JPH062059B2 - Method for producing chitosanase - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing chitosanase capable of degrading a polysaccharide such as chitosan, which is excellent in degradability of chitosan and can obtain chitosan oligosaccharide in a high yield. The present invention relates to a method for producing chitosanase capable of producing.

[Prior Art and Technical Background of the Present Invention]

Chitin is widely distributed in crustaceans, crustaceans, and other crustaceans, as well as in insects, shellfish, and fungi, and N-acetyl-
Molecular weight of β- (1 → 4) linked β-D-glucosamine
It is a straight-chain basic polysaccharide of over 1 million. Chitosan is deacetylated chitin and is a linear polysaccharide in which D-glucosamine is β- (1 → 4) linked.

In recent years, chitin and chitosan have been attracting attention as natural and abundant unused biological resources.Membranes, artificial skin, immobilized enzyme materials, affinity chromatography gels, contact lens forming raw materials, protein flocculating precipitants, etc. Used for.

Because of chitin and chitosan, which have versatility in this way, research and development on their effective use and practical application have recently been enthusiastically pursued, and along with this, chitosanase, a chitosan degrading enzyme, has been developed. Also, its application fields are being considered. For example, chitosan oligosaccharide, which is an enzymatic degradation product of chitosan, can be used for a wide range of applications such as food additives, cosmetic ingredients, pharmaceuticals, soil conditioners, antibacterial agents, plant seed germinants, plant virus disease control agents, etc. It is hoped that technological development for producing chitosan oligosaccharides is desired.

As chitosanases of microbial origin that have been reported so far, (a) Bacillus (Bacillus SP.) No. 99-5, (b) Bacillus (Bacillus SP.) R-4, (c) Bacillus Circulance Lcc -1, (d) Bacillus SP. No. 7-M, (e) Myxobacter SP. AL-1, (f) Streptomyces SP. WAK-83, (g) Strepto Myces (Streptomyces SP.) No.6, (h) Streptomyces griseus HUT 6037, (i) Penicillium ilandicum (Penicillium islandicum), (j) Alcaligenes SP. MHK-1, It is known that these produced chitosanases decompose chitosan to produce oligosaccharides.

However, the chitosanase obtained from the microorganisms (a) to (j) is too small in quantity for industrial use, and the decomposing power of chitosan was not always sufficient.

[Technical issues to be solved]

The present invention was made in view of the fact that the conventional method for obtaining chitosanase was immature for industrial use, and is excellent in the decomposing power of chitosan, and furthermore, it is possible to obtain chitosan oligosaccharide in a high yield. It is a technical object to provide a new method for producing chitosanase that can be used.

[Means adopted for solving the problem]

By the way, the microorganisms that produce chitosanase are (a) to
Although not limited to those listed in (j), it is extremely difficult to find a microorganism capable of efficiently producing chitosanase, which has a particularly excellent chitosan-decomposing power, among a myriad of microorganisms.

The present inventors' research started from trial and error work of searching for an unknown microorganism that produces chitosanase from a large number of kinds of microorganisms, and since the start of the research, a very empty experiment has been conducted. Was repeated. However, by chance, when the microorganism of the genus Alcaligenes was cultivated in a medium containing chitosan, it was found that chitosanase which very efficiently produces chitosan oligosaccharide is produced in the medium, and the present invention has been completed. It depends.

That is, the present invention belongs to the genus Alcaligenes, by culturing a strain producing chitosanase in a medium containing colloidal chitosan, to act on the strain colloidal chitosan, thus separating the chitosanase from the cultured strains The above-mentioned technical problems are solved by adopting the means.

Then, the above-mentioned strain producing chitosanase is Alcaligenes sp. IK-5 (Microtechnology Research Institute No. 9678).

The chitosanase obtained from the method of the present invention is characterized by acting on colloidal chitosan and having the highest enzyme activity at a temperature of 55 ° C to 60 ° C. Such chitosanase is a bacterium that extracellularly produces chitosanase belonging to the genus Alcaligenes and is produced in a culture medium by aerobically culturing in a liquid medium containing chitosan. For the chitosanase in the medium, after separating and removing the microbial cells, ammonium sulfate is added to the supernatant to salt out the protein component, and the chitosanase protein is concentrated, separated and purified by ion chromatography.

The liquid medium used for culturing the chitosanase-producing bacterium needs to contain colloidal chitosan in addition to a carbon source, a nitrogen source and other inorganic salts. Culturing is performed at a temperature of 30 ° C for 2 to
It is carried out under aerobic conditions of aerated shaking culture for 3 days.

Chitosanase-producing strains belong to the genus Alkaline Genes,
A bacterium that acts on colloidal chitosan and exhibits the highest enzyme activity at temperatures of 55 ° C to 60 ° C, and is Alcaligenes sp. IK-5 (Microtechnology Research Institute No. 9678)
Is preferred.

Alicarigenes sp. IK-5 is isolated from soil in the field of 3-chome Daiganji, Fukui City, which is a bacterium that grows in a medium containing chitosan as the sole carbon source. It has been deposited with the Institute for Microbial Engineering and Technology.

The mycological properties of Alcaligenes sp. IK-5 are listed below.

A. Morphological properties (1) Cell shape and size: 0.3-0.4 × 3-5 μm long rod.

(2) Cell polyplasia: Polymorphism is not shown with uniform size.

(3) Presence or absence of spores: None (4) Presence or absence of capsular membrane: None (5) Presence or absence of metachromatic bodies: None (6) Presence or absence of PHB: None (7) Motility presence: Pericardial movement (8) Proper motion: Small proper vibration (9) Dye production: No (10) Migration: Yes B. Properties on various media (1) Meat broth agar plate medium: A round, raised milky white colony is formed.

The surface of the colony is uneven and translucent.

The colony rises over time.

No pigment is produced.

(2) Broth agar slope medium: A milky white colony is formed.

The colony has a convex ridge and spreads over time.

No pigment is produced.

(3) Broth liquid medium: becomes cloudy as a whole over time.

A granular precipitate forms on the bottom, which increases over time.

C. Physiological properties (1) Nitrate reducing ability: Negative (Nitrate broth medium, 37 ° C, 3-7 days) (2) OF test: Negative (Hugh-heifson medium) (3) Cytochrome oxidase: Positive (4) Catalase: Positive (5) H ₂ S production: Negative (SIM and TSI medium) (6) Amino acid hydrolysis: Negative (lysine, arginine, ornithine) (7) Malonic acid availability: Negative (8) Citric acid availability: Negative ( 9) Esculin degradation: Positive (10) Phenylpyruvate production: Negative (11) Indole production: Negative (12) VP test (acetylmethylcarbinol production test): Negative (13) ONPG production: Positive ( 14) Urea production: Negative (15) Glycolytic ability: Glucose, lactose, sucrose, rhamnose, raffinose, adonite, arabinose and inositol are all negative.

For the above-mentioned mycological properties, see the Berseys Manual
Of Systematic Bacteriology (Bargey's M
The 1984 version of the “Anal of Systematic Bacteriology” was searched, and the IK-5 strain is considered to belong to the genus Alkaline Genes, and the present inventors named it the Alkaline Genes sp. IK-5 strain.

D. Enzymatic chemistry (1) It acts on working chitosan and decomposes it to produce chitosan oligosaccharides.

(2) Working temperature range and optimum working temperature Soluble chitosan acts as a substrate up to 80 ℃, and the optimum working temperature is 55 ℃.

Fig. 1 shows the relationship between the action temperature and the relative activity when the reaction was carried out at pH 6.0 for 10 minutes.

(3) Working pH range and optimum pH It works in the range of pH 3 to 11, and the optimum pH is pH 6.5.

FIG. 2 shows the relationship between pH and enzyme activity when a reaction solution obtained by adding soluble chitosan and an enzyme solution to buffer solutions of various pHs was reacted at 40 ° C. for 10 minutes.

(4) Thermal stability Stable by heating at 40 ° C for 10 minutes, and at higher temperature
It was gradually deactivated by heating for 15 minutes, and completely deactivated by heating at 55 ° C for 15 minutes.

The relationship between temperature and relative activity is shown in FIG.

(5) pH stability After standing in a 0.1 M buffer for 30 minutes at 30 ° C., the residual enzyme activity was measured, and it was stable in the range of pH 5 to pH 10.

The relationship between pH and relative activity is shown in FIG.

(6) Inhibitor The chitosanase obtained from the present invention is 5 × 10 ⁴ M CaC.
l, CdCl ₂ , KCl, MgCl ₂ , MgCl ₂ , NaCl, NiCl, and CoCl ₂ were not inhibited, and only about 30% was inhibited by FeCl ₂ .

(7) Substrate specificity When the activity specificity of soluble chitosan is 100% when acting on various substrates and the substrate concentration is 0.25%, the specificity is 71% for colloidal chitosan and 18% for glycol chitosan. However, colloidal chitin, microcrystalline cellulose, CM-
Cellulose was 0% and did not decompose the substrate at all.

(8) Molecular weight The molecular weight of the chitosanase obtained in the present invention was about 58,000 by SDS-polyacrylamide gel electrophoresis.

(9) Measurement of chitosanase titer Dissolve 1 g of powdered chitosan in 50 ml of 0.1 M acetic acid aqueous solution,
After adjusting to pH 6.0 with M sodium acetate solution,
Add 0.1 M acetate buffer to bring the total volume to 100 ml.
The% soluble chitosan solution serves as the substrate preparation. 1% of substrate
1 ml of enzyme solution is added to 1 ml of chitosan solution, and the enzyme reaction is performed at 37 ° C for 10 minutes. Then, the reaction solution is boiled for 3 minutes to stop the enzymatic reaction, and the reducing sugar produced in the reaction solution is treated with the Elson-Morgan method [Sakuzo Fukui: Quantitative method for reducing sugar, 112-
See page 119, Academic Publishing Center (1969)]. Under this reaction condition, the chitosanase activity is defined as one unit of the amount of enzyme that liberates a reducing sugar corresponding to 1 μmol of glucosamine.

〔Example〕

 Hereinafter, the present invention will be described in detail based on examples.

Preparation of seed culture: 100 ml of liquid medium (pH 7.3) containing 1.7% tryptone, 0.3% soypeptone, 0.25% glucose, 0.25% dipotassium phosphate and 0.5% NaCl was put into a 300 ml Erlenmeyer flask and sterilized. Alcaligenes SP. I
Inoculate K-5 and incubate at 30 ° C with shaking for 24 hours.

Preparation of culture solution for chitosanase production: 2 Erlenmeyer flask with 0.1% glucose, 0.5% colloidal chitosan, 0.5% peptone, 0.1% yeast extract, 0.1% K
Liquid medium (pH 7) containing H ₂ PO ₄ , 0.02% MgSO ₄ , 0.1% NaCl
After 1 was put in and sterilized, about 50 ml of the seed culture solution obtained above was inoculated into this and cultivated with shaking at 30 ° C. for 3 days. The culture broth was centrifuged at 6000 rpm to remove bacterial cells, and the chitosanase activity of the resulting supernatant was measured by the above-described method for measuring the chitosanase titer. 0.45 per mg of supernatant protein
It was a unit.

Purification of enzyme solution: Powdered ammonium sulfate was added to 1935 ml of the supernatant obtained above to 80% saturation, the mixture was centrifuged at 5000 rpm, and the obtained precipitate was dissolved in distilled water to make 300 ml. This enzyme solution and 0.02M
CM dialyzed against the phosphate buffer (pH 6.0), and then the resulting enzyme solution was equilibrated with 0.02M phosphate buffer (pH 6.0).
-Column packed with Sephadex C-50 [2.6 cm (diameter)
X 100 cm (length)] to adsorb chitosanase. After washing the column with about 300 ml of 0.02 M phosphate buffer, the enzyme protein was eluted with a linear gradient of 0 to 0.5 M sodium chloride. The enzyme solution eluted by this column chromatography operation had a chitosanase activity of 21.28 units / mg, which was about 47 times as purified as the supernatant.

Further, the eluate obtained by this column chromatography was placed in a column packed with CM-Sephadex C-50 by an ultrafiltration device using a membrane filter SM (product of Sartorius) [2.6 cm (diameter) × 100 cm (length)]. After adsorbing chitosanase, the enzyme protein was eluted with a linear concentration gradient of 0 to 0.5 M sodium chloride.

The chitosanase activity of the eluted enzyme solution obtained by this column chromatography was 38.60 units / mg, which was purified 86 times that of the initial supernatant.

The measured values in the above purification process are shown in Table 1 below.

[Effects of the present invention] As described in the above examples, according to the method of the present invention, a strain of the genus Alcaligenes obtained from among microorganisms existing in unrestricted condition in natural soil is used in a medium containing colloidal chitosan. Chitosanase can be efficiently produced by a relatively simple operation of culturing and separation treatment, and the chitosanase produced in this way has excellent chitosan decomposing ability to obtain chitosan oligosaccharide in high yield. Its industrial utility value is enormous.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between temperature and relative activity of chitosanase obtained by the present invention, and FIG. 2 is a graph showing the relationship between pH and relative activity of chitosanase obtained by the present invention,
FIG. 3 is a graph showing the thermostability of chitosanase obtained by the present invention, and FIG. 4 is a graph showing the pH stability of chitosanase obtained by the present invention.

Claims (1)

[Claims] 1. An Alkaline Genes sp. IK-5 strain that belongs to the genus Alkaligenes and produces chitosanase by acting on colloidal chitosan
No.), culturing in a medium containing colloidal chitosan,
Thereafter, a method for producing chitosanase, which comprises producing chitosanase by separating from this cultured strain.