기질 및 단백질 변형을 통한 Candida antarctica lipase B의 입체선택성 증가에 대한 연구

Abstract

Enzymes are highly efficient catalysts with extraordinary enantio- and regioselectivity, and they can accept a wide range of complex molecules as substrates. In particular, lipases are the most employed catalysts in organic synthesis to yield optically pure compounds through kinetic resolution. Obtaining enantiomerically pure compounds are important because they are a key factor in pharmaceutical industrial fields. Recently, advance in tailoring enzymes for improving activity and selectivity and combined use of enzymes with chemocatalytic reactions have expanded the role of biocatalysis to produce enantiopure compounds from racemic mixtures. Directed evolution or rational design techniques have proven to be successful for the development of enzymes with either enhanced or inverted enantioselectivity compared to their parental enzymes. Enantiopure tetrahydrofuran-2-carboxylate (THFC) is an important intermediate as well as a building block in the pharmaceutical industry and research. Especially (R)-tetrahydrofuran-2-carboxylate is the first compound that is incorporated into a penem skeleton to give furopenem, a clinically efficient non-natural β-lactam antibiotic. In this thesis, enzymatic kinetic resolution method is used to produce enantiopure tetrahydrofuran-2-carboxylate. Hydrolysis reaction of various esters of tetrahydrofuran-2-carboxylate (THFC) was performed by Candida antarctica lipase B (CAL-B). However, parental enantioselectivity of CAL-B is not high enough (E = 3.4) to produce enantiopure THFC. To improve enantioselectivity of CAL-B, enzyme engineering approach using site-directed mutagenesis is employed with substrate engineering. Through substrate engineering, enantiopreference of CAL-B wild-type is changed from (S)-selective to (R)-selective. And mutanted enzymes like I189Q (E = 13.5) and V190I (E = 11.3) showed higher enantioselectivity than wild-type (E = 5.6).