JPH0420290A - New chitosanase utk, production thereof and bacterium producing same enzyme - Google Patents

Abstract

NEW MATERIAL:Chitosanase UTK having following physicochemical properties; Action: Acting on chitosan and hydrolyzing said substance to produce oligochitosan; Optimum pH: 5.6; pH stability: stable at pH 5-9; Optimum temperature: 50 deg.C; Thermal stability: stable to 40 deg.C. USE:An enzyme for decomposition of chitosan. Oligochitosan, substance obtained by hydrolysis of chitosan has antibacterial properties, antifungal properties and antineoplastic properties and is useful as a preservative for foods, an anti- plant pathogenic fungi drug, etc. PREPARATION:A strain belonging to Bacillus genus, e.g. Bacillus sp. UTK (FERM p-11442) is aerobically cultured.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides a novel chitosan-degrading enzyme, chitosanase UTK.
This article relates to its production method and producing bacteria.

More specifically, the present invention relates to chitosanase LITK, a novel chitosanase degrading enzyme that hydrolyzes chitosan to produce chitooligosaccharides, its production method, and producing bacteria.

(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, x1-san has attracted attention as a substance useful in a wide range of applications, such as selective adsorbents for metals, flocculants for wastewater treatment, films, and surgical sutures.

In addition, chitooligosaccharides, which are hydrolysates of x1-san, have been found to have antibacterial, fungal, and antitumor activities, and are used as food preservatives, antiphytopathogenic preparations, and next-generation drugs. There are high hopes for the development.

Generally, chitooligosaccharides can be obtained by hydrolysis with acid or alkali, but the yield of oligomers is very poor. Also,
For example, when chitosan is hydrolyzed with hydrochloric acid, as a result of random decomposition, the amount of oligosaccharides obtained is mono-glucosamine, diglucosamine, tri-glucosamine, Te) ~
The higher the degree of polymerization, the lower the yield.

However, the physiological activity of chito-oligosaccharides is remarkable at a relatively high degree of polymerization, and chitosan can be decomposed to obtain oligosaccharides with a large degree of polymerization, and various oligosaccharides with different degrees of polymerization can be obtained. 1-san-degrading enzymes are in demand.

Conventionally, the production of Ki1-san degrading enzymes by microorganisms has already been reported in JP-A-62-201.571 and JP-A-63-94.
971, +1e+:1ges, A,, at, al
,,J,Bacteriol,, 120.844
(1974), Tominaga + y, et.
al,, Bjochim, Bj.

phys, Acta, 410.145 (+975)
However, there are no reports on chitosan-degrading enzyme-producing bacteria that can be used effectively in industry.

The present inventors discovered that the chitosan-degrading enzyme Bacillus sp. 1ITK produces a novel chitosan-degrading enzyme in a wide range of microorganisms, and this enzyme was
They decided to name it JTK.

The physicochemical properties of the x1-sanase UTK of the present invention are as follows.

(1) Action: Acts on x1-san to decompose it and produce chitooligosaccharide.

(2) Optimal pH: When using hydrochloric acid-sulphurium acetate buffer, acetate buffer, Atokj, ns &Pantin's buffer, optimal ρ11 is 5°6 (Figure 1) .

(3) pH stability: Hydrochloric acid-sodium acetate buffer, M
cl Jva]ne's buffer, ALokjns &
As a result of measuring the residual activity of the enzyme after incubation in Pantjn's buffer at 37°C for 3 hours, it was found to be stable at P115-9 (Figure 2).

(5) Thermal stability: 0.05M acetate buffer (P++ 5
．． 5) Insulate at each temperature of 20°C to 100°C for 15 minutes,
If residual activity! It is stable up to 40°C (Figure 4).

(6) Enzyme activity measurement method: Enzyme activity was measured using chitosan as a substrate in 1-%/LM sodium acetate buffer (pH 5, 7).

This substrate was mixed with imQ and enzyme solution J-IIQ at 37°C for 10 min.
After reacting for a minute, the mixture was boiled at 100° C. for 10 minutes to stop the reaction.

The generated glucosamine was obtained using a modified 5chales method (Imot
o.

T,, et, al,, Agric, Bjol,
Chem., 35.1154 (1971)). Note that 1 enzyme unit corresponds to the activity that increases the reducing power equivalent to 1 μmol of glucosamine per minute.

(7) Influence of metal ions, etc. To 2 mM of the enzyme solution diluted with 0.05 M acetate buffer (pH 5.5), add 1 mQ& of each 4X], 0-3M reagent solution, and incubate at 37°C for 15 minutes. Thereafter, 1 mQ of substrate was added, enzymatic reaction was performed for 10 minutes, and residual activity was examined. still,
Comparisons were made based on relative activity, with distilled water being used instead of the reagent as 100%.

Table 1 Inhibitors affected by metals, etc. Residual activity (%) Inhibitor Residual activity (%)
) Additive-free 100 KCQ
93.2AgNO36,1,K2CrO70,8A(
1, (SO,)33.4 Mg5o,
84.1BaCQ283.2MnCQ
, 1.05CaCQ, ] 10
NaCQ2106CdCQ, 26.3
NiCl1. , 34.0CoCQ2
88.9 PbC111,,9,2CLIC
Q7. 6.6 Zn(CHaCO
O) 26.5FeS045.8 PC
MB 11.9μgCQ, 11.9
EDTA 74.4ICH2COOH2
], 8 From the above results, this chitosan degrading enzyme is Ag+, AQ”
, Cu2+, Fe24, Hg 2'', p b 2 +,
It is inhibited by PCBM &.

(8) Enzyme purification method The enzyme can be isolated and purified according to conventional methods.

Bacterial cells were removed from the culture solution, the clear liquid was dialyzed against 0.02M phosphate buffer, the low molecular weight fraction was removed, the dialyzed fraction was adsorbed to colloidal chitosan, and the adsorbed fraction was centrifuged. Collect and elute the precipitate by adding 0.02M phosphate buffer containing 2M sodium chloride, and remove the supernatant by centrifugation.

This clear liquid is purified by a purification means such as C Tosephatex C-50 column chromatography (FIG. 5).

(9) Molecular weight The molecular weight of this enzyme was determined using Sephacryl 5-200.
When measured by gel filtration method, it is calculated to be 28,000. The results are shown in Figure 6.

The hesanase UTK-producing bacterium of the present invention, Bacillus sp.

It has been deposited with the IJT as FIERM P-11442 at the FIKEN, and the mycological properties of Bacillus sp, UTK are shown below.

(A) Morphological properties (broth agar medium) Cell size = 4 μ ~ 6 μ x 1 μ ~ 1.5 μ Cell shape: Presence or absence of pleomorphism of bacillus cells: Presence or absence of no motility: Presence of no spores: Yes Durham staining: Incubation 18-4 hours Positive (B) Physiological properties Catalase: + Oxidase: Ten Methyllets: ■-P test: + (pif 5.5) Liquefaction of gelatin; + Hydrolysis of starch: + Urease: Citrus Acid assimilation Christensen: Hydrogen sulfide production TSI: Ten-indole production: Denitrification reaction: Nitrite reduction: 0-17 test: Fermentation Oxygen influence: Microaerobic heat resistance 290°C Growth pH: pH 5 ~ 9 Production of acids and gases from sugars Types of sugars Production of gases Glucose Mannose Sucrose Rumnose Lactose Fur 1 Hose Cellobiose Raffinose Starch Glycerin Sorbit Inosiso 1- Salt tolerance 5% + 7% + Production of acids In the present invention, Bacillus sp. UTK can be cultured aerobically in culture medium and chitosanase UTK can be accumulated during culture.

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

Next, examples of the present invention will be shown.

Example 1 Bacillus sp, UTK, FIERM P-], ], 4
42 in an Erlenmeyer flask with 2.0% peptone and 1 part meat extract.
．． 0%, yeast extract 0.6%, salt 0.5% (pH 5,
Inoculate 50 mQ of a medium with the composition of 0) and incubate at 30°C for 2
Incubate for days. Using this as a starter culture medium IQ with the same composition.
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 obtained crude enzyme solution was dialyzed against 0.02M phosphate buffer to remove low molecular weight substances.

To 5 volumes of this dialysis fluid, add colloidal chitosan-1 hesane (preparation method: Chitosan Log to 500 mQ of deionized water to swell it, and add 90 mQ of IMM acid to dissolve it.

Add 250 mQ of 1M sulfurium acetate to this and
Adjust pH to 8 with potassium hydroxide. Next, stir with a blender to make a uniform colloid. Centrifuge this when necessary and use only the precipitate. ) was added and adsorbed.

The adsorbed material was centrifuged, the supernatant was discarded, and a volume of 0.02M phosphate buffer (pH 7.5) was added to centrifugally wash the precipitate.

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

Subsequently, cation exchange chroma I-fluorography was performed using CM-Sephadex C-50, and 0.05M phosphate buffer (O to 0.6M sodium chloride 11 linear concentration gradient: pl
i7.0) and the active fractions were collected. This active fraction was desalted by dialysis and lyophilized to give 25 mg.
Ki I-Zanase III TK was obtained.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the optimum temperature for xyl-sanase tlTK [11], Figure 2 is a diagram showing its pH stability, Figure 3 is a diagram showing its optimal temperature, and Figure 4 is a diagram showing its optimum temperature. Figure 5 shows the thermostability of x1-sanase L in the purification of Example 1.
FIG. 2 is a graph showing ITK's C tocefadesox C-50 column taroma h. FIG. 6 is a diagram showing the molecular weight of KiI-sanase UTK. Agent Patent Attorney Door ITI Parent Male Diagram i-1 Optimal 1- Atkins & Pant in' Suffer +2 Diagram pH Stability 1)- Ringe r uffer Diagram (℃) Optimal Temperature Diagram ('C) Heat Stability Procedure Amendment Band May 29, 1991

Claims (3)

[Claims] (1) Chitosanase UTK (
a) Action: Acts on chitosan and hydrolyzes it to produce chitooligosaccharide. (b) Optimum pH: pH 5.6 (hydrochloric acid-sodium acetate buffer, acetate buffer, Atokins &Pantin's
(c) pH stability: Hydrochloric acid-sodium acetate buffer, Mc
llvaine's buffer, Atokins & Pant
As a result of measuring the residual activity of the enzyme after incubation with in's buffer at 37°C for 3 hours, it was found to be stable at pH 5 to 9. (d) Optimal temperature: 50°C (e) Thermal stability: 0.05M acetate buffer (pH 5.5)
It is stable up to 40°C when the residual activity is measured by incubating at each temperature of 20°C to 100°C for 15 minutes. (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 sp. U.T.K.