Saccharomyces cerevisiae 에서 발현된 CycloinulooligosaccharideFructanotransferase 을 이용한 Cyclofructan의 생산
Production of Cyclofructan by Cycloinulooligosaccharide Fructanotransferase Expressed in Saccharomycescerevisiae.
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Paenibacillus ploymyxa 유래의 cycloinulooligosaccharide fructanotransferase(CFTase) 유전자(cft)를 Saccharomyces cerevisiae SEY2102에 발현시키기 위해 대장균과 효모의 shuttle vector인 pYES 2.0(GAL1 promoter)에 subcloning하였다. 구축된 pYGCFT(9.9 kb) plasmid를 S. cerevisiae SEY2102에 형질전환하였고, uracil이 결핍된 SD 배지에서 선별하였다. 선별된 형질전환체(S. cerevisiae SEY2102/pYGCFT)는 galactose 첨가에 의해 성공적으로 발현되어 cyclofructan(CF)을 생성함을 TLC로 확인하였다. 그러나 균체외로의 효소 분비는 이루어지지 않았고 cytoplasm 보다 periplasmic space에 많이 존재하였다. 효소반응 3시간부터 CF가 생성됨을 확인하였고, 최적온도와 최적 pH는 각각 45oC와 pH 8.0로 나타났으며, pH 10.0에서도 효소활성이 안정적으로 유지되었다. Inulin 기질에 따른 반응산물 분석 결과, Jerusalem artichoke와 dahlia tuber로부터 CF가 가장 효과적으로 생성되었다.
Abstract
The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus polymyxa was subcloned into the E. coli-yeast shuttle vector, pYES2.0 (GAL1 promoter). The constructed plasmid, pYGCFT (9.9 kb) was introduced into S. cerevisiae SEY2102 cell and then the yeast transformant was selected on the synthetic defined media lacking uracil. Based on the cyclofructan (CF) spots on thin-layer chromatogram, the cft gene under the control of GAL1 promoter was successfully expressed in the yeast transformant. The recombinant CFTase was not secreted into the medium and was predominantly localized in the periplasmic space. CF was started to be produced after 3 h of enzymatic reaction with inulin. The pH and temperature optimum for the CF production from inulin was pH 8.0 and 45oC, respectively. Enzyme activity was stably maintained up to the pH of 10.0. The examination of the inulin sources revealed that a dahlia tuber and Jerusalem artichoke were the best for the production of CF.
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