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Fast identification of thermostable beta-glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach

Authors

  • Wenjin Liu,

    1. Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, Virginia 24061; telephone: 540-231-7414; fax: 540-231-7414
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  • Jiong Hong,

    1. Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, Virginia 24061; telephone: 540-231-7414; fax: 540-231-7414
    2. School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, P.R. China
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  • David R. Bevan,

    1. Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
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  • Y.-H. Percival Zhang

    Corresponding author
    1. Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, Virginia 24061; telephone: 540-231-7414; fax: 540-231-7414
    2. Institute for Critical Technology and Applied Science (ICTAS), Virginia Polytechnic Institute and State University, Blacksburg, Virginia
    3. DOE BioEnergy Science Center (BESC), Oak Ridge, Tennessee
    • Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, Virginia 24061; telephone: 540-231-7414; fax: 540-231-7414.
    Search for more papers by this author

Abstract

Engineering costly cellulases on natural cellulosic substrates is of importance for emerging biomass-based biorefineries. Directed enzyme evolution is becoming a popular tool, but identification of desired mutants from a large mutant library remains challenging sometimes. In this work, we demonstrated a novel combinatorial selection/screening strategy for finding thermostable beta-glucosidase on its natural substrate—cellobiose. First, selection was conducted through complementation of beta-glucosidase for non-cellobiose-utilizing Escherichia coli so that only the cells expressing active beta-glucosidase can grow on a M9 synthetic medium with cellobiose as the sole carbon source (selection plate). Second, the clones on the selection plates were duplicated by using nylon membranes. After heat treatment, the nylon membranes were overlaid on M9/cellobiose screening plates so that remaining activities of thermostable beta-glucosidase mutants hydrolyzed cellobiose on the screening plates to glucose. Third, the growth of an indicator E. coli strain that can utilize glucose but not cellobiose on the screening plates helped detect the thermostable beta-glucosidase mutants on the selection plates. Several thermostable mutants were identified from a random mutant library of the Paenibacillus polymyxa beta-glucosidase. The most thermostable mutant A17S had an 11-fold increase in the half-life of thermoinactivation at 50°C. Biotechnol. Bioeng. 2009;103: 1087–1094. © 2009 Wiley Periodicals, Inc.

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