JPH0823992A - Production of hyaluronic acid - Google Patents

Abstract

PURPOSE:To produce a large amount of hyaluronic acid having high molecular weight in high efficiency at a low cost while preventing the intrusion of hemolytic streptolysin S and O by culturing a specific hyaluronic acid-producing bacterial strain and separating the acid from the cultured liquid. CONSTITUTION:A bacterial strain capable of producing hyaluronic acid, free from productivity of chondroitinase, exhibiting anhemolytic property and belonging to the genus Streptococcus [preferably Streptococcus equi NC93O627 (FERM P-14341)] is cultured in a medium and hyaluronic acid produced and accumulated in the cultured liquid is separated from the liquid by conventional method. The culture of Streptococcus equi NC93O627 is preferably carried out at 30-35 deg.C and pH 7-7.5 using glucose as the carbon source. The Streptococcus equi NC930627 can be produced e.g. by mutating Streptococcus equi ATCC 9527 and mutating again the produced strain free from chondroitinase productivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing hyaluronic acid. More specifically, having a hyaluronic acid-producing ability, culturing bacteria belonging to the genus Streptococcus showing chondroitinase non-producing and non-hemolytic properties in a medium,
The present invention relates to a method for efficiently producing high molecular weight hyaluronic acid free of hemolysin.

[0002]

Hyaluronic acid is a mucopolysaccharide present in all tissues of the living body. Hyaluronic acid alone or in the living body forms a complex by binding with proteins or other mucopolysaccharides, and acts as a retention of water in the living body, maintenance of tissue structure, lubrication, defense against bacterial invasion, etc. There is. Utilizing such a function, hyaluronic acid is used for medicines (eye drops, arthritis therapeutic agents, wound healing agents, etc.), cosmetics and the like. Industrially, it is obtained from living tissue such as chicken crowns and rhinoceros by an extraction method. In recent years, a method of culturing bacteria and obtaining hyaluronic acid from the culture medium has also been found.

However, in the production of hyaluronic acid by extraction from living tissues, a large amount of contaminants such as proteins and chondroitin sulfate exist, so separation and purification are very complicated and unsuitable for mass production. Therefore, the obtained hyaluronic acid is extremely expensive, and the function of hyaluronic acid cannot be fully utilized.

Regarding the production of hyaluronic acid by microorganisms, among the bacteria of the genus Streptococcus, A, C and D type bacteria of the Lansfield serogroup, for example,
Streptocossus pyoge
nes), Streptococcus zooepidemicus (Strept
ocossus zooepidemicus), Streptococcus equi (Streptocossus equi), Streptococcus equisimils, Streptocossus dysgalactia
e), Pasteurella multocida
Although S. pyogenes and Pasteurella are pathogenic bacteria to humans, they are not suitable for the production of hyaluronic acid. Industrially, a method of culturing a bacterium of the genus Streptococcus and extracting and purifying hyaluronic acid from the culture solution is disclosed in JP-A-63-141594. However, these methods have problems that the obtained hyaluronic acid has a low molecular weight and the yield is low.

On the other hand, bacterial chondroitinase is known to be an endo-β-hexosaminide eliminase having hyaluronidase activity [Science and Industry 4
9, 11 (1975)], the present inventors focused on this finding, and thought that a strain belonging to the genus Streptococcus that produces hyaluronic acid produces high molecular weight hyaluronic acid by making it a chondroitinase-non-producing strain. It was

[0006]

An object of the present invention is to obtain a chondroitinase-non-producing strain of a strain belonging to the genus Streptococcus that produces hyaluronic acid, and thereby efficiently obtain a large amount of high molecular weight hyaluronic acid. It is to provide a manufacturing method capable of

[0007]

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that Streptococcus equi ATCC9527 strain (Streptococcus equi A
The chondroitinase non-producing strain obtained by mutation treatment from (TCC9527) is subjected to mutation treatment again, lacking hemolytic activity, and a strain with extremely high productivity of high molecular hyaluronic acid,
Streptococcus equi NC930627 (Strept
ococcus equi NC930627) was obtained and it was found that high molecular weight hyaluronic acid can be produced by culturing the strain, and the present invention was completed.

That is, according to the present invention, a bacterium belonging to the genus Streptococcus, which has a hyaluronic acid-producing ability, is non-chondroitinase-producing and non-hemolytic, is cultured in a medium,
A method for producing hyaluronic acid, which comprises collecting and collecting hyaluronic acid in a culture solution.

The present invention will be described in detail below.

(1) Acquisition of mutant strain The chondroitinase non-producing mutant strain referred to in the present invention is a strain belonging to the genus Streptococcus that produces hyaluronic acid, for example, Streptococcus equi ATCC952.
7 strains were exposed to UV light and chemicals (N-methyl-N'-nitro-
Mutagenized with N-niorosoguanidine (NTG), methylmethanesulfonic acid, etc., and the treated cells were used for GYP agar medium (glucose 2%, yeast extract 1%, polypeptone 1).
%, Monopotassium phosphate 0.2%, agar 1.5%, pH
After inoculating and culturing in 6.3), a strain with good growth was further added to chondroitin sulfate agar medium (yeast extract 0.2%, polypeptone 0.2%, chondroitin sulfate 0.
4%, 0.1% monopotassium phosphate) to obtain a non-growing strain.

Next, this strain was subjected to mutation treatment again by a conventional method, and the treated bacterial cells were inoculated and cultured in equine defibrinated blood agar medium to obtain a non-hemolytic (streptricin-s deficient) mutant strain. To do. This strain is considered to lack the ability to produce streptolysin-o, which is one of the hemolysins, because it does not produce a hemolytic ring even when it is cultivated on blood agar medium, and it is a safer non-hemolytic strain. It can be said that there is.

(2) Bacteriological Properties The mutant strain of Streptococcus equi obtained in (1) (hereinafter referred to as the present bacterium) is a transparent colony having extremely strong viscosity on Brain Heart Infusion agar medium. , Which is non-hemolytic (β-hemolytic: negative), does not produce chondroitinase, and belongs to the Lancefield serogroup C. The mycological properties of this bacterium are as follows.

(A) Gram stainability: positive (b) 10 ° C growth: negative (c) 45 ° C growth: negative (d) 0.1% methylene blue resistance: negative (e) 6.5% salt resistance Sex: Negative (f) 40% Bile acid resistance: Negative (g) Bacitracin resistance: Negative (h) pH 9.6 Resistance: Negative (i) 60 ° C, 30 minutes Resistance: Negative (j) Gelatin degrading : Negative (k) Starch degradability: Positive (l) Esculin degradability: Negative (m) Sugar fermentability: Glucose (+), Galactose (-), Sucrose (+), Lactose (-), Maltose (+) , Sorbitol (-), salicin (+),
Glycerin (-), mannitol (-), trehalose (-) (n) Vitamin requirement: vitamin B ₁ (+), riboflavin (+), calcium pantothenate (-), pyridoxine hydrochloride (+), nicotinic acid ( +), P-aminobenzoic acid (+), folic acid (-), biotin (-), vitamin B
₁₂ (-) (o) Amino acid requirement: DL-alanine (-), L-arginine (-), DL-aspartic acid (+), L-cysteine (-), L-glutamic acid (-), glycine (+ ), L-histidine (+), DL-isoleucine (+), DL-leucine (+), L-lysine hydrochloride (-), DL-methionine (+), DL-phenylalanine (-), L-proline ( +), DL-serine (-), DL-threonine (+), DL-tryptophan (+), L-tyrosine (-), DL-valine (+) This strain was industrialized on May 27, 1994. Deposited as FERMP-14341 at Institute of Microbial Technology, Institute of Technology.

(3) Production of hyaluronic acid by the present bacterium The medium used in the present invention may be one used for ordinary culture, including carbon sources such as glucose, fructose, galactose, sucrose, monopotassium phosphate and diphosphate. In addition to inorganic salts such as potassium, magnesium sulfate, sodium sulfite, sodium thiosulfate, and ammonium phosphate, organic nutrient sources such as polypeptone, yeast extract, casamino acid, and corn steep liquor, various amino acids, vitamins and the like are appropriately used. These medium components may be added all at once or in divided additions.

In the culture of the present invention, since the bacterium used is a facultative anaerobic bacterium, either aeration culture or anaerobic culture may be used, and the culture temperature is 25 to 40 ° C., preferably 30 to 3
5 ° C is desirable.

Since the pH of the culture broth decreases with the growth of the bacterium, the pH of the culture broth is adjusted to 6.5 to 8.5, preferably 7 to 7, with a base such as sodium hydroxide, potassium hydroxide or ammonia.
Control to 7.5.

The present bacterium is a strain which produces high molecular weight hyaluronic acid with an extremely high yield and productivity, and particularly when glucose is used as a carbon source, good results can be obtained. When the amount of sugar added is 1%, the yield of sugar is 17%, but the yield of sugar decreases as the amount of sugar added increases. When the addition of sugar is 6% or more, the viscosity of the culture solution is 8,000 centipoise (c) at 35 ° C.
p), the culture fluid is not fluid, and increasing the stirring number has no effect. Culturing is carried out until the carbon source used is completely consumed in the culture solution.

Hyaluronic acid accumulated in the culture medium can be easily separated and purified, and a known separation method may be used.
That is, the hyaluronic acid concentration is 0.5 to
The culture solution is diluted to 1.0 g / L, and the pH is adjusted to acidic with an acid such as trichloroacetic acid, hydrochloric acid, or sulfuric acid. During this operation, contaminating proteins and low-molecular-weight hyaluronic acid form and form a complex called mucin clot, which precipitates.For example, centrifugation or microfiltration or filtration using activated carbon or Celite as a filter aid, etc. Bacteria and deproteinization become possible, facilitating subsequent purification operations.

After sterilization / protein removal, low-molecular compounds are removed by dialysis, and water-insoluble fine particles are removed by microfiltration, and then a hyaluronic acid-containing liquid is treated with a water-soluble organic solvent such as alcohol, acetone, or dioxane. Add hyaluronic acid to obtain hyaluronic acid, or obtain it directly by freeze-drying or spray-drying.

As a result of examining the hyaluronic acid obtained by extracting and purifying from the above-mentioned culture medium in comparison with the standard product, it can be confirmed that the obtained product is hyaluronic acid. The properties are shown below.

(1) Electrophoresis using a cellulose acetate membrane has a band at the same position as the standard. (2) Degraded by actinomycete hyaluronidase. (3) D-glucuronic acid and N-acetyl-D-glucosamine are present in a molar ratio of 1: 1. (4) The infrared absorption spectrum by the thin film method is the same as that of the standard product. (5) Measurement of molecular weight by limiting viscosity method (TCLaurent et a
l., Biochim. Biophys. Acta, 42, 476-485, 1960), resulting in 1.5 million.

[0022]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

[Example 1] (i) Acquisition of mutant strain Streptococcus equi ATCC9527 strain was GY
After culturing in P medium at 30 ° C. for 10 hours, cells in the logarithmic growth phase were collected by centrifugation and washed aseptically at 5 ° C. with 0.05 M phosphate buffer (pH 6.5). 1x
The cells were suspended in the same buffer solution so that the bacterial concentration was 10 ⁸ cells / ml, and NTG was added thereto at 200 μg / ml, and the mixture was shaken at 30 ° C. for 30 minutes. Immediately after the treatment, the cells were collected in a centrifuge, and 0.05M phosphate buffer (pH
After washing twice with 6.5), the cells were inoculated in a GYP medium and 37
After culturing at 2 ° C. for 2 hours and diluting the culture solution, the cells were inoculated again on GYP agar medium. After culturing at 30 ° C for 72 hours, bacteria are picked up from actively growing colonies, inoculated again on an agar medium containing chondroitin as a carbon source, and cultivated under the same conditions, and a strain that does not grow is a chondroitinase non-producing strain. Acquired as. The method of identifying the above-mentioned chondroitinase-producing / non-producing bacteria is devised by the present inventors.

Since the obtained strain exhibited β-hemolytic property (hemolysin: streptolysin-s), it was subjected to mutation treatment again in the same manner as above. After the treatment, the cells were inoculated / cultured on a horse defibrinated blood agar medium to obtain a strain that did not show hemolytic activity (Streptococcus equi NC930627, F).
ERM P-14341).

(Ii) Production of hyaluronic acid by the mutant strain Glucose 2%, polypeptone 1.5%, yeast extract 0.5%, monopotassium phosphate 0.2%, magnesium sulfate heptahydrate 0.05%, adecanol LG-109 (antifoaming agent; manufactured by Asahi Denka Kogyo) 0.005% composition medium (pH
7.0) 3 L was placed in a 5 L Jafermenter, sterilized and pre-cultured Streptococcus equi NC93
0627 was inoculated with 1% and the culture pH was kept constant at 7 with an aqueous 6N-sodium hydroxide solution at 48 at 30 ° C.
The cells were agitated for aeration (1 vvm) for a period of time.

Glucose is sterilized separately and then added at the start of the culture, or even during the culture, the glucose is added at an appropriate time while measuring the remaining amount of glucose.
Was added. Hyaluronic acid accumulates with the progress of culture,
After 32 hours of culturing, the viscosity of the culture reached 8,000 centipoise, and stirring was no longer effective. After 48 hours of culturing, glucose was completely consumed, so the culturing was terminated.

The brothed-out culture broth had a high viscosity, so it was diluted with water to 100 centipoise or less and adjusted to pH 4 or less with trichloroacetic acid. The precipitated mucin clot and other contaminants are removed together with the bacterial cells with a microfilter module (PW-103 Asahi Kasei),
Further, low molecular weight substances were removed by passing through an ultrafiltration module. After that, ethanol was added, and the precipitated solid matter was dried under reduced pressure to obtain 30 g (6 g per 1 L of culture solution) of hyaluronic acid. The obtained hyaluronic acid has a band at the same position as the standard in electrophoresis, and has an infrared absorption spectrum, decomposition of Streptomyces by hyaluronidase, and D-glucuronic acid and N-acetyl-D-glucosamine in a molar ratio. It was confirmed to be hyaluronic acid by virtue of its presence at 1: 1. The molecular weight was 1.5 million as a result of molecular weight measurement by the extreme root degree method.

EFFECT OF THE INVENTION By culturing the Streptococcus equi mutant strain according to the present invention, the yield and yield in the method for producing hyaluronic acid using Streptococcus spp.
In addition, hyaluronic acid, which is known as hemolysin and is free from streptolysin-s and streptolysin-o, can be obtained at low cost. The hyaluronic acid thus obtained can be used as a raw material for cosmetics and pharmaceuticals.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshiro Hatanaka 2-11-14 Aikawa, Higashiyodogawa-ku, Osaka City (72) Inventor Masayuki Sakai 1-8-7 Fukumachi, Nishiyodogawa-ku, Osaka City Nissei Chemical Industry Co., Ltd. (72 ) Inventor Seichika Okabe 1-8-7 Fukumachi, Nishiyodogawa-ku, Osaka City Nissei Chemical Industry Co., Ltd. (72) Inventor Hiroshi Akikaze 1-8-7 Fukumachi, Nishiyodogawa-ku, Osaka City Nissei Chemical Industry Co., Ltd. (72) Inventor Tomoaki Yada 1-8-7 Fukumachi, Nishiyodogawa-ku, Osaka City Nissei Chemical Industry Co., Ltd.

Claims (5)

[Claims] 1. A bacterium belonging to the genus Streptococcus, which has a hyaluronic acid-producing ability, is non-chondroitinase-producing and is non-hemolytic, is cultivated in a medium to produce and accumulate hyaluronic acid in the culture solution. A method for producing hyaluronic acid, which comprises collecting 2. The method according to claim 1, wherein the bacterium belonging to the genus Streptococcus is Streptococcus equi.
The method for producing hyaluronic acid according to the item. 3. The method for producing hyaluronic acid according to claim 1 or 2, wherein the bacterium belonging to the genus Streptococcus is Streptococcus equi NC930627. 4. A mutant strain belonging to the genus Streptococcus having hyaluronic acid-producing ability, non-chondroitinase-producing and non-hemolytic property. 5. The mutant strain according to claim 4, wherein the mutant strain belonging to the genus Streptococcus is Streptococcus equi NC930627.