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KMID : 0613820070170020241
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Journal of Life Science
2007 Volume.17 No. 2 p.241 ~ p.247
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Cell Surface Display of Cycloinulooligosaccharide Fructanotransferase Gene in Saccharomyces cerevisiae
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Kim Hyun-Jin
Lee Jae-Hyung
Kim Hyun-Chul
Kim Yeon-Hee
Kwon Hyun-Ju
Kim Byung-Woo
Nam Soo-Wan
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Abstract
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The cycloinulooligosaccharide fructanotransferase(CFTase) gene (cft) from Paenibacillus macerans was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTECFTN (9.0 kb) was introduced to S. cerevisiae EBY100 cell and then yeast transformants were selected on the synthetic defined medium lacking uracil and on the inulin containing medium. The surface display of CFTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form cycloinulooligosaccharides (cyclofructans, CFs) from inulin. The total activity of the CFTase was reached about 5.52 unit/l by cultivation of yeast transformant on YPDG medium. The optimized conditions determined were as follows; pH, 8.0; temperature, 50℃; substrate concentration, 5%; inulin source, Jerusalem artichoke. By the reaction with inulin, CFs consisting of cycloinulohexaose (CF6), cycloinuloheptaose (CF7), and cycloinulooctaose (CF8) were produced and CF6 was the major product.
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KEYWORD
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Cycloinulooligosaccharide fructanotransferase, Saccharomyces cerevisiae, cell surface display, cyclofructan, inulin
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