JP2885434B2 - Protease and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel proteolytic enzyme and a production method for producing the same using a novel producing bacterium.

2. Description of the Related Art Unsatisfactory effects may not be obtained with proteases that are generally widely used to degrade unused cortical proteins. Proteolytic enzymes characterized by their specificity for cortical proteins are expected. Conventional collagenases are one of them, but the discovery of proteolytic enzymes having characteristics with different specificities is expected.

Problem to be Solved by the Invention The present inventors have isolated a large number of microorganisms in order to obtain a strain capable of producing a novel protease when cultured under aerobic conditions, and obtained a cortical protein produced therefrom. As a result of examining the activity against, a strain belonging to the genus Chitofaga, the product of which has not been sufficiently examined so far, is compared with a denatured collagen substrate (azocol), which is a substrate for degradation of denatured collagen and / or ordinary protein degradation. To produce a proteolytic enzyme exhibiting remarkably strong activity (10 to 100 times the conventional literature value). Accordingly, an object of the present invention is to provide this novel protease and a method for producing the same.

Means for Solving the Problems The present invention relates to a novel proteolytic enzyme having remarkable activity on denatured collagen (azocol) obtained by culturing a production bacterium belonging to the genus Chitofaga, and a method for producing the same.

The microorganism used in the present invention is a collagen-degrading enzyme-producing bacterium belonging to the genus Citofaga, and a strain having a particularly high effect in the present invention is, for example, the Litrum L43-1.
This strain was deposited on August 22, 1989 with the Research Institute of Microbial Industry and Technology of the Ministry of International Trade and Industry under the accession number No. 10972 of the microtechnical laboratory.

This strain was isolated from the soil of Chiba Prefecture and its bacteriological characteristics are as follows.

1) Form A bacillus with a length of 2-3 μm and a width of about 0.5 μm. No flagella and spores.

2) Growing state Propagating on an ordinary agar medium, the colony is 1-2 mm in diameter,
Produces a translucent, glossy, convex, water-insoluble yellow pigment. Does not grow anaerobically on aerobic bacteria. The optimal growth temperature is 25
℃ -30 ℃, does not grow at 37 ℃. Growth requires peptones and amino acids.

3) Gram stain (-) 4) Physiological properties Glucose produces acid oxidatively. Urease (-), indole production (-), reduction of nitrate (-),
Gelatin liquefaction (+), oxidase (+), catalase (+) 5) Analysis of quinone Ubiquinone: There is no ubiquinone from Q-6 to Q-10.

Menaquinone: MK-6 as a main component and MK-7 as a subcomponent.

6) G + C content of DNA 33.07% 7) Cell fatty acids There are many branched fatty acids.

From the above properties, it was confirmed that this bacterium belongs to the genus Chitofaga.

In addition to the above-mentioned strains, any other strains or mutants thereof that exhibit the ability to produce the novel protease can be used for the enzyme production of the present invention.

This novel proteolytic enzyme is produced by growing the above-mentioned production strain under suitable aerobic culture conditions, for example, by a shaking culture method or an aeration stirring culture method.

The medium contains a nitrogen source and inorganic salts that can be taken by the production strain. Usually, it is desirable that the producing bacteria be cultured at 25 ° C to 30 ° C. All nitrogen sources used for bacterial growth are available to produce this enzyme. For example, peptone, meat extract, yeast, yeast extract, legume powder, corn steep liquor, rice bran, fu, gelatin, fish meal, meat meal, inorganic nitrogen and the like can be used. still,
A medium containing 0.1% to 0.4% gelatin is suitable for the production of collagenase. The cultivation is performed by inoculating a suitable medium that produces a large amount of the present protease, and the medium is preferably neutral.

Extraction and separation of the novel proteolytic enzyme can be performed by appropriately combining means commonly used to extract and separate products of microorganisms. For example, salting out method with ammonium sulfate etc., precipitation method with organic solvents such as acetone, methanol, isopropanol, adsorption and elution method with various adsorbents, adsorption and elution with ion exchanger, chromatography, gel filtration method with various carriers, various Electrophoresis method,
Ultrafiltration, lyophilization, dialysis, chromatofocusing, hydrophobic chromatography and the like. By combining and repeating these means, a novel protease can be isolated.

More specifically, the culture supernatant is filtered out, the solution is concentrated, and ammonium sulfate (ammonium sulfate) is added to collect a fraction which precipitates at 30-70% saturation. The precipitate is dialyzed against Tris buffer and applied to a column of DEAE Sepharose Cl-6B to collect a non-adsorbed fraction. This fraction is dialyzed against a phosphate buffer and adsorbed on a strong cation exchange resin (for example, Mono S HR5 / 5: Pharmacia) column. This column was extracted with a NaCl concentration gradient system by adding 0.5 M NaCl to the same buffer as the phosphate buffer used for dialysis, and the component eluted near the 0.1 M NaCl concentration was the enzyme of interest of the present invention. is there.

The physicochemical and enzymatic properties of the novel protease of the present invention are shown below.

1) Action: Shows remarkably high activity against the hydrolysis of denatured collagen (azocol: denatured collagen sample: manufactured by Sigma). In addition, it shows hydrolytic activity for casein, but hardly acts on synthetic substrates (Cbz-Gly-Pro-Leu-Gly-Pro) and insoluble collagen. Where Cb
z represents a carboxybenzoyl group.

2) Optimum temperature: around 35 ° C 3) Optimum pH: 7-8 (Tris / HCl buffer 1mM C
a ²⁺ ).

4) Thermal stability: stable at least below 37 ° C (1 mM Ca ²⁺ )
It is stable at 0 to 4 ° C (pH 7.5) for at least 5 days (1
mM Ca ²⁺ ).

5) pH stability: stable at 5 to 9 (1 mM Ca ²⁺ ).

6) Inhibitor: EDTA, Zn ²⁺ .

7) Purification method: This enzyme can be purified to a uniform degree by polyacrylamide electrophoresis by ammonium sulfate fractionation of the culture supernatant, DEAE Sepharose column chromatography, and strong cation exchange resin column chromatography.

8) Molecular weight: about 14,500 (depending on amino acid composition) to about 1
5,700 (Super Rose gel method).

9) Titer measurement method: 50 nM Tris / HCl buffer (pH 7.5, 4m
M CaCl ₂ containing) 0.45 ml of an enzyme solution (0.05 ml) and Azocoll (Sigma: 5 mg) and 10 minutes after the reaction at 30 ° C., 0.5 m
l of 10 mM EDTA and centrifuged.
Measure absorbance with. The titer is defined as one unit of the amount of the enzyme that changes _A530 by 1.00 per minute.

10) Isoelectric point: around pI8.3.

11) Ultraviolet absorption spectrum: as shown in FIG.

Absorption maximum (λ _max ): 276.3 nm, A ₂₈₀ / A ₂₆₀ : 1.5, E ^1% _{1 cm} ▼ (280): 101.5 12) Amino acid composition (number of amino acids per enzyme molecule): threonine 12 leucine 7 serine 7 tyrosine 8 glutamic acid 8 phenylalanine 5 glycine 14 bistidine 3 alanine 13 lysine 5 valine 9 arginine 3 half cystine 0 proline 13 methionine 1 tryptophan 5 (A288) Example An example of the present invention will be described below. Various methods can be used to collect the novel protease from the culture solution based on the properties clarified by the above-mentioned methods, and the present invention is not limited to the following examples.

Example 1 In the following medium (Table 1), a production strain belonging to the genus Chitofaga was cultured with shaking (110 rpm) at 25 ° C. for 18 hours. After culturing, Table 1 Ingredient weight polypeptone 5g yeast extract, 1g gelatin 2g monopotassium phosphate 0.5g dipotassium phosphate 0.5g magnesium 0.2g ferrous 0.01g manganese sulfate 0.01g sodium chloride 0.01g Calcium chloride sulfate 0.6 g Water 1 (pH before sterilization is 7.0) Remove the cells, concentrate liquid 750 ml by removing liquid 12 and removing those with a molecular weight of about 10,000 or less, and add ammonium sulfate to this to 30-70% The fractions that precipitate upon saturation are collected. Dissolve this and add 10 mM Tris / HCl buffer (pH 7.5) / 4 mM CaCl
_{2 was} dialyzed and applied to a column of DEAE Sepharose Cl-6B to collect non-adsorbed, passing-through fractions. Next, the collected fraction was subjected to 10 mM phosphate buffer (pH 6.5) / 1 mM C
Mono S HR5 / 5 dialyzed against aCl ₂ /10% glycerol
It was adsorbed on a column (Pharmacia). Elution was performed with the same buffer and a buffer obtained by adding 0.5 M NaCl to the same buffer, and an active fraction of the present enzyme eluted at around 0.1 M was collected. This fraction gave a single band by polyacrylamide gel (7.5%) electrophoresis. This fraction was further dialyzed against 25 mM Tris / HCl buffer (pH 7.5) / 2 mM CaCl ₂ and subjected to hydrophobic chromatography using phenyl superrose HR5 / 5. A fraction eluted as a single peak at around 0.75M was obtained by developing with a gradient system of 1M to 0M ammonium sulfate. This fraction shows remarkably high activity against azocol (manufactured by Sigma), but hardly acts on insoluble collagen and synthetic substrates (Cbz-Gly-Pro-Leu-Gly-Pro). showed that.

Example 2 Ammonium sulfate was added in the same manner as in Example 1 to collect a fraction which became a precipitate at 30 to 70% saturation. This was dissolved, dialyzed against 10 mM phosphate buffer (pH 6.5) / 1 mM CaCl ₂ /10% glycerol, and adsorbed on a column of CM Agarol Fast Flow. Elution was then carried out with the same buffer and a linear gradient system with 1M NaCl added to the same buffer. An active fraction of a novel protease was obtained around 0.15 M NaCl.

[Brief description of the drawings]

FIG. 1 is a chart showing an ultraviolet absorption spectrum of the novel protease of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A novel proteolytic enzyme derived from a microorganism belonging to the genus Chitofaga and having the following physicochemical properties. (1) Action: Shows remarkable activity in decomposing azochol and acts on casein, but hardly acts on insoluble collagen and synthetic substrates (Cbz-Gly-Pro-Leu-Gly-Pro). (2) Optimum pH: 7 to 8 (3) Optimum temperature: about 35 ° C (4) Molecular weight: about 14,500 to about 15,700 2. A method for producing a protease, comprising culturing a novel protease producing bacterium belonging to the genus Citofaga and having the following physicochemical properties, and collecting the protease from the culture. (1) Action: Shows remarkable activity in decomposing azochol and acts on casein, but hardly acts on insoluble collagen and synthetic substrates (Cbz-Gly-Pro-Leu-Gly-Pro). (2) Optimum pH: 7 to 8 (3) Optimum temperature: about 35 ° C (4) Molecular weight: about 14,500 to about 15,700