Biosynthesis Of R-PAC And Its Derivatives And Analogs

R-Phenylacetylcarbinol(R-PAC)is one of the important chiral intermediates in asymmetric synthesis,using as a precursor in the preparation of pharmaceuticals such as L-ephedrine,norephedrine,and phenylpropanolamine in the pharmaceutical industry.In this study,we prepared R-PAC under optimized conditions using the catalytic subunit of acetolactate synthase of Thermotoga maritime(TmAHAS-CSU)as a biocatalyst and pyruvate and benzaldehyde as substrates in the presence of the co-enzyme thiamine diphosphate.The isolated yield of the product R-PAC obtained was 83.6%with a purity of 98.3%determined by HPLC.Furthermore we also tested the substrate spectrum of TmAHAS-CSU by employing derivatives and analogs of benzylaldehyde and the results showed that the enzyme possessed fairly broad substrate adaptability.Firstly,we prepared the biocatalyst by autoinduction using the engineered E.coli strain EcR(DE3)-pET28a-TmAHAS-CSU which can overexpress TmAHAS-CSU.The strain was constructed in our lab in the previous work.After Ni-NTA purification,the purified enzyme was utilized to mediate the synthesis of R-PAC.In addition,we also used the whole cells of the EcR(DE3)-pET28a-TmAHAS-CSU strain as a biocatalyst to produce R-PAC.Secondly,we optimized the reaction conditions to maximize the yield of R-PAC in the reaction catalyzed by TmAHAS-CSU.We found that the optimum conditions for the preparation is as below:50 mM PBS buffer,pH 8.0,reaction time 40 min,temperature 80?,1%DMSO as co-solvent,and the concentrations of pyruvate and benzaldehyde are 50 mM and 40 mM,respectively(best ratio 1.25 to 1).We also found that better results could be generated when the whole cells were used as biocatalyst.Finally,we used the derivatives and analogs of benzaldehyde as the substrates to react with pyruvate under the catalysis of the engineered bacteria strain and the experiments were monitored by HPLC and LC-MS and the products were elucidated by 1H-NMR.The results showed that 13 compounds,such as the methoxylated and/or hydroxylated derivatives of benzaldehyde could be converted by the enzyme.These data not only expand the range of substrate spectrum of TmAHAS-CSU,but also lay a solid foundation for the further study of it.