Substrate Profiling And Molecular Evolution Of A Para-nitrobenzyl Esterase For Preparation Of Chiral Aryl Alcohols

The para-nitrobenzyl (pNP) esterase is a special kind of lipolytic enzymes. It belongs to Family VII, but shares the GGG(A)X motif in the oxyanion hole in the hormone-sensitive lipase (HSL) family.pNP esterases have high activity towards esters and promiscuous amidase activity. A pNP esterase from Bacillus subtilis ECU0554(BSE) was reported to exhibit high hydrolytic activity and excellent enantioselectivity towards l-menthyl ester.In this work, a pNP esterase from Bacillus amyloliquefaciens DSM7(BAE) was cloned and overexpressed in E. coli BL21(DE3), which showed62%identity with BSE on protein level. We fingerprinted the substrate profile of BAE, and examined its enantioselectivity in hydrolyzing esters of secondary aryl alcohols. Among them, optically pure1-(3’,4’-methylenedioxyphenyl)ethanol is a key chiral intermediate for synthesis of Steganacin, Podophyllotoxin and its derivatives. This alcohol was selected as the target product of biocatalytic process by BAE. The enantioselectivity of the BAE-catalyzed process was improved respectively by process engineering and protein engineering approaches. This dissertation was divieded into four parts as follows:Part I:Cloning and biochemical characterization of BAE. A "pnbA" gene was cloned from Bacillus amyloliquefaciens DSM7, and its enzyme (BAE) was expressed successfully in the recombinant host Escherichia coli BL21(DE3). BAE was purified using nickel affinity purification system. It fits the reaction temperature under30℃and shows the optimal activity at around pH8.0, and DMSO is a suitable cosolvent for the reaction. BAE shows high activity towards esters of secondary aryl alcohols, but the enantioselectivities are generally unsatisfactory. The effect of various substituents at different positions on the ring on the enantioselectivity was examined, indicates that a charged amino acid in the acitive pocket of BAE may be a key to control its enantioselectivity.Part II:Application of a high-throughput screening method with p-nitrophenol as pH indicator to fingerprint hydrolase by acetates of structurally diverse alcohols. CRL and PLE were used as reference enzymes to test the feasibility of the assay method. Influences of acyl groups with varied chain length on esterase/lipase activities were also investigated. Subsequently, the substrates of four home-made esterases (including BAE) were fingerprinted. Three parameters of these enzymes’substrate profiles were calculated and compared. Among them, the Shannon-Wiener index (usually adopted in papers concerning ecology) was adopted and confirmed feasible for the first time to evaluate the broad-spectrum activities of any enzymes in a quantitative manner.Part III:Optimization of enantioselectivity for BAE asymmetric hydrolysis of1-(3’,4’-methylenedioxyphenyl)ethyl acetate by process engineering strategy. BAE was employed for catalyzing the asymmetric hydrolysis of (RS)-1-(3’,4’-methylenedioxy phenyl)ethyl acetate to produce (R)-alcohol. Although BAE exhibited very high reaction rate, the enantioselectivity at30℃was merely moderate (E=35) like majority of other hydrolases documented. Simply by altering acyl group, adding surfactants and lowering reaction temperature, the enantioselectivity of BAE was remarkably improved. An excellent enantioselectivity and enantiomeric excess of product (E-140,97%ee) was achieved at0℃in the preparative synthesis of (R)-1-(3’,4’-methylenedioxyphenyl)ethanol in a decagram scale using0.5%Tween-80as emulsifier. Furtermore the ratio of substrate to catalyst (S/C, w/w) and reaction time are both better than literature data.Part IV:Improving BAE enantioselectivity for asymmetric hydrolysis of1-(3’,4’-methylenedioxyphenyl)ethyl acetate by molecular evolution approach. We built a homology model of BAE using the the cystale structure1C7I of pNP esterase BsubpNBE. Eight focused libraries around the complete binding pocket were chosen for saturation mutagenesis. The resultant best mutant BAE-V10had significantly improved the enantioselectivity and mantained the high activity level. Several selected mutants were purified and used as catalysts for the resolution of (R)-and (S)-enantiomers, and the kinetic parameters were analyzed. The tetrahedral intermediate of (R)-and (S)-enantiomers were constructed and molecular docking simulations were performed in the model of BAE-WT and mutants so as to analyze the reason for increase in enantioselectivity. Finally, the variant BAE-V10was used into the preparative synthesis of (R)-1-(3’,4’-methylenedioxyphenyl) ethanol in a decagram scale. Better enantioseclectivity (E-180) without using Tween-80and lowering temperature, shorter reaction time (=2.5h) and higher S/C (=40) were achieved, providing a more efficient and cost-effective bioprocess for the economic production of (R)-1-(3’,4’-methylenedioxy)ethanol.