JPH044868B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel Streptococcus zooepidemicus that has the ability to produce hyaluronic acid, does not produce hyaluronidase, and is non-hemolytic. More specifically, the present invention relates to a mutant strain of Streptococcus zooepidemicus that does not contain hemolysin and has the ability to efficiently produce high molecular weight hyaluronic acid. Conventional technology Today, hyaluronic acid is recognized to exist in all parts of the connective tissue of animals, and is industrially obtained from biological tissues such as chicken crests and umbilical cords by extraction methods. It retains water between cells, forms a jelly-like matrix within tissues to retain cells, controls material transfer between cells, and prevents physical shock or infection from outside by bacteria. are listed. Taking advantage of these functions, hyaluronic acid is used in medicines (arthritis treatments, eye drops, wound healing agents, etc.), cosmetics, and the like. However, the production of hyaluronic acid by extraction from living tissues is difficult and extremely expensive to mass produce due to the complexity of separation and purification. This has closed off the path to developing new uses for hyaluronic acid. Regarding the production of hyaluronic acid by microorganisms, bacteria of the genus Streptococcus, type A, C, and D of the Lancefield serogroup, such as Streptococcus pyogenes, Streptococcus zooepidemicus ( Streptococcus
zooepidemicus), Streptococcus equi, Streptococcus equisimilis, Streptococcus dysgalactiae and Streptococcus faecalis var. zymogenes) and Pasteurella multocida (Pasteurella
multocida) etc., are already known to produce hyaluronic acid; for example, Kendall et al. (FE
Kendall et al., J.Biol.Chem., 118, 61,
1937), WAPierce et al., J.
Baact., 63, 301, 1952), Pine Crenan (AP
MacLennan, J. Gen. Microbiol., 14, 134-142,
1956; J.Gen.Microbiol., 15, 485-491, 1956),
B. Holmström et al.
Appl.Microbiol., 15, 1409-1413, 1967), JBWoolcock, J.Gen.Microbiol.
85, 372-375, 1974), E. Kjems et al.
al., Acta Path.Microbiol.Scand.Sect.B, 84,
162-164, 1976), T. Bergan et al.
al., Acta Path.Microbiol.Scand., 75, 97−
103, 1969) and JACifonelli,
Carbohyd. Res., 14, 272-276, 1970). These reports were not aimed at mass production of hyaluronic acid, but were cultured using 1-1.5% glucose as a carbon source, and the amount of hyaluronic acid produced was 0.5-0.6g/
The yield based on sugar was 6% or less. In the above report, Matsukrennan reported that culturing a type of Lancefield serogroup C bacterium of the genus Streptococcus under aerobic conditions may promote the production of hyaluronic acid. Among the above-mentioned microorganisms that produce hyaluronic acid, Lancefield serogroup A bacteria of the genus Streptococcus and Pasteurella are known as pathogenic bacteria to humans, and are actually unsuitable for mass culture. JP-A-58-56692 discloses a method for industrially culturing hyaluronic acid-producing bacteria of the genus Streptococcus and extracting and purifying hyaluronic acid from the culture solution. This method is a method to obtain a large amount of hyaluronic acid by culturing Lancefield serogroup A and C bacteria of the genus Streptococcus.
g/g of hyaluronic acid is obtained. In this case, the yield of hyaluronic acid based on sugar was 5%, and remained unchanged even when the amount of glucose added was changed from 1% to 8%. Therefore, this sugar yield (5%) is almost the same as the sugar yield of hyaluronic acid producing bacteria in previous reports. In addition, there is a method for obtaining hyaluronic acid using Streptococcus bacteria.
JP-A-60-500597, JP-A-60-133894, JP-A-61
-15698, but there are problems such as the hyaluronic acid obtained has a low molecular weight and the yield is low. Furthermore, it is known that the hyaluronic acid-producing bacterial strains in both cases produce streptolysin (soluble hemolysin) and exhibit β-hemolytic properties. When attempting to produce hyaluronic acid by culturing a large amount of such bacteria, there is a risk that the hemolysin may be mixed into the hyaluronic acid product, and it is not preferable to incorporate such hyaluronic acid into cosmetics or pharmaceuticals. In order to improve this drawback, a method for obtaining hyaluronic acid by culturing a hyaluronic acid-producing strain that lacks the ability to produce streptolysin through mutation treatment with a chemical mutagen was proposed in Japanese Patent Application Laid-Open No. 60-251898.
has been disclosed. This contains 6 glucose
It is stated that 3.6g/of hyaluronic acid was obtained by adding 3.6% of hyaluronic acid, and the yield of hyaluronic acid based on sugar was 6%, which is still low productivity from the viewpoint of sugar yield. It is something. Problems to be Solved by the Invention In view of the above problems, as a result of intensive research aimed at creating a microorganism that does not produce hemolysin (streptolysin) and has a high ability to produce hyaluronic acid, the present invention has been isolated from nature. By mutating a strain that no longer exhibits hemolytic properties obtained through mutation from Streptococcus zooepidemicus that has the ability to produce hyaluronic acid, the strain loses the ability to produce hyaluronidase and produces high-molecular-weight hyaluronic acid. We succeeded in obtaining a new strain with extremely high virulence. The present invention was completed based on this knowledge, and therefore provides a mutant strain of Streptococcus zooepidemicus that does not contain hemolysin and has the ability to efficiently produce high-molecular-weight hyaluronic acid. It is something to do. Means for Solving the Problems (1) Obtaining a Mutant Strain In order to achieve the object of the present invention, the present inventors obtained a new mutant strain by the following method. First, Streptococcus zooepidemicus belongs to Lancefield serogroup C, which has a strong ability to produce hyaluronidase (hyaluronic acid degrading enzyme) and produces hyaluronic acid from bovine nasal mucosa.
of Determinative Bacteriology, 8th edition, 1974). This strain is known as MacLennan (MacLennan, J.Gen.
Microbiol., 14, 134-142, 1956) pointed out that when hyaluronic acid is produced well under aerobic conditions and glucose is used as the carbon source,
By adding 4% glucose, 2 g/hyaluronic acid was produced (yield of hyaluronic acid based on sugar was 5%). The molecular weight of the hyaluronic acid obtained at this time was 300,000 to 600,000. This strain was treated with ultraviolet rays and chemical agents (N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethylmethane, sulfonic acid, etc.), the treated bacterial cells are mixed and spread on blood agar medium, and colonies that do not show hemolytic properties are collected.The bacterial cells are then mutated again, and then treated with hyaluronic acid. Apply nutrient agar containing nutrient agar to the ground,
A mutant strain of Streptococcus zooepidemicus was obtained by collecting colonies that did not degrade hyaluronic acid (see Example 1 below). (2) Mycological properties Mutant strain of Streptococcus zooepidemicus obtained in Example 1 (hereinafter referred to as the main strain)
Todd Huyt Broth
Hewitt broth) forms transparent colonies with extremely strong viscosity on the agar plate, and is non-hemolytic (β-
Hemolytic: negative), non-production of hyaluronidase,
This bacterium is a streptococcus belonging to Lancefield serogroup C, and its bacteriological properties are as follows. (a) Gram staining: positive (b) 10℃ growth: negative (c) 45℃ growth: negative (d) 0.1% methylene blue resistance: negative (e) 6.5% salt resistance: negative (f) 40 % Bile resistance: Negative (g) Bacitracin resistance: Positive (h) PH9.6 resistance: Negative (i) 60℃, 30 minutes resistance: Negative (j) Gelatin decomposition: Negative (k) Starch decomposition :Positive (l) Sodium hippuric acid degradability: Negative (m) Aesculin degradability: Weakly positive (n) Arginine degradability: Positive (o) Sugar fermentability: Glucose, galactose,
seuucrose, lactose, maltose,
Positive for sorbitol and salicin, negative for glycerin, mannitol, trehalose and arabinose. Based on the mycological properties of this bacterium, the present inventors identified this bacterium as Streptococcus zooepidemicus.
It was named YIT2030 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Article No. 1305. (3) Production of hyaluronic acid using this bacterium The medium for culturing this bacterium to produce hyaluronic acid is
It is preferable that it contains a carbon source, an organic and inorganic nitrogen source, an inorganic salt, and other organic micronutrients as necessary. As the carbon source, those containing sugar such as glucose, galactose, sucrose, lactose, fructose, maltose, sorbitol, and starch hydrolyzate are preferable, and organic acids, aliphatic alcohols, and the like may also be used. As the nitrogen source, common organic and inorganic materials may be used, and various meat extracts, amino acid mixtures, peptone, yeast extract, etc. are preferable. Furthermore, chlorides, sulfates, phosphates, nitrates, carbonates, vitamins, etc. of sodium, potassium, calcium, magnesium, iron, etc. may be added as necessary. Aerobic conditions are essential for culturing, and it is better to increase the stirring speed as the viscosity of the culture solution increases, but excessive stirring is not preferred. The culture temperature is generally 25-38°C, which is the temperature at which bacteria grow. Furthermore, during culturing, this bacterium produces lactic acid, which inhibits the growth of the bacterium and the production of hyaluronic acid. Therefore, an alkaline aqueous solution is added to neutralize the lactic acid to maintain a pH within the range of 6-8. It is necessary to adjust within The alkaline aqueous solution used at this time may be an aqueous solution of sodium hydroxide, potassium hydroxide, or aqueous ammonia. This bacterium contains high molecular weight hyaluronic acid (molecular weight 200
-3 million) at an extremely high yield and production rate, and particularly good results are obtained when glucose is used as the carbon source. Added amount of sugar 3
% or less, the sugar yield was 14-15%, and if the amount added was more than that, the sugar yield tended to decrease slightly. When the addition of sugar is 6% (see Reference Example 1), the viscosity of the culture solution becomes 8000 centipoise (cp) at 36°C, and the culture solution becomes almost non-fluid, reaching the limit of culture without any effect even if the stirring speed is increased. Summer. Table 1 shows the amount of hyaluronic acid produced when culturing was performed with varying amounts of glucose added in the medium. That is, as a result of purifying the produced hyaluronic acid using a conventional method, the sugar yield was 15 when 1% glucose was added.
%, 1.5g/, 11% sugar yield when adding 6%
6.7 g/hyaluronic acid was obtained.

[Table] Next, by using a medium with a composition of 2% glucose and performing continuous culture at a dilution rate of 0.3 (hr ^-1 ), it is possible to achieve high yields and production rates continuously with a low viscosity that is easy to operate, and extremely stably. We were able to produce hyaluronic acid. The yield of hyaluronic acid based on sugar is 15%, and its productivity is 0.9g//hr, which is
It was possible to produce 21.6 g/hyaluronic acid (see Reference Example 2). As this bacterium is a polymeric substance, substances other than hyaluronic acid do not accumulate in the culture solution, so after culturing,
Separation and purification of hyaluronic acid accumulated in the culture solution is easy, and a known separation and purification method for polysaccharides may be used. An example of a method for separating and purifying hyaluronic acid is shown.
Dilute the culture solution with water to an appropriate viscosity (preferably 100 centibore or less), and adjust the pH to 4 or less with trichloroacetic acid. Then centrifugation or membrane filtration (pore size 0.2μm or less)
The bacterial cells are separated and removed. Next, the low molecular weight substances dissolved in the solution are removed by ultrafiltration, dialysis,
After removal by organic solvent precipitation, adsorption using an ion exchange resin, etc., hyaluronic acid can be obtained using methods such as organic solvent precipitation, freeze drying, or spray drying (see Reference Example 1). As a result of performing various studies on the hyaluronic acid obtained by extraction and purification from the above culture solution and comparing it with a hyaluronic acid standard (manufactured by Sigma), it was confirmed that this product is hyaluronic acid. . Its properties are shown below. (1) Shows the same mobility as the standard sample in electrophoresis using cellulose acetate membrane. (2) When it is degraded by actinomycete hyaluronidase (manufactured by Amano Pharmaceutical) and the degraded product is subjected to silica gel thin layer chromatography, two spots appear with the same mobility as the standard degraded product after treatment. (3) Analysis of the chemical composition reveals that N-acetyl-D-
Glucosamine and D-glucuronic acid in a molar ratio of 1:
1 exists. (4) The specific illuminance is [α] ²⁰ _D = -69°. (5) The infrared absorption spectrum measured by the thin film method is shown in Figure 3 and is the same as the standard product. (6) The ¹³ C-NMR spectrum measured after dissolving in heavy water is shown in Figure 4 and is the same as the standard. (7) Molecular weight is determined by viscosity measurement method (TCLaurent et al.,
Biochim. Biophys. Acta, 42, 476-485, 1960)
The result was 2-3 million. The present invention will be explained in more detail by showing Examples and Reference Examples below. Example 1 Streptococcus zooepidemicus, which exhibits β-hemolytic properties, produces hyaluronidase, and produces hyaluronic acid, collected from bovine nasal mucosa, was incubated at 37°C for 10 hours in Todd Huitt broth medium (manufactured by Difco). After culturing, the cells in the logarithmic growth phase were collected by centrifugation, and washed twice with 0.05M tris-maleate buffer (PH6.0) while repeating centrifugation at low temperature. ×
Suspend in the same buffer solution to a bacterial concentration of 10 ⁸ /ml,
Add NTG to this at 200μg/ml and hold at 37°C.
The mixture was shaken for 30 minutes. Next, the bacterial cells at low temperature were
2 with 0.05M Tris-maleate buffer (PH6.0)
After washing twice, the cells were inoculated into Todd Huyt's broth medium and cultured at 37°C for 18 hours. This culture solution was diluted with sterile physiological saline to a concentration of 1 x 10 ³ /ml, and 0.1 ml of the solution was poured onto blood (definated rabbit blood) agar (20 ml) and cultured. We sampled villages where there were no. The acquisition frequency of this mutant strain is approximately 4
It was ×10 ^-6 . Next, this non-hematopoietic strain was transferred to Totsudo Huyt Broth medium as above.
Culture at 37℃, collect cells in logarithmic growth phase, and add 0.05M
After washing with tris-maleate buffer (PH6.0),
The mixture was shaken at 37°C for 20 minutes in the same buffer containing 200 μg/ml of NTG. Next, after washing the microbial cells at low temperature with the same buffer solution, the ultrafiltration medium (Totdo Huyts Broth medium to ultrafiltration membrane YM made by Amicon) was used.
-10 to remove the polymer fraction).
Culture was carried out at 37°C for 18 hours. This culture solution was diluted with sterile physiological saline to a concentration of 1-5 x 10 ² /ml, and 0.1 ml of the solution was applied onto the ultrafiltrated medium agar (high purity agar) containing 0.1% sodium hyaluronate. do
Culture the bacteria in a moisture chamber at 37℃ for 20-40 hours, collect the grown bacteria using the replica method, and spray a 10% cetylpyridinium chloride aqueous solution onto the agar. We obtained a non-hyaluronidase-producing mutant strain Streptococcus zooepidemicus YIT2030, which forms colonies with cloudy surroundings, from among ten thousand bacterial strains. The above method for identifying hyaluronidase-producing and non-producing bacteria is based on the method of Erika Balke et al.
al., Zbl.Bakt.Hyg.A, 259, 194-200, 1985)
I did this by modifying. Reference example 1 Batch culture Glucose 6%, Polypeptone (Daigo Nutrient Chemical) 1.5%, Baker's yeast extract (Oriental Yeast Co., Ltd.) 0.5%, Potassium phosphate 0.2%, Magnesium sulfate heptahydrate 0.1%, Calcium chloride 0.005 % Adekanol LG-109 (antifoaming agent manufactured by Asahi Denka Kogyo) 0.001
% composition of medium (PH7.0) in 10 jars, and after sterilization, 1 portion of pre-cultured Streptococcus zooepidemicus YIT2030 was added.
% inoculation, and cultured with aeration and stirring at 37° C. for 42 hours while continuously controlling the culture pH to 7 with a 6N-sodium hydroxide aqueous solution. Glucose was sterilized separately and added all at once at the start of culture. The progress of the culture at this time is shown in FIG. As the culture progresses, hyaluronic acid accumulates, and after 29 hours of culture, the viscosity of the culture solution reaches nearly 8,000 centipoise, and it has almost no fluidity. After 42 hours of culture, when the glucose in the culture solution reaches zero, the culture is stopped. finished. Since the harvested culture solution had no fluidity, it was diluted with water to a viscosity of 100 centipoise or less. Next, PH this solution with trichloroacetic acid.
4 or less, pass through a hollow fiber microfilter module (PW-103 manufactured by Asahi Kasei) to remove bacterial cells and insoluble components, and then pass the filtrated liquid through a hollow fiber ultrafiltration membrane (HIP30-43 manufactured by Amicon). The low molecular weight substances in the solution were removed by adding water to the solution. This solution was then dried by freeze-drying to obtain 6.7 g of hyaluronic acid per culture solution. Reference example 2 Continuous culture Reference example 1 except that the glucose concentration was 2.5%
Pour 5 media of the same composition into 10 jar fermenters, and after sterilization (glucose is sterilized separately),
Inoculate 1% of the pre-cultured Streptococcus zooepidemicus YIT2030 and grow at 37°C while adjusting the culture pH to 7 with a 6N-sodium hydroxide aqueous solution.
Culture was carried out with aeration and stirring for 15 hours. Thereafter, a medium having the same composition as in Reference Example 1 except that the glucose concentration was changed to 2% was continuously added at a dilution rate of 0.3 (hr ^-1 ).
Continuous culture with aeration and stirring was performed at 37°C and pH 7 for one week. The progress of the culture at this time is shown in FIG. Collect the culture solution that has flowed out of the culture tank at regular intervals, and use it as reference example 1.
Hyaluronic acid was extracted and purified in the same manner. As a result, the yield of hyaluronic acid based on sugar was 15%, and its productivity was 0.9 g//hr, which was 21.6 g/hr/day.
of hyaluronic acid was obtained. Effects of the Invention By culturing the Streptococcus zooepidemicus mutant strain of the present invention, the yield of Streptococcus zooepidemicus far exceeds that of the previously reported method for producing hyaluronic acid using Streptococcus bacteria. High molecular weight hyaluronic acid without any lysine contamination can be obtained at high yield and production rate at low cost. Hyaluronic acid produced in this manner is optimal as a raw material for cosmetics and pharmaceuticals.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the culture progress for producing hyaluronic acid in Reference Example 1 of the present invention, FIG. 2 is a diagram showing the culture progress in Reference Example 2, and FIGS. 3 and 4 are diagrams showing the culture progress in Reference Example 2. 1 is a diagram showing an infrared absorption spectrum and an NMR spectrum of hyaluronic acid obtained in Example 1.

Claims (1)

[Scope of Claims] 1. Streptococcus zooepidemicus that has hyaluronic acid producing ability, non-hyaluronidase production, and non-hemolysis.
zooepidemicus). 2 Claim 1 showing the following mycological properties
Streptococcus zooepidemicus (a) Gram staining: positive (b) 10℃ growth: negative (c) 45℃ growth: negative (d) 0.1% methylene blue resistance: negative (e) 6.5% Salt resistance: Negative (f) 40% bile resistance: Negative (g) Bacitracin resistance: Positive (h) PH9.6 resistance: Negative (i) 60℃, 30 minutes resistance: Negative (j) Gelatin degradation Degradability of starch: negative (k) Degradability of starch: positive (l) Degradability of sodium hippurate: negative (m) Degradability of esculin: weakly positive (n) Degradability of arginine: positive (o) Sugar fermentability: glucose, galactose, sucrose , lactose, maltose, sorbitol, and salicin; negative for glycerin, mannitol, trehalose, and arabinose. 3 Streptococcus zooepidemicus is Streptococcus zooepidemicus YIT2030
(Feikoken Jokyo No. 1305).
Zooepidemicus.