JPH0595784A - New microorganism cell-lysing enzyme, its production and new streptomyces microorganism - Google Patents

Abstract

(57) [Summary] [Structure] The present invention relates to a microbial cell lysing enzyme derived from a microorganism of the genus Streptomyces, a method for producing the same, and a novel strain Streptomyces sp. 15001 producing the enzyme. [Effect] According to the present invention, it is possible to provide an enzyme having a broad lytic spectrum with yeast, filamentous fungi, and bacteria, and showing particularly high lytic activity against bacteria.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel microbial cell lysing enzyme, a method for producing the same and a bacterium producing the enzyme.

[0002]

2. Description of the Related Art It is known that microbial cell lytic enzymes are produced by many microorganisms including animals and plants. These enzymes are expected to be used for elucidation of cell wall structure, protoplast formation, and plasmid extraction, and are expected to be applied to bacterial cell components extraction, food disinfectants, detergents, and medical treatment.

Among those of microbial origin, many microbial cell lysing enzymes produced by strains belonging to the genus Streptomyces are known, for example, JP-A-61-135583 and JP-A-53-53.
No. 38687, JP-A-49-124280, JP-A-48-23988, JP-A-55-68289 and the like.

[0004]

Most of the above-mentioned conventional microbial cell lysing enzymes derived from Streptomyces microorganisms have a lytic activity against only yeast or only bacteria, and JP-A-55-68289. No. for yeast,
Although it has a broad lytic activity with filamentous fungi, bacteria, actinomycetes,
It was not active against Escherichia coli, Staphylococcus aureus, etc., and had a practical drawback.
Although many commercially available microbial cell lysing enzymes are known, many of them have weak lytic activity by themselves and often require a combination of a plurality of enzymes.

[0005]

[Means for Solving the Problems] The present inventors isolated a strain having the ability to produce an enzyme having a broad lytic spectrum from the sediments of mangrove mud wetlands on Iriomote Island, and identified the strains, culture conditions, and culture. The present invention has been completed as a result of earnestly investigating the method for producing a microbial cell lysing enzyme from a product and the properties of this enzyme.

Streptomyces which is the microorganism of the present invention
The sp. 15001 strain has the following properties. (a) Morphology The branching method of spore-forming hyphae is simple branching but forms pseudo-circle filaments. The form is straight to curved.

The spores are linked in long chains of 10 or more, the surface is smooth and elliptical, and the size is about 1.1 to 1.3 × 1.1 to 2.2 μm.
It does not form flagella spores, sporangia, sclerotia, conidia, and atypical mucus. The basal hyphae do not divide. Rarely, spores were observed on the basal hyphae. Although the spore stalk was observed to have settled on the basal hyphae, it was considered that the spores also settled on the aerial hyphae.

(B) Growth state in each medium

[0009]

[Table 1]

(C) Physiological properties

[0011]

[Table 2] (d) Assimilation of each carbon source

[0012]

[Table 3]

(E) Cell wall Simple method developed by Hasegawa et al. (J. Gen. Appl. Microbio
L., 29, 319 (1983)), the presence or absence of diaminopimelic acid in all bacterial cells, its isomers, and sugar composition were measured. As a result, cell wall type I and whole bacterial sugar type NC were found.
From the results of (a) to (e), the microorganism of the present invention was identified as a strain belonging to the genus Streptomyces according to the Burjay's Manual of Systematic Bacteriology 4th Edition (1989).

This strain is named Streptomyces sp. 15001 strain and has been deposited with the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology as Microorganism Research Institute No. 12545 (FERM P-12545). The present invention also belongs to the genus Streptomyces, the microorganism having the ability to produce the microbial cell lysing enzyme of the present invention having the enzyme chemical properties described below is cultured in a medium to produce and accumulate the enzyme, which is then accumulated. The present invention also provides a method for producing a microbial cell lysing enzyme, which comprises collecting seeds.

Examples of the microorganisms used in the production method of the present invention include Streptomyces sp.
01 shares. In the production method of the present invention, the medium containing seawater shown in Table 4 is usually used as the basal medium,
If seawater is not used, artificial seawater may be used. Of medium
pH 6 ~ 7, culture temperature 25 ~ 30 ℃, culture time 120 ~ 16
8 hours is desirable. Further, it is preferable to inoculate a precultured product for about 48 hours in advance.

The culture solution was sterilized by centrifugation, filtration, etc., desalted by ultrafiltration or dialysis, and freeze-dried to obtain a crude enzyme powder. The lytic activity was increased by desalting. It is also possible to appropriately purify and use by salting out, a method using an ion exchanger, or the like.

[0017]

[Table 4] Basic medium composition Sea water 900 ml Tap water 100 ml Polypeptone 5 g Yeast extract 3 g The lytic activity of the lytic enzyme was measured by the following method.

A small amount of distilled water is added to the washed cells of various microorganisms to suspend them, and the suspension is heated at 100 ° C. for 20 minutes to deactivate the autolyzing enzyme, and then heat-dead cells are obtained by centrifugation. Filamentous fungus is cultured in Ultra Disperser (Yamato Model LK-2
After crushing in 1), use a homogenizer (manufactured by Inai) to 500
A cell wall was obtained by subjecting the finely ground product at 0 rpm to the same heat treatment.

The microorganism prepared above was suspended in 1.0 ml of 50 mM Tris-HCl buffer (pH 8.5), and 0.5 ml of the crude enzyme solution and 1.5 ml of the same buffer solution were added to this substrate cell suspension. The reaction was allowed to proceed at 0 ° C., and the decrease in turbidity was measured at 570 nm. First shot (control)
The absorbance at 570 nm was 0.6 to 0.65. The activity to reduce the initial turbidity of the test bacterial cells or cell wall suspension by 1% in 30 minutes was defined as 1 unit.

The properties of the crude enzyme produced by this strain were examined by using Saccharomyces cerevisiae (IFO 0203) as a substrate. 1) Substrate specificity: This enzyme has protease activity, β-1,
It has 3-glucanase activity, chitinase activity, β-N-acetylglucosaminidase activity, amylase activity and cellulase activity. 2) The optimum pH of this enzyme is around 8-9. 3) This enzyme is stable at pH 6-9. 4) The optimum temperature for this enzyme is around 60 ° C. 5) This enzyme is stable up to 40 ° C, but is completely inactivated by heating at 70 ° C for 10 minutes. 6) Inhibited by 10 mM calcium chloride, magnesium chloride and sodium sulfate.

Furthermore, 0.1 mM leupeptin (Ac-L-Leu-L
-Leu-L-Argininal ・ 1 / 2H ₂ SO ₄・ H ₂ O), Phosphoramidon
(N- (α-rhamnopyranosyloxy-hydroxyphosphenyl) -L-Leu-L-Trp ・ 2Na ・ 2H ₂ O), antipain ([(S) -1
-Carboxy-2-phenylethyl] carbamoyl-L-Arg-L
-Val-Argiminal), E-64 ([L-3-trans-Carboxyoxirane-2-carbonyl] -L-Leu-Agmatine 1 / 2H
_It is also inhibited by protease inhibitors such as ₂ O). 7) The enzyme is relatively stable against 1 mM L-cysteine which is a reducing agent and stable against 1 mM 2-mercaptoethanol.

When the lysis spectrum was examined, it had a broad spectrum of yeast, filamentous fungi, and bacteria, and particularly had a high lytic activity against bacteria regardless of whether it was Gram positive or negative.

[0023]

The present invention will be described in more detail with reference to Reference Examples, Examples and Test Examples, but the scope of the present invention is not limited to these. In the following, unless otherwise indicated, Saccharomyces cerevisiae (IFO 0203) prepared by the above-mentioned method was used as a lyophilized powder as a substrate for lytic activity. Reference Example 1 Examination of culture time Shaking culture was performed in an Erlenmeyer flask at 30 ° C. for 48 hours in advance, and 2% of the culture solution was inoculated into 50 ml of a basic medium (medium shown in Table 4) in a 200 ml Erlenmeyer flask, and the temperature was set to 30 ° After culturing for 24 to 216 hours with shaking, the culture solution was centrifuged at 15000 rpm for 30 minutes to determine the wet weight of the cells, and the supernatant was used as a crude enzyme solution to measure the lytic activity by the method described above.

As a result, the growth amount of the bacterial cells was maximum at 72 to 96 hours, and the lytic activity was maximum at 120 hours or more (FIG. 1). After that, the cells were cultured for 120 to 144 hours. Reference Example 2 Examination of pH of medium When the culture was carried out in the same manner as in Reference Example 1 except that the pH of the basic medium was 5 to 9, it was found that high activity was obtained at pH 6 to 7 (Fig. 2). Reference Example 3 Examination of Seawater Concentration of Medium When the seawater concentration of the basic medium was set to 0, 25, 50, 90%, and other cultures were performed in the same manner as in Reference Example 1, high activity was obtained in 90% or more of seawater. Was found (Fig. 3). After that, the seawater concentration was set to 90%. Reference Example 4 Examination of culture temperature When the culture temperature was changed to 5 to 40 ° C. and other conditions were the same as in Reference Example 1, high activity was obtained at 25 to 30 ° C. (FIG. 4). After that, the culture was performed at 30 ° C.

[0025]

Example 1 (1) Method for Preparing Crude Enzyme Based on the culture conditions (basic medium pH 6 to 7, 30 ° C., 120 to 144 hours of culture) of Streptomyces sp. 15001 strain determined in Reference Examples 1 to 4. The cultivated culture solution was sterilized by centrifugation to obtain a crude enzyme solution (this crude enzyme solution can also be used as the microbial cell lysing enzyme of the present invention), and the dialysis membrane (manufactured by Sanko Junyaku) was used to remove the crude enzyme solution. After dialysis against salt water at 4 ° C. overnight and desalting treatment, the lytic activity increased up to about 2-fold. Further, the results were the same even when desalting treatment was carried out by ultrafiltration, and the activity did not change even after freeze filtration as a freeze-dried powder after ultrafiltration. Yield from 1 L culture
It was 0.4 to 0.5 g. In the following examples, the culture solution was sterilized, desalted and concentrated by ultrafiltration, sterilized by filtration, freeze-dried, and powdered to be used as the crude enzyme of the present invention in the following tests. Unless otherwise indicated, the activity of the lysis reaction was measured so that the final concentration of the crude enzyme was 0.05% (W / V). (2) Optimum pH and pH stability of the lytic activity of the crude enzyme of the present invention When the pH of the buffer solution used for measuring the lytic activity was changed and all other activities were measured in the same manner, the optimum pH of the enzyme was It was around 8-9 (Fig. 5). To examine pH stability, add 0.25 ml of 1.8% (W / V) crude enzyme aqueous solution to 0.5 ml of buffer solution at each pH, leave it at 23 ° C for 24 hours, and then remove each crude enzyme. The solution was diluted 2-fold with 50 mM Tris-HCl buffer (pH 8.5), and 0.5 ml of this was used as a crude enzyme solution and the lytic activity was measured in the same manner. The enzyme was stable at pH 6-9. There was (Fig. 6). The buffer used here was 50
mM acetate buffer (pH 4-6), phosphate buffer (pH 6-
7), Tris-HCl buffer (pH 7 to 9.5) and glycine-sodium hydroxide buffer (pH 9 to 13). (3) Optimum temperature and thermostability of the lytic activity of the crude enzyme of the present invention When the above-mentioned lytic reaction (pH 8.5) was carried out at each temperature of 30 ° C to 80 ° C and the activity was measured, the optimum temperature was 60 It was around ℃ (Fig. 7). The crude enzyme solution dissolved in Tris-HCl buffer (pH 8.5) was treated for 10 to 30 minutes at each temperature of 30 to 80 ° C, and the lytic activity was measured by the above method. Was stable, but was inactivated by treatment at 70 ° C for 10 minutes (Fig. 8). (4) Effect of Metal Salt, Protease Inhibitor and Reducing Agent on Lytic Activity of Crude Enzyme of the Present Invention Table 5 shows the results of measuring activity by adding metal salt, protease inhibitor and reducing agent to the lysis reaction solution. .

As a result, the lytic activity of this enzyme was inhibited by calcium, magnesium, sodium, and various protease inhibitors, but it was relatively stable against L-cysteine, which is a reducing agent, and against 2-mercaptoethanol. And stable.

[0027]

[Table 5]

(5) Substrate Specificity of the Crude Enzyme of the Present Invention Various activities were measured in order to elucidate the mechanism of the lytic reaction. As shown in Table 6, the enzyme was a protease, β-1,3-glucanase. , Chitinase, β-N
-It had various activities of acetylglucosaminidase, amylase and cellulase.

The methods for measuring various activities are as follows. Protease activity 1% casein solution (0.1M phosphate buffer pH 7) 0.1 ml in 1 ml.
After adding 1 ml of 1% crude enzyme solution and reacting at 35 ° C, after a certain time 5
The reaction was stopped by adding 3 ml of a% trichloroacetic acid solution.
After standing for 30 minutes to 1 hour, the mixture was centrifuged and the absorbance of the supernatant was measured at 280 nm. After mixing the substrate and the stop solution, the enzyme solution was added as a control, and an L-tyrosine solution was used as a calibration curve. The activity of releasing 1 µg of L-tyrosine equivalent per minute was defined as 1 unit (U). β-1,3-glucanase activity: 0.25 ml of 1.6% laminarin solution, 50 mM Tris-HCl buffer solution.
The reaction mixture consisting of 5 ml and 0.25 ml of 0.8% crude enzyme solution was heated at 35 ° C for 30
After reacting for -60 minutes, the reaction was stopped by heating at 100 ° C for 5 minutes, and the reducing sugar was quantified by the Somogi-Nelson method. A calibration curve is prepared using glucose, and 1 unit of the activity to release 1 μg of glucose equivalent per minute is defined as 1 unit.
(U) Amylase (α-glucanase) activity Using a 1.6% soluble starch solution as a substrate, the activity was measured in the same manner as described above. Cellulase activity Using a 1.6% carboxymethylcellulose (CMC) solution as a substrate, the activity was measured in the same manner as above. Chitinase activity After using 0.2% ethylene glycol chitin solution as a substrate and carrying out the reaction and reaction stop in the same manner as above, add 2 ml of 1.37 mM potassium ferricyanide solution dissolved in 0.5 M sodium carbonate solution and heat to 100 ° C for 15 minutes. After the reduction reaction, the absorbance at 420 nm was measured to determine the difference from the control. The calibration curve was prepared using N-acetylglucosamine, and the activity of releasing 1 μg of N-acetylglucosamine equivalent per minute was defined as 1 unit (U). β-N-acetylglucosaminidase activity 0.25% p-Nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside 0.5 ml, 50 mM Tris-HCl buffer (pH 7) 2.5 ml, 0.2% crude enzyme solution A reaction solution consisting of 1 ml was reacted at 35 ° C. for 90 minutes, 1 ml of a saturated potassium carbonate solution was added to stop the reaction, and the absorbance at 420 nm was measured. Absorption value of 0.01 at 420nm per minute
The increasing activity was defined as 1 unit (U).

[0030]

[Table 6]

From the above results, it is suggested that various activities have a complex effect on the lytic reaction. From this, it can be understood that this enzyme has a broad lysis spectrum. The protease activity was higher on the alkaline side. The high protease activity is in good agreement with the result in Example 8 that the lytic activity is inhibited by the protease inhibitor. (6) Lysis spectrum of crude enzyme of the present invention Table 9 shows the results of comparison of the lysis spectrum of the crude enzyme of the present invention with 7 kinds of commercial products. The enzyme had a broader lytic spectrum in all filamentous fungi, yeasts and bacteria than the commercially available products, and a strong activity was recognized especially against bacteria regardless of whether it was gram negative or positive.

The method for preparing the microorganism serving as a substrate is as follows. As yeast, Rhodotorula rubra (IFO 0001), Saccharomyces cerevisiae (IFO 0203), Schizosaccharomyces pombe (IFO 0354), Hansenula anomala
(IFO 0568), Candida Utilis (IFO 0639)
, Lipomyces starkiy (IFO 0678), and Tigosaccharomyces bailey (IFO0722) were used, and the medium was shake-cultured at 30 ° C. for 2 to 3 days in the medium shown in Table 7. Bacteria were collected from the culture solution, washed with sterilized water several times, suspended in a small amount of distilled water and heated at 100 ° C. for 20 minutes to obtain wet cells.

The bacteria include Pseudomonas aeruginosa (IFO12689), Vibrio parahaemoriticus, Escherichia coli (IFO 3301), Salmonella enteritidis, Staphylococcus aureus.
(ATCC 6538), Bacillus subtilis (IFO 13719), Micrococcus luteus (manufactured by Seikagaku Corporation),
The cells were cultured in the medium shown in Table 8 at 30 ° C for 1 to 2 days with shaking to obtain heat-dead cells in the same manner as yeast. As filamentous fungi, Aspergillus niger (OUT 5198), Alpergillus soya (IFO 4239), Mucor mused (IFO 7684), Penicillium caseicolum (AHU 8114), Penicillium
Grablum (AHU 8288), Rhizopus oryzae (IFO 5418)
Was shake-cultured in the medium shown in Table 7 at 30 ° C. for 2 to 7 days. The culture solution was changed to Ultra Disperser (Yamato Mod
(el LK-21) and the cells were crushed, and then further crushed with a rubbing type homogenizer (manufactured by Inouchi) at 5000 rpm. The crushed cells were treated in the same manner as yeast to collect wet cell walls.

[0034]

Table 7 medium Polypeptone yeast-fungi 5g K ₂ HPO ₄ 1g yeast extract 3g MgSO ₄ · 7H ₂ O 0.3g malt extract 3g water 1000ml glucose 10 g pH 7.0

[0035]

Table 8 Bacterial medium polypeptone 5 g * artificial seawater which may yeast extract 3g artificial seawater composition ^* seawater 900 ml NaCl 30 g tap water _{100ml KCl 0.7g pH7.0 MgCl 2 · 6H} 2 O 10.8g MgSO 4 · 7H 2 O 5.4g CaCl ₂ · 2H ₂ O 1 g Distilled water 1000 ml As a commercial product, Zymolyce 20T (Seikagaku Corporation), Novozyme 234 (Novo Biolab), Fungase I (Nagase Seikagaku), Usukizyme (Kyowa Kasei), Chitosanase (Wako Jun) Medicine), chitinase (Seikagaku Corporation), dolicerase (Kyowa Hakko), and cellulase (Wako Pure Chemical Industries) were used.

The lytic activity is based on the method described above, but the reaction is
It was carried out at 35 ° C, and different pH conditions and concentrations were used depending on the enzyme. For pH 5 and 6, 0.1M acetate buffer, pH 7.5
0.1M phosphate buffer was used for the above, and 0.05M Tris-HCl buffer was used for the one having pH 8.5.

[0037]

[Table 9]

[0038]

(7) Other properties By using the culture solution supernatant (crude enzyme solution) cultured in (1) in combination with the culture solution supernatant of a microorganism capable of degrading chitin, the lytic activity is about 1.5 times. Increased to.

[0040]

EFFECTS OF THE INVENTION According to the present invention, it is possible to provide an enzyme having a broad lytic spectrum with yeast, filamentous fungi, and bacteria, and particularly showing high lytic activity against bacteria, which enzyme has a cell wall structure of a microorganism. It can be widely applied to foods, pharmaceuticals, cosmetics, detergents, etc., including the research, protoplasting, and cell fusion.

[Brief description of drawings]

FIG. 1 is a diagram showing culture time and lytic activity of culture supernatant.

FIG. 2 is a diagram relating to lysing activity of medium pH and culture supernatant.

FIG. 3 is a diagram showing the concentration of seawater in a medium and the lytic activity of a culture supernatant.

FIG. 4 is a diagram showing culture temperature and lytic activity of culture supernatant.

FIG. 5 is a graph showing the effect of pH on the lytic activity of the crude enzyme of the present invention.

FIG. 6 is a diagram showing pH stability in lytic activity of the crude enzyme of the present invention.

FIG. 7 is a graph showing the effect of temperature on the lytic activity of the crude enzyme of the present invention.

FIG. 8 is a diagram showing thermostability in lytic activity of the crude enzyme of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1: 465)

Claims (3)

[Claims] 1. A microbial cell lysing enzyme having the following enzymatic chemistry: Substrate specificity: It has a protease activity using casein as a substrate. It has β-1,3-glucanase activity using laminarin as a substrate. It has a chitinase activity using ethylene glycol chitin as a substrate. p-nitrophenyl-
It has β-N-acetylglucosaminidase activity using 2-acetamido-2-deoxy-β-D-glucopyranoside as a substrate. It has α-glucanase (amylase) activity using soluble starch as a substrate. It has cellulase activity using carboxymethyl cellulose as a substrate. Optimum pH: around 8-9 Optimum temperature: around 60 ° C Stable pH: 6-9 Stable temperature: up to 40 ° C (10 minutes, pH8.5) Complete inactivation at 70 ° C, 10 minutes (pH8.5) .. Inhibitors: 0.1 mM leupeptin, 0.1 mM phosphoramidone, 0.1 mM antipain, 0.1 mM E-64, 10 mM calcium chloride, 10 mM magnesium chloride and 10 mM sodium sulfate, but relatively inhibited against 1 mM L-cysteine. Stable and stable to 1 mM 2-mercaptoethanol. 2. The method according to claim 1, which belongs to the genus Streptomyces.
The method for producing a microbial cell lysing enzyme according to claim 1, wherein the microbial cell lysing enzyme-producing bacterium according to claim 1 is cultured in a medium to produce and accumulate the microbial cell lysing enzyme according to claim 1, and the collected microbial cell lysing enzyme is collected. 3. A new strain of Streptomyces sp. 15001 which produces the microbial cell lysing enzyme according to claim 1.