JPS61280273A - Production of bacteriolytic enzyme - Google Patents

Abstract

PURPOSE:To obtain a bacteriolytic enzyme useful for the research of cell fusion, etc., and the disinfection of foods and pharmaceuticals, etc., by culturing a bacterial strain belonging to Flavobacterium genus and capable of producing an enzyme dissolving the cell wall of microorganisms and separating the enzyme from the culture product. CONSTITUTION:A bacterial strain belonging to Flavobacterium genus and capable of acting to the cell wall of microorganisms and dissolving the wall [e.g. Flavobacterium sp. SH-548 (FERM-P 8265)] is cultured in a medium and the culture liquid is centrifuged to remove the bacterial cells. The obtained supernatant liquid is added with ammonium sulfate to attain 60% saturation concentration and the precipitate is separated by centrifugation to obtain the objective bacteriolytic enzyme.

Description

[Detailed Description of the Invention] [Object of the Invention] An object of the present invention is to provide a method for producing a lytic enzyme by culturing a strain belonging to the genus Flavobacterium and having the ability to produce a microbial cell wall lytic enzyme. be.

(Industrial Application Field) Lytic enzymes are effectively used to elucidate the structure and physiological functions of microbial cell walls and to prepare grotoplasts necessary for research on cell fusion and the like.

Moreover, it is a useful substance not only in academic terms but also in practical terms, where it is applied to sterilize foods, medicines, etc., and to extract active ingredients from various microbial cells.

(Prior Art) (Problems to be Solved by the Invention) In addition to egg white lysozyme, various enzymes of microbial origin are known as lytic enzymes. However, the substrate specificity and the types of microorganisms that can be lysed vary; for example, the lytic enzyme is pl-4 glycosidase (N-acylhexosaminidase).

Amidase (N-acylmuramylalanine amidase)
Peptidases are mentioned, each of which is capable of lysing bacterial cell walls to a fairly wide range.

In research on the decomposition of persistent substances by microorganisms, the present inventors found that Rhodococcus erythropolis (
Rhodococcus erythro-poli
s) AN-13 was discovered, but since this bacterium cannot be lysed by lytic enzymes such as the above-mentioned lysozyme, a new lytic enzyme effective for the lysis of this bacterium was needed.

[Structure of the invention]

(Means for solving the problem) In view of the above situation, the present inventors screened enzyme-producing bacteria, and discovered new lytic enzyme-producing bacteria.
As a result of a search for organisms that have the ability to produce enzymes with high cell wall lytic activity that can lyse the Rhodococcus erythropolis AN-13 bacteria, one strain was isolated from soil, that is, one strain belonging to the genus Flavobacterium. The present invention was achieved based on the discovery that this produces an enzyme with strong bacteriolytic activity.

That is, the gist of the present invention is to culture a strain that belongs to the genus Flavobacterium and has the ability to produce an enzyme that acts on and dissolves the cell wall of microorganisms, and to collect the bacteria from the culture. This is a method for producing a lytic enzyme.

(Effect) The strain of the present invention has the following mycological properties.

(a) Morphology (1) Morphology of bacteria: Bacillus 0.5-Q, 8X2. O
~2.5μ (2) Flagella: None 3) Spores: None 4) Durham staining: Negative (b) Growth status in each medium (1) Broth agar plate culture: colony circular, convex circular,
Gold-rimmed, smooth, glossy, translucent, yellow (2) Broth agar slant culture: Good growth, insoluble yellow pigment production (3) Growth temperature: 10-30''C, does not grow at 35℃.

(C) Physiological Properties (1) Hydrolyzed Gelatin: + Casein: + Starch: + Cellulose - Chitin Ni Agar: - (2) Production of acids and gases from sugars Sugars Acid Gas Low Glucose - - Lactose - - Sucrose - Maldose - Bacteria with the above mycological properties are classified as flavobacteria according to the classification of Virginie's Manual of Deterministic Bacteriology, 8th edition (1974). This strain was identified as belonging to the genus P.
Labo Bacterium sp. S H-548 (Flav
obacterium sp QSH-548). This strain has been deposited with the National Institute of Microbiology and Industrial Technology as FAIKEN Bacteria No. 8265.

The culture conditions for obtaining a highly active lytic enzyme using the above bacterial strain are as follows. Examples of carbon sources used in the medium include glucose, mannose, lactose, maltose, and dextrin. Examples of nitrogen sources include meat extract.

Polypeptone, yeast extract, casamino acids, monosodium glutamate, etc. can be used.

Inorganic salts include MgSO4・7H20, Na2HPO
45c12H20゜KH2PO4, NaCf! Various inorganic salts such as the following can be used. The pH of the medium is 6-8. Preferably, the pH is 6.8°, the culture temperature is 20-30°C, and the culture time is preferably 15-48 hours.

After completion of the culture, the culture solution can be sterilized by means such as filtration or centrifugation to obtain a crude enzyme solution.

The lytic enzyme activity was measured by the following method. Washed microbial cells were washed with 25mM+J acid, buffer (pH 7,
1) was suspended so that the absorbance at 660 mist was 0.7, and 0.1- of the enzyme solution was added to this suspension 3-, and the mixture was reacted at 35°C for 30 minutes to measure the decrease in turbidity. . Enzyme activity 1
The unit was the amount of enzyme that reduced the turbidity by 0.001 per minute under the above conditions.

The properties of the lytic enzyme obtained by the present invention were investigated using a crude enzyme solution.

(1) Optimal pH The optimal pH for bacteriolytic activity is from pF7 to
It was 7.5.

(2) pH stability The pH stability of the crude enzyme after being kept at 4°C for 124 hours is
As shown in the figure, it was stable in the K pH range of 4.5 to 9.0.

(3) The residual activity of the thermostable crude enzyme was measured after it was treated at pH 7.1 and each temperature for 10 minutes. As shown in Figure 3, this enzyme
Although it was stable up to 5°C, about 50% of it was inactivated at 50°C.

(4) Lytic activity against various microorganisms The lytic ability of the crude enzyme against various microorganisms was investigated. Each strain was collected in the late logarithmic growth phase, washed three times with 0.1M phosphate buffer (pH 7, 1), and then used. As shown in Table 1, this enzyme dissolved Durham-positive bacteria other than Staphylococcus aureus and Streptomus rimosis, but had little effect on Durham-negative bacteria.

Table 1 This enzyme had the characteristic that it also acted on bacteria with glycolytic cell walls, which are indicated by the year in front of the strain. Kato et al. have reported a lytic enzyme produced by Flavobacterium sp. L-11 (Biken Journal
5 155.1962) This enzyme differs from this enzyme in that it acts on Staphylococcus aureus.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

〔Example〕

Dextrin 0.25%, meat extract 1%, polypebut 70.5%, NaCA Q, 2%, MgSO4-7
H200,025%, KH2PO40,2%, Na2H
Dispense 150rrL of a pH 6.8 culture medium containing 200.74% of PO4-12H into a 500ml volume Sakaro flask,
121" C1 was sterilized under pressure for 15 minutes. Bacterial solution 5-, which had been prepared by pre-cultivating Flavobacterium sp. After that, the bacterial cells were removed by centrifugation to obtain a culture supernatant.Next, ammonium sulfate was added to this culture supernatant to reach 60% saturation.The precipitate obtained by centrifugation was 10 m' tw(phosphate buffer (pi(6,
0) and dialyzed against the same buffer to obtain an enzyme solution. The lytic activity of the culture supernatant and the ammonium sulfate fractionated enzyme solution against freeze-dried bacterial cells of Rhodococcus erythropolis AN-13 and Yohihacilus subtilis was measured.

〔Effect of the invention〕

By using the enzyme produced by the 7Labobacterium sp. 5H-548 bacterium of the present invention, it is possible to lyse bacteria with glycolytic cell walls, which were not lysed by conventional zozyme, and to prepare protoplasts and intracellular cells. It is extremely effective for mild extraction of active ingredients, and can be expected to be used in this field.

[Brief explanation of the drawing]

Figures 1 to 3 are graphs showing the properties of the lytic enzyme produced by Flavobacterium sp. 5H-548 strain of the present invention, in which Figure 1 shows the optimum pH, Figure 2 shows the pH stability, Figure 3 shows thermal stability 0 Applicant: Kosai Sasaki, Representative, Steel Chemical Industry Co., Ltd. Figure 1: PH;? Figure 2 tj* ;l es ta
'/l (711ItH Third Calm Supervision Return ('c) Procedural Amendment (Spontaneous) 1. Indication of the case 1985 Patent Application No. 123873 @ 2. Name of the invention Process for producing lytic enzyme 3. Name of the person making the amendment Name: Steel Chemical Industry Co., Ltd. (0794-37-2151) 5. Contents of amendment Sp 5H-548) J [(Flavobacterium
riumsp, 5H-548) Corrected with J. that's all

Claims (1)

[Claims] (1) A method of producing a lytic enzyme characterized by culturing a bacterial strain that belongs to the genus Flavobacterium and having the ability to produce an enzyme that acts on and dissolves the cell wall of a microorganism, and collecting the enzyme from the culture. Production method.