JPS63279782A - Microorganism producing alkaline protease - Google Patents

Abstract

PURPOSE:To obtain an alkaline protease useful as an excellent enzyme for adding to detergent, by culturing a microbial strain belonging to Corynebacterium genus in an alkaline medium. CONSTITUTION:An alkaline protease having the following physical and chemical properties can be produced by culturing Corynebacterium sp. No.67 (FERM P-9335), Corynebacterium sp. No.68A (FERM P-9336) or Corynebacterium sp. No.68B (FERM P-9337) in a nutrient medium. Action, casein decomposition activity in a reaction solution containing a detergent component is >=70% of the activity in the absence of detergent; optimum pH and stable pH, optimum pH is 10-10.6 and stable in pH7-10.5; working temperature, about 50 deg.C; inhibition, activation and stabilization, thermal stability is increased by Ca<2+> and the activity is uninhibited by MIA and EDTA and inhibited by diisopropyl fluorophosphate; molecular weight, the molecular weight of an enzyme produced by 68A strain is about 14,600.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to novel bacteria belonging to the genus Corynebacterium, more specifically, novel bacteria belonging to the genus Corynebacterium that produce alkaline protease, and culturing these bacteria. The present invention relates to a method comprising obtaining alkaline protease from a culture thereof.

BACKGROUND OF THE INVENTION Alkaline protease is an enzyme that is used in large quantities in various fields such as the leather industry, food industry, and textile industry.

Furthermore, in recent years, various enzymes have been added to detergents for the purpose of improving their detergency. In particular, alkaline protease is known to decompose protein stains and greatly contribute to improving detergency. On the other hand, enzymes for detergents account for about 30% of the world's industrial enzyme market, and demand for alkaline protease, which is said to be essential for detergents, is extremely large.

Therefore, along with the improvement of alkaline protease productivity,
There is a desire to develop enzymes with properties not found in conventional products.

Under these circumstances, screening of alkaliphilic microorganisms as alkaline protease producing bacteria is currently being actively conducted. The discovery of a novel alkaline protease-producing bacterium is described in Japanese Patent Publication No. 55118/1983 for alkalophilic Bacillus bacteria, and Japanese Patent Publication No. 11396/1983 for alkalophilic actinomycetes.

Problems to be Solved by the Invention However, the conventional production of alkaline protease only from Bacillus and Streptomyces cannot adequately meet the demand. Furthermore, it is necessary to obtain alkaline proteases having various characteristics from various microorganisms in order to improve the detergency of detergents.

Means for Solving the Problems The present inventors have developed a highly stable and highly active alkali 10
As a result of screening the natural world for producing bacteria that produce tease at a high yield, a novel alkalophilic Corynebacterium was discovered and the present invention was completed. That is, Corynebacterium sp, No. 1, which belongs to the genus Corynebacterium. 6
7 strains (Feikoken Bacteria No. 9335), Corynebacterium sp, No.

68A strain (FEI Bacterium No. 9336), Corynebacterium sp.
337).

A further object of the present invention is to provide a method for culturing these strains and producing alkaline protease from the culture.

The present invention is the first to report the production of alkaline 10-tease by Corynebacterium bacteria.

Furthermore, alkaline protease obtained by culturing these bacteria in an alkaline medium exhibits casein-degrading activity even in highly basic detergent solutions, making it particularly suitable for use in detergent additives.

Next, the taxonomic properties of this strain isolated and collected from nature by the present inventors will be described in detail.

1) When observed, cells grown in a morphological medium have the following morphological characteristics.

■Cell shape and size All strains are short to long rods, with rounded cell ends.

The size is No. 67, short axis 0.5 to 0.6, long axis 1 to 4 (L
lllll) No, 68A short axis 0.3 to 0.6, long axis 1
~3.5(IIIIl)No, 68B short axis 0.5~0
．． 9, the major axis is 1 to 3 (lJm).

■ Cell pleomorphism No other features other than cell length are observed.

■ Motility All strains are motile. Strains 67 and 68A have several periflagella, and strain 68B has no flagella.

■ Spores Neither strain forms heat-stable spores.

■ Durham staining All strains are positive, but the positivity tends to become weaker as the culture progresses.

■ Anti-acidity Both strains are negative.

2) Growth status in the following media ■ Juice agar culture All strains grow well at pH 10 to 10.5, and in flat media the colonies are circular, ridged, shiny and opaque with golden edges. The color is between yellow and orange. No dye is released into the medium.

■ Broth liquid culture All strains grow well, and the medium is transparent with no turbidity, and there is no precipitate.

■ Broth gelatin needle puncture culture Both liquefy in layers at pH 10.5.

3) Physiological properties ■ Nitrate reduction Neither strain reduces under anaerobic conditions.

■　Denitrification reaction Neither strain grows under anaerobic conditions.

■ vp test All strains are negative when alkaline medium is used.

■ Starch hydrolysis Both strains hydrolyze.

■ Utilization of inorganic nitrogen sources (nitrates and ammonium salts) None of the strains utilizes either nitrates or ammonium salts.

■ Pigment production Neither strain produces anything.

■ Oxidase Both strains are negative.

■ Catalase Both strains are negative.

■Growth range i) Growth pH: All strains grow at a pH of 8.0 to 12.5, but pH around 10 is optimal. Almost no growth occurs at pH 7.

ii) Growth temperature: 67 strains grow below 42℃, 68A
The 68B strain grows at 45°C or lower. All stocks are 3
The optimum temperature is around 7°C.

[Phase] Attitude towards oxygen Both strains are aerobic.

■　O-F test Neither strain grows under anaerobic conditions.

[Stock] Production of acid from sugars + indicates that it is generated, and - indicates that it is not generated.

Saccharides 67 strains 68A strains 68B strains L-arabinose --- D-xylose --- D-glucose + + +D-mannose -10 - D-fructose + +10-galactose - --- Maltose + + +sucrose
+ ten + lactose
−10 − Trehalose + + +D-Sorvit −−− D−Mannit −−− Inosit −−− Glycerin Starch
+ + +4) Other properties ■ Liquefaction of gelatin All strains liquefy.

■ Sodium chloride resistance Both strains grow in the presence of 12% sodium chloride.

This strain is considered to belong to any of the genus Bacillus, Arthrobacterium, Crucia, and Corynebacterium since it is a bacillus with negative acid fastness, negative oxidase test, positive catalase test, and positive Durham.

Furthermore, since it does not produce heat-resistant spores, it does not belong to the genus Bacillus, and because it does not show significant polymorphism, it is not Arthrobacterium, and it is thought that it is not a Bacillus due to the state of acid production from carbohydrates. On the other hand, it is a bacterium of the genus Corynebacterium due to its nitrate reducing ability, carbonic acid production, and growth temperature.

However, growth at pH around neutrality is poor, and at pH 1, 0,
It is distinguished from known strains of the genus Corynebacterium because it grows best around 5. For the above search, please refer to Bergy's Manual of Sy.
Stematic Bacteriology No. 9
Edition, Volume 1 (1984), Volume 2 (1987) (The
Williams & Wilkins Company
y, U, S, ^, Publishing) was used.

As described above, the strain of the present invention is a novel strain belonging to the genus Corynebacterium, although it is different from conventional strains of the genus Corynebacterium.

Next, the novel strain of the present invention was cultured in an alkaline medium,
The characteristics of alkaline protease obtained by isolation from culture will be explained.

■ Action itself does not show protease activity In a reaction solution containing household detergent at the same concentration as under washing conditions, the alkaline protease obtained by culturing any strain of bacteria had a casein-degrading activity of more than 70% in the absence of detergent. shows.

■ Measurement of titer Measurement of alkaline protease activity Na2CO1-NaHCO3 containing 0.75% casein
Add 0.6 ml of enzyme solution to 0.6 ml of M solution (p Hlo, 6).
After adding 2 ml and reacting at 37°C for 10 minutes, 5%
Add 4 ml of trichloroacetic acid solution and further incubate at 37°C for 2
Leave for 0 minutes. The resulting precipitate was removed, and the supernatant was
Measure the absorbance at no+. One unit (IU) of enzyme is defined as the amount of enzyme that produces tyrosine equivalent to luH in one minute under the above conditions.

■ Optimum pH and stable pH rangei) Optimum pH: The enzyme produced by any strain has a
It is 10-10.6.

ii) Stable pH range: The enzyme produced from either strain has a pH of 7 to 10.5 when treated at 37°C. 1st
The figure shows the relationship between protease activity and reaction pH. For activity measurement, add acetic acid-sodium acetate buffer (.
-・) NaH2PO4-NaJPO1 at pH 6.3 to 7.3
Buffer solution (〇-〇) Tris-HCIM buffer solution (Δ
-Δ) pH 9.3 to 10.6, Na2CO, -NaH
Co, 1+! NaHCOs-NaOH buffer (Moum) with pH 10.3 to 10.7 Na2CO1-NaOH buffer (■-■) with pH 10.7 to 11.3 was used.

■ Suitable temperature range for action All strains were tested at 50% in a solution containing 2mM CaCl2.
It is around ℃. Figure 2 shows the relationship between protease activity and reaction temperature ■ Inactivation conditions such as pH 5 temperature The enzyme produced by strains 67 and 68B is
Na2C03 = NaHCO at pH 10,6 containing l2
When heat treated with :+ for 10 minutes, it is stable up to 40°C, shows 55% residual activity at 45°C, and is completely inactivated at 55°C.

The enzyme produced by strain 68A is stable up to 40°C, and
It shows 70% residual activity at 5°C and is completely inactivated at 55°C. FIG. 3 shows the relationship between treatment temperature and activity.

■ Inhibition, activation, and stabilization The thermostability of the enzyme produced by both strains is increased by Ca2+, and MIA (monoiodoacetic acid) and EDTA (
It is not inhibited by (ethylenediaminetetraacetic acid), but is inhibited by (diisopropylfluorophosphate).

■ The molecular weight of the enzyme produced by the 68A strain is approximately 14,600 (
(based on gel filtration method).

Next, examples of the present invention will be described.

A small amount of the sampled soil was suspended in sterilized water, and a loopful of the suspension was placed on an agar medium containing skim milk (10 g of skim milk).
, meat extract 5FI, Na2CO320FI, agar 20
g, consisting of 11 parts of water). Colonies were cultured at 37°C for several days, and those that dissolved the skim milk around the colonies were selected for the second screening. The strain obtained in this way was subjected to liquid culture, and the enzyme activity of the culture solution was measured.
This was a secondary screening. Furthermore, pH 10.3-10
．． 5. Enzyme activity was measured in detergent ingredients containing 0.332/1 surfactant, and 67 strains, 68A strains, and 68A strains were highly resistant.
68B stock was selected.

Example 1 Medium: 11 water, soybean peptone lh, yeast extract 5g
, glucose 2g, K2HPO41g, MgSO4.7
(2) A material containing 200.2 g and 20 g of Na2COs was used. Sterilize this at 121℃ for 15 minutes using NaCO.
5 was sterilized separately), 100 ml each was dispensed into Erlenmeyer flasks, inoculated with fresh bacteria that had been previously cultured in the same medium for 16 to 18 hours, and cultured with shaking at 37°C for 3 days.

The bacterial cells were removed from this culture, and the filtrate was obtained as an enzyme solution. The alkaline protease activity of this enzyme solution is 345U/
It was a company.

Reference Example 1 Ammonium sulfate was added to the culture supernatant obtained by the method shown in Example 1 to 60% saturation, and a precipitate was obtained by centrifugation and freeze-dried.

This was resuspended in a pH 7 or 10.6 buffer and used for experiments.

The influence of detergent components on the enzyme solution prepared as described above was investigated.

When we measured the casein decomposition ability of each enzyme in a reaction solution containing detergent components, the activity of each enzyme in the reaction solution without detergent components was 100%, and for the enzymes of strains 67 and 68A, it was 70.5%. , 68B strain enzyme showed 72% activity.

This result shows that these enzymes can be used as detergent ingredients.

[Brief explanation of the drawing]

FIG. 1(a) to FIG. 1(C) are graphs showing the relationship between the activity of the enzyme produced by the strain of the present invention and the pH of the reaction solution. Figures 2<a) to 2(c) are graphs showing the relationship between the activity of enzymes produced by the strain of the present invention and reaction temperature. Figures 3(a) to 3(C) are graphs showing the thermostability of enzymes produced by the strains of the present invention. In both figures, (a) is 67 strains, (b) is 68A.
strain, (c) relates to strain 68B. 41! I (Q) pH (b) pH (c) pH 2 times C0ノ (b) <C) Izumi 3 figure <Q) Ji9 bi [, 4 series C"Cn (b> (attached sheet) Procedure supplement Correct document July 2, 1988 1, Indication of the case Patent Application No. 115453 of 1988 2, Name of the invention Alkaline protease producing microorganism 3, Relationship with the case by the person making the amendment Patent applicant address name (024) Onoda Cement Co., Ltd. 4, Agent 5, Subject of amendment (51allll Breast Morikata Shin) (1) 67 strains of Corynebacterium sp, lro belonging to the genus Corynebacterium having the following mycological properties (Feikoken No. 9335), Corynebacterium sp.
No. 6), Corynebacterium sp, No. 68
An alkaline protease-producing microorganism selected from the group consisting of strain B (Feikoken Bibori No. 9337). 1) When observed, cells grown in a morphological medium have the following morphological characteristics. ■Cell shape and size All strains are short to long rods, with rounded cell ends. The size is No. 67, short axis 0.5 to Oy6, long axis 1 to 4 (μ
m) No. 68A Short axis 0J-0,6, Long axis 1-3-5
(μm) No, 68B Short axis 0.5~G, 9, Long axis 1
-3 (μm). (White) mtill! V) 5'/17n ■ Motility All strains are motile. Strains 67 and 68A have several periflagella, and strain 68B has no flagella. ■ Spores Neither strain forms heat-resistant spores. ■ Durham staining All strains are positive, but the positivity tends to become weaker as the culture progresses. ■ All acid-fast strains are negative. 2) Growth status in the following media ■ Juice agar culture All strains grow well at pH 10 to 10.5, and in flat media the colonies are circular, ridged, shiny and opaque with a golden edge. The color is between yellow and orange. No dye is released into the medium. ■ Broth liquid culture All strains grow well, and the medium is transparent with no turbidity, and there is no precipitate. ■ Meat juice gelatin puncture culture At pH 10.5, both liquefy in layers. 3) Physiological properties■ None of the strains reduce nitrate. ■ Denitrification reaction Neither strain grows under anaerobic conditions. ■ vp test All strains are negative when alkaline medium is used. ■ Hydrolysis of starch Both strains hydrolyze starch. ■ Utilization of inorganic nitrogen sources (nitrates and ammonium salts) None of the strains utilizes either nitrates or ammonium salts. ■ None of the strains produce pigment. ■ Oxidase All strains are negative. ■ Catalase All strains are l1. ■Growth range 1) Growth pH: All strains grow at a pH of 8.0 to 12.5, but optimally around I) HIO. I) Almost no growth in H7. i) Growth temperature: 67 strains grow at 42°C or lower, 688 strains and 611B strain grow at 45°C or lower. The optimum temperature for both strains is around 37°C. 0 Attitude towards oxygen Both strains are aerobic. 00-F Test None of the strains grow under anaerobic conditions. ■ Production of acid from sugars + indicates that it is produced, - indicates that it is not produced. 13= Saccharides 67 strains 68A strains 68B strains L-arabinose --- D-xylose --- D-glucose + + ten-mannose --- ten - D-fructose + + +D-galactose --- maltose + ten ten sucrose
＋ ＋ ＋Lactose −＋
− Trehalose + + +D-Sorvit −−− D-Mannit − −− Inosite − −− Glycerin + 10 + Starch
+ + 14) Other properties ■ Liquefaction of gelatin All strains liquefy. ■ Sodium chloride tolerance All strains grow in the presence of 12% sodium chloride. (2) A method for producing alkaline protease, which comprises culturing alkaline protease-producing bacteria belonging to the genus Corynebacterium and obtaining alkaline protease from the culture. (3) The alkaline glothiase-producing bacterium belonging to the genus Corynebacterium is Corynebacterium sp, No.
, strain 67 (Feikoken Bacterium No. 9335), Corynebacterium sp.
336), Corynebacterium sp, No. 3. The method for producing alkaline protease according to claim 2, wherein the strain is selected from the group consisting of strain 611B (Feikoken Bibori No. 9337). (2) Delete "under anaerobic conditions" from line 13 on page 11 of the specification. (3) "Negative" on page 12, line 10 of the specification is corrected to "positive."that's all

Claims (3)

[Claims] (1) Corynebacterium sp., which belongs to the genus Corynebacterium and has the following mycological properties. No. 67 strain (Feikoken Bacterium No. 9335), Corynebacterium sp. No
．． 68A strain (Feikoken Bacteria No. 9336), Corynebacterium sp. No. An alkaline protease-producing microorganism selected from the group consisting of strain 68B (Feikoken Bibori No. 9337). 1) Morphological cells grown in a medium have the following morphological characteristics when observed. [1] Cell shape and size All strains are short to long rods, with rounded cell ends. The size is No. 67 Minor axis 0.5 to 0.6, major axis 1 to 4 (μm)
No. 68A Minor axis 0.3 to 0.6, major axis 1 to 3.5 (
μm) No. 68B Minor axis 0.5 to 0.9, major axis 1 to 3
(μm). [2] Cell pleomorphism No other differences other than cell length are observed. [3] Motility All strains are motile. Strains 67 and 68A have several periflagella, and strain 68B has no flagella. [4] Spores Neither strain forms heat-resistant spores. [5] Gram staining All strains are positive, but the positivity tends to become weaker as the culture progresses. [6] Acid-fast All strains are negative. 2) Growth status in each of the following media [1] Broth agar culture All strains grow well at pH 10 to 10.5, and in flat media, the colonies are circular, ridged, and the entire periphery is glossy and opaque. be. The color is between yellow and orange. No dye is released into the medium. [2] Meat juice liquid culture All strains grow well, and the medium is transparent with no turbidity, and there is no precipitate. [3] Meat juice gelatin puncture culture At pH 10.5, both liquefy in a layered manner. 3) Physiological properties [1] Nitrate reduction None of the strains reduce nitrate under anaerobic conditions. [2] Denitrification reaction Neither strain grows under anaerobic conditions. [3] VP test All strains are negative when alkaline medium is used. [4] Hydrolysis of starch All strains hydrolyze starch. [5] Utilization of inorganic nitrogen sources (nitrates and ammonium salts) Neither strain utilizes either nitrates or ammonium salts. [6] Production of pigment None of the strains produces pigment. [7] Oxidase All strains are negative. [8] Catalase All strains are negative. [9] Growth range i) Growth pH: All strains grow at a pH of 8.0 to 12.5, but pH around 10 is optimal. Almost no growth occurs at pH 7. ii) Growth temperature: 67 strains grow below 42℃, 68A
The 68B strain grows at 45°C or lower. All stocks are 3
The optimum temperature is around 7°C. [10] Attitude towards oxygen All strains are aerobic. [11] O-F test None of the strains grow under anaerobic conditions. [12] Production of acid from saccharides + indicates that acid is produced, and - indicates that it is not produced. Saccharides 67 strains 68A strains 68B strains L-arabinose − − − D-xylose − − − D-glucose + + + D-mannose − + − D-fructose + + + D-galactose − − − Maltose + + + Sucrose + + + Lactose - + - Trehalose + + + D-Sorvit - - - D-Mannit - - - Inosit - - - Glycerin + + + Starch + + + 4) Other properties [1] Liquefaction of gelatin Both strains liquefy. [2] Sodium chloride tolerance All strains grow in the presence of 12% sodium chloride. (2) A method for producing alkaline protease, which comprises culturing alkaline protease-producing bacteria belonging to the genus Corynebacterium and obtaining alkaline protease from the culture. (3) The alkaline protease producing bacterium belonging to the genus Corynebacterium is Corynebacterium sp. No. 67
strain (Feikoken Bacteria No. 9335), Corynebacterium s
p. No. 68A strain (Feikoken Bacteria No. 9336), Corynebacterium sp. No. 68B strain (Feikoken Bacterium No. 9
3. The method for producing alkaline protease according to claim 2, wherein the strain is selected from the group consisting of (No. 337).