Direct Evolution Process for Robust Enzyme Catalysis in Organic Solvents

摘要

Industrial applications of enzymes have been limited by the availability ofrobust, reliable and economical protein-based catalysts. The goal of the work outlined in this proposal was to continue development and commercialization of an iterative process to rapidly evolve sequence variants of selected enzymes with enhanced industrial potential. This directed evolution approach involves the use of random mutagenic techniques followed by activity screening of thousands of resulting mutant clones under selected assay conditions. Screening for activity in successively higher concentrations of a selected solvent following sequential rounds of mutagenesis results in the accumulation of amino acid substitutions which synergistically contribute to optimum activity. Phase I results of this work have shown that a percentage of clones expressing the serine protease subtilisin, when subjected to error-prone PCR mutagenesis, and subsequently screened for improved ligase activity in dimethylformamide (DMF), produced enzyme which catalyzed the polymerization of amino acids into extended peptides.