JPH09327289A - Low-temperature active protease and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject new protease, derived from a bacterium of the genus Pseudomonas and useful as an enzyme for industrial use capable of manifesting high proteolytic activities even at low temperatures and retaining a flavor without deteriorating a taste due to the development of a detergent capable of well removing foulings even with water or temperature treatment. SOLUTION: This new low-temperature active protease is derived from a bacterium of the genus Pseudomonas and capable of hydrolyzing casein, producing amino acids and low-molecular peptides and manifesting >=20% relative activities at 0 deg.C and has about 25-30 deg.C optimal temperature, 45,000 molecular weight [measured by a sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) method] and great possibilities as an enzyme for industrial use such as the manifestation of high proteolytic activities even at low temperatures and impartment, etc., of a flavor without causing the deterioration in taste due to the development of a detergent capable of well removing foulings and temperature treatment. The protease is produced by culturing a strain, belonging to the genus Pseudomonas and having the ability to produce the protease having high activities even at low temperatures [e. g. Pseudomonas sp. PL-4 (FERM P-15410)].

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an enzyme having a high proteolytic activity under enzyme reaction conditions at low temperature and a method for producing the same.

[0002]

2. Description of the Related Art Enzymes usually have an optimum temperature for activity at 35 to 60 ° C. On the other hand, it is possible that some microorganisms that live in low-temperature environments such as cold regions produce enzymes having optimum temperatures at lower temperatures and low-temperature active enzymes. Due to its high enzymatic activity at low temperatures, low-temperature active enzymes are often used as industrial enzymes such as the development of detergents that stain well even with water and the addition of flavor without sacrificing the taste associated with temperature treatment. Have the potential. In particular, proteolytic enzymes and proteases are currently in great demand as industrial enzymes, but so far, several attempts have been made to isolate low temperature active proteases from cold-resistant bacteria, etc. A protease having a temperature in a low temperature range and having high activity at a low temperature has not been reported.

[0003]

Based on the above background, the present inventors have made various attempts to find a protease having a characteristic of showing high proteolytic activity at low temperature.

[0004]

As a result of earnest studies, the present inventors have found that Pseudomonas strains produce a novel enzyme having high proteolytic activity at low temperatures, Has been completed. That is,
The first of the present invention is a protease derived from a bacterium of the genus Pseudomonas, which shows a relative activity of 20% or more at 0 ° C at an optimum temperature around 25 to 30 ° C, and the second of the present invention is The following physicochemical properties: (1) Action: Casein is hydrolyzed to form an amino acid and a low molecular weight peptide.

(2) Optimum temperature: around 25 to 30 ° C. (3) Thermal stability: It is stable up to 30 ° C. when kept in 25 mM Tris-HCl buffer (pH 7.5) for 1 hour.

(4) Optimum pH: around 7.0 (5) Stable pH range: 6.0 to 11.0 (6) A protease having a molecular weight of about 45,000 (SDS-PAGE method). The third aspect of the present invention is the genus Pseudomonas. A method for producing a protease, which comprises culturing a strain that belongs to the above (1) and has the ability to produce the protease, and collecting the protease from the culture.

The first protease of the present invention need not have all of the above-mentioned physicochemical properties, but those having these properties are preferred. The second protease of the present invention is not limited to those derived from Pseudomonas bacteria, but proteases derived from Pseudomonas bacteria are preferred. The first and second proteases of the present invention are usually
Proteins such as casein and bovine serum albumin are hydrolyzed to produce amino acids such as tyrosine and low molecular weight peptides having a molecular weight of 3000 or less.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used for the production of the protease of the present invention may be any strain so long as it can produce the protease, preferably Pseudomonas bacteria, and variants of these strains. Alternatively, it may be a mutant strain. Preferable examples of Pseudomonas spp. Include Pseudomonas sp. PL-4
Is mentioned.

[0009] Pseudomonas sp. PL-4 is a strain newly isolated by the present inventors from the intestine of Hokke produced in Hokkaido, and its mycological properties are as follows. The results of observing the bacterium with an electron microscope are shown in FIG. This strain is a Gram-negative bacillus with flagella, size 1.0 × 2.0 μm, milky white,
Round, forming smooth colonies. It has no sporulation ability and has mycological properties such as a G + C content (mol%) of 58.7% in total DNA. As criteria for classification and identification,
"Bergey's Manual of Determinat
ive Bacteriology) ”, 8th edition (1974). Based on the above literature and mycological properties, this strain is considered to belong to the genus Pseudomonas, and this strain was designated as Pseudomonas s
It was named p. PL-4. This strain was transferred to the Institute of Biotechnology, Institute of Industrial Science and Technology on January 31, 1996 by FERM P-1.
Deposited as 5410.

The enzyme of the present invention can be produced by culturing the protease-producing bacterium in an enzyme-producing medium and collecting the protease from the culture. As the medium used for culturing the protease-producing bacterium, any of synthetic medium and natural medium can be used as long as it contains an appropriate amount of a carbon source, a nitrogen source, an inorganic substance and other necessary nutrients that can be assimilated by the strain used. As the carbon source, glucose, maltose or the like is used. As a nitrogen source,
For example, nitrogen-containing natural products such as peptones, yeast extract, and meat extract, and inorganic nitrogen-containing compounds such as sodium nitrate and ammonium chloride are used. As the inorganic substance, for example, potassium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, ferric chloride or the like is used. In addition, it is desirable to add proteins such as casein, gelatin and bovine serum albumin to the medium as a protease production inducer. The culture is usually performed by shaking culture or aeration-agitation culture. The culture temperature is usually controlled at 5 to 30 ° C, preferably 10 to 15 ° C. It can be carried out under conditions other than these as long as the strain used can grow. Culture period is usually 1 to
Protease is secreted outside the cells in 7 days.

For purification of the enzyme of the present invention, a generally used purification method may be used. For example, centrifugation, filtration, ultrafiltration or the like may be used for the method of separating the bacterial cells. Further, for the culture supernatant, salting out method using ammonium sulfate, sodium sulfate, etc., organic solvent precipitation method using acetone or ethanol, further ion exchange chromatography method using CM (carboxymethyl) carrier or DEAE (diethylaminoethyl) carrier, It can be purified by a gel filtration method using an agarose derivative or the like. The crude enzyme solution and purified enzyme solution obtained by these methods are added with stabilizers such as glycerol, sucrose, and ethylene glycol as a liquid enzyme agent, and a drying method such as spray drying or freeze drying is used. It can be used as a powdered enzyme preparation, or as an immobilized enzyme preparation after being immobilized on a suitable carrier.

Next, a method for measuring the activity of the protease of the present invention will be described. The reaction mixture is 50 mM containing 0.5% (w / v) casein.
200 μl of enzyme solution was added to 800 μl of phosphate buffer (pH 7.0). The reaction solution is allowed to react at 25 ° C for 60 minutes. After the reaction was completed, 5.4% (w / v) trichloroacetic acid was included 1
270 μl of M acetic acid-0.67 M sodium acetate aqueous solution is added to stop the reaction. After stopping the reaction, the reaction solution is centrifuged, and the free amino acid in the supernatant is measured by the phenol method. In the blind test, a reaction stop solution is added to an aqueous casein solution which is an enzyme reaction substrate before addition of the enzyme solution, and the same operation as described above is performed. Regarding the expression of the activity of protease, 1 unit (U) is the activity of releasing 1 micromole of tyrosine under the above-mentioned conditions.

[0013]

EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to the following examples.

(Example 1) Casein 1.0% (w / v), yeast extract 0.1% (w / v), magnesium sulfate 0.15% (w / v),
Calcium chloride 0.05% (w / v) and ferric chloride 0.0015% (w
The liquid medium (100 ml) containing / v) was transferred to a 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. Pseudomonas sp. PL-4 (FERM P-15410) was inoculated as an inoculum and cultured at 10 ° C for 72 hours to obtain a culture solution. The culture solution was centrifuged, and the resulting supernatant was dialyzed and fractionated by CM-biogel column chromatography and then Q-sepharose column chromatography to obtain an enzyme preparation. The obtained protease had the following properties.

(1) The following reaction was catalyzed. casein,
Proteins such as bovine serum albumin are hydrolyzed to produce amino acids such as tyrosine and low molecular weight peptides having a molecular weight of 3000 or less. (2) Optimum temperature The enzyme activity at each temperature was measured. The results are shown in Fig. 2, and the optimum temperature was 25 to 30 ° C. Further, it showed a relative activity of about 25% even at 0 to 4 ° C. The existence of an enzyme having a high proteolytic activity even at such a low temperature has not been reported so far. (3) Thermostability After the enzyme was kept warm in a 25 mM Tris-HCl buffer (pH 7.5) for 1 hour, the remaining activity was measured. The result is shown in FIG.
And was stable up to 30 ° C.

(4) Optimum pH Enzyme activity in Briton-Robinson buffer (pH 3.0 to 10.0) was measured. The results are shown in Fig. 4, and the optimum pH was around 7.0. (5) Stable pH range Briton-Robinson buffer (pH 3.0
~ 12.0) and stored at 20 ° C for 1 hour to measure the residual activity. The results are shown in Fig. 5, and the stable pH range was 6.0 to 11.0. (6) Molecular weight SDS-PAGE analysis showed about 45,0
00.

[0017]

According to the present invention, it is possible to provide a protease having high activity at low temperature.

[Brief description of drawings]

FIG. 1 is a photograph showing the morphology of an organism showing the result of observing Pseudomonas sp. PL-4 with an electron microscope.

FIG. 2 is a graph showing the relationship between reaction temperature and relative activity of the enzyme of the present invention.

FIG. 3 is a diagram showing the thermostability of the enzyme of the present invention.

FIG. 4 is a diagram showing the relationship between reaction pH and relative activity of the enzyme of the present invention.

FIG. 5 is a diagram showing pH stability of the enzyme of the present invention.

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[Procedure amendment]

[Submission date] June 17, 1996

[Procedure 3]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

Front page continuation (72) Inventor Isao Yumoto 2-17-1, Tsukikanto, Toyohira-ku, Sapporo-shi, Hokkaido Inside Institute of Industrial Science and Technology, Hokkaido Institute of Industrial Technology (72) Inventor Satoru Ogiya 2 Tsukikanto, Sapporo-shi, Toyohira-ku, Sapporo 17-2-1 No. 1 inside Hokkaido Institute of Industrial Technology

Claims (3)

[Claims] 1. A protease derived from a bacterium of the genus Pseudomonas, which has an optimum temperature of around 25 to 30 ° C. at 0 ° C.
A protease that exhibits a relative activity of 20% or more. 2. The following physicochemical properties: (1) Action: Casein is hydrolyzed to produce an amino acid and a low molecular weight peptide. (2) Optimum temperature: around 25 to 30 ° C (3) Thermal stability: It is stable up to 30 ° C when kept for 1 hour in 25 mM Tris-HCl buffer (pH 7.5). (4) Optimum pH: around 7.0 (5) Stable pH range: 6.0 to 11.0 (6) Protease having a molecular weight of about 45,000 (SDS-PAGE method). 3. A method for producing a protease, which comprises culturing a strain belonging to the genus Pseudomonas and having the ability to produce the protease according to claim 1 or 2, and collecting the protease from the culture.