JPH0746992A - Production of hyaluronic acid - Google Patents

Abstract

PURPOSE:To obtain in high yield hyaluronic acid useful for medicines, etc., easy to isolate and purify, by culturing microbes capable of producing hyaluronic acid in a culture fluid in the presence of amino acids such as arginine without being affected by the variation in the components in natural nutrients. CONSTITUTION:The objective compound, hyaluronic acid, can be obtained by culturing, under aerobic conditions, microbes capable of producing hyaluronic acid, a kind selected from Streptococcus equi and respective mutants thereof FM-100 (FERM P-9027) and FM-300 (FERM P-2319), in a culture fluid incorporated with 8 kinds of amino acids, i.e., arginine, glutamine, serine, glycine, methionine, leucine, isoleucine and lysine.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing medicinal hyaluronic acid by a fermentation method. More specifically, a microorganism capable of producing hyaluronic acid is cultured in a culture medium containing eight kinds of amino acids of arginine, glutamine, serine, glycine, methionine, isoleucine, leucine and lysine, and the medicinal hyaluronic acid is fermented by a fermentation method. Regarding how to get.

[0002]

2. Description of the Related Art Conventionally, the problems to be solved by the invention
Hyaluronic acid was obtained by extraction from the chicken of the chicken, the vitreous of the eye of the cow, and the like. However, the production of hyaluronic acid from the living body by the extraction method separates a large amount of hyaluronic acid from similar mucopolysaccharides and a large amount of contaminants such as proteins, and lowers the molecular weight due to hyaluronidase contained in tissues, etc. There was a problem that the purification was very complicated. In order to improve the problem, a method of culturing a microorganism of the genus Streptococcus, which is a microorganism capable of producing hyaluronic acid, and separating and purifying hyaluronic acid from the culture solution is disclosed. (Patent Fair 4-12
No. 960) Furthermore, in order to stabilize the hyaluronic acid productivity and improve the production yield, a method using a mutant strain (Japanese Patent Publication No. 4-43637).
Japanese Patent Publication No. 4-55675), a method of controlling culture conditions (Japanese Patent Laid-Open No. 3-35788, Japanese Patent Publication No. 4-64679), and a method of adding an active ingredient (Japanese Patent Laid-Open No. 62-289198, Japanese Patent Laid-Open No. 4-198198). -18839) is disclosed.

However, in any of these methods, due to the complexity of the auxotrophy, which is a property of a microorganism capable of producing hyaluronic acid, a mixture of natural organic nutrient sources rich in nutrients and less inhibitory to the growth of microorganisms, For example, it was necessary to add a large amount of yeast extract or polypeptone to the medium.

However, the natural organic nutrient mixture used in the conventional culture method contains various components such as sugars, proteins, amino acids, lipids, nucleic acids, vitamins and metals, among which hyaluronic acid is produced. On the other hand, it contains a lot of unnecessary ingredients. In particular, it is necessary to consider the removal of these unnecessary components in the purification step of hyaluronic acid for pharmaceuticals, and in addition to the difficulty due to the property of hyaluronic acid, the step becomes complicated, and as a result, maintenance of quality such as molecular weight and process Simplification becomes more difficult. Although the burden of the purification process can be reduced by reducing the amount of the natural organic nutrient mixture added, on the other hand, there are problems such as a decrease in the yield of hyaluronic acid due to a decrease in the components required for hyaluronic acid production. It was

As a result of intensive studies to solve the above problems, the present inventors have found that hyaluronic acid is added by adding eight kinds of amino acids, arginine, glutamine, methionine, serine, glycine, isoleucine, leucine and lysine. The amount of natural organic nutrient mixture added can be reduced without lowering the yield, the hyaluronic acid can be stably produced without being affected by the variation of the components in the natural nutrient source, the separation of hyaluronic acid, The inventors have found that the load of the refining process can be reduced, and completed the present invention.

[0006]

That is, the present invention is
(1) By a fermentation method, which comprises culturing a microorganism capable of producing hyaluronic acid by adding 8 kinds of amino acids of arginine, glutamine, serine, glycine, methionine, isoleucine, leucine and lysine to a culture solution Method for producing hyaluronic acid, (2) Microorganism capable of producing hyaluronic acid is a bacterium of the genus Streptococcus (1)
The method for producing hyaluronic acid according to (3), wherein the bacterium belonging to the genus Streptococcus is Streptococcus equi, a mutant strain FM-100 of Streptococcus equi (Microtechnology Research Institute Article 90
27) and a mutant strain FM- of Streptococcus equi
The method for producing hyaluronic acid according to (2), which is one kind selected from 300 (Microtechnical Laboratory Article No. 2319).

The present invention will be specifically described below.
Examples of the microorganism capable of producing hyaluronic acid used in the present invention include Streptococcus
equi), Streptococcus zooepidemicus (Strep
tococcus zooepidemicus), Streptococcus equisimilis, Streptococcus deygalactiae (Streptococcus dysgalactia)
e), Streptococcus p.
yogenes) and these mutants, among others, Streptococcus equi is preferable, and particularly Streptococcus equi mutant strain FM-100 (Mikokukenjojo 9027) or Streptococcus equi mutant strain FM-. 300 (Ministry of Industrial Science and Technology No. 2319) is preferable.

The medium used in the present invention is a carbon source consisting of sugar components such as glucose, fructose, galactose, sucrose, etc., primary potassium phosphate, secondary potassium phosphate, magnesium nitrate, sodium sulfite, sodium thiosulfate, and phosphoric acid. Inorganic salts such as ammonium, polypeptone, casamino acid, yeast extract, corn steep liquor, organic nutrient sources such as soybean hydrolyzate, and various vitamins and the like are preferably used if necessary. At this time, an appropriate amount of eight kinds of amino acids of arginine, glutamine, methionine, serine, glycine, isoleucine, leucine and lysine are added. There is no limit to the amount added, but it is preferable that the sugar component is 1%, since the effect above a certain level is not seen if added in excess.
0.001 to 0.1% each, more preferably 0.0
It is sufficient to add 01 to 0.05%. By adding these amino acids, it becomes possible to significantly reduce the amount of organic nutrient source in the medium that has been conventionally used, and it is possible to reduce the burden of the purification process without lowering the yield.

The culture of the present invention may be carried out by a known method such as stirring culture after aeration, and the culture temperature is preferably 30 to 35 ° C. Since the pH of the culture solution decreases with the growth of the bacterium, a pH adjusting agent such as sodium hydroxide, potassium hydroxide or ammonia is added to control the pH to 6.0 to 9.0. When culturing is performed in this manner, the viscosity of the culture solution gradually increases with the production of hyaluronic acid. Cultivation is stopped when the used carbon source is consumed in the culture solution, and high purity is achieved by a simple known purification method such as activated carbon treatment, sterilization by filtration, precipitation with an organic solvent such as alcohol, and desalting by ultrafiltration. Hyaluronic acid is obtained.

Focusing on amino acids that are essential for hyaluronic acid production as a component contained in a large amount in a mixture of natural organic nutrients, Streptococcus equi was cultivated, and the relationship between the change in concentration in the culture solution and the amount of hyaluronic acid produced was detailed. investigated. When the amino acid concentration in the culture was quantified over time, glutamine, arginine, serine, glycine, methionine, isoleucine, leucine, and lysine were consumed,
Valine, histidine, etc. are almost unchanged, and in addition to asparagine, Streptococcus eki NK
Glutamic acid (Japanese Patent Laid-Open No. 626-289198), which was said to be effective for hyaluronic acid production, was accumulated in -850215. The results are shown in Table 1.

[0011]

[Table 1]

[0012] Further, the amino acids consumed therein were examined in more detail. Generally, bacterial cell growth in culture can be divided into an induction phase, a logarithmic growth phase, a stationary phase, and a death phase. When the specific consumption rate of amino acids (consumption rate per cell) and the specific production rate of hyaluronic acid (production rate per cell) were calculated for each period, the specific production rate of hyaluronic acid was high, that is, hyaluronic acid. Streptococcus equi NK-850 during the stationary phase when production is most active
In 215, arginine (JP-A-62-289198), which was said to be effective for hyaluronic acid production, and glutamine (DSWILL, which was effective for streptococcus belonging to Group A) were used.
OUGHBY et al., 1964. J. Bacteriol. 87.1452-1456), and the specific consumption rate of 6 kinds of amino acids, serine, glycine, methionine, isoleucine, leucine and lysine, is also very high. It was found that these eight kinds of amino acids are essential for hyaluronic acid production.

Next, among the components contained in the mixture of natural organic nutrients, those that are essential for the production of hyaluronic acid, although they are trace amounts, were examined in detail as the minimum required amount of natural organic nutrients. Arginine, glutamine, serine, glycine,
Eight kinds of amino acids of methionine, isoleucine, leucine and lysine were added in sufficient amounts, and yeast extract and polypeptone as natural organic nutrient sources were adjusted to have gradual concentrations, and the yield of hyaluronic acid obtained from the culture solution was adjusted. Were compared. The yield of hyaluronic acid was about 7.5 g / l, which was 0.17% for yeast extract and 0.5% or more for polypeptone as natural organic nutrient sources. However, at lower concentrations, the hyaluronic acid yield decreased due to the restriction of trace metals and vitamins. The results of the relationship between the yeast extract and polypeptone concentration and the yield are shown in Table 2.

[0014]

[Table 2]

Furthermore, even if any one of the eight amino acids of arginine, glutamine, serine, glycine, methionine, isoleucine, leucine and lysine is deficient, the yield of hyaluronic acid is about 7.5 g / l to 4.5 g.
It was found that the amount decreased to 1 / l, and the yield could not be maintained with arginine and glutamine alone, which were conventionally considered to be effective for hyaluronic acid production.

Next, the purification process load was examined in detail by focusing on the degree of coloring of the culture broth derived from a natural organic nutrient source. The coloring of the medium is mainly derived from natural organic nutrient sources such as polypeptone and yeast extract, and the amount thereof directly imposes a burden on the purification process. (A) A medium containing 0.17% yeast extract, 0.5% polypeptone as a natural organic nutrient source, and eight kinds of amino acids of arginine, glutamine, serine, glycine, methionine, isoleucine, leucine and lysine (B) ) The degree of coloring of the culture solutions was compared using a medium containing yeast extract 0.5% and polypeptone 1.5% as an example. After adjusting the pH of the culture broth that was cultivated in each medium to 4 with hydrochloric acid, it was treated with activated charcoal and sterilized by filtration to give a coloring degree of 5
When calculated from the absorbance at 50 nm, the medium (A) was 0.000 and was colorless and transparent, and the medium (B) was 0.03.
It was 5. Since the absorbance was reduced to about 1/3, it was shown that the load on the purification step could be significantly reduced.

[0017]

EXAMPLES The present invention will now be described in more detail by way of examples, but the present invention is not limited thereto.

Example 1 Glucose 5%, dibasic potassium phosphate 0.2%, polypeptone 0.5%, yeast extract 0.17%, arginine 0.2%, glutamine 0.2%, methionine 0.05.
%, Serine 0.05%, glycine 0.05%, isoleucine 0.05%, leucine 0.05%, lysine 0.01%
% Inoculated with a pre-cultured Streptococcus eki FM-100 (Mikoken Kenjoyori No. 9027),
The culture was carried out at aeration of 1 vvm, agitation of 200 revolutions / minute, and a temperature of 33 ° C. while controlling the pH to 8.5 with sodium hydroxide. The culture time at the end was 14 hours.
After adjusting the pH of the culture solution to 4 with hydrochloric acid, dilute it twice with distilled water.
The bacteria were removed by treatment with activated carbon and filtration. Ethyl alcohol is added to the obtained sterilized solution to precipitate sodium hyaluronate. After this was filtered off, it was dissolved in water, cetylpyridinium chloride was added, and the resulting precipitate was collected by filtration and 2%
After redissolving in saline, precipitation with ethyl alcohol is repeated again. The obtained sodium hyaluronate was dried under reduced pressure at room temperature to obtain 7.5 g of white sodium hyaluronate per 1 L of the culture solution. The yield based on sugar was 15.0%. Further, after the pH of the culture solution was adjusted to 4 with hydrochloric acid, the absorbance at 550 nm at the time of treatment with activated carbon was 0.00
It was 0 and was colorless. The results are shown in Table 3.

Example 2 As a microorganism having a hyaluronic acid-producing ability, Streptococcus equi FM-300 (Microtechnical Lab.
No.) was used and the conditions were the same as in Example 1. The results are shown in Table 3.

Comparative Examples 1-8 Glucose 5%, dibasic potassium phosphate 0.2%, polypeptone 0.5%, yeast extract 0.17%, arginine 0.2%, glutamine 0.2%, methionine 0.2. 05
%, Serine 0.05%, glycine 0.05%, isoleucine 0.05%, leucine 0.05%, lysine 0.05%
% Of arginine, glutamine, methionine, serine, glycine, isoleucine, leucine, and lysine inoculated with pre-cultured Streptococcus eki FM-100 (Microtechnical Research Institute No. 9027). , Cultured. After the completion of the culture, the product was purified in the same manner as in Example. As shown in Table 3, the yield was poor in the medium lacking even one kind of each amino acid.

Comparative Example 9 Streptococcus equi FM-100 pre-cultured in a medium consisting of 5% glucose, 0.2% dibasic potassium phosphate, 1.5% polypeptone, 0.5% yeast extract.
Inoculated with (Microtechnology Research Institute No. 9027), aeration of 1vv
m, stirring 200 rev / min, temperature 33 ° C with caustic soda
The culture was performed while controlling H to 8.5. The culture time at the end was 14 hours. PH of the culture solution with hydrochloric acid
After adjusting to 4, the same activated carbon treatment as in Example 1 was carried out 55
The absorbance at 0 nm was measured. Absorbance is 0.035
It was colored with. The results are shown in Table 3.

[0022]

[Table 3]

[0023]

EFFECTS OF THE INVENTION According to the present invention, since it has the following effects, it is extremely useful as an industrial production method of hyaluronic acid. By adding eight amino acids of arginine, glutamine, methionine, serine, glycine, isoleucine, leucine and lysine in an appropriate amount, it is possible to reduce the natural organic nutrient mixture, and a culture method using a large amount of the natural organic nutrient mixture. Similarly, high yields of sodium hyaluronate can be obtained. By reducing the amount of natural organic nutrients, the load on the hyaluronic acid separation and purification process can be greatly reduced. By adding the nutrient source necessary for hyaluronic acid production, the culture yield is stabilized without being affected by the variation in the components in the natural nutrient source, and the management is easy.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Terumi Miyoshi 3-5-1, Asahimachi, Machida-shi, Tokyo Denka Kagaku Kogyo Co., Ltd.

Claims (3)

[Claims] 1. A microorganism capable of producing hyaluronic acid,
A method for producing hyaluronic acid by fermentation, which comprises adding 8 kinds of amino acids of arginine, glutamine, serine, glycine, methionine, isoleucine, leucine and lysine to a culture solution and culturing. 2. The method for producing hyaluronic acid according to claim 1, wherein the microorganism capable of producing hyaluronic acid is a bacterium belonging to the genus Streptococcus. 3. A bacterium belonging to the genus Streptococcus is Streptococcus equi, a mutant strain F of Streptococcus equi.
M-100 (Microtechnology Research Institute No. 9027) and Streptococcus equi mutant FM-300 (Microtechnology Research Laboratories No. 2)
319) and the method for producing hyaluronic acid according to claim 2.