Screening, Characterization And Application Of Enantioselective Esterases/lipases

Chirality is one of the important attributes in nature. The molecular distinguishing plays an important role in life activity. The two enantiomers of the same compound have not only different optical property and physical-chemistry property, but also have different bioactivity. The conventional methods for obtaining chiral compounds are kinetic resolution (one of the classical methods) of racemic substrates or asymmetric hydrolysis of the corresponding diesters. Nevertheless, when chemical separation reagents were used for the purpose of producing chiral compounds, large amount of chemical separation reagent is required, which is considered highly cost and not environment friendly. Recently, enzymatic kinetic resolution of racemic substrate or enzyme promoted asymmetric synthesis has become popular processes as they owning the property of environmental benign, being proceded under mild reaction conditions, and excellent enantioselectivity. Presently, among of the enzymes used in the preparation of chiral compounds, most are lipases. Nevertheless, the high price of lipases limits their application in large-scale production. For a long time, researchers have been exploring esterases for the purpose of replacing lipases as esterases are easily cloned and expressed at low cost. It is high possible for us to use esterases for the preparation of chiral compounds, which used to be produced by applying lipases as the biocatalysts.This thesis focuses on enzymatic preparation of two important chiral compounds, chiral secondary alcohols and chiral (S)-3-substituted glutaric acid monoester, high-throughput screening for obtaining enantioselective esterase/lipase and their characterization in catalyzing. There are six main aspects included in the thesis, the details are as following:(a) Screening for suitable enzymes from the enzyme pool is required before their application. For this purpose, we developed a high throughput screening method based on actual substrate and fluorescence. Compared with those conventional screening methods established, our developed method has the property of accuracy, reliability and sensibility.(b) An efficient hydrolase(Escherichia coli BioH) with high activity and enantioselectivity was obtained by the developed screening method mentioned above. In PBS solution, this enzyme could resolve an array of sec-alcohols efficiently, and ee values of the desired product reached to 98%. To our interested, the obtained hydrolase had the property of high concentration organic solvent tolerance, and esterses had such excellent performance has never been reported before. Later, this esterase was applied in the resolution of an array of secondary alcohols in non-aqueous media, and good to excellent results were achieved.(c) Several lipases and one esterase were attempt in reactions for asymmetric synthesis of chiral (S)-glutaric acid monoesters, and the initial results indicated that among these eight biocatalysts applied, two of them (Candida antarctic lipase B, (CALB), Lipozym TLIM) could catalyze the reaction of esterification. By comparing all parameters which can affect the enzyme efficiency including times, enzyme loadings, temperatures and solvents, CALB was chosen as the best one for asymmetric synthesis of the desired product. For alleviating labor force and expense as there are numerous alcohols in world, molecular docking was employed to aid and predict the possibility of reactions, and the result obtained indicated the result from reaction is well correlated to the prediction by molecular docking. In conclusion, molecular docking can predict the possibility of reaction, but could not predict the configuration selectivity of the enzyme applied in reaction accurately.(d) The combination of enantioselective esterase and lipase was employed in enzymatic and green resolution of chiral secondary alcohols. The approach was based on the following strategy:(1) In tranesterification procedure, the enzyme-accepted enantiomer was fully transformed to the corresponding esters, which ensured the other isomer leftover in the reaction system be with ee value of no less than 98%; (2) In the subsequent deacylation step, the acylated enantiomer was conducted to deacylation applying CALB as the biocatalyst in organic solvent, and free ammonia was applied as the deacylaton reagent. The enzymatic deacylation process was a highly selective one. After both acylation and deacylation, chiral secondary alcohols were afforded with ee values of more than 99% even the starting substrates for deacylation (the corresponding esters) were in low optical purities. The acyl donors, biocatalyst and organic solvent could be repeatly recycled. And enzymatic deacylation applying ammonia as deacylation reagent avoided the generation of waste salt stream when employing chemicals or in water solution, and meantime avoided the desired products be reacemized. In summary, the fully enzymatic and green resolution procedure was proved to be a green process for resolution of chiral secondary alcohols.(e) Based on the experimental results and results from literatures, we found an interesting distinction between enantioselective esterases and lipases in enantioselectivity. By analyzing and comparing the data obtained from literature and experiment, the enantioselective distinction between the two families of enzyme was laid in two main aspects:(1) Enantioselectivity fluctuation interval is broad for esterases when applying an array of the same type substrates with different substituents, nevertheless, for lipases, the fluctuation interval in enantioselectivity was narrow; (2) The enantioselectivity of esterases is more sensitive towards temperature and whose range is broader compared with lipases. Finally, we found E.coli BioH was indeed an enzyme described as "more esterase than lipase" because in the aspect of catalyzing, it displays the properties of esterase, and what is more, it owns the property of high concentration of organic solvent tolerance, which was thought exclusive for lipases.(f) And next, we investigate the enzyme promiscuity of esterases and lipases based on their accepted substrates and types of esterase/lipase promoted reaction, and the reaction catalyzed by enzymes mentioned in this thesis were ascribed in different types of enzyme promiscuity. Researches on enzyme promiscuity are thought to have potential applications, which will help us in preparation of chiral compounds at low cost.