Study On Microbial Synthesis Of(S)-4-chloro-3-hydroxybutyrate

(S)-4-chloro-3-hydroxybutyrate??S?-CHBE?is an important chiral building block in the enantioselective synthesis.It can be used to synthesis of HMG-CoA reductase inhibitors.Recently,with the development of biotechnology,a variety of biocatalysts have been developed to asymmetric synthesis of?S?-CHBE.The biological method for the synthesis of chiral compound has the simple process,high catalytic efficiency and mild reaction conditions and friendly to the environment,which was a promising method for the chemical synthesis.The key step for the synthesis of chiral compounds was the preparation of the biocatalyst.In this paper,the strain was firstly screened from contaminated soil samples which exhibited the highest activity and stereo-selectivity for the reduction of COBE to?S?-CHBE?e.e.>99%?.Then,the strain named ZJB-12028 was identified as the Rhodococcus qingsheng based on the morphology analysis,physiological characterisitics,and 16S rDNA sequencing.The species wasn't reported in the catalytic COBE to?S?-CHBE.To improve the enzyme production of ZJB-12028,the medium components and cultivation condition of ZJB-12028 included carbon source,nitrogen source,metal ions,culture temperature,pH,inoculums size and loading volume was optimized.It was proved that carbon resource,nitrogen source,culture temperature and pH was the significant impact factors of enzyme production,while the metal ions especially the Cu²⁺ and the phosphorus source can inhibited the activity of carbonyl reductase.A novel optimal method based on SVM-PSO theory was proposed.The results of SVM-PSO optimal model showed that optimal medium components are as follows?g/L?:glucose 47.7,yeast extract 28.7,the optimal cultivation conditions were initial pH 7.4,loading volume 80/500 mL,and inoculums size 6%.Under the optimal conditions,the biomass of ZJB-12028 reached to 11.3 g/L and the carbonyl reductase activity reached to 283.87 U/L,which increased 9.77%and 10.50%than before,respectively.At the same time,the condition of asymmetric reduction of COBE by the resting cell of R.qingsheng ZJB-12028 was optimized.The results showed the optimal reaction temperature was 30?,the optimal buffer was pH 7.0 phosphate buffer solution,and the cosbustrate glucose concentration was 20 g/L.In the case of fed-batch catalysis,240 mmol/L COBE was catalyzed to the?S?-CHBE,the concentration and the yield of production reached 184.15 mmol/L and 76.32%respectively.Simultaneously,the enzyme activity of carbonyl reductase was decrease 28.06%after preservation a week at 4?.The carbonyl reductase was purified and characterized in this experiment.The carbonyl reductase was purified to 4.6 fold with a yield of 6.91%from R.qingsheng ZJB-12028 through the ultrasonication,ammonium sulfate precipitation,DEAE-Sepharose,Butyl Sepharose and Bio-Scale CHT column chromatography.The carbonyl reductase showed a higher activity and good stability at pH 6.5 and 45?.While the half-life of enzyme was decreased with increasing temperature,which proved carbonyl reductase has the poor thermal stability.The result of experiment showed that Fe³⁺?Ag⁺?Cu²⁺ could severely inhibited the enzyme activity.Finally,the carbonyl reductase was found to have Km value of 8.991 mmol/L and Vm value of 0.359 mmol/?mg·min?by COBE as the substrate.