Functional Characterization Of Microbial Esterases And Their Application In Synthesis Of Chiral Compounds

Esterases are important industrial enzymes.They can catalyze several reactions,such as esterification,transesterification and hydrolysis.So,they are widely used in the fields of food,pharmaceuticals,fine chemical industry,agriculture and environmental management.In this study,two novel esterases from a bacterial strain from soil and a marine bacterium from Western Pacific,respectively,were cloned and characterized.At the same time,it was found that both of them have the ability of kinetic resolution.From the genome of Pseudomonas antitumoralis HUP007,an esterase gene EstP8 was cloned.According to the phylogenetic tree,esterase EstP8 belongs to the IV family of lipases and contains conserved HGGG motif.The optimal substrate of esterase EstP8 was p-NP C2,the optimal working temperature of esterase EstP8 was 50 °C and the optimal working pH of esterase EstP8 was 8.0.The hydrolysis activity of esterase EstP8 toward p-NP C2 was 105.19 U/mg,with Vmax and Km being 89.4 ?mol/min and 1.144 mM,respectively.EstP8 exhibited very good resistance to most metal ions tested.The addition of Cu2+,Mn2+ or Zn2+ of low concentrations could even stimulate the activity of EstP8.Organic solvents such as isooctane,methylbenzene,acetone and DMF could stimulate the activity of Est P8.Esterase EstP8 could generate chiral(R)-1-Phenylethanol through kinetic resolution.The addition of organic solvents could well stimulate the stereo-selectivity and conversion during kinetic resolution.The e.e.and conversion of generated(R)-1-Phenylethanol could reach 91% and 18%,respectively,in the presence of methylbenzene.The e.e.and conversion of generated(S)-styrallyl acetate could reach 98% and 60%,respectively,in the presence of DMSO.Psychrophilic esterase EstP8 possesses good potential in diverse industries such as stereo-selective biocatalysis.A novel esterase EstC10 from Bacillus sp.CX01 isolated from the deep sea of the Western Pacific Ocean and the functionalities of EstC10 was characterized.According to the phylogenetic tree,EstC10 was classified to the XIII esterase family with a typical ?/? hydrolase conservative pentapeptides GXSXG.The optimum substrate of EstC10 was p-NP C2 with hydrolytic activity of 310 U/mg.The optimum pH and pH of EstC10 were 8.0 and 35 °C,respectively.Isooctane and hexane did a positive activity toward EstC10 as well as Tween-20 and sodium tripolyphosphate,while Cu2+ and Zn2+ inhibited the activity of EstC10.Deep-sea microbial esterase EstC10 was developed as a novel biocatalyst in the kinetic resolution of racemic methyl 2-chloropropionate and generate(R)-methyl 2-chloropropionate with high enantiomeric excess(> 99%)after the optimization of process parameters such as pH,temperature,organic co-solvents,surfactants,substrate concentration and reaction time.At present,the reports about the kinetic resolution of racemic methyl 2-chloropropionate were quite rare.Therefore,esterase EstC10 has great potential value of application.