S-licarbazepine is the key intermediate of eslcarbazepine acetate.Oxcarbazepine was used as substrate to prepare S-licarbazepine in organic solvent/aqueous biphasic system.The strain was screened from soil with oxcarbazepine as the sole carbon source.The screened strain has the capability to reduce oxcarbazepine to S-licarbazepine.The DNA sequence of 16 S rDNA identification showed that the strain belongs to Bacillus anthrascis and preserved in china general microbiological culture collection center.It was named after Bacillus anthracis CGMCC No.12337.The fermentation medium and conditions for cultivation of Bacillus anthracis CGMCC No.12337 were optimized by response surface analysis method.The optimum fermentation conditions were as follows:42 g/L glucose,20 g/L peptone,pH 4.8,0.5 g/L K2HPO4,0.5 g/L KH2PO4,10% inoculation,33 0C,120 r/min for 36 h.Organic solvent/aqueous biphasic system was used as the biocatalytic reaction medium to prepare S-licarbazepine.DNS method was used to detect the toxicity of organic solvent to cells.The optimum reaction system were as follows: dibutyl phthalate/aqueous(1:1),pH 5.0,substrate concentration 3.97 mmol/L,cell concentration 30 g/L,30 g/L iospropanol as co-substrate,32 0C,120 r/min for 48 h.The conversion and enantiometric excess of S-licarbazepine reached 97.3% and 99.8%.In order to improve the utilization of oxcarbazepine,carbonyl reductase which exist in the cells of Bacillus anthracis CGMCC No.12337 was conjugated with polystyrene at the interface of organic solvent/aqueous biphasic system.Finally the best reaction conditions were as follows: carbonyl reductase 1.2 ml,polystyrene 3.75 mg/ml,toluene/Tris-HCl(12.5:10),substrate concentration 4.76 mmol/L,60 g/L isopropanol as co-substrate,30 0C,80 r/min for 6 h.The conversion and enantiometric excess of S-licarbazepine reached 97.3% and 99.8%. |