Saccharomyces cerevisiae에서 Bacillus CGTase의 표층발현

Surface Display of Bacillus CGTase on the Cell of Saccharomyces cerevisiae

B. stearothermophilus 유래의 CGTase 유전자(cgtS)를 보유하고 있는 재조합 plasmid pCGTS (4.8 kb)을 효모 표면 발현용 vector인 pYDl (GAL1 promoter)에 subcloning 하였다. 구축된 재조합 plasmid, pYDCGT (7.2 kb)는 S. cerevisiae EBY100에 형질전환하였고, tryptophan이 결여된 SD 배지에서 1차 선별된 형질전환체들을 YPGS배지에서 배양 후 활성 염색을 통하여 CD가 생 성 됨을 확인하였다. 배양시간과 효소반응시간에 따른 반응 산물을 TLC로 분석 한 결과, 배양 12시간째부터 효소활성이 나타났고, 반응 10분 이후부터 CD가 생성되어 시간이 지남에 따라 CD 생성양이 증가하는 것을 확인하였다. 회분 배양한 결과 $25^{\circ}C$와 $30^{\circ}C$에서 CGTase의 최대 활성이 각각 21.3 unit/1 와 16.5 unit/1로 나타났고, plasmid 안정성은 각각 $86\%$와 $82\%$로 나타나 배양온도에 상관없이 plasmid는 비교적 안정하게 유지되었다.

For the expression in Saccharomyces cerevisiae, Bacillus stearothermophilus cyclodextrin glucano­transferase gene (cgtS) in pCGTS (4.8 kb) was subcloned into the surface expression vector, pYD1 (GALl promoter). The constructed plasmid, pYDCGT (7.2 kb) was introduced into S. cerevisiae EBY100 cells, and then yeast transformants were selected on the synthetic defined media lacking tryptophan. The formation of cyclodextrin (CD) was confirmed with active staining of culture broth of transformant grown on starch medium. Enzymatic reaction products with respect to the culture time and the reaction time were examined by TLC analysis. The results indicated that the enzyme activity was exhibited after 12 h cultivation and CD was produced after 10min of enzymatic reaction. When the surface-engineered yeast cells were cultured on galactose medium, maximum activities of CGTase were about 21.3 unit/l and 16.5 unit/l at $25^{\circ}C\;and\;30^{\circ}C$, respectively. The plasmids stability showed about $80\%\;even\;at\;25^{\circ}C\;and\;30^{\circ}C$.