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KMID : 0613820070170091248
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Journal of Life Science
2007 Volume.17 No. 9 p.1248 ~ p.1254
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Secretory Overexpression of Clostridium Endoglucanase A in Saccharomyces cerevisiae Using GAL10 Promoter and Exoinulinase Signal Sequence
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Lee Jin-Woo
Lee Jae-Hyung
Kim Yeon-Hee
Seo Jin-Ho
Nam Soo-Wan
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Abstract
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The secretory overexpression of Clostridium thermocellum endoglucanase A gene (celA) was examined in Saccharomyces cerevisiae using Kluyveromyces marxianus exoinulinase (INU1) signal sequence and GAL10 promoter. The two plasmids, pYEG-CT1 with its own signal sequence, and pYInu-CT1 with INU1 signal sequence were introduced to S. cerevisiae SEY2102 and S. cerevisiae 2805 host strains, respectively, and then each transformant was selected on the synthetic defined media lacking uracil. The expression level and secretion efficiency of endoglucanase A was increased by 18~22% and 11%, respectively, by INU1 signal sequence over celA signal sequence. By considering the high level of expression (361 unit/l), plasmid stability (89%), and secretion efficiency (70%), S. cerevisiae 2805 harboring plasmid pYInu-CT1 was selected as the optimal host vector system for the production of cellulose-degrading enzyme and recombinant yeast probiotic. The total expression and secretion efficiency of endoglucanase A was 418 unit/l and 73%, respectively, in the batch fermentation of S. cerevisiae 2805/pYInu-CT1 on galactose medium. The molecular weight of secreted endoglucanase A was found to be greater than 100 kDa, presumably due to the N-linked glycosylation.
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KEYWORD
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Sawdust, rice straw, cotton waste, scanning electron microscope, transmission electron microscope, vacuolation, cytoplasmic material
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