JPS62104579A - Production of hyaluronidase - Google Patents

Abstract

PURPOSE:A specific microorganism in Streptococcus is cultured in a medium to inexpensively produce, in an industrial scale, hyaluronidase which is used in cosmetics, medicines, reagents for research, and so on. CONSTITUTION:A microorganism in group C of Streptococcus, such as Streptococcus dysgalactiae is cultured in a medium containing carbon and nitrogen sources, 1-4wt% of Tod-Hewitt broth, 0.01-0.1wt% of hyaluronic acid and so on, at 6-7pH and 25-35 deg.C for 1-2 days under weak aeration to accumulate hyaluronidase in the medium. The culture mixture is filtered and subjected to separation treatment to collect purified hyaluronidase which has a molecular weight of about 80,000, specifically acts on hyaluronic acid, but not other polysaccharides and heparin, optimally acts at a pH of 6-7, temperature of 35-45 deg.C and shows temperature stability up to 40 deg.C at 6pH for 15min.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing hyaluronidase (EC4,2,2,1).

More specifically, the present invention is characterized by culturing a microorganism belonging to Streptococcus @ group 6 and having the ability to produce hyaluronidase in a medium, accumulating hyaluronidase in the culture, and collecting the enzyme from the culture. The present invention relates to a method for producing hyaluronidase.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid present in animal tissues and mucus.

Hyaluronic acid is a trisaccharide polymer consisting of N-acetylglucosamine and glucuronic acid, and is a type of mucopolysaccharide with a molecular weight of several million. In living organisms, they form viscous solutions and gels together with proteins to maintain tissue structure, provide lubrication, and also protect against bacterial invasion. Therefore, it can be used in the fields of cosmetics, therapeutic agents, and research reagents.

Conventional technology Hyaluronidase is known to be widely distributed in animal testes, snake venom, bee venom, leeches, several types of pathogenic bacteria, and culture fluids of actinomycetes [Clinical Enzyme Handbook]
Shigeaki Baba et al.) 474-476, (1982), Connective Tissue Research (Connectiv
e Ti5sue Research), 3, 13
5-139 (1975), Japanese Patent Publication No. 46-1195, Japanese Patent Publication No. 54-1479973゜Problems to be Solved by the Invention When hyaluronic acid is hydrolyzed by hyaluronidase, the viscosity is moderately reduced, resulting in moderate water retention and moisturizing properties. It is possible to obtain basic materials for cosmetics that have sexual properties. It is also used as an absorption and diffusion enhancer for drugs in subcutaneous injections and as a research reagent.

Conventionally, as a method for producing hyaluronidase using microorganisms, a method using Streptomyces bacteria (Japanese Patent Publication No. 4
6-1195, JP-A-54-147997) are known. Also, Lancefield (Lancef 1el)
Serological classification according to d) [Berge's Manual of Determinative Batteriology]
y's Manual of Determinati
ve Bacteriol, ) 491, 1974)
A method using group A Streptococcus bacteria is known. In the method using Streptomyces bacteria, culture management is complicated and culture requires a long time. Furthermore, the method using microorganisms of the genus Streptococcus group A is a pathogenic bacterium that causes streptococcal infections in humans, and is difficult to use industrially. Therefore, it is desired to provide a method for industrially producing hyaluronidase using microorganisms that can be easily cultured.

Means for Solving the Problems As a result of repeated research into a method for industrially producing hyaluronidase at low cost, the present inventors have developed a method for producing hyaluronidase based on the serological classification by Lancefield [Bersy's Manual of Determination]. Native Reichteology (Bergey's Manualof)
Determinative Bacteriol,
) 491.1974] was cultured under appropriate conditions, a significant amount of hyaluronidase was accumulated in the culture, and the present invention was completed.

The present invention will be explained in detail below.

The present invention is directed to the production of hyaluronidase, which is characterized by culturing a microorganism belonging to Streptococcus group C and having the ability to produce hyaluronidase in a medium, accumulating hyaluronidase in the culture, and collecting the enzyme from the culture. Provide manufacturing method.

The microorganism used in the present invention may be any strain that belongs to group 0 of the genus Streptococcus and has the ability to produce hyaluronidase. Specifically, Streptococcus dysgalactiae (Streptococcus dysgalactiae)
ococcus dysgalactiae), Streptococcus equisimilis
Examples of microorganisms include microorganisms belonging to the genus Streptococcus equisimilis and Streptococcus equi. Preferably, Streptococcus dysgalactiae ATCC 27957, Streptococcus equisimilis ATCC 9542,
Streptococcus equi NCTC9682 and the like are used.

The mycological properties of the species to which these bacteria belong are described in the following literature.

Bergey's Manual of Determinative Batteriology
of Determinative Bacteriol
ogy) 8th edition (1974), Streptococcus dysgalactiae p, 498, Streptococcus
Equisimilis p. 497, Streptococcus equip. 498 The medium used in the present invention may be either a natural medium or a synthetic medium as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients. I can do it.

Carbon sources include various carbohydrates such as glucose, fructose, yucrose, starch, and molasses, various sugar alcohols such as glycerol, sorbitol, and mannitol, various organic acids such as acetic acid, lactic acid, and citric acid, and various types of methanol and ethanol. Alcohol, various glycols such as ethylene glycol and propylene glycol, various amino acids, or hydrocarbons are used.

Nitrogen sources include ammonia, various inorganic and organic ammonium salts such as ammonium chloride, ammonium phosphate, ammonium acetate, urea, amino acids and other nitrogen compounds, peptone, NZ-amine, meat extract, corn steep liquor, casein hydrolyzate, Nitrogen-containing organic substances such as finmil, defatted soybeans, etc. are used.

Examples of the inorganic substances used include primary potassium phosphate, secondary potassium phosphate, potassium chloride, magnesium sulfate, manganese sulfate, ferrous sulfate, sodium chloride, and calcium carbonate.

Todd Hewit Broth [: Todd Hewi] in the medium
ttf3roth, D7:ll (Difco)
By adding hyaluronic acid, corn steep liquor, etc., a larger amount of hyaluronidase can be produced.

At this time, the amount of each of these additives is 1 to 1 per weight of the medium.
4% (W/ν), 0.01-0.1% (W/V), 0.
Good results can be obtained at 1 to 3.0 w/v%V).

Cultivation was carried out at p ) (6, O ~ 7.0, temperature 25-35 °C).
~2 days with weak aeration stirring (0.1~Q, 5vvm).

100-300 rpm) Perform at low speed.

By culturing in this manner, hyaluronidase is produced and accumulated in the culture, mainly in the culture solution.

Hyaluronidase is collected from the culture as follows.

After culturing, the culture is filtered or centrifuged to remove the bacterial cells and obtain a liquid or supernatant. This P solution or supernatant solution is processed using methods normally used for enzyme purification, such as salting out, organic solvent precipitation, dialysis, ion exchange column chromatography, gel and filtration.
Process using methods such as freeze-drying. Purified hyaluronidase can be collected in this way.

For example, the culture solution is salted out with ammonium sulfate at 70% saturation, the resulting precipitate is dissolved in a buffer solution, and after dialysis,
DEAE-Sephadex (Pharmacia Fine
Chemical Co., Ltd.) and DEAE-Cellulose (Celha Co., Ltd.)
The adsorbed enzyme is adsorbed onto a column packed with an anion exchange resin such as , and the adsorbed enzyme is eluted using a concentration gradient of sodium chloride or ammonium sulfate. The eluted enzyme is further purified by a gel filtration method using Sephadex G-150 (manufactured by Farman Afine Chemicals) or Bio-Gel P-150 (manufactured by Bio-Rad).

The properties of the enzyme obtained in the present invention (hereinafter sometimes referred to as the present enzyme) will be described below using hyaluronidase produced from Streptococcus disgalactiae^TCC27957 as a representative.
Hyaluronidases produced from Streptococcus equisimilis ATCC9542 and Streptococcus equi NCTC9682 have almost similar properties.

(1) Action This enzyme acts on the α,4-3 bond between D-glucuronic acid and N-acetylglucosamine of hyaluronic acid, and catalyzes the reaction that liberates N-acetylglucosamine as a reducing end.

(2) Substrate specificity This enzyme acts specifically on hyaluronic acid and other polysaccharides,
It has almost no effect on chondroitin sulfate, heparin, chitin, etc. (see Table 1).

Table 1 Hyaluronic acid 100 Chondroitin sulfate AO Chondroitin sulfate CO, 9 Heparin
0 chitin (ethylene glycolization) 0 The Km value for hyaluronic acid was approximately 0.1 mg/ml.

(3) Optimum pH The optimum p)l of this enzyme is determined by the reaction at 37°C for 10 minutes.
It is around H6.0 to 7.0.

(4) Stable pH range The stable pH range of this enzyme is determined by treatment at 4°C for 24 hours.
It is between 5.0 and 8.0.

(5) Measurement method of titer The enzymatic activity of hyaluronidase is measured by measuring the number of N-acetylglucosamine residues produced by the enzymatic reaction.
gan-Elso oral method [Journal of Biological Chemistry (J, Biol.

[hem, ), 2nd, 959.1952].

(a) Reagent ■ Substrate 1 mg/ml hyaluronic acid aqueous solution Q, 5 ml ■ Buffer: 0. IM citric acid-0.2M disodium phosphate buffer (p, 86.0) Q, 2ml ■ Enzyme solution Q, 1ml ■ Borate solution + Dissolve 4.95 g of boric acid in 50 ml of water, After adjusting the pH to 9.1 with INN sicium hydroxide, the total volume was adjusted to 10 Qml, and 1 ml of p-dimethylaminobenzaldehyde (DMAB>Reagent: 10 g of DMAB was added to rice acetic acid containing 12.5% of 1ON hydrochloric acid. 10 Qml (b) Procedure Place the above reagents ① and ② in a test tube and preheat at 37°C for 5 minutes.Next, add the enzyme solution ② and dilute the entire solution with water. Adjust to Qml and shake at 37°C for 10 minutes to react. Stop the reaction by heating at 100°C for 3 minutes. Then add ■, cover with a glass bulb, and incubate in a boiling bath for exactly 3 minutes. Heat for 1 minute. Then immediately cool with cold water, add ① and leave at 37°C for 20 minutes.

After cooling to room temperature with cold water, absorbance at 585 nm is measured (ODtest). On the other hand, as a control, the same procedure as above is carried out using water instead of the enzyme, and the absorbance (ODblank) is measured.

(c) Calculation method of titer One unit of hyaluronidase is 1 μmo per minute at 37°C.
It is defined as the amount of enzyme that releases the N-acetylglucosamine residue of le.

The desired enzyme solution titer is determined by how many μmol the obtained 0Dtest-ODblank value is from the calibration curve prepared separately using N-acetylglucosamine.
Calculate whether or not you have a real shot at e.

(6) Range of suitable temperature for action The optimum temperature for this enzyme is around 35 to 45°C in a reaction at pH 6, 0, and for 10 minutes.

(7) Temperature stability This enzyme is stable up to 40°C when treated at pH 6.0 for 15 minutes, and is inactivated by about 60% at 45°C.

(8) Inhibition When this enzyme is reacted at 37°C and pH 6.0, S
inhibited by metal ions such as H inhibitors, silver, and mercury.

(9) Molecular Weight The molecular weight of this enzyme was calculated to be approximately 80.0 (IQ) by Sephadex G150 gel p-filtration method.

αO crystal structure and elemental analysis values were not measured because the enzyme was not crystallized.

The present invention will be specifically explained below with reference to Examples.

Example 1 Streptococcus dysgalactiae ATCC27
957wo) Lado Hevit Broth [Difco (Dif
Co., Ltd. 33g/d, Corn Steep Liquor 3
Inoculate 21 Erlenmeyer flasks containing 500 ml of medium (pH 6,8) consisting of g/J and incubate at 28°C for 7
Cultured with shaking for hours.

Transfer 50 Qml of this culture solution to 1 medium of the same composition as the above medium.
The cells were placed in a 30I2 jar fermenter containing 8I2, and cultured at 28°C for 16 hours with weak aeration and stirring (0.2vvm, 125rpm).

This culture is 18! was filtered through a large Nutsche filter using Radiolite #100 (manufactured by Showa Kagaku Kogyo Co., Ltd.) as a filter aid.
Culture liquid approx. 187! I got it.

Ammonium sulfate was added to these two culture solutions, and the portion that precipitated at 70% ammonium sulfate saturation was collected.

The yield of hyaluronidase activity in this precipitate was about 90%, and the specific activity was increased about 15 times.

This precipitate was dissolved in about 10 Qml of 0.01 MIJ acid buffer (pH 6.0).

Add this solution to the same buffer 5! Dialysis was performed for 24 hours.

The dialysate was passed through a DEAE-Sephadex column (11, diameter 6 cm) equilibrated with the same buffer.

In this operation, hyaluronidase is adsorbed onto DEAE-Sephadex. Further, impure proteins were washed away with about 1 ρ of the same buffer.

Next, from 0.01M phosphate buffer (pH 6,0), 1.
Elution was performed with a concentration gradient of sodium chloride up to the same buffer containing 0M IJ chloride at a flow rate of about 1 ml/min. The active fractions of about 20 Qml eluted were fractionated in about 20 ml portions, and ammonium sulfate was added to this, and the part that precipitated at 70% ammonium sulfate saturation was collected by centrifugation (12,000×g, 20 minutes). It was dissolved in 10 ml of 01M phosphate buffer (pH 6,0).

This solution was dialyzed against 5p of the same buffer for 24 hours.

After dialysis, the enzyme solution was equilibrated with the same buffer using a Sephadex G-150 column (500 ml, diameter 3.5 cm).
).

The eluate was collected in fractions of approximately 20 ml, and approximately 100 ml of fractions with high specific activity were collected and lyophilized to produce 40 mg of a powdered purified enzyme preparation of hyaluronidase (specific activity 10 units/m2).
g) was obtained.

The specific activity of the purified enzyme was about 200 times higher than that of the cell solution, and the yield of activity was about 60%.

Example 2 The strain used was Streptococcus equisimilis ATC.
C9542 was replaced with a fermentation medium consisting of Todd Hebisoto broth 3g/a and hyaluronic acid 0.1g/J (
The same procedure as in Example 1 was carried out except that pH 6, 8) was used. As a result, about 3 On+g of purified hyaluronidase with a specific activity of 9 fluorescence/mg was obtained. The yield of activity was approximately 40%.

Example 3 The strain used was Streptococcus equi NCTC968.
Example 1 was carried out in the same manner as in Example 1 except that Example 2 was replaced with Example 2.

As a result, about 30 mg of purified hyaluronidase with a specific activity of 8 units/mg was obtained. The yield of activity was approximately 40%.

Effects of the Invention According to the present invention, hyaluronidase can be produced industrially at low cost.

Claims (2)

[Claims] (1) Production of hyaluronidase, which is characterized by culturing a microorganism belonging to Streptococcus group C and having the ability to produce hyaluronidase in a medium, accumulating hyaluronidase in the culture, and collecting the enzyme from the culture. Law. (2) The method according to claim 1, wherein the microorganism belongs to Streptococcus dysgalactiae, Streptococcus equisimilis, or Streptococcus equi.