JPS6363382A - Production of chitosanase - Google Patents

Abstract

PURPOSE:To obtain chitosanase from a culture mixture efficiently, by using a novel bacterium belonging to the genus Bacillus pumilus, capable of producing chitosanase. CONSTITUTION:A bacterium [e.g. Bacillus pumilus BN-262 (FERM P-8814)] belonging to the genus Bacillus pumilus, capable of producing chitosanase, a variant thereof or a culture mixture obtained by cultivating the variant is cultivated and chitosanase is obtained from the culture mixture. The culture is preferably carried out at 27-32 deg.C at initial pH 6.5 for 40-96hr.

Description

[Detailed Description of the Invention] [Object of the Invention] (Industrial Application Field) The present invention is directed to the use of chitosanase.
Concerning a new manufacturing method.

(Prior art) As a method for producing chitosanase, Myxobacter (Myx
(obacter) [Journal of
Journal of Bacte
riology), Volume 120, No. 2.844-8
53 (1974)].

A method using bacteria belonging to the genus Bacillus [Biochimic
a et Biophysica Acta), 410
Vol., pp. 145-155 (1975);
No. 0580] is known.

(Problems to be Solved by the Invention) The object of the present invention is to efficiently produce chitosanase using microorganisms.

[Structure of the Invention] (Means for Solving the Problems) As a result of searching for various chitosanase-producing microorganisms, the present inventors discovered that a new strain belonging to the genus Bacillus efficiently produces chitosanase outside the bacterial body. As a result, the present invention was completed.

That is, the method for producing chitosanase of the present invention is characterized by culturing a chitosanase-producing bacterium belonging to Bacillus pamylus or a variant or mutant strain thereof, and collecting chitosanase from the culture.

The microorganism used in the present invention may be any strain of microorganism that belongs to the Bacillus Pamilus family and produces chitosanase, or may be a variant or mutant of these strains.

An example of a chitosanase-producing bacterium used in the present invention is strain BN-262, which was isolated from the soil of Yokohama City in the process of searching for antibiotics produced by bacteria. The mycological properties of the BN-262 strain are as follows.

■ Morphological properties The vegetative cells of this plant grown on broth agar were 0.5-0.
7 x 1.5-3.0 micron rods. 30℃, 2
Formation of endospores was observed after ~3 days of culture, and the size of the spores was 0.4 to 0.6 x 0.8 to 1.2 microns, forming an oval shape. Located from the center to the edge,
Swelling of the sporangium by spores is rarely observed.

It moves by flagella, and Durham staining is positive. anti-acid,
They do not show polymorphism and are usually isolated and not linked together for long periods of time.

(2) Cultural properties This strain grows well when cultured in a normal bacterial culture medium at 25-45°C for 1-3 days.

(1) Meat juice agar plate culture: Growth is good, colonies are pale yellowish brown and grow with small wrinkles, but no significant water-soluble or water-insoluble pigments are produced.The surface of the colonies is relatively dry. It shows the appearance.

(2) Meat juice agar slant culture: Same as (1) (3) Meat juice liquid culture: Forms a bacterial film and grows on the liquid surface.

(4) Meat juice gelatin puncture culture: Grows while liquefying gelatin.

(5) Lidomus milk: clearly liquefied, but no significant pH fluctuation or coagulation is observed.

■, Physiological properties (1) Nitrate reduction: negative (2) Denitrification reaction: negative (3) MR test: negative (4) VP test: positive (5) Indole formation: negative (6) Hydrogen sulfide formation : Negative (7) Hydrolysis of starch: Negative (8) Utilization of citric acid: Positive (9) Utilization of inorganic nitrogen source: Cannot grow using nitrate as the sole nitrogen source, but can grow with ammonium salt alone.

(IO) Pigment formation: Does not produce significant water-soluble and water-insoluble dyes.

(11) Urease: Negative (12) Oxidase: weakly positive (13) Catalase: Positive (14) Growth range: Grows in the temperature range of 15 to 45℃, but does not grow at 55℃, pH is neutral to slight Alkali is suitable for growth, and salt tolerance allows it to grow even in a medium containing 10% NaCu.

(15) Attitude towards oxygen: It is an aerobic bacterium and does not grow in anaerobic conditions.

(Is) O-F test: Type 0 (17) Sugar utilization 1) Growth of acid is observed from the following sugars, but no gas is generated.

L-arabinose, D-xylose, D-gurnice,
D-mannose, D-fructose, D-galactose, cibosugar, trehalose, D-sorbitol, D-mannite, glycerin ii) Neither acid nor gas is generated from the following sugars.

Strain BN-262, which has mycological properties superior to i-inositol, lactose, and starch, was identified in Bergey's Manual of Determinative Bacteriology, 8th edition, 1974 (Bergey's M
annual of Determinative Ba
cteriolog7゜8th edition, 19
74), and the following conclusions were obtained.

(1) Strain BN-262 is considered to belong to the genus Bacillus because it produces spores as an aerobic rod and is positive for catalase.

(2) Based on the morphology of the sporangium and spores, this strain belongs to the genus Bacillus according to Smith et al.

(3) Among Bacillus Ifi, Bacillus cereus and Bacillus megaterium differ in spore size and sporangium morphology. Limited to three species belonging to the butyl family.

(4) Due to the negative starch hydrolyzing ability and negative nitrate reducing ability, it is most appropriate to belong to Bacillus pamilus among these three species.

Based on the above results, the present inventors converted the BN-262 strain into Bacillus pamilus BN-262 (Bacillus pu.
milus BN-282) to distinguish it from known strains.

This strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as Fiber Science and Technology Research Institute No. 8814. BN-26
2 strains are susceptible to changes in their properties, as seen in other bacteria, and can be mutated by artificial mutation methods using ultraviolet rays, X-rays, chemicals, etc., and even with such mutant strains, BN Any strain of the genus Bacillus that has activity similar to strain -262 can be used in the method of the present invention.

As the medium used for chitosanase production, any synthetic or natural medium can be used as long as it contains an appropriate amount of one selected from carbon sources, nitrogen sources, inorganic substances, etc.

Nitrogen sources include, for example, Bacto soyton, corn gluten meal, corn or staple liquor, peptone, peanut meal, soybean meal, soluble vegetable protein, wheat germ, KNO3, NaNO3, C
&(NO3)2''H2O, etc. Examples of carbon sources include glucose, xylose, mannose, sucrose, glycerol, 7-rabinose, galactose, fructose, dextrin, etc. Examples of inorganic substances include sodium, Examples include salts such as potassium, magnesium, calcium, and manganese.

For example, chitosan is dissolved in the above medium using a dilute solution of an inorganic acid such as hydrochloric acid or sulfuric acid or an organic acid solution such as citric acid or acetic acid, or an enzyme such as chitosanase or an enzyme such as hydrochloric acid or sulfuric acid is added to the solution. The production of wood chitosanase can be significantly increased by adding more of this species and partially hydrolyzed chitosan.

The culture temperature is usually 25-37°C, preferably 27-32°C.
℃, and the initial pH is usually 6.0 to 7.0, preferably about 6.5.

The culture time is usually 20 to 96 hours, preferably 40 to 96 hours.
After 6 hours, chitosanase is produced and accumulated in the culture by aeration and agitation culture.

Chitosanase can be separated and purified by conventional enzyme methods such as column chromatography using Sr-Toyopearl 650M, fractional precipitation using ammonium sulfate, or gel filtration using Sephadex. can be obtained.

The chitosanase obtained as described above has the following physicochemical properties.

(1) Optimal pH and stable pH range The optimum pH in 0.2M acetate buffer is about 7.0.

In addition, the stable pH range can be adjusted by heating at 40℃ for 10 minutes.
It is in the range of H5.5 to 7.0.

(2) Measurement method of titer 2g of glycol chitosan was added to IN-HC statement 20d and 0
．． Dissolve in 2M #acid buffer 40-, adjust the pH to 5, 6, and make the total volume 100-. Add enzyme solution 1- to this 2% glycol chitosan solution 1- and react at 40°C for 10 minutes. 0 Reaction completed. Afterwards, Rohd Ie l! :Marg
The amount of glucosamine liberated is determined by the method of an. Under these reaction conditions, the amount of enzyme that releases 1 pmol of glucosamine per minute is defined as 1 unit.

(3) Range of suitable temperature for action The range of suitable temperature for action is 30 to 55°C, which was determined by adding this enzyme to a glycol chitosan solution at each temperature and measuring the enzyme activity when it was allowed to act for 10 minutes. It is.

(4) Conditions for inactivation by pH 1 temperature, etc. Heat treatment for 15 minutes in 0.2 M acetate buffer (pH 5, 6)
It is stable up to 5°C and shows 50% residual activity at 50°C.

(5) Inhibition, activation, and stabilization The residual activity after holding at 40°C for 30 minutes in a solution containing various total ions and inhibitors (containing 2.0 mM) (acetate buffer pH 5, 6) is as follows: As shown in the table below.

As is clear from the above, Fe2+, N i ”, Cu2
+, markedly inactivated by PCMB.

Activated by EDTA, N-ethylmaleimide.

(6) Molecular weight approximately 31,000 (using acrylamide gel (PH8, 8) with a pore size of 2.6%) (7) Polyacrylamide gel electrophoresis pore size 2.
When acrylamide electrophoresis is performed in a conventional manner using a 6% acrylamide gel (pH 8.8), a single band is shown.

(8) Isoelectric point approximately 9.3 (acrylamide gel focused electrophoresis method) Wood chitosanase enzymes are found in Rh 1zopus and Mucor, which belong to the order Mucoraleoles, and Trichoderma and Euascomycetes, which belong to Deuteromycetes.
It lyses the cell walls of Penicillium, which belongs to the genus Penicillium.

(Examples of the Invention) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example 1 Batato soiton 5%, peptone 1%, KNO30.2%
, glucose 1%, K H2P 040.1% and Mg
50 volumes of a pH 6.5 liquid medium containing 200.05% SO4.7H were prepared and sterilized at 120°C for 15 minutes.

As a pre-culture, 400 g of the above medium in a single Erlenmeyer flask
Bacillus pamilus BN-262 strain 1 platinum loop was inoculated in - and cultured with shaking at 28°C for 24 hours. This preculture solution 1.2
50 strains of the above-mentioned medium were inoculated, and cultured with aeration and agitation at 28° C. for 48 hours. After culturing, continuous centrifugation was performed at 6.00 rpm to obtain a culture supernatant.
．． Ammonium sulfate was added to the mixture to saturate the mixture with 75°C, and the mixture was allowed to stand overnight at 5°C for salting out. The resulting precipitate was collected by continuous centrifugation, dissolved in 1.8M deionized water, placed in a cellophane tube, and thoroughly dialyzed against deionized water. Apply this solution to Asahi Kasei Kogyo Forest's Ultra-Rapo Module 6000.
The product was ultra-passed and freeze-dried to obtain 10.7 g of crude enzyme powder. Chitosanase activity was 40 units/g.

Example 2 Batato soiton 5%, peptone 1%, KNO30.2%
A liquid medium with a pH of 6.0 containing 1% glucose, 0.2% chitosan (dissolved in hydrochloric acid and then neutralized), 1% KH2POa 0 and 5% MgSO4.7H was prepared and incubated at 120°C. Sterilized for 15 minutes. As a preculture, 40% of the above medium in a 250- Erlenmeyer flask
One platinum loop of Bacillus pamilus BN-262 strain was planted in - and cultured with shaking at 28°C for 48 hours.

Transfer this pre-culture solution 40 to the above culture i1 in a jar fermenter. ! The cells were inoculated in duplicate and cultured with aeration and stirring at 28°C for 48 hours. After culturing, continuous centrifugation was performed for 20 minutes at 8.00 rpm to obtain a culture supernatant.

After diluting 1.8 m of this supernatant by adding 1.8 m of deionized water, the pH was adjusted to 7.4 using hydrochloric acid.Then, the diluted liquid was diluted with 0.01 M phosphate buffer (pH 7.4). Column packed with equilibrated SP-Toyovar 650M (diameter 3
After adsorbing chitosanase, the column was washed with 0.01M phosphate buffer (PH 7.4) containing 0.085M salt, and then washed with 0.2M salt. 0.01M phosphate buffer containing (
500 sJ of PI(7,4) was passed through the above column to elute the adsorbed chitosanase to obtain a chitosanase-containing solution. Chitosanase activity was 450 units/g.

The chitosanase-containing solution was concentrated by ultrafiltration and equilibrated with 0.05M phosphate buffer (containing 0.1M sodium chloride). Column packed with Toyopearl HW55 (diameter 4
cm x length 100 cm), and then developed with 0.05 M phosphate buffer (containing 0.1 M sodium chloride), and the chitosanase active portion was collected and subjected to ultra7 filtration, concentration, desalting, and lyophilization to obtain a white powder. Chitosanase activity is 800 units/g
Met. When this white powder was subjected to acrylamide electrophoresis, a single band was detected.

[Effects of the Invention] According to the present invention, chitosanase can be efficiently produced.

Claims (1)

[Claims] A method for producing chitosanase, which comprises culturing a chitosanase-producing bacterium belonging to Bacillus pamilus or a variant or mutant strain thereof, and collecting chitosanase from the culture.