미생물학회지 (Korean Journal of Microbiology)

  • 제41권1호
  • /
  • Pages.87-92
  • /
  • 2005
  • /
  • 0440-2413(pISSN)
  • /
  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

Streptomyces griseus IFO13350 유래 sprA 및 sprB 유전자를 이용한 Pretense 생산균주 개발

Development of a Recombinant Streptomyces griseus with sprA and sprB Genes for Proteolytic Enzyme Production

  • 황지환 (경남대학교 식품생명공학부) ;
  • 이창권 (건국대학교 바이오 식의학 연구센터) ;
  • 이강무 (경남대학교 식품생명공학부) ;
  • 조병기 (경남대학교 식품생명공학부) ;
  • 박해룡 (경남대학교 식품생명공학부) ;
  • 황용일 (경남대학교 식품생명공학부)
  • Hwang Ji-Hwan (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Lee Chang-Kwon (Bio Food and Drug Research Center, Konkuk University) ;
  • Lee Kang-Mu (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Jo Byoung-Kee (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Park Hae-Ryong (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Hwang Yong-Il (Division of Food Science and Biotechnology, Kyungnam University)
  • 발행 : 2005.03.01
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초록

방선균 Streptomyces griseus에서 상업적 목적으로 생산되는 protease인 protease는 serine protease, alkaline protease, aminopeptidase및 carboxypeptidase로 구성되어 있는 복합체로서 의 약용 소염제로 널리 사용되어지고 있다. 본 연구에서는 기존에 개발되어 있는 방선균용 integration vector인 pSET152로부터 목적산물의 대량발현을 위해 방선균용 promoter ermE가 cloning된 새로운 integration vector인 pHJ101을 개발하였고, pretense의 생산량 증대에 사용하였다. 새로 개발된 integration vector에 S. griseus protease A를 코드하고 있는 유전자, sprA와 S. griseus pretense B유전자, sprB를 각각 cloning하여 plasmid pHJ201과 pHJ202를 구축하였다. 이들 plasmid들을 S. griseus IFO 13350에 형질전환하여 발현용plasmid가 chromosome에 integration된 재조합 균주 S. gliseus HA와 S. griseus HB를 얻었다. 이들 재조합균주로부터 전체 protease의 생산량을 확인한 결과, 모균주보다 각각 S. griseus HA는 약 5.3 배, S. griseus HB는 약 5 배 정도 생산량이 증대되었다. 이들 결과로부터 특정유전자의 고발현용 integration vector의 제작이 확인되었으며, 전체 protease의 생산량 증대의 가능성이 시사되었다.

Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp.

키워드

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