Wednesday, May 7, 2008 - 1:30 PM
12-01
Screening for new cellulase enzymes to enhance the synergistic effect by co-displaying cellulases on the surface of E.coli, LY01
Muhammad Nazmul Karim and Seunghyun Ryu. Chemical Engineering, Texas Tech University, 6th and Canton, MS: 43121, Lubbock, TX 79409
Abstract:
Simultaneous saccharification and fermentation (SSF) steps have been proposed in the literature to reduce the cost of bioethanol production process. A whole-cell biocatalyst system has been developed in our laboratories to directly produce ethanol from cellulose in a single step. The results were very successful in converting cellulose to ethanol. This whole cell biocatalyst was constructed with LY01 (provided by Dr. L. Ingram, University of Florida), which is one of the most developed ethanologenic Escherichia coli strains, used as a host cell. Previously, the cellulase genes, celCCA, celCCE and β-glucosidase from the mesophilic strain Clostridium cellulolyticum , were co-displayed. In this study, by displaying cDNA library of the C. cellulolyticum on the surface of the LY01, we screen for new cellulases, which show enhanced enzymatic synergistic effect. Moreover, cellulose hydrolysis rate is improved by selectively screening enzymes showing higher activity after performing directed evolution. The cell surface displayed enzymes are quantitatively measured using flow cytometric analysis. We are able to control the expression level of three different enzymes, which show higher synergistic activities. In this research we will also study the effects various substrates on the cell-surface display mechanism.
Simultaneous saccharification and fermentation (SSF) steps have been proposed in the literature to reduce the cost of bioethanol production process. A whole-cell biocatalyst system has been developed in our laboratories to directly produce ethanol from cellulose in a single step. The results were very successful in converting cellulose to ethanol. This whole cell biocatalyst was constructed with LY01 (provided by Dr. L. Ingram, University of Florida), which is one of the most developed ethanologenic Escherichia coli strains, used as a host cell. Previously, the cellulase genes, celCCA, celCCE and β-glucosidase from the mesophilic strain Clostridium cellulolyticum , were co-displayed. In this study, by displaying cDNA library of the C. cellulolyticum on the surface of the LY01, we screen for new cellulases, which show enhanced enzymatic synergistic effect. Moreover, cellulose hydrolysis rate is improved by selectively screening enzymes showing higher activity after performing directed evolution. The cell surface displayed enzymes are quantitatively measured using flow cytometric analysis. We are able to control the expression level of three different enzymes, which show higher synergistic activities. In this research we will also study the effects various substrates on the cell-surface display mechanism.
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See more of The 30th Symposium on Biotechnology for Fuels and Chemicals (May 4 -- 7, 2008)