778 A1 WO1994005778 A1 WO 1994005778A1 EP 9302352 W EP9302352 W EP 9302352W WO 9405778 A1 WO9405778 A1 WO 9405778A1
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WO
WIPO (PCT)
Prior art keywords
chitosan
new
strain
chitosanase
enzyme
Prior art date
Application number
PCT/EP1993/002352
Other languages
English (en)
Inventor
Bruno Maurice Léon BRODEL
Philippe Victor Jean Debeire
Pierre Frédéric Emmanuel MONSAN
François Marie Bernard PAUL
Jean-Pierre Marie Touzel
Original Assignee
Sandoz Ltd.
Sandoz-Patent-Gmbh
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
Priority claimed from GB929218512A external-priority patent/GB9218512D0/en
Priority claimed from GB929223493A external-priority patent/GB9223493D0/en
Application filed by Sandoz Ltd., Sandoz-Patent-Gmbh filed Critical Sandoz Ltd.
Priority to EP93919229A priority Critical patent/EP0659210A1/fr
Publication of WO1994005778A1 publication Critical patent/WO1994005778A1/fr

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Classifications

    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
    • D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
    • D21H17/005—Microorganisms or 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/736—Chitin; Chitosan; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/28—Polysaccharides or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
    • A61L17/06—At least partially resorbable materials
    • A61L17/10—At least partially resorbable materials containing macromolecular materials
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • 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)
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • 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)
    • C12Y302/01132—Chitosanase (3.2.1.132)

Definitions

  • the invention relates to a process for the production of depolymerized partially acetylated chitosan and to the new products obtained by this process, to the use of microorganisms of the genus Thermoactinomyces in a process for the production of an enzyme which depolymerizes partially acetylated chitosan and to a new strain of Thermoactinomyces .
  • Chitosan is the product of partial or complete deacetylation of chitin. Both chitinase (EC 3.2.1.14) and chitosanase act on partially acetylated chitosan. Such depolymerisation has been described with an enzyme of microbial origin, especially of the genus Bacillus and it has been tried to digest chitosan with chitinase produced by Streptomyces antibioticus. All these processes have not allowed commercial production of chitosan hydrolysates by enzymatic hydrolysis of industrial chitosans.
  • Such products are of interest in various fields, especially in the fields which require the use of concentrated chitosan solutions of low viscosity and/or the use of well characterized chitosan fractions (acetylation degree, molecular weight distribution, viscosity).
  • Thermoactinomyces produce substantial quantities of an enzyme which depolymerizes partially acetylated chitosan when cultivated under appropriate conditions and that a new strain of Thermoactinomyce s is of special interest in this respect.
  • This enzyme has some activity on colloidal chitin (i.e. 100% acetylated chitosan) and, therefore, acts as as chitinase.
  • the right substrate is a partially acetylated chitosan having an acetylation degree between 10% and 70% (which is the case with most industrial grades of chitosan).
  • the enzyme which depolymerizes partially acetylated chitosan will be called chitosanase.
  • the invention therefore, relates to a process for the production of such chitosanase which is characterized by the cultivation of Thermoactinomyces sp volunteer to a process for the production of such chitosan hydrolysates which is characterized by the use of such chitosanase and to the new strain of Thermoactinomyces which is identified by its deposition no. 1-1052 at the Institut Pasteur, Paris.
  • the invention further relates to the new products obtained by this process and which are characterized by their viscosity, molecular weight distribution and degree of acetylation. In general, the molecular weight distribution will be determined by capillary viscosimetry and can vary from 10,000 to 1,000,000. The degree of acetylation can vary between 10 and 70%.
  • Thermoactinomyces is a well known member of the actinomycetes family and is described in Bergey's Manual of Systematic Bacteriology, vol. 4 (1989), Williams & Wilkins, pages 2573-85.
  • One of its character istics is the fact that colonies grow at high temperatures (55°C) and that they are extremely heat resistant.
  • Typical species are T. vulgaris, T. thalpophilus, T. sacchari, T. dichotomicus, T. intermedius and T. putidus. (See the list of characteristics on pages 2583 and 2584 of the above-mentioned Manual).
  • the strains are cultivated at 55°C in a culture medium containing an inducer like N-acetyl-D-glucosamine (hereinafter N-AGA).
  • N-AGA N-acetyl-D-glucosamine
  • Other useful inducers are colloidal chitin, chitosan oligosaccharides and chitosan. The best inducer is N-acetylglucosamine.
  • a typical culture medium contains 2 g/1 of such inducer, 2 g/1 of yeast extract, 2 g/1 casein hydrolysate, 50 ml/1 of a solution of mineral salts, 10 ml/1 of vitamins, 10 ml 1 of trace minerals, 1 g/1 of ammonium chloride and 50 mM MOPS buffer.
  • the first step is a batch culture during which the strain is grown on the standard medium except that N-acetylglucosamine is substituted by glucose as a carbon source. Under such conditions, biomass is produced and no enzyme synthesis can be observed because no inducer is present in the culture medium.
  • the chitosanase synthesis is induced by adding sterile N-AGA at a constant flow rate of 0.6 g N-AGA/h into the fermentor with a peristaltic pump.
  • chitosanase After a period of 24 hours typical amounts of 0.1 to 2.0 U/ml of chitosanase can be obtained.
  • the culture broth can be used as such or the chitosanase is first isolated and concentrated before incubating it with the substrate.
  • the chitosanase activity of the culture broth is assayed at 60°C and pH 5 with a chitosan (20% acetylation degree) concentration of 1 % (w/v) in that the reducing power of the solution is measured after 5 and 10 minutes according to Kidby and Davidson, Anal. Biochem. 55(1), 321-25 (1973).
  • Standard solutions of N-acetyl-D-glucosamine (N-AGA) are used as reference.
  • One chito ⁇ sanase unit (U) corresponds to the appearance of one micromole of N-AGA per minute.
  • Thermoactinomyces which is identified by its deposition no. 1-1052 at the Institut Pasteur, Paris (deposition date March 1, 1991).
  • This new strain was isolated from a marshland in Hondschoote (France) in March 1989 and has the typical characteristics of the genus Thermoactinomyces as follows: thermophilic nature (growth at 55 ⁇ C), formation of aerial mycelium, Gram-positive reaction, single spores on both the aerial and substrate mycelium, resistance of the spores at lOO'C for 10 minutes and much ramification of the mycelium.
  • Biochemical characteristics of the new strain in comparison to other Thermoactinomyce s strains are summarised in Table 1. In its properties the new strain is most comparable to T. sacchari DSM 43356 and T. vulgaris DSM 43062 which are however, less productive in chitosanase.
  • acetic acid or any convenient organic acid e.g. formic or lactic acid
  • Partially deacetylated chitin polymers with a degree of acetylation above 25% are not industrially available at present.
  • an industrial grade of chitosan with a degree of acetylation of about 20% can be submitted to partial reacetylation according to the method of Hirano S., Tsuchida M. and Nagao N., Biomaterials (1989), Vol. 10 (October), 574- . More particularly, reacetylation can be performed under the following conditions:
  • the intrinsic viscosity ([ ⁇ ]) of each chitosan sample is determined using a capillary viscometer (Ubbelohde type, 25°C) at various polymer concentrations.
  • Intrinsic viscosity allows the determination of viscosimetric average molecular weight (Mv) by using the following relation:
  • the degree of acetylation (D.A.) is assayed by *H N.M.R. spectroscopy using an AC 300 Bruker model, at 30°C. Samples are purified by alkaline precipitation, lyophilized and solubilized in D 2 O.
  • D.A. is determined using the -CH 3 signal (1.91 ppm) and compared to Hj signal (reference).
  • the new products obtained according to the invention can be used in various industrial applications. They are useful additives for pharmaceutical compositions in which they act as an efficient promotor of wound healing and inhibitor of fibroplasia. They are also of interest for wound dressings and production of absorbable sutures.
  • the new products can be used for hair, skin and mouth care, where presently cosmetic formulations are limited by the poor solubility of high molecular weight polymers.
  • oligosaccharides are especially useful.
  • SUBSTITUTE SHEETs can also be used as auxiliaries in paper making in which they replace (in part or in total) synthetic polymers used as flocculating agents, fixing agents, drainage agents and retention agents.
  • thermophilic microorganisms Twenty samples collected from various ecological environments were screened for the isolation of chitosanase producing bacteria. This step of screening was performed at 55 ⁇ C in order to obtain thermophilic microorganisms.
  • the first step consisted of an enrichment investigated in liquid medium in erlenmeyer flasks of 125 ml containing 10 ml of medium in which was dispersed each sample.
  • the mineral salt solution contains 6g KH 2 PO 4 , 12g NaCl, 2.4g MgSO 4 and 1.6g CaCl s per liter H 2 O.
  • the vitamin solution contains 2mg biotin, 2mg folic acid, lOmg pyridoxin-HCl, 5mg thiamin-HCl, 5mg riboflavin, 5mg nicotinic acid, 5mg Calcium pantothenate, O.lmg vitamins B 12
  • the oligoelement solution contains 0.3g FeS0 4 .7 H 2 0, O.lg MnCl 2 , O.lg CoCl 2 .2 H 2 O, O.lg ZmCl 2 , 0.02g CuCl 2 , O.Olg H 3 BO 4 , 0.017g NA 2 SeO 3 , 0.026g NiS0 4 .6 H 2 O and 12.8g nitrilotriacetic acid per liter H 2 O.
  • each sample was assayed at 60°C for chitosanase activity with the procedure mentioned above.
  • the samples showing enzymatic activity towards chitosan were selected for isolation of microorganisms on agar plates containing collidal chitin.
  • the second step of the screening consisted of an isolation of the microorganisms on agar plates containing colloidal chitin.
  • the test for enzymic activity was based on the appearance of clearing zones around the active colonies. Due to its insolubility at pH 7.0, chitosan could not be used for agar plates and colloidal chitin was substituted for chitosan in this second step.
  • composition of the agar plates was: Nutritive Gelose supplemented with colloidal chitin (4 g/1) prepared by the method described in Methods in Enzymology (vol. 161 (1988)).
  • N-AGA N-acetyl-D-glucosamine
  • the activity of different Thermoactinomvces strains is determined according to the method described above using the culture broth after 24 hours of fermentation in erlenmeyer flasks. The results are summarised in Table 2
  • a batch culture using the medium described in Example 2 allows the production of 0.56 U/ml after 24 hours of fermentation.
  • the first step is a batch type culture during which the strain 1-1052 is grown on the medium described in Example 2 except that the N-acetylglucosamine is substituted by glucose. Under such conditions biomass is produced and no enzyme synthesis can be observed because no inducer is present in the culture medium.
  • the chitosanase synthesis is induced by adding sterile N-acetylglucosamine (N-AGA) at a constant flow rate of 0.48g N-AGA/h into the 2 liter fermentor with a peristaltic pump (fed-batch process).
  • N-AGA sterile N-acetylglucosamine
  • This (fed-batch) process allows the obtention of 2 U/ml of chitosanase after 24 hours of fermentation with the strain 1-1052.
  • a 10% chitosan solution of PROFLOC 340 (degree of acetylation ⁇ 30%; Protan Laboratories, Norway) is prepared using acetic acid with a final concentration of 4%. The pH of this solution is 4.8. The enzyme of Example 1 is added and the solution incubated at 60°C. The enzyme concentration is 0.25 U/ml, corresponding to 2.5 U/g of chitosan

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  • Health & Medical Sciences (AREA)
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Abstract

On a découvert que différentes souches du genre Thermoactynomyces produisent des quantités substantielles de chitosanase lorsqu'on les cultive dans des conditions appropriées, et qu'une de ces souches, identifiée I-1052, Institut Pasteur, Paris, présente un intérêt particulier. On a isolé une nouvelle enzyme thermostable dans le bouillon de culture de cette nouvelle souche. Cette nouvelle enzyme hydrolyse des chitosanes acétylés à des degrés divers. On obtient de nouveaux produits de chitosane partiellement acétylé dépolymérisé grâce à ces nouvelles enzymes, produits caractérisés par leur viscosité, leur répartition de poids moléculaires et leur degré d'acétylation. On peut utiliser ces nouveaux produits dans des compositions pharmaceutiques et cosmétiques et en tant qu'auxiliaires dans la fabrication du papier.
PCT/EP1993/002352 1992-09-01 1993-08-31 Nouveaux produits de depolymerisation de chitosane partiellement acetyle, nouvelles enzymes et nouvelles souches de thermoactinomyces destinees a leur production WO1994005778A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP93919229A EP0659210A1 (fr) 1992-09-01 1993-08-31 Nouveaux produits de depolymerisation de chitosane partiellement acetyle, nouvelles enzymes et nouvelles souches de thermoactinomyces destinees a leur production

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9218512.3 1992-09-01
GB929218512A GB9218512D0 (en) 1992-09-01 1992-09-01 Improvements in or relating to organic compounds
GB929223493A GB9223493D0 (en) 1992-11-10 1992-11-10 Improvements in or relating to organic compounds
GB9223493.9 1992-11-10

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Publication Number Publication Date
WO1994005778A1 true WO1994005778A1 (fr) 1994-03-17

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PCT/EP1993/002352 WO1994005778A1 (fr) 1992-09-01 1993-08-31 Nouveaux produits de depolymerisation de chitosane partiellement acetyle, nouvelles enzymes et nouvelles souches de thermoactinomyces destinees a leur production

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EP (1) EP0659210A1 (fr)
CA (1) CA2143120A1 (fr)
WO (1) WO1994005778A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2736835A1 (fr) * 1995-07-17 1997-01-24 Aber Technologies Pansement pour plaies chroniques, notamment escarres, en gel de chitine
DE102012009593A1 (de) 2012-05-07 2013-11-07 Innovent E.V. Verfahren zum Abbau von Polysacchariden

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402200A (zh) * 2018-10-02 2019-03-01 姜克忠 一种低聚壳聚糖的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS55122556A (en) * 1979-03-16 1980-09-20 Nippon Tennen Gas Kogyo Kk Sterilizing sheettlike substance
JPH0499493A (ja) * 1990-08-14 1992-03-31 Asahi Seibutsu Kogaku Kenkyusho:Kk 低分子化キトサンの製造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS55122556A (en) * 1979-03-16 1980-09-20 Nippon Tennen Gas Kogyo Kk Sterilizing sheettlike substance
JPH0499493A (ja) * 1990-08-14 1992-03-31 Asahi Seibutsu Kogaku Kenkyusho:Kk 低分子化キトサンの製造方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, vol. 93, no. 6, 1992, Philadelphia, PA, US; abstract no. 63873, D. FINK ET AL: "Cloning and expression in Streptomyces lividans of a chitosanase-encoding gene from the actinomycete Kitasatosporia N174 isolated from soil" page 409; *
BIOTECHNOL . LETT., vol. 13, no. 12, 1991, pages 845 - 850 *
DATABASE WPI Section Ch Week 8045, Derwent World Patents Index; Class A60, AN 80-79570C *
PATENT ABSTRACTS OF JAPAN vol. 16, no. 338 (C - 965)<5381> 22 July 1992 (1992-07-22) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2736835A1 (fr) * 1995-07-17 1997-01-24 Aber Technologies Pansement pour plaies chroniques, notamment escarres, en gel de chitine
WO1997003708A1 (fr) * 1995-07-17 1997-02-06 Aber Technologies S.A. Pansement pour plaies chroniques, notamment escarres, en gel de chitine
DE102012009593A1 (de) 2012-05-07 2013-11-07 Innovent E.V. Verfahren zum Abbau von Polysacchariden
DE102012009593B4 (de) 2012-05-07 2019-03-07 Innovent E.V. Verfahren zum Abbau von Polysacchariden ausgewählt aus den Stoffgruppen der Glykosaminoglykane und deren Derivaten sowie der Alginate

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EP0659210A1 (fr) 1995-06-28
CA2143120A1 (fr) 1994-03-17

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