Fast carbon-carbon bond formation by a promiscuous lipase

Lipase B from Candida antarctica was redesigned to catalyze the promiscuous reaction of carbon-carbon bond formation. Mutation of the catalytic serine to alanine afforded a mutant that catalyzed Michael additions of 1,3-dicarbonyls to alpha,beta-unsaturated carbonyl compounds at high specific rates, such as 4000 s-1. The enzyme-catalyzed Michael addition reaction followed saturation kinetics and showed substrate inhibition. The designed enzyme showed high rate enhancements with a catalytic proficiency higher than 108, which is on the same level as that observed for enzymes with native substrates.