Study On D-β-hydroxyisobutyrate Acid Synthetic Technique By Whole-cell Catalysis

D-(3-hydroxyisobutyrate (DHIBA) is the key intermediate for synthesis of captopril, and is also the important raw materials for vitamins, spices and antibiotics. As far as the biocatalysis of DHIBA be concerned, there are some advantages with raw material supplied easily, without separation and regeneration for coenzyme, disusing the separation for intermediate product. The purpose of the present work is to impove yield of DHIBA,the Specific method adopts candida rugosas to synthesize.At the same time, the preparation of biocatalyst,the selectivity of substrats, optimization of fermentation condition and use of reaction-Extraction technology are investigated.Due to the catalytic activity of Candida rugosas thalli for the important effects of DHIBA synthesis. In the experiment, the way of dilution coating plate was adopted to naturaly select microbial strains,and high active strain was obtained.The results showed that it is remarkable influence for thalli growth and DHIBA synthesis with different conversion substrates.the growing ability of thalli in culture medium including IBA is greater than culture medium including MA.However,the thalli for DHIBA synthesis with MA as substrate is more effective than with IBA as substrate.when concentration of IBA and MA in culture medium are 20g/L,cultured for 96h and the concentration of DHIBA is 3.98g/L and 4.56g/L respectively.Makeing use of MA as substrate to synthesize DHIBA in comparison with IBA as substrate,It can be got more fast response and high yield for DHIBA.Taking advantage of single factor experiment method to optimize reaction conditions,the adaptable initial reaction conditions was acquired. The results showed that the concentration of MA is lOg/L, concentration of glucose is 30g/L, pH 7.5, reaction temperature is 28℃and cultured thalli for 120h,the thalli biomass and the yield of DHIBA are both the best, right now the concentration of thalli is 13.23g/L and DHIBA reachs 4.99g/L.By means of response surface methodology analysis to further optimize reaction conditions, the optimum reaction conditions was determined.The optimum reaction conditions included that the concentration of MA is 13.62g/L, pH 7.57, the concentration of Glu is 33.22g/L, Cultured for 120h, the thalli biomass is15.64g/L and DHIBA reachs 4.99g/L.Using in situ reaction and extraction coupling technology to impove the DHIBA synthesis,the different extraction system for the effect of the growth of thalli and DHIBA synthesis was observed.The results showed that oleyl alcohol and trioctylamine (TOA) as extraction system is better.By way of optimizing experimental conditions, the optimum extraction conditions is attained. when pH is 5.8,the ratio of organic phase and water phase is 1:5,the content of TO A is 40%,cultured for 120h, the concentration of thalli can be got 11g/L and DHIBA reachs 4.45 g/L.The generated DHIBA is extracted immediately,and is not further oxidized and decomposed. The in situ reaction and extraction coupling technology simplifies the process of DHIBA synthesis,and heighten the yield of DHIBA.