WO2003054200A1 - Chitosanase et utilisation de celle-ci - Google Patents

Chitosanase et utilisation de celle-ci Download PDF

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Publication number
WO2003054200A1
WO2003054200A1 PCT/JP2002/012940 JP0212940W WO03054200A1 WO 2003054200 A1 WO2003054200 A1 WO 2003054200A1 JP 0212940 W JP0212940 W JP 0212940W WO 03054200 A1 WO03054200 A1 WO 03054200A1
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Prior art keywords
chitosan
aspergillus
molecular weight
activity
enzyme
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PCT/JP2002/012940
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English (en)
Japanese (ja)
Inventor
Tetsuya Fukazawa
Isshin Tanaka
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Sankyo Lifetech Company, Limited
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Priority to AU2002354461A priority Critical patent/AU2002354461A1/en
Publication of WO2003054200A1 publication Critical patent/WO2003054200A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01132—Chitosanase (3.2.1.132)
    • 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
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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)

Definitions

  • the present invention relates to a chitosan-degrading enzyme that is separated and purified from a culture of at least one of Aspergillus 'oryzae (Aspergillus oryzae), Aspergillus japonicus (Aspergillus japonicus), and Aspergillus' soperyae (Aspergillus sojae).
  • a method for producing low molecular weight chitosan and its salt using said chitosan degrading enzyme, low molecular weight chitosan and its salt produced by said method, and pharmaceutical composition comprising said low molecular chitosan and its salt, food The present invention relates to a method of producing a sample using the chitosan degrading enzyme, a sample produced by the method, and the like.
  • Chitin (] 3-1, 4-poly-N-acetyl dulcosamine) is a polysaccharide that is very abundant in nature, and is mainly produced from crustaceans such as E. flavum and squid molluscs on an industrial scale. It is extracted and produced.
  • Chitosan (3-, 4-polydarcosamine) is an amino-containing high-molecular-weight polysaccharide obtained by treating chitin with concentrated water, and is known to have various physiological activities. Among them, chitosan has a strong antibacterial action as a naturally occurring substance, and its safety is extremely high compared to synthetic antibacterial agents (“chitin”). ⁇ How to make use of chitosan ", Special edition of the business world Vol.
  • Chitosan produced by concentrated alkali treatment of chitin extracted from crustaceans such as Ebi and Ebi is a high molecular weight polysaccharide with a molecular weight of 1,000,000 or more and is soluble in water under acidic conditions, but it is neutral Or it does not dissolve in water under alkaline conditions. And, when it is not soluble in water, the physiological activity of chitosan can not be exerted sufficiently. Also, processing food materials including shellfish At the time of treatment, these high molecular weight polysaccharides resulted in very high viscosity, and there was also a problem that the subsequent processing takes time and effort.
  • chitosan in order to increase the solubility in water, it is considered desirable to lower the molecular weight, not the derivatization method.
  • the antibacterial activity of chitosan is known to be exerted not only with high molecular weight chitosan having a molecular weight of 1,000,000 or more but also with low molecular weight chitosan having a molecular weight of 10,000 or more. Lecture number 2 ⁇ 183 ⁇ , 2000 years).
  • chitosan having a molecular weight of 10,000 or less is reported to have side effects such as skin irritation (Japanese Patent Laid-Open No. 2000-169327), and methods for producing low molecular weight chitosan having a molecular weight of 10,000 or more are being studied. .
  • bacteria-derived chitinases include Bacillus, Corynebacterium (Veldkamp, H., Nature, 169, 500 (refer to 1952)), Cytophaga, and Acropocactor. ⁇ Heteropticum (Acromobacter hyteropticum: Campbell, Jr., LL et al., J. Gen. Microbiol., 5, 894 (1951)), the genus Flapobacterium (Flavobacterium) and Micrococcus colpogenes (Morococcus colpogenes) (Campbell, Jr., LL, et al., J. Gen. Microbiol., 5, 894 (1951)).
  • chitinase A GenBank Accession NO. D 13762
  • chitinases produced by actinomycetes As chitinases produced by actinomycetes, chitinases produced by the genera Streptomyces, Nocardiopsis and Actinomyces are known (Nakajin Teruta et al., Research Institute for Fermentation Research) Reported in the literature, 30, 19 (1966).
  • the chitinase produced by the genus Penicillium is known As the enzyme produced by the genus Aspergillus, Aspergillus sp. Sherif, AA, et al., Appl. Mcrobiol., Biotechnol., 35, 228 (1991)) and Aspergillus sp.
  • a chitinase produced by Gassus is known as an enzyme produced by yeast, for example, a chitinase produced by Saccharomyces 'Saccharomyces cerevisiae (Saccharomyces cerevisiae) can be exemplified, and preferably, Saccharomyces' cerevisise And chitinase (GenBank Accession No. M74070) and ORF D 9481.7 (GenBank Accession No. U28373).
  • chitinases derived from many organisms, but as a representative fermented food, Aspergillus japonicus (Aspergillus japonicus) and Aspergillus' Sojiyae, which are used for producing miso and soy sauce sake, etc. There is no report on the production of chitinase for (Aspergillus sojae), and there is no report on the properties or utilization of chitinases from these organisms. Similarly, with regard to Aspergillus oryzae contained in food, it has been reported that a conference presentation has been made on a gene that is considered to encode a chitinase (Chitin and Chitosan Research Vol.8, No.
  • the present inventors diligently studied a method of efficiently producing a low molecular weight chitosan which is soluble in water under neutral conditions and has an antibacterial activity.
  • Aspergillus' oryzae var. Sporof Enzymes using chitinase derived from the strain lavus Ohara J CM 2067) or chitinase derived from Aspergillus japonicus SANK 1 92 88 strain or chitinase derived from Aspergillus sogyae (Aspergillus soj.
  • non-derivative low molecular weight chitosan with a molecular weight distribution of 10,000 or more.
  • the present invention is a.
  • Chitosan-degrading enzymes which are separated and purified from at least one culture of Aspergillus oryzae (Aspergillus oryzae), Aspergillus' Japonics (Aspergillus ⁇ aponicus), and Aspergillus sojae (Aspergillus sojae);
  • Aspergillus 'Oryzae is a strain of Aspergillus oryzae var. Sporof lavus Ohara J CM 2067, and Aspergillus' Japonics (Aspergillus japonicus) is Aspergillus spiculis And Spergils sojae (Aspergillus sojae)
  • the isoelectric point shows an isoelectric point p I of 3.5 to 4.5 by isoelectric focusing method
  • a chitosan degrading enzyme which is a protein described in any one of the following a) to d): a) a protein consisting of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing;
  • amino acid sequence described in a) or b) it is characterized in that it comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added, and has chitosan degradation activity.
  • a chitosan degrading enzyme which is a protein described in any one of the following a) to c): a) amino acid sequence described in SEQ ID NO: 14 of the sequence listing, and A protein characterized in that the group is acetylated;
  • amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing, the amino acid sequence has one or several amino acids substituted, deleted, inserted or added, and a amino group of an N-terminal serine residue A protein characterized in that it is acetylated and has chitosan degrading activity;
  • a protein comprising the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing, and wherein the amino group of the serine residue at the N-terminus is acetylated, and is characterized by having chitosan degradation activity,
  • DNA consisting of a nucleotide sequence complementary to the nucleotide sequence possessed by the above-mentioned a) DNA, and which encodes a protein having a cleaving activity.
  • a DNA comprising the DNA according to the above a) and coding a protein having a chitosan degrading activity
  • a recombinant plasmid comprising the DNA according to (7) or (8),
  • a method for producing a chitosan degrading enzyme which comprises the following 1) to 2):
  • Aspergillus oryzae is a strain of Aspergillus oryzae (Aspergillus oryzae var. Sporof lavus Ohara J CM 2067), and Aspergillus Aspergillus oryzae (Aspergillus japonicus) is Aspergillus spl. Yes, Spergils sojae (Aspergillus sojae)
  • a pharmaceutical composition comprising the low molecular weight chitosan or the salt thereof according to any one of (21) to (24) as an active ingredient, (26)
  • the pharmaceutical composition according to (25) which is a therapeutic agent for trauma, bedsore or atopic dermatitis.
  • a cosmetic comprising the low molecular weight chitosan or a salt thereof according to any one of (21) to (24),
  • a treating agent for water or the like comprising the low molecular weight chitosan or a salt thereof according to any one of (21) to (24).
  • a method for producing a sample which substantially does not contain high molecular weight partially acetylated chitosan having a molecular weight of 1,000,000 or more in its liquid component which comprises the following steps 1) and 2):
  • the present invention relates to a chitinase derived from Aspergillus oryzae (Aspergillus oryzae) as a filamentous fungus useful for producing low molecular weight chitosan, or a chitinase derived from Aspergillus japonicus (Aspergillus japonicus) or a chitinase derived from Aspergillus sogyae (Aspergillus sojae)
  • the present invention relates to a chitinase, a low molecular weight chitosan produced by the chitinase, a pharmaceutical composition containing the low molecular weight chitosan and the like.
  • chitinase is an enzyme having an activity of hydrolyzing chitin or partially acetylated chitosan, and when chitin is hydrolyzed, N-acetyl darcosamine and its oligosaccharide are formed.
  • This "activity to hydrolyze chitin or partially acetylated chitosan" is defined as “chitosan degradation activity”.
  • Chitin is a partially acetylated chitosan having a high percentage of acetylation, and in the present invention, these are also collectively referred to as a partially acetylated chitosan unless otherwise noted.
  • both of the chitosan degrading enzyme and the chitinase refer to the enzyme having the above-mentioned chitosan degrading activity, and are used as synonyms.
  • the chitinases of the present invention include proteins having a chitosan degrading activity in cultures of microorganisms producing chitinases. Examples of such chitinases include chitinases derived from Aspergillus' oryzae (Aspergillus oryzae), chitinases derived from Aspergillus japonicus or chitinases derived from Aspergillus soae (Aspergillus sojae). . More preferred ⁇ Mr.
  • Aspergillus- Chimera derived from Aspergillus oryzae var. Sporof lavus Ohara J CM 2067, Aspergillus japonicus Chimera or Aspergillus japonicus Strain derived from Sank 19288 strain or Aspergillus japonicus 'Aspergillus sojae is a chitinase derived from SANK 22388 strain, and more preferably is a chitinase derived from Aspergillus oryzae (Aspergillus oryzae) var. Sporoflavus Ohara J CM 2067).
  • another example of the melaninase of the present invention includes a protein having all of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing and having chitosan degrading activity.
  • the protein may contain all of them as an internal amino acid sequence regardless of the order of the respective amino acid sequences.
  • preferred ones include all of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing, and the N-terminal sequence is “Ser-S er-Gly-Leu-Lys ", and a protein having chitosan decomposition activity can be mentioned.
  • amino acids shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing are more preferable.
  • a protein which contains all of the no acid sequence, whose N-terminal sequence consists of SerSerGly-Leu-Ly s-, and whose N-terminal is acetylated and which has chitosan degradation activity Can be mentioned.
  • chitinase of the present invention includes a protein having all of the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 in the sequence listing and having chitosan degrading activity.
  • one or several amino acid residues may be substituted, deleted, inserted or Z or at one or several sites.
  • the added protein is also included in the present invention as long as it has chitosan decomposition activity.
  • severe means a number not exceeding 10, and preferably a number not exceeding 5.
  • a protein having a substituted amino acid sequence has an activity equivalent to that of a naturally-occurring protein
  • a nucleotide sequence corresponding to serine of the nucleotide sequence corresponding to cysteine of the interleukin 1 (IL-2) gene It is known that a protein obtained by converting it into a protein has the activity of IL_2 (Wang, A. et al. (1984) Science 224,
  • a protein consisting of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing can be mentioned.
  • a protein comprising the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing is included in the present invention as long as it has a chitosan degrading activity.
  • chitinase of the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 14 in the sequence listing, in which the N-terminal amino group is acetylated.
  • a protein comprising such a protein is also included in the present invention as long as it has a chitosan degrading activity.
  • the protein consisting of the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing one or several amino acid residues were substituted, deleted, inserted and Z or added at one or several sites. Proteins are also included in the present invention as long as they have chitosan degrading activity.
  • Preferred as the chitinase of the present invention is a chitinase derived from Aspergillus oryzae (Aspergillus oryzae) var. Sporof lavus Ohara J CM 2067, azanhi derived from Aspergillus spp. Strain Aspergillus japonicus SANK 19288.
  • a chitinase derived from Aspergillus sojae SANK 22 388 strain a protein comprising the amino acid sequence as set forth in SEQ ID NO: 5 in the sequence listing, and an amino acid sequence as set forth in SEQ ID NO: 14 in the sequence listing;
  • a protein in which the amino group is acetylated is mentioned, but more preferable one is a chitinase derived from Aspergillus oryzae (Aspergillus oryzae) var. Sporof lavus Ohara J CM 2067 and SEQ ID NO: 14 in the Sequence Listing. It is a protein consisting of the described amino acid sequence and in which the N-terminal amino group is acetylated.
  • the "DNA of the present invention” refers to a DNA encoding the chitinase of the present invention.
  • the DNA may be in any form as currently known, such as cDNA, genomic DNA, artificially modified DNA, chemically synthesized DNA and the like.
  • DNA of the present invention is hybridization under stringent conditions with DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing, and chitosan degradation It includes DNA encoding a protein having activity.
  • DNA of the present invention is a nucleotide sequence homology of 70% or more, preferably 80% or more, more preferably 95% or more with the nucleotide sequence shown in nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing. DNA having sex is included. Such DNAs include mutant DNAs found in nature, artificially modified mutant DNAs, homologous DNAs from heterologous organisms, and the like.
  • nucleotide of the present invention a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing can be mentioned.
  • the codon corresponding to the desired amino acid may be selected optionally, and can be determined in a conventional manner, for example, in consideration of the codon usage frequency of the host to be used. (Grantham, R. et al. (1981) Nucleic Acids Res. 9, 143-174).
  • partial modification of the codons of these nucleotide sequences is carried out in a conventional manner by site-directed mutagenesis (site specific mutagenesis / Mark, DF et al.) Using a primer consisting of a synthetic oligonucleotide encoding the desired modification. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666) and the like.
  • DNA of the present invention is a DNA consisting of the nucleotide sequence shown by nucleotide numbers 214 to 1438 of SEQ ID NO: 4 in the sequence listing.
  • a DNA comprising a DNA consisting of a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing is also included in the present invention as long as it comprises a region encoding a protein having chitosan degradation activity. It is.
  • Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 National Institute of Advanced Industrial Science and Technology Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology-November 8, 2002-(1-1-1 Central, Tsukuba, Ibaraki Prefecture, Japan It has been deposited internationally in 6) and has the accession number FER M BP-8235. Therefore, the DNA of the present invention can also be obtained from the strain.
  • DNA hybridizing to the DNA inserted into the recombinant plasmid retained by this deposited strain under stringent conditions is also included in the present invention as long as the encoding protein has chitosan degrading activity.
  • a DNA comprising the above nucleotide sequence is also included in the present invention.
  • such DN The protein encoded by A is also included in the chitinase of the present invention.
  • Preferred as the DNA of the present invention is a DNA consisting of the nucleotide sequence shown by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing, and a transformed Escherichia coli Escherichia coli BM25.8 / pTriplEx-Citase-cDNA SANK 72102
  • the chitinase of the present invention can include a protein consisting of an amino acid sequence encoded by the DNA of the present invention.
  • a method of scraping the DNA from the end using Exonucleusase B a 131 or the like in order to produce a variant in which any one or two or more amino acids have been deleted. (Toshimitsu Kishimoto et al., "Science Chemistry Experiment Course 1 ⁇ Gene Research Method II" 335-354), cassette mutation (Toshimitsu Kishimoto, "New Chemical Experiment Course 2 ⁇ Nucleic Acid III Recombinant DNA Technology” 242-251) etc.
  • a protein obtained by genetic engineering based on the DNA of the present invention is included in the present invention as long as it has a chitosan degrading activity.
  • Such a chitinase need not necessarily have all of the amino acid sequences set forth in SEQ ID NO: 5 in the sequence listing, and even if it is a protein consisting of the partial sequence, for example, the protein has the ability to degrade chitosan.
  • DNA encoding such a chitinase is also included in the present invention.
  • hybridization under stringy end conditions may be carried out by combining hybridization with 5X SSC (0.75 M sodium chloride, 0.075 M sodium citrate) or a salt of the same. C. for about 12 hours in a single solution, and after prewashing with 5X SSC or a solution having a salt concentration equivalent thereto, if necessary, after 1 X SSC or 1 X SSC or The washing can be carried out by washing in a solution of the same salt concentration. Furthermore, washing can also be carried out in a solution of 0.1 ⁇ SSC or a salt concentration of this.
  • the chitinase used in the present invention may be one purified from chimpanzee-producing bacteria, one crudely purified, one from which a bacterial cell culture supernatant may be used as it is, as well as a cell lysate.
  • a cell lysate When culturing chitinase-producing bacteria, it is preferable to culture the medium by adding powdered chitin, powdered chitosan, colloidal chitin, colloidal chitosan or the like in addition to carbon source and nitrogen source. After completion of the culture of the kinase-producing bacteria, centrifugation is performed, and the culture supernatant from which the cells have been removed can be used as a crude enzyme solution as it is. Alternatively, crude enzyme solution may be roughly purified by ion exchange chromatography or the like or purified.
  • Chitinase-producing bacteria are microorganisms which intrinsically have the ability to produce chitinase, It includes microorganisms that accumulate chitinase inside cells, and microorganisms that secrete outside cells.
  • a culture supernatant of a chitinase-producing bacterium or a chitinase purified from the culture supernatant is used, a bacterium that secretes the chitinase out of the cells can be used.
  • Aspergillus oryzae is used as the chimera-producing bacteria used in the present invention.
  • Aspergillus oryzae or Aspergillus japonicus (Aspergillus japonicus) or Aspergillus 'Aspergillus sojae' can be mentioned, preferably Aspergillus oryzae (Aspergillus oryzae) var. Sporoflavus Ohara J CM 2067 strain or
  • Cultivation of chitinase-producing bacteria can be performed using a conventional culture medium and culture medium.
  • methods such as liquid culture and solid culture can be appropriately selected.
  • liquid culture flask culture or culture using a fermenter can be performed, and after the start of culture, a batch culture method without addition of a culture medium or a fed-batch culture method in which a culture medium is appropriately added during culture is used. be able to.
  • Carbon and nitrogen sources can be added to the medium, and vitamins and trace metals can be added as needed.
  • carbon sources examples include glucose, mannose, galactose, monosaccharides such as fructose, maltose, cellobiose, isoma! ⁇ Starch, lactose, disaccharides such as sucrose, polysaccharides such as starch, maltose extract
  • inorganic nitrogen such as ammonia, ammonium sulfate, ammonium nitrate, etc.
  • organic nitrogen such as yeast extract, maltose extract, corn stover plica, peptone, etc. are used, but as long as the chitinogenic bacteria grow It is not limited to these.
  • Powdered chitin or colloidal chitin can also be added to the culture medium to increase the amount of chitinase produced by chitinase-producing bacteria.
  • the amount of composition in these media can be selected appropriately.
  • the culture temperature, pH and aeration and agitation amount can be appropriately selected to be suitable for chitinase production.
  • chitinase used in the present invention, a chitinase derived from Aspergillus oryzae (Aspergillus oryzae) or Aspergillus 'Japonics (Aspergillus japonicus) or Aspergillus' Sopylase sojae (Aspergillus sojae) can be used, preferably Aspergillus oryzae (Aspergillus ) var. sporoflavus Ohara J CM 2067 strain or Aspergillus japonics
  • chitinase derived from strain SANK 22388.
  • the chitinases may be produced by these chitinase-producing bacteria themselves, or may be produced by mutants or modified products thereof.
  • the gene encoding the chitinases of these chitinase-producing bacteria may be used. It may be a recombinant protein produced from a transformant obtained by introduction into a host. Acquisition of chitinase-producing bacteria
  • Aspergillus oryzae Aspergillus oryzae (Aspergillus oryzae) var. Sporus lavus Ohara J CM 2067 is a strain of microbiology storage facility of the Research Institute of Biosciences, RIKEN (JCM: Japan Colllection of Microorganisms; Address 351— 0198 Wako, Hirosawa, Saitama, Japan 2 — 1, website address http: ww www. Jcm.riken.go.jp/ »Can be purchased from».
  • Aspergillus japonics S ANK 1 9 2 8 8 and Aspergillus-Sozyyae.
  • composition of the three types of media is as follows.
  • CYA medium Czapek Yeast Extract Agar medium
  • Zapeck concentrate 5 g of yeast extract, 30 g of sucrose, 15 g of agar, 1000 ml of distilled water
  • Zapeck concentrated solution (NaN03 30 g, KC1 5 g, MgS04-7H20 5 g, FeS04-7 H20 0.1 g, ZnS04-7 H20 0.1 g, CuS04-5H20 0.05 g distilled water 100 ml)
  • MEA medium ⁇ Malt Extract Agar medium ⁇ (Moltoex 20 g, Peptone 1 g, Darucose 20 g, Agar 20 g, Distilled water lGOO ml)
  • CY20S medium ⁇ Czapek Yeast Extract Agar with 203 ⁇ 4 Sucrose medium ⁇ (K2HPO4 1.0 g, * 10 ml of Zapeck concentrated solution, 5 g of yeast extract, 200 g of sucrose) , 15 g, 1000 ml of distilled water)
  • Colonies in CYA medium are 45 to 60% in diameter after one week of culture at 25 ° C.
  • Mycelium is white.
  • the formation of conidia is strong in the central part, and the color of the formation part shows olive brown (4F4) to black.
  • the color of the reverse side is from Gressi Yuello 1 (4B4) to Yellowish White (4A2). Sclerotia are not observed. No leachate or soluble pigment is observed.
  • Colonies in CY20S medium are 57 to 68 mm in diameter at 25 ° C. for 1 week of culture.
  • the colony resembles a colony of CYA medium.
  • the color of the back shows Pastor Ruio 1 (3A4).
  • Colonies in MEA medium are 51 to 57% in diameter after one week of culture at 25 ° C.
  • conidia The formation of conidia is strong, and the color of the formation is black. The color of the back is colorless.
  • the colony of CYA medium cultured at 37 ° C. for one week is 13 to 20 mm in diameter. It does not grow at 5 ° C.
  • Conidiophores are radial, conidiophores are smooth, colorless or colorless at the top, 4 to 11 wide.
  • the apex is spherical or oval, 19 to 50 wide.
  • Aspergilla is a single row. The phialide covers the top three quarters and is 7 to 10 x 3.5 to 4.5.
  • Conidia are oval to spherical, covered with a somewhat sparse ridge, and 3.5 to 4.5 x 3.5 to 4 in diameter.
  • Aspergillis' Japonics S ANK 1 9 2 8 8 shares, as S. Aspergills Japonics bar Akiuritus S ANK 1 9 2 8 8 shares, on September 13, 2013 National Institute of Advanced Industrial Science and Technology It was deposited internationally at the Patent Organism Depositary Center (1-1-1 Central 6th, Ibaraki Prefecture, 1-1-1 Central Ibaraki, Japan), and was assigned the accession number FE RM BP-7736.
  • Colonies in MEA medium have a diameter of 43 to 47 in 1 week culture at 25 ° C.
  • the colony is somewhat fluffy and somewhat sparse.
  • Mycelium is white and inconspicuous.
  • the formation of conidia is strong, and the color of the formation shows olive (1E8).
  • the color of the back is colorless.
  • the colony of CYA medium cultured at 37 ° (:, 1 week is 52 to 56 thighs in diameter. It does not grow at 5 ° C.
  • Conidiophores are radial, conidiophores are rough, colorless and 7 to 12, wide.
  • the peak is in the shape of an oval or spherical, 24 to 41 m wide.
  • Aspergilla is a single row, sometimes two rows.
  • the metre is 8 to 12 x 4 to 8 m.
  • the phialides are 10 to 11 x 4.5 to 6 J1.
  • Conidia are spherical, rough, 5 to 6.5 in diameter.
  • Aspergillus' Sojiyae S ANK 2 2 8 8 strains are designated as Aspergillus soyaeae S ANK 2 2 3 8 8 strains, and on September 13, 2013, National Institute of Advanced Industrial Science and Technology Patent Organism Depositary It was deposited internationally (at Ibaraki prefecture, Tsukuba, 1-1 1-1 Central No. 6) with the accession number FE RM BP-7738.
  • filamentous fungi are susceptible to mutation in nature or by artificial manipulation (eg, ultraviolet radiation, radiation, chemical treatment, etc.), and Aspergillus' Oryzae (Aspergillus oryzae var. Sporof lavus) of the present invention is susceptible to mutation.
  • Aspergillus' Oryzae Aspergillus oryzae var. Sporof lavus
  • Ohara J CM 2067 Aspergills japonicus S ANK 1 2 8 8 shares The same is true for the S. aspergillus S. sogyae strain S ANK 2 2 3 8 8 shares.
  • Aspergillus oryzae (Aspergillus oryzae var.
  • Sporoflavus Ohara J CM 2067 Aspergillus' Japonicus S ANK 1 9 2 8 8 strain Jaspergillus sogiyae S ANK 2 2 8 8 is a mutant of all of them Includes Further, among these mutant strains, those obtained by genetic methods such as recombination, transduction, transformation and the like are also contained. That is, Aspergillus oryzae (Aspergillus oryzae var. Sporoflavus Ohara J CM 2067) strains which produce chitinase, their mutants and strains which are not clearly distinguished from them are all Aspergillus-oryzae (Aspergillus oryzae var.
  • Sporoflavus Ohara J CM 20 67 is included in the strain.
  • Aspergillus japonicus strain SANK 19288 which produces chitinase, their mutants and strains which are not clearly distinguished from them are all included in the Aspergillus' japonicus strain SANK 19288 strain.
  • Aspergillus sogyae strain SANK 2 238, which produces chitinase, mutants thereof and strains which are not clearly distinguished from them are all included in the strain Aspergillus sp.
  • the chitinase of the present invention is a recombinant bran in which the DNA of the present invention has been incorporated into a vector.
  • the host cells can be transformed with Smid and obtained from culture products of the transformed cells.
  • a recombinant plasmid in which the DNA of the present invention is inserted into an appropriate vector is also included in the present invention.
  • vectors used for such purpose various generally known vectors can be used. Preferred examples include, but are not limited to, vectors for prokaryotic cells, vectors for eukaryotic cells, vector for cells derived from mammals, and the like.
  • Such recombinant plasmids can be used to transform other prokaryotic or eukaryotic host cells.
  • the gene is made into an expression vector, whereby the gene is expressed in each host. It is possible to express.
  • expression vectors are a preferred embodiment of the recombinant plasmids of the present invention.
  • Host cells can be obtained by introducing the recombinant plasmid of the present invention into various cells.
  • Such cells may be prokaryotes or eukaryotes as long as they can introduce a plasmid.
  • a prokaryotic host for example, Escherichia coli (Escherichia coli i), Bacillus subtilis (Baci 1 ls subtilis), and the like can be mentioned.
  • Escherichia coli Escherichia coli i
  • Bacillus subtilis Bacillus subtilis
  • vector I one having a sequence capable of imparting phenotypic (phenotype) selectivity to transformed cells is preferable.
  • E. coli, K12 strain and the like are often used, and as a vector, a plasmid of pBR322 or pUC type is generally used, but not limited thereto, various known strains and vectors are used. Both can be used.
  • a tryptophan (trp) promoter in E. coli, a tryptophan (trp) promoter, a leucine (lac) promoter, a lipoptophan laxase (tac) promoter, a lipoprotein (lpp) promoter, a polypeptide chain elongation factor Tu (tufB) Promoter Y. et al., and any promoter can be used for producing the chitinase of the present invention.
  • p TUB 2 2 8 As Bacillus subtilis, for example, the 2 0 7-25 strain is preferable, and as vector 1, p TUB 2 2 8 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) or the like is used. However, it is not limited to this.
  • Eukaryotic host cells include cells such as vertebrates, insects, and yeasts, and vertebrate cells include mammalian-derived cells, such as COS cells, which are monkey cells (Gluzman, Y. ( 1981) Cel l 23, 175-182, AT CCCRL— 1 6 5 0) and Chinese ⁇ Dihydrofolate reductase-deficient strain (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4126-4220) of hamster ovary cells (CH O cells, ATCC CCL-61), etc. It is often used but is not limited to these.
  • COS cells which are monkey cells (Gluzman, Y. ( 1981) Cel l 23, 175-182, AT CCCRL— 1 6 5 0) and Chinese ⁇ Dihydrofolate reductase-deficient strain (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4126
  • an expression promoter for vertebrate cells one having a promoter usually located upstream of a gene to be expressed, a splice site of RNA, a polyadenylation site, a transcription termination sequence and the like can be used, and further, it may be an origin of replication if necessary.
  • You may have Examples of the expression vector include p SV2 dhfr (Subramani, S. et al. (1981) MoI. Cell. Biol. 1, 854-864) having an SV40 early promoter, and the like. It is not limited to this.
  • COS cells As a host cell, when using COS cells as an example, as an expression vector,
  • the expression vector is a jetyl aminoethyl (DEAE) -dextran method (Lut man, H: and Magnus son, G. (1983) Nucleic Acids Res, 11,
  • E. et al. (1982) EMB 0 J. 1, 84 ⁇ 845) and the like can be incorporated into CO 2 S cells, and thus desired transformed cells can be obtained.
  • CHO cells when CHO cells are used as host cells, one vector capable of expressing an neo gene which functions as an antibiotic G418 resistant marker together with an expression vector, for example, pRSVne o
  • vector 1 using baculovirus P10 or the same basic protein promoter can also be used.
  • a recombinant protein is obtained by linking the secretion signal sequence of the envelope surface protein GP67 of Ac NPV to the N-terminal side of the target protein. It is also possible to express the quality as a secreted protein (Zhe-mei Wang, et al. (1998) Biol. Chem., 379, 167-174).
  • yeast As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known, and among them, Saccharomyces yeast, for example, baker's yeast Saccharomyces cerevisiae or petroleum yeast Pichia pastoris is preferable.
  • expression vectors of eukaryotic microorganisms such as yeast include, for example, promoters for alcohol dehydrogenase genes (Bennetzen, JL and Hal, BD (1982) J. Biol. Chem. 257, 3018-3025), and acidity. Promoters of phosphatase genes (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5) can preferably be used.
  • a secretory protein When expressed as a secretory protein, it can also be expressed as a recombinant having a secretory signal sequence and the cleavage site of an endogenous protease or known protease possessed by the host cell at the N-terminal side. It is. For example, in a system in which human mast cell tryptase of tryptic serine protease is expressed in petroleum yeast, a secretory signal sequence of ⁇ factor 1 of yeast and N-terminal 2 proteaase possessed by petroleum yeast are shown at the N-terminal side. It is known that active tryptase is secreted into the medium by tether expression of the cleavage site (Andrew, L. Niles' et al. (1998) Biotechnol. Appl. Biochem. 28, 125- 131).
  • the transformant obtained as described above can be cultured according to a conventional method, and the culture produces the chitinase of the present invention intracellularly or extracellularly.
  • the culture medium used for the culture various media commonly used can be appropriately selected depending on the host cell employed, and, for example, in the case of the above-mentioned COS cells, RPMI1 664 medium or Dulbecco's modified Eagle's medium It is possible to use a medium such as “DM EM”, to which a serum component such as bovine fetal serum is added as necessary.
  • the concentration of CO 2 may be in the range of 0 to 50%, preferably 1 to 10%, and more preferably 5%.
  • the culture temperature may be 0 to 99 ° C., preferably 20 to 50 ° C., and more preferably 35 ° to 40 ° C.
  • the chitinase of the present invention which is produced as a recombinant protein intracellularly or extracellularly of the transformant by the above-mentioned culture, has physicochemical properties, chemical properties and biochemical properties of the protein in the culture product (enzyme Various methods of separation using activities etc. ("Biochemical data collection II", 1157-1259, 1st edition, 1st print, June 23, 1980 Tokyo Chemical Co., Ltd. published; Biochemistry 25, No. 25, p8274-8277 (1986); Eur. J. Biochem., 163, p313-321 (1987), etc.) can be used for separation and purification.
  • the method specifically, for example, conventional reconstitution treatment, treatment with protein precipitant (salting out method), centrifugation, osmotic shock method, freeze-thawing method, ultrasonic disruption, ultrafiltration, gel filtration, Various liquid chromatography methods such as adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis methods, combinations thereof, and the like can be exemplified.
  • high yield and desired Recombinant proteins can be produced on an industrial scale.
  • histidine consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column.
  • the chitinase produced by the method as described above can also be mentioned as a preferred example of the present invention.
  • the low molecular weight chitosan or a salt thereof to be used in the present invention dissolves 10 g or more in 100 g of water at 0 to 1000C.
  • water should be distilled water, tap water, and well water.
  • low molecular weight chitosan or a salt thereof is dissolved in 100 g or more of water in an amount of 10 g or more under the condition of pH 1.0 to 7.0.
  • low molecular weight chitosan has antibacterial activity.
  • Partially acetylated chitosan refers to chitosan in which part of the amino group of chitosan is acetylated. Partially acetylated chitosan having a low degree of acetylation is produced by deacetylation of chitin with alkali Can. The degree of acetylation of partially acetylated chitosan can be controlled by the alkali concentration, reaction time and the like. Also, those having a molecular weight of 100,000 or more are particularly referred to as high molecular partially acetylated chitosan.
  • high molecular weight partially acetylated chitosan having a molecular weight of 100,000 or more examples include, for example, 30%, 20%, and 10% acetylated chitosan (trade name: chitosan 7 B, 8 B, 9 B) derived from Ebi-force 2 (All can be added by Tokichi Co., Ltd.), but the invention is not limited thereto.
  • the natural chitin contained in evi and power 2 is a high molecular weight partially acetylated chitosan having a higher degree of acetylation. However, these can also be used as partially acetylated chitosan.
  • the molecular weight of high molecular weight partially acetylated chitosan can be measured by high performance liquid chromatography using gel permeation chromatography (GPC) (with pullulan as a standard substance) (Takiguchi et al., Chitin 'chitosan research P 75-79, 1999).
  • the degree of acetylation of high molecularly partially acetylated chitosan can be determined by performing PVSK colloid titration with 1 Z 400 N polyvinyl sulfate solution using Toluidine 1 solution as an indicator (Toei K. et al. Anal. Chem. Acta 83, 59, 1976).
  • the degree of acetylation measured by this method is the percentage of amino groups that have been acetylated (for example, a degree of acetylation of 1% means that one out of 100 amino groups is acetylated). Is a value that represents
  • the degree of acetylation of chitosan can also be calculated from the integral ratio of 3 H of the acetyl group obtained by ⁇ -NMR to 7 H excluding hydroxyl groups other than the acetyl group.
  • the hydrolytic activity of chitinase can be measured according to the Randle-Morgan method (Randle CJ M et al. Biochem. J. GL 586-589, 1955). It can also be measured according to Schales's method (Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)).
  • the present invention relates to a method of producing low molecular weight chitosan.
  • the chitinase used in the method is based on 30% acetylated chitosan (viscosity 100 to 300 cps) under reaction conditions of pH 4.0 to 5.5 and temperature of 37 ° C. for 10 to 30 minutes.
  • the hydrolysis activity of said chitinase is less than 15%.
  • the amount and concentration of enzyme used in the method are not particularly limited.
  • the aqueous solution of the polymeric partially acetylated chitosan to be a substrate of chitinase in the method is 0.1 to 5% by mass, preferably 0.5 to 2% by mass, and more preferably 0.5 to 1%. It is mass% concentration.
  • the aqueous solution can also be used as a solution dissolved in water under acidic conditions (eg, ⁇ 1 to 6).
  • an alkali for example, sodium carbonate, sodium hydrogen carbonate, caustic soda, etc.
  • the acid used to acidify the polymer partially acetylated chitosan solution and dissolve it in water includes hydrochloric acid, acetic acid, trifluoroacetic acid, 'paratoluenesulfonic acid, acrylic acid, lactic acid, amino acid, citric acid, salicylic acid, glucuronic acid Although glycolic acid etc. can be mentioned, if it can produce an acidic solution, it will not be limited to these.
  • the pH range of the reaction solution for producing low molecular weight chitosan using the chitinase of the present invention may be in the range of pH 3 to 12, preferably in the range of pH 3 to 7, more preferably Is in the range of pH 4 to 6.
  • the temperature of the reaction solution may be 0 ° (: to 70 ° C., preferably 10 ° C. to 60 ° C., and more preferably 40 to 50 t.
  • the reaction time is 10 minutes to Seven days can be mentioned, preferably 3 hours to 5 days, more preferably 1 to 3 days.
  • various low molecular weight chitosan salts having a molecular weight of 10,000 or more and less than 1,000,000 can be obtained by carrying out a hydrolysis reaction by the chitosanase of the present invention.
  • Generate The type of salt depends on the acid used: hydrochloride, acetate, trifluoroacetate, p-toluenesulfonate, acrylate, lactate, amino acid salt, citrate, salicylate, glucuronate, glyco-one Examples include, but are not limited to, borate and the like.
  • the present invention provides a low molecular weight chitosan or a salt thereof which dissolves in 10 g or more in 100 g of water at 0 to 100 ° C.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising low molecular weight chitosan which dissolves 10 g or more in 100 g of water at 0 to 100 ° C.
  • the hydrolytic activity of chitinase is determined by the Randle-Morgan method (Randle CJM et al. It can be measured according to Biochem. J. GL 586-589, 1955). It can also be measured according to Schales's method (Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)).
  • Glycol Rechitin 1 ⁇ 2 can be prepared by the method described in R. Senzyu and S. Okimatsu, Nippon Nogeikagaku Kaishi, 23, 432 (1950).
  • the purified chitinase produced by the Aspergillus oryzae var. Sporof lavus Ohara J CM 2067 strain has the following properties.
  • the molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
  • the migration method shows an isoelectric point p I of about 3.5.
  • this enzyme has higher hydrolysis activity to partially acetylated chitosan (chitosan 9B, 8B, 7 B) with an acetylation degree of 10 to 30% than glycol chitin and an acetylation degree of 1% or less It can be seen that the hydrolytic activity for chitosan (chitosan 10B) is weak.
  • chitinase produced and purified by Aspergillus japonics strain SANK 19288 has the following properties.
  • the molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
  • the migration method shows an isoelectric point p I of about 3.5.
  • the pH value of the enzyme for other substrates is 100% when the pH of the enzyme for chitosan 7 B is 100% at 10 ° C at a temperature of 37 ° C, and the temperature is 37 ° C.
  • the hydrolytic activity in 10 minutes in Table 3 is shown as a relative value.
  • chitosan 10B is 1% acetylated chitin
  • chitosan 9 B is 10% acetylated chitin
  • chitosan 8 B is 20% acetylated chitin
  • chitosan 7 B is 30% acetylated chitin
  • CM-cellulose is sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.).
  • Glycol chitin is prepared by the method described above.
  • the purified chitinase derived from Aspergillus japonicus strain SANK 19288 is the most hydrolyzing active with respect to chitosan 7 B, and the hydrolytic activity decreases as the degree of acetylation decreases.
  • the purified chitinase has chitin hydrolysis activity and has no cellular hydrolysis activity.
  • the molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
  • An isoelectric focusing method shows a p I of about 4.5.
  • this enzyme has high hydrolysis activity for partially acetylated chitosan (chitosan 9B, 8B, 7B) having a degree of acetylation of 10 to 30%, and chitosan having a degree of acetylation of 1% or less (chitosan) It can be seen that there is little hydrolytic activity for 10B). There is no hydrolytic activity against propoxymethylcellulose-sodium sulfate.
  • chitinase produced and purified by Aspergillus sogyae strain SANK 22388 has the following properties.
  • the migration method shows an isoelectric point p I of about 4.5.
  • chitosan 10 B is 1% acetylated chitin
  • chitosan 9 B is 10% acetylated chitin
  • chitosan 8 B is 20% acetylated chitin
  • chitosan 7 B is 30% acetylated chitin (both are additive )).
  • CM-cellulose is carboxymethylcellulose sodium (manufactured by Wako Pure Chemical Industries, Ltd.).
  • Glycol chitin is prepared by the method described above.
  • purified chitinase derived from Aspergillus' Sojiyae strain SANK 22388 is the most hydrolyzing active with respect to chitosan 7B and 8B, and the hydrolyzing activity decreases as the degree of acetylation decreases.
  • the purified chitinase has chitin hydrolysis activity and does not have cellular hydrolysis activity. From the above, the properties of the chitinase of the present invention include, but are not limited to, the following.
  • the molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
  • the isoelectric point electrophoresis shows an isoelectric point p I of 3.0 to 5.0 (preferably p I of 3.5 to 4.5).
  • Chitosan 7B is hydrolyzed at pH 3.0 to pH 1.1 (preferably pH 3.5 to 10.5).
  • Also included in the present invention is a method of producing the chitinase of the present invention.
  • Aspergillus' Oryzae var. Sporoflavus Ohara J CM 2067, Aspergillus japonics S ANK 1 9 2 8 8, Aspergillus sogyae S ANK 2 2 3 8 8 and other chitinogenic microorganisms are cultured in a culture medium.
  • chitinase can be produced.
  • maltose extract manufactured by Difco
  • yeast extract manufactured by Difco
  • shake culture is carried out at 100-45 rmp for 1-15 days at 16-45 ° C in a medium of 0.05 to 1.0% chitosan.
  • the chitosan low molecular weight chitosan and various salt solutions obtained by the present invention can be used as they are, but can also be concentrated by ammonium sulfate precipitation, organic solvent precipitation, centrifugation, lyophilization, ultrafiltration, and the like.
  • purification, desalting and molecular weight fractionation can also be performed by dialysis, ultrafiltration, gel filtration, column chromatography, etc.
  • the low molecular weight chitosan hydrochloride prepared in this manner is excellent in water solubility and can be a solution with a concentration of up to about 10% by mass.
  • a 1% by mass aqueous solution of low molecular weight chitosan hydrochloride exhibits a pH of 4 to 5 and a precipitate (colloid) is precipitated when an alkali is added.
  • the pH is pH 6 to 7 It is four.
  • the physiological activity of the low molecular weight chitosan hydrochloride prepared here is examined using the antibacterial activity as an index.
  • the minimum inhibitory concentration (Minimum Inhibitory Concentration; MIC) of each of low molecular weight chitosan 9 B, 8 B and 7 B hydrochlorides to have antibacterial activity is measured.
  • MIC Minimum Inhibitory Concentration
  • low molecular weight chitosan hydrochloride has an activity equal to or higher than high molecular weight chitosan hydrochloride. Therefore, low molecular weight chitosan has superior physiological activity equal to or higher than high molecular weight chitosan.
  • the low molecular weight chitosan or its salt obtained in the present invention is, for example, an external preparation for wounds and wounds, a remedy for bed sores, a remedy for atopic dermatitis, Skin external preparations such as a remedy for acnes, bathing agents, cosmetics, face cleansers, basic cosmetics, oral hygiene agents such as dental caries preventives, liquid mouthwash, dental materials, enteric agents such as intestinal bacteria improvers, Medical field such as nosocomial infection preventive drug, food food field such as diet food, food preservative, preservative field such as animal and fish feed field, agricultural field such as plant activity enhancer, water treatment agent, metal adsorption
  • the low molecular weight chitosan of the present invention for an agent, a nuclear waste adsorbent, a drug separating agent, etc., the application is not particularly limited to these examples.
  • Also included in the present invention is a method for treating diseases such as trauma, bed sores, atopic dermatitis and the like, which comprises administering to an animal an effective amount of the low molecular weight chitosan of the present invention or a salt thereof.
  • the use of the low molecular weight chitosan or a salt thereof of the present invention for treating these diseases is also included in the present invention.
  • the usage of the low molecular weight chitosan or low molecular weight chitosan salt obtained by the present invention changes depending on the purpose, it may take various forms for use as described above. The forms are listed below, but are not particularly limited.
  • Low molecular weight chitosan can be used as a solid.
  • an aqueous solution of 10% by mass concentration or an organic solvent solution, more suitably diluted, may be used at an arbitrary concentration.
  • Diluent is water or organic solvent
  • pH is more acidic than 6.5, it can be used as a solution; if it is alkaline it can be used as a colloidal compound, but depending on the type of salt, it can be used as a solution even if it is weakly alkaline.
  • an inorganic substance or an organic substance it can be used as a solution at any pH.
  • it can be used as a cream agent by mixing low molecular weight chitosan solution or diluted solution with a cream base at an arbitrary concentration of 10% by mass or less.
  • a low molecular weight chitosan solution or dilution solution can be used as a film having any thickness.
  • Films having a plastic-like hardness are obtained by drying low molecular weight chitosan solutions in a frame having various thicknesses.
  • a flexible sheet having flexibility can be obtained by mixing a low molecular weight chitosan solution with a solvent such as dalyserine at an arbitrary ratio and drying it in a frame having various thicknesses.
  • the degree of flexibility is controlled, for example, by the rate of addition of a solvent such as glycerin.
  • a chitosan-methylcellulose composite film can be obtained by mixing methylcellulose with a low molecular weight chitosan solution and drying it in a frame having various thicknesses.
  • the dose varies depending on the type of disease, symptoms, patient's sex, age, dosage form and administration method, it is safe and any amount can be used.
  • 2 g / kg body weight or less is preferable, and in other cases it is adjusted appropriately.
  • the chitinase of the present invention can be effectively used to make an intractable sample easier to handle.
  • the viscosity was very high and difficult to handle. This was due to the high molecular weight polysaccharide (the main component being high molecular weight partially acetylated chitosan) contained in these crustaceans.
  • the chitinase of the present invention is added to the process of such food processing and reacted, the high molecular weight chitosan or high molecular chitin in the liquid component is divided, and the high molecular portion in the liquid component. It is possible to produce an easy-to-handle sample substantially free of acetylated chitosan, and a method for producing the easy-to-handle sample using a kita DNAase is also included in the present invention.
  • the raw material used for such a production method is not particularly limited as long as it is a sample containing a high molecular weight partially acetylated chitosan, but is preferably a sample containing crustaceans, and more preferably any one of Ebi and force 2 Or a sample containing one or both.
  • the reaction conditions for the raw material and chitinase the conditions for exhibiting the above-mentioned chitosan decomposition activity can be applied.
  • the reaction time is 10 minutes or more, and the time for the viscosity of the sample to be low There is no particular limitation, so long as it is preferably 10 minutes to 10 days, and more preferably 1 day to 5 days.
  • tes produced by such a method is also included in the present invention.
  • substantially free of partially polymerized acetylated chitosan in its liquid component means that the high viscosity is reduced or eliminated, which is caused by the inclusion of polymer partially acetylated chitosan in the sample. Strictly speaking, it does not indicate that the concentration is 0%, but preferably the content of high molecular partially acetylated chitosan is 20% or less, more preferably 10% or less, more preferably 5 It is less than%, and optimally 0%.
  • Example 1 Purification of a chitinase from Aspergillus sp. Oryzae var. Sporoflavus Ohara J'CM 2067
  • the hydrolysis activity of chitinase was measured as follows.
  • chitosan 7B (30% acetylated chitosan, manufactured by Tokichi Co., Ltd.) to completely dissolve powdered chitosan.
  • Chitosan 8B (20% acetylated chitosan, manufactured by Katokichi Co., Ltd.) 125 mg water 50 ml and acetic acid 50 The powder chitosan was completely dissolved by adding l.
  • chitosan 9 B (10% acetylated chitosan, manufactured by Tokichi Co., Ltd.) to completely dissolve powdered chitosan.
  • 5 Om 1 of water and 90 l of acetic acid were added to 125 mg of chitosan 10B (almost completely deacetylated chitosan, manufactured by Addo Corporation) to completely dissolve powdered chitosan.
  • the enzyme solution 1401 was added to a mixture of the chitosan aqueous solution 1601 and 40 OmM acetate buffer (PH5.01) 100 1 with stirring to homogenize, and the mixture was kept at 37 ° C. to carry out enzyme reaction for 20 minutes.
  • the reaction was stopped by adding a solution 4001 in which asetylaceton 101 was dissolved in 500 M aqueous solution of sodium carbonate 500 M to the enzyme reaction solution, and the mixture was incubated at 100 ° C. for 20 minutes. After ice-cooling, a mixed solution 400 1 in which N, N-dimethylaminobenzaldehyde 0. 8 g is dissolved in ethanol 30 ml and concentrated hydrochloric acid 3 O ml 400 1 and ethanol 1200 ⁇ 1 are added to the enzyme reaction solution and heated at 65 ° C for 10 minutes. The color reaction was allowed to occur. After ice-cooling, the precipitate was centrifuged and the absorbance at 530 nm of the supernatant was measured. Enzymatic reaction The enzyme activity that produces 1 mo 1 of darcosamine per minute was defined as 1 unit.
  • the column was thoroughly washed with 10 mM Tris ⁇ HCl buffer solution (pH 7.5), and then 0 to 0.4 M chloride was added in 600 ml of 1 O mM Tris ′ hydrochloric acid buffer solution (pH 7.5).
  • a linear concentration gradient of sodium was prepared to elute the components adsorbed on the column, and the chitosan decomposition activity was eluted in the fraction (135 ml) having a sodium chloride concentration of 0.20 to 0.30 M.
  • the crude purified enzyme fraction was thoroughly washed with 10 mM Tris ⁇ HCl buffer solution (pH 7.5), and then 0 to 0.4 M chloride was added in 600 ml of 1 O mM Tris ′ hydrochloric acid buffer solution (pH 7.5).
  • a linear concentration gradient of sodium was prepared to elute the components adsorbed on the column, and the chitosan decomposition activity was eluted in the fraction (135 ml) having a
  • the fraction was used as a purified enzyme solution. 5) Molecular weight measurement of purified enzyme
  • the molecular weight of the purified enzyme was determined by SDS-PAGE electrophoresis using a 5% polyacrylamide gel (see Laemmli, UK, Nature, 227, 680 (1970)).
  • the following proteins were used as standard proteins: a. Phospholase, molecular weight 94, 000: b. Albumin, molecular weight 67, 000: c. Ovalbumin, molecular weight 43000: d. Carbonic ' Anhydrase (carbonic anhydrase), molecular weight 30,000: e. Trypsin 'trypsin inhibitor, molecular weight 20, 100: f. Lactalbumin (-lactalbumin), molecular weight 14, 400.
  • the calibration curve obtained from the mobility of these standard proteins is shown in FIG. As shown in FIG. 1, the purified enzyme showed a single band with a molecular weight of about 40,000.
  • the purified enzyme was subjected to isoelectric focusing (see “Biochemistry Experimental Course 1 Protein Chemistry I Separation and Purification, Tokyo Kagaku Dojin (1976)” edited by the Japan Biochemical Society, p. 262 to 312).
  • the isoelectric point of the purified enzyme was about p 14.5.
  • the purified enzyme solution was concentrated to about 1 mg / ml using an ultrafiltration membrane (ULTRA FILTER UNIT USY_1 manufactured by ADVANTEC, molecular weight fractionation 10,000).
  • Denature buffer (6 M guanidinium hydrochloride, 1 OmM EDTA, 0.1 M ammonium bicarbonate pH 7.8) 400 1 and 5 OmM Di thiothreitol 8 1 were added to the concentrated enzyme solution 400 1 and reacted at 95 ° C. for 10 minutes. After cooling to room temperature, 5 OmM Iodoacetoamide 401 dissolved in Denature buffer solution was added to the reaction solution, and allowed to react at room temperature in the dark for 1 hour.
  • This solution was dialyzed against 1 L of water with Slide-A-Lyzer Mini Dialysis Units Plus Float (manufactured by PIERCE, molecular weight fraction: 10,000). Trypsin (Modified, Promega) 8n 1 was added to the obtained solution, and enzymatic reaction was performed at 37 ° C. for 7 hours. The reaction solution was subjected to a SMART system (manufactured by Pharmacia Biotech) to separate peaks of degraded amino acids. The separation conditions are shown below.
  • the volume was made up to 1,000 ml with pure water.
  • the molecular weight was measured in the same manner as in Example 15).
  • the enzyme showed a single band with a molecular weight of about 40,000.
  • the isoelectric point was measured in the same manner as in Example 1 6).
  • the isoelectric point of this enzyme is about! ) I was 3.5.
  • the volume was made up to 1,000 ml with pure water.
  • the column is thoroughly washed with 2 OmM Tris ⁇ HCl buffer solution (pH 7.5), and then 0 to 0.1 M of sodium chloride in 1 OmM Tris' hydrochloric acid buffer solution (pH 7.5) in 25 Oml. A linear concentration gradient was created to elute the components adsorbed on the column. Chitosan degradation activity was eluted in the fraction (10 ml) of sodium chloride concentration between 0. 0621 ⁇ and 0. 064M. This fraction was used as a purified enzyme solution.
  • the molecular weight was measured in the same manner as in Example 15).
  • the enzyme showed a single band with a molecular weight of about 40,000.
  • Example 4 Preparation of low molecular weight chitosan hydrochloride using chitinase derived from Aspergillus' oryzae var. Sporoflavus Ohara J CM 2067 strain
  • Chitosan 9 B or Chitosan 8 B or Chitosan 7 B, 2. 08 was suspended in 800 ml of water, concentrated hydrochloric acid was added to completely dissolve the powder as PH 2.5. After adjusting the pH to 5.0 with saturated aqueous sodium hydrogen carbonate solution, water is added to make the total volume 1,000 ml, 200 ml each is transferred to a 500 ml Erlenmeyer flask, and the enzyme prepared in Example 1 1) 10 ml of the solution was added, and the enzyme reaction was carried out by shaking at 80 rpm for 3 days at 40 ° C.
  • Low molecular weight chitosan 8 B hydrochloride was obtained as a white fluffy solid of 1.6 g.
  • Low molecular weight chitosan 7 B hydrochloride was obtained as a white fluffy solid of 0.6 g.
  • Example 5 Preparation of Low Molecular Weight Chitosan Hydrochloride Using Chitinase From Aspergillus japonics Strain SANK 19288
  • Low molecular weight chitosan hydrochloride was prepared in the same manner as in Example 4.
  • the enzyme solution was the one obtained in Example 21), and the enzyme reaction was performed at pH 4.0.
  • Low molecular weight chitosan 9 B hydrochloride was obtained as 1.3 g of a white fluffy solid.
  • the low molecular weight chitosan 8B hydrochloride was obtained as a white fluffy solid of 1.3 g.
  • Low molecular weight chitosan 7 B hydrochloride was obtained as a white fluffy solid of 0.9 g. '
  • Low molecular weight chitosan hydrochloride was prepared in the same manner as in Example 4. However, the enzyme solution was prepared in Example 3 and the enzyme reaction was performed at pH 4.5. The low molecular weight chitosan 9 B hydrochloride was obtained as 2.1 g of 'white fluffy solids. The low molecular weight chitosan 8B hydrochloride was obtained as a 1.7 g white fluffy solid. Low molecular weight chitosan 7 B hydrochloride was obtained as a white fluffy solid of 1.0 g.
  • Example 8 Determination of molecular weight fraction by gel filtration
  • dextran T10 molecular weight 10,000
  • T40 molecular weight 40,000
  • T70 molecular weight 70,000
  • T110 molecular weight 110, 000 0
  • ⁇ 250 molecular weight 250, 000
  • Example 9 Isolation of genomic DNA encoding chitinase from Aspergillus' oryzae (Aspergillus oryzae)
  • Oligoprimer PR50 which was identified from the Aspergillus 'oryzae genome' library, was synthesized using the primers C21 and F21.
  • A was amplified by approximately 800 bp by PCR using Aspergillus oryzae genomic DNA as a template. This DNA was separated by electrophoresis, cut out from an agarose gel, and purified using QIAquick Gel Extraction Kit (manufactured by Kyanagen). DNA purified in this way is DIG DNA Labeling Kit
  • Labeling was carried out using (Roche Diagnostic). The labeling method was as follows. First, the purified DNA was heated at 100 ° C. for 10 minutes to make it single-stranded and then quenched on ice. Then IO I Hexanucleotide 'mix (0.5 M Tris, 0.1 M MgCl 2 , ImM Dithioerithrito K 2 mg / ml BSA; pH 7.2), 10 ⁇ 1 dNTP labeling' Mix (Im M dATP, Im M dCTP, Im M dGTP, 0.65 mM dTTP , 0.35 mM DIG-11-dUTP; pH 7.5), 5 ⁇ 1 DNA polymerase (Klenow fragment) were added, and the total volume was adjusted to 100 1 with sterile water. The reaction solution was allowed to react at 37 ° C., and then the reaction was stopped by adding 10 10 0.2 M EDTA (pH 8.0). Then 10 1 N LiCl and 500 1 100% ethanol
  • the DNA was electrophoresed on a agarose gel, and the same position as the position of the band found by the above-mentioned Southern hybridization was excised from the gel and the DNA was extracted.
  • the extracted DNA was ligated to PUC118 EcoRI / BAP vector to obtain a DNA library containing a chitinase gene.
  • this membrane was prehybridized with DIG Easy Hyb for 1 hour. Thereafter, the membrane was subjected to hybridization overnight at 37 ° C. with a chitinase gene probe obtained in 9-13). After hybridization, the cells were washed three times with normal temperature 2 ⁇ SSC buffer, and further washed twice with 2 ⁇ SSC buffer warmed to 53 ° C. Thereafter, detection was performed using DIG Wash and Block Buffer Set (manufactured by Roche 'Dagnoistics Co., Ltd.). First, the membrane was acclimated with Wash buffer (0.1 M maleic acid, 0.15 M NACK 0.3% Tween 20: pH 7.5).
  • the membrane was immersed in a blocking solution (0.1 M maleic acid, 0.15 M NaCl: a solution containing 10% blocking reagent at pH 7.5) for blocking for 60 minutes to block nonspecific binding.
  • a blocking solution 0.1 M maleic acid, 0.15 M NaCl: a solution containing 10% blocking reagent at pH 7.5
  • the membrane was immersed in a blocking solution to which anti-digoxigenin antibody was added at a dilution of 10000, reacted at 37 ° C. for 30 minutes, and washed twice with Wash Buffer. Thereafter, the membrane was acclimated with Detection buffer (0.1 M Tris-HCU 0.1 M NaCl, pH 9.5).
  • the clone showing the positive signal obtained in 9-5) was cultured by L-broth (manufactured by Wako Pure Chemical Industries, Ltd.) for a while, and the DNA of the recombinant plasmid was extracted. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (manufactured by Qiagen). Then, the extracted DNA is sequenced using this plasmid-specific universal primer M13M4 and M13RV, and the result and the nucleotide sequence of the chitinase gene probe obtained in 9-13) are aligned. It was confirmed that it contains the sequence of chitinase.
  • each aliquot was aliquoted into 675 1 Rneasy Mini spin columns and centrifuged at 16000 rpm for 15 seconds at room temperature 700 1 of RW1 Wash buffer was added and similarly centrifuged at 16000 rpm for 15 seconds at room temperature 500 li ⁇ RPE Wash buf fer was added and RNA was washed by centrifuging at 16000 ⁇ ⁇ 1 ⁇ for 2 minutes at room temperature The spin column was set in a microtube, RNA was eluted with RNase free water, and collected by centrifugation at 10000 rpm at room temperature for 1 minute did.
  • RNA ljg was mixed with SMART IV Oligonucleotide, lil CDS III / 3 'PCR Primer in a sterile 0.5 ml tube and incubated at 72 ° C for 2 minutes. After that, it was placed on ice for 2 minutes and cooled. To this cooled sample was added 5X First-Strand Buffer, 1 n 1 20 mM DTT 1 1 n 1 lO mM dNTP Mix and 1 1 Power Script Reverse Transcriptase. The contents were mixed and allowed to react at 42 ° C.
  • PCR tube was set in a thermal cycler preheated to 95 ° C., reacted at 95 ° C. for 20 seconds, and subjected to 20 cycles of 95 ° C. for 5 seconds and 68 ° C. for 6 minutes.
  • Transfer 50 1 of this reaction solution (containing ds cDNA) into a sterile tube add 2 ° C / protase, and incubate at 45 ° C for 20 minutes to perform protease treatment, and then perform 50 1 deionization. Water was added.
  • IOO I phenol: black form: isoamyl alcohol mixture (25: 24: 1) was added, mixed for 1 minute, and centrifuged for 5 minutes in UOOOHD III.
  • the supernatant was transferred to a new tube and 1001 chloroform: isoamyl alcohol (24: 1) was added. After mixing for 1 minute, it was centrifuged again at 14000 rpm for 5 minutes. The supernatant was transferred to a new tube, added with 10 13 M sodium acetate, 1.2 nl glycogen and 260 1 95% ethanol (room temperature) and immediately centrifuged at room temperature with HOOO rpm for 20 minutes. After removing the supernatant with a pipet and washing the pellet with 80% ethanol 100 1, the pellet was dried. The pellet was dissolved in 79 1 deionized water. Subsequently, treatment with restriction enzyme Sfi I was performed.
  • the Sfi I digested cDNA sample was ablated in the center of the upper surface of the matrix and sufficiently absorbed on the surface of the matrix.
  • 600 1 column buffer was added and fractions were collected immediately in 16 tubes. Each 31 of this fraction was subjected to agarose gel electrophoresis to determine the peak fraction.
  • the fractions containing the cDNA were collected and collected in one new test tube. Add 1/10 volume of sodium acetate (3 M; pH 4.8), 1.3 ⁇ glycogen, 2.5 volumes of 95% ethanol (-20 ° C) to this test tube, mix gently, and mix at -20 ° C. Incubated.
  • the supernatant was removed by centrifuging at 14000 1 ⁇ ⁇ for 20 minutes at room temperature. After the pellet was completely dried, the pellet was resuspended in 7 ⁇ 1 deionized water.
  • the above-mentioned cDNA 0.5 ⁇ i was mixed with ⁇ TriplEx2 vector I, 0.5110 X Ligation Buffer, 0.5 il ATP, 0.5 / xl T4 DNA Ligase and 2 ⁇ 0 1 deionized water, and incubated at 16 ° C. Next, a ⁇ phage packaging reaction was performed on this linear sample.
  • Gigapack III Gold Packaging Extract manufactured by STRATAGENE was used for packaging.
  • the culture was carried out at 37 ° C. until it reached 2.0.
  • a period of 5 minutes cultures at 5000rpm were centrifuged and resuspended Peretsuto to LOMM MgSO 4 of 7.5 ml.
  • the packaging solution was diluted with IX ⁇ dilution buffer, the packaging solution was added to the suspension of 2001.E. coli XL1-Blue and adsorbed at 37 ° C. for 15 minutes.
  • the LB / MgSO 4 Totsupuaga of molten 2ml were mixed and spread evenly LB / MgSO 4 plates that had been warmed. After cooling at room temperature for 10 minutes, the top agar was solidified, and cultured at 37 ° C. for incubation. The resulting plaques were eluted by adding SM buffer and shaking to give a phage containing the Aspergillus oryzae cDNA library.
  • the plate is left for 5 minutes on a filter paper soaked with an alkaline denaturation solution (0.2 N NaOH, 1.5 M NaCl), and then neutralized solution (0.4 M Tris-HCl (pH 7.5), 2 ⁇ SSC ) Was placed on the soaked filter paper for 5 minutes. Further, the plate was washed with a neutralization solution for 1 minute while shaking, and then washed with 2 ⁇ SSC for another 5 minutes. The filter was dried on filter paper for about an hour, and baked in an oven at 80 ° C. for two hours. Thereafter, the membrane was subjected to hybridization with DNA probe obtained in X-3 at 37 ° C.
  • the plate was washed three times with 1 ⁇ 2 ⁇ SSC buffer at room temperature, and further washed twice with 2 ⁇ SSC buffer warmed to 53 ° C. Thereafter, detection was performed using DIG Wash and Block Buffer Set (manufactured by Roche 'Dagnoistics Co., Ltd.).
  • the membrane was acclimated with Wash buffer (0.1 M maleic acid, 0.15 M NaCl, 0.33 ⁇ 4 Tween 20; ⁇ 7 • 5). Then, the membrane is immersed in a blocking solution (0.1 M maleic acid, 0.15 M NaCl; a solution containing 10% blocking reagent in H7.5). Blocking was performed for 60 minutes to block nonspecific binding.
  • the membrane was immersed in a blocking solution to which anti-digoxigenin antibody was added at a dilution of 10000, reacted at 37 ° C. for 30 minutes, and washed twice with Wash Buffer. Then, the membrane was acclimated with Detection buffer (0.1 M Tris-HCK 0.1 NaCl; pH 9.5). After adding CSPD (Chemiluminescence substrate) to Detection buffer at 100-fold dilution and dropping it into purified membrane, color development was stabilized at 37 ° C for 15 minutes. After that, it was exposed to Lumi- Film Chemi luminescent Detecion Film (manufactured by Koni), developed, and detected.
  • Detection buffer 0.1 M Tris-HCK 0.1 NaCl; pH 9.5
  • the positive phage plaque showing this positive is removed from the agar medium, dissolved in SM buffer, transformed into E. coli BM25.8 strain, and transformed into E. coli strain Escherichia coli strain BM25.8 / pTri 1 Ex-Ch iti nas e-cDNA SANK 72102 strain was obtained. This strain was internationally deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary on January 8, 2002, under the Accession Number: FERM BP-8235. The infected phage DNA, ⁇ TriplEx-Chitinase-cDNA, is circularized in cells of E.
  • coli BM25.8 strain and exists as a recombinant plasmid pTriplEx- Chitinase-cDNA.
  • the resulting colonies of Escherichia coli BM25.8 / pTripI-cDNA-DNA SANK 7202 were cultured in LB / Ampicillin broth to extract DNA of the recombinant plasmid pTriplEx_Chitinase_cDNA. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (manufactured by Qiagen).
  • the plasmid DNA ⁇ 1 was diluted with 10XGPS Buffer (250 mM Tris-HCl; pH 8.0, 20 mM DTT, 20 mM 'ATP, 500 g / ml BSA), 1 ⁇ 1 GPSl (Transprimer-1 Donor plasmid), 11 xl sterile water
  • 10XGPS Buffer 250 mM Tris-HCl; pH 8.0, 20 mM DTT, 20 mM 'ATP, 500 g / ml BSA
  • 1 ⁇ 1 GPSl (Transprimer-1 Donor plasmid) 11 xl sterile water
  • TnsABC Transposase was added and allowed to bind at 37 for 10 minutes. Thereafter, 11 Start Solution (300 mM magnesium acetate) was added, reacted at 37 ° C. for 1 hour, and treated at 75 ° C. for 10 minutes to stop the reaction. Then, 'this reaction product 10 // 1 was transformed into E.
  • the binding frame has “ATG” as the initiation codon from nucleotide numbers 242 to 244, and nucleotide numbers 1436 to 1438. It is inferred that the amino acid sequence to be encoded is the sequence shown in SEQ ID NO: 5 in the sequence listing.
  • Example 11 Determination of the N-terminal amino acid sequence of the aspergillus oryzae origin chitinase The enzyme solution purified in Example 1 was concentrated to about 1 mg / m 1 using an ultrafiltration membrane (ULTRA FILTER UNIT USY-1, manufactured by ADVANTEC, molecular weight fraction 10,000).
  • Denatured buffer (6 M guanidinium hydrochloride, 1 OmM EDTA, 0.1 M ammonium bicarbonate pH 7.8) 400 1 addition 5011 ⁇ Dithiothreitol 8 1 was added to the concentrated enzyme solution 40 OI, and reacted at 95 ° C. for 10 minutes. After cooling to room temperature, 5 OmM Iodoacetoamide 401 dissolved in Denature buffer solution was added to the reaction solution, and allowed to react at room temperature in the dark for 1 hour. This solution was dialyzed against 1 L of water with Slide-A-Lyzer Mini Dialysis Units Plus Float (manufactured by PIERCE, molecular weight fraction: 10,000).
  • Trypsin (Modified, Promega) 81 was added to the resulting solution, and enzymatic reaction was performed at 37 ° C. for 7 hours.
  • the resulting enzyme reaction solution was measured by LC / MS (Pencon / Q-Tof 2).
  • the m / z 533 was set as the include mass, and the MS / MS spectrum was measured.
  • the assignment of the fragment ion shows that the N-terminal amino acid is Ser, the amino acid sequence Ser_Ser-Gly-Leu-Lys is contained at the N-terminus, and the presence of an acetyl group is suggested at the N-terminal (Sequence listing Amino acid sequences described in amino acid numbers 1 to 5 of SEQ ID NO: 14).
  • Primer 1 BamHI-cDNA and cDNA-BamHI, in which BamHI sites were respectively connected to the 5 'end and 3' end of chitinase 0RF, were designed and synthesized.
  • BamHI-cDNA 5'-gaggatccatgtci tccggactaaagtcgg-3, (Sequence Listing SEQ ID NO: 9)
  • cDNA-BamHI 5-ctggatccagcgaa aggggctcg caggattg- 3 (Sequence Listing Sequence Number 1)
  • the recombinant plasmid obtained in Example 10-3, pTriplEx-Chitinase-cDNA was used as a template, PCR was performed using the above-mentioned BamHI-cDNA and cDNA-BamHI as a primer, and a restriction enzyme site was introduced.
  • the DNA was amplified. Furthermore, it was inserted into pCR2.1-T0P0 vector (manufactured by In Vitrogen), transformed into E. coli dish 09, and amplified. Colonies grown on strain 1M109 were cultured in Lb / Ampicillin broth to extract plasmid DNA.
  • the plasmid DNA was treated with restriction enzyme BamHI to cleave chitinase 0RF.
  • pQE-60 manufactured by Qiagen
  • pQE-60 was digested with BamHI and further treated with alkaline phosphatase to remove the phosphate group at the 5 'end.
  • T0P0 TA Cloning Kit manufactured by Invitrogen Co.
  • the above-mentioned kit RF was used to carry out the ligation.
  • the reaction solution was transformed into E. coli JM109 strain and cultured overnight on Lb / Ampicillin agar plates. The grown colonies were inoculated into 3 ml of Lb / Ampicillin broth and cultured with shaking while shaking.
  • the plasmid DNA was extracted with QIAprep Spin Miniprep Kit (manufactured by Qiagen), and transformed into E. coli M15 [pREP4] strain. Incubate with Lb / Ampicin, Kanamaicin single plate and incubate the grown colony in 20 ml of Lb / Ampicillin, Kanamaicin broth and culture at 37 ° C with shaking. did. From this culture, 2.5 ml was inoculated into 100 ml of Lb / Ampic i 1 in, Kanamicin broth and cultured at 37 ° C. 0D 6 every 30 minutes from 1 hour after the start of culture.
  • the supernatant 100 1 is placed in a 1.5 ml tube, 1 denaturing agent (6% SDS, 243 ⁇ 4 glycerol, 12%) 3-mercaptoethanol, 0.3 M Tris-HCl (pH 6.8), 0.15% BPB) was added and treated at 100.degree. C. for 5 minutes.
  • the reaction solution was electrophoresed on each of samples 101 by e-PAGEL (manufactured by ATT0), and then stained and detected by Si immediately ly Blue SafeStain (manufactured by Invitrogen). As a result, a dark band was observed around about 44 kDa.
  • the supernatant of the cells cultured for a long time and collected was subjected to His tag purification.
  • the column tube was filled with Ni-NTA agarose and washed with about 10 ml of 0.1 M Tris-HCl (pH 8.0), and then the supernatant was adsorbed onto a column. Then, after washing away nonspecific binders with 8 ml of 10 mM imidazole, the protein adsorbed to the column is eluted with 8 ml of 200 mM imidazoyl. The eluate was electrophoresed on e-PAGEL and appeared as a single band.
  • the eluate and chitinase purified from the culture of Aspergillus oryzae obtained in Example 1 were subjected to SDS electrophoresis on a 12.5% acrylamide gel (shown in FIG. 30).
  • the molecular weight of the protein contained in the eluate is slightly larger than the purified enzyme of Aspergillus oryzae free, but this is considered to be due to the His tag, and the recombinant protein contained in the eluate is His. It was speculated to be a fusion recombinant chitinase.
  • the chitosan-degrading activity of this eluate was determined by the method described in Experimental Example 16) to be 2.
  • the chitinase from Aspergillus oryzae for which the amino acid sequence was identified in the present invention was composed of 39 9 amino acids (SEQ ID NO: 5 in the sequence listing) and post-translationally modified before being subjected to post-translational modification. It consists of eight amino acids (SEQ ID NO: 14 in the sequence listing). Is the gene derived from Aspergillus spp. Oryzae that has been reported as a chitinase gene (Chitin's Chitosan Research, Vol. 8, No. 2, page 238-239, 2002) 4 amino acids?
  • Test Example 1 Properties of crude enzyme solution of chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara J CM 20 67 strain
  • Example 1 2 quantification of reducing sugars was performed.
  • chitosan 7 B was measured as a substrate, it had an enzyme activity of 4.2 ⁇ 10 3 U / ml in the supernatant.
  • the relative value of chitosan 10B degradation activity is shown in Table 11 when the enzyme activity is 100% when chitosan 7 B is used as the substrate. It was found that it has chitosan 7 B degradation activity and almost no chitosan 10 B degradation activity.
  • the hydrolysis activity was measured at various temperatures under the condition of pH 5.0.
  • Enzyme solution 1401 was added to a mixture of chitosan 7 B solution 160 ⁇ 1 and 40 OmM acetate buffer (pH 5.0) 1001 prepared in Example 1 preheated for 5 minutes, and homogenized to obtain 10
  • the enzyme reaction was performed for a minute.
  • the quantification of the free reducing sugar after the enzyme reaction was according to Example 1.
  • Most The hydrolysis activity measured under the temperature condition showing high activity was taken as 100%, and the hydrolysis activity at each temperature was summarized in FIG. 6 as a relative value.
  • the enzymes in the culture supernatant were treated at various temperatures for 30 minutes and then their hydrolytic activity was measured.
  • the enzyme solution 2801 was added to 40OmM acetate buffer (pH 5.0) 2001 which was previously kept at the treatment temperature, stirred to make it uniform, and kept for 30 minutes.
  • the quantification of free reducing sugars was in accordance with Example 1. Assuming that the hydrolysis activity of a mixture of enzymes and buffers in the untreated (0 ° C. storage) group is 100%, the hydrolysis activity at each temperature is summarized in FIG. 7 as a relative value. ,
  • the enzyme reaction was performed for 20 minutes.
  • the quantification of free reducing sugar was according to Example 1.
  • the hydrolysis activity of the mixed solution of the enzyme and buffer solution of the untreated (0 ° C incubation) group is 100%, and the hydrolysis activity at each pH is shown in FIG. 8 as a relative value.
  • Test Example 2 Properties of crude purified chitinase derived from Aspergillus' Oryzae var. Sporoflavus O ara J CM 2067
  • Enzyme solution 1401 is added to the mixed solution of chitosan 7 B solution 160 ⁇ 1 and 40 OmM buffer solution 100 1 prepared in Example 1, and the mixture is stirred to homogenize and start the reaction.
  • the enzyme reaction was performed for a minute.
  • the quantification of the free reducing sugar after the enzyme reaction was according to Example 1.
  • the following buffer was used: pH 3. 3 to pH 5.
  • 9 to pH 8.5 MOPS-sodium carbonate buffer: pH 9.2 to ⁇ In the case of 3, sodium bicarbonate solution.
  • the hydrolytic activity under the pH condition where the activity was highest was taken as 100%, and the hydrolytic activity of the enzyme at each pH was shown as a relative value in FIG. The optimum pH was around pH 5.0.
  • the hydrolysis activity was measured at various temperatures under the condition of pH 5.0.
  • Enzyme solution 1401 was added to a mixture of 160 n 1 solution of chitosan 7 B prepared in Example 1 and 400 mM acetic acid buffer solution (pH 5.0) 100 1 which had been kept at the treatment temperature in advance, and the mixture was stirred to make it uniform.
  • the enzyme reaction was performed for 10 minutes.
  • the quantification of the free reducing sugar after the enzyme reaction was according to Example 1. Assuming that the hydrolysis activity measured under the temperature condition showing the highest activity was 100%, the hydrolysis activity at each temperature was summarized in FIG. 10 as a relative value.
  • the enzymes in the crude fraction were treated at various temperatures for 30 minutes and their hydrolytic activity was then measured.
  • the temperature was kept constant, 40OmM acetic acid buffer solution ( ⁇ 5.0) 200 ⁇ 1 and enzyme solution 280 X 1 were added and stirred to homogenize and kept for 30 minutes.
  • Heat-treated enzyme solution 240 1 was added to the chitosan 7 solution 160 1 prepared in Example 1, 20 at 37 ° C. The enzyme reaction was performed for a minute.
  • the quantification of free reducing sugars was in accordance with Example 1.
  • the hydrolytic activity at each temperature was summarized in FIG. 11 as a relative value, with the hydrolytic activity of the mixed solution of the enzyme and buffer solution of the untreated (o ° C. storage) group as 100%.
  • Example 1 2 Enzyme solution 50 n 1 is added to a mixture of chitosan 7 B solution 160 1 and 40 O mM buffer solution 100 1 and water 90 1 prepared by the method described above, and the mixture is homogenized to initiate the reaction.
  • the enzyme reaction was carried out for 40 minutes by incubating at 37 ° C.
  • the quantification of the free reducing sugar after the enzyme reaction was performed according to the method described in Example 12).
  • the following buffers were used: pH 3. 3 to pH 5. 9, sodium acetate monoacetate buffer: pH 5.9 to pH 8.4, MOPS-sodium carbonate buffer: pH 9.3 Sodium bicarbonate-sodium carbonate buffer for pH 10.4.
  • the hydrolytic activity under the pH condition where the activity was highest was taken as 100%, and the hydrolytic activity of the enzyme at each pH is shown as a relative value in FIG. The optimum pH was around pH 5.5.
  • the enzyme solution 75 n 1 is added to a mixed solution of 0.25 / 25% wt / v glycol chitin solution 240/1 and 40 OmM buffer 150 1 and water 135 ⁇ 1 to homogenize and start the reaction.
  • the enzyme reaction was carried out for 30 minutes by incubating at 37 ° C.
  • the quantification of the free reduced sugar after the enzyme reaction was performed by the Shells method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)).
  • the following buffer was used: pH 3.3 to pH 6.0, acetic acid-sodium acetate buffer: pH 6.3 to pH 7.8 2940
  • the hydrolysis activity of the purified enzyme was measured at various temperatures under the condition of pH 5.5.
  • Enzyme solution 40 H 1 was added and stirred to homogenize, and the enzyme reaction was performed for 10 minutes.
  • the quantification of the free reducing sugar after the enzyme reaction was according to Example 1.
  • the hydrolysis activity of the purified enzyme measured under the temperature condition showing the highest activity was taken as 100%, and the hydrolysis activity of the purified enzyme at each temperature is shown in FIG. 15 as a relative value. As shown in FIG. 15, the optimum temperature for the hydrolysis activity of the purified enzyme was 60 ° C. at PH 5.5.
  • the enzyme solution 1001 was added to a mixture of 40 mM acetic acid buffer (pH 5.5) 200 ⁇ 1 and water 180 1 which were previously maintained at the treatment temperature, stirred to homogenize, and incubated for 30 minutes.
  • Heat-treated enzyme solution 240 n 1 was added to chitosan 7 B solution 1601 prepared in the same manner as in 1), and an enzyme reaction was performed at 37 ° C. for 40 minutes.
  • the quantification of free reducing sugars was in accordance with Example 1.
  • the hydrolysis activity of the mixed solution of the purified enzyme and buffer solution of the non-treatment (0 ° C. incubation) group is taken as 100%, and the hydrolysis activity of the purified enzyme at each temperature is summarized in FIG. 16 as a relative value. As shown in FIG. 16, the purified enzyme was stable at 45 ° C. or less.
  • the reaction mixture was homogenized at 37 ° C. for 40 minutes, and free reducing sugar was quantified.
  • the hydrolysis activity of the mixture of the purified enzyme and buffer solution of the non-treatment (0 ° C. incubation) group is 100%, and the hydrolysis activity of the purified enzyme at each temperature is shown in FIG. 17 as a relative value.
  • the purified enzyme has a pH of 5 to pH 9. It was stable at 5.
  • the hydrolytic activity of the purified enzyme for various substrates was measured according to the method of Example 5.
  • the enzymatic reaction was carried out at pH 5.5 and ⁇ ⁇ 0.
  • the hydrolytic activity of the purified enzyme with respect to each of the other substrates is shown in Table 13 as a relative value, assuming that the hydrolytic activity of the purified enzyme with respect to chitosan 7 B is 100%.
  • Glycol chitosan 0 2. 1
  • the purified enzyme had the highest hydrolysis activity to chitosan 7 B, and the hydrolysis activity decreased as the degree of acetylation decreased. Also, the purified enzyme had chitin hydrolysis activity and no cellulase hydrolysis activity was observed.
  • Test Example 4 Various Properties of Chitinase Crude Enzyme Solution Derived from Aspergillus sp.
  • Example 1 2 According to the method described, quantification of reducing sugars was performed.
  • chitosan 7 B When chitosan 7 B was measured as a substrate, it had an enzyme activity of 3.4 ⁇ 10 ⁇ 3 U / ml in the supernatant.
  • the relative value of the chitosan 10 B degradation activity is shown in Table 14 when the enzyme activity is 100% when chitosan 7B is used as a substrate.
  • Example 1 2 140 x 1 of crude enzyme solution was added to a mixture of chitosan 7 B solution 160 1 and 40 OmM buffer solution 100 1 prepared according to the method described above, stirred to homogenize, and kept at 37 ° C. The enzyme reaction was carried out for 20 minutes, and the free reducing sugar after the enzyme reaction was quantified.
  • the following buffers were used: pH 3.7 to pH 6.0, acetic acid-sodium acetate buffer: pH 6.0 to pH 8.5 MOPS-sodium carbonate buffer: pH 9.3 to ⁇ In the case of 4, sodium bicarbonate-sodium carbonate buffer.
  • the hydrolysis activity under the highest activity pH condition is 100%, the hydrolysis activity of the enzyme at each pH is shown in FIG. 18 as a relative value. The optimum pH was around pH 4.0.
  • Substrate selectivity at pH 4.0 was measured. 10 ml of the prepared culture supernatant obtained in Example 2 was dialyzed three times every 12 hours against 1,000 ml of 1 mM mM Tris' hydrochloric acid buffer (pH 7.5) and used as an enzyme solution. . Enzyme solution 501 is added to a mixture of 25% wt / v substrate solution 1 Q 0 l and 40 OmM acetate buffer (pH 4.0) and water 160 I to homogenize and initiate the reaction, The enzyme reaction was carried out for 10 minutes by incubating at 37 ° C. The quantification of the free reducing sugar after the enzyme reaction was carried out by the Shells method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). The relative activities are shown in Table 15 when the hydrolysis activity is 100% when using a substrate exhibiting the maximum activity.
  • the reducing sugar was quantified according to the method described in Example 1 2).
  • Chitosan 7 B When Chitosan 7 B was measured as a substrate, it had an enzyme activity of 2.6 ⁇ 10 ⁇ 3 U / l in the supernatant.
  • the relative value of chitosan 10 B degradation activity is shown in Table 16 when the enzyme activity is 100% when chitosan 7 B is a substrate.
  • the measuring method is in accordance with Example 6.
  • the following buffers were used: pH 3. 8 to pH 6. 3 sodium acetate monoacetate buffer: pH 6. 2 to pH 8.6 MOP S-sodium carbonate buffer: pH 9 In the case of 3 to ⁇ ⁇ . 4 sodium bicarbonate sodium bicarbonate sodium bicarbonate buffer.
  • the hydrolysis activity under the highest activity pH condition was set to 100%, and the hydrolysis activity of the enzyme at each pH was shown as a relative value in FIG. The optimum pH was around pH 4.5.
  • Substrate selectivity at pH 4.5 was measured. 10 mM of the culture supernatant prepared in Example 4 was dialyzed three times every 12 hours against 1 mM of Tris ⁇ HCl buffer (pH 7.5) and 1,000 ml, and used as an enzyme solution. Enzyme solution 501 is added to a mixture of 5% wt Zv of substrate solution 16 O 1 and 400 mM acetate buffer ( ⁇ ⁇ 4.5) and water 160 ⁇ 1 to homogenize and initiate the reaction, The mixture was incubated at 37 ° C and subjected to enzyme reaction for 10 minutes. The quantification of the free reducing sugar after the enzyme reaction was carried out by the Shiers method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). Shows maximum activity P0212940
  • the relative activity is shown in Table 17 when the hydrolysis activity with 100 mg of the substrate is 100%.
  • chitosan 9B 1 g was suspended in water of 20 Om 1 and adjusted to pH 2.5 with concentrated hydrochloric acid to dissolve it completely. This was dialyzed against water and lyophilized to obtain 537 mg of a white cotton-like solid, high molecular weight chitosan 9 B hydrochloride.
  • the polymeric chitosans 10B and 8B and 7B hydrochlorides were also prepared in the same way. Distilled water for each polymer chitosan hydrochloride 10 Omg The product was dissolved in 50 ml, and 20 U of chitosanase (Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 3 hours. The reaction solution was freeze-dried, the dried solids was measured 1 H- NMR dissolved in D 2 ⁇ . .
  • the degree of ascetylation was calculated from the integral ratio of 3 H of the acetyl group to 7 H excluding the hydroxyl group other than the acetyl group. The results are shown in Table 19.
  • Test Example 8 Antibacterial Activity of Low Molecular Weight Chitosan
  • Staphylococcus aureus' Aureus (Staphylococcus aureus) 209 P JC-1 and Pseudomonas aeruginosa PA 01 as test bacteria and Example 4
  • the MIC of the low molecular weight chitosan hydrochloride prepared in Examples 5 and 6 using chitinases derived from various Aspergillus species was measured. The results are shown in Table 18. '
  • the bacteria to be used for the test were cultured in the above culture medium for ⁇ , and diluted with the same culture medium so that the number of bacteria was 2 ⁇ 10 6 .
  • Each low molecular weight chitosan hydrochloride 2 Omg was dissolved in distilled sterile water to make 1 Oml.
  • Diluted sterile water was used as a dilution solution beforehand to prepare a multiple dilution series of the sample solution, to which the above culture medium 501 and test bacteria solution 501 were added, and cultured at 37 ° C. for 18 hours to determine M I C.
  • the results are shown in Table 20.
  • Test Example 9 Properties of purified chitinase derived from Aspergillus japonics strain SANK 1928 1118 222252552111
  • the PH activity of the hydrolytic activity possessed by purified chitinase from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 3 1).
  • the hydrolytic activity under the pH condition where the activity was the highest was defined as 100%, and the hydrolytic activity of the enzyme at each pH was shown as a relative value in FIG.
  • the optimum pH was around pH 5.0.
  • the temperature activity of the hydrolytic activity possessed by the purified chitinase derived from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 32).
  • the hydrolysis activity under the temperature condition at which the activity was the highest was regarded as 100%, and the hydrolysis activity of the enzyme at each temperature was summarized in Figure 21 as a relative value.
  • the optimum temperature was 65 ° C. at PH 5.0.
  • the pH stability of the purified chitinase derived from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 3 4).
  • the hydrolysis activity of the mixture of the purified kitinase and buffer solution of the non-treatment (0 ° C. incubation) group is defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 23 as a relative value.
  • the purified chitinase was stable at pH 4 to pH 9.5.
  • the substrate specificity of the purified chitinase derived from Aspergillus' Japonics SANK 19288 strain was measured by the method described in Test Example 35).
  • the hydrolysis activity of the purified biotinylase for each of the other substrates is 100% when the hydrolysis activity of the purified chitinase at pH 5.0 at 37 ° C. for 10 minutes is 100% relative to chitosan 7B.
  • the relative values are shown in Table 21. 21] Relative activity (%) Chitosan 10B 0
  • purified chitinase from Aspergillus' Japonicas SANK 19288 strain is the highest hydrolyzing activity with respect to chitosan 7 B, and the degree of acetylation is TJP02 / 12940
  • the hydrolysis activity decreased as 58 decreased.
  • the purified chitinase had chitin hydrolysis activity, and no cellulase hydrolysis activity was observed.
  • the partial amino acid sequence of purified chitinase derived from Aspergillus japonicus SANK 19288 strain was determined by the method described in Example 1. 7). Among the separated degraded amino acids, three peaks of B buffer concentration of about 28%, about 30% and about 35% were collected. The sequences of the three were analyzed by an amino acid sequence analyzer (Procise cI, manufactured by Applied Biosystem). The results are described from the N-terminal side.
  • the hydrolytic activity of purified chitinase from Aspergillus sogyae strain SANK 22388 is as follows: The H activity was measured by the method described in Test Example 3.1). The hydrolysis activity under the highest activity pH condition was taken as 100%, and the hydrolysis activity of the enzyme at each pH was shown as a relative value in FIG. The optimum pH was around pH 5.5 and pH 9.0.
  • the purified chitinase derived from Aspergillus sogyae strain SANK 22388 is different from the chimeras derived from aspergillus oryzae and aspergillus japonics, and there are two optimum pH values (see Example 10 1).
  • ⁇ Circle around (2) ⁇ The temperature activity of the purified chitinase derived from Sogyae SANK 22388 strain was measured at these two pH values by the method described in Test Example 32).
  • Figure 25 temperature activity at pH 5.5
  • Figure 26 pH 9.0
  • the optimum temperature was 60 ° C. at pH 5.5 and 35 ° C. at pH 9.0.
  • the purified chitinase derived from Aspergillus sogyae strain SANK 22388 is different from the chimeras derived from Aspergillus perylase oryzae and Aspergillus japonics, and there are two optimum pH values (see Example 10 1).
  • the thermal stability of the purified chitinase derived from JAIE SANK 22388 strain was measured at these two pH points by the method described in Test Example 3.3). Taking the hydrolysis activity of the mixture of purified chitinase and buffer solution of the untreated (0 ° C incubation) group as 100% and the hydrolysis activity of the enzyme at each temperature as a relative value, the temperature at pH 5.5 is shown in Fig. 27. Stability) and FIG. 28 (temperature stability at pH 9.0). The purified chitinase was stable at 40 ° C. or less at any pH.
  • the pH stability of the purified chitinase derived from Aspergillus' soji SANK 22388 strain was measured by the method described in Test Example 3.4).
  • the hydrolysis activity of the mixture of the purified chitinase and buffer solution of the non-treatment (0 ° C. incubation) group is defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 29 as a relative value.
  • the purified kinase was stable at pH 4.5 to pH 10.
  • the purified chitinase derived from Aspergillus' Sojiyae strain SANK 22388 is the most hydrolyzing activity with respect to chitosan 7B and 8B, and the hydrolysis activity decreases as the degree of acetylation decreases. Also, the purified chitinase had chitin hydrolysis activity and no cellulase hydrolysis activity was observed.
  • Formulation example 1 Preparation of 5% chitosan ointment formulation
  • a solution of 3.0 g of propylene glycol and 0.1 g of methyl parabenzoate in 80 ml of a purified water solution is heated to 70 ° C., and 5.0 g of the low molecular weight chitosan 9 B hydrochloride prepared in Example 4 is added and dissolved. It was a molecular chitosan 9 B solution.
  • 3.0 g of stearic acid 1.0 g of setal, 6.0 g of glycerol monostearate 6.0 g of liquid paraffin, 0.1 g of propyl parapoxybenzoate, and 2.5 g of polyoxyl stearate 40 at 70 ° C.
  • a trauma healing test was conducted to predict the efficacy of 5% chitosan ointment in a rat skin defect model.
  • the 5% chitosan ointment prepared in Preparation Example 1 was used.
  • u-pasta ointment 70 g of purified white sugar and 3 g of popydonide in 100 g, polyethylene glycol as an additive, concentrated glycerin, polyoxyethylene [160] polyoxypropylene [30] glycol, Pullulan containing potassium iodide, serial number BA1S, manufactured by Kowa Co., Ltd.
  • animals used in the test 6-week-old Sprague-Dawley (SD) male rats (Japan SLC Co., Ltd.) were purchased and used.
  • the animals are fed with chow (FR-2 for rearing mice and rats, Funabashi Farm Co., Ltd.) and tap water in an environment controlled rearing apparatus (CLEA Japan, Inc.) with an average temperature of 23 ° C and an average humidity of 55%. The conditions were adjusted for 6 days under conditions of 19 o'clock and used for experiments. 0212940
  • the area change during the treatment process can be calculated from the area ratio () by the formula of (major axis x minor axis after minor defect creation on the measurement day) major axis x minor axis x 100
  • the treatment rate was calculated as the number of treatment days, assuming that the area ratio was 5% or less.
  • the experimental results are expressed as mean soil standard deviation.
  • the 5% chitosan ointment group reduced the number of treatment days to 12.6 ⁇ 1.9 days, compared to 13.7 ⁇ 1.3 days in the untreated group. There was no improvement in the number of treatment days, 13.6 ⁇ 0.4 days in the control group.
  • the low molecular weight chitosan of the present invention which has a molecular weight of 10,000 or more and is soluble even under neutral conditions. Furthermore, the low molecular weight chitosan of the present invention exhibits excellent antibacterial and wound healing effects, and the pharmaceutical composition containing the low molecular weight chitosan of the present invention as an active ingredient is useful as a therapeutic agent for trauma, bed sores, atopic dermatitis and the like. It is.
  • SEQ ID NO: 10 PCR primer cDNA-BamHI [Brief description of the drawing]
  • FIG. 1 shows the molecular weight of the purified chitinase from Aspergillus' Oryzae var. Sporoflavus Ohara J CM2067.
  • the plot of a to f is a marker, and the point shown by the arrow is a purified chitinase derived from Aspergillus oryzae var. sporoflavus Ohara J CM2067.
  • FIG. 2 shows the molecular weight distribution of low molecular weight chitosan prepared using chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara J CM2067.
  • FIG. 1 shows the molecular weight of the purified chitinase from Aspergillus' Oryzae var. Sporoflavus Ohara J CM2067.
  • the plot of a to f is a marker, and the point shown by the arrow is a purified chit
  • FIG. 3A shows the distribution of low molecular weight chitosan 9B hydrochloride
  • FIG. 2B shows the distribution of low molecular weight chitosan 8 B hydrochloride
  • FIG. 2C shows the distribution of low molecular weight chitosan 7B hydrochloride.
  • Sporoflavus Ohara J CM 2067 is shown by a solid circle in a solid line, and all dotted lines show the distribution of marker molecules ( Fill small circle: Low molecular weight chitosan with molecular weight 10000, Filled square: Low molecular weight chitosan with molecular weight 40000, Fill triangle: Low molecular weight chitosan with molecular weight 70000, Open circle: Low molecular chitosan with molecular weight 110000, Open square: Molecular weight 250000 Low molecular weight chitosan).
  • FIG. 3 shows the molecular weight distribution of low-molecular-weight chitosan prepared using chitinase derived from Aspergillus japonics strain SANK 19288.
  • Fig. 3A shows the distribution of low molecular weight chitosan 9B hydrochloride
  • Fig. 3B shows the distribution of low molecular weight chitosan 8B hydrochloride
  • Fig. 3C shows the distribution of low molecular weight chitosan 7B hydrochloride.
  • Fig. 6 shows the molecular weight distribution of low molecular weight chitosan prepared by using Fig. 4A shows the distribution of low molecular weight chitosan 9B hydrochloride, Fig. 4B shows the distribution of low molecular weight chitosan 8B hydrochloride, and Fig. 4C shows the distribution of low molecular weight chitosan 7B hydrochloride.
  • FIG. 5 shows the relationship between the activity of chitinase in the culture supernatant of Aspergillus oryzae var. Sporoflavus Ohara J CM 2067 strain and pH.
  • FIG. 6 shows the relationship between temperature and the activity of chitinase in the culture supernatant of Aspergillus oryzae var. Sporoflavus Ohara J CM 2067 strain.
  • FIG. 7 shows the temperature stability of chitinase in the culture supernatant of Aspergillus oryzae var. Sporoflavus Ohara J CM2067 strain.
  • FIG. 8 shows the pH stability of chitinase in the culture supernatant of Aspergillus oryzae var.
  • FIG. 9 shows the relationship between the activity and PH of crude purified chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara J CM2067.
  • FIG. 10 shows the relationship between activity and temperature of crude purified chitinase from Aspergillus' Oryzae var. Sporoflavus Ohara J CM2067.
  • FIG. 11 shows the PH stability of crude chitinase from Aspergillus' Oryzae var. Sporoflavus Ohara J CM2067.
  • FIG. 12 shows the temperature stability of crude purified chitinase from Aspergillus oryzae var. Sporoflavus Ohara J CM2067.
  • FIG. 13 shows the relationship between the activity and the pH of purified chitinase derived from Aspergillus' Oryzae var. Sporoflavus Ohara J CM2067 when chitosan 7 B is used as a reaction substrate.
  • FIG. 14 shows the relationship between the activity of purified chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara J CM2067 and pH when glycol chitin is used as a reaction substrate.
  • Figure 1'5 shows the relationship between temperature and the activity of purified chitinase from Aspergillus oryzae var. Sporoflavus Ohara J CM2067.
  • FIG. 16 shows the temperature stability of purified chimeras derived from Aspergillus oryzae var. Sporoflavus Ohara J CM2067.
  • FIG. 17 shows the pH stability of purified chitinase from Aspergillus oryzae var.
  • FIG. 18 shows the relationship between the activity of PH and the activity of the kinase in the culture supernatant of Aspergillus / Japonics SANK 19'288 strain.
  • FIG. 19 shows the relationship between the activity of chitinase in the culture supernatant of Aspergillus sogyae strain SANK 22388 and pH.
  • FIG. 20 shows the relationship between the activity of purified chitinase derived from Aspergillus japonicus strain SANK 19288 and pH when using glycol chitin as a reaction substrate.
  • FIG. 21 shows the relationship between temperature and the activity of purified chitinase derived from Aspergillus' Japonics strain SANK 19288.
  • FIG. 22 shows the temperature stability of purified chitinase derived from Aspergillus japonics strain SANK 19288.
  • FIG. 23 shows the pH stability of the purified chitinase derived from Aspergillus japonics strain SANK 19288.
  • FIG. 24 shows the relationship between the activity of purified chitinase derived from Aspergillus sogyae strain SANK 22388 and pH when using glycol chitin as a reaction substrate.
  • FIG. 25 shows that Aspergillus' Sojiyae SANK22 in the condition of pH 5.5.
  • the relationship between temperature and the activity of purified chitinase derived from strain 388 is shown.
  • FIG. 26 shows the relationship between temperature and the activity of purified chitinase derived from Aspergillus' Sogyae strain SANK 22 388 under conditions of pH 9.0.
  • FIG. 27 shows the temperature stability of purified chitinase derived from Aspergillus' Sogyae strain SANK 22 388 under conditions of pH 5.5.
  • FIG. 28 shows the temperature stability of purified chitinase derived from Aspergillus' Sogyae strain SANK 22 388 under conditions of pH 9.0.
  • FIG. 29 shows the pH stability of purified chitinase derived from Aspergillus sogyae strain SANK 22388.
  • Figure 30 shows the SDS electropherogram of chitinase. (12% acrylamide gel, C BB staining, lane M: marker, lane 1: His fusion recombinant chitinase, lane 2 ': purified chitinase from Aspergillus oryzae var. Sporoflavus Ohara J CM2067)

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Abstract

L'invention concerne une chitosanase soluble dans l'eau, même dans des conditions neutres, et permettant de produire un chitosane à faible masse moléculaire présentant un effet antimicrobien, un ADN codant pour cette enzyme, un procédé permettant de produire un chitosane à faible masse moléculaire au moyen de cette enzyme, le chitosane à faible masse moléculaire obtenu par ce procédé, des compositions médicinales et des aliments contenant ce chitosane à faible masse moléculaire en tant que principe actif, un procédé permettant de produire un échantillon facile à manipuler en utilisant l'enzyme décrit etc..
PCT/JP2002/012940 2001-12-11 2002-12-10 Chitosanase et utilisation de celle-ci WO2003054200A1 (fr)

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CN101962666A (zh) * 2010-09-06 2011-02-02 湖北工业大学 一种利用甲壳废弃物制备甲壳素、l-乳酸钙和复合氨基酸或肽的方法
CN110554176A (zh) * 2019-09-04 2019-12-10 南通大学 利用分子量、乙酰化程度线性调控壳聚糖生物材料降解速度的方法

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JPH11322809A (ja) * 1998-05-18 1999-11-26 Mikio Ito 抗ヘリコバクター・ピロリ剤
JP2000169327A (ja) * 1998-12-03 2000-06-20 Pias Arise Kk 低刺激性皮膚外用剤及び浴用剤
WO2000056762A2 (fr) * 1999-03-22 2000-09-28 Novozymes Biotech, Inc. Methodes de surveillance de l'expression genique multiple

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JPH11322809A (ja) * 1998-05-18 1999-11-26 Mikio Ito 抗ヘリコバクター・ピロリ剤
JP2000169327A (ja) * 1998-12-03 2000-06-20 Pias Arise Kk 低刺激性皮膚外用剤及び浴用剤
WO2000056762A2 (fr) * 1999-03-22 2000-09-28 Novozymes Biotech, Inc. Methodes de surveillance de l'expression genique multiple

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ZHANG X.Y. ET AL.: "Cloning and characterization of a chitosanase gene from the koji mold aspergillus oryzae strain IAM2660", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 65, no. 4, April 2001 (2001-04-01), pages 977 - 981, XP002966334 *
ZHANG X.Y. ET AL.: "Purification and characterization of chitosanase and exo-beta-D-glucosaminidase from a koji mold, aspergillus oryzae IAM2660", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 64, no. 9, 2000, pages 1896 - 1902, XP002966333 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101962666A (zh) * 2010-09-06 2011-02-02 湖北工业大学 一种利用甲壳废弃物制备甲壳素、l-乳酸钙和复合氨基酸或肽的方法
CN110554176A (zh) * 2019-09-04 2019-12-10 南通大学 利用分子量、乙酰化程度线性调控壳聚糖生物材料降解速度的方法

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