Cloning, Expression, Screening And Directed Evolution For Enhanced Enantioselectivity Of Esterases

Esterases (EC3.1.1.x) are the most predominant class of enzymes employed in biocatalysis research. They can hydrolyze a broad range of ester bonds, are usually stable in organic solvents and exhibit good to excellent activity and stereoselectivity. Since then, they have already been applied in food, agriculture, medicine and other industry fields for the synthesis of a series of important compounds. In this article, a series of esterases from Escherichia coli K-12 were cloned and their properties were studied. In addition, the directed evolution strategy was applied to improve esterases'properties and the mutants with enhanced enantioselectivity were obtained.Through GenBank database analysis, thirty one genes predicted to encode esters were collected from Escherichia coli K-12. After PCR, the products were purified and digested by two restriction enzymes. Then the ligation was carried out with vector pET-30a (+) which is digested by the same restriction enzymes. The recombinant expression vectors were transformed into E. coli BL21 cells. The target expressed protein was obtained after induced by IPTG.An efficient screening strategy, combining activity screen and enantioselectivity screen, was established. By applying p-nitrophenyl butyrate as substrate, five enzymes (XL3, XL 10, XL15, XL27, XL31)showing relative high activity were obtained according to the change of absorbance at 405 nm. For the enantioselectivity screening. (R)-and (S)-1-phenylethyl acetates were used as substrate and pH indicator-bromothymol blue was used to monitor hydrolase-catalyzed reaction. According to the change of the absorbance at 630 nm, one enzyme XL15, showing relative high enantioselectivity toward (R)-1-phenylethyl acetate was obtained. The enantioselectivity (E value) is>100 and the conversion can reach to 31.2% and 36.8% respectively both in hydrolysis and esterification of 1-phenylethyl acetates.The study of the enzymes'characteristics showed that the esterases exhibited high activity towards short-chain fatty acids as p-nitrophenyl butyrate (C4), p-nitrophenyl caproate (C6) and the activity of these proteins dropped abruptly toward p-nitrophenyl myristate (C14) and p-nitrophenyl palmitate (C16). These enzymes have maximum activity under different reaction temperature and reaction pH value.The protein expression condition is optimized as below:IPTG,0.1 mmol/L; cell growth rate before induction, OD600=0.7; induce temperature,30℃. The only one target protein (28kDa) was obtained after further purification of XL15. By sequence analysis and made comparison of XL15 with other esterases, it is understood that the enzyme XL15 belongs toα/β/αhydrolase family and with a typical Ser-His-Asp active site. We now predicted that near the active site serine triad, there formed two "pockets"-the large pocket and the medium pocket. The orientation of the methyl group of the chiral carbon for the two enantiomers was related to enzyme's enantio selectivity. During the hydrolysis reaction of 1-phenylethyl acetate, the phenyl group pointed out toward the protein surface, into the large hydrophobic pocket, and the methyl group of the chiral carbon for the R enantiomer binded nicely into the medium hydrophobic pocket and then the enzyme showed high enantioselectivity towards R enantiomer.To enhance enantioselectivity of esterase, the method of directed evolution was applied. The target of esterase herein is RSP₂728 from Rhodobacter sphaeroides. An efficient directed evolution strategy and a high-throughput screening method were established. After four cycles of error-prone PCR technology, four mutants A6A5, B7F11, C8G1 and D3E11 were obtained. Compared with the wild-type esterase, the enantioselectivity of the four mutants was improved from E=3.1 to E=5.3,8.8,14.0 and 30.8 in the hydrolysis of Methyl mandelate and the activity was improved from 10U/mg to 43 U/mg,142 U/mg,88 U/mg and 24 U/mg. According to the sequence analysis, the mutant A6A5 showed amine acid Asn62 was changed into Tyr62; mutant B7F11 showed amine acid Asn62 was changed into Tyr62 and amine acid Leu145 was changed into His145; mutant C8G1 showed amine acid Asn62 was changed into Cys62 and amine acid Leu145 was changed into His145; mutant D3E11 showed amine acid Asn62 was changed into Cys62, amine acid Met121 was changed into Tyr121 and amine acid Leu145 was changed into His145. This result demonstrated that enzyme directed evolution is able to be applied to improve enantioseletivity of enzyme.