Investigating the Structural and Functional Consequences of Circular Permutation on Lipase B from Candida Antarctica

摘要

The engineering of lipase B from Candida antarctica (CALB) by circular permutation has yielded over sixty hydrolase variants,and several show significantly improved catalytic performance.Here we report a detailed characterization of ten selected enzyme variants by kinetic and spectroscopic methods to further elucidate the impact of circular permutation on the structure and function of CALB.Our experiments identify lipase variants with up to 175-fold enhanced K_(cat)/k_M values over wild-type.In addition,circular permutation does not change the enzymes' enantio-preference and preserves or even improves their enantioselectivity compared to that of the wild-type enzyme.Finally,our spectro-scopic analyses suggest that the structural effects of circular per-mutation on CALB are mostly local,concentrating on regions near the native and new protein termini.The observed changes in secondary structure and protein thermostability vary amonq enzyme variants but directly correlate with the locations of the new termini,a first step towards a predictive framework.