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Saccharomyces cerevisiae에서 발현된 Endoinulinase를 이용한 Inulooligosaccharides의 생산

Production of Inulooligosaccharides by Endoinulinase Expressed in Saccharomyces cerevisiae

김현진|권현주|김병우|남수완|김현철
한국미생물·생명공학회 (The Korean Society For Microbiology And Biotechnology)|2005.12|한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)|제33권 제4호|pp. 281-287

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초록

Paenibacillus polymyxa 유래의 endoinulinase 유전자(inu,2.733 kb, E.C. 3.2.1.7)를 Saccharomyces cerevisiae에 발현시키기 위해 대장균과 효모의 shuttle vector (GAL1promoter 함유)에 subcloning하여 구축된 pYGENIU27 (8.6kb) plasmid를 S. cerevisiae SEY2102에 형질전환하였다.Uracil이 결핍된 SD 배지와 inulin이 포함된 배지에서 효모형질전환체를 선별하였다. 효모 형질전환체의 배양에서endoinulinase는 periplasmic space에 다량 존재함이 확인되었다. YPDG 배지에서 재조합 endoinulinase는 총 활성 1.81unit/ml으로 생산되었다. Inulooligosaccharides (IOSs) 생산을 위한 효소의 최적 반응 조건으로 pH 8.0, 반응온도 45oC,기질은 Jerusalem artichoke로 결정되었다. 효소활성은 pH10.0에서도 안정적으로 유지되었으며, 최적 반응 조건 (ginulin당 36 unit 효소 이용)에서 반응 10분 후부터 IOSs가생산되기 시작하였다. 생성된 IOSs의 구성분으로 inulobiose(F2), inulotriose (F3)와 inulotetraose (F4)가 생성되었고, 이중에서 F3가 주성분이었다. 이상의 결과는 효모에서 생산된재조합 endoinulinase를 이용하여 inulin으로부터 기능성 감미소재인 IOSs의 산업적 생산을 위한 기초결과로 활용될 것이다.

Abstract

The endoinulinase gene (inu, 2.733 kb, EC 3.2.1.7) from Paenibacillus polymyxa was subcloned into an Escherichia coli-yeast shuttle vector with GAL1 promoter for the expression in Saccharomyces cerevisiae. The constructed plasmid, pYGENIU27 (8.6 kb) was introduced into S. cerevisiae SEY2102 cell and then the yeast transformant was selected on the synthetic defined media lacking uracil and on the inulin-containing media. The recombinant endoinulinase was predominantly localized in the periplasmic space of the yeast cell. The total activity of the endoinulinase reached 1.81 unit/ml by cultivation of yeast transformant on YPDG medium. The optimized conditions determined for the inulooligosaccharides (IOSs) production from inulin were as follows; pH, 8.0; reaction temperature, 45oC; inulin source, Jerusalem artichoke. Enzyme activity was stably maintained up to the pH of 10.0. Under the optimized condition and with endoinulinase of 36 unit/g-inulin, IOSs started to be produced after 10 min of enzymatic reaction. By the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), and inulotetraose (F4) were produced and F3 was the major product. Consequently, these data would be used as a fundamental parameters for the production of functional sweetener IOSs from inulin by recombinant yeast endoinulinase.

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