Rational enhancement of the enantioselectivity of Candida antarctica lipase B in kinetic resolution of N-(2-ethyl-6-methylphenyl) alanine

Conference Dates

September 24-28, 2017


The lipase B from Candida antarctica (CalB)-catalyzed enantioselective resolution of N-(2-ethyl-6-methylphenyl)alanine (NEMPA) from the corresponding racemic methyl ester has been investigated. However, the enantioselectivity of CalB towards the resolution is not high enough to obtain enantiomerically pure products. With the aim to improve lipase CalB enantioselectivity, the effects of microenvironment on lipase CalB-catalyzed enantioselective resolution were initially investigated, and the high enantioselective ratio of CalB (e.e.p=90.5%) is achieved in THF/buffer (35% v/v). Introduce additives such as amides and amino acids in THF/water reaction system, the enantioselective ratio of CalB can be improved further (e.e.p is up to 98.6%). Structural studies with IR spectroscopy and Michaelis–Menten kinetics show that the established strong hydrogen bonds interactions between enzyme and solvents are the main driving force that affects the spatial conformation of the enzyme, which further affect the affinity and enantioselectivity of lipase CalB towards the substrate. Based on these results, we modified the lipase CalB with amine-rich molecules to increase the hydrogen interactions between lipase CalB, reaction solvents and substrates. The resulted lipase CalB tagged with 6-histidine at the N terminal and 10-lysine at the C terminal (6His-CalB-10Lys) indeed show the better enantioselectivity than that of commercial lipase CalB in enantioselective resolution of NEMPA in pure buffer. In particular, polyamine tags fused in CalB can also help achieve high soluble expression of CalB in E. coli and can directly mediate silicification, which leads to rapid formation of a CalB-silica particle complex through a one-step approach. The strategies described are practical process for obtaining high enantiomerically pure (S)-NEMPA using lipase CalB.

This abstract is an original research focused on the enhancement of the enantioselectivity of lipase CalB-catalyzed hydrolysis reaction by rational change of solvents, addition of additives and modification of enzyme. All the research strategies belong to the topics of Biocatalysis and Enzyme engineering. So, it is suitable for publication in Enzyme Engineering XXIV Conference

This document is currently not available here.