Site-directed Mutation Of Lipase CAL-B And Its Enantionselectivity In Kinetic Resolution

Recently, the engineering of enzyme, especially the site-specific mutation, has attracted more and more attention, due to the increasingly understanding of the micro-structure and catalytic mechanism of this biocatalyst. Among all the enzyme reported in the literature, lipase B from Candida antarctic (CAL-B) was one of the most widely investigated enzyme, both academically and industrially, because of its high stereoselectivity towards secondary alcohols and amines. The active site is characterized by the catalytic triad Ser(105)-His(224)-Asp(187), an oxyanion hole assembled by Thr(40) and Gln(106) that stabilizes the transition state of the reaction, and a binding pocket composed of two domains, one for the acyl moiety of the ester and another one for the alcohol part, respectively. This thesis mainly focused on the site specific mutagenesis of the key residue of substrate binding pocket and its influence on the stereoselectivity of the mutant.At first, Trp(104), which is located at the bottom of the alcohol binding pocket, was mutated into smaller amino acid residues, and 29 CAL-B mutants of WB series were acquired. Among them, WB2-WB8 were mono-site mutated mutants; WB9-WB12 were WB7 based mutants of which the amino acid residue at the entrance of the substrate binding pocket (281 and 282) were mutated; WB14 was based on WB9 and the amino acid residue at the acid pocket of enzyme (149) was also mutated; WB22 was based on WB9 and the amino acid residue 144 was mutated as well; WB15-WB25 were WB9 based mutants of which the amino acid residue at the acid binding pocket (144 and 149) were mutated. The gene sequences of these mutants were characterized and confirmed. Five mutants were purified by a nickel column. The SDS-PAGE electrophoresis test confirmed that the amount of other proteins was greatly decreased and the molecular weight of CAL-B or its mutants was around 33kDa. Into the solution of pure enzyme was added surfactants and after lyophilization enzyme powder was successfully obtained.Then, the stereoselectivity of the mutants were thoroughly investigated. The hydrolization of 1-Phenylethanol acetate was chosen as the model reaction and chiral GC was used to analyze the reaction results. An inversion of stereoselectivity was observed, as wild-type CAL-B only catalyzed the hydrolization of R-substrate while most of mutants were S-substrate specific. Ten mutants, which were WB2, WB7, WB11, WB14, WB15, WB16, WB17, WB18, WB20, WB21, displayed adequate catalytic activity and stereoselectivity. After a certain reaction time, the yield was in a range within 30-50%, and ee value could reach up to 99%, with E value above 20. The relation between the stereoselectivity of the mutants and the structure of the substrate was further investigated. For aliphatic substrates, when the length of the aliphatic chain was increased to five carbon atoms, an increasing in catalytic activity and a decreasing in stereoselectivity was observed for the mutants, while the catalytic activity of wild-type CAL-B was decreased rapidly. For aromatic substrates, when the benzene group was substituted by bromo, nitro, or methoxy group, the catalytic activity of the mutants decreased dramatically. It could be deduced that, the substrate binding pocket greatly affected both the catalytic activity and the stereoselectivity of the enzyme. The mutants of CAL-B showed a broad substrate scope, and a series of substrate could be resolved to produce chiral secondary alcoho. The kinetic profile of WB26 and WB27 which were mutanted from WB14 and WB11 catalyzed hydrolization of 1-Phenylethanol acetate was investigated as well.This thesis also investigated the catalytic behave of the CAL-B mutants towards the acyl resolution of 1-Phenylethanol with Vinyl acetate as the acyl donor and toluene as the reaction media. The catalytic activity and stereoselectivity of mutant WB2, WB7, WB 11, WB 14 were investigate in detail. It was found that, all the four mutants were S-substrate specific while wild-type CAL-B only catalyzed the acylation of R-substrate. This phenomenon was in congruent with the observation in the hydrolization experiment. WB7 and WB11 showed the best catalytic activity. Vos were determined with different concentration of substrate for mutant WB2 and WB11. Km and Ktwere then calculated and compared with those of wild-type CAL-B.