Kinetic Studies And Processing Optimization Of Stereoinversion Catalyzed By Candida Parapsilosis

Stereoinversion process catalyzed by microorganism whole cell is one of the most important and best industrial application prospect methods in biocatalyst chiral synthesis. The optically phenyl-1,2ethanediol (PED), key intermediates in the synthesis of pharmaceuticals and fine chemicals, is choosed as the model substrate in this thesis. For the common shortcoming of stereoinversion such as long reaction time and low substrate concertration, the studies of reaction mechanism of stereoinversion, fermentation of biocatalyst and optimization of process are carried out to reveal the factors which hinder the improvement of reaction efficiency. Furthermore, the processes for preparation of different isomers of PED by oxidoreductase coupled with the regeneration of coenzyme are established. The stereoinversion process catalyzed by whole cell is succeeded in pilot scale for the production of optically PED with high efficiency.Firstly, the fermentation culture medium, especially the effect of metal ions, was observed by Plackett-Burman design and optimized by response surface analysis while biomass and catalytic ability of cell as the indicator. The cultues conditions including pH, temperature and culture time were also optimized by orthogonal experimental design. At the optimized condition, the biomass and optical purity of product reached 33.3 g/L and 97.0%e.e. respectively.Secondly, the effect dissolved oxygen to the fermentation of biocatalyst was carried out in the 7 L and 150 L fermentor. It was proved that high oxygen throguout was benifitful to the biomass and harmful to the stereoselectity of cell. The biomass and optical purity of product reached 47 g/L and 99.3% e.e. at the controlled dissolved oxygen by adjusting of stirring speed and air volume in 150 L fermentor, respectively.Thirdly, the dehydrogenase, CPADH, which catalyzed the stereoselectly oxidation reaction of stereoinversion, was purified and characteristiced including the substrate specificity and coenzyme independent. CPADH catalyzed the reversible oxidation reaction was inhibited by the high concerntration of substrate. It was indicated that the oxidation reaction followed Theorell-Chance BiBi mechanism by the studies of apparent michaelis constant Km'at different substrate concerntration without product inhibitation, studies of dead-end inhibition and studies of product inhibitation. Based on the ability of catalyzing reduction reaction of CPADH, the asymmetric reduction process for R-PED production coupled with NADH regeneration catalyzed by formate dehydrogenase was estabolished with high yeild and optical purity of 95.8% and 100%e.e. at the substrate concerntration of 60 mmol·l-1, respectively.Forthly, the carbonyl reductase, CPCR, which catalyzed the asymmetric reduction reaction of stereoinversion, was purified and characteristiced including the substrate specificity and coenzyme independent. CPCR catalyzed the irreversible reduction reaction was inhibited by the high concerntration of coenzyme. It was indicated that the reduction reaction also followed Theorell-Chance Bi Bi mechanism by the studies of apparent michaelis constant Km'at different substrate concerntration without product inhibitation and studies of product inhibitation. The asymmetric reduction processes for S-PED production coupled with NADPH regeneration catalyzed by glucose dehydrogenase or glucose-6-phosphate dehydrogenase were established with high yeild of 91.3% and 86.8% and the same optical purity of 100%e.e. at the substrate concerntration of 60 mmol·l-1, respectively.Fifthly, it was suggested that the oxidation reaction was the restrict step of the whole stereoinversion process after the comparasion of kinetic and thermodynamic studies of CPCR and CPADH. The effct of enviromental factors includes optimal reaction temperature and pH value to the kinetic and thermodynamic parameters shown that the great difference between CPCR and CPADH caused the low efficiency of stereoinversion reaction. The optimal eaction temperature and pH value of CPADH was 35℃and 9.0 while it of CPCR was 45℃and 4.5.Sixly, it was found that thefactors including pH value, reaction temperature, dissolved oxygen and biomass to the stereoinversion cataylzed by whole cell was quite different with it in the reaction catalyzed by the purified enzyme. The optimal substrate concerntration was improved from 35 mmol·l-1 to 70 mmol·l-1 with high yeild and optical purity of more than 90% and 99%e.e. Based on the fact that the restrictive step of stereoinversion reaction was inhibited by high concerntration of substrate, in situ product removal (ISPR) techniche was applied to decrease the concerntration of substrate in reaction phase and remove the inhibition of substrate. The substrate concerntration was improved from 70 mmol·l-1 to 220 mmol·l-1 with high yeild and optical purity of more than 90% and 99%e.e. in the bi-aqueous phase system and resin-aqueous phase system. The process of resin-aqueous phase system was succeeding to scale up to 100L reactor with high yeild and optical purity of 94% and 99%e.e. after 48 h.