Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Characterization of Achlya bisexualis $\beta$-Amylase Expression in an Amylolytic Industrial Strain of Saccharomyces cerevisiae

전분 분해성 산업용 Saccharomyces cerevisiae에서 Achlya bisexualis $\beta$-Amylase의 발현 특성 규명

  • Lee, Ok-Hee (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Lim, Mi-Hyeon (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Kim, Ji-Hye (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Ryu, Eun-Hye (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Ko, Hyun-Mi (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Chin, Jong-Eon (Department of Cosmetology, Dongkang College University) ;
  • Bai, Suk (Department of Biological Sciences, College of Natural Sciences, Chonnam National University)
  • 이옥희 (전남대학교 자연과학대학 생물학과) ;
  • 임미현 (전남대학교 자연과학대학 생물학과) ;
  • 김지혜 (전남대학교 자연과학대학 생물학과) ;
  • 유은혜 (전남대학교 자연과학대학 생물학과) ;
  • 고현미 (전남대학교 자연과학대학 생물학과) ;
  • 진종언 (동강대학 피부미용계열) ;
  • 배석 (전남대학교 자연과학대학 생물학과)
  • Published : 2008.09.30
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Abstract

To develop an amylolytic industrial yeast strain producing $\beta$-amylase, the BAMY gene encoding Achlya bisexualis $\beta$-amylase was constitutively expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) in an industrial strain of Saccharomyces cerevisiae. Yeast transformation was carried out by an integration system containing $\delta$-sequences as the recombination site. The integrative cassette devoid of bacterial DNA sequences was constructed that contains the BAMY gene and $\delta$-sequences. Industrial S. cerevisiae transformed with this integrative cassette secreted 45 kDa $\beta$-amylase into the culture medium. The $\beta$-amylase activity of the transformant was approximately 18.5-times higher than that of A. bisexualis. The multi-integrated BAMY genes in the transform ant were stable after 100 generations of growth in nonselective medium. Hydrolysis of soluble starch and various starches with the enzyme released maltose but not glucose or oligosaccharides.

$\beta$-Amylase를 생산하여 전분 분해능을 갖는 산업용 효모를 개발하기 위해 산업용 Saccharomyces cerevisiae에서 Achlya bisexualis $\beta$-amylase (BAMY)유전자를 ADC1 promoter에 연결하여 구성적으로 발현시켰다. 효모의 형질전환은 $\delta$-서열을 재조합 부위로 하는integration 시스템을 이용하였다. Integration 시스템의 세균 유전자 부분은 제거되고 BAMY 유전자와 $\delta$-서열을 갖고 있는 짧은integrative cassette를 제조하였다. BAMY 유전자를 발현하는 재조합 S. cerevisiae 형질전환체는 세포외 배지로 45 kDa의 $\beta$-amylase를 분비하였고, $\beta$-amylase 활성은 A. bisexualis에 비해 약 18.5배 높았다. 형질전환체에 다중도입된 BAMY 유전자는 비선택배지에서 100세대 생장 후에도 안정되게 유지되었다. 각종전분을 기질로 했을 매 $\beta$-amylase의 활성은soluble starch를 기질로 했을 경우와 유사하게 높았고, 가수분해산물 분석 결과 maltose가 주 분해산물이었다.

Keywords

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