Secretion of thermophilic bacterial cellobiohydrolase in Saccharomyces cerevisiae |
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| Institution: | 1. Department of Biotechnology, Faculty of Engineering, Nagoya University, Chikusa-ku 464-01, Japan;2. Center of Gene Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 724, Japan;1. Institute for Food, Nutrition and Well-being and Department of Food Science, University of Pretoria, Private Bag X20, Hatfield 0028, South Africa;2. Department of Agricultural and Biological Engineering, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47907, USA;3. Department of Food Science, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47907, USA;1. Department of Environmental and Bio-chemical Engineering, Sun Moon University, Chungnam 31460, Republic of Korea;2. Department of Life Science and Bio-chemical Engineering, Sun Moon University, Chungnam 31460, Republic of Korea;1. Tomas Bata University in Zlín, Department of Food Technology, nám. T. G. Masaryka 5555, Zlín, Czech Republic;2. Tomas Bata University in Zlín, Department of Food Analysis and Chemistry, nám. T. G. Masaryka 5555, Zlín, Czech Republic;3. Mendel University in Brno, Department of Crop Science, Breeding and Plant Medicine, Zemědělská 1, 61300 Brno, Czech Republic;1. Department of Chemistry, Faculty of Science, Atatürk University, 25240 Erzurum, Turkey;2. Research Laboratory of Advanced Water and Wastewater Treatment Processes, Department of Applied Chemistry, Faculty of Chemistry, University of Tabriz, 51666-16471 Tabriz, Iran |
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| Abstract: | The partial DNA sequence corresponding to the N-terminal amino acid sequence of cellobiohydrolase derived from a thermophilic anaerobe NA10 was determined. The cellobiohydrolase gene fused to the secretion signal (signal peptide and T-S region) from Saccharomyces diastaticus was expressed in an ethanologenic yeast, S. cerevisiae YIY345, under control of the glucoamylase promoter. The recombinant yeast produced cellobiohydrolase: approximately 40% of the total cellobiohydrolase activity was detected in the medium, and the remaining cellobiohydrolase was localized in the intracellular fraction. An analysis of the N-terminal amino acid sequence of the main intracellular cellobiohydrolase revealed that the signal peptide and T-S region were removed proteolytically. Alteration of the amino acid residues at the cleavage site by insertion of a Bgl II linker led to an approximately 3.5-fold increase in the total cellobiohydrolase production, but did not affect the efficiency of secretion into the medium. Cellobiohydrolase production was not repressed in the presence of glucose. The recombinant yeast hydrolyzed carboxymethyl cellulose in the medium. The results suggest the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast. |
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