WO2003054200A1 - Chitosanase and use thereof - Google Patents

Chitosanase and use thereof 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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French (fr)
Japanese (ja)
Inventor
Tetsuya Fukazawa
Isshin Tanaka
Original Assignee
Sankyo Lifetech Company, Limited
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Priority to AU2002354461A priority Critical patent/AU2002354461A1/en
Publication of WO2003054200A1 publication Critical patent/WO2003054200A1/en

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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

It is intended to provide a chitosanase which is soluble in water even under neutral conditions and capable of efficiently producing a low-molecular weight chitosan having an antimicrobial effect; a DNA encoding this enzyme; a process for producing a low-molecular weight chitosan with the use of the enzyme; the low-molecular weight chitosan produced by this process; medicinal compositions and foods containing the low-molecular weight chitosan as the active ingredient; a process for producing a sample easy to handle with the use of the above enzyme; and so on.

Description

明細書 キトサン分解酵素及びその用途  Specification Chitosan decomposing enzyme and use thereof
[技術分野] [Technical field]
本発明は、 ァスペルギルス 'オリザェ （Aspergillus oryzae) 、 ァスペルギルス · ジャポニクス （Aspergillus japonicus)、 ァスペルギルス 'ソジヤエ（Aspergillus sojae) の少なくとも 1種の培養物から分離精製されるキトサン分解酵素に関する。 また、 該キトサン分解酵素を用いた低分子キトサン及びその塩の製造方法、 該方法に より製造される低分子キトサン及びその塩、および該低分子キトサン及びその塩を含 む医薬組成物、 食品、 該キトサン分解酵素を用いた試料の製造方法、 該方法により製 造される試料等に関する。  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). Also, 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.
[背景技術] [Background technology]
キチン （]3— 1， 4—ポリ一 N—ァセチルダルコサミン) は天然に非常に多く存在す る多糖であり、 主として力二 'ェビ等の甲殻類およびイカ軟甲から工業的規模で抽出 され生産されている。 キトサン （3— 1， 4—ポリダルコサミン） は、 キチンを濃ァ ル リ水で処理することにより得られる、 アミノ基を有する高分子多糖であり、 さま ざまな生理活性を有することが知られている （技報堂「キチン ·キトサンハンドブッ ク J302- 376頁） 。 なかでも、 キトサンは天然物由来物質としては強力な抗菌作用を有 し、 かつ安全性が合成抗菌剤と比較して非常に高い （ 「キチン ·キトサンの活用法」 財界特別増刊第 46巻 28号 106-111頁 1998年） ことが知られている。 特に、 メチシ リンセフエム而す性黄色ブドウ球菌 （methichilin resistants aureus) (以下、 「MRSA」 と記す）を含むス夕ヒロコッカス 'ァゥレウス （Staphylococcus aureus) (加工技術 33巻 512- 515頁 1998年、名城大学農学部学術報告 34巻 25- 34頁 1998年、 繊維科学 35卷 37_39頁 1993年、大阪府立公衆衛生研究所研究報告 薬事指導編 No.29, 7- 12頁 1995年、 加工技術 32巻 339-343頁 1997年、 加工技術 33巻 530-532頁 1998 年） 、 ェシエリキア 'コリ （Escherichia coli) (Tokura S et al. Macromol. Symp. 120, 1-9, 1997) 、 シユードモナス ' エルジノーサ （Pseudomonas aeruginosa) (Loke WK et al. J. Bioraed. Mater. Res. 53, 8-17, 2000, Kim H. J. et al. J. Biomater. Sci. Polym. Ed. 10， 543-56, 1999) 等の細菌に対して効果があることが知られている。 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. 46, No. 28, page 106-111 (1998) is known. In particular, S. entericus Staphylococcus aureus (processing technology, vol. 33, 512-515), Meijo University, Faculty of Agriculture, which includes methicillin resistance bacteria (methichilin resistant aureus) (hereinafter referred to as "MRSA"). Journal of Scientific Research Vol. 34, pp. 25-34 1998, Textile Science 35, pp. 37-39 1993, Research report of the Osaka Prefectural Public Health Research Institute Pharmaceutical Affairs No. 29, pp. 7-12 1995, Processing Technology Vol. 32 pp. 339-343 1997, Processing Technology 33: 530-532 (1998), Escherichia coli 'Escherichia coli' (Tokura S et al. Macromol. Symp. 120, 1-9, 1997), Pseudomonas' Erginosa (Pseudomonas aeruginosa) (Loke) Res. 53, 8-17, 2000, Kim HJ et al. J. Biomater. Sci. Polym. Ed. 10, 543-56 (1999), etc. The effect on bacteria etc. It is known that there is.
力二 ·ェビ等の甲殻類から抽出されたキチンを濃アルカリ処理することによって製 造されたキトサンは分子量 100万以上の高分子多糖であり、酸性条件では水,に溶け るが、 中性あるいはアルカリ性の条件では水に溶けない。 そして、水に溶けない状態 ではキトサンの生理活性は十分に発揮できない。 また、 甲殻類を含む食品材料を加工 する際、 これら高分子多糖が原因で非常に粘度が高いものになってしまい， その後の 処理に手間がかかるという問題もあった。 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.
そこで、 水に対する溶解性を増すために、 高分子キトサンの誘導体化 （ 「キチン · キトサンの活用法」財界特別増刊第 46巻 28号 78— 8 1頁 1998年）あるいは、 高分子キトサンが有する生理活性を損わずに低分子化するといった試みがなされて いる。  Therefore, in order to increase the solubility in water, derivatization of high molecular weight chitosan ("How to use chitin · chitosan" special edition of the business community No. 46, 28, 78-81, 1998) or the physiological state that high molecular weight chitosan has Attempts have been made to reduce the molecular weight without losing the activity.
誘 体化を行うと、天然物とは異なる構造となる可能性があるので天然型のキトサ ンとは異なる性質を有するようになったり、使用に際しては安全性試験データ等が必 要になる場合がある。 更に誘導体化反応に使用した有機溶媒の除去、 処理が生産コス トを引き上げるという問題もある。  If derivatized, it may have a structure different from that of natural products, so it may have different properties from natural chitosan, or when it is necessary to use safety test data etc. There is. Furthermore, there is also a problem that removal of the organic solvent used for the derivatization reaction and processing increase the production cost.
そこで、 水に対する溶解性を上げるためには誘導体化する方法ではなく、 低分子化 が望ましいと考えられている。 キトサンが有する抗菌活性は、 分子量が 100万以上 の高分子キトサンだけではなく分子量が 1万以上の低分子キトサンでも発揮されるこ とが知られている （日本農芸化学会誌 74巻大会講演要旨集 講演番号 2Ε183α、 2 000年） 。  Therefore, 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 Ε 183α, 2000 years).
一方、 分子量 1万以下のキトサンには、 皮膚刺激性等の副作用があることが報告さ れており （特開 2000— 169327)、 分子量 1万以上の低分子キトサンの製造方法が検討 されている。  On the other hand, 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. .
低分子キトサンの製造方法としては、化学的あるいは酵素的手法による高分子キト サンの加水分解が検討されてきた。 低分子キトサンの化学的製造方法としては、 過酢 酸等の酸化剤で処理する方法（特許第 3076212号公報、特開平 5— 65368公報）、 超音波処理する方法（キチン ·キトサン研究 5巻 75-79頁 1999年）等が挙げられる。 しかし、 酸化剤で処理する方法では分子量 500 - 30, 000のキトサンが大量に 生成することが知られており、 分子量 1万以上のキトサンの割合は定かではない。 ま た、 超音波で処理する方法では装置の大型化が困難であり、 大量製造に不向きである と考えられる。低分子キトサンの酵素的製造方法としてはキトサナーゼを用いる方法 (特許第 2763112号公報、 特開平 11-322809公報） 、 キチナ一ゼを用いる方法 （日本 農芸化学会誌 74巻大会公演要旨集 講演番号 2F313Q!、 2000年） などが挙げら れる。  As a method for producing low molecular weight chitosan, hydrolysis of high molecular weight chitosan by chemical or enzymatic method has been studied. Chemical production methods for low molecular weight chitosan include: treatment with an oxidizing agent such as peracetic acid (Japanese Patent No. 3076212, JP-A-5-65368), Method of ultrasonic treatment (chitin · chitosan study 5) 75 P.-79, 1999). However, it is known that large amounts of chitosan having a molecular weight of 500 to 30,000 are produced by the method of treatment with an oxidizing agent, and the proportion of chitosan having a molecular weight of 10,000 or more is unknown. In addition, it is difficult to enlarge the size of the device by ultrasonic treatment, which is considered unsuitable for mass production. Methods using chitosanase as an enzymatic method for producing low molecular weight chitosan (Japanese Patent No. 2763112, JP-A-11-322809), Method using chitinase (Abstracts of the Meeting of the Japan Society for Agrochemical Science, Volume 74 Conference No. 2F313Q! , 2000) and others.
また、 細菌由来のキチナ一ゼとしては、 バチルス （Bacillus) 属、 コリネバクテリ ゥム (Corynebacterium) 属 (Veldkamp, H. , Nature, 169, 500 (1952)参照） 、 サイ卜ファーガ（Cytophaga)属、ァクロモパクター ·ヒテロプティカム（Acromobacter hyteropticum: Campbell, Jr. , L. L. et al. , J. Gen. Microbiol. , 5, 894 (1951)参照）、 フラポパクテリゥム （Flavobacterium) 属及びミクロコッカス · コルポケネス (Micrococcus colpogenes) (Campbell, Jr., L. L. , et al. , J. Gen. Microbiol., 5, 894(1951)) .の生産するキチナーゼ並びに、 アルテロモ ナス 'エスピー （Alteromonas sp.) の生産するキチナ—ゼ A (GenBank Accession NO.D13762) 等が知られている。 放線菌の生産するキチナーゼとしては、 ストレプト ミセス （Streptomyces) 属、 ノカルディオプシス (Nocardiopsis) 属及びァクチノミ セス （Actinomyces) 属の生産するキチナーゼが知られている （中神照太ら， ェ技院 醱酵研究所報告， 30， 19 (1966)。 糸状菌の生産するキチナ一ゼとしては、 ァスペル ギルス （Aspergillus) 属、 リゾプス （Rhizopus) 属、 タラ口ミセス （Talaromyces) 属、 トリコデルマ （Trichoderma) 属及びぺニシリウム （Penici Ilium) 属の生産する キチナ一ゼが知られている。 ァスペルギルス属の生産する酵素としては、 ァスペルギ ルス.ニジュランス（Aspergillus nidulans)、ァスペルギルス'ニガ一（Aspergillus niger)、ァスペルギルス ·カンディドス（Aspergillus candidus： Sherif, A. A. , et al.， Appl. Mcrobiol., Biotechnol. , 35, 228 (1991)) 及びァスペルギル ス 'フミガッス （Aspergillus fumigatus) の生産するキチナ一ゼが知られている。 酵母の生産する酵素としては、 サッカロミセス 'セレヴイシェ （Saccharomyces cerevisiae) の生産するキチナーゼを例示することができ、 好適にはサッカロミセ ス 'セレヴイシェの生産するキチナーゼ（GenBank Accession No.M74070)及び ORF D9481.7 (GenBank Accession No.U28373) である。 Furthermore, examples of 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)). It is known that chitinase A (GenBank Accession NO. D 13762) and the like produced by eggplant 'sp. (Alteromonas sp.). 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). As chitinases produced by filamentous fungi, Aspergillus (Aspergillus), Rhizopus (Rhizopus), Talaromyces (Talaromyces), Trichoderma and Penicillium (Penici Ilium) 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 (Aspergillus fumigatus) 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).
このように、 多くの生物由来のキチナーゼが知られているが、 代表的な発酵食品で ある、 味噌やしようゆ日本酒等の生産に使用されているァスペルギルス ·ジャポニク ス（Aspergillus japonicus)およびァスペルギルス'ソジヤエ（Aspergillus sojae) についてはキチナーゼを生産するとの報告はなく、 これら生物由来のキチナ一ゼ性質 や利用についての報告もない。 また、 同様に食品に含まれるァスペルギルス ·オリザ ェ （Aspergillus oryzae)に関しては、 キチナ一ゼをコードすると考えられる遺伝子 について学会発表があつたとの報告 （キチン ·キトサン研究 第 8巻 第 2 号 238- 239頁 2002年） があるものの、 その遺伝子がコードする蛋白質の活性について は報告されておらず、 現在までにデータべ一ス等でその配列が確認されていない。 一方、 キトサナーゼを用いて低分子キトサンを製造する方法は、 常に同じ分子量の 低分子キトサンを得るための反応制御が煩雑であるという問題点もあった。  As such, there are known 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. 2 238-239 Although there is a page 2002), the activity of the protein encoded by the gene has not been reported, and so far the sequence has not been confirmed in databases etc. On the other hand, the method of producing low molecular weight chitosan using chitosanase also has a problem that reaction control for always obtaining low molecular weight chitosan having the same molecular weight is complicated.
[発明の詳細な説明] . Detailed Description of the Invention.
[発明の開示]  Disclosure of the Invention
本発明者らは、 中性条件下でも水に可溶であり、 抗菌作用を有する低分子キトサン を効率よく生産する方法について鋭意検討を行ったところ、 ァスペルギルス 'オリザ ェ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2067) 株 由来のキチナ一ゼあるいはァスペルギルス ·ジャポニクス（Aspergillus japonicus) SANK 1 92 88株由来のキチナーゼあるいはァスペルギルス · ソジヤエ (Aspergillus soj.ae) SANK 22388株由来のキチナ一ゼを用いた酵素反応 により、 力二，ェビ由来の高分子キトサンから分子量 1万以上の低分子非誘導体のキ トサンを作製することに成功し、 本発明を完成するに至った。 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. ae) SANK 22388 reaction As a result, the present inventors have succeeded in producing a low molecular weight non-derivative chitosan having a molecular weight of 10,000 or more from high molecular weight chitosan derived from phi-2 and hibi, and the present invention has been completed.
本発明により、 非誘導体の低分子キトサンを、 1万以上の分子量分布を有する状態 で供給できることが可能となる。  According to the present invention, it is possible to supply non-derivative low molecular weight chitosan with a molecular weight distribution of 10,000 or more.
[課題を解決するための手段] [Means for Solving the Problems]
本発明は、  The present invention
(1) ァスペルギルス ·オリザェ （Aspergillus oryzae) 、 ァスペルギルス 'ジ ャポニクス （Aspergillus 〗aponicus) 、 ァスペルギルス ·ソジヤエ （Aspergillus sojae) の少なくとも 1種の培養物から分離精製されるキトサン分解酵素、  (1) 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);
(2) ァスペルギルス 'オリザェ （Aspergillus oryzae) がァスペルギルス ·ォ リザェ (Aspergillus oryzae var. sporof lavus Ohara J CM 2067) 株であり、 ァスペルギルス 'ジャポニクス （Aspergillus japonicus) がァスペルギ ルス 'ジャポニクス （Aspergillus japonicus) SANK 19288株であり、 ァ スペルギルス · ソジヤエ (Aspergillus sojae) がァスペルギルス · ソジヤエ(2) Aspergillus 'Oryzae (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)
(Aspergillus sojae) SANK 22388株である （ 1 ) 記載のキトサン分解酵 素、 (Aspergillus sojae) The chitosan degrading enzyme according to (1), which is SANK 22388 strain,
(3) 配列表の配列番号 1乃至 3の部分アミノ酸配列を有することを特徴とする、 (1) または （2) に記載のキトサン分解酵素、 (3) The chitosan degrading enzyme according to (1) or (2), which has a partial amino acid sequence of SEQ ID NO: 1 to 3 in the sequence listing,
(4) 以下の性質を示す （1) 乃至 （3) のいずれか 1つに記載のキトサン分解酵 素： (4) The chitosan degrading enzyme according to any one of (1) to (3), which exhibits the following properties:
1) SDS— PAGE電気泳動法にて分子量約 40, 000を示す；  1) It shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis;
2) 等電点電気泳動法にて等電点 p I 3. 5乃至 4. 5を示す；  2) The isoelectric point shows an isoelectric point p I of 3.5 to 4.5 by isoelectric focusing method;
3) 30 %ァセチル化キトサン （粘度 100〜300 c p s) を、 ρΗ3.' 5乃至 ρ HI 0. 5にて加水分解する；  3) Hydrolysis of 30% acetylated chitosan (viscosity 100-300 cps) with に て 3.'5 to HI HI 0.5
4) グリコールキチンを、 pH3. 0乃至 ρΗΙ Ο. 5にて加水分解する；  4) hydrolyze glycol chitin at pH 3.0 to Ο5.
5) 0°C乃至 80°Cで 3) 記載の加水分解活性を発揮する；  5) exert the hydrolysis activity described in 3) at 0 ° C to 80 ° C;
6) 45 °C以下の温度で安定である；  6) stable at temperatures below 45 ° C;
7) pH5乃至pH9. 5の pH条件下で安定である、  7) Stable under pH conditions of pH 5 to pH 9.5,
(5) 下記の a)乃至 d)のいずれか一つに記載の蛋白質であるキトサン分解酵素： a) 配列表の配列番号 5に記載のアミノ酸配列からなる蛋白質； (5) 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;
b)配列表の配列番号 4のヌクレオチド番号 242乃至 1438に示されるヌクレオ チド配列によりコードされるアミノ酸配列からなる蛋白質； b) Nucleotides shown in nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing A protein consisting of an amino acid sequence encoded by a nucleotide sequence;
c) a) または b) に記載のアミノ酸配列において、 一つまたは数個のアミノ酸が置 換、 欠失、 挿入または付加したアミノ酸配列からなり、 かつ、 キトサン分解活性を有 することを特徴とする蛋白質； c) In the 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. protein;
d) a) または b) に記載のアミノ酸配列を含むことからなる蛋白質、 d) a protein comprising the amino acid sequence of a) or b),
(6) 下記の a)乃至 c)のいずれか一つに記載の蛋白質であるキトサン分解酵素： a)配列表の配列番号 14に記載のアミノ酸配列からなり、 N末端のセリン残基の —ァミノ基がァセチル化されていることを特徴とする蛋白質； (6) 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;
b) 配列表の配列番号 14に記載のアミノ酸配列において、 一つまたは数個のァミノ 酸が置換、 欠失、 挿入または付加したアミノ酸配列からなり、 且つ、 N末端のセリン 残基のひーァミノ基がァセチル化されており、 且つ、 キトサン分解活性を有すること を特徴とする蛋白質； b) In the 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;
c) 配列表の配列番号 14に記載のアミノ酸配列を含み、 且つ、 N末端のセリン残基 のひーァミノ基がァセチル化されており、 且つ、 キトサン分解活性を有することを特 徴とする蛋白質、 c) 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,
(7) 下記に記載のいずれか一つに記載の DN A： (7) The DN A described in any one of the following:
a)配列表の配列番号 4のヌ,クレオチド番号 242乃至 1438に示されるヌクレオ チド配列からなる DNA ;  a) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, nucleotide nucleotides 242 to 1438 in the nucleotide list;
b) 上記 a) に記寧の DNAの有するヌクレオチド配列と相補的なヌクレオチド配列 からなる DNAとストリンジェントな条件下でハイブリダィズし、 かつ、 キ卜サン分 解活性を有する蛋白質をコードすることを特徴とする DNA； b) A protein which hybridizes under stringent conditions with a 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. To be DNA;
c) 上記 a) に記載の DNAと 95%以上のヌクレオチド配列相同性を有するヌクレ ォチド配列からなり、 かつ、 キトサン分解活性を有する蛋白質をコードすることを特 徴とする DNA；  c) DNA characterized by comprising a nucleotide sequence having a nucleotide sequence homology of 95% or more with the DNA described in a) above, and encoding a protein having a chitosan degrading activity;
d) 配列表の配列番号 5に記載のアミノ酸配列からなる蛋白質をコ一ドする DNA； e)配列表の配列番号 4のヌクレオチド番号 242乃至 1438に示されるヌクレオ チド配列を含むことからなる D N A、 d) a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing; e) a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 at nucleotide numbers 242 to 1438 in SEQ ID NO: 4;
(8) 下記の a) 乃至 c) のいずれか一つに記載の DNA： (8) The DNA according to any one of the following a) to c):
a) 形質転換大腸菌 Escherichia coli BM25.8/pTriplEx-Chitinase-cDNA S A NK 72102 (FERM B P— 8235)により保持される組換えプラスミド、 pTriplEx- Chitinase- cDNAに挿入されている DNA；  a) Recombinant plasmid carried by transformed E. coli Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA S A NK 72102 (FERM BP-8235), DNA inserted into pTriplEx- Chitinase-cDNA;
b) b) 上記 a) に記載の DNAの有するヌクレオチド配列と相補的なヌクレオチド 配列からなる DN Aとストリンジェントな条件下でハイブリダィズし、 かつ、 キトサ ン分解活性を有する蛋白質をコードすることを特徴とする DNA； b) b) hybridizing under stringent conditions with DNA comprising a nucleotide sequence complementary to the nucleotide sequence possessed by the DNA described in a) above, and A DNA characterized by encoding a protein having degradative activity;
c) 上記 a) に記載の DNAを含み、 かつ、 キトサン分解活性を有する蛋白質をコ一 ドすることを特徴とする DNA、 c) A DNA comprising the DNA according to the above a) and coding a protein having a chitosan degrading activity,
(9) (7) または （8) に記載の DNAにコードされる蛋白質であるキトサン分 (9) Chitosan, which is a protein encoded by the DNA according to (7) or (8)
(10) 形質転換大腸菌 Escherichia coli BM25.8/pTriplEx-Chitinase-cDNA SANK 72102 (FERM B P _ 8235 ) により保持される組換えプラス ミド、 pTriplEx- Chitinase- cDNAに揷入されている DNAによりコードされる蛋白質 であるキトサン分解酵素、 (10) A recombinant plasmid carried by transformed Escherichia coli Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235), encoded by DNA incorporated into pTriplEx- Chitinase-cDNA Chitosan-degrading enzyme, which is a protein
(11) 下記の a) 乃至 b) に示される活性を有することを特徴とする、 （5) 、 (6) 、 (9) または （10) に記載のキトサン分解酵素； (11) The chitosan degrading enzyme according to (5), (6), (9) or (10), which is characterized by having the activity shown in a) to b) below;
a) 30 %ァセチル化キトサン （粘度 100〜300 c p s) を、 pH3. 5乃至 p HI 0. 5、 0 °C乃至 80 °Cの条件にて加水分解する活性； a) activity to hydrolyze 30% acetylated chitosan (viscosity 100-300 cps) under conditions of pH 3.5 to pH 0.5, 0 ° C to 80 ° C;
b) グリコールキチンを、 pH4. 0乃至 ρΗΙ Ο. 5にて加水分解する活性、 b) activity to hydrolyze glycol chitin at pH 4.0 to pH 5.
(12) (7) または （8) に記載の DNAを含む組換えプラスミド、 (12) A recombinant plasmid comprising the DNA according to (7) or (8),
(13) 発現ベクターであることを特徴とする、 （12) に記載の組換えプラスミ ド、 (13) The recombinant plasmid according to (12), which is characterized by being an expression vector
(14) (12) または （13) に記載の組換えプラスミドで形質転換された宿主 細胞、 (14) A host cell transformed with the recombinant plasmid according to (12) or (13),
(15) 原核細胞または真核細胞であることを特徴とする、 （14) に記載の宿主 細胞、 (15) The host cell according to (14), which is characterized by being a prokaryotic cell or a eukaryotic cell.
(16) 形質転換大腸菌 Escherichia coli BM25.8/pTriplEx-Chitinase-cDNA SANK 72102 (FERM B P— 8235 )である（ 14)記載の宿主細胞、 (16) The host cell according to (14), which is transformed E. coli Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM B P-8235),
(17) 下記 1) 乃至 2) を含む、 キトサン分解酵素の製造方法： (17) A method for producing a chitosan degrading enzyme, which comprises the following 1) to 2):
1) 下記の a) 乃至 e) のいずれか一つに記載の細胞を、 キトサン分解酵素を産生す る条件下で培養する工程；  1) culturing the cells according to any one of the following a) to e) under conditions for producing a chitosan degrading enzyme;
a) ァスペルギルス ·オリザェ （Aspergillus oryzae) ； b) ァスペルギルス ·ジャポニクス （Aspergillus japonicus) ； c) ァスペルギルス 'ソジヤエ （Aspergillus sojae) ； a) Aspergillus oryzae (Aspergillus oryzae); b) Aspergillus japonicus (Aspergillus japonicus); c) Aspergillus 'Aspergillus sojae';
d) (1.4) 乃至 （16) のいずれか一つに記載の宿主細胞；  d) A host cell according to any one of (1.4) to (16);
e) (5) 、 （6) 、 （9) 乃至 （1.1) のいずれか一つに記載のキトサン分解酵 素を産生する細胞；  e) a cell that produces the chitosan-degrading enzyme according to any one of (5), (6), (9) to (1.1);
2) 1) の培養産物からキトサン分解酵素を分離，精製する工程。  2) A process of separating and purifying a chitosan degrading enzyme from the culture product of 1).
(18) ァスペルギルス ·オリザェ （Aspergillus oryzae) がァスペルギルス · オリザェ (Aspergillus oryzae var. sporof lavus Ohara J CM 2067) 株であり、 ァスペルギルス ·ジャポニクス （Aspergillus japonicus) がァスペルギ ルス 'ジャポニクス （Aspergillus japonicus) SANK 19288株であり、 ァ スペルギルス · ソジヤエ （Aspergillus sojae) がァスペルギルス · ソジヤエ(18) Aspergillus oryzae (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)
(Aspergillus soj e) SANK 22388株である （17) 記載の方法、 (Aspergillus soje) The method according to (17), which is SANK 22388 strain,
(19) (17)または（18)に記載の方法により製造されるキトサン分解酵素、 (19) A chitosan degrading enzyme produced by the method according to (17) or (18),
(20) 以下の工程 1) および 2) を含む低分子キトサンの製造方法： (20) A process for producing low molecular weight chitosan comprising the following steps 1) and 2):
1 )ァセチル化度が 10%乃至 30 %のァセチル化キトサン水溶液に (1)乃至（6)、 (9) 乃至 （11) および （19) のいずれか一,つに記載のキトサン分解酵素を添加 する；  1) The chitosan degrading enzyme according to any one of (1) to (6), (9) to (11) and (19) is added to an aqueous acetylated chitosan solution having an acetylation degree of 10% to 30%. Do;
2) pH3乃至 12、 10°C乃至 60°Cの条件で 10分以上酵素反応を行なう、  2) Perform the enzyme reaction for 10 minutes or longer under conditions of pH 3 to 12, 10 ° C to 60 ° C,
(21) (20) に記載の方法により製造される低分子キトサン又はその塩、 (21) Low molecular weight chitosan or a salt thereof produced by the method according to (20)
(22) 分子量が 4万〜 25万でァセチル化度が 10%である （21) 記載の低分 子キトサン又はその塩、 ' (22) The low molecular chitosan or the salt thereof according to (21), wherein the molecular weight is 40,000 to 250,000 and the degree of acetylation is 10%.
(23) 分子量が 1万〜 1 1万でァセチル化度が 20%である （21) 記載の低分 子キトサン又はその塩、 (23) The low molecular chitosan or the salt thereof according to (21), which has a molecular weight of 10,000 to 10,000 and an acetylation degree of 20%.
(24) 分子量が 1万〜 7万でァセチル化度が 30%である （21) 記載の低分子 キトサン又はその塩,、 (24) The low-molecular-weight chitosan or a salt thereof according to (21), which has a molecular weight of 10,000 to 70,000 and an acetylation degree of 30%.
(25) (21) 乃至 （24) のいずれか一つに記載の低分子キトサン又はその塩 を有効成分として含有する医薬組成物、 (26) 外傷、 床ずれまたはアトピー性皮膚炎の治療剤であることを特徴とする、 (25) 記載の医薬組成物 (25) 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.
(27) (21) 乃至 （24) のいずれか一つに記載の低分子キトサン又はその塩 を有する化粧料、 (27) A cosmetic comprising the low molecular weight chitosan or a salt thereof according to any one of (21) to (24),
(28) (21) 乃至 （24) のいずれか一つに記載の低分子キトサン又はその塩 を有する食品、 (28) A food having the low molecular weight chitosan or a salt thereof according to any one of (21) to (24),
(29) (21) 乃至 （24) のいずれか一つに記載の低分子キトサン又はその塩 を有する水等処理剤、 (29) 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).
(30) 以下の工程 1) および 2) を含む、 その液体成分中に分子量 100万以上 の高分子部分ァセチル化キトサンを実質的に含有しない試料の製造方法： (30) 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):
1)分子量 100万以上の高分子部分ァセチル化キトサンを含む試料に（1)乃至（6)、 (9) 乃至 （1 1) および （19) のいずれか一つに記載のキトサン分解酵素を添加 する；  1) Addition of the chitosan-degrading enzyme according to any one of (1) to (6), (9) to (11) and (19) to a sample containing high molecular weight partially acetylated chitosan having a molecular weight of 1,000,000 or more Do;
2) pH3乃至 12、 10 乃至 60°Cの条件で 10分間以上酵素反応を行なう、  2) carry out the enzyme reaction at pH 3 to 12, 10 to 60 ° C. for 10 minutes or longer,
(31) 分子量 100万以上の高分子部分ァセチル化キトサンを含有する試料が、 甲殻類を含む試料であることを特徴とする、 （30) 記載の方法、 (31) The method according to (30), wherein the sample containing high molecular weight partially acetylated chitosan having a molecular weight of 1,000,000 or more is a sample containing crustaceans,
(32) 甲殻類がェピおよび力二のいずれか一つまたは両方である （31) 記載の 方法、 (32) The method according to (31), wherein the crustacean is either one or both of Epi and power-2;
(33) (30) 乃至 （32) のいずれか一つの方法により製造される、 その液体 成分中に分子量 100万以上の高分子部分ァセチル化キトサンを実質的に含有しな い試料、 (33) A sample produced substantially by the method according to any one of (30) to (32), wherein the liquid component does not substantially contain high molecular weight partially acetylated chitosan having a molecular weight of 1,000,000 or more.
(34) (21) 乃至 （24) 記載の低分子キトサン又はその塩の有効量を動物に 投与することを含む、 外傷、 床ずれおよびアトピー性皮膚炎から選択される一つまた は複数の疾患の治療方法、 (34) (21) to (24), which comprises administering an effective amount of the low molecular weight chitosan or a salt thereof according to any one or more diseases selected from trauma, bedsore and atopic dermatitis Method of treatment,
(35) 外傷、 床ずれおよびァトピ一性皮膚炎から選択される一つまたは複数の疾 患を治療するための、 （21) 乃至 （24) のいずれか一つに記載の低分子キトサン 又はその塩の使用、 に関する。 (35) The low-molecular-weight chitosan or a salt thereof according to any one of (21) to (24), for treating one or more diseases selected from trauma, bedsore and atopic dermatitis. Use of, About.
[発明を実施するための最良の形態] BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
本発明は低分子キトサンを製造するために有用な糸状菌として、 ァスペルギルス · オリザェ （Aspergillus oryzae) 由来のキチナーゼあるいはァスペルギルス .ジャ ポニクス （Aspergillus japonicus) 由来のキチナーゼあるいはァスペルギルス ·ソ ジヤエ （Aspergillus sojae) 由来のキチナ一ゼ、 該キチナーゼによって製造される 低分子キトサン、 該低分子キトサンを含む医薬組成物等に関する。  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.
本発明においてキチナ一ゼとはキチン又は部分ァセチル化キトサンを加水分解す る活性を有する酵素をいい、キチンを加水分解すると N—ァセチルダルコサミンとそ のオリゴ糖が生成する。 この 「キチン又は部分ァセチル化キトサンを加水分解する活 性」 を 「キトサン分解活性」 と定義する。 キチンとは、 部分ァセチル化キトサンのう ち、 ァセチル化の割合が高いものをいい、 本発明においては、 特に断りが無い限り、 これらもあわせて部分ァセチル化キトサンと呼ぶ。 また、 本発明において、 キトサン 分解酵素とキチナーゼはいずれも上記キトサン分解活性を有する酵素を指し、同義語 として用いる。 本発明のキチナ一ゼには、 キチナーゼを生産する微生物の培養物中の キトサン分解活性を有する蛋白質が含まれる。 このようなキチナーゼの例としては、 ァスペルギルス 'オリザェ （Aspergillus oryzae) 由来のキチナ一ゼ、 ァスペルギ ルス ·ジャポニクス （Aspergillus japonicus) 由来のキチナーゼまたはァスペルギ ルス 'ソジヤエ （Aspergillus sojae) 由来のキチナ一ゼが挙げられる。 より好適な もの【ま、 ァスペルギルス -： リザェ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2067) 由来のキチナ一ゼ、 ァスペルギルス ·ジャポニクス (Aspergillus japonicus) SANK 19288株由来のキチナ一ゼまたはァスぺ ルギルス 'ソジヤエ （Aspergillus sojae) SANK 22388株由来のキチナ一 ゼであり、 さらにより好適には、 ァスペルギルス ·オリザェ （Aspergillus oryzae) var. sporoflavus Ohara J CM 2067) 由来のキチナ一ゼである。  In the present invention, 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. In the present invention, 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).
また、 本発明の午チナーゼの別の例としては、 配列表の配列番号 1、 配列番号 2お よび配列番号 3に示されるアミノ酸配列を全て含み、 且つ、 キトサン分解活性を有す る蛋白質が挙げられる。 該蛋白質は、 各々のアミノ酸配列の順序によらず、 内部アミ ノ酸配列としてこれらの全てを含むものであればよい。 このようなキチナ一ゼの中で 好ましいものは、 配列表の配列番号 1、 配列番号 2および配列番号 3に示されるアミ ノ酸配列を全て含み、 且つ、 N末端の配列が "S e r— S e r— G l y— L e u— L y s"からなり、 且つ、 キトサン分解活性を有する蛋白質が挙げられる。 より好まし いものとしては、 配列表の配列番号 1、 配列番号 2および配列番号 3に示されるアミ ノ酸配列を全て含み、 且つ、 N末端の配列が S e r-S e r-G l y-Leu-Ly s—からなり、 且つ、 N末端がァセチルイ匕されており、 且つ、 キトサン分解活性を有 する蛋白質が挙げられる。 In addition, 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. Be The protein may contain all of them as an internal amino acid sequence regardless of the order of the respective amino acid sequences. Among such chitinases, 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. More preferably, the 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.
また、 本発明のキチナーゼの別の例としては、 配列表の配列番号 11、 配列番号 1 2および配列番号 13に示されるアミノ酸配列を全て含み、 且つ、 キトサン分解活性 を有する蛋白質が挙げられる。  In addition, another example of the 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.
また、 配列表の配列番号 5に記載のアミノ酸配列からなる蛋白質において、 一個ま たは数個の部位に、 1個または数個のアミノ酸残基が、 置換、 欠失、 挿入および Zま たは付加した蛋白質も、 キトサン分解活性を有する限り、 本発明に含まれる。 数個と は 10個を超えない個数を意味し、 好適には 5個を超えない個数をいう。 置換したァ ミノ酸配列を有する蛋白質が、天然型蛋白質と同等の活性を有する例として、例えば、 インタ一ロイキン 2 (I L-2)遺伝子のシスティンに相当するヌクレオチド配列を セリンに相当するヌクレオチド配列に変換して得られた蛋白質が、 I L_2活性を保 持することが知られている （Wang, A. et al. (1984) Science 224,  In the protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing, 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. The term "several" means a number not exceeding 10, and preferably a number not exceeding 5. As an example in which a protein having a substituted amino acid sequence has an activity equivalent to that of a naturally-occurring protein, for example, 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,
1431-1433) 。 1431-1433).
また、 本発明のキチナーゼの別の例としては、 配列表の配列番号 5に記載のァミノ 酸配列からなる蛋白質を挙げる事ができる。 また、 配列表の配列番号 5に記載のアミ ノ酸配列を含むことからなる蛋白質であっても、キトサン分解活性を有する限り本発 明に含まれる。  In addition, as another example of the chitinase of the present invention, a protein consisting of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing can be mentioned. In addition, even 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.
また、本発明のキチナ一ゼの別の例としては配列表の配列番号 14に記載のァミノ 酸配列からなり、 N末端のひーァミノ基がァセチルイ匕されている蛋白質が挙げられる。 また、 このような蛋白質を含むことからなる蛋白質も、 キトサン分解活性を有する限 り本発明に含まれる。 また、 配列表の配列番号 14に記載のアミノ酸配列からなる蛋 白質において、 一個または数個の部位に、 1個または数個のアミノ酸残基が、 置換、 欠失、 揷入および Zまたは付加した蛋白質も、 キトサン分解活性を有する限り、 本発 明に含まれる。  In addition, another example of the 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. In addition, a protein comprising such a protein is also included in the present invention as long as it has a chitosan degrading activity. In 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.
本発明のキチナ一ゼとして好適なものは、ァスペルギルス ·オリザェ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2067) 由来のキチナ一ゼ、 ァ スペルギルス 'ジャポニクス （Aspergillus japonicus) SANK 19288株由 来のキチナ一ゼ、 ァスペルギルス ·ソジヤエ （Aspergillus sojae) SANK 22 388株由来のキチナーゼ、配列表の配列番号 5に記載のアミノ酸配列からなる蛋白 質および配列表の配列番号 14に記載のアミノ酸配列からなり、 N末端のひーァミノ 基がァセチル化されている蛋白質が挙げられるが、 より好適なものは、 ァスペルギル ス -オリサェ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2 067) 由来のキチナーゼおよび配列表の配列番号 14に記載のアミノ酸配列からな り、 且つ N末端のひーァミノ基がァセチル化されている蛋白質である。 本発明において、 「本発明の DNA」 とは、 本発明のキチナ一ゼをコ一ドする DN Aをいう。 DNAとしては、 cDNA、 ゲノム DNA、 人工的に改変された DN A、 化学的に合成された DNAなど、現在知られる限りどのような形態をとつていても良 い。 Preferred as the chitinase of the present invention is a chitinase derived from Aspergillus oryzae (Aspergillus oryzae) var. Sporof lavus Ohara J CM 2067, a chimichi 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. In the present invention, 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.
本発明の D N Aの例としては、配列表の配列番号 4のヌクレオチド番号 242から 1438に示されるヌクレオチド配列と相補的なヌクレオチド配列からなる DN A とストリンジェントな条件下でハイブリダィズし、 且つ、 キトサン分解活性を有する 蛋白質をコードする DNAが挙げられる。  An example of the 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.
本発明の DNAの別の例としては、配列表の配列番号 4のヌクレオチド番号 242 から 1438に示されるヌクレオチド配列と 70%以上、 好ましくは 80%以上、 よ り好ましくは 95 %以上のヌクレオチド配列相同性を有する DNAが挙げられる。 こ のような DNAとしては、 自然界で発見される変異型 DNA、 人為的に改変した変異 型 DNA、 異種生物由来の相同 DNAなどが含まれる。  Another example of the 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.
また、 本発明のヌクレオチドのさらに他の例としては、 配列表の配列番号 5に記載 のアミノ酸配列からなる蛋白質をコードする DNAが挙げられる。 なお、 所望のアミ ノ酸に対応するコドンは、 その選択も任意でよく、 例えば利用する宿主のコドン使用 頻度を考慮して常法に従い決定できる。 （Grantham, R. et al. (1981) Nucleic Acids Res. 9, 143-174) 。 さらに、 これらヌクレオチド配列のコドン の一部改変は、 常法に従い、 所望の改変をコードする合成オリゴヌクレオチドからな るプライマ一を利用した、 部位特異的変異導入法 （site specific mutagenesis/ Mark, D. F. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666) などに従うことができる。  Furthermore, as yet another example of the 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). Furthermore, 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.
本発明の DNAのまた別の例としては、配列表の配列番号 4のヌクレオチド番号 2 42から 1438に示されるヌクレオチド配列からなる DN Aが挙げられる。 又、 配 列表の配列番号 4のヌクレオチド番号 242から 1438に示されるヌクレオチド 配列からなる DNAを含むことからなる DNAも、キトサン分解活性を有する蛋白質 をコードする領域を含む限り、 本発明に含まれるものである。  Another example of the 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. In addition, 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.
本発明の D N Aが挿入された組換えプラスミドを保持する形質転換大腸菌  A transformed E. coli carrying a recombinant plasmid into which the DNA of the present invention has been inserted
Escherichia coli BM25.8/pTriplEx-Chitinase-cDNA SANK 72102は、 平成 14年 11月 8日付けで独立行政法人産業技術総合研究所特許生物寄託センタ ― (日本国茨城県つくば市東 1-1-1 中央第 6) に国際寄託され、 受託番号 FER M BP- 8235が付されている。 したがって、 本発明の DN Aは、 該菌株からも 取得することが可能である。 また、 この寄託菌株の保持する組換えプラスミドに挿入 された DNAとストリンジェントな条件下でハイブリダィズする D N Aも、 コードす る蛋白質がキトサン分解活性を有する限り、 本発明に含まれる。 また、 上記のヌクレ ォチド配列を含むことからなる DNAも本発明に含まれる。 さらに， このような DN Aによってコードされる蛋白質も本発明のキチナ一ゼに含まれる。 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. In addition, 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. In addition, a DNA comprising the above nucleotide sequence is also included in the present invention. Furthermore, such DN The protein encoded by A is also included in the chitinase of the present invention.
本発明の DNAとして好適なものは、配列表の配列番号 4のヌクレオチド番号 24 2乃至 1438に示されるヌクレオチド配列からなる DNA、 および、 形質転換大腸 菌 Escherichia coli BM25.8/pTriplEx-C itinase-cDNA SANK 72102 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
(FERM BP— 8235) により保持される組換えプラスミド、 The recombinant plasmid carried by (FERM BP-8235),
pTriplEx-Chitinase-cDNAに挿入されている DNAであるが、 最適には形質転換大腸 菌 Escherichia coli BM25.8/pTr iplEx-Chi t inase-cDNA SANK 72102Although it is DNA inserted into pTriplEx-Chitinase-cDNA, it is optimal that the transformed colon bacillus Escherichia coli BM25.8 / pTr iplEx-Chitinase-cDNA SANK 72102
(FERM BP— 8235) により保持される組換えプラスミド、 The recombinant plasmid carried by (FERM BP-8235),
pTriplEx-Chitinase-cDNAに揷入されている DNAである。 It is DNA inserted into pTriplEx-Chitinase-cDNA.
また、 本発明のキチナ一ゼには、 本発明の DNAによりコードされるアミノ酸配列 からなる蛋白質を挙げることができる。 また、 本発明のキチナーゼにおいて、 任意の 一つもしくは二つ以上のアミノ酸を欠失させた改変体を作製するためには、ェキソヌ クレア一ゼ B a 1 31等を用いて DNAを末端から削る方法 （岸本 利光ら "続生化 学実験講座 1 ·遺伝子研究法 I I" 335-354)、 カセット変異法 （岸本 利光、 "新生 化学実験講座 2 ·核酸 I I I 組換え DNA技術 " 242-251) などに従うことがで きる。 このように、 本発明の DNAを元に遺伝子工学的手法により得られる蛋白質で あっても、 キトサン分解活性を有する限り本発明に含まれる。 このようなキチナ一ゼ は、必ずしも配列表の配列番号 5に記載のアミノ酸配列の全てを有するものである必 要はなく、 例えばその部分配列からなる蛋白質であっても、 該蛋白質がキトサン分解 活性を示す限り本発明のキチナーゼに包含される。 また、 このようなキチナ一ゼをコ ―ドする DNAも本発明に含まれる。  Further, the chitinase of the present invention can include a protein consisting of an amino acid sequence encoded by the DNA of the present invention. In addition, in the chitinase of the present invention, in order to produce a variant in which any one or two or more amino acids have been deleted, a method of scraping the DNA from the end using Exonucleusase B a 131 or the like. (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. You can Thus, even 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. Is included in the chitinase of the present invention as long as In addition, DNA encoding such a chitinase is also included in the present invention.
本発明において、 ストリンジエンドな条件下でのハイブリダィゼ一シヨンは、 ハイ ブリダィゼーシヨンを、 5XSSC (0. 75M 塩化ナトリウム、 0. 075M ク ェン酸ナトリウム） またはこれと同等の塩濃度のハイブリダィゼ一シヨン溶液中、 3 7°C乃至 42°Cの温度条件下、 約 12時間行い、 5XSSCまたはこれと同等の塩濃 度の溶液等で必要に応じて予備洗浄を行った後、 1 X S S Cまたはこれと同等の塩濃 度の溶液中で洗^"を行うことにより実施できる。 また、 さらに 0. 1XSSCまたは これと同等の塩濃度の溶液中で洗浄を行うこともできる。  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.
本発明に用いるキチナ一ゼはキチナ一ゼ生産菌から精製したもの、粗精製したもの、 菌体の破碎液の他、 菌体の培養上清をそのまま用いたものでも良い。 キチナーゼ生産 菌を培養する際には、 培地中に炭素源、 窒素源の他に粉末キチン、 粉末キトサン、 コ ロイダルキチン、 コロイダルキトサン等を添加して培養するのが好ましい。 キチナ一 ゼ生産菌の培養終了後に遠心分離を行ない、菌体を除いた培養上清をそのまま粗酵素 液として用いることができる。 また、 粗酵素液をイオン交換クロマトグラフィー等に よって粗精製したり、 精製したりしたものを用いることもできる。  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. 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.
キチナ—ゼ生産菌とはキチナ—ゼ生産能を本質的に先天的に有する微生物をいい、 キチナ一ゼを菌体内に蓄積する微生物や、 菌体外に分泌する微生物等を含む。 キチナ ーゼ生産菌の培養上清又は培養上清から精製したキチナ一ゼを用いる場合には菌体 外にキチナ一ゼを分泌する菌を用いることができる。 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. When 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.
本発明に用いる'キチナ一ゼ生産菌としては、 ァスペルギルス · オリザェ As the chimera-producing bacteria used in the present invention, Aspergillus oryzae is used.
(Aspergillus oryzae) あるいはァスペルギルス ·ジャポニクス （Aspergillus japonicus) あるいはァスペルギルス 'ソジヤエ （Aspergillus sojae) を挙げるこ とができ、 好ましくはァスペルギルス ·オリザェ （Aspergillus oryzae) var. sporoflavus Ohara J CM 2067株あるいはァスペルギルス'ジャポニクス(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
(Aspergillus japonicus) SANK 19288あるいはァスペルギルス ·ソジャ ェ （Aspergillus sojae) SANK 22388株を挙げることができる。 キチナ一 ゼ生産菌の培養は通常の培地を培養装置、 培地を用いて行なうことができる。培養は 液体培養、 固体培養等の方法を適宜選択できる。 液体培養の場合はフラスコ培養や発 酵槽を用いた培養を行なうことができ、培養開始後は培地の追加のないバッチ培養法 や培養中に適宜培地を添加していく流加培養法を用いることができる。培地には炭素 源、 窒素源を添加し、 必要に応じてビタミン、 微量金属等を添加することができる。 炭素源としては、グルコース、マンノース、ガラクトース、フルクト一ス等の単糖類、 マル] ス、 セロビオース、 イソマル! ^一ス、 ラク ] ス、 スクロース等の二糖類、 デンプン等の多糖類、マルトエキストラクト等を挙げることができるがキチナーゼ生 産菌が生育する限りこれらに限定されない。 窒素源としてはアンモニア、 硫酸アンモ 二ゥム、 硝酸アンモニゥム等の無機窒素、 イースト ·ェクストラクト、 マルトェクス トラクト、 コーンスティ一プリカ一、ペプトン等の有機窒素が用いられるがキチナ一 ゼ生産菌が生育する限りこれらに限定されない。 また、 キチナ一ゼ生産菌のキチナー ゼの生産量を増やすために培地中に粉末キチンやコロイダルキチンを添加すること もできる。これらの培地中の組成物量は適宜選択することができる。培養温度、 pH、 通気攪拌量はキチナ一ゼ生産に適するように適宜選択することができる。 Aspergillus japonicus SANK 19288 or Aspergillus sojae SANK 22388 strain can be mentioned. Cultivation of chitinase-producing bacteria can be performed using a conventional culture medium and culture medium. For culture, methods such as liquid culture and solid culture can be appropriately selected. In the case of 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. Examples of carbon sources include glucose, mannose, galactose, monosaccharides such as fructose, maltose, cellobiose, isoma! ^ Starch, lactose, disaccharides such as sucrose, polysaccharides such as starch, maltose extract However, as long as the chitinase-producing bacteria grow, they are not limited thereto. As the nitrogen source, 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.
本発明に用いるキチナーゼとしては、 ァスペルギルス ·オリザェ （Aspergillus oryzae) あるいはァスペルギルス 'ジャポニクス （Aspergillus japonicus) あるい はァスペルギルス 'ソジヤエ （Aspergillus sojae) 由来のキチナーゼを用いること ができ、 好ましくはァスペルギルス ·オリザェ （Aspergillus oryzae) var. sporoflavus Ohara J CM 2067株あるいはァスペルギルス.ジャポニクス As the 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
(Aspergillus japonicus) SANK 19288あるいはァスペルギルス 'ソジャ ェ （Aspergillus sojae) SANK 22388株由来のキチナ一ゼを用いることが できる。 キチナーゼは、 これらキチナ一ゼ生産菌自身が生産するものでもよいし、 そ の変異体または修飾体が生産するものであってもよく、 更に、 これらキチナ一ゼ生産 菌のキチナーゼをコードする遺伝子を宿主に導入して得られた形質転換体から生産 される組換えタンパク質であってもよい。 キチナーゼ生産菌の入手 Aspergillus japonicus SANK 19288 or Aspergillus sojae It is possible to use a 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. Furthermore, 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
ァスペルギルス ·オリザェ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2067株は理化学研究所 生物基盤研究部 微生物系統保存施設 （ J C M： Japan Col lection of Microorganisms；住所 〒 351— 0198 日本国 埼玉県和光巿広沢 2— 1、 ホームページアドレス く http：〃 ww. jcm.riken.go.jp/ » より購入することができる。  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 ww. Jcm.riken.go.jp/ »Can be purchased from».
ァスペルギルス ·ジャポニクス （Aspergillus japonicus) SANK 19288 株とァスペルギルス 'ソジヤエ （Aspergillus sojae) S ANK 22388株の菌 学的性質を以下に示す。  The bacteriological properties of Aspergillus japonicus SANK 19288 strain and Aspergillus' Sasperae S ANK 22388 strain are shown below.
ァスペルギルス ·ジャポニクス S ANK 1 9 2 8 8株とァスペルギルス - ソジヤエ . S ANK 2 2 3 8 8株をクリックとピッ トの文献 （Klich, M. A. and Pitt, J. I. (1988) A laboratory guide to the common Aspergillus species and their teleomorphs.  Aspergillus japonics S ANK 1 9 2 8 8 and Aspergillus-Sozyyae. S ANK 2 2 3 8 8 clicks and the document of the pit (Klich, MA and Pitt, JI (1988) Laboratory guide to the common Aspergillus species and their teleomorphs.
CSIRO, Division of Food Processing, North Ryde N.S.W. , Austraria.)に従い、 3種類の培地 （CYA培地， MEA培地， CY20S培地） に接種して、 菌学的性状を観察した。 According to CSIRO, Division of Food Processing, North Ryde N. S. W., Australia., Three types of medium (CYA medium, MEA medium, CY20 S medium) were inoculated to observe mycological characteristics.
色調の表示は「メチューン ·ハンドブック ·ォブ ·カラ一」 (Kornerup, A. and Wanscher , J . H. (1978) Methuen handbook of  The display of the color tone is "Metune Handbook · Ob · Cala I" (Kornerup, A. and Wanscher, J. H. (1978) Methuen handbook of
colour (3rd. edition). Erye Methuen, London. )に従った。 color (3rd. edition). Erye Methuen, London.
3種類の培地 （CYA培地， MEA培地， CY20S培地） の組成は以下の通 りである。  The composition of the three types of media (CYA media, MEA media, CY20S media) is as follows.
CYA培地 {ザペックィ一ストアガー（Czapek Yeast Extract Agar) 培 地 } (K2HP04 1.0 g, *ザペック濃縮液 10 ml, イーストエキス 5 g， シュ一クロース 30 g, 寒天 15 g, 蒸留水 1000 ml)  CYA medium (Czapek Yeast Extract Agar medium) (K2HP04 1.0 g, * 10 ml of Zapeck concentrate, 5 g of yeast extract, 30 g of sucrose, 15 g of agar, 1000 ml of distilled water)
*ザペック濃縮液 （NaN03 30 g, KC1 5 g, MgS04 -7H20 5 g, FeS04 -7H20 0.1 g, ZnS04 -7H20 0.1 g， CuS04 -5H20 0.05 g 蒸留水 100ml) * 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培地 {モルトエキスァガー（Malt Extract Agar)培地 } (モルトェキ ス 20 g, ペプトン 1 g， ダルコ一ス 20 g, 寒天 20 g， 蒸留水 lGOOml)  MEA medium {Malt Extract Agar medium} (Moltoex 20 g, Peptone 1 g, Darucose 20 g, Agar 20 g, Distilled water lGOO ml)
CY20S培地 {20%シユークロースザペックィ一ストアガー（Czapek Yeast Extract Agar with 20¾ Sucrose)培地 } (K2HPO4 1.0 g, *ザぺッ ク濃縮液 10 ml, イーストエキス 5 g, シュ一クロース 200 g， 15 g, 蒸留水 1000 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)
1 ) 菌学的性状 . a) ァスペルギルス ·ジャポニクス S ANK 1 9 2 8 8株の菌学的性状1) Mycological characteristics. a) Mycological characteristics of Aspergillus japonics S ANK 1 9 2 8 8
CYA培地でのコロニーは、 25°C、 1週間の培養で直径 45乃至 60 匪である。 菌糸 は白色である。 分生子形成は中心部で旺盛、 形成部の色はオリ一ブブラウン （4F4) 乃至黒色をを呈する。 裏面の色はグレイッシユイエロ一 （4B4) 乃至イェローイツシ ュホワイト （4A2) を呈する。 菌核は観察されない。 浸出液や可溶性色素は観察され ない。 CY20S培地でのコロニ一は、 25°C、 1週間の培養で直径 57乃至 68 mmである。 コロニーは CYA培地のコロニーに似る。 裏面の色はパストラルイエロ一 （3A4) を呈 する。 MEA培地でのコロニーは、 25°C、 1週間の培養で直径 51乃至 57 讓である。 分生子形成は旺盛、 形成部の色は黒色である。 裏面の色は無色である。 37°C、 1週間 培養した CYA培地 のコロニーは、直径 13乃至 20 mmである。 5°Cでは生育しない。 分生子頭は放射状、 分生子柄は平滑、 無色または頂部で褐色、 幅 4乃至 11 で ある。 頂のうは球形乃至楕円形、 幅 19乃至 50 である。 ァスペルジラは単列であ る。 フィアライドは頂のうの 3/4を覆い、 7乃至 10 X 3.5乃至 4.5 である。 分生子は楕円乃至球形、やや疎な棘で覆われ、直径 3.5乃至 4.5 X 3.5乃至 4 である。 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. 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.
以上の菌学的性状より、 本菌に該当する菌を検索したところ、 クリツ クとピッ トの文献に記載されているァスペルギルス ジャポニクス ノ —ル アキユリ—タス (Aspergillus j aponi cus var. aculeatus (Iizuka) Al-Musallam) の性状とほぼ一致した。 よって本菌株をァスぺ' ルギルス ジャポニクス バール アキユリ—タス (Aspergillus j aponi cus var. aculeatus (Iizuka) Al-Musal 1 am) と同定した。  According to the above-mentioned bacteriological characteristics, when the bacteria corresponding to this bacterium were searched, Aspergillus japonica nor. Acriuritus (Aspergillus j aponi cus var. Aculeatus (Iizuka) described in the literature of Kuritsu and Pit) It almost agrees with the properties of Al-Musallam). Therefore, the strain was identified as Aspergillus japonicus bark auritus (Aspergillus j aponi cus var. Aculeatus (Iizuka) Al-Musal 1 am).
尚、 ァスペルギルス 'ジャポニクス S ANK 1 9 2 8 8株は、 ァスペル ギルス ジャポニクス バール アキユリ—タス S ANK 1 9 2 8 8株として、 平成 1 3年 9月 1 3 日に独立行政法人産業技術総合研究所特許生 物寄託センター （日本国茨城県つくば巿東 1-1-1 中央第 6) に国際寄託され、 受 託番号 F E RM B P— 7 7 3 6が付された。  In addition, 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.
b) ァスペルギルス ·ソジヤエ S ANK 2 2 3 8 8株の菌学的性状 CYA培地でのコロニーは、 25°C、 1週間の培養で直径 46乃至 52 mmである。 コロニ —は綿毛状である。 菌糸は白色、 密である。 分生子形成は旺盛、 形成部の色はオリー ブ（2F-E7) を呈する。 裏面の色はイエロ一イツシュホワイト （2A2) を呈する。 CY20S 培地でのコロニーは、 25°C、 1週間の培養で直径 42乃至 45 腿である。 コロニ一は CYA培地のコロニーに似る。分生子形成は旺盛、形成部の色はオリーブイエロ一（3D6) 乃至オリーブ （3E7) を呈する。 MEA培地でのコロニーは、 25°C、 1週間の培養で直径 43乃至 47 匪である。 コロニ一はやや綿毛状、 やや疎である。 菌糸は白色、 目立た ない。 分生子形成は旺盛、 形成部の色はォリーブ （1E8) を呈する。 裏面の色は無色 である。 37° (：、 1週間培養した CYA培地のコロニーは、 直径 52乃至 56 腿である。 5°Cでは生育しない。 b) Mycological characteristics of Aspergillus sogyae strain S ANK 2 2 8 8 8 The colonies in CYA medium are 46 to 52 mm in diameter after 1 week of culture at 25 ° C. The colony is fluffy. Mycelium is white and dense. The formation of conidia is strong, and the color of the formation is olive (2F-E7). The color of the back is yellowish 1 itsh white (2A2). Colonies in CY20S medium are 42-45 thighs in diameter at 25 ° C for 1 week in culture. The colony resembles a colony of CYA medium. The formation of conidia is strong, and the color of the formation part is olive yellow (3D6) to olive (3E7). 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.
分生子頭は放射状、 分生子柄は粗面、 無色、 幅 7乃至 12, ιである。 頂のうは洋 なし形乃至球形、 幅 24乃至 41 mである。 ァスペルジラは単列、 ときどき二列であ る。メトレは 8乃至 12 X 4乃至 8 mである。フィアライドは 10乃至 11 X 4.5 乃至 6 J1である。 分生子は球形、 粗面、 直径 5乃至 6.5 である。  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.
以上の菌学的性状より、 本菌に該当する菌を検索したところ、 クリツ クとピッ トの文献に記載されているァスペルギルス ソジヤエ  Based on the above-mentioned mycological characteristics, when a bacterium corresponding to this bacterium was searched, Aspergillus sp.
(Aspergillus so j ae Sakaguchi & Yamada) の性状とほほ一致した。 よって本菌株をァスペルギルス ソジヤエ （Aspergillus sojae  The characteristics were in agreement with those of (Aspergillus so j ae Sakaguchi & Yamada). Therefore, the strain is used as Aspergillus sojae (Aspergillus sojae).
Sakaguchi & Yamada と同疋した。 I co-hosted with Sakaguchi & Yamada.
尚、 ァスペルギルス 'ソジヤエ S ANK 2 2 3 8 8株は、 ァスペルギ ルス ソジヤエ S ANK 2 2 3 8 8株として、 平成 1 3年 9月 1 3 日に独立行政法人産業技術総合研究所特許生物寄託センター （日本国茨城県つくば 巿東 1-1- 1 中央第 6) に国際寄託され、 受託番号 F E RM B P— 7 7 3 8が付された。  In addition, 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.
周知の通り、 糸状菌は自然界において、 または人工的な操作 （例えば、 紫外線照射、 放射線照射、 化学薬品処理等） により、 変異を起こしやす く、本発明のァスペルギルス'オリザェ（Aspergillus oryzae var. sporof lavus Ohara J CM 2067) 株、 ァスペルギルス ·ジャポニクス S ANK 1 9 2 8 8株ゃァスペルギルス ·ソジヤエ S ANK 2 2 3 8 8株もその点 は同じである。 本発明にいうァスペルギルス ·オリザェ （Aspergillus oryzae var. sporoflavus Ohara J CM 2067 ) 株、 ァスペルギルス 'ジャポニク ス S ANK 1 9 2 8 8株ゃァスペルギルス ·ソジヤエ S ANK 2 2 3 8 8株はその全ての変異株を包含する。 また、 これらの変異株の中に は、 遺伝的方法、 たとえば組み換え、 形質導入、 形質転換等によりえら れたものも含有される。 即ち、 キチナ一ゼを生産するァスペルギルス ·オリ ザェ (Aspergillus oryzae var. sporoflavus Ohara J CM 2067)株、 それらの変異株およびそれらと明確に区別されない菌株は全てァスペルギ ルス -オリザェ (Aspergillus oryzae var. sporoflavus Ohara J CM 20 67) 株に包含される。 キチナーゼを生産するァスペルギルス ·ジャポニクス SANK 19288株、 それらの変異株およびそれらと明確に区別されな い菌株は全てァスペルギルス 'ジャポニクス SANK 19288株に包含さ れる。 キチナ一ゼを生産するァスペルギルス ·ソジヤエ S ANK 2 2 3 8 8株、それらの変異株およびそれらと明確に区別されない菌株は全てァ スペルギルス 'ソジヤエ S ANK 2 2 3 8 8株に包含される。  As is well known, 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. 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) strain according to the present invention, 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.
また、 本発明のキチナーゼは、 ベクターに本発明の DN Aが揷入された組換えブラ スミドで宿主細胞を形質転換し、該形質転換された細胞の培養産物から得る事もでき る。 このように適当なベクタ一に本発明の D N Aが挿入された組換えプラスミドも本 発明に含まれる。 このような目的に用いるベクターとしては、 一般に知られているさ まざまなベクターを用いることができる。好適なものとしては、原核細胞用ベクター、 真核細胞用ベクター、 哺乳動物,由来の細胞用べクタ一などが挙げられるが、 これに限 定されない。 このような組換えプラスミドにより、 他の原核生物、 または真核生物の 宿主細胞を形質転換させることができる。 さらに、 適当なプロモー夕一配列および Z または形質発現に関わる配列を有するベクターを用いるか、 もしくはそのような配列 を導入することにより、 発現べクタ一とする.ことで、 それぞれの宿主において遺伝子 を発現させることが可能である。 このような発現ベクターは、 本発明の組換えプラス ミドの好適な態様である。 In addition, 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. Thus, a recombinant plasmid in which the DNA of the present invention is inserted into an appropriate vector is also included in the present invention. As 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. Furthermore, by using a vector having an appropriate promoter sequence and Z or a sequence relating to expression, or by introducing such a sequence, the gene is made into an expression vector, whereby the gene is expressed in each host. It is possible to express. Such expression vectors are a preferred embodiment of the recombinant plasmids of the present invention.
本発明の組換えプラスミドを、 各種細胞に導入することにより、 宿主細胞を得るこ とができる。 このような細胞は、 プラスミドを導入することができる細胞であれば原 核細胞であっても真核細胞であってもよい。  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.
原核細胞の宿主としては、例えば、大腸菌（Escher ichi a col i)や枯草菌（Baci 1 lus subt i l is) などが挙げられる。 目的の遺伝子をこれらの宿主細胞内で形質転換させる には、 宿主と適合し得る種由来のレブリコンすなわち複製起点と、 調節配列を含んで いるプラスミドベクターで宿主細胞を形質転換させる。 また、 ベクタ一としては、 形 質転換細胞に表現形質 （表現型） の選択性を付与することができる配列を有するもの が好ましい。  As a prokaryotic host, for example, Escherichia coli (Escherichia coli i), Bacillus subtilis (Baci 1 ls subtilis), and the like can be mentioned. In order to transform a gene of interest in these host cells, host cells are transformed with a plasmid vector that contains regulatory sequences and a Lebricon or replication origin from a species compatible with the host. In addition, as vector I, one having a sequence capable of imparting phenotypic (phenotype) selectivity to transformed cells is preferable.
例えば、 大腸菌としては K 1 2株などがよく用いられ、 ベクターとしては、 一般に p B R 3 2 2や p U C系のプラスミドが用いられるが、 これらに限定されず、 公知の 各種菌株、 およびベクターがいずれも使用できる。  For example, as 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.
プロモーターとしては、大腸菌においては、 トリプトファン（trp) プロモーター、 ラク 1 ス（l ac)プロモータ一、 卜リプトフアン ·ラク 1 ^一ス （tac)プロモーター、 リポプロテイン （lpp) プロモーター、 ポリペプチド鎖伸張因子 Tu (tufB) プロモー 夕一等が挙げられ、 どのプロモーターも本発明のキチナ一ゼの産生に使用することが できる。  As a 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.
枯草菌としては、 例えば 2 0 7 - 2 5株が好ましく、 ベクタ一としては p TUB 2 2 8 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) などが用いられる が、 これに限定されるものではない。  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.
プロモーターとしては、枯草菌の 一アミラーゼのシグナルぺプチド配列をコード する D N A配列を連結することにより、 菌体外での分泌発現も可能となる。  By linking a DNA sequence encoding the signal peptide sequence of Bacillus subtilis monoamylase as a promoter, it is also possible to carry out secretory expression outside of the cell.
真核細胞の宿主細胞には、 脊椎動物、 昆虫、 酵母などの細胞が含まれ、 脊椎動物細 胞としては、 哺乳動物由来の細胞、 例えば、 サルの細胞である C O S細胞 （Gluzman, Y. (1981) Cel l 23, 175-182, AT C C C R L— 1 6 5 0 ) やチャイニーズ · ハムスター卵巣細胞 （CH〇細胞、 ATCC CCL-61) のジヒドロ葉酸還元酵 素欠損株 （Urlaub, G. and Chasin, L. A. (1980) Proc. Natl. Acad. Sci. USA 77, 4126-4220) 等がよく用いられているが、 これらに限定されない。 脊椎動物細胞の発現プロモーターとしては、通常発現しょうとする遺伝子の上流に 位置するプロモーター、 RNAのスプライス部位、 ポリアデニル化部位、 および転写 終結配列等を有するものを使用でき、 さらにこれは必要により複製起点を有してもよ い。 該発現べクタ一の例としては、 S V40の初期プロモーターを有する p S V2 d h f r (Subramani, S. et al. (1981) Mol. Cell. Biol. 1， 854-864) 等 が挙げられるが、 これに限定されない。 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. As 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.
宿主細胞として、 COS細胞を用いる場合を例に挙げると、発現べクタ一としては、 As a host cell, when using COS cells as an example, as an expression vector,
SV40複製起点を有し、 COS細胞において自立増殖が可能であり、 さらに、 転写 プロモーター、 転写終結シグナル、 および RN Aスプライス部位を具えたものを用い ることができる。 該発現べクタ一は、 ジェチルアミノエチル （DEAE) —デキスト ラン法 (Lut man, H: and Magnus son, G. (1983) Nucleic Acids Res, 11,It has an SV40 origin of replication, is capable of autonomous growth in COS cells, and may further comprise a transcription promoter, a transcription termination signal, and an RNA splice site. The expression vector is a jetyl aminoethyl (DEAE) -dextran method (Lut man, H: and Magnus son, G. (1983) Nucleic Acids Res, 11,
1295-1308)、 リン酸カルシウム— DNA共沈殿法 （Graham, F. L. and van der1295-1308), calcium phosphate-DNA coprecipitation method (Graham, F. L. and van der
Eb, A. J. (1973) Virology 52, 456 - 457)、および電気パルス穿孔法（Neumann,Eb, A. J. (1973) Virology 52, 456-457), and electrical pulse drilling (Neumann,
E. et al. (1982) EMB0 J. 1, 84卜 845)などにより C O S細胞に取り込ませ ることができ、 かくして所望の形質転換細胞を得ることができる。 また、 宿主細胞と して CHO細胞を用いる場合には、 発現べクタ一と共に、 抗生物質 G418耐性マー 力一として機能する n e o遺伝子を発現し得るベクタ一、 例えば pRSVn e oE. et al. (1982) EMB 0 J. 1, 84 卜 845) and the like can be incorporated into CO 2 S cells, and thus desired transformed cells can be obtained. In addition, 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
(Sambrook, J. et al. (1989) ： 'Molecular Cloning A Laboratory(Sambrook, J. et al. (1989): 'Molecular Cloning A Laboratory
Manual " Cold Spring Harbor Laboratory, NY)や p S V 2— n e o (Southern,Manual "Cold Spring Harbor Laboratory, NY) or p S V 2-n e o (Southern,
P. J. and Berg, P. (1982) J. Mol. Appl. Genet. 1, · 327-341)などを コ ' トランスフエクトし、 G418耐性のコロニーを選択することにより、 本発明の キチナ—ゼを安定に産生する形質転換細胞を得ることができる。 PJ and Berg, P. (1982) J. MoI. Appl. Genet. 1, · 327-341), etc. to stabilize the chitinase of the present invention by selecting G418 resistant colonies. Transformed cells can be obtained.
昆虫細胞を宿主細胞として用いる場合には、 鱗翅類ャガ科の Spodoptera  When insect cells are used as host cells, Spodoptera spp.
frugiperdaの卵巣細胞由来株化細胞 (S： f 一 9または S f - 21) や Trichoplusia niの卵細胞由来 High Five細胞（Wickham, T. J. et al, (1992) Biotechnol. Prog. I: 391- 396) などが宿主細胞としてよく用いられ、 バキュロウィルストラン スファ一ベクタ一としてはオートグラファ核多角体ウィルス （AcNPV) のポリへ ドリン蛋白質のプロモーターを利用した pVL 1392/1393がよく用いられ る (Kidd, I. M. and V. C. Emery (1993) The use of baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 42, 、37-159)。 この他にも、 バキュロウィルスの P 10や同塩基性蛋白質のプロモ一夕一 を利用したベクタ一も使用できる。 さらに、 Ac NPVのエンベロープ表面蛋白質 G P 67の分泌シグナル配列を目的蛋白質の N末端側に繋げることにより、組換え蛋白 質を分泌蛋白質として発現させることも可能である （Zhe- mei Wang, et al. (1998) Biol . Chem: , 379, 167 - 174)。 Frugiperda cell line derived from ovarian cells (S: f 19 or S f-21) or Trichoplusia ni egg cell-derived High Five cells (Wickham, TJ et al, (1992) Biotechnol. Prog. I: 391- 396), etc. Is often used as a host cell, and pVL 1392/1393 using the promoter of the polyprotein of autographer nuclear polyhedrosis virus (AcNPV) is often used as a baculovirus transfer vector (Kidd, IM and VC Emery (1993) The use of baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 42, 37-159). In addition to this, vector 1 using baculovirus P10 or the same basic protein promoter can also be used. Furthermore, 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).
真核微生物を宿主細胞とした発現系としては、 酵母が一般によく知られており、 そ の中でもサッカロミセス属酵母、例えばパン酵母 Saccharomyces cerevis i aeや石油 酵母 Pichia pas tori sが好ましい。 該酵母などの真核微生物の発現べクタ一として は、例えば、アルコール脱水素酵素遺伝子のプロモーター（Bennetzen, J. L. and Hal l, B. D. (1982) J. Biol . Chem. 257, 3018-3025)や酸性フォスファタ ーゼ遺伝子のプロモータ— （Miyanohara, A. et al. (1983) Proc. Nat l . Acad. Sci . USA 80， 1-5) などを好ましく利用できる。 また、 分泌型蛋白質とし て発現させる場合には、分泌シグナル配列と宿主細胞の持つ内在性プロテア一ゼある いは既知のプロテアーゼの切断部位を N末端側に持つ組換え体として発現すること も可能である。例えば、 トリプシン型セリンプロテア一ゼのヒトマスト細胞トリプタ ーゼを石油酵母で発現させた系では、 N末端側に酵母の αファクタ一の分泌シグナル 配列と石油酵母の持つ Κ Ε Χ 2プロテア一ゼの切断部位をつなぎ発現させることに より、 活性型トリプターゼが培地中に分泌されることが知られている （Andrew, L. Ni les' et al. (1998) Biotechnol. Appl. Biochem. 28, 125—131)。  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. Examples of 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. 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 α 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).
上記のようにして得られる形質転換体は、 常法に従い 養することができ、 該培養 により細胞内、 または細胞外に本発明のキチナ一ゼが産生される。 該培養に用いられ る培地としては、 採用した宿主細胞に応じて慣用される各種のものを適宜選択でき、 例えば、 上記 C O S細胞であれば、 R P M I 1 6 4 0培地やダルベッコ改変イーグル 培地 （以下 「DM E M」 という） などの培地に、 必要に応じゥシ胎児血清などの血清 成分を添加したものを使用できる。 培養条件としては、 C O 2濃度は 0乃至 5 0 %の 範囲であればよく、 好適には 1乃至 1 0 %でありより好適には 5 %である。 培養温度 は 0乃至 9 9 °Cであればよく、 好適には 2 0乃至 5 0 °Cであり、 より好適には 3 5乃 至 4 0 °Cである。  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. As 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. As culture conditions, 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.
上記培養により形質転換体の細胞内または細胞外に組換え蛋白質として産生され る本発明のキチナーゼは、 培養産物中から、 その蛋白質の物理化学的性質、 化学的性 質、 生化学的性質 (酵素活性など） 等を利用した各種の分離操作 （ 「生化学デ一タブ ック II」 、 1175- 1259項、 第 1版第 1刷、 1980年 6月 23日株式会社東京化学同人発 行； Biochemistry, vo l . 25, No. 25, p8274-8277 (1986)； Eur. J. Biochem. , 163, p313-321 (1987)等参照） により分離、 精製することができる。 該方法としては、 具体的には例えば通常の再構成処理、 蛋白沈殿剤による処理 （塩析 法）、遠心分離、浸透圧ショック法、凍結融解法、超音波破碎、 限外ろ過、ゲル濾過、 吸着クロマトグラフィー、 イオン交換クロマトグラフィー、 アツフィニティークロマ トグラフィー、 高速液体クロマトグラフィー （H P L C) 等の各種液体クロマトダラ フィ一、 透析法、 それらの組み合わせ等 ¾例示できる。 上記により、 高収率で所望の 組替え蛋白質を工業的規模で製造できる。 また、 発現させる組換え蛋白質に 6残基か らなるヒスチジンを繋げることにより、ニッケルァフィ二ティーカラムで効率的に精 製することができる。 上記方法を組み合わせることにより容易に高収率、 高純度で本 発明のキチナーゼを大量に製造できる。 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. As 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. By the above, high yield and desired Recombinant proteins can be produced on an industrial scale. In addition, by linking histidine consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column. By combining the above methods, the chitinase of the present invention can be easily produced in large quantities with high yield and high purity.
以上のような方法により製造されたキチナ一ゼも本発明の好適な例としてあげる 事ができる。 本発明に用いる低分子キトサン又はその塩は、 0〜1 0 0 °Cの水 1 0 0 gに対して 1 0 g以上溶解する。 ここでいう水とは、 蒸留水、 水道水、 井戸水であるとよい。 さ らに、 低分子キトサン又はその塩は、 P H I . 0〜7 . 0の条件下で、 1 0 0 gの水 に 1 0 g以上溶解する。 また、 低分子キトサンは抗菌活性を有する。  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. Here, water should be distilled water, tap water, and well water. Furthermore, 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. In addition, low molecular weight chitosan has antibacterial activity.
部分ァセチル化キトサンとは、キトサンのァミノ基の一部がァセチル化されている ものをいい、 ァセチル化度が低い部分ァセチル化キ卜サンは、 キチンをアルカリで脱 ァセチル化することによって製造することができる。部分ァセチル化キトサンのァセ チル化度はアルカリ濃度、 反応時間等により制御することができる。 また、 分子量が 1 0 0万以上のものを、 特に高分子部分ァセチル化キトサンと呼ぶ。 分子量 1 0 0万 以上の高分子部分ァセチル化キトサンとしては例えば、 ェビ ·力二由来の 3 0 %、 2 0 %、 1 0 %ァセチル化キトサン （商品名 キトサン 7 B、 8 B、 9 B (いずれも加ト 吉 （株） 製） を用いることができるが、 これらに限定されず、 ェビ、 力二に含まれる 天然のキチンは、 よりァセチル化度が高い高分子部分ァセチル化キトサンであるが、 これらも部分ァセチル化キ卜サンとして用いることができる。  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. Examples of high molecular weight partially acetylated chitosan having a molecular weight of 100,000 or more 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.
高分子部分ァセチル化キトサンの分子量は、ゲルパーミエ一ションクロマトグラフ ィ （G P C) を用いる高速液体クロマトグラフィー （プルランを標準物質とする） に より、 測定することができる （滝口ら、 キチン'キトサン研究 P . 75- 79, 1999) 。 高分子部分ァセチル化キトサンのァセチル化度は、指示薬としてトルイジンブル一 溶液を用い、 1 Z 4 0 0 Nポリビニル硫酸力リゥム溶液で PVSKコロイド滴定を行 うことにより測定することができる（Toei K. et al . Anal . Chem. Acta 83, 59, 1976) 。 この方法で測定したァセチル化度は、 ァセチル化されているアミノ基 の％ (例えば、 ァセチル化度が 1 %とは、 1 0 0個のアミノ基のうち 1個がァセチル 化されていることを意味する） を表す値である。  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
キトサンのァセチル化度は、 Ή-NMRにより得られるァセチル基の 3 Hとァセチル基 以外の水酸基を除く 7 Hとの積分比からも算出することができる。  The degree of acetylation of chitosan can also be calculated from the integral ratio of 3 H of the acetyl group obtained by Ή-NMR to 7 H excluding hydroxyl groups other than the acetyl group.
キチナーゼの加水分解活性は、 Randle-Morgan 法 （Randle C. J. M et al . Bi ochem. J. GL 586-589, 1955)に従って、測定することができる。また、 Schales 変法 （ Imoto, T. and Yagashi ta, K.， Agric. Biol . Chem. , 35, 1154 (1971) ) に従っても測定できる。 本発明は低分子キトサンの製造方法に関する。 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.
該方法において使用されるキチナーゼは、 PH4. 0〜5. 5、 温度 37°Cで 10 〜 30分間の反応条件下での 30%ァセチル化キトサン （粘度 100〜300 cps) を基 質とした場合の加水分解活性を 100%とした場合に、 グリコールキチンを基質とし た場合の加水分解活性が 100%より小さく、 ァセチル化度が 1%以下のキトサン (粘度 50〜200 cps) を基質とした場合の前記キチナ一ゼの加水分解活性が 1 5 %以下である。  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. When the hydrolysis activity of 100% is used and the hydrolysis activity is lower than 100% when glycol chitin is used as the substrate and when the chitosan with a degree of aserylation of 1% or less (viscosity 50 to 200 cps) is used as the substrate The hydrolysis activity of said chitinase is less than 15%.
該方法における酵素の使用量及び使用濃度については、 特に限定されない。 ' 該方法において、キチナ一ゼの基質となる高分子部分ァセチル化キトサンの水溶液 は、 0. 1〜5質量％、 好適には 0. 25〜2質量％、 さらに好適には 0. 5〜1質 量％濃度である。 該水溶液は、 酸性条件下 （例えば、 ρΗ1〜6) で水に溶解した溶 液として用いることもできる。 また、 この溶液に更にアルカリ （例えば、 炭酸ナトリ ゥム、 炭酸水素ナトリウム、 苛性ソーダなど） を加えて弱酸性 （例えば、 PH4. 0 〜6. 0) にした溶液として、 該方法に用いる事もできる。  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, Η 1 to 6). In addition, an alkali (for example, sodium carbonate, sodium hydrogen carbonate, caustic soda, etc.) may be further added to this solution to make it weakly acidic (for example, pH 4.0 to 6. 0) and used as the solution. .
高分子部分ァセチル化キトサン溶液を酸性にして水に溶解させるために使用する 酸としては、塩酸、酢酸、 トリフルォロ酢酸、'パラトルエンスルホン酸、アクリル酸、 乳酸、 アミノ酸、 クェン酸、 サリチル酸、 グルクロン酸、 グリコール酸等を挙げるこ とができるが酸性溶液を作ることができればこれらに限定されない。  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.
本発明のキチナーゼを用いて低分子キトサンを製造する際の反応溶液の PH範囲 としては、 pH3〜l 2の範囲を挙げることができるが、 好適には pH3〜7の範囲 であり、さらに好適には pH4〜 6の範囲である。該反応溶液の温度は、 0° (：〜 70°C であればよく、 好適には 10°C〜60°Cであり、 さらに好適には 40〜50tである。反 応時間は 10分〜 7日間を挙げることができるが、 好適には 3時間〜 5日間であり、 よ り好適には 1〜3日間である。  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.
高分子部分ァセチル化キトサンを様々な酸を用いて水溶液にした後に、本発明のキ チナ一ゼによる加水分解反応をおこなうことにより、 分子量 1万以上、 100万未満 の様々な低分子キトサン塩が生成する。塩の種類としては使用した酸に応じて塩酸塩、 酢酸塩、 トリフルォロ酢酸塩、 パラトルエンスルホン酸塩、 アクリル酸塩、 乳酸塩、 アミノ酸塩、 クェン酸塩、 サリチル酸塩、 グルクロン酸塩、 グリコ一ル酸塩等が挙げ られるが、 これらに限定されない。 本発明によって、 0〜100°Cの水 100 gに対して 10 g以上溶解する低分子キ トサン又はその塩が提供される。  After making high molecular partially acetylated chitosan into an aqueous solution using various acids, 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.
さらに、 本発明は、 0〜100°Cの水 100 gに対して 10 g以上溶解する低分子 キトサンを含む医薬組成物を提供する。  Furthermore, the present invention provides a pharmaceutical composition comprising low molecular weight chitosan which dissolves 10 g or more in 100 g of water at 0 to 100 ° C.
キチナーゼの加水分解活性は、 Randle- Morgan 法 （Randle C. J.M et al. Biochem. J. GL 586-589, 1955)に従って、測定することができる。また、 Schales 変法 （ Imoto, T. and Yagashita, K. , Agric. Biol. Chem. , 35, 1154(1971)) に従っても測定できる。 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)).
グリコ一レキチン ½：、 R. Senzyu and S. Okimatsu, Nippon Nogeikagaku Kaishi, 23, 432 (1950)に記載されている方法によって製造することができる。 ァスペルギルス ·オリザェ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2067) 株により生産され、 精製されたキチナ一ゼは、 以下の性質を有す る。  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.
1 ) SDS-PA G E電気泳動法にて分子量約 40, 000を示す。  1) It shows about 40,000 molecular weight by SDS-PAGE electrophoresis.
2) 等電点電気泳動法にて等電点 p I 4. 5を示す。  2) The isoelectric point p I 4.5 is indicated by isoelectric focusing method.
3) キトサン 7B (加ト吉 （株） 製） を、 pH3. 5乃至 ρΗΙ Ο. 5にて加水分解 する。  3) Hydrolyze chitosan 7B (manufactured by Katokichi Co., Ltd.) at pH 3.5 to pH 5.
4) グリコールキチンを、 pH3. 0乃至 ρΗΙ Ο. 5にて加水分解する。  4) Hydrolyze glycol chitin at pH 3.0 to Ο5.
5) 80°C以下で 3) 記載の加水分解活性を発揮する。  5) It exerts the hydrolysis activity described in 3) below 80 ° C.
6) 3) 記載の加水分解活性の最適 pHは pH 5. 5である。  6) 3) The optimum pH of the hydrolytic activity described is pH 5.5.
7) 4) 記載の加水分解活性の最適 pHは pHl 0. 0である。  7) 4) The optimum pH of the hydrolytic activity described is pH 10.0.
8) 3) 記載の加水分解活性の最適温度は pH 5. 5では 60°Cである。  8) 3) The optimum temperature for the hydrolysis activity described is 60 ° C at pH 5.5.
9) 45 °C以下の温度で安定である。  9) Stable at temperatures below 45 ° C.
10) pH5乃至 pH9. 5の pH条件下で安定である。  10) Stable under pH conditions of pH 5 to pH 9.5.
11) キトサン 7 Bに対する酵素の pH5. 5あるいは pH10. 0、 温度 37°Cに おける 10分間の加水分解活性を 100%としたときの、他の基質に対する酵素の p H5. 5あるいは PH10. 0、 温度 37 °Cにおける 10分間の加水分解活性が相対 値として表 1の値を示す。 表中、 キトサン 10Bは 1 %ァセチル化キチン、 キトサン 9 Bは 10 %ァセチル化キチン、 キトサン 8 Bは 20%ァセチル化キチン、 キトサン 7 Bは 30%ァセチル化キチンである （いずれも加ト吉 （株） 製） 。 CM—セル口一 スは、 カルポキシメチルセルロースナトリウムである （和光純薬社製） 。 グリコール キチンは、 前述の方法により調製する。  11) When the hydrolysis activity of the enzyme for chitosan 7 B at pH 5.5 or pH 10.0 and temperature 37 ° C. for 10 minutes is 100%, pH H5. The hydrolysis activity for 10 minutes at a temperature of 37 ° C. is shown as a relative value in Table 1. In the table, 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 (all are Tokichi Co., Ltd.) Made). The CM cell is sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.). Glycol chitin is prepared by the method described above.
[表 1] 相対活性 (%)  [Table 1] Relative activity (%)
PH5. 5 pHl 0. 0 キトサン 10 B 2. 3 0 PH5. 5 pHl 0. 0 Chitosan 10 B 2. 3 0
キトサン 9B 62. 8 29 キトサン 8B 83. 7 44. 6 Chitosan 9B 62. 8 29 Chitosan 8B 83. 7 44. 6
キトサン 7B 100 00 Chitosan 7B 100 00
グリコールキトサン 0 2 1 Glycol chitosan 0 2 1
グリコールキチン 28. 3 64 2 Glycol Chitin 28. 3 64 2
CM—セルロース 0 0 CM—cellulose 0 0
リケナン 0. 9 3 6 表 1から、 この酵素は、 pH5. 5においてはグリコールキチンよりもァセチル化 度が 10〜 30 %の部分ァセチル化キトサン （キトサン 9B、 8B、 7 B) に対する 加水分解活性が高く、 ァセチル化度が 1%以下のキトサン （キトサン 10B) に対す る加水分解活性がほとんどないことがわかる。力ルポキシメチルセル口一スナトリウ ムに対する加水分解活性はない。 Lichenan 0.93 6 From Table 1, at pH 5.5 this enzyme has a hydrolytic activity towards partially acetylated chitosan (chitosan 9B, 8B, 7 B) with an acetylation degree of 10 to 30% than glycol chitin It is high that there is almost no hydrolytic activity for chitosan (chitosan 10B) having a degree of degree of acetylation of 1% or less. There is no hydrolytic activity against sodium propoxymethylcellulose.
12) 下記に示す部分アミノ酸配列を有する。 配列は、 N末端側から記す。  12) It has a partial amino acid sequence shown below. The sequences are described from the N-terminal side.
Glu-Met-Thr-Pro-Tyr-Leu-Asp-Phe-Tyr-Arg-Leu-Met-Ala-Tyr-Gln (配列表の配列 番号 1) Glu-Met-Thr-Pro-Tyr-Leu-Asp-Phe-The-Tyr-Arg-Leu-Met-Ala-Tyr-Gln (Sequence Listing SEQ ID NO: 1)
11 e-Va 1 -Va 1 -G 1 y-Me t -P r o- 11 e-Ty r-G 1 y-Ar g-Ly s-G 1 u (配列表の配列番号 2 ) Ala-Leu-Pro-Lys-Pro-Gly-Ala-Thr-Glu-Tyr-Val-Asp-Pro-Ser-Ile [配列表の酉 S列 番号 3) ァスペルギルス ·ジャポニクス SANK 19288株により生産されるキチナ一 ゼは、 租酵素液において、 以下の性質を有する。  11 e-Va 1 -Va 1 -G 1 y-Me t -P r o 11 e-T y r G 1 y-Ar g -L y sG 1 u (Sequence Listing SEQ ID NO: 2) Ala-Leu-Pro-Lys -Pro-Gly-Ala-Thr-Glu-Tyr-Val-Asp-Pro-Ser-Ile [S-sequence No. 3 of the sequence listing 3) Aspergillus japonics The chitinase produced by strain SANK 19288 is In the following properties.
1) SDS-PAGE電気泳動法にて分子量約 40， 000を示す。  1) The molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
2) 等電点転記泳動法にて等電点 p I約 3. 5を示す。  2) Isoelectric point transcription The migration method shows an isoelectric point p I of about 3.5.
3) キトサン 7Bを、 pH3. 5乃至 ρΗΙ Ο. 5にて加水分解する。  3) Hydrolyze chitosan 7B at pH 3.5 to Ο5.
4) 3) 記載の加水分解活性の最適 pHは pH4. 0である。  4) 3) The optimum pH of the described hydrolytic activity is pH 4.0.
5) キトサン 7 Bに対する酵素の p H 4. 0、 温度 37 °Cにおける 1 0分間の加水分 解活性を 100%としたときの、 他の基質に対する酵素の pH 4. 0、 温度 37。 こ おける 10分間の加水分解活性が相対値として表 2の値を示す。  5) pH 4.0 of the enzyme for other substrates, temperature 37, assuming that the pH of the enzyme for chitosan 7 B is 100% and the hydrolysis activity for 10 minutes at 37 ° C. is 100%. The hydrolytic activity for 10 minutes in this case is shown as a relative value in Table 2.
[表 2] [Table 2]
相対活性 （％) キトサン 10 B 8 2 Relative activity (%) Chitosan 10 B 8 2
キトサン 9B 67 6 キトサン 8B 74. 4 Chitosan 9B 67 6 Chitosan 8B 74.4
キトサン 7B 100 Chitosan 7B 100
グリコールキトサン 8. 2 Glycol chitosan 8.2
グリコ一ルキチン 23. 3 Glycol ruchitin 23. 3
CM—セルロース 1 1. 4  CM—Cellulose 1 1. 4
リケナン 25. 3 Lickenan 25.3
表 2から、 この酵素は、 グリコールキチンよりもァセチル化度が 10〜 30%の部 分ァセチル化キトサン （キトサン 9B、 8B、 7 B) に対する加水分解活性が高く、 ァセチル化度が 1%以下のキトサン （キトサン 10B) に対する加水分解活性は弱い ことがわかる。 また、 ァスペルギルス ·ジャポニクス SANK 19288株により生産され、 精 製されたキチナーゼは、 以下の性質を有する。 From Table 2, 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. In addition, chitinase produced and purified by Aspergillus japonics strain SANK 19288 has the following properties.
1) SDS-P AGE電気泳動法にて分子量約 40, 000を示す。  1) The molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
2) 等電点転記泳動法にて等電点 p I約 3. 5を示す。  2) Isoelectric point transcription The migration method shows an isoelectric point p I of about 3.5.
3) キトサン 7Bを、 pH3. 5乃至 ρΗΙ Ο. 5にて加水分解する。  3) Hydrolyze chitosan 7B at pH 3.5 to Ο5.
4) 3) 記載の加水分解活性の最適 pHは pH 5. 0である。  4) 3) The optimum pH of the described hydrolytic activity is pH 5.0.
5) 3) 記載の加水分解活性の最適温度は pH 5. 0では 65°Cである。  5) 3) The optimum temperature for the hydrolysis activity described is 65 ° C at pH 5.0.
6) 50°C以下の温度で安定である。  6) Stable at temperatures below 50 ° C.
7) pH4乃至 pH9. 5の pH条件下で安定である。  7) Stable under pH conditions of pH 4 to pH 9.5.
8 ) キトサン 7 Bに対する酵素の p H 5. 0、 温度 37 °Cにおける 10分間の加水分 解活性を 100 %としたときの、 他の基質に対する酵素の p H 5. 0、 温度 37でに おける 10分間の加水分解活性が相対値として表 3の値示す。 表中、 キトサン 10B は 1%ァセチル化キチン、 キトサン 9 Bは 10%ァセチル化キチン、 キトサン 8 Bは 20%ァセチル化キチン、 キトサン 7 Bは 30 %ァセチル化キチンである (いずれも 加ト吉 (株) 製） CM—セルロースは、 カルポキシメチルセルロースナトリウムで ある （和光純薬社製） 。 グリコールキチンは、 前述の方法により調製する。  8) 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. In the table, 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 (all are Tokichi Co., Ltd.) CM-cellulose is sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.). Glycol chitin is prepared by the method described above.
[表 3] 相対活性 (%) キトサン 10 B 0  [Table 3] Relative activity (%) Chitosan 10 B 0
キトサン 9 B 58 6 Chitosan 9 B 58 6
キトサン 8 B 72 4 キトサン 7B 100 Chitosan 8 B 72 4 Chitosan 7B 100
ダルコ一ルキトサン 0 Dharco Chitosan 0
グリコールキチン 28 3 Glycol Chitin 28 3
CM—セルロース 0  CM—cellulose 0
リケナン 4 2 Lickenan 4 2
表 3に示すとおり、 ァスペルギルス ·ジャポニクス SANK 19288株由来 の精製キチナ一ゼはキトサン 7 Bに対して最も加水分解活性が高く、 ァセチル化度が 小さくなるにつれて加水分解活性は低下する。 また、 該精製キチナ一ゼはキチン加水 分解活性を有し、 セルラ一ゼ加水分解活性は無い。' As shown in Table 3, 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. In addition, the purified chitinase has chitin hydrolysis activity and has no cellular hydrolysis activity. '
9) 下記に示す部分アミノ酸配列を有する。 配列は、 N末端側から記す。 9) It has a partial amino acid sequence shown below. The sequences are described from the N-terminal side.
His-Tyr-Pro-Thr-Asp-Ser-Trp-Asn-Asp-Val-Gly-Thr-Asn-Val-Tyr (配列表の配列番 号 11) His-Tyr-Pro-Thr-Asp-Ser-Trp-Asn-Asp-Val-Gly-Thr-Asn-Val-Tyr (SEQ ID NO: 11)
Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (配列表の配列番 号 12)  Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (SEQ ID NO: 12)
Leu-Ser-Gln-Met-Thr-Pro-Tyr-Leu-Asp-P e-Tyr-Asn-Leu-Met-Ala-Tyr-Asp-Tyr-Ala -Gly (配列表の配列番号 13) · ァスペルギルス ·ソジヤエ SANK 22388株により生産されるキチナーゼ は、 粗酵素液において、 以下の性質を有する。  Leu-Ser-Gln-Met-Thr-Pro-Tyr-Leu-Asp-Pe-Tyr-Asn-Leu-Met-Ala-Tyr-Asp-Tyr-Ala-Gly (SEQ ID NO: 13) · Aspergillus · Chitinase produced by Sozyyaea strain SANK 22388 has the following properties in a crude enzyme solution.
1) SDS— PAGE電気泳動法にて分子量約 40, 000を示す。  1) The molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
2 ) 等電点電気泳動法にて p I約 4. 5を示す。 2) An isoelectric focusing method shows a p I of about 4.5.
3) キトサン 7 Bを、 pH3. 5乃至 pH9. 5にて加水分解する。  3) Hydrolyze chitosan 7 B at pH 3.5 to pH 9.5.
4) 3) 記載の加水分解活性の最適 pHは pH4. 5である。  4) 3) The optimum pH of the hydrolytic activity described is pH 4.5.
5) キトサン 7 Bに対する酵素の pH4. 5、 温度 37°Cにおける 10分間の加水分 解活性を 100%としたときの、 他の基質に対する酵素の pH4. 5、 温度 37 に おける 10分間の加水分解活性が相対値として表 4の値を示す。  5) When the hydrolysis activity for 10 minutes at a temperature of 37 ° C. is 100%, the hydrolysis for 10 minutes at pH 37, a temperature of 37 ° C., when the pH of the enzyme for chitosan 7 B is 100%. The decomposition activity shows the values in Table 4 as relative values.
[表 4]  [Table 4]
相対活性 （％) キトサン 10 B 0 Relative activity (%) Chitosan 10 B 0
キトサン 9 B 67 7 Chitosan 9 B 67 7
キトサン 8B 71 6 キ卜サン 7B 100 Chitosan 8B 71 6 Yellow 7B 100
グリコールキトサン 2. 9 Glycol chitosan 2.9
グリコールキチン 81. 9 Glycol Chitin 81.9
CM—セルロース 0  CM—cellulose 0
リケナン 11. 9 Lickenan 11. 9
表 4から、この酵素は、ァセチル化度が 10〜30%の部分ァセチル化キトサン（キ トサン 9B、 8B、 7 B).に対する加水分解活性が高く、 ァセチル化度が 1%以下の キトサン （キトサン 10B) に対する加水分解活性がほとんどないことがわかる。 力 ルポキシメチルセル口一スナトリゥムに対する加水分解活性はない。 また、 ァスペルギルス ·ソジヤエ SANK 22388株により生産され、 精製 されたキチナーゼは、 以下の性質を有する。 From Table 4, 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. In addition, chitinase produced and purified by Aspergillus sogyae strain SANK 22388 has the following properties.
1 ) SDS-P A G E電気泳動法にて分子量約 40, 000を示す。  1) It shows about 40,000 molecular weight by SDS-PAGE electrophoresis.
2) 等電点転記泳動法にて等電点 p I約 4. 5を示す。  2) Isoelectric point transcription The migration method shows an isoelectric point p I of about 4.5.
3) キトサン 7 Bを、 pH3. 5乃至： pH9. 5にて加水分解する。  3) Chitosan 7 B is hydrolyzed at pH 3.5 to pH 9.5.
4) 3) 記載の加水分解活性の最適 pHは pH 5. 5および pH9. 0である。 4) 3) The optimum pH of the described hydrolytic activity is pH 5.5 and pH 9.0.
5) 3) 記載の加水分解活性の最適温度は p H 5. 5では 60 °C、 pH9. 0では 3 5°Cである。 5) 3) The optimum temperature for the described hydrolytic activity is 60 ° C for pH 5.5 and 35 ° C for pH 9.0.
6) pH 5. 5および pH9. 0において、 40 °C以下の温度で安定である。  6) Stable at temperatures below 40 ° C at pH 5.5 and pH 9.0.
7) pH4. 5乃至 pHl 0の pH条件下で安定である。  7) Stable under pH conditions of pH4.5 to pH10.
8) キトサン 8 Bに対する酵素の pH 5. 0、 温度 37 °Cにおける 10分間の加水分 解活性を 100%としたときの、 他の基質に対する酵素の pH 5. 0、 温度 37でに おける 10分間の加水分解活性が相対値として表 5の値を示す。 表中、 キトサン 10 Bは 1 %ァセチル化キチン、 キトサン 9 Bは 10 %ァセチル化キチン、 キトサン 8 B は 20 %ァセチル化キチン、 キトサン 7 Bは 30 %ァセチル化キチンである （いずれ も加ト吉 （株） 製） 。 CM—セルロースは、 カルボキシメチルセルロースナトリウム である （和光純薬社製） 。 グリコールキチンは、 前述の方法により調製する。  8) Enzyme activity against other substrates at pH 5.0 at a temperature of 37 with a pH of 5.0 for the enzyme to chitosan 8 B and 100% hydrolysis activity for 10 minutes at a temperature of 37 ° C. The hydrolytic activity in minutes is shown in Table 5 as a relative value. In the table, 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.
[表 5] 相対活性 {%) キトサン 10B 14. 3 [Table 5] Relative activity {%) Chitosan 10B 14. 3
キトサン 9B 73. 6 キトサン 8B 100 Chitosan 9B 73.6 Chitosan 8B 100
キトサン 7B 97 4 Chitosan 7B 97 4
ダルコールキトサン 0 Dalcor Chitosan 0
グリコ一ルキチン 23 8 Glycol ruchitin 23 8
CM—セルロース 0  CM—cellulose 0
リケナン 0 Likenan 0
表 5に示すとおり、 ァスペルギルス 'ソジヤエ SANK 22388株由来の精 製キチナーゼはキトサン 7Bおよび 8Bに対して最も加水分解活性が高く、 ァセチル 化度が小さくなるにつれて加水分解活性は低下する。 また、 該精製キチナーゼはキチ ン加水分解活性を有し、 セルラ一ゼ加水分解活性はない。 . 以上のことから、本発明のキチナーゼの有する性質としては以下のようなものが挙 げられるが、 これに限定されるものではない。 As shown in Table 5, 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. In addition, 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.
1) SDS-PAGE電気泳動法にて分子量約 40， 000を示す。  1) The molecular weight is approximately 40,000 by SDS-PAGE electrophoresis.
2)等電点電気泳動法にて等電点 p I 3. 0乃至 5. 0 (好適には p I 3. 5乃至 4. 5) を示す。  2) 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).
3) キトサン 7Bを、 pH3. 0乃至 pHl l. 0 (好適には pH3. 5乃至 10. 5) にて加水分解する。  3) Chitosan 7B is hydrolyzed at pH 3.0 to pH 1.1 (preferably pH 3.5 to 10.5).
4) グリコ一ルキチンを、 pH3. 0乃至 pHl l. 5 (好適には pH4乃至 pH 1 0. 5) にて加水分解する； '  4) hydrolyzing glycolluctin at pH 3.0 to pH 1.5 (preferably pH 4 to pH 10.5);
5) 0°〇乃至80°〇で3) 記載の加水分解活性を発揮する；  5) exert the hydrolysis activity described in 3) at 0 ° 〇 to 80 °°;
6) 50°C以下 （好適には 45°C以下） の温度で安定である；  6) stable at temperatures below 50 ° C. (preferably below 45 ° C.);
7) pH4. 0乃至 pHI O (好適には pH5乃至 pH9. 5) の pH条件下で安定 である。  7) Stable under pH conditions of pH 4.0 to pHIO (preferably pH 5 to pH 9.5).
また、 本発明のキチナ一ゼを製造する方法も本発明に含まれる。  Also included in the present invention is a method of producing the chitinase of the present invention.
ァスペルギルス 'オリザェ var. sporoflavus Ohara J CM 2067株、 ァスペルギルス ·ジャポニクス S ANK 1 9 2 8 8株、 ァスペルギルス ·ソ ジヤエ S ANK 2 2 3 8 8株を初めとするキチナ一ゼ産生微生物を培地で培 養することにより、 キチナーゼを生産することができる。 例えば、 0. 1〜5. 0% マルトェクストラクト （Difco (株） 製） 、 0. 1〜1. 0%イーストエキストラク ト （Difco (株） 製） 、 0. 05〜1. 0%キチンあるいは 0. 05〜1. 0%キト サンの培地で、 16〜45°Cで 1〜15日間、 100〜250 rmpで振とう培養す る。  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. By doing this, chitinase can be produced. For example, 0.1 to 5.0% maltose extract (manufactured by Difco), 0.1 to 1.0% yeast extract (manufactured by Difco), 0. 05 to 1.0% chitin Alternatively, 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.
ァスペルギルス 'オリザェ var. sporoflavus Ohara J CM 2067株、 ァ スペルギルス ·ジャポニクス S A N K 1 9 2 8 8株、 ァスペルギルス ·ソジヤエ S A N K 2 2 3 8 8株由来の酵素は、 次の各点が共通である。 Aspergillus' Oryzae var. Sporoflavus Ohara J CM 2067 stock, The following enzymes are common to Spergilus japonicus strain SANK 1 928 and Aspergillus sogyae strain SANK 2 288.
1 ) 弱酸性で最も高い部分ァセチル化キトサン分解活性を有する。  1) It is weakly acidic and has the highest partially acetylated chitosan degradation activity.
2 ) 3 0 %ァセチル化キトサンに対する、 p H 4. 0〜6 . 0、 温度 3 7 °Cにおける 1 0〜3 0分間の加水分解活性を 1 0 0 %とすると、 1 %ァセチル化キトサンに対す る該活性は 1 5 %以下である。  2) Assuming that the hydrolysis activity for 10 to 30 minutes at 30 ° C acetylated chitosan, pH 4.0 to 6.0, temperature 37 ° C is 100%, 1% acetylated chitosan The activity against it is 15% or less.
本発明によって得られるキトサン低分子キトサン及び各種塩溶液は、そのまま用い ることもできるが、 硫安沈殿 ·有機溶媒沈殿 ·遠心分離 ·凍結乾燥 ·限外濾過等によ り、 濃縮することもできる。 また、 透析 ·限外濾過'ゲル濾過'カラムクロマトダラ フィ一等により精製と脱塩及び分子量分画をすることもできる。  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. In addition, purification, desalting and molecular weight fractionation can also be performed by dialysis, ultrafiltration, gel filtration, column chromatography, etc.
このようにして作製した低分子キトサン塩酸塩は、 水に対する溶解性に優れ、 最大 約 10質量％濃度の溶液とすることができる。また、低分子キトサン塩酸塩の 1質量％ 水溶液は、 p H 4. 9〜5 . 6を示し、 アルカリを添加した場合に沈殿物（コロイド） が析出する p Hは p H 6 . 6〜7 . 4である。  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. In addition, 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.
ここで作製した低分子キトサン塩酸塩が有する生理活性を、抗菌活性を指標として 調べる。 抗菌活性を有するための低分子キトサン 9 B、 8 B、 7 B各塩酸塩の最小阻 止濃度 （Minimum Inhibi tory Concentrat ion; MIC) を測定する。 高分子キトサン 塩酸塩の坊菌活性と比較すると、 グラム陽性菌'陰性菌にかかわらず、 低分子キトサ ン塩酸塩は高分子キトサン塩酸塩と同等以上の活性を有している。 したがって、 低分 子キトサンは高分子キトサンと同等以上の優れた生理活性を有している。 このことか ら高分子キトサンが有するとされている抗菌活性は、本発明の低分子キトサンにおい ても保持されているものと考えられる。また、皮膚欠損傷動物の治癒試験においても、 治癒日数の短縮効果がみられ、 動物における生理活性も有している .  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. In comparison with the Bacillus subtilis activity of high molecular weight chitosan hydrochloride, regardless of the gram-positive bacteria 'negative bacteria, 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. From this, it is considered that the antibacterial activity which is considered to be possessed by high molecular weight chitosan is also retained in the low molecular weight chitosan of the present invention. In addition, in the healing test of skin-deficient injured animals, the effect of shortening the healing days was also observed, and they also have physiological activity in animals.
このように優れた生理活性を有していることから、本発明で得られる低分子キトサ ンまたはその塩は、 例えば、 創傷 ·褥創用外用剤、 床ずれ治療薬、 アトピー性皮膚炎 治療薬、 二キビ治療薬等の皮膚外用剤、 入浴剤、 化粧料、 洗顔剤、 基礎化粧品、 虫歯 予防薬等の口腔衛生剤、 液状洗口剤、 歯科向けの素材、 腸内細菌改善薬等の整腸剤、 院内感染予防薬等の医療分野、 ダイエットフード、 食品保存剤、 防腐剤等の食品添加 物分野、 動物 ·魚類用飼料等の分野、 植物活性増強剤などの農業分野、 水等処理剤、 金属吸着剤、原子力廃棄物吸着剤薬剤除法剤等に本発明の低分子キトサンを使用する ことが考えられるが、 用途は特にこれらの例に限定されるものではない。 また本発明 の低分子キトサンまたはその塩の有効量を動物に投与することを含む、外傷、床ずれ， アトピー性皮膚炎等の疾患の治療方法も本発明に含まれる。 また、 これらの疾患を治 療するための本発明の低分子キトサンまたはその塩の使用も本発明に含まれるもの とする。 本発明によって得られる低分子キトサン又は低分子キトサン塩の用法は目的に応 じて変わるが、 上述のような用途に使用するには、 さまざまな形態をとり得る。 以下 に形態を列挙するが、 特に限定されるわけではない。 低分子キトサンは、 固体のまま 用いることができる。 また、 10質量％濃度の水溶液あるいは有機溶媒溶液、 さらに適 宜これを希釈して任意の濃度で用いることができる。 希釈剤は水あるいは有機溶媒Due to such excellent physiological activity, 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 Although it is conceivable to use 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. In addition, 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. Although 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. In addition, 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
(アルコール類など） 等を使用することができる。 最終的に pHが 6. 5より酸性であ れば溶液として、 アルカリ性であればコロイド状化合物として使用できるが、 塩の種 類によっては弱アルカリ性でも溶液として使用できる。 さらに、 無機物あるいは有機 物を共存させることにより、 任意の pHで溶液として用いることができる。 また、 低 分子キトサン溶液あるいは希釈溶液を 10質量％以下の任意の濃度においてクリーム 基材と混合することにより、 クリ一ム剤として使用することができる。低分子キトサ ン溶液あるいは希釈溶液を、任意の厚さを有するフィルムとして使用することができ る。 プラスチック様の硬さを有するフィルムは、 低分子キトサン溶液を、 さまざまな 厚さを有する枠の中で乾燥させることにより得られる。 また、 柔軟性を有する軟質シ ートは、 低分子キトサン溶液に例えばダリセリン等の溶剤を任意の割合で混合して、 さまざまな厚さを有する枠の中で乾燥させることにより得られる。柔軟性の度合いは、 例えばグリセリン等の溶剤を加える割合で調節される。 さらに、 キトサンーメチルセ ルロース複合フィルムは、 低分子キトサン溶液にメチルセルロースを混合し、 さまざ まな厚さを有する枠の中で乾燥させることにより得られる。 (Alcohols etc.) etc. can be used. Finally, if the 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. Furthermore, by coexisting an inorganic substance or an organic substance, it can be used as a solution at any pH. In addition, 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. In addition, 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. Furthermore, 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.
用量は、 疾患の種類、 症状、 患者の性別、 年令、 剤型、 投与法に応じて変わるが、 安全性が高いので、 任意の量を用いることができる。 生体に投与する場合には 2g/kg 体重以下が好ましく、 それ以外の場合には、 適宜調節される。 また、 本発明のキチナ一ゼは、 扱いにくい試料をより扱い易くする為にも有効に用 いる事ができる。 従来、 甲殻類などを含む試料を、 粉碎する， すり潰す、 ミキサーに かけるなどの加工をした場合、 粘度が非常に高くなり、 扱いにくかった。 これは、 こ れら甲殻類に大量に含まれる高分子多糖 （主成分は高分子部分ァセチル化キトサン） が原因であった。 たとえば、 このような食品加工の過程に、 本発明のキチナ一ゼを加 えて反応させ、 その液性成分中の高分子キトサンまたは高分子キチンを分癣し、 その 液性成分中に高分子部分ァセチル化キトサンを実質的に含まない、扱い易い試料を製 造することが可能であり、キチナ一ゼを用いた該扱い易い試料の製造方法も本発明に 含まれる。 このような製造方法に用いる原材料としては、 高分子部分ァセチル化キト サンを含む試料であれば特に限定されないが、好ましくは、甲殻類を含む試料であり， より好ましくはェビおよび力二のいずれか一つまたは両方を含む試料であ,る。原材料 とキチナ一ゼの反応条件としては上記のキトサン分解活性を発揮する条件を適用す ることができる。 反応時間は 1 0分以上であって、 試料の粘度が低い状態になる時間 であれば特に限定されないが、 好ましくは 10分間から 10日間程度であり、 より好 ましくは 1日間から 5日間程度である。 また、 このような方法によって製造された te 料も本発明に含まれる。 Although 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. When administered to a living body, 2 g / kg body weight or less is preferable, and in other cases it is adjusted appropriately. In addition, the chitinase of the present invention can be effectively used to make an intractable sample easier to handle. In the past, when processing samples such as crustaceans, grinding, grinding, and mixing in a mixer, 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. For example, 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. As 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. In addition, tes produced by such a method is also included in the present invention.
「その液性成分中に高分子部分ァセチル化キトサンを実質的に含まない」 とは、 試 料に高分子部分ァセチル化キトサンが含有される事によりもたらされる、高い粘性が 減少または消失した状態を指し、 厳密に濃度として 0%であることを指さないが、 好 ましくは高分子部分ァセチル化キトサンの含有量が 20%以下であり、より好ましく は 10%以下であり、 さらに好ましくは 5%以下であり、 最適には 0%である。  The phrase "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%.
[実施例] [Example]
以下に、 実施例及び試験例を挙げるが、 本発明の範囲はこれらに限定されるもので はない。  Examples and test examples are given below, but the scope of the present invention is not limited to these.
実施例 1. ァスペルギルス 'オリザェ var. sporoflavus Ohara J'CM 2 067株からのキチナーゼの精製  Example 1. Purification of a chitinase from Aspergillus sp. Oryzae var. Sporoflavus Ohara J'CM 2067
1) 粗酵素液の調製  1) Preparation of crude enzyme solution
滅菌した表 6の組成の培地 100mlが入っている 500ml容の三角フラスコ （種 フラスコ） にァスペルギルス 'オリザェ var. sporoflavus Ohara J CM 20 67株の菌体を接種し、 26°Cにて 8日間、 100 r pmの振とう培養を行った。 Inoculate cells of Aspergillus' Oryzae var. Sporoflavus Ohara J CM 20 67 strain into a 500 ml Erlenmeyer flask (seed flask) containing 100 ml of the culture medium having the composition shown in Table 6 for 8 days at 26 ° C. The shaking culture was performed at 100 rpm.
[表 6] [Table 6]
培地 マルトェクストラクト 10 g  Medium Malt Quent 10g
イースト ·ェクストラクト 5 g East Abstract 5 g
粉末キチン 2 g Powdered chitin 2 g
あるいは コロイダルキトサン （キミツ化学 （株） 製） 2 g Or Colloidal chitosan (Kimitsu Chemical Co., Ltd.) 2 g
純氷で 1, 000mlとした。 Made up to 1,000 ml with pure ice.
培養終了後、 4°C、 10， 000 XGにて 10分間の遠心分離を行った。 得られた上 清を粗酵素液とした。 After completion of the culture, centrifugation was performed at 10,000 XG for 10 minutes at 4 ° C. The obtained supernatant was used as a crude enzyme solution.
2) 酵素活性測定法  2) Enzyme activity measurement method
キチナーゼの加水分解活性は以下のようにして測定した。 The hydrolysis activity of chitinase was measured as follows.
①部分ァセチル化キ卜サンの加水分解反応  Hydrolysis reaction of one-part acetylated silica.
キトサン 7B (30%ァセチル化キトサン、 加ト吉 （株） 製） 125mgに水 50m 1および酢酸 30 lを加え、 粉末キトサンを完全に溶解させた。 キトサン 8B (2 0%ァセチル化キトサン、 加ト吉 （株） 製） 125mgに水 50mlおよび酢酸 50 lを加え、 粉末キトサンを完全に溶解させた。 キトサン 9 B (10%ァセチル化キ トサン、 加ト吉 （株） 製） 125mgに水 50m 1および酢酸 50 ^1を加え、 粉末 キトサンを完全に溶解させた。 キトサン 10B (ほぼ完全な脱ァセチル化キトサン、 加ト吉 （株） 製） 125mgに水 5 Om 1および酢酸 90 lを加え、 粉末キトサン を完全に溶解させた。 これら キトサン水溶液 160 1および 40 OmM酢酸緩衝 液 （PH5. 0) 100 1の混合液に酵素液 140 1を加え撹拌して均一にし、 37 °Cで保温して 20分間酵素反応を行った。 50 ml of water and 30 liters of acetic acid were added to 125 mg of 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. Water (50 ml) and acetic acid (50 ^ 1) were added to 125 mg of 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.
②ランドル ·モーガン法による遊離還元糖の定量 、  2 Determination of free reducing sugars by Rundle Morgan method
ァセチルァセトン 10 1を 0. 5 M炭酸ナトリゥム水溶液 500 1に溶かした溶 液 400 1を酵素反応液に加えて反応を停止させた後、 100°Cで 20分間保温し た。 氷冷後、 N, N-ジメチルァミノべンズアルデビド 0. 8 gをエタノール 30m 1及び濃塩酸 3 Omlに溶解した混合溶液 400 1とエタノール 1200 ^ 1を酵 素反応溶液に加え、 65°Cで 10分間発色反応をさせた。 氷冷後、 沈殿物を遠心分離 し上清の 530 nmにおける吸光度を測定した。酵素反応 1分間当たり 1 mo 1 のダルコサミンを生成する酵素活性を 1単位とした。 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.
3) 粗精製酵素液の調製  3) Preparation of crudely purified enzyme solution
1) で得られた粗酵素液 50 Omlに、 氷冷下攪拌しながら硫酸アンモニゥム 280 gを徐々に加えた。 混合溶液をー晚 4 °Cで静置したのち、 10 mMトリス ·塩酸緩衝 液 （PH7. 5) 3, 00 Om 1に対して 12時間ずつ 5回透析した。 これを、 予め 1 OmMトリス ·塩酸緩衝液（pH7. 5)で平衡化した D EAEトヨパール （東ソ一 (株） 製） カラム （直径 2. 2 (：111ズ長さ20 ( 111) に添加し、 吸着させた。 10m Mトリス ·塩酸緩衝液 （pH7. 5) で該カラム十分洗浄した後、 1 OmMトリス ' 塩酸緩衝液 （pH 7. 5) 600ml中に 0乃至 0. 4 Mの塩化ナトリウムの直線的 濃度勾配を作製して該カラムに吸着した成分を溶出させた。キトサン分解活性は塩化 ナトリウム濃度が 0. 20 乃至0. 30Mの画分 （135m 1) に溶出された。 こ れを粗精製酵素画分とした。  To 50 O ml of the crude enzyme solution obtained in 1), 280 g of ammonium sulfate was gradually added while stirring under ice-cooling. The mixed solution was allowed to stand at −4 ° C., and then dialyzed 5 times for 12 hours each against 10 mM Tris · HCl buffer solution (PH 7.5), 3, 00 Om 1. Add this to a D EAE Toyopearl (manufactured by Tosoh Co., Ltd.) column (diameter: 2.2 (: 111 length: 20 (111)) equilibrated with 1 OmM Tris · HCl buffer (pH 7.5) in advance. 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.
4) 精製酵素溶液の調製  4) Preparation of purified enzyme solution
3) で得られた活性画分 135 m 1を 2 OmMトリス ·塩酸緩衝液 （pH7. 5) 3, 000 m 1に対して 12時間ずつ 3回透析した後、 予め 20 mMトリス '塩酸緩 衝液 （PH7. 5) で平衡化した MonoQ (フアルマシア社製） カラム （直径 7m mX長さ 5 cm)に添加し、吸着させた。該カラムを 2 OmMトリス'塩酸緩衝液（p H7. 5) で十分洗浄した後、 1 OmMトリス ·塩酸緩衝液 （pH7. 5) 250m 1中に 0乃至 0. 15 Mの塩化ナトリウムの直線的濃度勾配を作製して該カラムに 吸着した成分を溶出させた。 キトサン分解活性は塩化ナトリゥム濃度が 0. 054M 乃至 0. 057Mの画分 （1 Om 1) に溶出された。 この  3) After dialysing the active fraction 135 m 1 obtained in 3 times against 2 O mM Tris · hydrochloric acid buffer (pH 7.5) and 3,000 m 1 for 12 hours three times, 20 mM Tris' hydrochloric acid buffer It was added to and adsorbed on a MonoQ (manufactured by Pharmacia) column (diameter 7 m m × length 5 cm) equilibrated with (PH 7.5). After thoroughly washing the column with 2OmM Tris'HCl buffer (pH 7.5), linear solutions of 0 to 0.15 M sodium chloride in 1OmM Tris · HCl buffer (pH 7.5) 250 ml A concentration gradient was created to elute the components adsorbed on the column. The chitosan decomposition activity was eluted in the fraction (1 Om 1) with sodium chloride concentration of 0.504 M to 0.57 M. this
画分を精製酵素溶液とした。 5) 精製酵素の分子量測定 The fraction was used as a purified enzyme solution. 5) Molecular weight measurement of purified enzyme
12. 5%ポリアクリルアミドゲルを用いた SDS— PAGE電気泳動法（Laemmli, U.K., Nature, 227, 680(1970)参照） により、 精製酵素の分子量を求めた。 標準 タンパク質として次のものを用いた： a. ホズホリラーゼ （phosphorylase) 、 分子 量 94, 000 ： b. アルブミン （albumin) 、 分子量 67， 000 ： c . ォバルブ ミン（ovalbumin)、分子量 43000： d.カルボニック'アンヒドラ一ゼ（carbonic anhydrase)、分子量 30， 000： e. トリプシン'インヒビ夕一（trypsin inhibitor), 分子量 20， 100 : f . ひーラクタルブミン （ -lactalbumin) 、 分子量 14, 4 00。  12. 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.
これら標準タンパクの移動度から求められた検量線を図 1に記載した。図 1に示す通 り、 精製酵素は分子量約 40, 000の単一バンドを示した。 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.
6) 精製酵素の等電点  6) Isoelectric point of purified enzyme
精製酵素を等電点電気泳動 （日本生化学会編 「生化学実験講座 1 タンパク質の化学 I 分離精製、 東京化学同人刊 （1976年） 」 262乃至 312頁参照） に供した。 精製酵 素の等電点は約 p 14. 5であった。 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.
7) 部分アミノ酸配列の決定  7) Partial amino acid sequence determination
精製レた酵素溶液を約 lmg/m l程度まで限外濾過膜 （ADVANTEC 社製 ULTRA FILTER UNIT USY_1、 分子量分画 1万） を用いて濃縮した。 濃縮酵素液 400 1 に Denature buffer (6M塩酸グァニジン、 1 OmM EDTA、 0. 1M炭酸水素アンモ ニゥム pH 7.8) 400 1および 5 OmM Di thiothrei tol 8 1を加え、 95°Cで 10分間反応させた。 室温まで冷却後、 反応液に Denature buffer溶液に溶解した 5 OmM Iodoacetoamide40 1を加え、 暗所室温で 1時間反応させた。 この溶液 を Slide- A- Lyzer Mini Dialysis Units Plus Float (PIERCE社製、 分子量分画 1 万） で水 1L に対して透析した。 得られた溶液にトリプシン （Modified, Promega 社製） 8 n 1を加え、 37°Cで 7 時間酵素反応させた。 反応液を SMART system (Pharmacia Biotech社製）にかけて分解アミノ酸のピークを分離した。分離条件を 以下に示す。  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.
カラム： Nomura Chemical Develosil 300 CDS-HG-5 (lmmX 150mm) Column: Nomura Chemical Developil 300 CDS-HG-5 (lmmX 150mm)
A buffer： 0.1¾TF A/Water  A buffer: 0.13⁄4 TF A / Water
B buffer： 0.1%TF A/Ace t oni tri 1  B buffer: 0.1% TF A / Ace t oni tri 1
グラジェント： 5→60%B 1%Z分. Gradient: 5 → 60% B 1% Z minutes.
Flow : 50 1 /ml  Flow: 50 1 / ml
分離した分解アミノ酸のうち、 B buffer 濃度 15 %程度の 2 つのピークおよび 3 5 %程度のピークを分取した。 この 3つ （No. ：！〜 No. 3) についてアミノ酸配 列解析装置 （Procise cIX、 Applied Biosystem社製） でアミノ酸配列を解析した。 その結果得られた部分ァミノ酸配列をァミノ末端側から記す。 No. 1 ： Glu Met Thr Pro Tyr Leu Asp Phe Tyr Arg Leu Met Ala Tyr Gin (配列表の配列番号 1) ； Among the separated degraded amino acids, two peaks of B buffer concentration of about 15% and peaks of about 35% were separated. The amino acid sequences of the three (No .:! To No. 3) were analyzed with an amino acid sequence analyzer (Procedice IX, manufactured by Applied Biosystem). The partial amino acid sequence obtained as a result is described from the amino terminal side. No. 1: Glu Met Thr Pro Tyr Leu Asp Phe Tyr Arg Leu Met Ala Tyr Gin (SEQ ID NO: 1);
No, 2 ： He Val Val Gly Met Pro lie Tyr Gly Arg Lys Glu (配列表 の配列番号 2) ；  No, 2: He Val Val Gly Met Pro lie Tyr Gly Arg Lys Glu (SEQ ID NO: 2);
No. 3 ： Ala Leu Pro Lys Pro Gly Ala Thr Glu Tyr Val Asp Pro No. 3: Ala Leu Pro Lys Pro Gly Ala Thr Glu Tyr Tyr Asp Pro
Ser lie (配列表の配列番号 3) 。 Ser lie (SEQ ID NO: 3).
' 実施例 2. ァスペルギルス 'ジャポニクス SANK 19288株からのキチナ ーゼの精製  'Example 2. Purification of chitinase from Aspergillus sp. Japonics strain SANK 19288
1) 粗酵素液の調製  1) Preparation of crude enzyme solution
滅菌した表 7の組成の培地 100mlが入っている 500ml容の三角フラスコ （種 フラスコ） にァスペルギルス ·ジャポニクス SANK 19288株の菌体を接種 し、 26 °Cにて 8日間、 100 r pmの振とう培養を行った。 培養終了後、 4°C条件 下、 10, 000 XGにて 10分間の遠心分離を行った。 得られた上清を粗酵素液と した。 Inoculate cells of Aspergillus japonicus strain SANK 19288 into a 500 ml Erlenmeyer flask (seed flask) containing 100 ml of the culture medium having the composition shown in Table 7, and shake at 100 rpm for 8 days at 26 ° C. Culture was performed. After completion of the culture, centrifugation was carried out at 10,000 XG for 10 minutes under 4 ° C. conditions. The obtained supernatant was used as a crude enzyme solution.
[表 7]  [Table 7]
培地 マルトェクストラクト 10 g  Medium Malt Quent 10g
イースト ·ェクストラクト 5 g East Abstract 5 g
粉末キチン 5 g Powdered chitin 5 g
純水で 1, 000mlとした。 The volume was made up to 1,000 ml with pure water.
2) 精製酵素溶液の調製 2) Preparation of purified enzyme solution
1) で得られた粗酵素液 200m 1に、 氷冷下攪拌しながら硫酸アンモニゥム 112 gを徐々に加えた。 混合溶液を一晩 4°Cで静置したのち、 20 mMトリス ·塩酸緩衝 液 （PH7. 5) 3， 000m 1に対して 12時間ずつ 5回透析し、 これを粗精製酵 素画分とした。 予め 20 mMトリス ·塩酸緩衝液 （ p H 7. 5) で平衡化した M o n oQ (フアルマシア社製） カラム （直径 7mmX長さ 5 cm) に粗精製酵素画分を添 加し、 吸着させた。 該カラムを 2 OmMトリス ·塩酸緩衝液 （pH7. 5) で十分洗 浄した後、 1 OmMトリス ·塩酸緩衝液 （pH 7. 5) 250ml中に 0乃至 0. 1 5 Mの塩化ナトリゥムの直線的濃度勾配を作製して該カラムに吸着した成分を溶 出させた。 キトサン分解活性は塩化ナトリゥム濃度が 0. 063 M乃至 0. 066 M の画分 （10ml) に溶出された。 この画分を精製酵素溶液とした。  To 200 ml of the crude enzyme solution obtained in 1), 112 g of ammonium sulfate was gradually added while stirring under ice-cooling. The mixed solution is allowed to stand overnight at 4 ° C., and then dialyzed 5 times for 12 hours each against 20 mM Tris · HCl buffer (PH 7.5) and 3,000 ml, and this is combined with the crude purified enzyme fraction and did. The crude enzyme fraction was added to a MonoQ (manufactured by Pharmacia) column (diameter 7 mm × length 5 cm) equilibrated with 20 mM Tris · HCl buffer solution (pH 7.5) and adsorbed. . After thoroughly washing the column with 2 OmM Tris · HCl buffer solution (pH 7.5), a straight line of 0 to 0.1 M sodium chloride in 250 ml of 1 OmM Tris · hydrochloric acid buffer solution (pH 7.5) A concentration gradient was created to elute the components adsorbed on the column. The chitosan decomposition activity was eluted in a fraction (10 ml) of sodium chloride at a concentration of 0.63 M to 0. 066 M. This fraction was used as a purified enzyme solution.
3) 精製酵素の分子量  3) Molecular weight of purified enzyme
実施例 1 5) と同じ方法で分子量を測定した。 本酵素は分子量約 40， 000の単 一バンドを示した。 4) 精製酵素の等電点 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. 4) Isoelectric point of purified enzyme
実施例 1 6) と同じ方法で等電点を測定した。 本酵素の等電点は約！） I 3. 5であ つた。 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.
実施例 3. ァスペルギルス ·ソジヤエ SANK 2238, 8株からのキチナーゼ の精製 」  Example 3. Purification of chitinase from Aspergillus spp. SANK 2238 strain 8 "
1) 粗酵素液の調製 1) Preparation of crude enzyme solution
滅菌した表 8の組成の培地 100mlが入っている 500m 1容の三角フラスコ （種 フラスコ） にァスペルギルス 'ソジヤエ SANK 22388株の菌体を接種し、 26 にて 8日間、 100 r pmの振とう培養を行った。 培養終了後、 4°C条件下、 10, 000 X Gにて 10分間の遠心分離を行った。得られた上清を粗酵素液とした。 Inoculate cells of Aspergillus' Sojiyae strain SANK 22388 into a 500-m 1-volume Erlenmeyer flask (seed flask) containing 100 ml of the culture medium having the composition shown in Table 8, and shake culture at 100 rpm for 26 days at 26 days. Did. After completion of the culture, centrifugation was carried out at 10,000 × G for 10 minutes at 4 ° C. The obtained supernatant was used as a crude enzyme solution.
[表 8] [Table 8]
培地 卜 10 g  Medium 卜 10 g
イースト ·ェクストラクト 5 g East Abstract 5 g
粉末キチン 5 g Powdered chitin 5 g
純水で 1, 000mlとした。 The volume was made up to 1,000 ml with pure water.
2) 精製酵素溶液の調製 2) Preparation of purified enzyme solution
1) で得られた粗酵素液 20 Om 1に、 氷冷下攪拌しながら硫酸アンモニゥム 1 12 gを徐々に加えた。 混合溶液をー晚 4°Cで静置したのち、 20mMトリス ·塩酸緩衝 液 （pH7. 5) 3, 00 Omlに対して 12時間ずつ 5回透析し、 これを粗精製酵 素画分とした。 予め 20 mMトリス■塩酸緩衝液 （p H 7. 5) で平衡化した M o n oQ (フアルマシア社製） カラム （直径 7mmX長さ 5 cm) に粗精製酵素画分を添 加し、 吸着させた。 該カラムを 2 OmMトリス ·塩酸緩衝液 （pH7. 5) で十分洗 浄した後、 1 OmMトリス '塩酸緩衝液 （pH 7. 5) 25 Oml中に 0乃至 0. 1 5 Mの塩化ナトリゥムの直線的濃度勾配を作製して該カラムに吸着した成分を溶 出させた。 キトサン分解活性は塩化ナトリウム濃度が 0. 0621^乃至0. 064M の画分 （10ml) に溶出された。 この画分を精製酵素溶液とした。  To the crude enzyme solution 20 Om 1 obtained in 1), 12 g of ammonium sulfate 1 was gradually added while stirring under ice-cooling. The mixed solution was allowed to stand at 4 ° C. and dialyzed 5 times for 12 hours each against 20 mM Tris · hydrochloric acid buffer (pH 7.5) 1.00 Oml to obtain a crudely purified enzyme fraction. . The crude enzyme fraction was added to and adsorbed onto a MonoQ (manufactured by Pharmacia) column (diameter 7 mm × length 5 cm) equilibrated with 20 mM Tris · HCl buffer (pH 7.5) in advance. . 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.
3) 精製酵素の分子量 3) Molecular weight of purified enzyme
実施例 1 5) と同じ方法で分子量を測定した。 本酵素は分子量約 40， 000の単 一バンドを示した。 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.
4) 精製酵素酵素の等電点 実施例 1 6) と同じ方法で等電点を測定した。 本酵素の等電点は約 p I 4. 5であ つた。 実施例 4. ァスペルギルス 'オリザェ var. sporoflavus Ohara J CM 2067株由来キチナーゼを用いた低分子キトサン塩酸塩の調製 4) Isoelectric point of purified enzyme The isoelectric point was measured in the same manner as in Example 1 6). The isoelectric point of this enzyme was about p I 4.5. Example 4 Preparation of low molecular weight chitosan hydrochloride using chitinase derived from Aspergillus' oryzae var. Sporoflavus Ohara J CM 2067 strain
キトサン 9 Bあるいはキトサン 8 Bあるいはキトサン 7 B、 2. 08を水800 m 1に懸濁し、 濃塩酸を加えて PH2. 5として粉末を完全に溶解した。 飽和炭酸水素 ナトリウム水で pHを 5. 0に合わせた後、水を加えて全量を 1, 000 mlとし、 200 mlずつを 500 m 1三角フラスコに移し、 実施例 1 1) で調製した酵 素液 10mlを加え、 40°Cで 3日間 80 r pmで振とうして酵素反応を行わせた。 反応終了後、 全ての反応液を回収し、 氷冷下 1%苛性ソーダ水で pHl 2以上とした のち、 10, 000 r pm、 10分間の遠心分離で沈殿物を得た。 回収した沈殿物を 200mlの水に懸濁し、 氷冷下濃塩酸を加えて pH 2. 5として溶解し、 分子量分 画 10， 000の透析膜を用いて、 水に対して透析を行なった。 透析終了後、 溶液を 濾過し、 濾液を凍結乾燥した。 低分子キトサン 9 B塩酸塩は、 2. 0 gの白色綿状 固体として得られた。 低分子キトサン 8 B塩酸塩が 1. 6 gの白色綿状固体として 得られた。 低分子キトサン 7 B塩酸塩が 0. 6 gの白色綿状固体として得られた。 実施例 5. ァスペルギルス ·ジャポニクス SANK 19288株由来キチナ ーゼを用いた低分子キトサン塩酸塩の調製 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. After completion of the reaction, all the reaction solution was recovered, adjusted to pH 2 or higher with 1% sodium hydroxide aqueous solution under ice-cooling, and then centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The collected precipitate was suspended in 200 ml of water, concentrated hydrochloric acid was added under ice cooling to dissolve it to pH 2.5, and dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 100,000. After dialysis, the solution was filtered and the filtrate was lyophilized. Low molecular weight chitosan 9 B hydrochloride was obtained as 2.0 g of a white fluffy solid. 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
実施例 4と同様の方法で低分子キトサン塩酸塩を調製した。 酵素'液は実施例 2 1) で得られたものを用い、 酵素反応は pH4. 0で行った。 低分子キトサン 9 B塩酸塩 は、 1. 3 gの白色綿状固体として得られた。 低分子キトサン 8B塩酸塩が 1. 3 g の白色綿状固体として得られた。 低分子キトサン 7 B塩酸塩が 0. 9 gの白色綿状固 体として得られた。 ' 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. '
実施例 6. ァスペルギルス ·ソジヤエ SANK 22388株由来キチナーゼ を用いた低分子キトサン塩酸塩の調製  Example 6 Preparation of low molecular weight chitosan hydrochloride using chitinase derived from Aspergillus sogyae strain SANK 22388
実施例 4と同様の方法で低分子キトサン塩酸塩を調整した。但し、 酵素液は実施例 3 で調整したものを用い、 酵素反応は pH4. 5で行った。 低分子キトサン 9 B塩酸塩 は、 2. 1 gの'白色綿状固体として得られた。 低分子キトサン 8B塩酸塩が 1. 7 g の白色綿状固体として得られた。 低分子キトサン 7 B塩酸塩が 1. 0 gの白色綿状固 体として得られた。 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.
実施例 7. 低分子キトサン塩酸塩のァセチル化度  Example 7. Degree of acetylation of low molecular weight chitosan hydrochloride
実施例 4〜6にて調製した低分子キトサン塩酸塩 10 Omgを蒸留水 10mlに溶 解し、 キトサナーゼ 10U (和光純薬 （株） 製） を加えて室温で 3時間攪拌した。 反 応溶液を凍結乾燥し、 乾燥固体を D20に溶かして1 H— NMRを測定した。 10 mg of low molecular weight chitosan hydrochloride prepared in Examples 4 to 6 was dissolved in 10 ml of distilled water, 10 U of chitosanase (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was lyophilized, the dry solids was measured 1 H- NMR dissolved in D 2 0.
ァセチル基の 3 Hとァセチル基以外の、.水酸基を除く 7 Hとの積分比から、 ァセチル 化度を算出した。 結果は表 9に示す。 From the integral ratio of the 3 H of the acetyl group to the 7 H other than the acetyl group except. The degree of conversion was calculated. The results are shown in Table 9.
[表 9] ァセチル化度 （％) 実施例 4 実施例 5 実施例 6 低分子キトサン 9 B塩酸塩 10. 7 12. 8 12. 7 [Table 9] Degree of acetylation (%) Example 4 Example 5 Example 6 Low Molecular Weight Chitosan 9 B Hydrochloride 10. 7 12. 8 12. 7
低分子キトサン 8 B塩酸塩 16. 4 20. 1 20. 1 Low molecular weight chitosan 8 B hydrochloride 16. 4 20. 1 20. 1
低分子キトサン 7 B塩酸塩 20. 0 29. 7 30. 4 Low molecular weight chitosan 7 B hydrochloride 20. 0 29. 7 30. 4
実施例 8. ゲル濾過法での分子量分画の決定 Example 8. Determination of molecular weight fraction by gel filtration
分子量マーカーとして、 デキストラン T 10 (分子量 10, 000) 、 T40 (分子 量 40， 000) 、 T 70 (分子量 70, 000) 、 T110 (分子量 110, 00 0) 、 Τ250 (分子量 250, 000) (フアルマシア （株） 製） の各 1 %溶液 2 00 1を、 あらかじめ ΡΗ4. 5の 20 OmM酢酸緩衝液で膨潤させたゲル濾過材As molecular weight markers, dextran T10 (molecular weight 10,000), T40 (molecular weight 40,000), T70 (molecular weight 70,000), T110 (molecular weight 110, 000 0), Τ 250 (molecular weight 250, 000) (harmacia Gel filter medium in which each 1% solution 2001 (manufactured by Co., Ltd.) was swelled with 20 mM mM acetate buffer in advance.
(Toyopearl HW55 Fine) をつめたカラム （86X2 cm) に展開し、 1. 75m 1ずつに分画した。水 1容に濃硫酸 8容を加えた硫酸液 1. 8 m 1、力ルバゾ一ル 0. 25 gをエタノール 50m 1に溶かした溶液 60 n 1を各々の分画液 200 1に 加え、 100°Cで 10分間保温した。 氷冷後 420 nmにおける吸光度を測定した。 実施例 4、 5、 6で作製した低分子キトサン塩酸塩の各 5%溶液 200 ^ 1を、 同 様にして 1. 75m 1ずつに分画した。 各分画 280 1に pH6. 5の 40 OmM MOP S一炭酸ナトリゥム緩衝液 100 1およびキトサナ一ゼ 10 U (和光純薬The column (86 × 2 cm) packed with (Toyopearl HW55 Fine) was developed and fractionated into 1. 75 m 1 aliquots. Add 1 volume of water, 1 volume of concentrated sulfuric acid, 8 volumes of sulfuric acid solution 1. 8 m 1 solution, 0.25 g of force rubazol in 50 mL of ethanol 60 n 1 solution is added to each fraction 200 1 The mixture was kept at ° C for 10 minutes. After ice cooling, the absorbance at 420 nm was measured. Each 5% solution 200 ^ 1 of the low molecular weight chitosan hydrochloride prepared in Examples 4, 5 and 6 was similarly fractionated into 1. 75 ml. Each fraction 280 1 to 40 OmM MOP S sodium bicarbonate monocarbonate buffer 100 1 and Chitosanase 10 U (Wako Pure Chemical Industries, Ltd.)
(株） 製） を加えて、 37°Cで 10分間酵素反応を行なった。 遊離還元糖の定量は実 施例 1にしたがった。 分子量マーカ一と各低分子キトサンについて、 最大吸光度を示 す分画を 100%とし同一グラフ上にプロットした（図 2〜4)。図から推定した低分 子キトサンの分子量を表 10にまとめた。 Co., Ltd.) was added, and the enzyme reaction was carried out at 37 ° C. for 10 minutes. The quantification of free reducing sugars was according to Example 1. The fraction showing the maximum absorbance for the molecular weight marker 1 and each low molecular weight chitosan was 100% and plotted on the same graph (FIGS. 2 to 4). The molecular weight of low molecular weight chitosan estimated from the figure is summarized in Table 10.
[表 10] 分子 実施例 4 実施例 5 実施例 6 低分子キトサン 9B塩酸塩 4万一 11万 4万一 11万 1万一 25万 低分子キトサン 8B塩酸塩 1万 _3万 ' 4万 4万一 11万 低分子キトサン 7B塩酸塩 1万 4万 7万 [Table 10] Molecule Example 4 Example 5 Example 6 Low Molecular Weight Chitosan 9B Hydrochloride 40,101,110,110,120 15,000 Low Molecular Weight Chitosan 8B Hydrochloride 10,000, 30,000, 40,000 1 110,000 Low-molecular-weight chitosan 7B hydrochloride 10,40,000
実施例 9. ァスペルギルス 'オリザェ（Aspergillus oryzae)由来のキチナ一ゼを コードするゲノム DN Aの分離 Example 9. Isolation of genomic DNA encoding chitinase from Aspergillus' oryzae (Aspergillus oryzae)
9- 1) ァスペルギルス ·オリザェ（Aspergillus oryzae)のゲノム DNAの分離 ァスペリレギ Jレス -才1 Jザ： £ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2067株を培地 （1% Mali Extract, 0.5% Yeast Extract) 100mlに て 30°Cで 4日間振盪培養した後、 この培養液をろ過装置を用い、菌体を集菌した。続 いて、 この菌体を -80°Cに冷却した乳鉢に入れ、 液体窒素を注いだ。 液体窒素を加え ながら乳棒を用いて菌体が粉末状になるまで十分碎いた。 粉末状の菌体約 1.5g を 20mlの溶菌バッファー（62.5mM EDTA、 50mM Tris-HCK 50mM SDS : pH8.0)に加え、 穏やかに攪拌した。 次に、 これに 10mlの 9.5M 酢酸アンモニゥムを添加し、 さらに 15mlの TE飽和フエノールを添加し穏やかに攪拌した。この溶液を高速冷却遠心機（日 立製ローター No.3)を用い、 4°C， 5000卬 mで 5分間遠心し、上清を約 25ml回収した。 回収した上清に 15mlのクロ口ホルムを添加し、 穏やかに攪拌した後、 4°C， 5000rpm で 10分間遠心した。 上清を新しいチューブに回収し、 上清と等量の 2-プロパノール を添加し攪拌した後、 4°C， lOOOOrpmで 10分間遠心した。 沈殿を 400 1の TEに溶 解した後、 1mlのマイクロチュ一ブに移し、 10 Iの lOmg/ml リボヌクレア一ゼを添 加し 37。Cで 20分処理した。 次いで、 40 1の 3M 酢酸ナトリウム、 400 l クロ口 ホルムを添加し攪拌し、 室温で lOOOOrpm, 5分間遠心分離した。 分離した上清をマ イク口チューブに回収し、 1mlの 100%エタノール （- 20°Cで冷却） を添加して穏やか に攪拌した。 ガラス棒で糸状になった DNAを巻き取り、 エタノールをきつた後に 200 lの TEに溶解させた。 9- 1) Isolation of Genomic DNA of Aspergillus oryzae (Aspergillus oryzae) Aspergillus sp. J Les-Age 1 J: £ (Aspergillus oryzae) var. Sporof lavus Ohara J CM 2067 medium (1% Mali Extract, 0.5% Yeast) Extract) After shaking culture at 30 ° C. for 4 days in 100 ml, cells were collected from the culture using a filtration apparatus. Subsequently, the cells were placed in a mortar cooled to -80 ° C and liquid nitrogen was poured. Using a pestle while adding liquid nitrogen, the cells were thoroughly ground until powdery. About 1.5 g of powdered cells was added to 20 ml of lysis buffer (62.5 mM EDTA, 50 mM Tris-HCK 50 mM SDS: pH 8.0) and gently stirred. Next, 10 ml of 9.5 M ammonium acetate was added to this, and another 15 ml of TE saturated phenol was added and gently stirred. This solution was centrifuged for 5 minutes at 4 ° C. and 5000 μm using a high-speed cooling centrifuge (Rotor No. 3 manufactured by Nichi-sha), and about 25 ml of the supernatant was recovered. To the collected supernatant, 15 ml of chloroform was added, gently stirred, and then centrifuged at 5000 rpm for 10 minutes at 4 ° C. The supernatant was collected in a new tube, and after adding and stirring an equal volume of 2-propanol as the supernatant, it was centrifuged at 4 ° C, lOOOOrpm for 10 minutes. The precipitate is dissolved in 400 1 TE and transferred to a 1 ml microtube, and 10 I of lOmg / ml ribonuclease is added 37. Treated with C for 20 minutes. Next, 40 1 of 3 M sodium acetate, 400 l chloroform was added and stirred, and the mixture was centrifuged at room temperature for 5 minutes with lOOOO rpm. The separated supernatant was collected in a microtube, 1 ml of 100% ethanol (cooled at -20 ° C) was added and gently stirred. The threading DNA was taken up with a glass rod, and after ethanol was fixed, it was dissolved in 200 l of TE.
9-2) ァスペルギルス ·オリザェ ·ゲノム DNAライブラリーの作製 9-2) Aspergillus oryzae · Preparation of genomic DNA library
9- 1)で得られたァスペルギルス ·オリザェ ·ゲノム DNA10 gを、制限酵素 EcoRI (宝酒造社製) 10// 1を用い、 50mM Tris-HCl (ρΗ7·5)、 10mM MgCl2、 ImM DTT及び lOOmM NaClからなる溶液中で、 37°Cで約 2時間切断処理した後、 DNAを回収した。 ァスペルギルス ·オリザェ ·ゲノム MAの EcoRI切断産物と、 同じく EcoRIで切断さ れたべクタ一、 PUC118 EcoRI/BAP (宝酒造社製） を Rapid DNA Ligation Kit (口 シュ ·ダイァグノスティック社製）を用いてライゲ一シヨンを行い、ァスペルギルス ' オリザェのゲノム DNAライブラリ一とした。 9-1) using the restriction enzyme EcoRI (manufactured by Takara Shuzo) 10 // 1, 50 mM Tris-HCl (Η 7 · 5), 10 mM MgCl 2 , ImM DTT and 100 mM After digestion for about 2 hours at 37 ° C. in a solution consisting of NaCl, DNA was recovered. The EcoRI digested product of Aspergillus oryzae genome MA, and the vector digested with EcoRI, PUC118 EcoRI / BAP (Takara Shuzo), using the Rapid DNA Ligation Kit (Gaus d'Aignostic) Perform a Leigation series and make it the Aspergillus' Oryzae genomic DNA library.
9-3) DIG (ジゴキシゲニン） 標識 DNAプローブの作製 9-3) Preparation of DIG (digoxigenin) labeled DNA probe
実施例 1で同定した内部アミノ酸配列 （配列表の配列番号 2) を元に作製したミツ クスプライマ一 F21、 および F21 を用いてァスペルギルス 'オリザェのゲノム'ライブ ラリーから同定したオリゴプライマー PR50を合成した。 A Mitsu produced based on the internal amino acid sequence (SEQ ID NO: 2 in the sequence listing) identified in Example 1. Oligoprimer PR50, which was identified from the Aspergillus 'oryzae genome' library, was synthesized using the primers C21 and F21.
F21 : 5' -attggcataccaacaactatcttagagc-3' (配列表の配列番号 7)  F21: 5'-attggcataccaacaactatcttagagc-3 '(SEQ ID NO: 7)
P 50： 5' -taaattgacccatatcctctatgcatt-3' (配列表の配列番号 8)  P 50: 5'-taaattgacccatatcctctatgcatt-3 '(SEQ ID NO: 8 in the sequence listing)
この 2種類のプライマーを用いて、 ァスペルギルス ·オリザェのゲノム DNAをテン プレ一トとした PCRにより、.約 800bpの Aを増やした。 この DNAは電気泳動により 分離し、 ァガ口一スゲルから切り出した後、 QIAquick Gel Extraction Kit (キ ァゲン社製） を用いて精製した。 こうして精製した DNAを DIG DNA Labeling Kit Using these two types of primers, 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
(ロシュ ·ダイァグノスティック社製） を用いて標識した。 標識の方法は以下の様に 行った。 まず、 精製した DNAを 100°Cで 10分間加熱し、 1本鎖にした後、 氷上で急冷 した。 次いで IO I へキサヌクレオチド 'ミックス（0.5M Tris、 0.1M MgCl2、 ImM DithioerithritoK 2mg/ml BSA； pH7.2), 10^1 dNTPラベリング 'ミックス（ImM dATP, ImM dCTP, ImM dGTP, 0.65mM dTTP, 0.35mM DIG - 11- dUTP; pH7.5)、 5^1 DNAポリメラーゼ (Kl enow fragment)を加え、滅菌水で全量が 100 1になるよ うに調整した。 この反応液を 37°Cでー晚反応させた後、 10 1 0.2M EDTA(pH8.0) を添加して反応を停止させた。 次いで、 10 1 N LiClと 500 1 100%エタノール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
(- 20°Cで冷却させたもの） を加え、 _70°Cで 30分間静置し、 標識された DNAを沈殿 させた。 冷却遠心機で 4°C、 15000rpm、 10分間遠心した。 エタノールを捨て、 100 /1 70%エタノール（- 20°Cで冷却したもの）を添加してペレツトを洗浄した後、 15000rpm、 10分間遠心し、 ピペットで完全にエタノールを除去した。 ペレットを乾燥した後、 50 li\ TEに溶解し、 25ml DIG Easy Hyb (ロシュ 'ダイァグノスティック社製） に 加え、 キチナーゼ遺伝子プローブとした。 (The one cooled at -20.degree. C.) was added and allowed to stand at _70.degree. C. for 30 minutes to precipitate labeled DNA. The mixture was centrifuged at 15000 rpm for 10 minutes at 4 ° C. in a refrigerated centrifuge. After discarding the ethanol and adding 100/170% ethanol (cooled at -20 ° C) to wash the pellet, it was centrifuged at 15000 rpm for 10 minutes, and the ethanol was completely removed with a pipette. After the pellet was dried, it was dissolved in 50 li \ TE and added to 25 ml DIG Easy Hyb (Roche 'Dagnoistics Co., Ltd.) to make a chitinase gene probe.
9-4) キチナ一ゼ遺伝子を含む部分 DNAライブラリ一の作製 9-4) Preparation of partial DNA library containing chitinase gene
9— 1 )で得られたァスペルギルス'オリザェ ·ゲノム DNA 20 gを制限酵素 Ec o l で切断処理を行った後、 精製し DNAを回収した。 精製した DNAをァガロースゲルで電 気泳動により分離し、 ナイロンメンブレンにブロッテイングを行った。次いで、 9— 3) で得られたキチナ一ゼ遺伝子プローブを用い、 サザンハイブリダィゼーシヨンを 行った結果、 キチナ一ゼ遺伝子を含む一本の単一のバンドが得られた。 そこで、 次に 9— 1) で得られたァスペルギルス ·オリザェ*ゲノム DNA 80 gを制限酵素 EcoRI で切断処理を行った後、 精製し DNAを回収した。 この DNAをァガロースゲルで電気泳 動し、上記のサザンハイブリダイゼ一シヨンによって見られたバンドの位置と同じ位 置をゲルから切り出し、 DNAを抽出した。 抽出した DNAを PUC118 EcoRI/BAPベクタ —にライゲ一ションを行い、 キチナ一ゼ遺伝子を含む DNAライブラリーとした。  After 20 g of Aspergillus sp. Oryzae genomic DNA obtained in 9-1) was cleaved with restriction enzyme Ecol, it was purified and the DNA was recovered. The purified DNA was separated by electrophoresis on a agarose gel and blotted on a nylon membrane. Subsequently, Southern hybridization was performed using the chitinase gene probe obtained in 9-3). As a result, a single single band containing the chitinase gene was obtained. Therefore, 80 g of Aspergillus oryzae * genomic DNA obtained in 9-1) was cleaved with restriction enzyme EcoRI and then purified to recover the DNA. 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.
9- 5) キチナ一ゼ遺伝子含有 DNAのスクリーニング 9 -4) で得た DNAライブラリ一により、 大腸菌 JM109株 （宝酒造社製） を形質転 換させた。 寒天培地上に形質転換体コロニーを形成させ、 その上に、 ナイロントラン スフアーメンプレン （アマシャム社製） を重ねて、 メンブレン上にコロニーの一部を 移行させた。 このメンブレンをアルカリ変性溶液 （0.5M NaOH、 3.0M Nad) を浸し たろ紙上に 20分間静置し、 その後中和溶液 （1M Tris-HCl (pH7.5)) を浸したろ紙 上に 10分間置いた。 さらに、 2XSSC溶液で振盪しながら洗浄した。 次に、 このメン ブレンを DIG Easy Hybでプレハイブリダィゼ一シヨンを 1時間行った。 その後、 メンブレンを 9一 3)で得られたキチナーゼ遺伝子プローブと 37°Cで一晩ハイブリダ ィゼ一シヨンを行つた。 ハイブリダイゼーション後、 常温の 2 X SSCバッファーで三 回洗浄し、 さらに 53°Cに温めた 2 X S S Cバッファーで二回洗浄した。 その後、 DIG Wash and Block Buffer Set (ロシュ 'ダイァグノスティック社製） を用いて検 出を行った。 まず、 Wash buffer (0.1M マレイン酸、 0.15M NaCK 0.3% Tween 20： pH7.5)でメンブレンを順化した。 次いで、 このメンブレンをブロッキング溶液 (0.1M マレイン酸、 0.15M NaCI :pH7.5に 10%ブロッキング試薬を含む溶液）に浸し て 60分間ブロッキングを行い、 非特異的な結合をブロッキングした。 次に、 メンブ レンをブロッキング溶液に抗ジゴキシゲニン抗体を 10000倍希釈で加えた溶液に浸し、 37°Cで 30 分間反応させた後、 Wash Buffer で二回洗浄した。 その後、 Detection buffer (0.1M Tris-HCU 0.1M NaCI : pH9.5)でメンブレンを順化した。 Detection bufferに CSPD (Chemi luminescence substrate)を 100倍希釈で加え、 純化したメ ンブレンに滴下した後、 37°Cで 15 分間発色を安定させた。 その後 Lumi- Film Chemi luminescent Detecion Film (コニ力社製）に感光させ、現像した。その結果、 ポジティブシグナルを示すクローンを確認できた。 9-5) Screening of DNA containing chitinase gene E. coli JM109 strain (Takara Shuzo) was transformed with the DNA library I obtained in 9-4). A transformant colony was formed on an agar medium, and nylon transfer mass (manufactured by Amersham) was overlaid thereon to partially transfer the colony onto the membrane. The membrane was allowed to stand for 20 minutes on a filter paper soaked with alkaline denaturing solution (0.5 M NaOH, 3.0 M Nad), and then for 10 minutes on a filter paper soaked with a neutralization solution (1 M Tris-HCl (pH 7.5)). placed. Further, it was washed with 2X SSC solution while shaking. Next, 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). Then, 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. Next, 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). 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. Thereafter, it was exposed to light and developed with Lumi-Film Chemi luminescent Detecion Film (manufactured by Koni). As a result, a clone showing a positive signal could be confirmed.
9 - 6) 組換え体プラスミド DNAの抽出と配列解析 9-6) Extraction and sequence analysis of recombinant plasmid DNA
9 - 5)で得たポジティブシグナルを示すクローンを L-broth (和光純薬工業社製） で一晚培養し、組換え体プラスミドの DNAを抽出した。 QIAprep Spin Miniprep Kit (キアゲン社製）を用い、 プラスミド DNAを抽出した。 次いで、 抽出した DNAをこのプ ラスミド特有のユニバーサルプライマ一、 M13M4, Ml 3RVを用いて配列解析を行い、 その結果と 9一 3)で得られたキチナーゼ遺伝子プローブのヌクレオチド配列とをァ ラインメントし、 キチナ一ゼの配列を含むことを確認した。 キチナーゼの配列を含む ことが確認できたプラスミド DNAを GPS- 1 Genome Priming System (ニューイング ランド バイオ ·ラボ社製） を用いて、 全体の配列を決定した。 手順は、 まず上記で 抽出したプラスミド DNA に 2 1 10XGPS Buffer (250raM Tr is - HC1 ;pH8.0、 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. The entire sequence of the plasmid DNA, which was confirmed to contain the chitinase sequence, was determined using the GPS-1 Genome Priming System (New England Bio-Lab Co., Ltd.). The procedure is as follows. First, the plasmid DNA extracted above is prepared from 2 1 1 0XGPS Buffer (250raM Tris-HC1; pH 8.0,
20mM DTT、 20mM ATP、 500 μg/ml BSA)、 1 1 GPSl (Transprimer-1 Donor plasmid) 、 \\u\ 滅菌水を加え混合した後、 1^1 TnsABC Transposaseを添加し 37°Cで 10 分間結合させた。 その後、 1 1 Start Solution (300mM magnesium acetate)を添加し、 37°Cで 1時間反応させた後、 75°Cで 10分間処理することで、 反 応を停止した。 次いで、 この反応産物 10 ilを大腸菌 JM109株に形質転換し、 アンピ シリンとカナマイシンを含む LB寒天培地に塗布し、 37°Cでー晚培養した。 生育して きたコロニーを LB液体培地でー晚培養し、 プラスミド DNAを抽出した。 この抽出し たプラスミド DNAを pGPSlのプライマーである Nプライマーおよび Sプライマーで配 列をよみ、 キチナーゼをコ一ドするゲノム配列の全配列を決定した。 実施例 10. ァスペルギルス ·オリザェ由来のキチナ一ゼをコードする cDNAの 同定 Add 20mM DTT, 20mM ATP, 500 μg / ml BSA), 1 GPSl (Transprimer-1 Donor plasmid), \\ u \ sterile water and mix, add 1 ^ 1 TnsABC Transposase and keep at 37 ° C for 10 minutes It was combined. Then 1 1 Start Solution (300mM magnesium Acetate was added, reacted at 37 ° C. for 1 hour, and treated at 75 ° C. for 10 minutes to stop the reaction. Then, 10 l of this reaction product was transformed into E. coli JM109 strain, coated on LB agar medium containing ampicillin and kanamycin, and cultured at 37 ° C. for 晚. The grown colonies were cultured in LB liquid medium for sputum to extract plasmid DNA. The extracted plasmid DNA was sequenced with pGPSl primers N primer and S primer, and the entire sequence of the chitinase encoding genomic sequence was determined. Example 10. Identification of a cDNA encoding a chitinase from Aspergillus oryzae
10— 1) ァスペルギルス ·オリザェの全 RNAの精製  10 — 1) Purification of total RNA of Aspergillus oryzae
ァスぺレキノレス ·オリザェ (Aspergillus oryzae) var. sporof lavus Ohara J CM 2067株を Potato Dextrose培地 20mlで 30° (：、 3日間前培養した。 その 後、 液体培地 （1% Malt Extract, 0.5¾ Yeast Extract) に 1 %植菌し、 30°Cで 3 日間培養した。 培養した菌体を吸引集菌し、 -80°Cで冷やした乳鉢 （ォ一トクレーブ 滅菌済） に移した。 液体窒素を加えながら、 乳棒で菌体を破砕し、 粉末状にした。 完 全に粉末状になった菌体を Rneasy Plant Mini Kit (キアゲン社製）を用いて次 のように全 RNAの精製を行った。まず粉末状の菌体約 lgを 4.5ml RLT buffer (j3 - Mercaptoeihanol)に加え、 激しく振とうし菌体を溶解させ、 氷上に置いた。 0.5ml ずつ QIAshredderスピンカラムに分注し、 室温で 15000rpm、 2分間遠心した。 通過液 に 0.5倍量のエタノールを添加し、 ピペッティングで攪拌した。その後、 675 1ずつ Rneasy Miniスピンカラムに分注し、 室温で 16000rpm、 15秒間遠心した。 700 1の RW1 Wash bufferを加え、 同じように室温で 16000rpm、 15秒間遠心した。 次に 500 li\ RPE Wash buf ferを添加し、室温で 16000ι·ρπι、 2分間遠心して RNAを洗浄した。 スピンカラムをマイクロチューブにセットし、 の Rnase free waterで RNA を溶出させ室温で 10000rpm、 1分間遠心して回収した。  Speroflavus Ohara J CM 2067 strain was precultured with 20 ml of Potato Dextrose medium at 30 ° (:, for 3 days. Thereafter, liquid medium (1% Malt Extract, 0.53⁄4 Yeast). The extract was inoculated with 1% and cultured for 3 days at 30 ° C. The cultured cells were collected by suction and transferred to a mortar (sterilized with Klebe) which had been cooled at -80 ° C. Liquid nitrogen was transferred. While adding, the cells were crushed with a pestle and powdered The cells completely powdered were purified using the Rneasy Plant Mini Kit (manufactured by Qiagen) as follows for purification of total RNA. First, about 1 g of powdered cells was added to 4.5 ml of RLT buffer (j3-Mercaptoeihanol), the mixture was vigorously shaken to dissolve the cells, and placed on ice 0.5 ml each was dispensed onto a QIAshredder spin column and allowed to stand at room temperature. The mixture was centrifuged at 15000 rpm for 2 minutes 0.5 volume of ethanol was added to the flow through and stirred by pipetting. Then, 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 16000ι · 1πι 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.
10 -2) ァスペルギルス ·オリザェ cDNAライブラリ一の作製 10 -2) Preparation of the Aspergillus oryzae cDNA library
ァスペルギルス ·オリザェの cDNA ライブラリ一は、 SMART cDNA Library Construction Kit (クロンテック社製）を用いて作製した。 まず、 滅菌した 0.5ml チューブに 10— 1)で精製した全 RNA lj gと SMART IV Oligonucleotide, l i l CDS III/3' PCR Primerを混合し、 72°Cで 2分間インキュベートした。 そ の後、 氷上に 2分間置き、 冷却した。 この冷却した試料に 5X First- Strand Buffer, 1 n 1 20mM DTTゝ 1 n 1 lOmM dNTP Mix および 1 1 Power Script Reverse Transcriptaseを添加した。 内容物を混合し、 42°Cで 1時間反応させた後、 氷上に置き、 first-strand DNA合成を停止させた。 次いで、 LD PCRによる cDNAの 増幅を行った。 PCRチューブに前記の First-Strand DNAを 2 1添加し、さらに次の 試薬を添加した： 80 1 脱イオン水、 10 1 10XAdvantage2 PGR Buffer, 2^1 50XdNTP Mix. 5， PCR Primer, CDS 111/3， PCR Primer 2^1 50An Aspergillus oryzae cDNA library was prepared using the SMART cDNA Library Construction Kit (Clontech). First, 10 l) purified total 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. for 1 hour, and then placed on ice to stop first-strand DNA synthesis. Next, amplification of cDNA by LD PCR was performed. Add 2 1 of the above First-Strand DNA to the PCR tube, and then add the following: Reagents were added: 80 1 deionized water, 10 1 10 X Advantage 2 PGR Buffer, 2 ^ 1 50 X dNTP Mix. 5, PCR Primer, CDS 111/3, PCR Primer 2 ^ 1 50
X Advantage 2 Polymerase Mix。 この PCRチューブを 95°Cに予熱したサーマルサ ィクラ一にセットし、 95°Cで 20秒反応させた後、 95°Cで 5秒、 68°Cで 6分のサイク ルを 20サイクル行った。 この反応液 （ds cDNAを含む） 50 1を滅菌したチューブ に取り、 2ιι\ プロテア一ゼ Κを添加して 45°Cで 20分間インキュベートし、 プロテ ァーゼ処理を行った後、 50 1の脱イオン水を添加した。 IOO I フエノール：クロ 口ホルム：イソアミルアルコール混合液 （25 : 24 : 1) を添加し、 1分間混合し て UOOOHDIIIで 5分間遠心分離した。 上清を新しいチューブに移し、 100 1 クロ口 ホルム：イソアミルアルコール（ 24： 1 )を添加した。 1分間混合した後、再度 14000rpm で 5分間遠心分離した。上清を新しいチューブに移し、 10 1 3M 酢酸ナトリウム、 l.2,nl グリコーゲン及び 260 1 95%エタノール （室温） を加え、 直ちに室温で HOOOrpmで 20分間遠心分離した。 ピぺットで上清を除去し、 80%エタノール 100 1 でペレットを洗浄した後、 ペレットを乾燥させた。 ペレットを 79 1 脱イオン水に 溶解した。 続いて、 制限酵素 Sfi I による処理を行った。 79 1 CDNA、 IO I 10 X Sfi Buffer, 10^1 Sfi Enzymeおよび 1 I 100X BSAを混合し、試験管を 50°Cで 2時間インキュベートした。その後、 1% xylene cyanol染色液を添加 した。 CHROMA SPIN- 400カラムを使い、 cDNAを分子ふるいにかけた。 CHROMA SPIN カラムは数回転倒混和してゲルマトリックスを懸濁させ、 さらにピぺットマンで懸濁 した。 カラムの蓋を取り、 カラムが自然にドリップするように設置した。 次にカラム バッファーを静かに添加し、 重力で自然にドリップさせた。 このバッファ一のドリツ プが終了した後、マトリックスの上面中央に Sfi Iで分解した cDNAサンプルをアブ ライし、 マトリックスの表面に十分吸収させた。 表面から液体がなくなり、 ドリップ が終了したら、 600 1 カラムバッファーを添加し、 直ちに試験管 16本に 1滴ずつ フラクションを収集した。 このフラクションの各 3 1 をァガロースゲル電気泳動に かけ、 ピ一クフラクシヨンを判定した。 cDNAを含むフラクションを収集し、 1本の新 しい試験管に集めた。 この試験管に 1/10倍量の酢酸ナトリウム （3M ;pH4.8) 、 1.3 \ グリコーゲン、 2.5倍量の 95%エタノール（- 20°C) を加え、軽く振って混合し、 -20°Cでー晚インキュベートした。 室温で 14000ι·ρπι、 20分間遠心分離し、 上清を除去 した。 ペレットを完全に乾燥させた後、 7^1 脱イオン水にペレットを再懸濁した。 上記の cDNA 0.5 ^ i を λ TriplEx2 ベクタ一、 0.5 1 10X Ligation Buffer, 0.5 il ATP, 0.5/xl T4 DNA Ligaseおよび 2· 0 1 脱イオン水と混合 し、 16ででー晚インキュベートした。 次に、 このライゲ一シヨンサンプルに対して λ ファ一ジパッケージング反応を行った。 パッケージングには Gigapack III Gold Packaging Extract (STRATAGENE社製） を用いた。 Packaging Extractが入ったマ イク口チューブに上記でライゲーションした DNAベクタ一を 加え、静かにピ ベッティングで混合した後、室温で 2時間インキュベートした。 2時間後、 500 1 SM bufferを加え、さらに 20 1のクロ口ホルムを混合した後、軽く遠心分離することに よりパッケージング液を調製した。 次に、 パッケージングしたファージを感染させる ホスト細胞を培養した。 E. coli XL1- Blueの凍結ストックを LB/tetracyclineァガ —プレートに植菌し、 37°Cでー晚培養した。 生育したシングルコロニ一を MgS04と tetracyclineを含むプレートに移し、 37°Cでー晚培養した。生育したシングルコロニ —を 15mlの LB/MgS04 /マル! ^一スブロスに植菌し、 培養液の 0DS。。が 2.0になるまで 37°Cで培養した。培養液を 5000rpmで 5分間を遠心分離し、ペレツトを 7.5mlの lOmM MgS04に再懸濁した。パッケージング液を IX λ希釈バッファーで希釈した後、該パッ ケージング液 を、 200 1 Ε. coli XL1- Blue懸濁液に添加し、 37°Cで 15分 間吸着させた。該反応液に、 融解した 2mlの LB/ MgS04トツプアガーを混合し、 温め ておいた LB/ MgS04プレートに均等に広げた。 室温で 10分間冷やし、 トツプアガー を固めた後、 37°Cでー晚培養した。 得られたプラークを SMバッファ一を加えてー晚 振とうして溶出させ、 ァスペルギルス ·オリザェ cDNAライブラリ一を含むファージ を得た。 X Advantage 2 Polymerase Mix. The 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. 79 1 CDNA, IO I 10 X Sfi Buffer, 10 ^ 1 Sfi Enzyme and 1 I 100 X BSA were mixed, and the test tube was incubated at 50 ° C. for 2 hours. Thereafter, 1% xylene cyanol staining solution was added. The cDNA was molecular sieved using a CHROMA SPIN-400 column. The CHROMA SPIN column was inverted several times to suspend the gel matrix, and further suspended with a pipetman. Remove the lid of the column and set the column to drip naturally. The column buffer was then added gently and allowed to drip naturally with gravity. After completion of this buffer dripping, 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. When the surface was drained and dripping was complete, 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 · ιπι 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 λ 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 λ phage packaging reaction was performed on this linear sample. Gigapack III Gold Packaging Extract (manufactured by STRATAGENE) was used for packaging. Add the above-ligated DNA vector into the microtube containing the packaging extract and gently After mixing by betting, it was incubated at room temperature for 2 hours. After 2 hours, 500 1 SM buffer was added, and after further mixing with 201 aliquots, the packaging solution was prepared by light centrifugation. Next, host cells infected with the packaged phage were cultured. Frozen stocks of E. coli XL1-Blue were inoculated on LB / tetracycline agarose plates and cultured at 37 ° C. for 7 days. Grown single colonies one was transferred to a plate containing MgSO 4 and tetracycline, and 37 ° C De晚culture. The grown single colonies - the 15ml of LB / MgS0 4 / Mar ^ one Suburosu in inoculated, 0D S of the culture solution. . 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. After the packaging solution was diluted with IX λ dilution buffer, the packaging solution was added to the suspension of 2001.E. coli XL1-Blue and adsorbed at 37 ° C. for 15 minutes. To the reaction mixture, 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.
10-3) キチナ一ゼ遺伝子含有 cDN Aのスクリーニング 10-3) Screening for cDNAs containing chitinase gene
10-2) で得た cDNAライブラリ一を含むファージを大腸菌 XL1- Blue株 （クロン テック社製） に感染させた。 希釈したファージ 1 1を培養した 200 1 XL1- Blueに 添加し、 37°Cで 10分から 15分間吸着させた。 2mlの融解した LB/ MgS04トップアガ 一を添加し、 混合し、 温めておいた LB/ MgS04プレートに注ぎ、 プレートをまわして トツプアガーを均等に広げた。室温で 10分間冷やし、 トップァガ一を固めた後、 37°C でー晚インキュベートし、 寒天培地上にプラークを形成させた。 その上に、 ナイロン ファージの一部
Figure imgf000043_0001
20分間乾燥させ た後、アル力リ変性溶液（0.2N NaOH、 1.5M NaCl)を浸したろ紙上に 5分間静置し、 その後中和溶液 （0.4M Tris-HCl (pH7.5)、 2XSSC) を浸したろ紙上に 5分間置い た。 さらに、 中和溶液で 1分間振盪しながら洗浄した後、 2XSSCでさらに 5分間洗浄 した。 フィルタ一をろ紙上で 1時間程乾燥させ、 80°Cのオーブンで 2時間べ一キング を行った。 その後、 メンブレンを X— 3で得られた DNAプローブと 37°Cでー晚ハイブ リダイゼ—シヨンを行つた。 ハイブリダイゼーション後、 常温の 2 X SSCバッファ一 で三回洗浄し、 さらに 53°Cに温めた 2XSSCバッファーで二回洗浄した。その後、 DIG Wash and Block Buffer Set (ロシュ 'ダイァグノスティック社製） を用いて検 出を行った。 まず、 Wash buffer (0.1M マレイン酸、 0.15M NaCl、 0.3¾ Tween 20； ρΗ7·5)でメンブレンを順化した。 次いで、 このメンブレンをブロッキング溶液 (0.1M マレイン酸、 0.15M NaCl； H7.5に 10%ブロッキング試薬を含む溶液）に浸し て 60分間ブロッキングを行い、 非特異的な結合をブロッキングした。 次に、 メンブ レンをブロッキング溶液に抗ジゴキシゲニン抗体を 10000倍希釈で加えた溶液に浸し、 37°Cで 30 分間反応させた後、 Wash Buffer で二回洗浄した。 その後、 Detection buffer (0.1M Tris-HCK 0.1 NaCl ; pH9.5)でメンブレンを順化した。 Detection bufferに CSPD (Chemi luminescence substrate)を 100倍希釈で加え、 純化したメ ンブレンに滴下した後、 37°Cで 15 分間発色を安定させた。 その後 Lumi- Film Chemi luminescent Detecion Film (コニ力社製） に感光させ、 現像し、 検出した結 果、 ポジティブシグナルを示すクロ一ンを検出できた。 このポジティブを示すファ一 ジプラークを寒天培地ごとくり抜き、 SMバッファーに溶解させた後、 大腸菌 BM25.8 株 に 形 質 転 換 さ せ 、 形 質 転 換 大 腸 菌 株 Escherichia coli BM25.8/pTr i 1 Ex-Ch i t i nas e-cDNA SANK 72102株を得た。 この菌株は平成 14年 1 1月 8日付けで、独立行政法人産業技術総合研究所特許生物寄託センターに 国際寄託され、 受託番号： FERM BP— 8235を付与された。 感染したファー ジ DNAである λ TriplEx-Chitinase-cDNAは、 大腸菌 BM25.8株の細胞内で環状化し、 組換えプラスミド pTriplEx- Chitinase- cDNAとして存在する。 得られた Escherichia coli BM25.8/pTr iplEx-Chi t inase-cDNA SANK 7 2 1 02株のコロニーを LB/Ampicilin ブロスでー晚培養し、 組換え体プラスミド pTriplEx_Chitinase_cDNA の DNAを抽出した。 QIAprep Spin Miniprep Kit (キアゲン社製）を用い、 プラス ミド DNAを抽出した。 次いで、 該プラスミド DNA μ1 に 10XGPS Buffer (250mM Tris-HCl； pH8.0, 20mM DTT、 20mM' ATP, 500 g/ml BSA)、 1^1 GPSl (Transprimer-1 Donor plasmid)、 11 xl 滅菌水を加え混合した後、 TnsABC Transposase を添加し 37でで 10分間結合させた。 その後、 1 1 Start Solution (300mM magnesium acetate)を添加し、 37°Cで 1時間反応させた後、 75°Cで 10分間 処理することで、 反応を停止した。 次いで、'この反応産物 10//1を大腸菌 IM109株に 形質転換し、 アンピシリンとカナマイシンを含む LB寒天培地に塗布し、 37Cでー晚 培養した。 生育してきたコロニーを LB液体培地でー晚培養し、 プラスミド DNAを抽 出した。 この抽出したプラスミド DNAを pGPSlのプライマ一である Nプライマ一およ び Sプライマーで配列をよみ、 ァスペルギルス ·オリザェのキチナ一ゼをコードする DNAのヌクレオチド配列を決定した （配列表の配列番号 4) 。 また、 配列表の配列 番号 4に記載のヌクレオチド配列から、ォ一プンリ一ディングフレーム（O R F)は、 ヌクレオチド番号 242から 244番目の "ATG" を開始コドンとし、 ヌクレオチ ド番,号 1436から 1438番目の "TAG" を終止コドンとする領域であり、 コ一 ドするアミノ酸配列は配列表の配列番号 5に示される配列であることが推察された。 実施例 11. ァスペルギルス ·オリザェ由来キチナ一ゼの N末端アミノ酸配列の決 実施例 1で精製した酵素溶液を約 1 mg/m 1程度まで限外濾過膜 （ADVANTEC社製 ULTRA FILTER UNIT USY- 1、 分子量分画 1万） を用いて濃縮した。 濃縮酵素液 40 O Iに Denature buffer (6M塩酸グァニジン、 1 OmM EDTA、 0. 1M炭酸水素 アンモニゥム pH 7.8) 400 1ぉょび5011^ Dithiothreitol 8 1を加え、 95°Cで 10分間反応させた。 室温まで冷却後、 反応液に Denature buffer溶液に溶 解した 5 OmM Iodoacetoamide40 1を加え、 暗所室温で 1時間反応させた。 こ の溶液を Slide- A- Lyzer Mini Dialysis Units Plus Float (PIERCE社製、 分子 量分画 1 万） で水 1L に対して透析した。 得られた溶液にトリプシン （Modified, Promega社製） 8 1を加え、 37°Cで 7時間酵素反応させた。 得られた酵素反応液を LC/MS (Pencon/Q-Tof 2)で測定した。 Include massとして m/z 533を設定し、 MS/MS スぺクトルを測定した。フラグメン卜イオンの帰属から N末端アミノ酸は Serである こと、 N末端に Ser_Ser-Gly- Leu- Lysというアミノ酸配列を含むこと、 および、 N末 端にァセチル基の存在が示唆された（配列表の配列番号 14のアミノ酸番号 1から 5 に記載のアミノ酸配列） 。 この結果を配列表の配列番号 5のアミノ酸配列と照らし合 わせると、 ァスペルギルス ·オリザェが産生するキチナ一ゼは、 その N 末端、 "Met-Ser-Ser" 、 が翻訳後修飾をうけて、 "Acety卜 Ser-Ser" の形 （配列表の配列 番号 14) で産生されていることが示唆された。 実施例 1 2. ァスペルギルス ·オリザェのキチナーゼ遺伝子の発現 10-2) was used to infect E. coli strain XL1-Blue (manufactured by Krontech). The diluted phage 11 was added to cultured 200 1 XL1-Blue and adsorbed at 37 ° C. for 10 to 15 minutes. Was added molten LB / MgSO 4 Toppuaga one 2 ml, mixed and warmed poured into LB / MgSO 4 plates had been, spread equally Totsupuaga Turn the plate. After cooling at room temperature for 10 minutes, the top agarose was solidified and incubated at 37 ° C. to form plaques on the agar medium. On top of that, part of the nylon phage
Figure imgf000043_0001
After drying for 20 minutes, 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. After hybridization, 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.). First, the membrane was acclimated with Wash buffer (0.1 M maleic acid, 0.15 M NaCl, 0.33⁄4 Tween 20; Η 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. Next, 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. As a result, a clone showing a positive signal could be detected. 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, λ 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). Then, the plasmid DNA μ1 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 After addition and mixing, 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. coli strain IM109, coated on LB agar medium containing ampicillin and kanamycin, and cultured at 37C. The grown colonies were cultured in LB liquid medium for sputum and plasmid DNA was extracted. The extracted plasmid DNA was sequenced with the primers N-primer and S-primers of pGPSl, and the nucleotide sequence of the DNA encoding the Aspergillus oryzae chitinase was determined (SEQ ID NO: 4 in the Sequence Listing). . In addition, from the nucleotide sequence set forth in SEQ ID NO: 4 in the sequence listing, the binding frame (ORF) 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). When this result is compared with the amino acid sequence of SEQ ID NO: 5 in the Sequence Listing, the chitinase produced by Aspergillus oryzae is post-translationally modified at its N-terminus, “Met-Ser-Ser”, It was suggested that the protein is produced in the form of "Acety 卜 Ser-Ser" (SEQ ID NO: 14 in the Sequence Listing). Example 1 2. Expression of the chitinase gene of Aspergillus oryzae
キチナーゼ 0RFの 5' 末端と 3' 末端にそれぞれ BamHIサイトを接続させたプライ マ一、 BamHI- cDNAおよび cDNA- BamHIを設計し， 合成したた。  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， (配列表の配列番号 9) cDNA-BamHI： 5 -ctggatccagcgaaaccggctcgcaggttg- 3 (配列表の配列畨号 1 BamHI-cDNA: 5'-gaggatccatgtci tccggactaaagtcgg-3, (Sequence Listing SEQ ID NO: 9) cDNA-BamHI: 5-ctggatccagcgaa aggggctcg caggattg- 3 (Sequence Listing Sequence Number 1)
0) 0)
実施例 10— 3) で得られた組換えプラスミド、 pTriplEx-Chitinase- cDNAを鐃型 とし、 プライマ一として上記の BamHI- cDNAおよび cDNA- BamHIを用いて PCRを行い、 制限酵素サイトを導入したキチナ一ゼ DNAを増幅させた。さらに pCR 2.1-T0P0べク ター （インヴィトロジェン社製） に挿入し、 大腸菌皿 09株に形質転換させ、 増幅さ せた。； 1M109株を生育させたコロニ一を Lb/Ampicilinブロスでー晚培養し、プラスミ ド DNAを抽出した。 このプラスミド DNAを制限酵素 BamHIで処理し、 キチナーゼ 0RF を切断した。 また、 この断片を連結させるベクターとして、 pQE- 60 (キアゲン社製） を BamHIで切断し、 さらにアルカリフォスファタ一ゼで 5' 末端にあるリン酸基を除 去する処理を行った。 その後、 T0P0 TA Cloning Kit (インヴィトロジェン社製） を用いて上記のキチナ一ゼ 0RFとライゲ一シヨンを行った。 該反応液を大腸菌 JM109 株に形質転換させ、 Lb/Ampicilinァガープレートで一晩培養した。生育してきたコロ ニーを Lb/Ampicilinブロス 3mlに植菌し、 振とうしながらー晚培養した。 該培養液 からプラスミド DNAを QIAprep Spin Miniprep Ki t (キアゲン社製）で抽出し、 大 腸菌 M15 [pREP4]株に形質転換した。 Lb/Ampic i l in、 Kanamaicinァガ一プレートで ー晚培養し、 -,生育してきたコロニ一を Lb/Ampici l in、 Kanamaicinブロス 20mlに植 菌し、振とうしながら 37°Cでー晚培養した。この培養液から 2. 5mlを Lb/Ampic i 1 in、 Kanamicinブロス 100mlに植菌し、 37°Cで培養した。培養開始後 1時間後から 30分お きに 0D6。Bを測定し、 0. 6— 0. 7の間になるまで培養後、 ImM IPTGを加えた。 IPTG添 加後、 18°Cで培養を続け、 4及び 6時間後に集菌した。菌体に 10ml PBS (137mM NaCK 8. ImM Na2HP04 ·12Η20 2. 68mM KC1、 1. 47raM KH2P04)を添加し、 ヴオルテックスをか けよく懸濁した。 この懸濁液をソニケーシヨンにより、 菌体を破碎した後、 4°Cで 8000rpm、 5分間遠心分離した。 上清 100 1を 1. 5mlチューブに取り、 1変性剤 (6% SDS、 24¾ glycerol, 12% )3—メルカプトエタノール、 0. 3M Tri s-HCl (pH6. 8)、 0. 15% BPB) を添加し 100°Cで 5分間処理した。 この反応液を各サンプル 10 1ずつ e- PAGEL (ATT0社製） で電気泳動した後、 Si即 ly Blue SafeStain (イン ヴィトロジェン社製） で染色、 検出した。 その結果、 約 44kDa付近に濃いバンドが見 られた。 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. Further, as a vector to which this fragment is ligated, pQE-60 (manufactured by Qiagen) was digested with BamHI and further treated with alkaline phosphatase to remove the phosphate group at the 5 'end. Thereafter, using the 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. Said culture fluid 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. B was measured and cultured until it was between 0.6 and 0.7, then ImM IPTG was added. After the addition of IPTG, the culture was continued at 18 ° C., and after 4 and 6 hours, the cells were harvested. The cells 10ml PBS (137mM NaCK 8. ImM Na 2 HP0 4 · 12Η 2 0 2. 68mM KC1, 1. 47raM KH 2 P0 4) was added and Keyoku or suspended the Vuorutekkusu. The suspension was disrupted by sonication and centrifuged at 8000 rpm for 5 minutes at 4 ° C. 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.
6時間培養し集菌した菌体の上清中にキチナ一ゼが存在することは、試験例 1一 6 ) に記載のシエールズ法により確認した。  The presence of chitinase in the supernatant of the cells cultured and collected for 6 hours was confirmed by the Schierz method described in Test Example 1-16).
時間培養し集菌した菌体の上清は、 His タグ精製を行った。 カラムチューブに Ni-NTAァガロースを詰め、 0. 1M Tris-HCl (pH8. 0)約 10mlで洗浄した後、上清を力 ラムに吸着させた。次いで、 8mlの 10mMイミダゾールで非特異的な結合物を洗い流し た後、 8mlの 200mMイミダゾ一ルでカラムに吸着している蛋白質を溶出させ、 。 この 溶出液を e- PAGELで電気泳動した結果、 単一のバンドとして現れた。 また、 この溶出 液と実施例 1で得られたァスペルギルス ·オリザェの培養物から精製したキチナーゼ を 12. 5 %アクリルアミドゲルによって S D S電気泳動を行なった （図 3 0に示す） 。 溶出液に含まれる蛋白質の分子量ば、 ァスペルギルス ·オリザェ由籴の精製キチナ一 ゼよりも少し大きくなつているが、 これは H i sタグによるものと考えられ、 溶出液 に含まれるリコンビナント蛋白質が H i s融合リコンビナントキチナーゼであるこ とが推察された。 この溶出液のキトサン分解活性を試験例 1一 6 ) に記載のシエール ズ法により測定したところ、 2 . O U/mlであり、 この溶出液に含まれるリコンビナン ト蛋白質がキチナーゼであることが確認された。 なお、 1分間に 1 molのァセチル ダルコサミンを生成する酵素量を 1 Uと定義した。  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. OU / ml, and it was confirmed that the recombinant protein contained in this eluate was chitinase. The The amount of enzyme that produces 1 mol of acetyl dulcosamine per minute was defined as 1 U.
本発明においてアミノ酸配列が同定されたァスペルギルス ·オリザェ由来のキチナ —ゼは、翻訳後修飾を受ける前の 3 9 9アミノ酸からなるもの（配列表の配列番号 5 ) および翻訳後修飾を受けた 3 9 8アミノ酸からなるもの （配列表の配列番号 1 4 ) で ある。キチナ一ゼ遺伝子として報告 (キチン'キトサン研究 第 8巻 第 2号 238-239 頁 2002年）されているァスペルギルス.オリザェ由来の遺伝子は 4 3 1アミノ酸か らなる蛋白質をコードするものであり、 コードする蛋白質の活性も不明であるので、 本発明のァスペルギルス.オリザェ由来のキチナ一ゼとは異なる分子であると推測さ れる。 試験例 1. ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM 20 67株由来のキチナ一ゼの粗酵素液の諸性質 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? Since it encodes a protein consisting of the following and the activity of the encoding protein is unknown, it is presumed that it is a molecule different from the Aspergillus oryzae-derived chitinase of the present invention. Test Example 1. Properties of crude enzyme solution of chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara J CM 20 67 strain
実施例 1. 1) で得られた粗酵素液について、 活性測定を行なった。 The activity of the crude enzyme solution obtained in Example 1. 1) was measured.
1) 加水分解活性  1) Hydrolysis activity
実施例 1 2) の方法にしたがって、 還元糖の定量を行った。 キトサン 7 Bを基質と して測定した場合、 上清中 lmlあたり 4. 2 X 10_3Uの酵素活性を有していた。 キトサン 7 Bを基質としたときの酵素活性を 100%としたときの、キトサン 10B 分解活性の相対値を表 11に示す。 キトサン 7 B分解活性を有し、 キトサン 10B分 解活性がほとんどないことが判明した。 According to the method of Example 1 2), quantification of reducing sugars was performed. When 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.
[表 11] [Table 11]
相対活性 （％) キトサン 7 B分解活性 100 Relative activity (%) Chitosan 7 B degradation activity 100
キトサン 10B分解活性 1. 3 Chitosan 10B degradation activity 1.3
2) pH活性 2) pH activity
キトサン 7 B溶液 160 1および 40 OmM緩衝液 100 1の混合液に、 酵素液 140 lを加え撹拌して均一にし、 37°Cで 20分間酵素反応を行った。 酵素反応 後の遊離還元糖の定量は実施例 1 2) 記載の方法にしたがった。 緩衝液は次のもの を用いた： PH3. 6乃至 pH5.9の場合、酢酸—酢酸ナトリウム緩衝液： pH 5. 9乃至 pH8. 2の場合、 3- (N-Morpholine) propanesul fonic acid (以下 「M 〇PS」 と記す）一炭酸ナトリウム緩衝液： pH 9. 0乃至 pH10. 2の場合、 炭 酸水素ナトリゥムー炭酸ナトリゥム緩衝液。最も活性が高かった p H条件での加水分 解活性を 100%とし、各 pHにおける酵素の加水分解活性を相対値として図 5に記 載した。 至適 pHは pH5. 0付近であった。 140 l of the enzyme solution was added to a mixture of chitosan 7 B solution 160 1 and 40 O mM buffer solution 100 1 and stirred to homogenize, and the enzyme reaction was performed at 37 ° C. for 20 minutes. The quantification of the free reducing sugar after the enzyme reaction was performed according to the method described in Example 12). The following buffer was used: pH 3. 6 to 5.9, acetic acid-sodium acetate buffer: pH 5.9 to pH 8.2 2- (N-Morpholine) propanesulfonic acid (below) Sodium monocarbonate buffer solution, which is referred to as “M o PS”: sodium hydrogen carbonate carbonate buffer solution in the case of pH 9.0 to pH 10.2. The hydrolysis activity under the highest activity pH condition was defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 5 as a relative value. The optimum pH was around pH 5.0.
3) 培養上清中の酵素の温度活性  3) Temperature activity of enzyme in culture supernatant
PH5. 0の条件下において、 種々の温度で加水分解活性を測定した。 5分間予熱し た実施例 1で作製したキトサン 7 B溶液 160 ^ 1および 40 OmM酢酸緩衝液（p H5. 0) 100 1の混合液に、 酵素液 140 1を加え撹拌して均一にし、 10 分間酵素反応を行った。 酵素反応後の遊離還元糖の定量は実施例 1にしたがった。 最 も高い活性を示した温度条件で測定された加水分解活性を 100%とし、各温度にお ける加水分解活性を相対値として図 6にまとめた。 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.
4) 培養上清中の酵素の温度安定性  4) Temperature stability of enzyme in culture supernatant
培養上清中の酵素を種々の温度で 30分間処理した後、その加水分解活性を測定した。 あらかじめ処理温度に保持した 40 OmM酢酸緩衝液 （pH5. 0) 200 1に、 酵素液 280 1を加え撹拌して均一にし、 30分間保温した。 実施例 1で作製した キトサン 7 B溶液 160 1に加温処理した酵素液 240 i lを加え、 37 °Cで 20 分間酵素反応を行った。 遊離還元糖の定量は実施例 1にしたがった。 無処理 （0°C保 温）群の酵素および緩衝液の混合液が有する加水分解活性を 100%とし、 各温度に おける加水分解活性を相対値として図 7にまとめた。 ， 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. To the chitosan 7 B solution 160 1 prepared in Example 1, 240 I l of the enzyme solution obtained by heating was added, and an enzyme reaction was performed at 37 ° C. for 20 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. ,
5) 培養上清中の酵素の pH安定性  5) pH stability of the enzyme in the culture supernatant
培養上清 400 1に、以下に述べる各 pHの 40 OmM緩衝液 70 1及び水 90 H 1を添加し、 37 °Cにて 1'诗間保温した。 緩衝液は次のものを用いた.： H3. 9 乃至 pH7. 3の場合、 酢酸一酢酸ナトリウム緩衝液： pH 6. 5乃至 pH8. 6の 場合、 MOPS—炭酸ナトリウム緩衝液： pH9. 3乃至 ρΗΙ Ο. 4の場合、 炭酸 水素ナトリウム一炭酸ナトリウム緩衝液： PH10. 0乃至 pHl l. 4の場合、 リ ン酸水素 2ナトリウム—水酸化ナトリゥム緩衝液。 400 mM酢酸緩衝液 （ p H 5. 0) 100 1および実施例 1 2)記載の方法で作製したキトサン 7 B溶液 160 1の混合液に、 加温した酵素混合液 140 PL 1を加え撹拌して均一にし、 37。 で 20分間酵素反応を行つた。遊離還元糖の定量は実施例 1にしたがつた。無処理（ 0 °C 保温）群の酵素および緩衝液の混合液が有する加水分解活性を 100%とし、 各 pH における加水分解活性を相対値として図 8にまとめた。 To the culture supernatant 4001, 40 OmM buffer solution 70 1 and water 90 H 1 of each pH described below were added, and the mixture was incubated at 37 ° C. for 1 ′. The following buffer was used: H 3 .9 to pH 7.3 sodium acetate monoacetate buffer: pH 6.5 to pH 8.6 MOPS-sodium carbonate buffer: pH 9.3 to In case of ΗΙ Ο 4. Sodium hydrogencarbonate sodium bicarbonate buffer: In case of pH 10. 4 to pH 1.4, disodium hydrogenphosphate-sodium hydroxide buffer. Heated enzyme mixture 140 PL 1 was added to a mixture of 400 mM acetate buffer (pH 5.0) 100 1 and chitosan 7 B solution 160 1 prepared by the method described in Example 1 2) and stirred. And uniform, 37. 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.
6) 培養上清中の酵素の基質選択性  6) Substrate selectivity of enzyme in culture supernatant
次に、 pH5. 0における基質選択性を測定した。 実施例 2で調製した培養上清 10 mlを 1 OmMトリス '塩酸緩衝液 （pH 7. 5) 1， 000 mlに対して 12時 間ごとに 3回透析したものを酵素液として用いた。 0. 25% wt/vの基質溶液 160 X 1および 400 mM酢酸緩衝液 (pH5. 0 ) および水 160 1の混合液 に酵素液 50 I 1を加え撹拌して均一にし反応を開始し、 37°Cで保温して 10分間 酵素反応を行った。酵素反応後の遊離還元糖の定量はシェ一ルズ法（Imoto, T. and Yagashita, K. , Agric. Biol. Chem. , 35, 1154(1971)参照） により行った。 最大活性を示す基質を用いたときの加水分解活性を 100%としたときの、相対活性 を表 12に示す。 Next, substrate selectivity at pH 5.0 was measured. 10 ml of the culture supernatant prepared in Example 2 was dialyzed 3 times every 12 hours against 1,000 ml of 1 OmM Tris' hydrochloric acid buffer (pH 7.5), and used as an enzyme solution. Enzyme solution 50 I 1 is added to a mixture of 25% wt / v substrate solution 160 X 1 and 400 mM acetate buffer (pH 5.0) and water 160 1 to homogenize and start the reaction, 37 The enzyme reaction was carried out for 10 minutes by incubating at ° 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 12 when the hydrolysis activity is 100% when using a substrate showing the maximum activity.
[表 12] 相対活性 （％) キトサン 1 OB 12. 7 [Table 12] Relative activity (%) Chitosan 1 OB 12.7
キトサン 9B 78. 9 Chitosan 9B 78.9
キトサン 8B 82. 3 Chitosan 8B 82.3
キトサン 7B 100 Chitosan 7B 100
ダルコールキトサン 17. 7 Dalcor Chitosan 17. 7
ダルコールキチン 52. 5 Dharcor Chitin 52. 5
CM—セルロース 2. 2 CM—Cellulose 2.2
リケナン 31. 6 Lickenan 31.6
試験例 2. ァスペルギルス 'オリザェ var. sporoflavus O ara J CM 2 067株由来の粗精製キチナ一ゼの諸性質 Test Example 2. Properties of crude purified chitinase derived from Aspergillus' Oryzae var. Sporoflavus O ara J CM 2067
実施例 1. 2) で得られたァスペルギルス 'オリザェ var. sporoflavus Ohara J CM 2067株由来の粗精製キチナ一ゼの諸性質を調べた。 Various properties of crude purified chitinase derived from Aspergillus sp. Oryzae var. Sporoflavus Ohara J CM 2067 obtained in Example 1. 2) were examined.
1 ) 粗精製画分中の酵素が有する加水分解活性の p H活性  1) The pH activity of the hydrolytic activity possessed by the enzyme in the crudely purified fraction
実施例 1で作製したキトサン 7 B溶液 160 ^ 1および 40 OmM緩衝液 100 1の混合液に、 酵素液 140 1を加え撹拌して均一にし反応を開始し、 37°Cで保 温して 20分間酵素反応を行った。酵素反応後の遊離還元糖の定量は実施例 1にした がった。 緩衝液は次のものを用いた： pH3. 3乃至 pH5. 9の場合、 酢酸一酢酸 ナトリウム緩衝液： PH5. 9乃至 pH8. 5の場合、 MOPS—炭酸ナトリウム緩 衝液： PH9. 2乃至 ρΗΙ Ο. 3の場合、 炭酸水素ナトリウム一炭酸ナトリウム緩 衝液。 最も活性が高かった PH条件での加水分解活性を 100%とし、 各 pHにおけ る酵素の加水分解活性を相対値として図 9に記載した。 至適 pHは pH5. 0付近で あった。 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: sodium acetate monoacetate buffer: pH 5. 9 to pH 8.5: MOPS-sodium carbonate buffer: pH 9.2 to ΗΙ 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.
2) 粗精製画分中の酵素が有する加水分解活性の温度活性  2) Temperature activity of hydrolysis activity of the enzyme in crude purified fraction
PH5. 0の条件下において、 種々の温度で加水分解活性を測定した。 あらかじめ処 理温度に保持した実施例 1で作製したキトサン 7 B溶液 160 n 1および 400m M酢酸緩衝液 （pH 5. 0) 100 1の混合液に、 酵素液 140 1を加え撹拌 して均一にし、 10分間酵素反応を行った。 酵素反応後の遊離還元糖の定量は実施例 1にしたがった。 最も高い活性を示した温度条件で測定された加水分解活性を 10 0%とし、 各温度における加水分解活性を相対値として図 10にまとめた。  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.
3) 粗精製画分中の酵素の温度安定性  3) Temperature stability of enzyme in crude purified fraction
粗精製画分中の酵素を種々の温度で 30分間処理した後、その加水分解活性を測定 した。 処理温度に保持した、 40 OmM酢酸緩衝液 （ρΗ5. 0) 200 ^ 1および 酵素液 280 X 1を加え撹拌して均一にし、 30分間保温した。 実施例 1で作製した キトサン 7 Β溶液 160 1に加温処理した酵素液 240 1を加え、 37°Cで 20 分間酵素反応を行った。 遊離還元糖の定量は実施例 1にしたがった。 無処理 （o°c保 温） 群の酵素および緩衝液の混合液が有する加水分解活性を 100%とし、 各温度に おける加水分解活性を相対値として図 11にまとめた。 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 (Η 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%.
4) 粗精製画^^中の酵素の pH安定性  4) pH stability of the enzyme in crude purified fraction ^^
粗精製画分中の酵素 400 1に、以下に述べる各 pHの 40 OmM緩衝液 1 0 1および水 20 1を添加し、 37 にて 1時間保温した。緩衝液は次のものを用い た： ρΗ3. 1乃至 ρΗ6. 1の場合、 酢酸—酢酸ナトリウム緩衝液： pH 6. 1乃 至 ρΗ8. 4の場合、 MOPS—炭酸ナトリウム緩衝液： ρΗ9. 0乃至 ρΗΙ Ο. 3の場合、 炭酸水素ナ卜リウムー炭酸ナトリウム緩衝液： pH 10. 4乃至 pHl 1. 4の場合、 リン酸水素 2ナトリゥム一水酸化ナトリゥム緩衝液。 400 mM酢酸 緩衝液（pH 5.0) 100 1および実施例 1 で作製したキトサン 7B溶液 160 H 1混合液に、 加温した酵素混合液 140 n 1を加え撹拌して均一にし、 37°Cで 2 0分間酵素反応を行った。 遊離還元糖の定量は実施例 1にしたがった。 無処理 （0°C 保温） 群の酵素および緩衝液の混合液が有する加水分解活性を 100%とし、 各温度 における加水分解活性を相対値として図 12にまとめた。 ' 試験例 3. ァスペルギルス 'オリザェ var. sporoflavus Ohara J CM 2 067株由来の精製キチナ一ゼの諸性質 To the enzyme 4001 in the crude purified fraction was added 40 OmM buffer solution of each pH described below and 101 of water and 201, and the mixture was kept at 37 for 1 hour. The following buffers were used: acetic acid-sodium acetate buffer in the case of Η3.1 to Η6.1; MOPS-sodium carbonate buffer in the case of pH 6.1 to Η8.4: Η9.0 to In the case of ΗΙ Ο 2.3, sodium bicarbonate-sodium carbonate buffer: pH 10.4 to pH 14. In the case of 1.4 hydrogen phosphate 2 sodium phosphate monohydroxide sodium buffer. To a mixed solution of 400 mM acetate buffer (pH 5.0) 100 1 and chitosan 7B solution 160 H 1 prepared in Example 1, add 140 n 1 of the heated enzyme mixture and stir to homogenize the mixture at 37 ° C. 2 The enzyme reaction was performed for 0 minutes. The quantification of free reducing sugars was in accordance with Example 1. The hydrolysis activity of the mixture of the non-treated (0 ° C. incubation) group of enzymes and buffer was 100%, and the hydrolysis activity at each temperature was summarized in FIG. 12 as a relative value. Test example 3. Properties of purified chitinase derived from Aspergillus sp. Orio var. Sporoflavus Ohara J CM 2067
実施例 1. 3) で得られたァスペルギルス 'オリザェ var. sporoflavus Ohara J CM 2067株由来の精製キチナ一ゼの諸性質を調べた。 Various properties of the purified chitinase derived from Aspergillus sp. Oryzae var. Sporoflavus Ohara J CM 2067 obtained in Example 1. 3) were examined.
1) 精製酵素が有する加水分解活性の pH活性  1) pH activity of the hydrolysis activity of the purified enzyme
実施例 1 2)記載の方法によって作製したキトサン 7 B溶液 160 1および 4 0 OmM緩衝液 100 1および水 90 1の混合液に、酵素液 50 n 1を加え撹拌 して均一にし反応を開始し、 37°Cで保温して 40分間酵素反応を行った。 酵素反応 後の遊離還元糖の定量は実施例 1 2) 記載の方法にしたがった。 緩衝液は次のもの を用いた： PH3. 3乃至 pH5. 9の場合、酢酸一酢酸ナトリウム緩衝液： pH 5. 9乃至 pH8. 4の場合、 MOPS-炭酸ナトリウム緩衝液： pH 9. 3乃至 pH10. 4の場合、 炭酸水素ナトリウム—炭酸ナトリウム緩衝液。最も活性が高かった pH条 件での加水分解活性を 100%とし、各 pHにおける酵素の加水分解活性を相対値と して図 13に記載した。 至適 pHは pH5. 5付近であった。  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.
また、 0. 25% w t/vのグリコールキチン溶液 240 / 1および 40 OmM緩 衝液 150 1及び水 135 ^ 1の混合液に、酵素液 75 n 1を加え撹拌して均一に し反応を開始し、 37°Cで保温して 30分間酵素反応を行った。酵素反応後の遊離還 元糖の定量はシェ—ルズ法 （Imoto, T. and Yagashita, K.， Agric. Biol. Chem. , 35, 1154(1971)参照）により行った。緩衝液は次のものを用いた： pH 3. 3乃至 pH6. 0の場合、 酢酸—酢酸ナトリウム緩衝液： pH 6. 3乃至 pH7. 8 2940 In addition, 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
50 の場合、 リン酸水素 1ナトリゥム-リン酸水素 2ナトリゥム緩衝液： H7. 3乃至 pH8. 7の場合、 トリス '塩酸緩衝液： PH9. 3乃至 ρΗΙ Ο. 4の場合、 炭酸 水素ナトリウム—炭酸ナトリウム緩衝液： PHI 0. 6乃至 ρΗ11· 5の場合、 リ ン酸水素 2ナトリゥム一水酸化ナトリゥム緩衝液。最も活性が高かった p H条件での 加水分解活性を 100%とし、各 pHにおける酵素の加水分解活性を相対値として図 14に記載した。 至適 pHは pHl 0付近であった。  In the case of 50, hydrogen phosphate 1 sodium-hydrogen phosphate 2 sodium buffer: H7.3 to pH8.7, Tris' hydrochloric acid buffer: PH9.3 to ΗΙ4. Sodium hydrogencarbonate-carbonate Sodium buffer: In the case of PHI 0.6 to Η11 · 5, hydrogen phosphate 2 sodium hydroxide monohydroxy sodium buffer. Assuming that the hydrolysis activity under the highest activity pH condition is 100%, the hydrolysis activity of the enzyme at each pH is shown in FIG. 14 as a relative value. The optimum pH was around pH10.
2) 精製酵素が有する加水分解活性の温度活性  2) Temperature activity of hydrolysis activity of the purified enzyme
PH5. 5の条件下において、 種々の温度で精製酵素の加水分解活性を測定した。 あ らかじめ処理温度に保持した 1) と同様に調製したキトサン 7 B溶液 1 60 1およ び 400 mM酢酸緩衝液（p H 5. 5) 100 ^ 1および水 100 1の混合液に、 酵素液 40 H 1を加え撹拌して均一にし、 10分間酵素反応を行った。酵素反応後の 遊離還元糖の定量は実施例 1にしたがった。最も高い活性を示した温度条件で測定さ れた精製酵素の加水分解活性を 100%とし、各温度における精製酵素の加水分解活 性を相対値として図 15にまとめた。 図 15に示す通り、 精製酵素の加水分解活性の 最適温度は、 PH 5. 5において 60°Cであった。  The hydrolysis activity of the purified enzyme was measured at various temperatures under the condition of pH 5.5. A mixture of chitosan 7 B solution 1601 and 400 mM acetate buffer (pH 5. 5) 100 ^ 1 and water 100 1 prepared in the same manner as 1) kept at the treatment temperature 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.
3) 精製酵素の温度安定性  3) Temperature stability of purified enzyme
精製酵素を種々の温度で 30分間処理した後、 その加水分解活性を測定した。 あらか じめ処理温度に保持した 40 OmM酢酸緩衝液 （pH 5. 5) 200 ^ 1および 水 180 1の混合液に、 酵素液 100 1を加え撹拌して均一にし、 30分間保温 した。 1) と同様に調製したキトサン 7 B溶液 1 6 0 1に加温処理した酵素液 24 0 n 1を加え、 37 °Cで 40分間酵素反応を行った。遊離還元糖の定量は実施例 1に したがった。 無処理 （0°C保温） 群の精製酵素および緩衝液の混合液が有する加水分 解活性を 100%とし、各温度における精製酵素の加水分解活性を相対値として図 1 6にまとめた。 図 16に示す通り、 精製酵素は 45 °C以下で安定であった。 After treating the purified enzyme at various temperatures for 30 minutes, its hydrolytic activity was measured. 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.
4) 精製酵素の pH安定性  4) pH stability of purified enzyme
精製酵素 150 1に、 以下に述べる各 pHの 40 OmM緩衝液 50 a 1を添加し、 37°Cにて 1時間保温した。 緩衝液は次のものを用いた： PH3. 2乃至 pH6. 2 の場合、 酢酸一酢酸ナトリウム緩衝液： pH 6. 1乃至 pH8. 2の場合、 MOPS —炭酸ナトリウム緩衝液： PH8. 8乃至 ρΗΙ Ο. 1の場合、 炭酸水素ナトリウム 一炭酸ナトリウム緩衝液： PH9. 6乃至 pHl l. 3の場合、 リン酸水素 2ナトリ ゥムー水酸化ナトリウム緩衝液。 40 OmM酢酸緩衝液 （pH 5, 5) 100 / お よび実施例 1で作製したキトサン 7 B溶液 160 1及び水 70 a 1の混合液に、加 温した酵素混合液 70 a 1を加え撹拌して均一にし、 37°Cで 40分間酵素反応を行 レ 遊離還元糖の定量をした。 無処理 （0°C保温） 群の精製酵素および緩衝液の混合 液が有する加水分解活性を 1 00 %とし、各温度における精製酵素の加水分解活性を 相対値として図 1 7にまとめた。 図 17に示す通り、 精製酵素は pH5乃至 pH9. 5で安定であった。 To the purified enzyme 1501, 40 OmM buffer solution 50 a 1 of each pH described below was added, and incubated at 37 ° C. for 1 hour. The following buffers were used: pH 3. 2 to pH 6.2 sodium acetate monoacetate buffer: pH 6.1 to pH 8.2 MOPS-sodium carbonate buffer: pH 8.8 to ΗΙ水 素 1. In case of 1, sodium bicarbonate sodium bicarbonate buffer: in case of pH 9. 6 to pH 13. 3 hydrogen phosphate 2 sodium mousse sodium hydroxide buffer. To a mixture of 40 mM acetic acid buffer solution (pH 5, 5) 100 / and chitosan 7 B solution 160 1 prepared in Example 1 and water 70 a 1, add heated enzyme mixture 70 a 1 and stir. 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. As shown in FIG. 17, the purified enzyme has a pH of 5 to pH 9. It was stable at 5.
5) 精製酵素の基質特異性  5) Substrate specificity of the purified enzyme
精製酵素の種々の基質に対する加水分解活性を実施例 5の方法にしたがつて測定した。 伹し酵素反応は、 pH5. 5及び ρΗΙ Ο. 0で行なった。 キトサン 7 Bに対する精 製酵素の加水分解活性を 100%としたときの、他の各基質に対する精製酵素の加水 分解活性を相対値として表 13に記載した。 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 ΗΙ Ο 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%.
[表 13] , 相対活性 （％) [Table 13], Relative activity (%)
PH5. 5 H 10. 0 キトサン 10B 2. 3 0 PH5. 5 H 1 0 0 Chitosan 10 B 2. 3 0
キトサン 9B 62. 8 29. 1 Chitosan 9B 62. 8 29. 1
キ卜サン 8B 83, 7 44. 6 Yellow 8B 83, 7 44. 6
キトサン 7 B 100 100 Chitosan 7 B 100 100
グリコ一ルキトサン 0 2. 1 Glycol chitosan 0 2. 1
グリコ一ルキチン 28 3 64. 2 Glycol ruchitin 28 3 64. 2
CM—セルロース 0 0  CM—cellulose 0 0
リケナン 0 9 3. 6 表 11に示す通り、 精製酵素はキトサン 7 Bに対して最も加水分解活性が高く、 ァ セチル化度が小さくなるにつれて加水分解活性は低下した。 また、 精製酵素はキチン 加水分解活性を有し、 セルラーゼ加水分解活性は見られなかった。 試験例 4. ァスペルギルス ·ジャポニクス SANK19288株由来のキチナ一 ゼ粗酵素液の諸性質 Likenan As shown in Table 11, 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.
1) 実施例 1 2) 記載の方法にしたがって、 還元糖の定量を行った。 キトサン 7 B を基質として測定した場合、 上清中 lmlあたり 3. 4X 10—3Uの酵素活性を有し ていた。 キトサン 7Bを基質としたときの酵素活性を 100%としたときの、 キトサ ン 10 B分解活性の相対値を表 14に示す。 1) Example 1 2) According to the method described, quantification of reducing sugars was performed. 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.
[表 14] [Table 14]
相対活性 (%) キトサン 7 B分解活性 100 Relative activity (%) Chitosan 7 B degradation activity 100
キトサン 10B分解活性 1. 5 キトサン 7 B分解活性を有し、 キトサン 10B分解活性がほとんどないことから、 上 清中にはキチナーゼが生産されていることが判明した。 Chitosan 10B degradation activity 1.5 Chitosan 7B degradation activity was observed, and it was found that chitinase was being produced in the supernatant, because chitosan 10B degradation activity was scarce.
2) 粗酵素液の pH活性  2) pH activity of crude enzyme solution
実施例 1 2)記載の方法にしたがって調製したキトサン 7 B溶液 160 1および 40 OmM緩衝液 100 1の混合液に粗酵素液 140 x 1を加え撹拌して均一に し、 37 °Cで保温して 20分間酵素反応を行い、 酵素反応後の遊離還元糖の定量を行 なった。 緩衝液は次のものを用いた： PH3. 7乃至 pH6. 0の場合、 酢酸—酢酸 ナトリウム緩衝液： PH6. 0乃至 pH8. 5の場合、 MOPS—炭酸ナトリウム緩 衝液： pH9. 3乃至 ρΗΙ Ο. 4の場合、 炭酸水素ナトリウム—炭酸ナトリウム緩 衝液。 最も活性が高かった pH条件での加水分解活性を 100%とし、 各 pHにおけ る酵素の加水分解活性を相対値として図 18に記載した。 至適 pHは pH4. 0付近 であった。 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 乃至In the case of 4, sodium bicarbonate-sodium carbonate buffer. Assuming that 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.
3) 粗酵素の基質選択性  3) Substrate selectivity of crude enzyme
PH4. 0における基質選択性を測定した。 実施例 2で得られた調製した培養上清 1 0m 1を 1 OmMトリス '塩酸緩衝液 （pH7. 5) 1， 000 mlに対して 12 時間ごとに 3回透析したものを酵素液として用いた。 0. 25% wt/vの基質溶 l Q 0 il lおよび 40 OmM酢酸緩衝液 （pH4. 0) および水 160 Iの混合 液に酵素液 50 1を加え撹拌して均一にし反応を開始し、 37°Cで保温して 10分 間酵素反応を行った。 酵素反応後の遊離還元糖の定量はシェ一ルズ法 （Imoto, T. and Yagashita, K.， Agric. Biol. Chem. , 35, 1154(1971)参照）により行つ た。 最大活性を示す基質を用いたときの加水分解活性を 100%としたときの、 相対 活性を表 15に示す。  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.
[表 15] 相対活性 （％) キトサン 10 B 8 2 [Table 15] Relative activity (%) Chitosan 10 B 8 2
キトサン 9B 67, 6 Chitosan 9B 67, 6
キトサン 8 B 74 4 Chitosan 8 B 74 4
キ卜サン 7B 00 Yellow 7B 00
グリコールキトサン 8 2 Glycol chitosan 8 2
グリコ一ルキチン 23 3 Glycol ruchitin 23 3
CM—セルロース 11 4 リケナン 2 5. 3 CM—Cellulose 11 4 Lickenan 2 5. 3
試験例 5. ァスペルギルス 'ソジヤエ SANK 22388株由来のキチナーゼ 粗酵素液の諸性質 Test Example 5. Properties of Chitinase Crude Enzyme Solution Derived from Aspergillus' Sojiyae Strain SANK 22388
1) 加水分解活性 ·  1) Hydrolytic activity ·
実施例 1 2) に記載の方法にしたがって還元糖の定量を行った。 キトサン 7 Bを基 質として測定した場合、 上清中 lm lあたり 2. 6X 10—3Uの酵素活性を有してい た。 キトサン 7 Bを基質としたときの酵素活性を 100 %としたときの、 キトサン 1 0 B分解活性の相対値を表 16に示す。 The reducing sugar was quantified according to the method described in Example 1 2). 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.
[表 16] [Table 16]
相対活性 （％) キトサン 7 B分解活性 100 Relative activity (%) Chitosan 7 B degradation activity 100
キトサン 10B分解活性 0 Chitosan 10B degradation activity 0
キトサン 7 B分解活性を有し、 キトサン 10 B分解活性がほとんどないことから、 上清中にはキチナ一ゼが生産されていることが判明した。 ' It has been found that chitinase is produced in the supernatant, since it has chitosan 7 B degradation activity and almost no chitosan 10 B degradation activity. '
2) 培養上清中の酵素の pH活性  2) pH activity of the enzyme in the culture supernatant
測定法は実施例 6にしたがつた。緩衝液は次のものを用いた： p H 3. 8乃至 p H 6. 3の場合、 酢酸一酢酸ナトリウム緩衝液： PH6. 2乃至 pH8. 6の場合、 MOP S—炭酸ナトリウム緩衝液： pH9. 3乃至 ρΗΙ Ο. 4の場合、 炭酸水素ナトリウ ムー炭酸ナトリウム緩衝液。 最も活性が高かった pH条件での加水分解活性を 10 0%とし、 各 pHにおける酵素の加水分解活性を相対値として図 1 9に記載した。 至 適 pHは pH4. 5付近であった。 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 ΗΙ Ο. 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.
3) 培養上清中の酵素の基質選択性  3) Substrate selectivity of enzyme in culture supernatant
PH4. 5における基質選択性を測定した。 実施例 4で調製した培養上清 10m 1を 1 OmMトリス ·塩酸緩衝液（pH7. 5) 1, 000 m 1に対して 12時間ごとに 3 回透析したものを酵素液として用いた。 0. 2 5%wtZvの基質溶液 1 6 O 1お よび 400 mM酢酸緩衝液（ρ Η 4.5)および水 160 ^ 1の混合液に酵素液 50 1を加え撹拌して均一にし反応を開始し、 37°Cで保温して 10分間酵素反応を行つ た。 酵素反応後の遊離還元糖の定量はシエールズ法 （Imoto, T. and Yagashita, K., Agric. Biol. Chem. , 35, 1154(1971)参照） により行った。 最大活性を示 P0212940 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 (Η Η 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
54 す基質を用いたときの加水分解活性を 100%としたときの、相対活性を表 17に示 す。  The relative activity is shown in Table 17 when the hydrolysis activity with 100 mg of the substrate is 100%.
[表 17] . 相対活性 (%) キトサン 10B 0  [Table 17] Relative activity (%) Chitosan 10B 0
キトサン 9 B 67. 7 Chitosan 9 B 67. 7
キトサン 8 B 71. 6 Chitosan 8 B 71.6
キトサン 7B 100 Chitosan 7B 100
ダルコールキトサン 2. 9 Dalcor Chitosan 2.9
グリコ一ルキチン 81. 9 Glycol ruchitin 81.9
CM—セルロース 0  CM—cellulose 0
リケナン 11. 9 Lickenan 11. 9
試験例 6. 各低分子キトサン塩酸塩の水に対する溶解性 Test Example 6. Water solubility of each low molecular weight chitosan hydrochloride
実施例 4〜 6にて調製した各低分子キトサン塩酸塩 10 Omgを水 1 Om 1に溶か し、 pHを測定した。 この溶液に 1 N苛性ソーダ水を少しずつ加え、 溶液が白濁して コロイドが析出する pHを測定した。 結果を表 18に示す。 10 mg of each low molecular weight chitosan hydrochloride prepared in Examples 4 to 6 was dissolved in 1 Om 1 of water, and the pH was measured. To this solution was added 1 N caustic soda solution little by little, and the solution became cloudy, and the pH was measured to precipitate colloid. The results are shown in Table 18.
[表 18] ' [Table 18] '
.1 %水溶液の pH コロイド析出 pH 実施例 4 5 6 4 5 6 低分子キトサン 9 B塩酸塩 4. 9 5. 6 4. 9 6. 7 6. 6 6. 8 低分子キトサン 8 B塩酸塩 4. 9 5. 5 5. 2 7. 1 7. 0 7. 1 低分子キトサン 7 B塩酸塩 5. 0 5. 5 5. 4 7. 3 7. 4 7. 4 .1% aqueous solution pH colloidal precipitation pH Example 4 5 6 4 5 6 low molecular weight chitosan 9 B hydrochloride 4. 9 5. 6 4. 6 6. 7 6. 6 6. 8 low molecular chitosan 8 B hydrochloride 4 9 5. 5 5. 2 7. 1 7. 0 7. 1 Low molecular weight chitosan 7 B hydrochloride 5. 0 5. 5 5. 4 7. 3 7. 4 7. 4
試験例 7. 高分子キトサン塩酸塩の調製 Test Example 7. Preparation of Polymeric Chitosan Hydrochloride
キトサン 9B、 1 gを 20 Om 1の水に懸濁し、 濃塩酸で pH2. 5に合わせて、 完 全に溶解した。 これを水に対して透析後、 凍結乾燥を行い、 537mgの白色綿状固 体として、 高分子キトサン 9 B塩酸塩が得られた。 高分子キトサン 10B及び 8Bお よび 7 B塩酸塩も同じ方法で調製した。各高分子キトサン塩酸塩 10 Omgを蒸留水 50mlに溶解し、 キトサナ一ゼ 20U (和光純薬 （株） 製） を加えて室温で 3時間 攪拌した。反応溶液を凍結乾燥し、乾燥固体を D2〇に溶かして1 H— NMRを測定し た。 . 1 g of chitosan 9B 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 〇. .
ァセチル基の 3 Hとァセチル基以外の、 水酸基を除く 7 Hとの積分比から、 ァセチル 化度を算出した。 結果を表 19に示す。 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.
[表 19] ァセチル化度 (%) 高分子キトサン 10B塩酸塩 く 1 [Table 19] Degree of asset (%) Polymeric chitosan 10B hydrochloride 1
高分子キトサン 9 B塩酸塩 14 High molecular weight chitosan 9 B hydrochloride 14
高分子キトサン 8 B塩酸塩 23 High molecular weight chitosan 8 B hydrochloride 23
高分子キトサン 7 B塩酸塩 34 High molecular weight chitosan 7 B hydrochloride 34
試験例 8. 低分子キトサンの抗菌活性 Test Example 8. Antibacterial Activity of Low Molecular Weight Chitosan
被検菌としてス夕フイロコッカス 'ァゥレウス （Staphylococcus aureus) 209P JC-1株及びシユードモナス ·ァエルギノーサ （Pseudomonas aeruginosa) PA01株 を被検菌とし、 試験例 7で作製した高分子キトサン塩酸塩並びに実施例 4、 実施例 5 および実施例 6にて各種ァスペルギルス族由来のキチナ一ゼを用いて調製した低分 子キトサン塩酸塩の MI Cを測定した。 結果は表 18に示した。 ' Polymer chitosan salt salt prepared in Test Example 7 with the test bacteria bacteria 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. '
使用培地  Medium used
Mueller Hinton Broth (Difco (株) 製） を用い、 いずれも pH6. 5に調整した。 試料菌液の調製  All were adjusted to pH 6.5 using Mueller Hinton Broth (manufactured by Difco Co., Ltd.). Preparation of sample bacteria liquid
試験に使用する菌を上記培地でー晚培養し、 菌数が 2 X 106になるように同培地で 希釈した。 The bacteria to be used for the test were cultured in the above culture medium for 晚, and diluted with the same culture medium so that the number of bacteria was 2 × 10 6 .
検体の調製 Sample preparation
各低分子キトサン塩酸塩 2 Omgを蒸留滅菌水に溶かし、 1 Omlとした。 Each low molecular weight chitosan hydrochloride 2 Omg was dissolved in distilled sterile water to make 1 Oml.
試験方法  Test method
あらかじめ蒸留滅菌水を希釈液とし、 検体溶液の倍数希釈系列を作製しておき、 これ に上記培地 50 1及び試験菌液 50 1を加え、 37 °Cで 18時間培養し、 M I C を判定した。 結果は表 20に示した。 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.
[¾20]  [3⁄420]
M I C ( g/raL) スタフイロコッカス シユードモナス MIC (g / raL) Staphylococcus siudomonas
•ァゥレウス •ァエルギノ一サ 高分子キ卜サン 9 B塩酸塩 (試験例 7) 15 6 5 高分子キトサン 8 B塩酸塩 (試験例 7) 31 2 5 高分子キ卜サン 7 B塩酸塩 (試験例 7) 31 2  • Aureus • Aeruginosa Polymer Xanthine 9 B Hydrochloride (Test Example 7) 15 6 5 Polymer Chitosan 8 B Hydrochloride (Test Example 7) 31 2 5 Polymer Xyzsan 7 B Hydrochloride (Test Example 7) 31 2
低分子キトサン 9 B塩酸塩 (実施例 4) 15 6 5 低分子キトサン 8 B塩酸塩 (実施例 4) 15 6 5 低分子キトサン 7 B塩酸塩 (実施例 4) 31 2 Low Molecular Weight Chitosan 9 B Hydrochloride (Example 4) 15 6 5 Low Molecular Weight Chitosan 8 B Hydrochloride (Example 4) 15 6 5 Low Molecular Weight Chitosan 7 B Hydrochloride (Example 4) 31 2
低分子キトサン 9 B塩酸塩 (実施例 5) 15 6 2 低分子キトサン 8 B塩酸塩 (実施例 5) 15 6 5 低分子キトサン 7 B塩酸塩 (実施例 5 ) 31 2 Low Molecular Weight Chitosan 9 B Hydrochloride (Example 5) 15 6 2 Low Molecular Weight Chitosan 8 B Hydrochloride (Example 5) 15 6 5 Low Molecular Weight Chitosan 7 B Hydrochloride (Example 5) 31 2
低分子キトサン 9 B塩酸塩 (実施例 6) 15 6 2 低分子キトサン 8 B塩酸塩 (実施例 6) 15 6 2 低分子キトサン 7 B塩酸塩 (実施例 6) 31 2
Figure imgf000057_0001
5 Low Molecular Weight Chitosan 9 B Hydrochloride (Example 6) 15 6 2 Low Molecular Weight Chitosan 8 B Hydrochloride (Example 6) 15 6 2 Low Molecular Weight Chitosan 7 B Hydrochloride (Example 6) 31 2
Figure imgf000057_0001
Five
試験例 9. ァスペルギルス ·ジャポニクス SANK 1928 1118株由来の精製キ チナーゼの諸性質 222252552111 Test Example 9. Properties of purified chitinase derived from Aspergillus japonics strain SANK 1928 1118 222252552111
実施例 2. 2) で得られたァスペルギルス ·ジャポニクス SANK 19.288 株由来の精製キチナ一ゼの諸性質を調べた。  Various properties of purified chitinase derived from Aspergillus japonics SANK 19.288 strain obtained in Example 2. 2) were examined.
1) ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチナ一 ゼが有する加水分解活性の p H活性  1) The pH activity of the hydrolytic activity possessed by the purified chitinase from Aspergillus sp. SANK 19288 strain
ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチナ一ゼが 有する加水分解活性の PH活性を、 試験例 3 1) に記載の方法により測定した。 最 も活性が高かった pH条件での加水分解活性を 100 %とし、 各 pHにおける酵素の. 加水分解活性を相対値として図 20に記載した。至適 pHは pH5. 0付近であった。  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.
2) ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチナ一 ゼが有する加水分解活性の温度活性 '  2) Temperature activity of hydrolysis activity of purified chitinase from Aspergillus sp. Strain SANK 19288
ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチナーゼが 有する加水分解活性の温度活性を、 試験例 3 2) に記載の方法により測定した。 最 も活性が高かった温度条件での加水分解活性を 100%とし、各温度における酵素の 加水分解活性を相対値として図 2 1にまとめた。 至適温度は PH5. 0において 6 5°Cであった。  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.
3) ァスペルギルス 'ジャポニクス SANK 19288株由来の精製キチナ一 ゼの温度安定性 ァスペルギルス ·ジャポニクス SANK 19288株'由来の精製キチナーゼの 温度安定 を、 試験例 3 3) に記載の方法により測定した。 無処置 （0°C保温） 群 の該精製キチナ一ゼおよび緩衝液の混合液が有する加水分解活性を 100 %とし、各 温度における酵素の加水分解活性を相対値として図 22にまとめた。該精製キチナ一 ゼは 50°C以下で安定であった。 3) Thermal stability of purified chitinase from Aspergillus' Japonics strain SANK 19288 The temperature stability of the purified chitinase derived from Aspergillus japonicus strain SANK 19288 'was measured by the method described in Test Example 3 3). The hydrolytic 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 hydrolytic activity of the enzyme at each temperature is summarized in FIG. The purified chitinase was stable at 50 ° C. or less.
4) ァスペルギルス 'ジャポニクス SANK 19288株由来の精製キチナ一 ゼの PH安定性  4) PH stability of purified chitinase from Aspergillus' Japonics strain SANK 19288
ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチナーゼの pH安定性を、 試験例 3 4) に記載の方法により測定した。 無処置 （0°C保温） 群 の該精製キチナ一ゼおよび緩衝液の混合液が有する加水分解活性を 100 %とし、各 pHにおける酵素の加水分解活性を相対値として図 23にまとめた。該精製キチナ一 ゼは PH4乃至 pH9. 5で安定であった。  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.
5) ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチナ一 ゼの基質特異性  5) Substrate specificity of purified chitinase from Aspergillus japonics strain SANK 19288
ァスペルギルス'ジャポニクス SANK 19288株由来の精製キチナーゼの 基質特異性を、試験例 3 5)に記載の方法により測定した。キトサン 7Bに対する、 PH5. 0、 37°C、 10分間の条件における該精製キチナ一ゼの加水分解活性を 1 00%としたときの、他の各基質に対する該精製キチナ一ゼの加水分解活性を相対値 として表 21に示した。 21] 相対活性 （％) キトサン 10B 0  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
キトサン 9B 58 6 Chitosan 9B 58 6
キトサン 8B 72 4 Chitosan 8B 72 4
キトサン 7B 100 Chitosan 7B 100
ダルコールキトサン 0 Dalcor Chitosan 0
グリコールキチン 28 3 Glycol Chitin 28 3
CM—セル口一ス 0  CM-Cell Pass 1
4 2  4 2
表 21に示すとおり、 ァスペルギルス 'ジャポニクス SANK 19288株由 来の精製キチナ一ゼはキトサン 7 Bに対して最も加水分解活性が高く、 ァセチル化度 TJP02/12940 As shown in Table 21, 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
58 が小さくなるにつれて加水分解活性は低下した。 また、 該精製キチナ一ゼはキチン加 水分解活性を有し、 セルラーゼ加水分解活性は見られなかつた。  The hydrolysis activity decreased as 58 decreased. In addition, the purified chitinase had chitin hydrolysis activity, and no cellulase hydrolysis activity was observed.
6) ァスペルギルス 'ジャポニクス SANK 19288株由来の精製キチナ一 ゼの部分アミノ酸配列の決定  6) Determination of partial amino acid sequence of purified chitinase from Aspergillus' Japonics strain SANK 19288
ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチナ一ゼの 部分アミノ酸配列の決定を、 実施例 1. 7) 記載の方法でおこなった。 分離した分解 アミノ酸のうち、 B buffer濃度 28%程度、 30 %程度、 35%程度の 3つのピ一 クを分取した。 この 3 つについてアミノ酸配列解析装置 （Procise cI 、 Applied Biosystem社製） で配列を解析した。 結果を N末端側から記す。  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.
His-Tyr-Pro-Thr-Asp-Ser-Trp-Asn-Asp-Val-Gly-Thr-Asn-Val-Tyr (配列表の配列番 号 11) His-Tyr-Pro-Thr-Asp-Ser-Trp-Asn-Asp-Val-Gly-Thr-Asn-Val-Tyr (SEQ ID NO: 11)
Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (配列表の配列番 号 12)  Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (SEQ ID NO: 12)
Leu-Ser-Gln-Met-Thr-Pro-Tyr-Leu-Asp-Phe-Tyr-Asn-Leu-Met-Ala-Tyr-Asp-Tyr-Ala -Gly (配列表の配列番号 13) 試験例 10. ァスペルギルス 'ソジヤエ SANK 22388株由来の精製キチ ナーゼの諸性質  Leu-Ser-Gln-Met-Thr-Pro-Tyr-Leu-Asp-Phe-Tyr-Asr-Let-Leu-Met-Ala-Tyr-Asp-Tyr-Ala-Gly (Sequence Listing SEQ ID NO: 13) Test Example 10 Properties of the purified chitinase from Aspergillus' Sojiyae strain SANK 22388
実施例 3. 2) で得られたァスペルギルス 'ソジヤエ SANK 22388株由 来の精製キチナ一ゼの諸性質を調べた。  Various properties of purified chimeras derived from Aspergillus sp. 'Sojiyae SANK 22388 strain obtained in Example 3.2) were examined.
1) ァスペルギルス 'ソジヤエ SANK 22388株由来の精製キチナ一ゼが 有する加水分解活性の P H活性  1) PH activity of hydrolytic activity possessed by purified chitinase derived from Aspergillus' Sojiyae SANK 22388 strain
ァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナーゼが有す る加水分解活性の： H活性を、 試験例 3. 1) に記載の方法により測定した。 最も活 性が高かった pH条件での加水分解活性を 100%とし、各 pHにおける酵素の加水 分解活性を相対値として図 24に記載した。 至適 pHは pH 5. 5および pH9. 0 付近であった。  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.
2) ァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナーゼが 有する加水分解活性の温度活性  2) temperature activity of hydrolytic activity possessed by purified chitinase derived from Aspergillus sogyae strain SANK 22388
ァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナ一ゼはァス ペルギルス ·オリザェおよびァスペルギルス ·ジャポニクス由来のキチナ一ゼと異な り、 至適 pHが 2点存在する （試験例 10 1) 参照） ため、 ァスペルギルス■ソジ ヤエ SANK 22388株由来の精製キチナ一ゼの温度活性はこの 2点の pH において、 試験例 3 2) に記載の方法により測定した。 最も活性が高かった温度条 件での加水分解活性を 100%とし、各温度における酵素の加水分解活性を相対値と して図 25 (pH 5. 5における温度活性） および図 26 (pH9. 0における温度 活性）にまとめた。至適温度は pH 5. 5において 60°C、 pH9. 0において 35°C であった。 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) and Figure 26 (pH 9.0), where the hydrolysis activity under the temperature condition where the activity was the highest was 100% and the hydrolysis activity of the enzyme at each temperature was a relative value. Temperature at Active). The optimum temperature was 60 ° C. at pH 5.5 and 35 ° C. at pH 9.0.
3) ァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナーゼの 温度安定性  3) Thermal stability of purified chitinase derived from Aspergillus sogyae strain SANK 22388
ァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナーゼはァス ペルギルス ·オリザェおよびァスペルギルス ·ジャポニクス由来のキチナ一ゼと異な り、 至適 pHが 2点存在する （試験例 10 1) 参照） ため、 ァスペルギルス ·ソジ ヤエ SANK 22388株由来の精製キチナーゼの温度安定性はこの 2点の p Hにおいて、 試験例 3. 3) に記載の方法により測定した。 無処置 （0°C保温） 群の 該精製キチナーゼおよび緩衝液の混合液が有する加水分解活性を 100%とし、各温 度における酵素の加水分解活性を相対値として図 27 (pH5. 5における温度安定 性） および図 28 (pH9. 0における温度安定性） にまとめた。 該精製キチナーゼ はいずれの pHにおいても 40°C以下で安定であった。  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.
4) ァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナ一ゼの pH安定性  4) pH stability of purified chitinase from Aspergillus spp. SANK 22388
ァスペルギルス'ソジヤエ SANK 22388株由来の精製キチナ一ゼの pH 安定性を試験例 3. 4) に記載の方法により測定した。 無処置 （0°C保温） 群の該精 製キチナーゼおよび緩衝液の混合液が有する加水分解活性を 100%とし、 各 pHに おける酵素の加水分解活性を相対値として図 29にまとめた。該精製キチナ一ゼは p H4. 5乃至 pH 10で安定であった。  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.
5) ァスペルギルス 'ソジヤエ SANK 22388株由来の精製キチナ一ゼの ァスペルギルス'ソジヤエ SANK 22388株由来の精製キチナ一ゼの基質 特異性を試験例 3. 5) に記載の方法により測定した。 キトサン 8Bに対する、 pH 5. 0、 37°C、 10分間の条件における該精製キチナーゼの加水分解活性を 100% としたときの、他の各基質に対する該精製キチナーゼの加水分解活性を相対値として 表 22に示した。  5) Purification of the purified chitinase derived from Aspergillus 'Sojiae SANK 22388 strain The substrate specificity of the purified chitinase derived from Aspergilli' s soy bean SANK 22388 was measured by the method described in Test Example 3.5). When the hydrolysis activity of the purified chitinase at pH 5.0, 37 ° C., 10 minutes to the chitosan 8 B is 100%, the hydrolysis activity of the purified chitinase to each of the other substrates is shown as a relative value. 22 shows.
[表 22] 相対活性 （％) キトサン 10B 14. 3 [Table 22] Relative activity (%) Chitosan 10B 14. 3
キトサン 9B 73. 6 Chitosan 9B 73.6
キトサン 8 B 00 Chitosan 8 B 00
キトサン 7 B 97. 4 0 Chitosan 7 B 97.4 0
60 ダルコ一ルキトサン 0  60 Darukol Chitosan 0
グリコールキチン 23. 8 Glycol Chitin 23. 8
CM—セルロース 0  CM—cellulose 0
リケナン 0 Likenan 0
表 22に示すとおり、 ァスペルギルス 'ソジヤエ SANK 22388株由来の 精製キチナーゼはキトサン 7Bおよび 8Bに対して最も加水分解活性が高く、 ァセチ ル化度が小さくなるにつれて加水分解活性は低下した。 また、 該精製キチナ一ゼはキ チン加水分解活性を有し、 セルラーゼ加水分解活性は見られなかつた。 製剤例 1. 5%キトサン軟膏製剤の作製 As shown in Table 22, 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
プロピレンダリコール 3.0g、パラォキシ安息香酸メチル 0. lgの精製水 80ml溶液を 70°Cに加温し、 実施例 4にて調製した低分子キトサン 9 B塩酸塩 5.0gを加えて溶解 し、 低分子キトサン 9 B溶液とした。 ステアリン酸 3.0g、 セタール 1.0g、 モノステ ァリン酸グリセリン 6.0g流動パラフィン 5.0g、パラォキシ安息香酸プロピル 0. lg、 ステアリン酸ポリオキシル 40 2.5gを 70°Cにて加温溶解後、 同温度で上記の低分子 キトサン 9 B溶液を加え、 精製水をくわえて全量を lOOgとし、 混合乳化した。 乳化 後攪拌しながら室温まで冷却し、 白色の 5%低分子キトサン 9 B塩酸塩クリーム (5% キトサン軟膏と呼ぶ） を lOOg得た。 試験例 10. 5 %キトサン軟膏の外傷治癒試験  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. After dissolving 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. and heating at 70 ° C., Low-molecular-weight chitosan 9 B solution was added, and purified water was added to make the total amount 100 g, and mixed and emulsified. After emulsification, the mixture was cooled to room temperature with stirring to obtain 100 g of white 5% low molecular weight chitosan 9B hydrochloride cream (referred to as 5% chitosan ointment). Test Example 10. Trauma healing test of 5% chitosan ointment
ラット皮膚欠損傷モデルでの 5%キトサン軟膏の有効性を予知する目的で外傷治癒 試験を行なった。  A trauma healing test was conducted to predict the efficacy of 5% chitosan ointment in a rat skin defect model.
1) 試験材料 1) Test material
被験物質としては、 製剤例 1で調製した 5%キトサン軟膏を用いた。 対照薬として は、 ユーパスタ軟膏 （100 g中に精製白糖 70 g、 ポピドンョ一ド 3 gを含有、 添 加物としてポリエチレングリコール、 濃グリセリン、 ポリオキシエチレン [160]ポ リオキシプロピレン [30]グリコール、 プルラン、 ヨウ化カリウムを含有、 製造番号 BA1S、 興和株式会社製） を使用した。 試験に用いる動物としては、 6週齢の Sprague-Dawley (SD) 系雄性ラット （日本エスエルシー株式会社） を購入して使用し た。 動物は平均温度 23°C、 平均湿度 55%の環境制御飼育装置 （日本クレア株式会社） で固形飼料 （マウス ·ラット飼育用 FR-2、 株式会社船橋農場） および水道水を与え、 照明時間 7時— 19時の条件下で 6日間馴化飼育管理を行い実験に用いた。 0212940 As the test substance, the 5% chitosan ointment prepared in Preparation Example 1 was used. As a control drug, 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.) was used. As 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
61  61
2) 実験方法 2) Experimental method
7週齢の SD系雄性ラット （体重 157.6g乃至 235.9g) を 1群 5匹で 6群用いた。 ラ ットの背部被毛を電気バリカンで除毛した後、， EBAエバクリーム （東京田辺製薬株式 会社製） を塗布し、 1 5分後に洗浄して脱毛した。 背部脱毛部位を 7 0%エタノール で消毒後、 ペントパルビタール 'ナトリウム （40mg/kg， に p.) 麻酔下に円形ポンチ (内径 15min)を用いて背部正中線で対称の打ち抜き創を 2個所作製した。欠損傷作製 24時間後に無処置群、 基剤群、被験物質群および対照薬群に各群 5匹で群分けし、 そ の後被験物質および対照薬を欠損傷部 2箇所に、 約 0. lg/部位、 一日 2回、 1 0日間 塗布した。 ラットは実験期間中、 個別ケージで飼育した。 効果の判定はノギスを用い て欠損傷部の長径と短径を測定した。  Seven-week-old SD male rats (body weight 157.6 g to 235.9 g) were used in six groups of five animals per group. After the hair on the back of the rat was removed with an electric clipper, EBA Eve cream (manufactured by Tokyo Tanabe Seiyaku Co., Ltd.) was applied, and after 15 minutes, it was washed and depilated. After disinfecting the back hair loss site with 70% ethanol, make two punched wounds symmetrical on the back midline using circular punch (15 min inside diameter) under anesthesia with pentoparbital 'sodium (40 mg / kg, p.) did. Preparation of Defective Wound 24 hours after the treatment group is divided into no treatment group, base group, test substance group and control drug group with 5 animals each, and then test substance and control drug are placed in 2 parts of defect wound area, approximately 0. The site was applied twice a day for 10 days. The rats were kept in individual cages for the duration of the experiment. The evaluation of the effect was performed by using a caliper to measure the major axis and minor axis of the defect.
治療過程における面積変化は、 （測定日の長径 X短径 欠損傷作製後の長径 X短 径） X100の式で面積比 ( ) を求め、 面積比から治療面積 （5-15日目） を、 完治率 は面積比が 5%以下を完治例とし治療日数を算出し、 表 23に示した。 実験結果は平 均値土標準偏差で表した。 無処理群の 13.7±1.3 日に対して、 5%キトサン軟膏群は 12.6±1.9日と治療日数を短縮した。 対照薬群では 13.6±0.4日と治療日数の改善は みられなかった。  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.
[表 2 3]ラット皮膚欠損傷モデルの治療日数 被験物質 治療日数 無処置 13.7±1.3 [Table 2 3] The number of days of treatment for rat skin defect wound model Test substance Treatment days No treatment 13.7 ± 1.3
5%キトサン 12.6±1.9  5% chitosan 12.6 ± 1.9
対照薬 13.6±0.4 Control drug 13.6 ± 0.4
[産業上の利用の可能性] [Possibility of industrial use]
以上述べたように、 本発明のキチナーゼを用いる事により、 分子量 1万以上で、 中 性条件でも可溶な、 本発明の低分子キトサン製造することができる。 更に本発明の低 分子キトサンは優れた抗菌性および外傷治癒効果を示し、本発明の低分子キトサンを 有効成分として含有する医薬組成物は、 外傷、 床ずれ、 アトピー性皮膚炎などの治療 剤として有用である。 また、 本発明のキチナーゼを用いて、 高分子ァセチル化キトサ ンを含有することにより高い粘度を示す試料を原料として、粘度の低い扱い易い試料 を製造することができる。 [配列表フリ一ワード] As described above, by using the chitinase of the present invention, it is possible to produce 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. Moreover, by using the chitinase of the present invention, by using a polymeric acetylated chitosan, it is possible to produce an easy-to-handle sample having a low viscosity, using a sample exhibiting high viscosity as a raw material. [Sequence chart free one word]
配列番号 6 キチナ一ゼ遺伝子プローブ SEQ ID NO: 6 chitinase gene probe
配列番号 7 PCRプライマー F21 SEQ ID NO: 7 PCR primer F21
配列番号 8 PCRプライマー PR50 SEQ ID NO: 8 PCR primer PR50
配列番号 9 PCRプライマー BamHI- cDNA SEQ ID NO: 9 PCR primer BamHI-cDNA
配列番号 10 ： P C Rプライマー cDNA- BamHI [図面の簡単な説明] SEQ ID NO: 10: PCR primer cDNA-BamHI [Brief description of the drawing]
図 1は、 ァスペルギルス 'オリザェ var. sporoflavus Ohara J CM2067由来 の精製キチナーゼの分子量を示す。 a〜； f のプロットはマーカ一であり、 矢印に示す 点は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由来の精 製キチナ一ゼである。 図 2は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由来 のキチナーゼを用いて作製した低分子キトサンの分子量分布を示す。図 3 Aは低分子 キトサン 9 B塩酸塩の分布を示し、図 2 Bは低分子キトサン 8 B塩酸塩の分布を示し、 図 2 Cは低分子キトサン 7 B塩酸塩の分布を示す。 各グラフにおいて、 ァスペルギル ス ·オリザェ var. sporoflavus Ohara J CM2067由来のキチナ一ゼを用いて作 製した低分子キトサンの分布は実線に塗りつぶしの円で示し、点線は全てマーカー分 子の分布を示す （塗りつぶし小さい円：分子量 10000の低分子キトサン、 塗りつぶし 四角：分子量 40000の低分子キトサン、 塗りつぶし三角：分子量 70000の低分子キト サン、 白抜き円：分子量 110000の低分子キトサン、 白抜き四角：分子量 250000の低 分子キトサン） 。 ' 図 3は、 ァスペルギルス ·ジャポニクス SANK19288株由来のキチナーゼ を用いて作製した低分子キトサンの分子量分布を示す。図 3 Aは低分子キトサン 9 B 塩酸塩の分布を示し、 図 3 Bは低分子キトサン 8 B塩酸塩の分布を示し、 図 3 Cは低 分子キトサン 7 B塩酸塩の分布を示す。 各グラフにおいて、 ァスペルギルス ·ジャポ ニクス SANK1 9288株由来のキチナーゼを用いて作製した低分子キトサン の分布は実線に塗りつぶしの円で示し、 点線は全てマーカー分子の分布を示す （塗り つぶし小さい円：分子量 10000の低分子キトサン、 塗りつぶし四角：分子量 40000の 低分子キトサン、 塗りつぶし三角：分子量 70000の低分子キトサン、 白抜き円：分子 量 110000の低分子キトサン、 白抜き四角：分子量 250000の低分子キトサン） 。 図 4は、 ァスペルギルス ·ソジヤエ SANK 22388株の培養上清中のキチナ PC画 2/12940 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. 3A shows the distribution of low molecular weight chitosan 9B hydrochloride, FIG. 2B shows the distribution of low molecular weight chitosan 8 B hydrochloride, and FIG. 2C shows the distribution of low molecular weight chitosan 7B hydrochloride. In each graph, the distribution of low-molecular-weight chitosan produced by using a chitinase derived from Aspergillus oryzae var. 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, and Fig. 3C shows the distribution of low molecular weight chitosan 7B hydrochloride. In each graph, the distribution of low molecular weight chitosan prepared by using chitinase derived from Aspergillus japonicus SANK1 9288 strain is indicated by a solid circle in a solid line, and all dotted lines indicate the distribution of marker molecules (small circle: molecular weight 10000) Low molecular weight chitosan, Filled square: Low molecular weight chitosan with molecular weight 40000, Filled triangular: Low molecular weight chitosan with molecular weight 70000, Open circle: Low molecular weight chitosan with molecular weight 110000, Open square: Low molecular chitosan with molecular weight 250000). Fig. 4 shows chitina in the culture supernatant of Aspergillus sogyae strain SANK 22388. PC drawing 2/12940
63 ーゼを用いて作製した低分子キトサンの分子量分布を示す。図 4 Aは低分子キトサン 9 B塩酸塩の分布を示し、 図 4 Bは低分子キトサン 8 B塩酸塩の分布を示し、 図 4C は低分子キトサン 7 B塩酸塩の分布を示す。 各グラフにおいて、 ァスペルギルス 'ソ ジヤエ SANK 22388株の培養上清中のキチナ一ゼを用いて作製した低分子 キトサンの分布は実線に塗りつぶしの円で示し、点線は全てマーカー分子の分布を示 す （塗りつぶし小さい円：分子量 10000の低分子キトサン、 塗りつぶし四角：分子量 40000の低分子キトサン、 塗りつぶし三角：分子量 70000の低分子キトサン、 白抜き 円：分子量 110000の低分子キトサン、白抜き四角：分子量 250000の低分子キトサン）。 図 5は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067株培 養上清中のキチナ一ゼの活性と pHとの関係を示す。 図 6は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067株培 養上清中のキチナ一ゼの活性と温度との関係を示す。 図 7は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067株培 養上清中のキチナ一ゼの温度安定性を示す。 図 8は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067株培 養上清中のキチナ一ゼの pH安定性を示す。 図 9は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由来 の粗精製キチナーゼの活性と P Hとの関係を示す。 図 10は、 ァスペルギルス 'オリザェ var. sporoflavus Ohara J CM2067由 来の粗精製キチナ一ゼの活性と温度との関係を示す。 図 1 1は、 ァスペルギルス 'オリザェ var. sporoflavus Ohara J CM2067由 来の粗精製キチナーゼの P H安定性を示す。 図 12は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由 来の粗精製キチナーゼの温度安定性を示す。  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. In each graph, the distribution of low-molecular-weight chitosan prepared using chitinase in the culture supernatant of Aspergillus' Sojiae strain SANK 22388 is indicated by a solid circle in a solid line, and all dotted lines indicate the distribution of marker molecules ( Fill small circle: low molecular weight chitosan with molecular weight 10000, Fill 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: low with 250000 Molecular chitosan). 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. Sporoflavus Ohara J CM2067 strain. 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.
' 図 13は、 キトサン 7 Bを反応基質としたときのァスペルギルス 'オリザェ var. sporoflavus Ohara J CM2067由来の精製キチナ一ゼの活性と p Hとの関係を示 す。 PC漏 2/12940 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. PC leak 2/12940
64  64
図 14は、 グリコールキチンを反応基質としたときのァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由来の精製キチナーゼの活性と pHとの関 係を示す。 図 1'5は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由 来の精製キチナーゼの活性と温度との関係を示す。 図 16は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由 来の精製キチナ一ゼの温度安定性を示す。 図 17は、 ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由 来の精製キチナーゼの p H安定性を示す。 図 18は、 ァスペルギルス ·ジャポニクス SANK 19'288株培養上清中のキ チナ—ゼの活性と p Hとの関係を示す。 図 19は、 ァスペルギルス ·ソジヤエ SANK 22388株の培養上清中のキチ ナーゼの活性と p Hとの関係を示す。 図 20は、 グリコールキチンを反応基質としたときのァスペルギルス ·ジャポニク ス SANK19288株由来の精製キチナーゼの活性と pHとの関係を示す。 図 21は、 ァスペルギルス 'ジャポニクス SANK 19288株由来の精製キチ ナーゼの活性と温度との関係を示す。 図 22は、 ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチ ナーゼの温度安定性を示す。 図 23は、 ァスペルギルス ·ジャポニクス SANK 19288株由来の精製キチ ナーゼの p H安定性を示す。 図 24は、 グリコールキチンを反応基質としたときのァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナーゼの活性と p Hとの関係を示す。 図 25は、 pH5. 5の条件における、 ァスペルギルス 'ソジヤエ SANK22 388株由来の精製キチナーゼの活性と温度との関係を示す。 図 26は、 pH9. 0の条件における、 ァスペルギルス 'ソジヤエ SANK 22 388株由来の精製キチナーゼの活性と温度との関係を示す。 図 27は、 pH5. 5の条件における、 ァスペルギルス 'ソジヤエ SANK22 388株由来の精製キチナーゼの温度安定性を示す。 図 28は、 pH9. 0の条件における、 ァスペルギルス 'ソジヤエ SANK22 388株由来の精製キチナーゼの温度安定性を示す。 図 29は、 ァスペルギルス ·ソジヤエ SANK 22388株由来の精製キチナ一 ゼの pH安定性を示す。 ' 図 30は、 キチナ一ゼの SDS電気泳動図を示す。 （12%アクリルアミドゲル、 C BB染色、 レーン M：マ一カー、 レーン 1 ： H i s融合リコンビナントキチナーゼ、 レーン 2 '：ァスペルギルス ·オリザェ var. sporoflavus Ohara J CM2067由来 の精製キチナーゼ） 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. Sporoflavus Ohara J CM2067. 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)

Claims

； 請求の範囲 ; The scope of the claims
1. ァスペルギルス 'オリザェ （Aspergillus oryzae) 、 ァスペルギルス 'ジャ ポニクス （Aspergillus japonicus) 、 ァスペルギルス ·ソジヤエ (Aspergillus sojae) の少なくとも 1種の培養物から分離精製されるキトサン分解酵素。 1. Chitosan-degrading enzyme isolated and purified from at least one culture of Aspergillus sp. Oryzae, Aspergillus sp. Aspergillus or Aspergillus sop (Aspergillus sojae).
2. ァスペルギルス■オリザェ （Aspergillus oryzae) がァスペルギルス ·オリ ザェ (Aspergillus oryzae var. sporof lavus Ohara J CM 2067) 株 であり、 ァスペルギルス 'ジャポニクス （Aspergillus japonicus) がァスペルギル ス 'ジャポニクス （Aspergillus japonicus) SANK 19288株であり、 ァス ペルギルス · ソジヤエ （Aspergillus sojae) がァスペルギルス · ソジヤエ2. Aspergillus sp. Oryzae (Aspergillus oryzae) is a strain of Aspergillus oryzae (Aspergillus oryzae var. Spore of lavus Ohara J CM 2067), and Aspergillus sp. Aspergillus sojae (Aspergillus sojae)
(Aspergillus sojae) SANK 22388株である請求項 1記載のキトサン分解 (Aspergillus sojae) The degradation of chitosan according to claim 1, which is SANK 22388 strain.
3. 配列表の配列番号 1乃至 3の部分アミノ酸配列を有することを特徴とする、 請 求項 1または請求項 2に記載のキトサン分解酵素。 3. The chitosan degrading enzyme according to claim 1 or 2, which has a partial amino acid sequence of SEQ ID NO: 1 to 3 in the sequence listing.
4. 以下の性質を示す請求項 1乃至請求項 3のいずれか 1つに記載のキトサン分解 酵素： 4. The chitosan degrading enzyme according to any one of claims 1 to 3, exhibiting the following properties:
1) ' SDS— PAGE電気泳動法にて分子量約 40， 000を示す；  1) It shows a molecular weight of about 40,000 by 'SDS-PAGE electrophoresis';
2) 等電点電気泳動法にて等電点 p I 3. 5乃至 4. 5を示す；  2) The isoelectric point shows an isoelectric point p I of 3.5 to 4.5 by isoelectric focusing method;
3) 30%ァセチル化キトサン （粘度 100〜300 c p s) を、 pH3. 5乃至 p HI 0. 5にて加水分解する；  3) hydrolyze 30% acetylated chitosan (viscosity 100-300 cps) at pH 3.5 to pH 0.5;
4) グリコ一ルキチンを、 pH3. 0乃至 pH10. 5にて加水分解する；  4) hydrolyze glycolluctin at pH 3.0 to pH 10.5;
5) 0°C乃至 80°Cで 3) 記載の加水分解活性を発揮する；  5) exert the hydrolysis activity described in 3) at 0 ° C to 80 ° C;
6) 45 °C以下の温度で安定である；  6) stable at temperatures below 45 ° C;
7) pH5乃至pH9. 5の pH条件下で安定である。  7) Stable under pH conditions of pH 5 to pH 9.5.
5. 下記の a) 乃至 d) のいずれか つに記載の蛋白質であるキトサン分解酵素： a) 配列表の配列番号 5に記載のアミノ酸配列からなる蛋白質； 5. 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;
b)配列表の配列番号 4のヌクレオチド番号 242乃至 1438に示されるヌクレオ チド配列によりコードされるアミノ酸配列からなる蛋白質；  b) a protein consisting of an amino acid sequence encoded by the nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing;
c) a) または b) に記載のアミノ酸配列において、. 一つまたは数個のアミノ酸が置 換、 欠失、 揷入または付加したアミノ酸配列からなり、 つ、 キトサン分解活性を有 することを特徴とする蛋白質；  c) In the 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 decomposition activity. With protein;
d) a) または b) に記載のアミノ酸配列を含むことからなる蛋白質。 d) A protein comprising the amino acid sequence described in a) or b).
6. 下記の a) 乃至 c) のいずれか一つに記載の蛋白質であるキトサン分解酵素： a) 配列表の配列番号 14に記載のアミノ酸配列からなり、 N末端のセリン残基の α ーァミノ基がァセチル化されていることを特徴とする蛋白質； 6. A chitosan decomposing enzyme which is a protein according to any one of the following a) to c): a) an amino acid sequence according to SEQ ID NO: 14 in the sequence listing, and an α-amino group of a serine residue at the N-terminus A protein characterized in that it is acetylated;
b) 配列表の配列番号 14に記載のアミノ酸配列において、 一つまたは数個のァミノ 酸が置換、 欠失、 挿入または付加したアミノ酸配列からなり、 且つ、 N末端のセリン 残基のひーァミノ基がァセチル化されており、 且つ、 キトサン分解活性を有すること を特徴とする蛋白質； b) In the 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;
c) 配列表の配列番号 14に記載のアミノ酸配列を含み、 且つ、 N末端のセリン残基 のひーァミノ基がァセチル化されており、 且つ、 キトサン分解活性を有することを特 徴とする蛋白質。 c) A protein which comprises the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing, and in which the amino group of the serine residue at the N-terminus is acetylated and which has chitosan degrading activity.
7. 下記に記載のいずれか一つに記載の DNA： 7. The DNA described in any one of the following:
a)配列表の配列番号 4のヌクレオチド番号 242乃至 1438に示されるヌクレオ チド配列からなる DN A； a) A DNA comprising the nucleotide sequence shown by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing;
b) 上記 a) に記載の DNAの有するヌクレオチド配列と相補的なヌクレオチド配列 からなる DNAとストリンジェントな条件下でハイブリダィズし、 かつ、 キトサン分 解活性を有する蛋白質をコードすることを特徴とする DN A； b) a DNA which hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above under a stringent condition, and which encodes a protein having a chitosan degrading activity A;
c) 上記 a) に記載の DNAと 95%以上のヌクレオチド配列相同性を有するヌクレ ォチド配列からなり、 かつ、 キトサン分解活性を有する蛋白質をコードすることを特 徴とする DNA；  c) DNA characterized by comprising a nucleotide sequence having a nucleotide sequence homology of 95% or more with the DNA described in a) above, and encoding a protein having a chitosan degrading activity;
d) 配列表の配列番号 5に記載のアミノ酸配列からなる蛋白質をコードする DNA； e)配列表の配列番号 4のヌクレオチド番号 242乃至 1438に示されるヌクレオ チド配列を含むことからなる DNA。 d) a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing; e) a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 at nucleotide numbers 242 to 1438 in SEQ ID NO: 4.
8. 下記の a) 乃至 c) のいずれか一つに記載の DNA： 8. DNA according to any one of the following a) to c):
a) 形質転換大腸菌 Escherichia coli BM25.8/pTr iplEx-Chi t inase-cDNA S A NK 72102 (FERM B P— 8235 )により保持される組換えプラスミド、 pTriplEx-Chitinase-cDNAに揷入されている DNA；  a) A recombinant plasmid carried by transformed E. coli Escherichia coli BM25.8 / pTripIEx-Chitinase-cDNA S A NK 72102 (FERM B P-8235), DNA incorporated into pTriplEx-Chitinase-cDNA;
b) b) 上記 a) に記載の DNAの有するヌクレオチド配列と相補的なヌクレオチド 配列からなる DNAとストリンジエンドな条件下でハイブリダィズし、 かつ、 キトサ ン分解活性を有する蛋白質をコードすることを特徴とする DN A； b) b) A protein that hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above under stringent conditions and encodes a protein having a chitosan degradation activity. To do DN A;
c) 上記 a) に記載の DNAを含み、 かつ、 キトサン分解活性を有する蛋白質をコー ドすることを特徴とする D N A。  c) A DNA comprising the DNA described in a) above and coding a protein having a chitosan degrading activity.
9. 請求項 7または請求項 8に記載の DNAにコードされる蛋白質であるキトサン 分解酵素。 9. A chitosan-degrading enzyme which is a protein encoded by the DNA according to claim 7 or 8.
10. 形質転換大腸菌 Escherichia coli BM25.8/pTr i p ΙΕχ-Ch i t i nas e-cDNA SANK 72102 (FERM B P— 8235 ) により保持される組換えプラス ミド、 pTriplEx- Chitinase- cDNAに挿入されている DNAによりコードされる蛋白質 であるキトサン分解酵素。 10. A recombinant plasmid, pTriplEx- Chitinase-cDNA, which is carried by the transformed E. coli Escherichia BM25.8 / pTripΙΕχ-Chitinas-cDNA SANK 72102 (FERM BP-8235) Chitosan-degrading enzyme which is a protein encoded.
11. 下記の a) 乃至 b) に示される活性を有することを特徴とする、 請求項 5、 請求項 6、 請求項 9または請求項 10に記載のキトサン分解酵素； 11. The chitosan-degrading enzyme according to claim 5, claim 6, claim 9 or claim 10, having the activity shown in the following a) to b):
a) 30%ァセチル化キトサン （粘度 100〜300 c p s) を、 pH3. 5乃至 p HI 0. 5、 0 °C乃至 80 °Cの条件にて加水分解する活性； a) activity to hydrolyze 30% acetylated chitosan (viscosity 100-300 cps) under conditions of pH 3.5 to pH 0.5, 0 ° C to 80 ° C;
b) グリコールキチンを、 PH4. 0乃至 ρΗΙ Ο. 5にて加水分解する活性。 b) The activity of hydrolyzing glycol chitin at pH 4.0 to Ο5.
12. 請求項 7または請求項 8に,記載の DNAを含む組換えプラスミド。 12. A recombinant plasmid comprising the DNA according to claim 7 or 8.
13. 発現べクタ一であることを特徴とする、 請求項 12に記載の組換えプラスミ ド。 13. The recombinant plasmid according to claim 12, characterized in that it is an expression vector.
14. 請求項 12または請求項 13に記載の組換えプラスミドで形質転換された宿 主細胞。 14. A host cell transformed with the recombinant plasmid according to claim 12 or 13.
15. 原核細胞または真核細胞であることを特徴とする、 請求項 14に記載の宿主 細胞。 15. The host cell according to claim 14, which is a prokaryotic cell or a eukaryotic cell.
1 6. 形質転換大腸菌 Escherichia coli BM25.8/pTr iplEx-Chi t inase-cDNA SANK 72102 (FERM B P— 8235 ) である請求項 14記載の宿主細 胞。 1 6. The host cell according to claim 14, wherein the host cell is transformed Escherichia coli BM25.8 / pTripI-Ex- Chitinase-cDNA SANK 72102 (FERM B P-8235).
17. 下記 1) 乃至 2) を含む、 キトサン分解酵素の製造方法： 17. A process for producing a chitosan degrading enzyme, which comprises the following 1) to 2):
1) 下記の a) 乃至 e) のいずれか一つに記載の細胞を、 キトサン分解酵素を産生す る条件下で培養する工程；  1) culturing the cells according to any one of the following a) to e) under conditions for producing a chitosan degrading enzyme;
a) ァスペルギルス 'オリザェ （Aspergillus oryzae) ；  a) Aspergillus' Oryzae (Aspergillus oryzae);
b) ァスペルギルス 'ジャポニクス （Aspergillus japonicus) ；  b) Aspergillus' Aspergillus japonicus;
c) ァスペルギルス 'ソジヤエ （Aspergillus sojae) ；  c) Aspergillus sojae (Aspergillus sojae);
d) 請求項 14乃至請求項 16のいずれか一つに 3載の宿主細胞；  d) The host cell according to any one of claims 14 to 16;
e) 請求項 5、 請求項 6、 請求項 9乃至請求項 11のいずれか一つに記載のキトサ ン分解酵素を産生する細胞；  e) A cell producing the chitosan degrading enzyme according to any one of claim 5, claim 6, claim 9 to claim 11;
2) 1) の培養産物からキトサン分解酵素を分離 ·精製する工程。 2) A step of separating and purifying chitosan degrading enzyme from the culture product of 1).
18. ァスペルギルス ·オリザェ （Aspergillus oryzae) がァスペルギルス ·ォ リザェ (Aspergillus oryzae var. sporof lavus Ohara J CM 2067) 株であり、 ァスペルギルス 'ジャポニクス （Aspergillus japonicus) がァスペルギ ルス 'ジャポニクス （Aspergillus japonicus) SANK 19288株であり、 ァ スペルギルス · ソジヤエ （Aspergillus sojae) がァスペルギルス · ソジヤエ18. Aspergillus oryzae (Aspergillus oryzae) is a strain of Aspergillus oryzae var. Spore of lavus Ohara J CM 2067, and Aspergillus 'Japonicus (Aspergillus japonicus) is Aspergillus' japonicus (Aspergillus japonicus Yes, Spergils sojae (Aspergillus sojae)
(Aspergillus sojae) SANK 22388株である請求項 17記載の方法。 (Aspergillus sojae) The method according to claim 17, which is SANK 22388 strain.
19. 請求項 17または請求項 18に記載の方法により製造されるキトサン分解酵 素。 19. A chitosan degrading enzyme produced by the method according to claim 17 or 18.
20. 以下の工程 1) および 2) を含む低分子キトサンの製造方法： 20. A process for the preparation of low molecular weight chitosan comprising the following steps 1) and 2):
1)ァセチル化度が 10%乃至 30%のァセチル化キトサン水溶液に請求項 1乃至請 求項 6、請求項 9乃至請求項 11および請求項 19のいずれか一つに記載のキトサン 分解酵素を添加する；  1) The chitosan decomposing enzyme according to any one of claims 1 to 6, 9 to 11 and 19 is added to an aqueous solution of acetylated chitosan having a degree of acetylation of 10% to 30%. Do;
2) pH3乃至 12、 10°C乃至 60°Cの条件で 10分以上酵素反応を行なう。  2) Perform the enzyme reaction for 10 minutes or longer under conditions of pH 3 to 12 and 10 ° C to 60 ° C.
21. 請求項 20に記載の方法により製造される低分子キトサン又はその塩。 21. Low molecular weight chitosan or a salt thereof produced by the method according to claim 20.
22. 分子量が 4万〜 25万でァセチル化度が 10%である請求項 21記載の低分 子キトサン又はその塩。 22. The low molecular chitosan or the salt thereof according to claim 21, which has a molecular weight of 40,000 to 250,000 and an acetylation degree of 10%.
23. 分子量が 1万〜 11万でァセチル化度が 20%である請求項 21記載の低分 子キトサン又はその塩。 23. The low molecular chitosan or the salt thereof according to claim 21, which has a molecular weight of 10,000 to 110,000 and an acetylation degree of 20%.
24. 分子量が 1万〜 7万でァセチル化度が 30%である請求項 21記載の低分子 キトサン又はその塩。 24. The low molecular weight chitosan or a salt thereof according to claim 21, which has a molecular weight of 10,000 to 70,000 and an acetylation degree of 30%.
25. 請求項 21乃至請求項 24のいずれか一つに記載の低分子キトサン又はその 塩を有効成分として含有する医薬組成物。 25. A pharmaceutical composition comprising the low molecular weight chitosan or a salt thereof according to any one of claims 21 to 24 as an active ingredient.
26. 外傷、 床ずれまたはァトピー性皮膚炎の治療剤であることを特徴とする、 請 求項 25記載の医薬組成物。 26. The pharmaceutical composition according to claim 25, which is a therapeutic agent for trauma, bedsore or atopic dermatitis.
27. 請求項 21乃至請求項 24のいずれか一つに記載の低分子キトサン又はその 塩を有する化粧料。 27. A cosmetic comprising the low molecular weight chitosan or a salt thereof according to any one of claims 21 to 24.
2 8 .. 請求項 2 1乃至請求項 2 4のいずれか一つに記載の低分子キトサン又はその 塩を有する食品。 Food having the low molecular weight chitosan or a salt thereof according to any one of claims 2 to 24.
2 9 . 請求項 2 1乃至請求項 2 4のいずれか一つに記載の低分子キトサン又はその 塩を有する水等処理剤。 A water or the like treating agent having low molecular weight chitosan or a salt thereof according to any one of claims 2 to 24.
3 0 . 以下の工程 1 ) および 2 ) を含む、 その液体成分中に分子量 1 0 0万以上の 高分子部分ァセチル化キトサンを実質的に含有しない試料の製造方法： 3 0. A method for producing a sample substantially free of partially partially acetylated chitosan having a molecular weight of 100,000 or more in its liquid component, comprising the following steps 1) and 2):
1 ) 分子量 1 0 0万以上の高分子部分ァセチル化キトサンを含む試料に請求項 1、 請 求項 2、 請求項 3、 請求項 4、 請求項 5、 請求項 6、 請求項 9、 請求項 1 0、 請求項 1 1および請求項 1 9のいずれか一つに記載のキトサン分解酵素を添加する； 1) Claim 1, claim 2, claim 3, claim 4, claim 5, claim 6, claim 9, claim 9 for samples containing high molecular weight partially acetylated chitosan having a molecular weight of 100,000 or more. 10, adding the chitosan degrading enzyme according to any one of claims 1 1 and 19;
2 ) p H 3乃至 1 2、 1 0 °C乃至 6 0 °Cの条件で 1 0分間以上酵素反応を行なう'。 2) pH 3 to 12, perform enzyme reaction at 10 ° C to 60 ° C for 10 minutes or more '.
3 1 . 分子量 1 0 0万以上の高分子部分ァセチル化キトサンを含有する試料が、 甲 殻類を含む試料であることを特徴とする、 請求項 3 0記載の方法。 31. The method according to claim 30, wherein the sample containing high molecular weight partially acetylated chitosan having a molecular weight of 100,000 or more is a sample containing crustaceans.
3 2 . 甲殻類がェビおよび力二のいずれか一つまたは両方である請求項 3 1記載の 方法。 32. The method according to claim 31, wherein the crustacean is either one or both of evi and power.
3 3 . 請求項 3 0乃至請求項 3 2のいずれか一つの方法により製造される、 その液 体成分中に分子量 1 0 0万以上の高分子部分ァセチル化キトサンを実質的に含有し ない試料。 3 3. A sample produced by the method according to any one of claims 3 0 to 3 2 and containing substantially no polymeric partially acetylated chitosan having a molecular weight of 100,000 or more in its liquid component. .
3 4. 請求項 2 1乃至請求項 2 4記載の低分子キトサン又はその塩の有効量を動物 に投与することを含む、 外傷、 床ずれおよびアトピ一性皮膚炎から選択される一つま たは複数の疾患の治療方法。 3. One or more selected from trauma, bed sores and atopic dermatitis, comprising administering to the animal an effective amount of the low molecular weight chitosan or a salt thereof according to claim 1 to 24. For the treatment of diseases.
3 5 . '外傷、 床ずれおよびアトピー性皮膚炎から選択される一つまたは複数の疾患 を治療するための、請求項 2 1乃至請求項 2 4のいずれか一つに記載の低分子キトサ ン又はその塩の使用。 3 5 'The low-molecular-weight chitosan or the low-molecular-weight chitosan according to any one of claims 21 to 24 for treating one or more diseases selected from trauma, bedsore and atopic dermatitis. Use of that salt.
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CN110554176A (en) * 2019-09-04 2019-12-10 南通大学 method for linearly regulating degradation speed of chitosan biomaterial by using molecular weight and acetylation degree

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