Rational enhancement of enzyme-catalyzed enantioselective reaction by construction of recombinant enzymes based on additive strategy.

A rational enhancement of kinetic resolution process for producing (S)-N-(2-ethyl-6-methylphenyl) alanine from racemic methyl ester using lipase B from Candida antarctica (CalB) was investigated. With the benefit results that lipase CalB-catalyzed reactions can be effectively regulated using amino acids (such as histidine and lysine) as additives, CalBs modified (mCalBs) by n-histidines at the N terminal and n-lysines at the C terminal were constructed and expressed. The results show that both soluble and precipitated mCalBs can effectively catalyze the hydrolysis reaction without adding any extra additives. The enantioselective ratio (E value) of soluble and precipitated mCalBs could be improved from 12.1 to 20.3, which were higher than that (E value was only 10.2) of commercial Novozym 435 (immobilized CalB). The study indicated that the amino acid-rich molecules introduced on lipase CalB can produce positive effects on enantioselectivity of enzyme. It provides unusual ideas for reasonable regulation of enzyme-catalyzed reactions.