Formation Of C-C Bonds Catalyzed By Hydrolytic Enzyme And Its Applications Study

Enzymes are expected to be fast, specific and highly stereoselectivity biocatalysts. Research in the area of biocatalytic promiscuity has attracted significant attention from chemists and biochemists in recent years. The term "promiscuity" refers to secondary activities of an enzyme in addition to its primary physiological activity. Enzyme promiscuity provides new possibilities for exploiting enzymatic synthesis in organic chemistry. In this thesis, we developed enzyme catalysis in organic medium to catalyze a series of reactions by screenning several enzymes e.g. lipase, amylase and cellulase. Through the promiscuity enzyme, C-C bond and C=C double bond derivatives were synthesized, and at the same time, the reaction mechanism was study.It was observed that cellulase had a promiscuous ability to catalyze aldol reaction in the organic media. The influence factors including enzyme sources, solvents, temperature, water content and enzyme concentration were systematically examined. It was found that Cellulase from trichoderma viride had the highest catalytic activity in the Aldol reaction of benzaldehyde and acetone with DMSO as the solvent, while the denatured enzyme and the control (in the absence of enzyme) demonstrated low activity in this reaction. When the aldol reaction was achieved at 50 ℃ for 24 h under 15% water content and 10mg/mL enzyme concentration. The yield of 85% was got. These results indicate that the enzymatic tertiary structure plays a critical role, then the mechanism for aldol reaction catalyzed by cellulase was also proposed.In Knoevenagel condensation, we found lipase and amylase showed excellent catalytic activity. The influence factors including enzyme sources, solvents, temperature, water content and enzyme concentration were systematically examined. porcine pancreas a-amylase (PPA) and porcine pancreas lipase (PPL) had high catalytic activity. The experimental parameters of PPA-catalyzed Knoevenagel reaction were optimized and a best condition was selected: the reaction temperature of 30℃, the reaction time of 12 h, water content of 10%, enzyme concentration of 10mg/mL. The yield of 85% was got in ethanol solvent. This protocol has a good adaptability to different substrates in the reaction and good recyclability of enzyme (reused for 6 times without significant loss of activity). A reaction mechanism was supposed to elucidate the catalytic process that the transfer of proton was taken place by the His-Asp dyad.To our surprised, in the process of the lipase-catalyzed Knoevenagel condensation between benzaldehyde and methyl cyanoacetate, the transes-terification of the product in the presence of an alcoholic cosolvent was taken place. PPL was found to tolerate a variety of functional groups on the aromatic ring and produce the highest yields by screenning twelve lipases. The reaction kinetic study showed that the condensation proceeded much faster than the esterification reaction. It was to be proved that lipase from porcine pancreas has higher activity for the promiscuous reaction than naturally occurring esterification catalysis in the organic media.Robinson annulation catalyzed by lipase and amylase in organic media was reported. A one-pot synthesis of the Wieland-Miescher ketone catalyzed by PPL and PPA was presented with high yield. Finally the mechanism for PPL-catalyzed Robinson annulation was also proposed.