JPH0391478A - Production of collagenase - Google Patents

Abstract

PURPOSE:To produce a partially purified product of enzyme by culturing a fungi such as Cytophaga L43-1 strain, belonging to the genus Cytophaga, capable of producing and collecting collagenase from the culture solution. CONSTITUTION:A bacterium such as Cytophaga L43-1 strain (FERM P-10792) belonging to the genus Cytophaga, capable of producing collagenase, is inoculated into a proper medium containing a nitrogen source such as peptone or gelatin and an inorganic salt. Then the bacterium is cultured under a proper aerobic culture condition by shaking culture or aerated spinner culture. A partially purified product of collagenase is extracted and separated by proper combination of means conventionally used in extraction of separation of product of microorganism. Then partially purified product of collagenase can be further purified by combining the above-mentioned purifying means and the partially purified product can practically used for hydrolysis of collagen.

Description

[Detailed Description of the Invention] [Industrial Application Field] [Prior Art] Collagenase having insoluble collagen degrading activity is widely used as a reagent for cell dispersion and various other biochemical reagents. On the other hand, it is also used to treat illnesses such as burns, pressure sores, hernias caused by decomposition of intervertebral disc collagen, and lower back pain. Up until now, Clostridium histolytecum has been most commonly used to produce collagenase, but it is difficult to culture because it is an anaerobic bacterium, and it is dangerous because it is a pathogenic bacterium. A production method using Streptomyces is known as a production method using aerobic culture to solve these problems.

[Problems to be Solved by the Invention] In order to obtain a strain capable of producing a novel collagenase by culturing it under aerobic conditions, the present inventor isolated a large number of microorganisms, and investigated the collagenase produced by them. The activity was investigated. As a result, it was discovered that a strain belonging to Cytophaga, whose products had not been sufficiently investigated, exhibited high activity, and the production of a new collagen-degrading enzyme was expected. The present invention relates to a method for producing a partially purified preparation of a collagen degrading enzyme using a newly produced bacterium, Cytophaga.

[Means for Solving the Problems] The present invention is a method for culturing production bacteria belonging to Cytophaga.

The microorganism used in the present invention is a collagen-degrading enzyme-producing bacterium belonging to Cytophaga, and a strain that is particularly effective in the present invention is, for example, Cytophaga strain L43-1. This strain was deposited on August 22, 1989, at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under accession number 10972. This strain was isolated from soil in Chiba Prefecture, and its mycological characteristics are as follows.

l) Morphology It is a bacterium with a length of 2 to 3 μs and a width of about 0.5 parts. It does not have flagella or spores.

2) Growth condition Proliferates on normal agar medium, colonies are 1-2++ u in diameter
a. Produces a semi-transparent, glossy, convex, water-insoluble yellow pigment. It is an aerobic bacterium and does not grow anaerobically.

The optimum growth temperature is 25°C to 30°C, and it does not grow at 37°C. Requires peptone and amino acids for growth.

3) Durham staining 0 4>Physiological properties Acid is produced oxidatively from glucose. Urease 0,
Indole production 0, nitrate reduction 0, gelatin liquefaction (→
, Oxidase (→, Catalase (→5) Analysis of quinones Ubiquinone: There are no ubiquinones from Q-6 to Q-10.

Menaquinone: MK-6 is the main component and MK-7 is the subcomponent.

8) G+C content of DNA 33.07% 7) Bacterial fatty acids High in branched fatty acids.

Based on the above characteristics, this bacterium was recognized to belong to Cytophaga.

Bacterial strains other than those mentioned above, including those belonging to Cytophaga that exhibit the ability to produce collagen-degrading enzymes, or mutant strains thereof, can be used.

Collagen-degrading enzymes are produced by growing the above-mentioned production strains under appropriate aerobic culture conditions, such as shaking culture or aerated agitation culture. The medium contains a nitrogen source, inorganic salts, etc. that can be ingested by the production strain. It is generally desirable to culture the producing bacteria at 25°C to 30°C. To produce this enzyme, any nitrogen source used for bacterial growth can be used. For example, peptone, meat extract,
Yeast, yeast extract, legume powder, corn/steve/liquor, rice bran, wheat gluten, gelatin, fish meal, meat meal, inorganic nitrogen, and the like can be used. Note that a medium containing 0.1 to 0.4% gelatin is suitable for producing collagen degrading enzyme.

Cultivation is carried out by inoculating and culturing in an appropriate medium that increases the production amount of this collagen degrading enzyme, but it is preferable that the medium be around neutrality.

Extractive separation of a partially purified preparation of collagen degrading enzyme can be carried out by appropriately combining means commonly used for extracting and separating products of microorganisms. For example, salting out with ammonium sulfate, precipitation with organic solvents such as acetone, methanol, isopropanol, adsorption with various adsorbents, elution, adsorption with ion exchangers, elution, chromatography, and gel filtration with various carriers. , various electrophoresis methods, ultrafiltration methods, freeze-drying methods, dialysis methods, chromatofocusing, hydrophobic chromatography, etc.

The partially purified sample obtained in the present invention can be further purified by combining the above-mentioned purification means, and the partially purified sample of the present enzyme can be practically used for collagen degradation.

Incidentally, the activity of collagen degrading enzyme is measured by the following method.

i.e. 50mM) Lis HC, Q buffer (pH 7,
Contains 5.4mM CaCρ2) 0.9ml, enzyme solution 0.1ml and bovine Achilles key collagen 10 x 3
React at 0°C for 60 minutes. 0.1 m of 1.0 ml after reaction
Add M acetic acid, centrifuge and collect the supernatant. Part 0.1
ml is colored with ninhydrin and measured at a wavelength of 57 Qnm. The activity is expressed as one unit, which is the amount of enzyme that gives the color development of 1 μM leucine equivalent in 1 minute.

Also, 50mM) Li-HC, Q buffery-(pH 7,
Add enzyme solution to 1% bovine Achilles key collagen (containing 5°C, 4mM Ca C, Q 2 ) and leave it at 25°C.
The degree of collagen digestion was observed.

Next, the method for producing a partially purified preparation of collagen degrading enzyme of the present invention will be explained in more detail with reference to Examples.

[Example] A production strain belonging to Cytophaga was cultured with shaking at 25°C for 18 hours in a medium (Table 1) consisting of the following components (11
0rpn+). After culturing, the bacterial cells were removed, and the furnace liquid 12ρ was ultrafiltered to remove those with a molecular weight of about 10,000 or less and concentrated to 75%.
Ammonium sulfate was added to the solution, and the precipitate was collected at 30 to 70% saturation to obtain a partially purified sample of the enzyme. The total protein amount, total activity, specific activity, and recovery rate are shown in Table 2 below. The specific activity increased by about 3.7 times, and the recovery rate was 86.3%. When this fraction was applied to a DEAE Sepharose column, a non-adsorbed fraction and several adsorbed fractions were obtained.

No. table Polypeptone yeast extract Zen La Chin monopotassium phosphate dipotassium phosphate magnesium sulfate ferrous sulfate manganese sulfate sodium chloride calcium chloride water g g g 0.5g 0.5g 012g 0.01g 0.01g 0.01g 0.6g% = 1ρ (Pl-4tx 7.0 for the reception)

Claims (1)

[Claims] A method for producing a collagen-degrading enzyme preparation, which comprises culturing collagen-degrading enzyme-producing bacteria belonging to Cytophaga and collecting a partially purified collagen-degrading enzyme preparation from the culture solution.