5-02: Improving the synthetic activity of Candida antarctica lipase B via mutagenesis

The growth of the biodiesel industry has resulted in an excess of glycerol, driving down its market value. Low-cost glycerol offers an interesting opportunity to produce biomass-derived raw materials and chemical intermediates that have previously been manufactured from petroleum. Converting the surplus glycerol into a higher value product will enhance the economics of biodiesel production. It was found that Candida antarctica lipase B effectively catalyzes the conversion of glycerol to glycerol carbonate, a versatile chemical that may be used as a solvent or additive by itself, or as a platform chemical for other industries. Under the right conditions, nearly 100% glycerol conversion was achieved after 12 hours of reaction time, with the primary product being glycerol carbonate.

Two lipase properties were selected for improvement by protein engineering: product selectivity and enzyme activity under a broader range of reaction conditions. Candida antarctica lipase B was cloned into the pET22-b(+) vector and expressed in Escherichia coli Rosetta-gami B(DE3). The lipase was modeled in Molecular Operating Environment modeling program, and residues affecting the shape of the active site were identified as candidates for site-directed mutagenesis. Surface residues were targeted to decrease the effect of reaction water concentration on enzyme activity. We will be reporting the identity of beneficial mutations, the degree of enhancement obtained, and postulate structure-function mechanisms.