Enhanced Stereoselectivity And Broadened Substrate Scope Of Rhodobacter Sphaeroides Esterase And A Study On Their Mechanism

Carboxyl esterase （EC3.1.1.1） can hydrolyze ester bonds with good activity and high stereoselectivity, which is also stable in organic solvents. Since then, they have already been applied in food, agriculture, medicine and other industry fields for the synthesis of a series of important chiral compounds. In this study, a rarely reported esterase was cloned from Rhodobacter sphaeroides by molecular biological technology and taken into directed evolution to obtain desired stereoselectivity and broadened substrate scope.The directed evolution of esterase RSP₂728for advanced enantioselectivity was carried out. An efficient high-throughput screening method which based on pH indicator-bromothymol blue’s sensitivity to the formation of acid following the hydrolysis of R, S-methyl mandelate were established. After three rounds of error-prone PCR, three mutants YH （Asn62Tyr, Leu145His）, CH （Asn62Cys, Leu145His） and CVH （Asn62Cys, Met121Val and Leu145His） were obtained. Compared with the wild-type esterase, the enantioselectivity of the three mutants has been improved2.8,4.5and9.8folds, which was from3.13to8.77,14and30.8.To elucidate if the above approach was possible to improve the esterase with desired property, all of the three variants were taken into substrate scope investigation. And it was intriguing that variant CH acquired from the second round mutagenesis became a more attractive mutant rather than triple mutant CVH. In an effort to throw some light on the origin of enhanced enantioselectivity and broadened substrate scope by directed evolution, the X-ray structure of the WT esterase was newly acquired.Based on the crystal structure, molecular docking and molecular dynamics simulation were taken into study. Theoretical analysis of MD results showed that the catalytic residues and mutated residues were not the crucial factors for the origin of enhanced selectivity. Residues within a distance of10A to the binding esters were taken into investigation, and the interaction energies between those residues and the esters were seem to be related to the desired property. The energy differences between the R and S-methyl mandelate were increased from0.8kJ/mol for the WT to9.4kJ/mol for the third generation variant CVH, which intuitively indicated that S-methyl mandelate was more likely to be catalyzed than R-methyl mandelate along the evolutionary step. Hence, the energy partitioning was consistent with the experimental findings and directed evolution turned out to be a useful and efficient mean. In conformation analysis, the adjacent residues were taken into consideration and it was found that Phe116and Gly201moved more close, thus increased the intra-molecular interaction of respective atoms between each other. Same phenomenon was found between Val170and Trp73. These additional interactions derived from a strong cooperative effect of two mutations resulted in decreasing the size of binding pocket, thus it strengthened the competition between two enantiomers.Finally, the trans-esterification catalyzed by Rsp esterase was carried out and the optimal reaction condition was obtaind, while the catalytic property was found to be different from hydrolysis. The wild type esterase turned out to the most active and got the highest enantioselectivity. Therefore, the screening method used in directed evolution should be modified in order to obtain excellent enzyme in organic solvents.