JPH0367579A - Enzyme for resolving microbial cell wall and its microbial product - Google Patents

Abstract

NEW MATERIAL: An alkalophilic genus Bacillus microorganism KFCC 10671 (FERM BP-2841) producing a microbial cell wall lytic enzyme.

USE: Used as a microorganism for producing a large amount of new microbial cell wall lytic enzyme.

PREPARATION: A microorganism growing at pH10.2 is separated from the soil and a cultured supernatant of the microorganism is dropped on an agar-agar medium suspended with a microbial bodies, then a strain generating a round transparent zone in the dropped site is separated as the objective strain.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a microbial cell wall lytic enzyme that reacts with the cell wall of a microorganism and lyses the microorganism, and a microorganism that produces the same.

(Prior art and problems to be solved by the invention) Peptidoglycan, which is a major component of microbial cell walls, consists of peptides of N-acetylglucosamine and N-acetylmuramic acid that form peptide bonds with the lactic acid residue of muramic acid. Peptide bridges are formed between the peptide chains, creating strong bonds.

The amino acid sequence of the peptide chain differs slightly depending on the fungus.

Microbial cell wall lytic enzymes that degrade peptidoglycan in cell walls are classified into three types: glycosidase, which cleaves the polysancharide chain of peptidoglycan, and acetylmura ξ, which degrades polysaccharide and peptide bonds.
Examples include lu L-alanine ξdase and endopeptidase that cleaves the polypeptide chain itself.

Until now, research on these enzymes has mainly focused on staphylococcal enzymes that are resistant to lysozyme.
This was applied to enzymes that dissolve genus microorganisms and enzymes that dissolve mold and yeast.

Among some microorganisms, Streptomyces griseus (Streptomyces griseus)
tol IIces riseus), Stre tom ces albu
s), Streptomyces 0 erythreus (Stre
tom ces er threus), Streptomyces luterzenis
r enesis), Stretomyces orientalis (Stretomyces orientalis)
is) etc. and Staphylococcus aureus (Sta
h lococcus (Myxococcus xan
tus) s Xutomonas 6 aeropinosa (Pseu
It is known that the genus Dos+onas aeuro 1nosa) S Chlorapis q) produces this lytic enzyme.

Microbial cell wall lytic enzymes are widely used to investigate the structure of microbial cell walls, and are often added to cheese, sausage, potato salad, alcoholic beverages, etc. as food preservatives to protect foods from microbial contamination. It is used for purposes such as dissolving and separating intracellular substances.

The present inventors isolated from soil an alkalophilic microorganism that produces a novel microbial cell wall lytic enzyme that has different physicochemical properties from previously known microbial cell wall lytic enzymes,
After identifying it as a Bacillus sp. microorganism and examining the strain's properties and enzymatic characteristics, we discovered that it had different characteristics from existing production strains.Based on this, we completed the present invention. .

Accordingly, an object of the present invention is to provide a microbial cell wall lytic enzyme that reacts with microbial cell walls and lyses microorganisms, and a microorganism that produces this enzyme.

(Means for Solving the Problems) The present invention uses alkalophilic Bacillus sp. KFCC1 that produces a microbial cell wall lytic enzyme.
0671 (Feikoken Jokyo No. 2841).

The present invention also provides a method for producing a microbial cell wall lytic enzyme, which comprises culturing the microorganism in a nutrient medium having a pH of 7.5 to 11.5, and recovering the microbial cell wall lytic enzyme from the culture solution.

The present invention will be explained in detail below.

Isolation of bacterial strains with enzyme activity Among the microorganisms grown in alkaline conditions (pH 1O, 2) from soil, in order to isolate bacterial strains with microbial cell wall lytic activity, we cultured them on an agar medium in which the bacterial cells were suspended. Drop clear liquid and create a circular clear zone at the site.
e) The strain that produced the following was isolated as a strain with cell wall lytic activity. In addition, as a method for confirming cell wall lytic enzymes, alkaliphilic Bacillus sp.
(A) and Bacillus megatherium (Ba-cillu
s me aterium) While reacting this enzyme on the microbial cell wall of (B) at 45°C for 80 minutes, samples were taken every 20 minutes, and the absorbance at 660°C and the free amino acids and reducing power in the reaction product were measured. did. The results are shown in Figure 1.

As the reaction time of this enzyme progressed, the absorbance of the cell wall at 660 nm decreased and the amount of mild amino acids increased, but no change in reducing power was observed. This enzyme is a microbial cell wall lytic enzyme presumed to be an endopeptidase that cleaves the peptide bonds of peptidoglycan, which is a major component of microbial cell walls.

Identification of microbial strain The strain of the present invention is Durham positive, and as shown in the micrograph in Figure 2, its morphology is that of a bacillus (0.4pX1.7//a
), it was identified as a microorganism belonging to the genus Becillus based on its catalase positivity and spore-forming characteristics. In addition, Table 1 shows the results of experiments and studies on various culture and physiological characteristics.

Table 1 1. Morphological characteristics Morphology Bacillus Motility Positive Durham staining positive 2. Culture characteristics Nutrient medium pH 7.0 pH10.0 Glucose nutrient medium pH 7.0 ptl　10.0 Basic medium (pH 10,2) Basal medium (containing 10% NaCl) Maximum temperature for growth + + ＋　＋ + 42℃ :Cannot grow, +:Grows, ++: Grows well.

3.Biochemical characteristics Starch hydrolysis Casein hydrolysis Gelatin hydrolysis νP test Catalase oxidase positive positive positive negative positive positive indole test gelatin degradation positive positive The microbial characteristics of such strains are known as microbial cell wall lytic enzyme producing strains. As a result of comparison with the characteristics of microorganisms such as Streptomyces, Staphylococcus, Achromonas, Myxococcus, and Xutomonas, the strain of the present invention has characteristics different from those of known microorganism cell wall lytic enzyme producing seedlings, and has a pH of 7. It was found that it is an alkalophilic Bacillus species that grows at a temperature of 5 to 11.5 cm.

Therefore, the present inventors named the strain producing this novel microbial cell wall lytic enzyme as Bacillus MFCC10671.

This strain was submitted to the Institute of Microbiology and Industrial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under Microtechnology Research Institute No. 2841, and to the Korean Seed Bacteria Association.
It has been deposited under accession number FCC10671.

Starch, glucose, sugar, molasses, galactose, fructose, maltose, arabinose, raffinose, etc. are used as carbon sources for culturing the above-mentioned strain Bacillus KFCC10671.

As a nitrogen source, urea, nitrate, ammonium salt, ammonia, protein, amino acid, peptone, yeast, yeast extract, etc. can be used. In addition to these, inorganic salts such as phosphates, carbonates, and sulfates can be used. The pH of the medium is 7.0-12.
0 is appropriate, and the culture temperature is 20-45°C.

The strain of the present invention was mixed with 2% soluble starch, 0.5% yeast extract,
Polypeptone 0.5%, dipotassium hydrogen phosphate 0.1%,
Magnesium sulfate heptahydrate 0.2%, sodium carbonate 1
Enzyme production was good when cultured under alkaline conditions at 30°C in a pH 10.2 medium in which 50% of the enzyme was dissolved in water.

As shown in Examples 3 and 4 below, the cell wall lytic enzyme produced by Bacillus KFCC10671 of the present invention is an enzyme having a single subunion with a molecular weight of about 27,000 Daltons, and the optimum reaction pH is 10. It is a cell wall lytic enzyme of nearby alkali-resistant living microorganisms.

This enzyme has the effect of dissolving microbial cell walls even in the genus Shutomonas, and is a novel enzyme with different physicochemical properties from existing microbial cell wall-dissolving enzymes.

The microorganism that produces this enzyme is an alkalophilic Bacillus genus, and the enzyme of the present invention is novel, as the nature of the strain and enzyme conditions are different from those of known microbial enzymes. .

(Examples) Hereinafter, the bacterial strain of the present invention and the microbial cell wall lytic enzyme produced by this strain will be described in detail based on the following examples.

Example 1 The strain of the present invention was used as a carbon source such as starch and sugars, a nitrogen source such as urea, ammonium salts, protein, and acetic acid, a nutrient source such as vitamins, nucleic acids, yeast extract, peptone, and malt extract, and phosphoric acid. When cultured in a medium composed of inorganic salts such as salts, carbonates, and sulfates, novel microbial cell wall lytic enzymes can be obtained. For example, soluble starch) 2%, yeast extract 0.5%,
Polypeptone 0.5%, K12P0.0.1%, Mg5
The strain of the present invention was grown at 30% in a medium (pH 10.2) containing 0.02% O<・711200, Na, and 1% CO dissolved in water.
Incubate with shaking water for 30 hours under aerobic conditions at ℃. On this occasion,
1.20 x 103 U/+d of enzyme is obtained from the culture solution.

In addition, soluble starch 1.0%, yeast extract 0.5%, polypeptone 0.5%, JPO40.1%, Mg5O, 7
NagCOi or NaHCO to a medium containing H200.02%.

was added at varying concentrations of 0.25%, 0.5%, 1.0%, and 2.0% to adjust the initial pl+ differently, and then cultured with shaking at 37°C for 24 hours to determine its activity. It was measured.

Table 2 shows the measurement results of enzyme activity.

Table 2 Effects of carbonate concentration and pH at the initial stage of culture on lytic enzyme production 0010,49,2 NaHCOz O,257,88,52B0.50
8.3 9.0 311.00
B, 8 9.2 802.00
9.1 9.3 97 Regarding the production of cell wall lytic enzymes, better results were obtained when carbonate was added at 2.0% than when carbonate was added at a low concentration.

The pH at the initial stage of culture was better at pH 10 than at the neutral side. The enzyme production at p old 0.4 with the addition of NazCOz at a concentration of 2.0% was more than three times higher than on the neutral side with the addition of carbonate at a concentration of 0.25%.

In order to measure the activity of the above enzyme, a substrate and bacterial cells were prepared.

The alkaliphilic Bacillus microorganism used as the substrate was inoculated into a liquid medium, cultured at 37°C for 16 hours, centrifuged, and the aggregates were collected, washed twice with 0.9% NaCl, and mixed with a buffer solution (pl+10. 0) at OD &60 = 1.0
The bacterial cells were suspended so that

Add 0.1- of the enzyme solution to 2 d of bacterial cells with 0 Diio = 1.0, react at 45°C for 10 minutes, and then reduce the absorbance by 6
Measured at 60 nm. At this time, one unit of enzyme activity is
OD, reacted for 1 minute under the given conditions.

It was defined as the amount of enzyme that reduces the absorbance of =1.0 by 0.001.

Example 2 2% soluble starch, 0.5% yeast extract, 0 polypeptone
．． 5%, potassium phosphate 0.1%, Mg5O, 7H20
Bacillus FCC10671 of the present invention was cultured in a medium containing 0.02% NazCO3 and 1% NazCO3 dissolved in distilled water.

Figure 3 shows the results of observing the growth, enzyme production, and pH changes of the strain over time.

As shown in FIG. 3, the growth of the bacteria increased until 18 hours after inoculation and reached the normal phase. Moreover, it gradually decreased after 18 hours. The pH of the medium was initially 1O02 and gradually decreased to 24
The pH decreased to 8.6 over time, and then increased. After 60 hours, a fermentation phenomenon of alkaliphilic bacteria with a pH of 9.4 was observed. Enzyme production gradually increased after 12 hours and reached maximum activity at 36 hours (1.2X103
U/mff), but the activity subsequently decreased.

Example 3 As a result of examining the characteristics of the enzyme produced by this strain, 5DS-
Polyacrylamide gel electrophoresis revealed that this enzyme is a type of enzyme having a single subunit with a molecular weight of about 27,000 Daltons, and the optimum pH was around 10.0. The results of this experiment are shown in FIG.

A is after (NH4)zsO4 fractionation, B is after 0M cellulose column chromatography, and C is Sephadex G-10.
0 after gel filtration, D after hydroxylapatite column chromatography, and E standard protein molecular weight marker.

This includes Streptomyces erythreus, Streptomyces luterzenis, Streptomyces orientalis, which have been reported as microbial cell wall producing strains.
The molecular weights of Achromobacter lunatus, Shutomonas aerobinosa, etc. are 18.500.22,000.
There is a slight difference between 33,000.16,000.24 and 500, and the optimum pH is 6.0 for Streptomyces luterzenis and 6.0 for Achromobacter.
Lunatos is 8.5, Myxococcus xanthos is 7.
5. The results show that the optimal pH is highly alkaline compared to 6.4 for Shutomonas aeropinosa, and this enzyme has novel properties that are different from other cell wall lytic enzymes. It was confirmed that

Example 4 In order to examine the range of action of the cell wall lytic enzyme produced by this strain, the test strain used in this experiment was treated with malt extract 0.
5%, hepton 1%, NaC 10.5% medium at 30°C.
After culturing until the normal phase, O
D&&. = 1.0, 0.1 m of enzyme solution was added to this bacterial cell 2-, and the reaction was carried out at 45°C for 15 minutes at 660 r.
The decrease in absorbance and luminous intensity of u++ was measured and the results of conversion in % are shown in Table 3.

Among the test strains used in the experiment, Bacillus microorganisms include Bacillus megaterium and Bacillus brevis (Bacillus megaterium).
illus brevis), Bacillus am Ioli ue
faciens) etc. were lysed by this enzyme. It was observed that the genus Shutomonas and other microorganisms were lysed.

It is known that the cell walls of microorganisms are not only not pure glycopeptides, but also exhibit properties of cell wall lytic enzymes, depending on the degree of polymerization, cross-linking and charge of the peptidoglycan.

Known cell wall lytic enzymes cannot lyse microorganisms of the genus Shutomonas, but this enzyme was confirmed to lyse microorganisms of the genus Shutomonas.

Table 3 Sp・00 (Effects of the Invention) The novel microorganism cell wall lytic enzyme can be produced in large quantities by the microorganism of the present invention. This is expected to be used as a new material for investigating the structure of microbial cell walls and for dissolving and separating intracellular substances.

[Brief explanation of drawings]

Figure 1 shows the reaction product obtained by reacting the enzyme of this strain with the microbial cell wall over the course of reaction time (minutes).
Absorbance at 0 nm (・〉 and free agaric acid (μmol/-))
(b) and reducing power (μmol/-) (black Δ); (A) is a graph showing changes in the alkaliphilic Bacillus genus (
Bacillus sp,) VC-335, (B) Bacillus megaterium μ±1 vomitus)
The case of KFCC32320 is shown. FIG. 2 is an electron micrograph (X4.
000). Figure 3 is a graph showing the results of observing the growth, enzyme production, and pH changes of this strain over time, and the horizontal axis is the culture time (
), ○ is dry cell weight (0 to g), ・is activity (U/d
) xlO', Δ indicates pH. FIG. 4 shows the results of 5DS-polyacrylamide gel electrophoresis of purified enzyme protein, and A is (NH
4) After tS04 fractionation, 8 is after CM total cellulose column chromatography, C is after Sephadex G-100 gel filtration, D is after hydroxylapatite column chromatography, and E is a standard protein molecular weight marker. Reducing power (μmole/ml I im) Diagram Incubation time (hours) Drafting procedure amendment for drawings, 7. Hei rfc2 August 13th

Claims (1)

[Claims] 1. Alkaliphilic Bacillus sp. KFCC106 that produces a microbial cell wall lytic enzyme
71 (Feikoken Joyori No. 2841) microorganisms. 2. Alkaliphilic Bacillus (￥Bacillus￥s
p. ) KFCC10671 (Feikoken Joyori No. 2841)
of microorganisms are cultured in a nutrient medium with a pH of 7.5 to 11.5,
A method for producing a microbial cell wall lytic enzyme, which comprises recovering the microbial cell wall lytic enzyme from a culture solution. 3. Alkaliphilic Bacillus (￥Bacillus￥s
p. ) KFCC10671 (Feikoken Joyori No. 2841)
A microbial cell wall lytic enzyme produced by microorganisms with the following characteristics. Molecular weight: Approximately 27,000a Dalton Optimum activity: Around pH 10 Reaction temperature: 40-80°C Optimum reaction temperature: 50°C Stable pH: pH 5-11 Optimum stable pH: pH 10 Alkali resistance