JPH0675503B2 - Novel chitosanase UTK, its production method and production strain - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel chitosan-degrading enzyme, chitosanase UTK, a method for producing the same, and a bacterium producing the same.

More specifically, the present invention relates to a novel chitosan-degrading enzyme, chitosanase UTK, which hydrolyzes chitosan to produce chitooligosaccharide, a method for producing the same, and a bacterium producing the same.

(Prior Art) Chitosan is a β-1,4 polymer of D-glucosamine obtained by deacetylating chitin contained in the shells of crabs and shrimps.

In recent years, chitosan has been attracting attention as a substance useful in a very wide range of applications such as a metal selective adsorbent, a coagulant for wastewater treatment, a film, and a surgical suture.

In addition, it was revealed that chitooligosaccharide, which is a hydrolyzate of chitosan, also has antibacterial, antifungal, and antitumor activity, and is expected to be used as a food preservative, antiphytopathogenic drug, and as a drug. It is sent.

Generally, a method for obtaining a chitooligosaccharide can be obtained by hydrolysis with an acid or an alkali, but the yield of the oligomer is very poor. Also,
For example, when hydrolyzing chitosan with hydrochloric acid, as a result of random decomposition, the amount of oligosaccharide obtained is in the order of mono-glucosamine, di-glucosamine, tri-glucosamine, tetra-glucosamine, the higher the degree of polymerization, Yield is reduced.

However, the bioactivity of chito-oligosaccharides is remarkable in the portion with a relatively high degree of polymerization, and it is possible to decompose chitosan to obtain oligosaccharides with a high degree of polymerization and different degrees of polymerization, and chitosan with high potency. Degrading enzymes are needed.

Conventionally, the production of chitosan degrading enzymes by microorganisms has already been disclosed in JP-A-62-201571, JP-A-63-94971, Hedges, A., e.
t.al., J.Bacteriol., 120,844 (1974), Tominaga, Y., e
Although there are reports such as t.al., Biochim. Bio-phys. Acta, 410, 145 (1975), there is no report on a chitosan-degrading enzyme-producing bacterium that can be effectively used industrially.

The present inventors searched for a chitosan-degrading bacterium for a wide range of microorganisms, and as a result, a bacterium belonging to the genus Bacillus, Bacillus sp.U.
We found that TK (currently fixed as Bacillus licheniformis UTK) produces a novel chitosan-degrading enzyme, and named this enzyme chitosanase UTK. In addition, chitosanase UTK, chitosan in the medium,
It is an enzyme that is produced constitutively without adding inducers such as colloidal chitosan.

The physicochemical properties of the chitosanase UTK of the present invention are as follows.

(1) Action and substrate specificity: It acts on chitosan and decomposes it to produce chitooligosaccharide. It also works on powdered chitosan and 50% deacetylated chicken, but not on carboxymethylcellulose (CMC) at all.

(2) Optimum pH: Optimum pH when using hydrochloric acid-sodium acetate buffer, acetate buffer, Atokins &Pantin's buffer
Is 5.6 (Fig. 1).

(3) pH stability: hydrochloric acid-sodium acetate buffer, Mcllva
ine's buffer, Atokins &Pantin's buffer at 37 ℃
As a result of measuring the residual activity of the enzyme which was kept warm for a time, pH 5-9
It is stable (Fig. 2).

(5) Thermal stability: 20 ℃ to 100 with 0.05M acetate buffer (pH5.5)
When the residual activity is measured by incubating for 15 minutes at each temperature of ℃ 40
It is stable up to ° C (Fig. 4).

(6) Method for measuring enzyme activity: For enzyme activity, chitosan was used as a substrate in 1% / 1M sodium acetate buffer (pH 5.7).

After reacting 1 ml of this substrate with 1 ml of the enzyme solution at 37 ° C. for 10 minutes, the reaction was stopped by boiling at 100 ° C. for 10 minutes.

The glucosamine produced was modified by the Schales modified method (Imoto, T., et.
l., Agric. Biol. Chem., 35, 1154 (1971)). The enzyme unit was defined as 1 unit of activity for increasing the reducing power corresponding to 1 μmol of glycosamine per minute.

(7) Effect of metal ions, etc. 2 ml of enzyme solution diluted with 0.05 M acetate buffer (pH 5.5)
To this, add 1 ml of 4 × 10 ⁻³ M reagent solution and incubate at 37 ° C. for 15 minutes. After that, 1 ml of the substrate was added, an enzymatic reaction was carried out for 10 minutes, and the residual activity was examined. In addition, the comparison was carried out by the relative activity with 100% using distilled water instead of the reagent.

From the above results, this chitosan degrading enzyme is Ag ⁺ , Al ³⁺ , Cu
^{Inhibited by 2+} , Fe ²⁺ , Hg ²⁺ , Pb ²⁺ , PCBM.

(8) Method for Purifying Enzyme The enzyme can be isolated and purified according to a conventional method.
Bacteria were removed from the culture solution, and the supernatant was dialyzed against 0.02M phosphate buffer to remove low molecular weight fraction, and the dialyzed fraction was adsorbed on colloidal chitosan, and the adsorbed fraction was centrifuged. The precipitate is collected, 0.02M phosphate buffer containing 2M sodium chloride is added to the precipitate to elute, and the supernatant is collected by centrifugation.

This supernatant is purified by a purification means such as CM-Sephadex C-50 column chromatography (Fig. 5).

(9) Molecular weight The molecular weight of this enzyme is calculated to be 28,000 when measured by the gel filtration method using Sephaacrylic s-200. The results are shown in Fig. 6.

The chitosanase UTK-producing bacterium of the present invention, Bacillus sp. UTK, has been deposited at MIC as FERM P-11442 (currently,
Bacillus licheniformi UTK
s UTK) and transferred from the previous FERM P-11442
Deposited as FERM BP-3794. ), Bacillus sp.
The mycological properties of UTK (ie Bacillus licheniformis UTK) are shown below.

(A) Morphological properties (meat agar medium) Cell size: 3.5 μ ~ 5.0 μ x 0.5 μ ~ 0.9 μ Cell shape: Bacillus Cell polymorphism: No motility: Peripheral flagella spores Presence or absence: Yes Gram stain: 18 to 48 hours of culture Positive (B) Physiological properties Catalase: + Oxidase: + Methyl red: − VP test: + (pH 5.5) Liquefaction of gelatin: + Hydrolysis of starch: + Urease: -Assimilation of citric acid Christensen: + Purification of hydrogen sulfide TSI: + Formation of indole: -Denitrification reaction: + Reduction of nitrite: -OF test: Fermentation Oxygen effect: Microaerobic heat resistance : 90 ℃ Growth pH: pH5 ~ 9 Acid and gas production from saccharides Sugar type Gas production Acid production Glucose- + mannose- + sucrose- + rhamnose-lactose-maltose-cellobiose- + rough Inose --- Starch-+ Glycerin-+ Sorbit --- Inosit --- Salt tolerance 5% + 7% + In the present invention, Bacillus licheniformis UTK is cultivated aerobically in a culture medium and chitosanase UTK is added during the culture. Can be accumulated.

The obtained culture solution is centrifuged, the supernatant is used as a crude enzyme solution, and the crude enzyme solution is dialyzed to remove low-molecular substances, and purified by an adsorption method using colloidal chitosan. High titer chitosanase UTK can be obtained by purification by exchange column chromatography.

Next, examples of the present invention will be described.

Example 1 50 ml of a medium having the composition of Bacillus licheniformis UTK (FERM BP-3794) of peptone 2.0%, meat extract 1.0%, yeast extract 0.6%, salt 0.5% (pH 5.0) in an Erlenmeyer flask.
And incubate at 30 ° C for 2 days. With this as an inoculum,
The medium 1 having the same composition was inoculated and cultured at 30 ° C. for 2 days.

Next, this culture solution was centrifuged (8,000 rpm × 15 minutes), and the supernatant was collected to obtain a crude enzyme solution. The crude enzyme solution obtained was 0.
It was dialyzed against 02M phosphate buffer to remove low molecular weight substances.

For 5 volumes of this dialysate, colloidal chitosan (preparation method: 10 g of chitosan was added with 500 ml of deionized water to swell,
Add 90 ml of 1M acetic acid to dissolve. To this, 250 ml of 1M sodium acetate is added, and the pH is adjusted to 8 with 1N potassium hydroxide. Next, the mixture is agitated with a blender to form a uniform colloid. Centrifuge this when necessary and use only the precipitate. ) Was added for adsorption.

The adsorbate was centrifuged, the supernatant was discarded, 1 volume of 0.02 M phosphate buffer (pH 7.5) was added, and the precipitate was washed by centrifugation.

Subsequently, 2 volumes of 0.02M phosphate buffer (pH 7.5) containing 3M sodium chloride was added to the precipitate, and the mixture was shaken at 37 ° C for 1 hour to elute the adsorbed enzyme. The eluted fraction was dialyzed against 0.05M phosphate buffer (pH 7.0) to desalt.

Subsequently, cation exchange chromatography with CM-Sephadex C-50 was performed to obtain 0.05M phosphate buffer (0 to 0.6M).
Elution was performed with a linear concentration gradient of sodium chloride: pH 7.0), and active fractions were collected. This active fraction was desalted by dialysis and lyophilized to obtain 25 mg of chitosanase UTK.

[Brief description of drawings]

Figure 1 shows the optimum pH of chitosanase UTK, Figure 2 shows its pH stability, Figure 3 shows its optimum temperature, and Figure 4 shows its thermal stability. FIG. 5 is a diagram showing CM-Sephadex C-50 column chromatography of chitosanase UTK in the purification of Example 1. FIG. 6 is a measurement diagram of the molecular weight of chitosanase UTK.

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Claims (3)

[Claims] 1. A chitosanase UT having the following physicochemical properties:
K (a) Action and substrate specificity: acts on chitosan and hydrolyzes it to produce chitooligosaccharide. Powdered chitosan, 50
It also acts on% deacetylated chitin, but not on carboxymethylcellulose (CMC) at all. (B) Optimum pH: pH 5.6 (when using hydrochloric acid-sodium acetate buffer, acetate buffer, Atokins &Pantin's buffer) (c) pH stability: hydrochloric acid-sodium acetate buffer, Mcllva
ine's buffer, Atokins &Pantin's buffer at 37 ℃
As a result of measuring the residual activity of the enzyme which was kept warm for a time, pH 5-9
And stable. (D) Optimum temperature: 50 ° C (e) Thermal stability: 20 ° C to 100 with 0.05M acetate buffer (pH 5.5)
When the residual activity is measured by incubating for 15 minutes at each temperature of ℃ 40
Stable up to ℃. 2. A method for producing chitosanase UTK, which comprises culturing a chitosanase UTK-producing bacterium belonging to the genus Bacillus and collecting chitosanase UTK from the culture. 3. Bacillus licheniformis producing chitosanase UTK.