Research On Asymmetric Addition Reaction Catalyzed By Thermophilic Hydrolases

C-C bond forming reaction is one of the most important reaction in the organic synthetic field because it can be the intermediates for synthetizing some important compounds. This reaction were used to be catalyzed by using metal, strong acid and base as catalysts. It was not only harmful to environment, but also difficult to obtain the high enantioselective products. The traditional approachs to get the chiral compounds were the separation or hydrolysis of racemic substrates by chemical separation reagents, which was considered as a bad effect on environment, so more and more researchers pay attention to find an efficient and safe method used in organic synthetic reaction. In the recent years, enzymatic catalytic promiscuity has been widely used in organic synthesis because of their properties in high effiency, economically feasible and high enantioselectivity.In this work, we used a series of hydrolases cloned by our group to explore enzymatic catalytic promiscuity which was applied to Aldol and Henry reaction. The result presented an Acylaminoacyl Peptidase ST0779 from Sulfolobus tokodaii could be thought as a promising biocatalyst to catalyze the Henry reaction. This enzyme brought out excellent catalytic efficiency kcat/Km(6–8 fold higher) and enantioselectivity ee%(90–99%) after comparing with Porcine Pancreatic Lipase(PPL). The catalytic versatility of ST0779 was identified as the enzyme displayed activity towards a lot of substituted benzaldehydes with different electron donating group and electron withdrawing group, and then the electron effects of the benzaldehyde substituents were analyzed by Hammett plotting. Besides, we investigated the effect of catalytic triad or the amino acid close to active site on catalytic efficiency and enantioselectivity by site directed mutagenesis.In addition, as for the aldol reaction, we found a novel thermostable esterase Tnap0664, which was cloned from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 brought out the high catalytic activity towards the reaction of 4-nitrobenzaldehyde with 2-Butanone. The excellent results were obtainedwith the yield(82%) and the higher enantioselectivity(96%) after optimizing the condition parameters of the reaction. Tnap0664 could be the promising biocatalysts in the field of C-C bond formation reaction.Therefore, this research not only presents the novel enzymes can catalyze the Henry reaction with higher yield and enantioselectivity than ever reported, but also showsthe huge potential of thermophilic archaea to be an optimal source for mining novel enzymes for biocatalytic promiscuity, which could provide a variety of potent biosynthesis approachs to produce different kinds of molecules.