Engineering Nonaqueous Solvent-Compatible Enzymes

Given current powerful tools for protein molecular engineering and a continually improving understanding of protein folding and stability, it is possible to construct vastly improved industrial enzyme catalysts. Enzymes are better suited to industrial syntheses if they can be made stable and active in high concentrations of polar organic solvents. Sitedirected mutagenesis has been used to test two "design rules" for stabilizing enzymes in organic media. We have shown that surface charge substitution and the introduction of metal-chelating sites provide simple and generally-applicable mechanisms for enzyme stabilization. A variant of α-lytic protease containing two surface charge substitutions is 27 times more stable than wild-type enzyme in 84% DMF. Random mutagenesis and rapid screening techniques have been used to isolate enzyme variants with enhanced catalytic activity in polar organic solvents. Our first experiments resulted in a variant of subtilisin E that is 38 times more active in 85% DMF than the wild-type enzyme. Subsequent rounds of random mutagenesis and screening have yielded a variant 256 times more active in 60% DMF.