KR20120119060A - Streptococcus equisubsp. zooepidemicus and method for preparing hyaluronic acid using the same - Google Patents

Abstract

PURPOSE: A streptococcus zooepidermicicus and a manufacturing method of hyaluronic acid using the strain is provided to manufacture the high molecular weight of hyaluronic acid at high yield. CONSTITUTION: A streptococcus zooepidemicus KCTC 11898BP produces hyaluronic acid. The streptococcus zooepidermicicus KCTC 11898BP is obtained by inducing mutation on the streptococcus zooepidermicicus KCTC 3318 strain using NTG and ultraviolet ray. A manufacturing method of the hyaluronic acid comprises a step of separating the hyaluronic acid from the obtained culture fluid by culturing the streptococcus zooepidermicicus KCTC 11898BP. The produced hyaluronic acid has the average molecular weight of 4,500,000 Da. [Reference numerals] (AA) Streptococcus zooepidemicus KCTC 3318; (BB) Varied strain in which hemolytic dielectric character of SZ1 (KCTC 3318) mother strain is converted into non-hemolytic; (CC) SZN2-1(varied strain which does not manufacture hyaluronic acid); (DD) SZN2U1-1(varied strain which manufactures hyaluronic acid at high yield); (EE) SZN2U2-1(varied strain which is not influenced by organic nitrogen source while manufacturing the hyaluronic acid at high yield); (FF) SZN3U2-2(varied strain which manufactures hyaluronic acid at high yield); (GG) SZN4U2P-2(strain which is not interrupted at the initial glucose concentration of 80g/l or higher); (HH) SZN5U2P-2 (Streptococcus zooepidemicus KCTC 11898BP)

Description

Streptococcus jupitemicus and a method for producing hyaluronic acid using the strain {Streptococcus equisubsp. zooepidemicus and Method for preparing hyaluronic acid using the same}

The present invention relates to a novel hyaluronic acid-producing strain Streptococcus equisubsp . Zooepidemicus KCTC 11898BP and a method for producing hyaluronic acid using the strain.

Hyaluronic acid is a colorless, transparent, high-viscosity polysaccharide with a molecular weight of 50,000 to 13,000,000 Da, in which glucuronic acid and N-acetyl glucosamine are alternately bonded with (1-3) and (1-4). Hyaluronic acid is a substance secreted by the self-protective needs of living organisms, and is widely used in the medical and cosmetics fields because of its excellent moisturizing effect, lubrication effect, and protection against invasion of bacteria.

Hyaluronic acid can be extracted from biological tissues such as chicken crust, skin, umbilical cord, joint fluid, bovine eye, etc., but these tissues contain impurities such as chondroitin sulfate and glucose aminoglycans, which are expensive to separate and purify. The yield is also low, which is uneconomical. In contrast, the production of hyaluronic acid using microorganisms can produce high yield hyaluronic acid at a relatively low cost.

The microorganisms known as hyaluronic acid producing strains include disgalactier, St. epidemius, Iqui, piogenes, Iquisimilli, and Pacalis in Streptococcus, but most of the hyaluronic acid produced by these microorganisms has a molecular weight of less than 3,000,000 Da. Productivity is also known to be relatively low. In addition, there is a problem in the reproducibility, such as the difference in yield depending on the type of medium in the production process, in particular, it is known that the change in production yield according to the type of organic nitrogen source is large.

The present invention is to provide a novel Streptococcus equisubsp. Zooepidemicus strain capable of producing high molecular weight hyaluronic acid in a high yield without being substantially influenced by an organic nitrogen source, and a method for producing hyaluronic acid using the same.

In order to solve the above problems, the present invention is a novel Streptococcus juepidemicus ( Streptococcus) having a hyaluronic acid production capacity equisubsp . zooepidemicus ) provides a KCTC 11898BP strain.

The present invention also provides a method for producing hyaluronic acid, which comprises separating hyaluronic acid from a culture obtained by culturing Streptococcus equisubsp . Zooepidemicus KCTC 11898BP and hyaluronic acid produced by the above method.

Streptococcus juice epi Demi kusu (Streptococcus in accordance with the present invention equisubsp . zooepidemicus ) KCTC 11898BP can be used to produce high molecular weight hyaluronic acid without substantially being affected by organic nitrogen sources.

1 is a flow chart illustrating a mutant process for preparing a Streptococcus jupitemicus mutant strain according to the present invention. N means NTG treated strain, U means UV treated strain, P means osmotic resistance strain (excellent growth strain at 4% sodium chloride), and the number means the number of treatments.

The present invention provides a hyaluronic acid producing strain Streptococcus equisubsp. Zooepidemicus KCTC 11898BP.

The present inventors have invented a novel mutant strain Streptococcus juepidemicus SZN5U2P- by mutating NTG and ultraviolet light to Streptococcus juepidemicus KCTC 3318 parent strain to produce a strain capable of producing high molecular weight hyaluronic acid. 2 was obtained. The Streptococcus jupitemicus SZN5U2P-2 was deposited on March 21, 2011 at the Korea Institute of Bioscience and Biotechnology, and received the accession number KCTC 11898BP.

As can be seen through the following examples, the Streptococcus juepidemicus KCTC 11898BP strain according to the present invention can produce a high yield of high molecular weight hyaluronic acid having an average molecular weight of 4,500,000 Da at a high yield of 6.0 to 6.5 g / L on average . In addition, unlike the parent strain that hyaluronic acid production yield is greatly affected by the type of organic nitrogen source of 2.0 to 3.3 g / ℓ, the strain Streptococcus juepidemicus KCTC 11898BP according to the present invention is 6.0 regardless of the type of organic nitrogen source Constant production yield of from 6.5 g / l. That is, it was confirmed that the Streptococcus jupitemicus KCTC 11898BP strain according to the present invention had significantly improved hyaluronic acid productivity compared to the parent strain without substantially being affected by the type of the organic nitrogen source.

Therefore, the present invention is Streptococcus Streptococcus equisubsp . zooepidemicus ) Provided is a method for producing hyaluronic acid comprising separating hyaluronic acid from a culture obtained by culturing KCTC 11898BP.

Separation of hyaluronic acid from the culture solution of Streptococcus juepidemicus KCTC 11898BP may be a conventional method known in the art, including, but not limited to, for example, J. Soc. Cosmet. Japan. 22, 35-42 (1988) can be isolated and purified by the method.

The present invention also provides a hyaluronic acid produced by the above method. The hyaluronic acid produced by the above method has an average molecular weight of 4,500,000 Da and may be used as a therapeutic agent and an adjuvant in cosmetics and medicine. In particular, hyaluronic acid having a molecular weight of about 4,500,000 Da may be usefully used as a moisturizing agent.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Example  1: Production of Mutant Strains

Mutant strains were obtained by mutating with N1-methyl-N1-nitro-N1-nitrosoguanidine (NTG) and UV light using Streptococcus juepidemicus KCTC 3318 with hyaluronic acid production capacity as a parent strain (see FIG. 1). ).

Platinum was inoculated with Streptococcus jupitemicus KCTC 3318 in 25ml / 250ml of tote hew medium, incubated at 37 ° C and 200rpm until the logarithmic growth phase, and 1 ml of the culture solution was centrifuged at 4 ° C, followed by 2-3 times tris maleic acid buffer. After washing, NTG was added at a concentration of 200-250ug / ml and then mutated at 37 ° C, 100rpm, and 30-60 minutes. Thereafter, after washing 2-3 times with TM buffer and washing twice with saline buffer, diluted to a certain concentration, and then cultured in blood agar medium, strain SZ1 without hemolysis was selected.

Mutant strain SZ1, which does not cause hemolysis, was mutated to NTG in the same manner as above to obtain a mutant strain, and then each single mutant strain was transplanted to a tote hew agar medium containing 400ug / ml of hyaluronic acid and 1% albumin fraction V, respectively. After incubation for 24 hours in a 37 ℃ incubator. 10 ml of 2N acetate solution was added thereto and reacted for 10 minutes to obtain a mutant strain SZN2-1 having an opaque periphery of colony, that is, no hyaluronidase. The mutant strain was added to 1.8 L of fermentation production medium (60 g of glucose per 1 L of water, 0.6 g of magnesium sulfate, 6 g of yeast extract, 2.5 g of potassium diphosphate, 5 g of sodium chloride, 6 g of glutamic acid, 17 g of casein peptone) and added to a 5 L small fermenter. After sterilization, 200ml of the seed culture was inoculated and fermented at 35 ° C for 24 hours under conditions of 600rpm and 1vvm. As a result of confirming the fermentation productivity by adjusting the pH of the fermentation broth with 5-7N NaOH to 7.00, it was confirmed that hyaluronic acid was produced at 2 to 4 g / l, and thus the fermentation yield was low and the reproducibility was not suitable as an industrial production strain.

SZN2-1 mutant strains having a genotype that was not hemolytic and did not produce hyaluronidase were inoculated with platinum to 25ml / 250ml of tote hew medium again and incubated at 37 ° C and 200rpm until logarithmic growth period. The culture solution was centrifuged at 4 ° C., washed 2-3 times with sterile saline solution and diluted to a predetermined concentration to give a flat medium (2 g of glucose per 1 liter of water, 5 g of ammonium sulfate, 0.5 g of potassium phosphate, 0.5 g of potassium phosphate). , 0.5g of magnesium sulfate, 2g of sodium chloride, 1g of yeast extract, 50ug of biotin, 100ug of thiamine hydrochloride, 10mg of iron sulfate, 10mg of manganese sulfate, 3g of calcium carbonate, 15g of agar), and then UV light of 265nm at 30cm height using UV lamp Was irradiated for 20-80 seconds. After ultraviolet irradiation, it was left in the dark for 2-4 hours and then incubated for 2-6 days in a 37 ℃ incubator.

The highly grown and transparent colonies were screened and subjected to repeated UV treatment and NTG treatment to obtain high hyaluronic acid SZN3U2-2 strain which was not substantially affected by the type of organic nitrogen source.

SZN3U2-2 strain is affected by high glucose concentration at the initial stage, which leads to a long fermentation time. To solve this problem, NTG is further subjected to mutant treatment and then diluted to a certain concentration. SZN5U2P-2 strain with high yield after clearing and spreading) was obtained. The strain was deposited on March 21, 2011 at the Korea Institute of Bioscience and Biotechnology, Biotechnology Center (Accession No. KCTC 11898BP).

Experimental Example  1: Hyaluronic Acid Productivity Assay According to Organic Nitrogen Source I

Fermentation production medium using organic nitrogen sources of manufacturers A (Bio springer), B (Difco) and C (Solabia) (80 g of glucose per 1 liter of water, 0.6 g of magnesium sulfate, 6 g of yeast extract, 2.5 g of potassium diphosphate, 5 g of sodium chloride, 6 g of glutamic acid and 17 g of casein peptone) were added to 1.8 L and sterilized in a 5 L small fermenter, followed by inoculation with 200 ml of the strain SZN5U2P-2 seed culture and fermented at 35 ° C. under 600 rpm and 1 vv for 18 hours. The pH of the fermentation broth was adjusted to 7.00 with 5-7N NaOH, and hyaluronic acid present in the fermentation broth was recovered by a known polysaccharide separation and purification method (J. Soc. Cosmet. Japan. 22, 35-42 (1988)). The productivity of hyaluronic acid was quantitatively analyzed by the carbazole method (Z. Dische, J. Biol. Chem. 167, 189 (1947)).

Final fermentation productivity is as shown in Table 1 below. The mutant strain of the present invention was confirmed to be a stable strain producing hyaluronic acid in high yield compared to the parent strain (KCTC 3318) with little effect of organic nitrogen sources by manufacturer.

division KCTC3318 SZN5U2P-2 Company A 3.1 6.5 Company B 2.0 6.0 Company C 2.3 6.3

(Hyaluronic acid g / L)

Experimental Example  2: Hyaluronic Acid Productivity Assay According to Organic Nitrogen Source II

As a result of comparing the fermentation productivity according to the type of organic nitrogen source in the same manner as in Experimental Example 1, the results shown in Table 2 were obtained. As in the following fermentation experiments, the parent strain (KCTC 3318) confirmed that the production of hyaluronic acid was greatly affected by 2.0 to 3.3 g / l depending on the type of organic nitrogen source. In contrast, the mutant strain of the present invention produced hyaluronic acid at a high yield of 6.0 to 6.5 g / l with little effect from the type of organic nitrogen source. In addition, as a result of measuring the molecular weight of hyaluronic acid produced by a capillary viscometer (Narlin, Analytical Biochemistry 147, 347-395 (1985)), the average molecular weight of the produced hyaluronic acid was 4,500,000 Da.

division KCTC3318 SZN5U2P-2 Casein 3.3 6.2 Tryptone 2.2 6.4 Yeast Extract 2.0 6.1

(Hyaluronic acid g / L)

Korea Biotechnology Research Institute KCTC11898BP 20110321

Claims (3)

Streptococcus Streptococcus zooepidemicus ) KCTC 11898BP. Streptococcus Streptococcus zooepidemicus ) A method for producing hyaluronic acid comprising separating hyaluronic acid from a culture obtained by culturing KCTC 11898BP. Hyaluronic acid produced by the method according to claim 2 and having an average molecular weight of 4,500,000 Da.

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