JPH08275776A - New chitinase and its production - Google Patents

Abstract

PURPOSE: To obtain a new chitinase, capable of decomposing chitin and important for effective utilization of the chitin. CONSTITUTION: This chitinase C-l or C-3 is obtained by culturing a Vibrio sp. H-8 strain (FERM P-14737), etc., and having the following properties: (l) each capable of acting on chitin and hydrolyzing the chitin into a soluble N- acetylchitooligosaccharide; (2) optimal pH7.8-8.2 (40 deg.C), 50 deg.C optimal temperature (pH8.0), stable by treatment at pH8.0 and 50 deg.C for 1hr, about 69000 molecular weight [measured according to a sodium dodecyl sulfate-polyacrylamide gel electrophoretic method (SDS-PAGE)] and 3.6 isoelectric point for the chitinase C-l; (3) optimal pH7.8-8.2 (40 deg.C), 50 deg.C optimal temperature (pH8.0), stable by treatment at pH8.0 and 50 deg.C for 1hr, about 81000 molecular weight (measured according to the SDS-PAGE method) and 3.9 isoelectric point for the chitinase C-3, etc., and (4) each having high activities.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to novel chitinase C-1 and chitinase C-3 produced by a microorganism belonging to the genus Vibrio. The enzyme of the present invention is
It is extremely important for effective utilization of chitin because it efficiently decomposes chitin used for various purposes.

[0002]

Chitin is a polysaccharide having a large number of β- (1,4) -linked N-acetyl-D-glucosamines and is widely distributed in nature. Chitinase hydrolyzes chitin and decomposes it into N-acetylchitobiose via N-acetylchitooligosaccharide. Chitinase (EC3.2
・ 1.14) is widely distributed in the microbial world, and research is being conducted mainly on Streptomyces microorganisms. On the other hand, research on chitinase produced by Vibrio spp.
It was first reported by Uchida et al. (Fermentation Engineering Vol. 57, No. 3, 131-140 1979). Furthermore, recently, Takahashi et al. (J.
Ferment. Bioeng. Vol-75 457-459 1993).

However, the above chitinase was not satisfactory in terms of chitinase activity and thermostability. That is, the activity of each of these enzymes was less than 10 units per mg of protein, and the thermal stability could not be maintained for 1 hour or more at 50 ° C.

[0004]

An object of the present invention is to provide a novel chitinase having high chitinase activity and high thermostability.

[0005]

Means for Solving the Problems The inventors of the present invention searched for a chitinase derived from a marine bacterium in order to solve the above problems, and as a result, the H-8 strain belonging to the genus Vibrio has a high chitinase activity. In addition, the present invention was completed by the discovery that it produces a novel chitinase that is also excellent in thermostability.

That is, the present invention is chitinase C-1 or chitinase C-3 having the following enzymatic chemical properties. A. Chitinase C-1 (1) Acts on chitin and hydrolyzes chitin to soluble N-acetylchitooligosaccharide. (2) Optimum pH: 7.8-8.2 at 40 ° C (3) Optimum temperature: 50 ° C at pH 8.0 (4) pH stability: Stable at 5.5-9.0 at 40 ° C ( 5) Thermal stability: stable at 50 ° C for one hour at pH 8.0 (6) Molecular weight: about 69000 (SDS-PAGE method) (7) Isoelectric point: 3.6 B. Chitinase C-3 (1) Acts on chitin and hydrolyzes chitin to soluble N-acetylchitooligosaccharide. (2) Optimum pH: 7.8-8.2 at 40 ° C (3) Optimum temperature: 50 ° C at pH 8.0 (4) pH stability: Stable at 5.5-9.0 at 40 ° C ( 5) Thermal stability: stable at 50 ° C for one hour at pH 8.0 (6) Molecular weight: about 81000 (SDS-PAGE method) (7) Isoelectric point: 3.9 Further, the present invention is of the genus Vibrio. Belongs to Chitinase C-1
Alternatively, it is a method for producing chitinase C-1 or chitinase C-3, which comprises culturing a microorganism capable of producing chitinase C-3 and collecting chitinase C-1 or chitinase C-3 from the culture.

Hereinafter, the present invention will be described in detail. H-8 strain was isolated from the bottom mud collected in Lake Hamana, Shizuoka Prefecture. H
The bacteriologic properties of strain -8 are shown below. The medium used is Marine Agar (Bacto MarineAgar 2216) (manufactured by Difco) or Marine Broth (Bacto Marine Broth 2216) (manufactured by Difco).
Was used. Inoculate this medium with these strains at 30 ° C from 24
After culturing for 48 hours, the morphology was observed using an optical microscope and a transmission electron microscope. Since this strain requires sodium chloride, the biochemical properties were tested by preparing a medium using 75% artificial seawater or adding 3% sodium chloride to the medium. (1) Morphology (under aerobic conditions) Cell shape and size: Unfolded bacilli, 0.7-1.1x
1.3-1.8 μm Presence or absence of cell polymorphism: None Motility: Yes Flagella epidemic state: polar hairs, periflagellates are observed around cells when cultured in solid medium.

Swarming: Yes Presence of spores: No Gram stainability: Negative (2) Growth state in each medium Marine agar plate culture: Good growth, colonies grow in convex circles, full edge wet light, opaque It is milky white and has a smooth surface.

Marine agar slope culture: good growth, filamentous, milky white marine broth culture: good growth, culture solution is uniformly cloudy. Marine broth gelatin stab culture: Liquefaction (after 1 week). Litmus milk: Digest (3) Physiological properties Reduction of nitrates: Reduction Indole formation: formation.

Production of hydrogen sulfide: Not produced in TSI medium. Decomposition of starch: It hydrolyzes. Use of citric acid: Use Simons medium. : Use Cristensen medium. Use of inorganic nitrogen source: Use.

Formation of dye: No formation. Urease activity: Negative Oxidase activity: Positive Catalase activity: Positive Growth range (temperature, pH): Temperature 12-45 ℃ Best growth at 30 ℃: Best growth at pH 3-10 6-8 Attitude toward oxygen: normal Sex anaerobic OF test: Fermenting glucose.

Methyl red test: Positive VP reaction: Presence or absence of acid and gas generation from positive saccharides After culturing at 30 ° C. for 1 week Acid generation Gas generation L-arabinose + -D-xylose-D-glucose + -D -Mannose + -D-galactose + -D-fructose + -maltose + -sucrose --- lactose --- trehalose + -D-sorbit --- D-mannitol + -inosit --- glycerin + -starch + ---: Not generated, +: Generated Esculin degradation: Negative Arginine degradation: Negative DNA degradation: Positive PHB accumulation: No accumulation Saltiness: Growing in the range of 1% to 10% ONPG test: Positive Tween80 Degradability: Positive GC content: 45.8% Isoprenoid quinone: Main quinone Ubiquinone Q-8 Minor quinone Ubiquinone -7, Bajizu Manual of from Q-6, Q-9 menaquinone MK-7 de-methyl-menaquinone DMK-8 or more of the microbiological properties
As a result of comparison with known strains according to the classification criteria of Determinative Bacteriology 8th Edition, it is a Gram-negative bacillus, has polar flagella, facultatively anaerobic, and fermentable in OF test, This strain was considered to belong to the genus Vibrio because of its saltiness, no luminescence, oxidase positivity, and GC content of 45.8%. And this strain is Vibrio sp. It has been deposited as the H-8 strain at the Institute of Biotechnology, Institute of Industrial Science, under the deposit number FERM P-14737 (deposit date: January 19, 1995).

Chitinase C-1 and chitinase C-3
Can be obtained from the culture by culturing a chitinase-producing bacterium belonging to the genus Vibrio in a medium. As the chitinase-producing bacterium, any strain can be used as long as it belongs to the genus Vibrio and produces chitinase C-1 and chitinase C-3. Moreover, chitinase C-1 or chitinase C-3 can also be used in artificial mutation methods of these microorganisms, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagen, or naturally occurring mutant strains, or genetically engineered or cell fusion mutant strains. Any of the above can be used in the present invention. In order to select a strain having such a chitinase-producing ability, the culture of the strain is taken out,
It can be performed by measuring its chitinase activity. Among the chitinase-producing strains, typical strains are
Vibrio sp. H-8 strain (FERM P-14737) can be mentioned.

For culturing the microorganism of the present invention, a culture method generally used for culturing microorganisms can be used. As the medium, both synthetic medium and natural medium can be used as long as they can contain assimilable carbon sources, nitrogen sources, inorganic substances and necessary growth and production promoting substances. Since this enzyme is an inducing enzyme, it is essential to use chitin or chitin oligosaccharide as a carbon source. In addition, other carbon sources such as glucose, starch, dextrin, maltose,
Mannose, fructose, sucrose, arabinose, mannitol, molasses, etc. may be used alone or in combination. Furthermore, depending on the assimilation capacity of the bacterium, hydrocarbons,
Alcohols and organic acids are also used. As the nitrogen source, ammonium chloride, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steve liquor, soybean powder, casamino acid, etc. may be used alone or in combination. In addition, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate,
Inorganic salts such as copper sulfate and seawater are added as needed. Further, a trace amount component that promotes the growth of the used bacterium and the production of chitinase can be appropriately added.

As a culturing method, a general culturing method is used, but a liquid culturing method, particularly a deep agitation culturing method is most suitable. A culture temperature of 16 to 60 ° C., particularly 30 to 42 ° C., is suitable, and the pH of the medium during the culture is 6 to 11, especially 7 by adding ammonia water, ammonium carbonate solution, hydrochloric acid solution or the like.
It is desirable to maintain ~ 9. Usually 6 to 96 in liquid culture
When cultured for a long time, chitinase C-1 and chitinase C-3 which are the target substances are produced outside the cells. The culture is stopped when the maximum production in the culture is reached.

Separation and purification of chitinase C-1 and chitinase C-3 from the culture are carried out according to a conventional method for isolating and purifying the enzyme from the culture. For example, the culture is separated into a culture filtrate and cells by filtration, and the culture filtrate is subjected to salting out using ammonium sulfate or the like to obtain a precipitate. This precipitate can be purified by procedures such as dissolution, dialysis, gel filtration chromatography and ion exchange chromatography.

Chitinase C-1 produced by the above procedure
Has the following enzymatic chemistry: (1) It acts on chitin and hydrolyzes chitin to soluble N-acetylchitooligosaccharide. (2) Optimum pH: 7.8-8.2 at 40 ° C (3) Optimum temperature: 50 ° C at pH 8.0 (4) pH stability: Stable at 5.5-9.0 at 40 ° C ( 5) Thermostability: stable at 50 ° C for one hour at pH 8.0 (6) Molecular weight: about 69000 (SDS-PAGE method) (7) Isoelectric point: 3.6 Chitinase C-3 It has enzymatic chemistry. (1) It acts on chitin and hydrolyzes chitin to soluble N-acetylchitooligosaccharide. (2) Optimum pH: 7.8-8.2 at 40 ° C (3) Optimum temperature: 50 ° C at pH 8.0 (4) pH stability: Stable at 5.5-9.0 at 40 ° C ( 5) Thermostability: Stable at 50 ° C for one hour at pH 8.0 (6) Molecular weight: about 81000 (SDS-PAGE method) (7) Isoelectric point: 3.9 Enzyme chemical properties above Regarding the enzyme activity of the known chitinase, the chitinase of the present invention was compared with the known chitinase, and it was stable at 50 ° C. for 1 hour or more, and the enzyme activity was 10 units or more per 1 mg of protein, and thus the known chitinase. Is clearly different from. Therefore, chitinase C-1 and chitinase C-3 were identified as new enzymes.

The enzyme of the present invention can be used, for example, as a reagent for forming mold protoplasts or for producing N-acetylchitobiose.

[0019]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. [Example 1] Chitin powder 5 g / L, peptone 5 g / L, yeast extract 1 g / L, glucose 5 g / L, ferric phosphate 0.01 g / L
L, pre-culture medium with composition of 1000 ml natural seawater (before sterilization
pH7.5) Add 30 ml to a 300 ml flask and add Vibrio s
p. The H-8 strain was inoculated as an inoculum and cultured at 37 ° C for 24 hours. The preculture liquid thus obtained was inoculated at a ratio of 5% v / v into 30 L of a medium having the same composition as the above composition in a 100 L fermentor, and aerated and agitated at 40 ° C (rotation speed 100 rpm, The culture was performed for 18 hours with an aeration rate of 0.7 vvm). At this time, the pH is 7.
Culture was performed while adjusting to 8. [Example 2] 30 L of the culture solution obtained in Example 1 was added to 1000 rpm.
The culture supernatant was obtained by centrifugation. The supernatant was salted out with ammonium sulfate (80% saturation), the precipitate was filtered, suspended in 2 L of 20 mM sodium acetate buffer (pH 5.0), filtered again, and the filtrate was used as a crude enzyme solution. Next, 50FT-C-110
Put the above crude enzyme into a dialysis tube (manufactured by Sanko Junyaku) and add 20mM
Dialysis was performed using 40 L of sodium acetate buffer as a dialysate. The dialyzed enzyme suspension was added to DEAE Toyopearl HW-55.
(Manufactured by Tosoh Corporation) was used to fractionate the chitinase active fraction. By carrying out the fractionation with DEAE Toyopearl HW-55 twice, 140 mg of purified chitinase C-1 and 120 mg of purified chitinase C-3 could be obtained. [Example 3] To measure the activities of chitinase C-1 and chitinase C-3, a modified Petri dish (Agr. Biol. Chem. V.
35, No. 7, 1154-1156, 1971). It should be noted that one unit of chitinase is an amount having an activity of releasing 1 μmol of N-acetylglucosamine per minute.

According to the above method, the activities of chitinase C-1 and chitinase C3 were measured. As a result, the activity of chitinase C-1 was 11.5 units and the activity of chitinase C-3 was 10.8 units per mg of protein.

[0021]

INDUSTRIAL APPLICABILITY The present invention provides a novel chitinase having high activity and excellent thermostability.

 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Hiroshi Sano 1900 Sodeshi-cho, Shimizu-shi, Shizuoka Kaiyo Biotechnology Research Institute Co., Ltd. Shimizu Laboratory (72) Inventor Miyuki Nishijima 1900, Sodeshi-cho, Shimizu-shi, Shizuoka (72) Inventor Kazuo Oishi 7-1 Sanzonocho, Numazu City, Shizuoka Prefecture 303 (72) Inventor Masaaki Yamagishi 256-2 Ogawa, Yaizu City, Shizuoka Prefecture (72) Inventor Suzuki Mitsuaki 16-6, Izumi-cho, Numazu-shi, Shizuoka Room 202, Ooka Dormitory (72) Inventor, Atsuhiro Katsuyama 2-12-17, Tonooka, Shimizu-shi, Shizuoka (72) Inventor, Miwa Ha, 328 Shiodatahamano, Fukuda-cho, Iwata-gun, Shizuoka

Claims (2)

[Claims] 1. Chitinase C-1 or chitinase C-3 having the following enzymatic chemistry. A. Chitinase C-1 (1) Acts on chitin and hydrolyzes chitin to soluble N-acetylchitooligosaccharide. (2) Optimum pH: 7.8-8.2 at 40 ° C (3) Optimum temperature: 50 ° C at pH 8.0 (4) pH stability: Stable at 5.5-9.0 at 40 ° C ( 5) Thermal stability: stable at 50 ° C for one hour at pH 8.0 (6) Molecular weight: about 69000 (SDS-PAGE method) (7) Isoelectric point: 3.6 B. Chitinase C-3 (1) Acts on chitin and hydrolyzes chitin to soluble N-acetylchitooligosaccharide. (2) Optimum pH: 7.8-8.2 at 40 ° C (3) Optimum temperature: 50 ° C at pH 8.0 (4) pH stability: Stable at 5.5-9.0 at 40 ° C ( 5) Thermal stability: stable at pH 8.0 at 50 ° C for one hour treatment (6) Molecular weight: about 81000 (SDS-PAGE method) (7) Isoelectric point: 3.9 2. A microorganism belonging to the genus Vibrio and capable of producing chitinase C-1 or chitinase C-3 is cultured,
Chitinase C-1 or chitinase C-from the culture
3. A method for producing chitinase C-1 or chitinase C-3, which comprises collecting 3.