Wenjin Liu1, Xiaozhou Zhang1, Zuoming Zhang1, and Percival Zhang2. (1) Biological Systems Engineering, Virginia Tech, 411 Latham Hall, Blacksburg, VA 24061, (2) Biological Systems Engineering Department, Institute for Critical Technology and Applied Science (ICTAS), Virginia Tech University, 210-A Seitz Hall, Blacksburg, VA 24061
Engineering costly cellulases on natural cellulosic substrates is of importance for emerging biomass-based biorefineries. Complicated relationship among heterogeneous cellulose and different action mode cellulase components results in great challenges for cellulase engineering. Most times, cellulase activities on cellulose analog substrates (e.g., soluble substrate or chromogenic substrates) have no relationship with their activities on natural substrates. Directed enzyme evolution is becoming a popular tool, but identification of the desired mutants from a large mutant library remains challenging sometimes. For beta-glucosidase, we have designed a novel combinatorial selection/screening approach for fast identification of thermostable beta-glucosidase mutants on cellobiose. 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 thermostability at 50°C. In addition, we also attempted to improve the family 48 exoglucanase and the family 5 endoglucanase through directed evolution. The mutant libraries are cell-surface displayed in E. coli or secretory across membrane in Bacillus. Our preliminary results clearly supported the technical feasibility of cellulase engineering through directed evolution.
