JPH06121676A - Novel alkaline lytic enzyme and method for producing the same - Google Patents

Abstract

PURPOSE:To obtain a lytic enzyme having optimum pH in the alkali side, showing lytic activity against a wide range of bacteria in the presence of a surfactant, to produce a strain capable of producing the enzyme and the lytic enzyme by the strain. CONSTITUTION:A bacterium belonging to the genus alkaliphilic Bacillus or alkaliphilic Nocardiopsis or an actinomycetes belonging to the genus alkaliphilic Streptomyces is cultured to produce the objective lytic enzyme in the medium. The lytic enzyme is recovered from the medium and optionally purified to give a new lytic enzyme.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel alkaline lytic enzyme obtained by culturing an alkalophilic Bacillus bacterium, an alkalophilic Nocardiopsis actinomycete, or an alkalophilic Streptomyces genus actinomycete, and its production. The present invention relates to a strain and a method for producing the enzyme. More specifically, the present invention provides a novel alkaline lytic enzyme, a new producing strain, and a method for producing the enzyme, which are incorporated into all detergents for clothes to contribute greatly to the improvement of detergency.

[0002]

2. Description of the Related Art Clothes that are worn daily vary considerably depending on the type, individual differences, environmental differences, etc., but are formed in association with sweat and sebum secreted in large amounts from the skin surface and keratinization of cells on the skin surface layer. In addition to the dirt on the skin and dust from the outside, it is also contaminated by microorganisms such as bacteria and mold.
In particular, natural fibers such as cotton, wool, and silk, which have high hygroscopicity and have a well-developed fiber structure and have many voids, have significantly more bacteria attached than chemically synthesized fibers. From a hygienic point of view, it would be highly desirable if many bacteria that adhered to the worn clothing could be easily removed, along with other common stains, by regular washing. It is one of the original purposes of cleaning. The removal of bacteria adhering to the fabric by washing varies considerably depending on the type of bacteria and the growth state of the bacteria, but simply increasing the detergent concentration or the washing time does not substantially increase the disinfection efficiency of the fabric. On the other hand, by increasing the washing temperature, increasing the number of rinses, or drying the fabric with hot air, the sterilizing or sterilizing effect can be remarkably enhanced, and the effect can be further enhanced by combining these conditions. Is recognized (Minagawa; Antibacterial and antifungal, Vol.4, p.15)
(1976)). However, unlike the Western countries (washing temperature of 60 to 100 ° C), the most common method of washing clothes in Japan is low-temperature washing (room temperature to 40 ° C). Is difficult and leaves many problems from a hygienic standpoint. As a countermeasure from the viewpoint of cleaning contaminated bacteria adhering to clothes, a method of decomposing and removing microorganisms by adding a lytic enzyme to detergent can be considered. As the lytic enzyme, egg white-derived lysozyme is widely known, but the optimum pH is neutral to weakly acidic, and the bacterial species to lyse are limited (the lytic spectrum is narrow), and the surface activity is also high. It is not suitable as an enzyme to be added to detergents because it has low activity in the agent. In addition, properties different from lysozyme
Although some lytic enzymes having a lytic spectrum, an optimum pH, etc. have been reported, none of them satisfy the properties as a detergent enzyme. Enzymes of the genus Nocardiopsis (PCT Int.Appl.WO 91 10723) show lytic activity against a limited number of strains in detergents, but the optimum pH is around neutral and Considering the medium pH (around 10), it has disadvantages. For this reason,
It has an optimum pH on the alkaline side, and is also used as a detergent.
The appearance of a lytic enzyme having improved properties as a detergent enzyme, such as stability against various surfactants and a broader lytic spectrum, has been strongly desired.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel alkaline lytic enzyme which has a broad lytic spectrum from gram positive bacteria to negative bacteria and is stable among various surfactants, and a method for producing the same.

[0004]

[Means for Solving the Problems] The present inventors are capable of producing a lytic enzyme having novel properties, particularly an enzyme having a wide range of activity from Gram-positive bacteria to negative bacteria and having an optimum pH on the alkaline side. In order to find such a microorganism, as a result of intensive research, a bacterium belonging to the alkalophilic Bacillus genus, an alkalophilic Nocardiopsis genus, and a lytic enzyme derived from an actinomycete belonging to the alkalophilic Streptomyces genus,
We found that it has an optimum pH on the alkaline side and has new properties such as being stable in various surfactants, and that it can lyse a wide range of bacteria from Gram positive bacteria to negative bacteria in the presence of surfactants. The present invention has been completed by taking out the target enzyme from the culture solution of the strain and elucidating its properties. The bacterium producing the lytic enzyme of the present invention was newly isolated by the present inventors, and each of them has NSE-11.
4, there are 2 strains named NSE-565, but they have the following mycological properties. For testing and classification of mycological properties, "Classification and Identification of Microorganisms" (edited by Hasegawa) and Berg
ey's Manual of Systematic Bacteriology (1986). Since both strains hardly grow in a neutral medium, a medium adjusted to pH 9 with 0.2% sodium carbonate was used in the test described below.

1A. Morphological properties The following morphological characteristics are observed when cultured on broth agar plate medium at 30 ° C for 3 days. (a) Cell shape and size NSE-114: Bacillus, in the early stage of culture, the filaments are continuous up to a length of 30 μm or more and gradually divided, so the size is variable. 0.6-1.0 μm x 2.5-5 in the latter stage of culture.
It becomes 0 μm. NSE-565: Bacillus, 0.6–1.0 μm × 2.0–4.0 μm (b) Motility NSE-114: Not observed. NSE-565: Yes. (c) Spore NSE-114: An elliptical spore having a size of 0.6 to 0.8 μm × 1.2 to 2.0 μm is formed slightly in the center. Almost no swelling of sporangia is observed. NSE-565: 0.5-0.8 μm x 1.0-2.0 μm elliptical spores are formed from the ends. Almost no swelling of sporangia is observed. (d) Gram stainability Both strains were positive.

1B. Culture properties (a) Meat agar plate culture NSE-114: A round, flat, and full-edge colony is formed.
The surface of the colony is rough and whitish.
It has a musky color. NSE-565: Form a circular, flattened, full-edge colony.
The surface of the colony is smooth and shiny, and has a yellow to yellowish brown color. (b) Meat juice liquid culture Both strains grow well, but no pellicle is formed. (c) Meat juice gelatin Both strains liquefy. (d) Litmus milk Both strains grow but no coagulation is observed. Discoloration of litmus is unknown because the medium is alkaline.

1C. Physiological properties (a) The results of the following physiological tests were negative for all strains. Denitrification reaction, VP test, urease and indol production. (b) The results of the following physiological tests were positive for all strains. Reduction of nitrate, hydrolysis of starch, hydrolysis of casein, use of citric acid, use of oxidase, catalase, inorganic nitrogen source (nitrate, ammonium salt). (c) MR test Cannot judge because the medium is alkaline. (d) Hydrogen sulfide generation NSE-114: Positive. NSE-565: Negative. (e) Growth temperature range NSE-114: Can be grown at 50 ° C or lower. NSE-565: Can grow at temperatures below 35 ℃. (f) Growth pH range NSE-114: Can grow within the pH range of 7-11. NSE-565: Can grow in the range of pH 7.5 to 10.5. (g) Attitude toward oxygen Both strains can grow under aerobic and anaerobic conditions. (h) Salt tolerance Both strains can grow in a broth medium containing 7% NaCl. (i) Utilization of sugars

[0008]

[Table 1]

Both strains are gram-staining positive aerobic bacilli, and are bacteria belonging to the alkalophilic Bacillus genus because of their characteristics of forming spores with catalase-positive and having an optimum pH range for growth on the alkaline side. Was identified. Both strains are named as follows and have been deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology. NSE-114: Alkalophilic Bacillus sp. NSE-114 (Microtechnical Research Institute No. 13181) NSE-565: Alkalophilic Bacillus sp. NSE-565 (Microtechnical Research Institute No. 13182)

On the other hand, the actinomycetes used in the present invention were newly isolated by the inventors and have the isolation number NSC.
There are two strains, -529 and NSC-1203, and their mycological forms are as shown below. In addition, since both bacteria hardly grow in neutrality, a medium to which pH was adjusted to about 9 by adding 0.2% sodium carbonate was used in the test described below.

2A. Morphological properties NSC-529: Vegetative hyphae are well developed and irregularly branched on synthetic and natural agar. In addition, fragmentation of the base hypha is observed. Spores are well formed on yeast malt agar medium, tyrosine agar medium and the like. According to microscopic observation, the branching method of sporulated hyphae is simple branching,
Spores are linearly formed. Spores usually have three or more linkages and show long chains at the latter stage of culture. The surface of the spores is smooth and the shape is cylindrical. NSC-1203: Vegetative hyphae are well-developed and irregularly branched on synthetic and natural agar. Spores are well formed on yeast malt agar medium, oatmeal agar medium and the like. According to the microscopic observation, the branching method of the sporulated hyphae is simple branching, and the spores are linearly formed.
Spores usually have 3 or more chains, the surface is smooth and the shape is cylindrical.

2B. Growing state on medium The following are the results of macroscopic observation after culturing in various mediums at 30 ° C for 14 days.

[0013]

[Table 2]

[0014]

[Table 3]

2C. Physiological properties (1) Optimum temperature for growth NSC-529: 20 to 30 ° C. It grows slightly even at 37 ℃. Does not grow at 40 ° C. NSC-1203: 20-30 ° C. It grows slightly even at 37 ℃. (2) Optimal growth pH NSC-529: pH 9.5 to 10.5 NSC-1203: pH 9.0 to 10.0 (3) Biochemical properties (a) The results of the following physiological tests were positive for both strains.
Liquefaction of gelatin and hydrolysis of starch. (b) Melanin-like pigment formation NSC-529: Negative. NSC-1203: Positive. (c) Reduction of nitrate NSC-529: Positive. NSC-1203: Negative. (d) Resistance to salt Both strains grow on an agar medium containing 4% NaCl. (4) carbon source availability

[0016]

[Table 4]

(5) Cell wall diaminopimelic acid (DAP)
Type NSC-529: meso-DAP NSC-1203: LL-DAP (6) Sugar composition in all bacterial cells NSC-529: C type NSC-1203: (not identified) It is clear that the strain belongs to the bacterium, and as a result of examining the taxonomic position with reference to the "Experimental method for identifying actinomycetes" (edited by the Japanese Society of Actinomycetes, 1985), the NSC-529 strain is The alkalophilic Nocardiopsis genus NSC-1203 was identified as a strain belonging to the alkalophilic Streptomyces genus. These two strains of actinomycetes are named as follows and have been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology. NSC-529: Alkalophilic Nocardiopsis sp. NSC-529
(Microtechnology Research Institute, No. 13183) NSC-1203: Alkalophilic Streptomyces sp. NSC-1203
(Microtechnology Research Institute, No. 13184)

Next, a method for culturing the above-mentioned microorganism and collecting a novel lytic enzyme from the culture solution will be described. Usable medium is a medium used for culturing ordinary microorganisms, and is not particularly limited as long as the strain of the present invention can grow. For example, gluco- is used as an organic carbon source and a nitrogen source.
Sodium, starch, dextrin, peptone, yeast extract and the like are suitable, and addition of potassium phosphate, sodium phosphate, magnesium sulfate, salt and the like is effective as the inorganic salt. In addition, various organic and inorganic substances necessary for the growth of bacteria and the production of enzymes can be added as required. Although it is desirable to add bacterial cells to the medium, it is not always necessary for enzyme production. As a method for culturing in the above-mentioned medium, shaking culture or aeration stirring culture using a fermenter or the like is suitable. The culture temperature is not particularly limited as long as it can grow, but is preferably around 30 ° C. Further, since the optimum growth pH of the microorganism used in the present invention is within the basic range, it is necessary to adjust the pH value of the medium using an appropriate alkaline reagent. Therefore, 0.2% sodium carbonate can be mentioned as a typical example, but needless to say, sodium hydroxide,
Alkaline reagents such as potassium hydroxide, sodium hydrogen carbonate, sodium phosphate and calcium hydroxide can also be used.

After culturing as described above, the lysing enzyme, which is the target substance, can be collected and purified from the culture medium according to a general method for collecting and purifying the enzyme. That is, solids such as bacterial cells are first removed from the culture solution by a method such as filtration or centrifugation to obtain a crude enzyme solution. The crude enzyme solution thus obtained can be used as it is, but this crude enzyme solution is subjected to a conventional enzyme purification method, that is, salting out with ammonium sulfate, precipitation with an organic solvent such as acetone or alcohol, ultrafiltration, ion By using techniques such as an exchange resin and a gel filtration method alone or in combination, it is possible to obtain a lysing enzyme with higher purity. The activity of lytic enzyme is measured by the following method. That is, heat-treated freeze-dried cells of Corynebacterium aquaticum IFO 12154 were added to 25 mM CHES buffer (pH 9) at OD.
_{Suspend to 660} = 1.0. To 3 ml of this cell suspension,
Add 0.2 ml of the enzyme solution, react at 37 ° C for 30 minutes, and measure the decrease in absorbance at 660 nm. Regarding the display of the enzyme activity, the amount of enzyme that reduces the absorbance of 0.001 per minute under the above conditions was defined as 1 unit.

Next, the physicochemical properties of the lytic enzyme obtained in the present invention will be shown. (a) Optimal pH It was measured using a buffer system of Miller & Golder with a constant ionic strength (I = 0.05). The value at the optimum pH is 100%, and each pH is
The relative activity was expressed. The results are shown in Fig. 1. The optimum pH of the enzyme produced by NSE-565 strain and NSC-529 strain is
About 9 and about NSE-114 strain and NSC-1203 strain enzyme were around 9.5. (b) Stable pH Add 500 units of enzyme solution to each buffer solution (100 mM), and at 30 ℃
Leave for 16 hours. This treatment solution is used as a 25 mM CHES buffer solution.
It was diluted appropriately with (pH 9.0) and the residual activity was measured according to the method described above. The enzyme activity before treatment was defined as 100%, and the activity of the sample treated at each pH was expressed as a relative value, and the results are shown in FIG. The relationship between pH and buffer is pH 4.0,
At 5.0, acetate buffer, at pH 6.0, 7.0, 8.0 phosphate buffer, at pH 9.0, 10.0, 11.0 borate buffer, p
For H12.0 and 13.0, potassium chloride buffer was used, respectively. As is clear from FIG. 2, all the enzymes were stable in the pH range of 5 to 11 under the above conditions. (c) Optimum temperature At each temperature, the activity was measured according to the method described above. Taking the activity value at the optimum temperature as 100%, the activity at each temperature was relatively expressed, and the results are shown in FIG. The optimum temperature of each enzyme was around 60 ° C. (d) Stable temperature About 500 units of enzyme solution was added to 25 mM CHES buffer (pH 9.0) and treated at each temperature for 10 minutes. The activity of this treatment liquid
It measured according to the method mentioned above. Enzyme activity before treatment 10
Fig. 4 shows the results of relatively expressing the activity of the treatment liquid at each temperature, which was set to 0%. All of the enzymes retained almost 100% activity when treated at 50 ° C. (e) Effect of surfactant Add about 500 units of lytic enzyme to 25 mM CHES buffer solution (pH 9.0) in which 0.1% (w / v) of various surfactants were dissolved, and add at 20 ° C.
Let stand for 30 minutes. This treatment solution was appropriately diluted with the same buffer solution except for the surfactant, and the residual activity was measured according to the method described above. Table 5 shows the results in which the residual activity was expressed as a relative value, with the enzyme activity when treated in the same manner without the addition of a surfactant as 100%. All four enzymes have sufficient stability against these surfactants.

[0021]

[Table 5]

(F) Effect of Metal Ions 25 mM CHES buffer solution (p
H9) and add about 500 units of lytic enzyme,
Minute processing was performed. This treated solution was appropriately diluted with the same buffer, and the residual activity was measured according to the method described above. The effect of each metal ion was expressed as a relative value, with the activity of the system containing no metal salt as 100%.

[0023]

[Table 6]

(G) Molecular Weight The molecular weight of the enzyme was examined by SDS-polyacrylamide gel electrophoresis. Both LE-114 and LE-565 are about 30,0
00, LE-529, LE-1203 were all about 20,000. (h) Isoelectric point The isoelectric point of the enzyme was examined by isoelectric focusing. LE-11
4, LE-565 is about pH around 10, LE-529 is around pH 9.5,
LE-1203 had a pH around 11.

[0025]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention.

Example 1 For the NSE-114 strain and NSE-565 strain, a medium having the following composition was prepared and cultured. That is, polypeptone 1.0%, yeast extract 0.5%, dihydrogen phosphate 1 potassium 0.1%, magnesium sulfate 0.02%, sodium carbonate (separate sterilization) 0.2%. One platinum loop was inoculated into a 500 ml Erlenmeyer flask containing 150 ml of the above-mentioned medium, and shake-cultured at 30 ° C. and 210 rpm for 24 hours to obtain a seed culture solution. Then, a 30 L jaw mentor containing 15 L of the same medium
Then, inoculate 150 ml of the seed culture solution, stir speed 200 rpm, aeration 15 L / m
Incubated at 30 ° C for 40 hours. The lytic activity at the end of culture is
860 units / ml (NSE-114) and 1,200 units / ml (NSE-565), respectively
Met. The bacterial cells and the like were filtered from this culture solution to obtain a crude enzyme solution. Then, the crude enzyme solution was salted out with ammonium sulfate, the 80% saturated fraction was redissolved, desalted by an ultrafiltration module (manufactured by Asahi Kasei) and concentrated, and then freeze-dried, respectively, about 1.5 g (N
SE-114) and about 2.3 g (NSE-565) of enzyme powder were obtained. The lytic activity per mg of the obtained enzyme powder was 2,200 units (N
SE-114) and 1,000 units (NSE-565).

Example 2 For the NSC-529 strain and NSC-1203 strain, a medium having the following composition was prepared and cultured. That is, 1.0% soluble starch,
Polypeptone 0.5%, yeast extract 0.2%, dipotassium hydrogenphosphate 0.1%, magnesium sulfate 0.02%, sodium carbonate (other sterilization) 0.2%. One platinum loop was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the above-mentioned medium, and cultured by shaking at 30 ° C. and 210 rpm for 48 hours. 10 ml of this culture solution was inoculated into a 5 L Erlenmeyer flask containing 1 L of the same medium, and cultured by shaking at 30 ° C. and 210 rpm for 32 hours to obtain a seed culture solution. Next, a 200 L jar fermenter containing 100 L of the same medium was inoculated with 2 L of the seed culture, and the stirring number was 200 r.
Culture was carried out at 30 ° C. for 64 hours at pm, aeration of 100 L / min. The lytic activity at the end of the culture was 500 units / ml (NSC-529) and 1,200 units / ml (NSC-1203), respectively. From this culture solution, bacterial cells and the like were removed by a filtration method to obtain a crude enzyme solution. Then, the crude enzyme solution obtained here was salted out with ammonium sulfate, the redissolved 80% saturated fraction was dialyzed, desalted and then freeze-dried, respectively, about 2.7 g (NSC-529), and About 8.5 g (NSC-1203) of enzyme powder was obtained. The lytic activity per mg of the crude enzyme powder obtained here was 1,600 units (NSC-529) and 1,600 units (NSC-
1203).

Example 3 To examine the range of action of the lytic enzyme produced by this strain, Micrococcus luteus ATCC- was used as a test strain.
4698, Staphylococcus epidermidis JCM-241
4, Gram-positive bacteria such as Corynebacterium aquaticum IFO-12154, Bacillus subtilis IAM-1028, Lactobacillus casei AHU-1697, and Escherichia
Coli IFO-12734, Citrobactor Diver Suspension JCM-16
59, Gram-negative bacteria such as Proteus vulgaris IFO-3167, Acinetobacter calcoaceticus IFO-12552, and Propionibacterium avidum ATCC-25577 were selected. Propionibacterium avidum was statically cultivated in GAM medium under anaerobic conditions at 30 ° C for 48 hours. Other strains were meat extract 0.3%, polypeptone 1.5%, yeast extract.
After culturing in a medium containing 0.3%, 0.3% salt, and pH 7.2 for 16 hours at 30 ° C and washing the cells once with physiological saline, 25 mM CHES
It was suspended in a buffer solution (pH 9.0) so that OD ₆₆₀ = 1.0.
An enzyme solution (0.2 ml) was added to this bacterial cell solution (3 ml), and the mixture was reacted at 37 ° C. for 30 minutes, and the amount of decrease in absorbance at 660 nm was measured. This reaction was performed in the presence of 0.05% DBS. The results are shown in Table 7.

[0029]

[Table 7]

As is clear from Table 7, the enzyme of the present invention retains the ability to lyse a wide range of bacteria, both Gram-positive and negative bacteria. Moreover, it was not only stable to a surfactant, but also exhibited sufficient lytic activity in the presence thereof. Moreover, the bacteriolytic action was shown against Staphylococcus genus and various Gram-negative bacteria resistant to lysozyme, demonstrating the high utility of the enzyme of the present invention.

Example 4 Gram-positive such as Staphylococcus epidermidis JCM-2414 and Corynebacterium aquaticum IFO-12154 were used as test strains in order to examine the bactericidal effect of the lytic enzyme produced by the strain of the present invention. Bacteria and Escherichia coli IAM-1132, Proteus vulgaris IFO-316
Gram-negative bacteria such as Acinetobacter calcoaceticus IFO-12552 are selected, and each of them is a medium of meat extract 0.3%, polypeptone 0.5%, salt 0.5%, pH 7.2 at 30 ° C, 16 ° C.
Culture was performed with shaking for an hour. To the obtained culture solution, filter-sterilized enzyme (1,000 units) was added, and in the presence of 0.01% DBS,
The reaction was carried out at pH 9.0 and 37 ° C for 30 minutes. This reaction solution was aseptically diluted, and the viable cell count was counted by the plate method. The killing rate was calculated assuming that the viable cell count when the enzyme was not added was 100%, and the results are shown in Table 8. As is clear from Table 8, the enzyme of the present invention has not only bacteriolytic action but also bactericidal action against a wide range of microbial strains.

[0032]

[Table 8]

[0033]

EFFECT OF THE INVENTION By culturing the newly isolated bacteria and actinomycetes of the present invention, it is possible to provide a novel lytic enzyme having an optimum pH on the alkaline side and high stability against various surfactants. It has become possible. Further, since the enzyme of the present invention can dissolve a wide range of bacteria from Gram-positive bacteria to negative bacteria in the presence of a surfactant, by adding it to a detergent or the like, it has an unprecedented excellent ability to remove adherent bacteria in terms of health and hygiene. Can be given. Furthermore, by utilizing its strong and wide bacteriolytic activity and bactericidal action, it is possible to provide a useful means for dissolving and extracting various intracellular substances and preventing various bacterial contaminations.

[Brief description of drawings]

FIG. 1 shows the relationship between enzyme activity and pH of the enzyme of the present invention.

[Explanation of symbols]

 1. Enzyme LE-114 2. Enzyme LE-565 3. Enzyme LE-529 4. Enzyme LE-1203

FIG. 2 shows the pH stability of the enzyme of the present invention.

[Explanation of symbols]

 1. Enzyme LE-114 2. Enzyme LE-565 3. Enzyme LE-529 4. Enzyme LE-1203

FIG. 3 shows the relationship between enzyme activity and temperature of the enzyme of the present invention.

[Explanation of symbols]

 1. Enzyme LE-114 2. Enzyme LE-565 3. Enzyme LE-529 4. Enzyme LE-1203

FIG. 4 shows the temperature stability of the enzyme of the present invention.

[Explanation of symbols]

 1. Enzyme LE-114 2. Enzyme LE-565 3. Enzyme LE-529 4. Enzyme LE-1203

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C12R 1:07) (C12N 9/14 C12R 1: 465)

Claims (13)

[Claims] 1. A novel alkaline lytic enzyme having the following physicochemical properties. _{(1) It has an} optimum _{pH of 9.0} to _9.5 , and has an activity of _50% or more of the optimum _{pH in} the range of _{pH 6.5} to ₁₁ . _{(2) The} optimum temperature is around ₆₀ ° C. _{(3) pH} stability When heat treatment at ₃₀ ° C _{for 16} hours, _{pH 5}
Stable in the range of ~ ₁₁ and _{pH 9} at ₅₀ ℃ _{, 10}
The residual activity after ₉₀ minutes heat treatment is _90% or more. _(Four)
Effect of metal ions _{pH 9, 20} ℃ _{, 1mM} metal salt concentration,
A ₃₀ min _{treatment, MgCl2, CaCl 2, BaCl 2} , MnCl 2, FeCl 2, F
eCl ₃ , CoCl ₂ , NiCl ₂ , CuCl ₂ , ZnCl ₂ , AlCl ₃ , CdCl ₂ , Hg
It has a residual activity of 80% or more with respect to Cl ₂ . (5) Substrate specificity 0.05% sodium dodecylbenzene sulfonate (DBS)
Resistant to lysozyme in the presence Staphylococcus epidermidis JCM-2414, Micrococcus luteus ATCC-4698, Corynebacterium aquaticum
Gram-positive bacteria such as IFO-12154, Bacillus subtilis IAM-1028, Lactobacillus casei AHU-1697, and Escherichia coli IFO-12734, Citrobacter diversus JCM-1659, Proteus vulgaris IFO-316
7, Acinetobacter Calcoaceticus IFO-12552,
It shows lytic action against Gram-negative bacteria such as Propionibacterium avidum ATCC-25577. 2. The alkaline lytic enzyme according to claim 1, which further has the following properties:
E-114). (6a) Temperature stability pH9, heat treatment for 10 minutes, 6
Stable up to 0 ° C. (7a) Molecular weight 30,000 (8a) Isoelectric point around pH10 3. The alkaline lytic enzyme according to claim 1, which further has the following properties:
E-565). (6b) Temperature stability pH9, heat treatment for 10 minutes, 5
It is stable up to 0 ° C and has a residual activity of about 80% even at 60 ° C. (7b) Molecular weight 30,000 (8b) Isoelectric point around pH10 4. The alkaline lytic enzyme according to claim 1, which further has the following properties:
E-529). (6c) Temperature stability pH9, 5 minutes against heat treatment for 10 minutes
It is stable up to 0 ° C and has a residual activity of about 60% even at 60 ° C. (7c) Molecular weight 20,000 (8c) Isoelectric point around pH 9.5 5. The alkaline lytic enzyme according to claim 1, which further has the following properties:
E-1203). (6d) Temperature stability pH9, heat treatment for 10 minutes, 5
It is stable up to 0 ° C and has a residual activity of about 60% even at 60 ° C. (7d) Molecular weight 20,000 (8d) Isoelectric point around pH 11 6. Alkalophilic Bacillus sp. NSE-114, which produces a novel alkaline lytic enzyme, having the properties described in claims 1 and 2 (Microtechnology Research Institute No. 13181). 7. A bacterium of the genus Alkalophilic Bacillus sp. NSE-565, which produces a novel alkaline lytic enzyme, having the properties according to claims 1 and 3 (Microtechnology Research Institute No. 13182). 8. Alkalophilic Nocardiopsis actinomycetes having the properties according to claims 1 and 4, which produce a novel alkaline lytic enzyme.
sp. NSC-529) (Microtechnology Research Institute, No. 13183). 9. Alkalophilic Streptomyces, which has the properties of claims 1 and 5 and produces a novel alkaline lytic enzyme.
sp. NSC-1203) (Ministry of Industrial Science, Myc. No. 13184). 10. A bacterium which produces a novel alkaline lytic enzyme having the properties of claims 1 and 2 and which belongs to the alkalophilic Bacillus genus, or the strain of claim 6 is cultivated, and the bacterium is placed in a culture solution. A method for producing a novel alkaline lytic enzyme, which comprises producing and accumulating a novel alkaline lytic enzyme having the properties of items 1 and 2 and collecting the enzyme from the culture solution. 11. A bacterium belonging to the alkalophilic Bacillus genus or a strain according to claim 7, which produces a novel alkaline lytic enzyme having the properties according to claims 1 and 3, or is cultured in a culture solution. A method for producing a novel alkaline lytic enzyme, which comprises producing and accumulating a novel alkaline lytic enzyme having the properties of items 1 and 3 and collecting the enzyme from the culture solution. 12. An actinomycete that produces a novel alkaline lytic enzyme having the properties according to claims 1 and 4 and belongs to the alkalophilic Nocardiopsis genus, or the strain according to claim 8, and culture medium. A method for producing a novel alkaline lytic enzyme, characterized in that the novel alkaline lytic enzyme having the properties of claims 1 and 4 is produced and accumulated therein, and the enzyme is collected from the culture solution. 13. A culture solution obtained by culturing a novel alkaline lytic enzyme having the properties according to claims 1 and 5 and culturing an actinomycete belonging to the alkalophilic Streptomyces genus or the strain according to claim 9. A method for producing a novel alkaline lytic enzyme, characterized in that the novel alkaline lytic enzyme having the properties according to claims 1 and 5 is produced and accumulated therein, and the enzyme is collected from the culture solution.