Fermentation, Purification Of An Esterase From Microbacterium Sp. SIT101 And Chemo-enzymatic Synthesis Of Chiral α-halogenated Aryl Alcohols

The pros of biocatalytic method is environmentally friendly, mild conditions, high selectivity and so on. So biocatalysis has been widely applied in the production of high value-added chiral compounds, as a green, efficient synthetic pathway. This thesis consists of two parts, the first part is the optimization of fermentation of Microbacterium SIT101 in both shake flasks and a fermentor, and the target esterase from Microbacterium SIT101 was purified in order to lay the foundation for further heterologous expression and improvement of the enzyme specific activity at the molecular level. In the second part, the asymmetric reduction of a-halogenated aryl ketones to chiral a-halogenated aryl alcohols catalyzed by resting cells of Rhodotorula sp. AS2.2241 was studied in"one pot"system.In the second chapter of this thesis, the production of esterase of Microbacterium SIT101 was optimized both in shaking flask and in a fermentor. The nutrient media components (carbon source) and the fermentation condition (amount of inoculum) of shake flask were investigated. The results showed that the optimal carbon source component was soluble starch. The optimum inoculum volume was 10% and fermentation time was 45 h. Under the optimum conditions, the maximum esterase activity was 2.09 U/g which was increased nearly 1 fold relative to the initial activity. And the fermentation conditions (dissolved oxygen, amount of inoculum, speed) of fermentor were also investigated. The results showed that the optimum fermentation conditions were 10% of inoculum volume,60% dissolved oxygen, speed 800 rpm and fermentation time was 32 h. The maximum esterase activity was 2.21 U/g which was higher than that of shaking flask. Then the cells after fermentation were patially purified by cell sonication, ammonium sulfate precipitation, dialysis, ultrafiltration and gel filtration.The results showed that the specific activity of enzyme after purification was 10.5 U/g which was increased nearly 10 times of the initial specific activity. And the enzyme activity recovery was 5.03%.In the third chapter of the thesis, a novel one-pot two-step chemo-enzymatic method was developed, by coupling chemical halogenating reaction of aryl ketones and biocatalytic reduction of Rhodotorula sp. AS2.2241, for the preparation of chiral a-halogenated aryl alcohols with high yield and high optical purity. The aryl ketones as substrates were catalyzed by p-toluenesulfonic acid to get a-halogenated aryl ketones. After adjusting the pH value of reaction mixture to 7.0, resting cells of Rhodotorula sp. AS2.2241 were added in the reaction system to catalyze the asymmetric reduction of a-halogenated aryl ketones and chiral a-halogenated aryl alcohols were obtained in high yield (95%) and optical purity (>99% ee).