KR100472007B1 - Microorganism producing hyaluronic acid and method of producing hyalironic acid using thereof - Google Patents

Microorganism producing hyaluronic acid and method of producing hyalironic acid using thereof Download PDF

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KR100472007B1
KR100472007B1 KR10-2002-0048915A KR20020048915A KR100472007B1 KR 100472007 B1 KR100472007 B1 KR 100472007B1 KR 20020048915 A KR20020048915 A KR 20020048915A KR 100472007 B1 KR100472007 B1 KR 100472007B1
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hyaluronic acid
producing
streptococcus
acid
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박흥순
장성홍
김을제
박중경
한영진
이충
한희용
김윤철
박호진
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주식회사 코오롱
주식회사 백텍
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Priority to CNB03819581XA priority patent/CN100342004C/en
Priority to EP03788171A priority patent/EP1543103B1/en
Priority to US10/523,769 priority patent/US7575914B2/en
Priority to AU2003252566A priority patent/AU2003252566A1/en
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Abstract

본 발명은 히아루론산 생산 균주 및 상기 균주를 이용한 히아루론산 생산방법에 관한 것이다. 특히 본 발명은 고분자의 히아루론산을 고수율로 생산할 수 있는 비용혈성 균주 스트렙토코커스 에스피 KL0188에 관한 것이다. The present invention relates to a hyaluronic acid producing strain and a method for producing hyaluronic acid using the strain. In particular, the present invention relates to a non-hemolytic strain Streptococcus sp KL0188 capable of producing a high yield of hyaluronic acid of a polymer.

Description

히아루론산 생산 균주 및 상기 균주를 이용한 히아루론산 생산방법{MICROORGANISM PRODUCING HYALURONIC ACID AND METHOD OF PRODUCING HYALIRONIC ACID USING THEREOF}Hyaluronic acid production strain and hyaluronic acid production method using the strain {MICROORGANISM PRODUCING HYALURONIC ACID AND METHOD OF PRODUCING HYALIRONIC ACID USING THEREOF}

[발명이 속하는 기술분야][TECHNICAL FIELD OF THE INVENTION]

본 발명은 히아루론산 생산 균주 및 상기 균주를 이용한 히아루론산 생산방법에 관한 것으로, 보다 상세하게는 고분자의 히아루론산을 고수율로 생산할 수 있는 스트렙토커커스 에스피 KL0188 및 상기 균주로부터 생산된 히아루론산에 관한 것이다.The present invention relates to a hyaluronic acid-producing strain and a method for producing hyaluronic acid using the strain, and more particularly, to a streptococcus sp. KL0188 capable of producing a high yield of polymer hyaluronic acid and hyaluronic acid produced from the strain.

[종래기술][Private Technology]

히아루론산(Hyaluronic acid)은 분자량이 50,000 내지 13,000,000 Da에 이르는 무색의 고점도 다당류로, 반복단위인 글루쿠론산과 N-아세틸 글루코즈아민이 (1-3)과 (1-4)로 번갈아 결합되어 있다. 히아루론산은 보습효과를 가지며, 물리적 마찰에 대한 윤활효과 및 세균 등의 침입에 대한 보호효과가 우수하여 화장품 첨가제, 관절염치료제, 안과수술용 수술보조제 및 외과수술후의 유착저해제 등으로 광범위하게 사용된다. 이러한 히아루론산은 소의 안구, 닭벼슬, 동물의 완충조직, 태반, 암세포 및 피부 등에 다량 함유되어 있다. Hyaluronic acid is a colorless, high-viscosity polysaccharide with a molecular weight of 50,000 to 13,000,000 Da, in which glucuronic acid, a repeating unit, and N-acetyl glucosamine are alternately combined with (1-3) and (1-4). Hyaluronic acid has a moisturizing effect, has excellent lubricating effect against physical friction and protection against invasion of bacteria and the like, and is widely used as a cosmetic additive, an arthritis treatment agent, an ophthalmic surgery surgical aid, and an adhesive inhibitor after surgery. The hyaluronic acid is contained in a large amount of eyeballs, chicken rice, animal buffer tissues, placenta, cancer cells and skin.

히아루론산의 수득방법은 상기에서 언급한 생체조직으로부터 추출하거나(미국특허 4,141,973 및 미국특허 4,303,676), 미생물을 발효시켜 발효산물로서 회수하는 방법이 있다. 그러나, 추출방법에 의하여 수득한 히아루론산에는 콘드로이친 설페이트, 글리코사미노 글리간설페이드 등의 불순물이 섞여 있어 이들을 제거하기 위한 복잡한 정제과정이 요구될 뿐만 아니라 그에 따른 고가의 비용으로 대량생산하기 어렵다. 반면에 미생물을 이용한 히아루론산의 생산은 상대적으로 생산비용이 적게들고, 고분자량의 히아루론산을 비교적 간단한 방법으로 수득할 수 있다(일본특허공개 58-056692 및 미국특허86-00066).The method of obtaining hyaluronic acid may be extracted from the aforementioned biological tissues (US Pat. No. 4,141,973 and US Pat. No. 4,303,676), or the fermentation of microorganisms may be recovered as a fermentation product. However, the hyaluronic acid obtained by the extraction method is mixed with impurities such as chondroitin sulfate and glycosamino glycan sulfate, which requires a complicated purification process to remove them and is difficult to mass produce at a high cost. On the other hand, the production of hyaluronic acid using microorganisms is relatively low in production cost, and high molecular weight hyaluronic acid can be obtained by a relatively simple method (Japanese Patent Laid-Open No. 58-056692 and US Patent 86-00066).

히아루론산 생산에 사용되는 미생물로는 스트렙토코커스 속의 파이오제넥스, 패칼리스, 디스갈락티에, 주에피데미쿠르, 이퀴, 이퀴시밀리스 등이 있으며, 이들은 버기스(Bergy's) 편람에 의하면 란세필드 혈청군 A 또는 C 타입에 속한다. 이러한 미생물들은 용혈성을 가지는 연쇄상 구균으로, 베타-용혈작용을 갖는 것으로 보고되어 있다. 그러나 스트렙토코커스 속 미생물을 이용하여 생산된 히아루론산(일본특허공개 58-566922, 미국특허공개 60-500997, 대한민국 특허등록 10-250573, 대한민국 특허공개 10-250573)은 평균분자량이 300,000 내지 3,500,000 Da으로 비교적 작아 의약용 치료제 및 보조제로 사용하기 어려우며, 화장품용도로 사용하기에 보습력이 낮은 단점이 있다. 또한 미국특허 제 6090596호에서는 분자량이 6,300,000 내지 9,500,000 Da의 고분자량 히아루론산을 생산하는 방법을 기재하였으나, 히아루론산이 생산성이 배양액 1L 당 0.35 g 으로 매우 낮은 수준이다. The microorganisms used for the production of hyaluronic acid include Piogenex, Pakalis, Disgalactier, Jupitemicur, Iqui, and Iquicimilis in Streptococcus, according to Bergy's handbook Lancefield serogroup A Or C type. These microorganisms are hemolytic streptococci, which have been reported to have beta-hemolytic action. However, hyaluronic acid produced using microorganisms of Streptococcus (Japanese Patent Publication 58-566922, US Patent Publication 60-500997, Republic of Korea Patent Registration 10-250573, Republic of Korea Patent Publication 10-250573) has an average molecular weight of 300,000 to 3,500,000 Da It is difficult to use as a medical treatment and adjuvants because it is small, has a low moisturizing power for use in cosmetics. In addition, US Patent No. 6090596 describes a method for producing high molecular weight hyaluronic acid having a molecular weight of 6,300,000 to 9,500,000 Da, but hyaluronic acid productivity is very low, 0.35 g per 1 L of culture medium.

상기 종래기술의 문제점을 해결하기 위하여 안출된 것으로서, 본 발명은 고분자량의 히아루론산을 고수율로 생산할 수 있는 균주를 제공하는 것을 목적으로 한다.In order to solve the problems of the prior art, an object of the present invention is to provide a strain capable of producing a high yield of high molecular weight hyaluronic acid.

또한 본 발명은 히알루로니다제 효소가 없고 비용혈성을 가지는 히아루론산 생산 균주를 제공하는 것을 목적으로 한다.It is also an object of the present invention to provide a hyaluronic acid producing strain which is free of hyaluronidase enzyme and which is non-hemolytic.

또한 본 발명은 비용혈성 균주로부터 생산되고 순수 정제된 고분자량의 히아루론산을 제공하는 목적으로 한다.It is another object of the present invention to provide a high molecular weight hyaluronic acid produced from a non-hemolytic strain and purified.

상기 목적을 달성하기 위하여 본 발명은 히아루론산 생산균주 스트렙토코커스 에스피(Streptococcus sp.) KL0188(KCTC10248BP)를 제공한다.In order to achieve the above object, the present invention provides a hyaluronic acid producing strain Streptococcus sp . KL0188 (KCTC10248BP).

또한 본 발명은 스트렙토코커스 에스피 KL0188(KCTC1024BP)를 배양하여 수득한 배양액으로부터 히아루론산을 분리하는 것을 포함하는 히아루론산 생산방법을 제공한다.The present invention also provides a method for producing hyaluronic acid comprising separating hyaluronic acid from the culture obtained by culturing Streptococcus sp KL0188 (KCTC1024BP).

또한 본 발명은 상기의 스트렙토코커스 에스피 KL0188(KCTC1024BP)로부터 생산된 히아루론산을 제공한다.The present invention also provides hyaluronic acid produced from the Streptococcus sp KL0188 (KCTC1024BP).

이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.

본 발명자들은 고분자량의 히아루론산을 고수율로 생산할 수 있으며, 비용혈성을 가지는 균주를 제조하기 위하여, 스트렙토코커스 주에피데미쿠스에 돌연변이를 유발하여 스트렙토코커스 에스피 KL0188를 제조하였다. The present inventors can produce high molecular weight hyaluronic acid in high yield, and in order to prepare a strain having a non-hemolytic strain, Streptococcus sp. KL0188 was prepared by inducing mutations in the main epidemius.

본 발명의 스트렙토코커스 에스피 KL0188은 2002년 5월 10일자로 대한민국에 소재한 유전자은행(Korean Collection for Type Culture)에 기탁번호 KCTC10248BP로 기탁하였다. 스트렙토코커스 에스피 KL0188은 비용혈성 균주이며, 히아루로니다제 효소가 없어 히아루론산을 고수율로 생산할 수 있다.Streptococcus sp KL0188 of the present invention was deposited on May 10, 2002 to the Korean Bank for Korean (Korean Collection for Type Culture) Accession No. KCTC10248BP. Streptococcus sp KL0188 is a non-hemolytic strain and lacks a hyaluronidase enzyme, which can produce hyaluronic acid in high yield.

스트렙토코커스 에스피 KL0188은 탄소원, 질소원, 무기염류 및 비타민 등의 미량원소가 포함된 배지에서 배양할 수 있다. 탄소원으로는 글루코즈, 슈크로즈, 갈락토즈 또는 푸룩토즈가 사용될 수 있으나, 바람직하게는 글루코즈이다. 질소원으로는 암모늄 나이트레이트, 암노늄 설페이트, 트립톤, 펩톤, 이스트엑기스 또는 카사미노산이 사용될 수 있으며, 무기염류로는 염화나트륨, 인산 제일 나트륨, 인산 제이 나트륨, 황산 제일철 또는 황산마그네슘이 사용될 수 있다. Streptococcus sp KL0188 can be cultured in a medium containing trace elements such as carbon sources, nitrogen sources, inorganic salts and vitamins. Glucose, sucrose, galactose or fructose may be used as the carbon source, but preferably glucose. As a nitrogen source, ammonium nitrate, ammonium sulfate, tryptone, peptone, yeast extract or casamino acid may be used, and as inorganic salts, sodium chloride, sodium phosphate, dibasic sodium phosphate, ferrous sulfate or magnesium sulfate may be used.

본 발명에서 일예로 사용한 스트렙토코커스 에스피 KL0188 배양배지조성물은 다음과 같다: 20 내지 80 g/L의 글루코즈, 5 g/L의 이스트엑기스, 17 g/L의 카제인 펩톤, 7 g/L의 글루탐산, 0.7 g/L의 황산마그네슘, 2.5 g/L의 인산제이칼륨 및 5.0 g/L의 염화나트륨.Streptococcus sp KL0188 culture medium composition used as an example in the present invention is as follows: 20 to 80 g / L glucose, 5 g / L yeast extract, 17 g / L casein peptone, 7 g / L glutamic acid, 0.7 g / L magnesium sulfate, 2.5 g / L dipotassium phosphate and 5.0 g / L sodium chloride.

스트렙토코커스 에스피 KL0188은 30 내지 37 ℃에서 호기성 배양할 수 있으며, 배양시 배양액의 pH는 6.5 내지 7.5로 유지시켜주는 것이 좋다. 상기 pH는 배양중 변동되는 인자로 인위적으로 조절하는 것이 좋다. 상기 pH 범위를 벗어날 경우 생산되는 히아루론산의 생산량과 분자량이 달라질 수 있는 문제점이 있다. pH의 조정은 5N NaOH 용액 또는 1N HCl 용액으로 실시할 수 있다.Streptococcus sp KL0188 can be aerobic culture at 30 to 37 ℃, it is good to maintain the pH of the culture medium at 6.5 to 7.5 when incubating. The pH is artificially adjusted to the factors that vary during the culture. If the pH is out of the range there is a problem that the production amount and molecular weight of the hyaluronic acid produced. The pH can be adjusted with 5N NaOH solution or 1N HCl solution.

스트렙토코커스 에스피 KL0188으로부터 생산된 히아루론산은 통상의 방법(J. Soc. Cosmet. Japan. 22, 35-42(1988))으로 배양액으로부터 분리 및 정제할 수 있다. 스트렙토코커스 에스피 KL0188은 약 6.0 내지 7.5 g/L의 히아루론산을 생산하며, 상기 히아루론산은 평균 분자량이 5,000,000 Da 이상의 고분자이다. Hyaluronic acid produced from Streptococcus sp KL0188 can be isolated and purified from the culture by conventional methods (J. Soc. Cosmet. Japan. 22, 35-42 (1988)). Streptococcus sp KL0188 produces about 6.0-7.5 g / L of hyaluronic acid, the hyaluronic acid having an average molecular weight of 5,000,000 Da or more.

따라서, 본 발명의 스트렙토코커스 에스피 KL0188은 저가의 비용으로 고수율로 히아루론산을 생산할 뿐만 아니라 비교적 간단한 방법으로 히아루론산을 정제할 수 있다. 또한 이로부터 생산된 히아루론산은 화장품, 의약용 치료제 및 보조제로 사용할 수 있다. Therefore, the Streptococcus sp KL0188 of the present invention can produce hyaluronic acid at high yield at low cost and can purify hyaluronic acid by a relatively simple method. In addition, hyaluronic acid produced therefrom can be used as cosmetics, medicaments and adjuvants.

이하 본 발명의 실시예를 기재한다. 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명이 하기 실시예에 한정되는 것은 아니다. Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

실시예 1. 돌연변이 균주의 스크리닝Example 1. Screening of Mutant Strains

스크렙토코커스 주에피데미쿠스에 돌연변이를 유발하여 비용혈성을 가지며, 히알루로니다제 활성이 없는 돌연변이주를 선별하였다.Mutants that were non-hemolytic by inducing mutations in S. aureus epidemicus were selected.

스트렙토코커스 주에피데미쿠스(KCTC 3318)을 50 ml의 토드휴이트 배지(DIFCO사의 Bacto Todd Hewitt Broth)에 접종하여 37 ℃에서 대수성장기까지 배양했다. 배양액 1 ml을 저온에서 원심분리하여 침전된 세포를 수거하고, 여기에 50 mM 트리스-말레산 완충액(pH 6.0)을 첨가하여 2회 세척하였다. 세척한 세포는 트리스-말레산 완충액에 1×103 세포/ml의 농도로 분산시키고, NTG(N-메틸-N'-니트로소구아니딘)를 200 ug/ml 농도로 혼합하였다. 혼합물은 37 ℃에서 30분 동안 교반하고, 세포는 50 mM의 트리스-말레산 완충액(pH 6.0)으로 2회 세척하였다. 세포는 토드-후이트 배지에 접종하여 37 ℃에서 18시간 동안 배양하였다. 배양액을 수득한 다음 멸균 식염수로 1×103 세포/ml 농도로 희석하고, 0.1 ml을 혈액 한천(Blood Agar) 배지상에 배양하여 용혈 현상을 나타내지 않는 콜로니를 선별하였다.Streptococcus epidemius (KCTC 3318) was inoculated in 50 ml of Todd hewitt medium (Bacto Todd Hewitt Broth, DIFCO) and incubated at 37 ° C. until logarithmic growth. 1 ml of the culture was centrifuged at low temperature to collect the precipitated cells, which were washed twice with 50 mM tris-maleic acid buffer (pH 6.0). The washed cells were dispersed in tris-maleic acid buffer at a concentration of 1 × 10 3 cells / ml, and NTG (N-methyl-N′-nitrosoguanidine) was mixed at a concentration of 200 ug / ml. The mixture was stirred at 37 ° C. for 30 minutes and the cells were washed twice with 50 mM tris-maleic acid buffer (pH 6.0). Cells were inoculated in Todd-Height medium and incubated at 37 ° C. for 18 hours. Cultures were obtained and then diluted to 1 × 10 3 cells / ml with sterile saline, and 0.1 ml were cultured on Blood Agar medium to select colonies that did not exhibit hemolysis.

비용혈성 돌연변이 균주는 다시 상기한 방법과 동일하게 실시하여 돌연변이를 유발시켰고, 배양액 1 ml 당 400 ug의 히아루론산과 1 %의 알부민 분획V를 함유하는 토드휴이트 한천배지에 단일 콜로니가 형성되도록 도말하였다. 이를 37 ℃의 습실에서 2 내지 5일간 정치 배양한 다음, 여기에 2N 아세테이트 용액 10 ml을 가하여 10분간 정치시켰다. 이때 성장이 빠르고 점액성물질을 크게 형성하는 콜로니들을 선별하였다. The non-blood mutant strain was again subjected to mutation in the same manner as described above, and plated to form a single colony in Todd Hewitt agar medium containing 400 ug of hyaluronic acid and 1% albumin fraction V per ml of culture. This was incubated for 2 to 5 days in a 37 ° C. wet room, and then 10 ml of 2N acetate solution was added thereto for 10 minutes. At this time, the colonies were selected for rapid growth and large formation of mucus.

선별한 콜로니들은 각각 히아루론산 생산용 배지 1.5 L에 접종하여 35 ℃, pH 6.95 내지 7.05에서 20시간 동안 호기배양하였다. 배양액을 회수하여 25 ℃에서 디지털 점도계(Brookfield DVII+, 4 spin, 30rpm)를 사용하여 절대점도를 측정하고, 일부는 유기용매로 침전시킨 후 다시 증류수에 녹여 카바졸방법(Z. Dische, J. Biol. Chem. 167, 189(1947))에 의하여 히아루론산의 생산량을 정량하였다. 상기의 분석을 통해 배양액의 절대점도가 높고, 히아루론산의 생산량이 높은 균주를 선별하였다.Selected colonies were inoculated in 1.5 L of hyaluronic acid production medium and incubated for 20 hours at 35 ° C., pH 6.95 to 7.05. Recover the culture and measure the absolute viscosity using a digital viscometer (Brookfield DVII +, 4 spin, 30rpm) at 25 ℃, part of it precipitated with an organic solvent and dissolved in distilled water again Carbazole method (Z. Dische, J. Biol) Chem. 167, 189 (1947)) was used to quantify the production of hyaluronic acid. Through the above analysis, strains with high absolute viscosity and high yield of hyaluronic acid were selected.

선별한 균주는 16S rDNA의 염기서열을 분석하여 동정하였고(Jukes, T. H. & C. R., (1969). In mammalian protein metabolism, pp. 21-132; Edited by H. N. Munro., Saito, N. & Nei, M.(1987) Mol Biol vol 4, 406-425), 이는 스트렙토코커스 속에 속한 균주임을 확인하여 스트렙토코커스 에스피(Streptococcus sp.) KL0188로 명명하였다.Selected strains were identified by analyzing the nucleotide sequence of 16S rDNA (Jukes, TH & CR, (1969) .In mammalian protein metabolism, pp. 21-132; Edited by HN Munro., Saito, N. & Nei, M (1987) Mol Biol vol 4, 406-425), which identified it as a strain belonging to the Streptococcus genus and named it Streptococcus sp. KL0188.

상기 스트렙토코커스 에스피 KL0188은 2002년 5월 10일자로 대한민국에 소재한 유전자 은행에 기탁번호 KCTC 10248BP로 기탁하였다.The Streptococcus sp KL0188 was deposited on May 10, 2002 with the accession number KCTC 10248BP to the Gene Bank, South Korea.

실시예 2. 히아루론산 생산성 검증Example 2. Hyaluronic Acid Productivity Verification

스트렙토코커스 에스피 KL0188을 배양하여 히아루론산 생산효율 및 생산된 히아루론산 분자량을 측정하였다.Streptococcus sp KL0188 was cultured to measure hyaluronic acid production efficiency and produced hyaluronic acid molecular weight.

상기에서 분리한 미생물은 100 ml의 토드-휴이트 배지에 접종하고, 35℃로 대수기까지 배양하여 이를 1차 종 배양액으로 사용하였다. 1차 종배양액은 1 L의 트립틱 소이 브로스(Difco, USA)에 접종하고, 35℃로 대수기까지 배양하여 2차 종 배양액으로 준비하였다. The microorganisms isolated above were inoculated in 100 ml of Todd-Hewitt medium, incubated at 35 ° C. to log phase and used as the primary species culture. Primary seed culture solution was inoculated in 1 L of Tryptic Soy Broth (Difco, USA), and incubated at 35 ° C. to log phase to prepare a secondary seed culture solution.

30 L 발효조에 60 g/L의 글루코즈, 5 g/L의 이스트엑기스, 17 g/L의 카제인 펩톤, 7 g/L의 글루탐산, 0.7 g/L의 황산마그네슘, 2.5 g/L의 인산제이칼륨 및 5.0 g/L의 염화나트륨을 포함하는 히아루론산 생산용 배지 20 L를 넣어 멸균하고, 2차 종배양액 1000 ml을 접종하였다. 배양액의 pH는 6.95 내지 7.05, 온도는 35 ℃, 통기량은 1.0 VVM으로 유지하면서 호기 배양하였다. 배양기간 중 일정량의 시료를 취하여 배양액의 점도를 측정하였고, 더이상 배양액 점도가 증가하지 않을 때까지 배양하였다. 20시간 배양하였을 때 배양액 점도가 더 이상 증가하지 않는 것으로 측정되어 배양을 종결하였다. 최대 점도는 약 29,000 cps였다. 60 g / L glucose, 5 g / L yeast extract, 17 g / L casein peptone, 7 g / L glutamic acid, 0.7 g / L magnesium sulfate, 2.5 g / L dipotassium phosphate in a 30 L fermenter And 20 L of hyaluronic acid production medium containing 5.0 g / L sodium chloride were sterilized and inoculated with 1000 ml of the secondary seed culture solution. The pH of the culture was 6.95 to 7.05, the temperature was 35 ° C., and the aeration was incubated while maintaining the airflow at 1.0 VVM. During the incubation period, a certain amount of sample was taken to measure the viscosity of the culture solution, and cultured until the culture medium viscosity no longer increased. Cultures were determined to no longer increase when incubated for 20 hours to terminate the culture. Maximum viscosity was about 29,000 cps.

배양액중에 존재하는 히아루론산은 공지된 다당류 분리정제방법(J. Soc. Cosmet. Japan. 22, 35-42(1988))에 따라 실시하여 회수하였다. 이후 히아루론산은 카바졸 방법(Z. Dische, J. Biol. Chem. 167, 189(1947))에 의하여 정량하였고, 모세관 점도계에 의한 방법(Narlin, Analytical Biochemistry 147, 347-395(1985))으로 생산된 히아루론상의 평균 분자량을 측정하였다. 그 결과 히아루론산 생산량은 7.0 g/L으로 확인되었으며, 평균 분자량은 5,500,000 달톤이었다. Hyaluronic acid present in the culture was recovered by carrying out the known polysaccharide separation and purification method (J. Soc. Cosmet. Japan. 22, 35-42 (1988)). Subsequently, hyaluronic acid was quantified by the carbazole method (Z. Dische, J. Biol. Chem. 167, 189 (1947)), and produced by a capillary viscometer method (Narlin, Analytical Biochemistry 147, 347-395 (1985)). The average molecular weight of the prepared hyaluronic phase was measured. As a result, hyaluronic acid production was confirmed to be 7.0 g / L, the average molecular weight was 5,500,000 Daltons.

비교예 3. 스트렙토코커스 주에피데미쿠스의 히아루론산 생산성 검정Comparative Example 3. Hyaluronic Acid Productivity Assay of Streptococcus Jupitemicus

스트렙토코커스 주에피데미쿠스(KCTC3318)을 상기 실시예 2와 동일한 방법으로 배양하고, 수득한 히아루론산의 생산성 및 분자량을 측정하였다. 스트렙토코커스 주에피데미쿠스(KCTC3318)은 실시예 2와 동일한 조건에서 배양하였을 때 배양 24시간 후 배양액의 점도가 더 이상 증가하지 않아 배양을 종결하였다. 이때 측정된 점도는 약 4,000 cps였고, 히아루론산 생산량은 3.0 g/L이었으며, 평균 분자량은 2,500,000 달톤이었다. Streptococcus epidemicus (KCTC3318) was incubated in the same manner as in Example 2, and the productivity and molecular weight of the obtained hyaluronic acid were measured. Streptococcus epidemicus (KCTC3318) was incubated in the same conditions as in Example 2 after 24 hours of incubation and the viscosity of the culture solution no longer increased, the culture was terminated. The viscosity measured at this time was about 4,000 cps, the yield of hyaluronic acid was 3.0 g / L, and the average molecular weight was 2,500,000 Daltons.

본 발명의 스트렙토코커스 에스피 KL0188는 스트렙토코커스 주에피데미쿠스에 비하여 히아루론산 생산량이 매우 우수하고, 히아루론산의 평균 분자량 역시 높은 것으로 확인되었다. Streptococcus sp KL0188 of the present invention was very excellent in hyaluronic acid production compared to Streptococcus epidemius, and the average molecular weight of hyaluronic acid was also confirmed.

본 발명의 스트렙토코커스 에스피 KL0188은 비용혈성 균주로, 고분자의 히아루론산을 고수율로 생산한다. 따라서, 스트렙토코커스 에스피 KL0188로부터 생산된 히아루론산은 화장품 및 약제로 사용할 수 있다.Streptococcus sp KL0188 of the present invention is a non-hemolytic strain, producing a high yield of hyaluronic acid of the polymer. Therefore, hyaluronic acid produced from Streptococcus sp KL0188 can be used in cosmetics and pharmaceuticals.

Claims (3)

히아루론산 생산균주 스트렙토코커스 에스피(Streptococcus sp.) KL0188(KCTC10248BP).Hyaluronic acid producing strain Streptococcus sp . KL0188 (KCTC10248BP). 제 1항의 스트렙토코커스 에스피 KL0188(KCTC1024BP)를 배양하여 수득한 배양액으로부터 히아루론산을 분리하는 것을 포함하는 히아루론산 생산방법.A method for producing hyaluronic acid comprising separating hyaluronic acid from a culture obtained by culturing Streptococcus sp KL0188 (KCTC1024BP) of claim 1. 삭제delete
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