Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Expression of a $\beta$-1,3-Glucanase Gene from Bacillus circulans in B. subtilis and B. megaterium

Bacillus subtilis와 Bacillus megaterium에서의 $\beta$-1,3-glucanase 유전자의 발현

  • 김기훈 (인제대학교 의생명공학대학 임상병리학과) ;
  • 김지연 (인제대학교 바이오헬스 소재 연구센터) ;
  • 김한복 (호서대학교 자연과학부 생명과학전공) ;
  • 이동석 (인제대학교 의생명공학대학 임상병리학과v)
  • Published : 2001.12.01
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Abstract

A Bacillus circulans KCTC3004 $\beta$-1,3-glucanase gene contained in a recombinant plasmid pLM460 derived from subcloning the original recombinant plasmid pLM530 was trasferred into a new shuttle vector plasmid pLMS1180 by ligating linearized DNAs of pLM460 and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLMS1180 produced the $\beta$-1,3-glucanase substantially. Most of the enzyme was produced during the exponential growth period. The maxium activities of the $\beta$-1,3-glucanase produced by the Bacillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125 (pLM1180) enzyme showed the activity 14 times higher than that of the gene donor cells, followed by the B. megaterium ATCC14945 (pLMS 1180) enzyme with activity 5 times higher than that of the gene donor cells. While E. coli secreted about 7% of the produced enzyme, B. subtilis excreted the enzyme into the medium wholly and B. megaterium about 97% of the total product. The SDS-PAGE of this enzyme produced in E. coli (pLMS1180), B subtilis (pLMS1180) or B. megaterium (pLMS1180) indicated a molecular weight of 38,000. The enzymes overproduced in three different host cells hydrolyzed laminarin to produce mainly laminaribiose, laminaritriose, and laminarioligosaccharides. The plasmid pLMS1180 was stable in B. megaterium, E. coli, but was unstable in B. subtilis.

Bacillus circulans KCT3004 기원의 $\beta$-1,3-glucanase 유전자를 함유한 재조합 플라스미드 pLM460과 pUB110을 이용하여 shuttle 플라스미드 pMLS1180을 제작하고 Bacillus 세포에 이동.발현시켰다. pLMS1180으로 형질전환된 B. subtilis와 B. megaterium은 효율적으로 $\beta$-1,3-glucanase를 생산하였고, 이 효소들은 세포의 증식과 비례하여 생산되었다. 형질전환체가 생산하는 $\beta$-1,3-glucanase의 최대 활성을 유전자 공여 균주인 B. circulans와 비교하여 보니, B. subtilis는 14배, B. megaterium은 5배 정도의 높은 활성을 나타내었다. 그리고 대장균 형질전환체는 분비율이 7% 정도인데 반하여 B. subtilis 형질전환체는 생산된 효소를 전부, B. megaterium 형질전환체는 약 97%를 세포 외로 분비하는 것을 알 수 있었다. SDS-PAGE를 통해 대장균과 B. subtilis, B. megaterium에서 발현된 효소의 분자량을 분석해 보니 약 38,000으로 추정되었다. 또한, 이들 형질전환체가 생산하는 $\beta$-1,3-glucanase는 laminarin에 작용하여 주된 산물로서 laminaribiose (G2), laminaritriose (G3) 이상의 다양한 laminarioligosaccharide들을 생산함이 확인되었다. pLMS1180의 각 숙주 내에서이 안정성을 살펴본 결과 B.megaterium에서는 88%, 대장균에서는 75%, B. subtilis에서는 48%로 나타났다.

Keywords

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