JPH1175827A - New chitosanase-producing microorganism and production of chitosanase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain chitosanase-producing microorganisms, capable of producing highly active chitosanase suitable for production of a low molecular chitosan or a chitosan oligosaccharide useful for medicine, and belonging to the genus Arthrobacter, and further to provide a method for producing the chitosanase by using the chitosanase-producing microorganisms. SOLUTION: This new chitosanase-producing microorganisms are chitosanase- producing microorganisms A1 strain (FERM 16197), chitosanase-producing microorganisms A2 strain (FERM 16198) and chitosanase-producing microorganisms A3 strain (FERM 16199). The method for producing a chitosanase comprises culturing at least one kind of the chitosanase-producing microorganisms and separating and collecting the chitosanase from the cultured material.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel chitosanase belonging to the genus Arthrobacter which produces highly active chitosanase suitable for use in the production of low molecular weight chitosan or chitosan oligosaccharide useful for pharmaceuticals and the like. The present invention relates to a fungus and a method for producing chitosanase using the novel chitosanase-producing fungus.

[0002]

2. Description of the Related Art Chitosan is a deacetylated product of chitin, which is a kind of polysaccharide composed of amino sugars contained in shells such as crabs and shrimps. The chitosan has been used in various fields such as a metal adsorbent, a biodegradable film, a coagulant for an organic substance, a carrier for immobilizing enzymes, and the like.In recent years, chitosan has been used in blood cholesterol. It has been found that it has various physiological actions such as an increase suppression effect, an improvement in immunity, a blood pressure lowering action, and the like, and is attracting attention. In addition, low molecular weight chitosan and chitosan oligosaccharide also have antitumor properties, drugs,
Applications in various fields such as food and cosmetics are in the spotlight. Furthermore, low molecular weight products of various degrees of polymerization of chitosan are soluble in water and do not have the astringent taste peculiar to chitosan, so that they can be easily added to foods and various bioactive effects are expected.

[0003] In order to reduce the molecular weight of chitosan, it is common to use an acid or an enzyme, but the enzymatic method is effective in terms of stability of product quality and prevention of side reactions. As a microorganism producing chitosanase, for example, Bacillus
Bacillus pumilus BN-262 (JP-A-63-63)
382) and Bacillus circulans (Bacillus
circulans) LCC-1 strain (Japanese Patent Publication No. Hei 7-16402)
And Bacillus genus No. 7-
No. 311), a Bacillus genus PI-7S strain (Japanese Patent Application Laid-Open No. 1-247081), alkaligenes
(Alcaligenes) MHK-1 strain (Japanese Patent Application Laid-Open No. 62-201571), Pseudomonas H-14 strain (Japanese Patent Application Publication No. 5-23740), and Aspergillus fumigatus mutation Shares (Japanese Unexamined Patent Publication
No. 22554).

[0004] The above-mentioned conventional chitosanase-producing bacteria are not satisfactory in cost, culturing days and efficiency, and are not sufficiently satisfactory in chitosanase activity.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a highly active chitosanase having properties different from those of chitosanase produced by conventional chitosanase-producing bacteria and having a high activity suitable for producing low molecular weight chitosan. The purpose is to obtain a high yield in time.

In order to achieve the above object, the present inventor has searched for microorganisms producing chitosanase more broadly in nature, and as a result, found bacteria having high chitosanase activity from wastewater of crab and shrimp processing plants and the like. Three strains from
A phylogenetic study was performed. As a result, it was found that the isolated strain produced a large amount of chitosanase in a new species of the genus Arthrobacter and that the chitosanase had high activity. The present inventors have completed the present invention based on this newly found chitosanase-producing bacterium and new findings.

[0007]

The first invention for which a patent is sought is a chitosanase-producing bacterium A1 belonging to the genus Arthrobacter (Microorganism No. 16197).
It is.

[0008] The second invention to be patented is a chitosanase-producing bacterium A2 belonging to the genus Arthrobacter (No. 16198, Microtechnical Laboratories).

The third invention for which a patent is sought is a chitosanase-producing bacterium A3 belonging to the genus Arthrobacter (No. 16199, Microtechnical Laboratory).

[0010] The fourth invention to be patented is a chitosanase-producing bacterium A1 belonging to the genus Arthrobacter (Art. No. 16197), and a chitosanase-producing bacterium A2 (Art. Chitosanase characterized by culturing at least one kind of a chitosanase-producing bacterium A3 strain (Microtechnical Laboratories No. 16199) and separating and collecting chitosanase from the culture. -A method for producing zeolites.

The Arthrobacter used in the present invention (Arthrob)
The chitosanase producing strains A1, A2 and A3 belonging to the genus acter) have been deposited with the National Institute of Technology (FERM P-16197, 16198, 16199) and have the following mycological properties. Less than,
Unless otherwise indicated, all three strains show similar properties.

(I) Form Polymorphic bacilli having a size of 2.0 × 0.5 μm to 0.8 × 0.6 μm. Flagella, no motility. The colony is colorless and shiny on a broth agar medium.

(Ii) Growing state 3.0% triptyce soy broth (BBL)
0.5% yeast extract and 1.5% agar medium (pH
7.0), a clear colony after culturing at 30 ° C for 24 hours.
Is generated.

(Iii) Physiological properties (1) Gram stain: positive (2) Spores: none (3) Attitude to oxygen: aerobic (4) Oxidase: positive (5) Catalase: positive (6) Nitrate reduction: Negative (7) Denitrification reaction: Negative (8) Methyl red test: Negative (9) VP reaction: Negative (10) OF test: Negative (11) Indole formation: Not generated. (12) Antiacid: Negative (13) Starch decomposition: Not decomposed. (14) Decomposition of gelatin: Decomposes. (15) Urease: positive (16) Production of hydrogen sulfide TSI medium: not produced. Acetate liquid medium: not generated. (17) Use of citrate Koser's medium: Use. Christensen's medium: use. (18) Use of inorganic nitrogen source Nitrate: Slightly used. Ammonia salt: Use. (19) Dye formation: Not generated. (20) Village color tone: No characteristic pigment is formed. (21) Elongation of surrounding cells of colony: negative (22) Viable temperature range: grow at 7-35 ° C,
It shows good growth at 5-30 ° C. (23) Viable pH range: A1 and A2 strains have a pH of 4.5
~ 10.5, A3 strain grows at 4.5 ~ 11, and all 3 strains
It shows good growth at pH 5-6. (24) NaCl resistance: Grow at 7% or less. (25) Growth of acids and gases from carbon sources: as shown in Table 1. (26) Utilization of carbon source: See Table 2.

[0015]

[Table 1] Generation of acids and gases from carbon sources

[0016]

[Table 2] Utilization of carbon sources Part 2. Utilization of carbon sources

(Iv) Chemotaxonomic properties (1) Diamino acid of cell wall: L-Lys (2) Amino acid composition of cell wall: Lys-Ala ₂ (3) Glycolyl test: acetyl type (4) Arabinogalactan of cell wall : None (5) Mycolic acid: None (6) Quinone: MK-9 (H ₂ ) (7) DNA G + Cmol%: 64%

The above result and the "verges manual
Of Systematic Bacteriology, Volume 1 (1
984)] [Bergey's Manual of Systematic Bacter
iology, Volume 1 (1984)] [William & Wilkins]) and "The Version of the Manual of Ditaminative Bacteriology, Vol. 9, 1994"["Be
rgey's Manual of Determinative Bacteriology, Volum
e 9 (1994)] [William & Wilkins]), these strains are considered to be new species of the genus Arthrobacter.

[0019]

Next, a chitosanase-producing bacterium A1 strain belonging to the genus Arthrobacter (No. 16197 of Microtechnical Laboratories), a chitosanase-producing bacterium A2 strain (No. 16198 of Microtechnical Laboratories), chitosanase A method for producing chitosanase using the A-producing bacterium A3 strain (Microtechnical Laboratories No. 16199) will be described.

First, as a medium used for the production of chitosanase, any medium such as a synthetic medium or a natural medium may be used as long as the medium contains an appropriate amount selected from a carbon source, a nitrogen source, an inorganic substance and the like. Can be used. For example,
Commercially available triptyce soy broth (BBL)
Chitosanase production is possible even with a 3%, 0.5% yeast extract medium. In any medium, the production of the present chitosanase can be significantly increased by adding chitosan dissolved in an acid, or water-soluble chitosan, chitin flake, or chitosan flake. The culture temperature is preferably 25 ° C.
30 ° C., pH about 5-6. The culture time is 1 to 2 days, and chitosanase is secreted and produced in the culture by aeration and agitation culture or the like.

Next, the culture thus obtained can be used as it is as a crude enzyme solution by removing the cells by filtration or centrifugation. Furthermore, when chitosanase is isolated from the crude enzyme solution, high purity can be easily obtained by ordinary purification means, for example, salting out, solvent precipitation, electrophoresis, gel filtration, ammonium sulfate precipitation, and the like. You can get a sample.

The physical properties of chitosanase produced from the strain of the present invention are shown below. Action: Acts on and breaks down chitosan. Working temperature and optimum working temperature: When dissolved chitosan is used as a substrate, the working temperature is 10 to 60 ° C, but the optimum working temperature is 50 ° C. Thermal stability: almost stable at 40 ° C for 30 minutes, 50
Approximately 60% of the enzyme is inactivated by treatment at 30 ° C for 30 minutes.
It was completely inactivated by the treatment for 30 minutes. Working pH Range and Optimum pH: It works in the range of pH 3-10, the optimum pH being 5.

[0023]

[Effect] Arthrobacter of the present invention.
Strains 1, A2 and A3 are bacteria belonging to the novel genus Arthrobacter that can produce high-activity chitosanase in large quantities. Chitosanase obtained from this bacterium can convert chitosan into small molecules with high efficiency or decompose it into chitosan oligosaccharides.
These low molecular weight chitosan and chitosan oligosaccharide have various physiological actions and are water-soluble, so they are easy to handle and useful for various products.

[0024]

EXAMPLES The present invention will be described in further detail with reference to examples, but the present invention is not limited to these examples. First, a medium of 3% tryptic soy broth (BBL) and 0.5% yeast extract was prepared.
5 ml thereof was dispensed into a medium-sized test tube and sterilized. Next, a loopful of Arthrobacter sp., A1 strain (FERM P-16197) was inoculated from the stock medium and cultured with shaking at 27 ° C. for 24 hours. In the main culture, 100 ml of the same medium as above was dispensed into 300 ml Erlenmeyer flasks and sterilized.
For 1-2 days with shaking. In this case, the jar
A large amount may be prepared using a mentor or the like. After completion of the culture, the mixture was centrifuged, and the supernatant was filtered through a 0.45 μm membrane filter, lyophilized, and used as crude chitosanase.

<Measurement of Activity> The crude chitosanase was appropriately diluted, and 1.5 ml of a 0.5% chitosan solution was added to 0.5 ml of the diluted solution, followed by reaction at 37 ° C. for 30 minutes. The chitosan solution is obtained by dissolving chitosan in a 0.1 M sodium acetate buffer. After completion of the reaction, the reaction was stopped by immersion in boiling water for 5 minutes.
Activity was measured.

The chitosanase activity was measured by adding 0.5 ml of a sample to a test tube with a glass stopper, adding 1.0 ml of acetylacetone reagent (1.5 ml of acetylacetone to 50 ml with 1.25N sodium carbonate) and 90 ° C. And heat for 1 hour. After cooling with water, 10 ml of 96% ethanol was gently added, and then Ehrl
ich reagent (9-dimethylaminobenzaldehyde 1.6
g are dissolved in 30 ml of concentrated hydrochloric acid and 30 ml of 96% ethanol. ) Add 1.0 ml and stir and mix. After mixing 1
The mixture was left at room temperature for a time, and the absorbance at 530 nm was measured.

The amount of reducing sugar was determined from a calibration curve prepared using glucosamine as a standard substance. Under these conditions, 1
The amount of enzyme that releases reducing sugars corresponding to μmol glucosamine is defined as one unit of chitosanase activity.

<Enzyme activity> After culturing as in the Examples, the chitosanase activity was measured and found to be 42.75 U / l. Furthermore, when 1% of flake chitosan or 0.75% of dissolved chitosan was added to the above medium and cultured for 1 day, the chitosanase activity increased remarkably (about 50 times in the former and about 70 times in the latter).

According to the present invention, highly active chitosanase can be obtained in a short time, and a chitosan oligosaccharide having three or more saccharides can be obtained in a high yield. As an effective use of chitosan that has fallen to the floor or the like during the production of chitosan and cannot be added to food, it can be added to a culture medium, and thereby a high yield of chitosanase is obtained. This can reduce the overall cost of chitosan and chitosanase production.

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[Procedure amendment]

[Document name] Notification of change of accession number

[Submission date] September 30, 1997

[Name of the deposited institution] Institute of Biotechnology, Industrial Technology Institute

[Accession number] FERM P-16197

[Accession number] FERM P-16198

[Accession number] FERM P-16199

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takashi Imamura 7-36-5 Kurihama, Yokosuka City, Kanagawa Prefecture Inside Nichiro Central Research Institute Co., Ltd. (72) Tadao Tanimura 7-36-5, Kurihama, Yokosuka City, Kanagawa Prefecture Shares Nichiro Central Research Laboratory

Claims (4)

[Claims] 1. A chitosanase producing strain A1 belonging to the genus Arthrobacter (Art.
issue). 2. A chitosanase producing strain A2 belonging to the genus Arthrobacter (Art.
issue). 3. A chitosanase-producing bacterium A3 belonging to the genus Arthrobacter (Art.
issue). 4. A chitosanase-producing bacterium A1 belonging to the genus Arthrobacter (Art.
No. 16), chitosanase-producing bacterium A2 strain
No. 16), chitosanase-producing bacterium A3 strain
And a method for producing chitosanase, comprising culturing at least one kind of chitosanase, and separating and collecting chitosanase from the culture.