Study On The Synthesis Of Chiral Drug Intermediates By Microbial Enzyme

Due to the high catalytic efficiency, high stereo specificity and mild reactionconditions, biocatalysis successfully meet the green industrial requirements ofenvironmental friendly and resource conservation compared with traditional chemicalcatalysis. Chiral compounds, including chiral drugs, chiral materials and chiralintermediates, as the main products by biocatalysis have been widely applied inpharmaceutical, chemical and food industries. Whole-cell biocatalysis, as one of themain methods of biocatalysis, uses whole cells of microorganisms as catalyst withoutadding cofactors for coenzyme regeneration in-vitro. Since these cofactors such asNADP+and NADPH are very expensive, whole-cell biocatalysis becomes moreconvenient and economic.In this essay, two kinds of intermediates of chiral drugs, levetiracetam acid and(R)-α-phenylethanol, were synthesized in high conversion and ee value by two kindsof whole cells of microorganisms, respectively. In the second part, the method tosynthesize levetiracetam acid by biocatalysis was studied. According to the structureof levetiacetam, a novel path to hydrolyze levetiracetam ester in high stereospecificity by screening microorganisms from nature was designed. Following thepath, a strain which can synthesize levetiracetam acid in high enantioselectivity wassuccessfully screened out from the soils. Combined with the patterns of colony, itsphysio-biochemical properties and16S rDNA sequencing analysis, the strain wasnamed as Sphingobacterium sp. SIT102. In the third part, the optimum conditions offermentation of Sphingobacterium sp. SIT102were tested and resulted in glucose10g/L, peptone20g/L, MgSO40.5g/L，KH2PO40.5g/L，K2HPO40.5g/L，pH7.0, at30°C,180rpm for36h. In addition, the biomass and specific activity of enzyme wasincreased from2.41g/L to3.53g/L and1.74U/g to3.16U/g, repectively. What’smore, the optimum conditions of reaction were also tested in the fourth part. Underthe conditions of substrate content5mM, biomass10gdcw/L, pH7.5, at35°C,180rpmfor20h, the conversion, ee value of product and E value can reach48.3%,96.5%and175.7, respectively. In the fifth part, a new method was designed to enhance the ee value of(R)-α-phenylethanol reduced from acetophenone. This reaction was catalyzed bypichia etchellsii AS2.625which was an available strain in our group. Several kinds oforganic solvents, surfactants and cyclodextrins (CDs) were chosen to add into thisreduction reaction as co-solvents, such as Cyclohexane,2-Propanol, Tween80,[BMIM][BF4], DMSO, α-CD, β-CD, γ-CD, Methyl-β-CD, Hydroxypropyl-β-CD andSulfobutylether-β-CD. As a result, β-CD performed the best enhancement in the eevalue of (R)-α-phenylethanol due to CD’s better biocompatibility and bio-stabilitycompared with other additives. The optimum substrate concentration, β-CDconcentration, pH and reaction temperature were20mM,30mM,7.0and30oC,respectively. Under the optimum conditions, the ee value was enhanced from80%to97%at a conversion of98%. For other aryl ketones, CD can also improve the ee valueof their products in varying degree. This new method may have a brightly applicationin the future.