Study On Kinetic Resolution Of 1-Phenyl-1,2-ethanediol Catalyzed By Kurthia Gibsonii SC0312 Cells

Enantiomerically pure chiral alcohols and their derivatives are important precursors and chiral intermediates in the field of fine chemistry,pharmaceutical,and functional material.Optically pure 1-phenyl-1,2-ethanediol?PED?is a crucial chiral structural unit.?R?-PED can be used to synthesize chiral drugs for the treatment of psychotic disorders,metabolic problems and hyperparathyroidism.?S?-PED is an essential raw material and chiral additive in liquid crystal materials.With the rise of the concept of clean green preparation,biocatalysis has gradually become a main means to obtain enantiomerically pure chiral alcohols.Currently,it is mainly divided into enzymatic method and whole-cell method.Compared with enzyme catalysis,advantages of the whole-cell catalysis are more significant,such as the higher stability,the decrease of catalyst costs,and no addition of external cofactors.In this study,a novel strain screened by our laboratory-Kurthia gibsonii SC0312 was used as the biocatalyst to prepare optically pure?S?-PED by asymmetric oxidation of racemic PED.Firstly,characteristics of the catalytic reaction in different reaction media were studied,and it revealed the effects of different reaction media on the cell viability,cell membrane integrity and permeability.Then,the immobilization technology was introduced into the DESs-containing system to effectively improve the stability and repeat utilization of the biocatalyst,thereby establishing a highly efficient and highly selective biocatalytic system for preparing enantiomerically pure?S?-PED.In this study,phase transfer strategy was used to investigate the effects of nine organic solvents?tert-amyl alcohol,n-hexanol,2-methyltetrahydrofuran,dibutyl phthalate,dimethyl carbonate,butyl acetate,2,2,4-trimethylpentane,n-decane,dipentene?on the asymmetric oxidation of racemic PED by Kurthia gibsonii SC0312 cells.It was found that the catalytic activity of microbial cells maintained well in the dibutyl phthalate/buffer two-phase system,the initial reaction rate,yield and?S?-PED e.e.value were 2.35 mmol/L/h,48.2%,>99.9%,respectively.Simultaneously,the partition coefficient of toxic substances-HAP?oxidation product?was higher in dibutyl phthalate/buffer system?6.37?.It could effectively alleviate product inhibition by the extraction of organic solvent,and eventually increased cell activity and substrate concentration of this reaction.Further study of the influence mechanism of organic solvents on microbial cells showed that dibutyl phthalate not only exhibited good biocompatibility,but also had influence on the integrity and permeability of cell membranes.Under the volume ratio of organic solvent and buffer of 5:5,buffer pH of 6.5,reaction temperature of 35o?,rotation speed of 180 r/min,cell concentration of 25 mg/mL,and substrate concentration of 90 mmol/L,the initial reaction rate,yield and the?S?-PED e.e.value reached 3.90 mmol/L/h,48.4%,99.0%,respectively,and the E value was above 40.The lower repeated utilization rate and poor stability of bio-catalyst limited the further development of free-cell catalysis.Therefore,it investigated four immobilization methods,such as calcium alginate embedding,calcium alginate-gelatin embedding,calcium alginate embedding-polydopamine coating,and calcium alginate embedding-chitosan coating.Results showed that the immobilization method of calcium alginate embedding-polydopamine coating could markedly improve the cell stability and catalytic performance.After 8 batches,the yield and the?S?-PED e.e.value of this reaction reached 48.9%,88.1%,and the immobilized structure remained intact.With the immobilized conditions of sodium alginate content of 2%,calcification time of 2 h,3-hydroxytyramine hydrochloride concentration of 2 mg/mL,coating time of 4 h,the pH stability,temperature stability and operational stability of cells were improved significantly.In order to further improve the catalytic efficiency of this reaction,the influences of ILs?C₄MIM·BF₄,C₄MIM·NO₃,C₄MIM·Br,C₄MIM·H₂PO₄?and DESs-[ChCl][U]on asymmetric oxidation of racemic PED catalyzed by immobilized Kurthia gibsonii SC0312 cells was investigated.It pointed out that the highest initial reaction rate?4.90 mmol/L/h?,yield?49.3%?and?S?-PED e.e.value?>99.9%?were obtained in the system containing[ChCl][U].Compared with the buffer system,[ChCl][U]-containing system could appropriately improve the integrity and permeability of cell membranes,and manifested good biocompatibility to Kurthia gibsonii SC0312 cells.Under the conditions of[ChCl][U]content of 4%?v/v?,buffer pH of 7.5,reaction temperature of 35o?,rotation speed of 180 r/min,and substrate concentration of 100 mmol/L,the initial reaction rate reached up to 8.25 mmol/L/h,with excellent yield?49.6%?and?S?-PED e.e.value?>99.9%?.Moreover,when the cell concentration was increased to 60 mg/mL,the reaction time was shortened from 24 h to 7 h,indicating that under the conditions of high substrate concentration,the increase of cell concentration obviously accelerated the reaction rate and shortened the reaction process.This study enriched the theoretical basis for efficient conversion of biocatalytic chiral aromatic alcohols and provided an effective new way for highly efficient and selective resolution of chiral aromatic alcohols.