Study On α-ketoisocaproate Biosynthesis By Rhodococcus Opacus DSM 43250

The a-ketoisocaproate (KIC) is a intermediary metabolite of branched-chain amino acid, and plays an important role within a living organism. It has been implicated not only as a regulating factor in metabolism but also as a key intermediate of organic synthesis and biosynthesis. Because of favorable functional properties in medical industry, biological industry, and food industry, a-ketoisocaproate is fast emerging in the last few years.In this paper, the Rhodococcus opacus DSM 43250 strain which can synthesize L-amino acid oxidase was selected for a-ketoisocaproate production. A series of feasible approaches or strategies were carried out to achieve high product concentration, in the optimization of Rhodococcus opacus cultivation processes, Main research of this dissertation include following aspects:1. A high performance liquid chromatography method was developed to the determination of a-ketoisocaproate in biotransformatic mixture. The effects of concentration of methanol, temperature, pH value and flow rate on retention time and peek height were studied. The separation of the reaction mixture was conducted on a ZORBAX SB-Aq (4.6×250 mm,5μm) column and the detection was carried out at 203 nm. The mobile phase employed was 0.01 mol/L ammonium phosphate dibasic with 10% methanol working at flow rate of 0.8 mL/min at 35℃for the whole run.2. This work aims to optimize the culture medium for biotransformation of a-ketoisocaproate via a statistical analysis based approach. Finally a second-order polynomial model was constructed via Box-Behnken design and regression analysis by SAS software. The optimal culture medium consisted of:(NH4)2SO4 0.23 g/L, malt extract 2.42 g/L, and NaNO3 1.43 g/L. With the obtained culture medium, the maximal a-ketoisocaproate production reached 23.93 mg/L.3. This work aims to optimize the conditions for biotransformation of a-ketoisocaproate via a statistical analysis based approach. Finally a second-order polynomial model was constructed via Box-Behnken design and regression analysis by SAS software. The optimal conditions consisted of:inoculum size 6.93%, liquid volume 33.38 mL, and initial pH7.6. With the obtained conditions, the maximal a-ketoisocaproate production reached 30.97 mg/L.4. The researches showed the effects of the different pH, aeration ratio and agitation speed on fermentation efficiency in the fermentation of a-ketoisocaproate in 3L automatic fermentor. The results showed that the final optimal conditions for batch fermentation were determined as followed:pH7.25, aeration ratio 1.0vvm, agitation speed 300 r/min. With the obtained conditions, the maximal a-ketoisocaproate production reached 267 mg/L. 5.The effects of production parameters on the biotransformation were investigated to enhance the yield of a-ketoisocaproate using whole cells. The influence of several parameters has been studied, such as temperature, pH, cell concentration, substrate concentration and rotating speed. Using response surface methodology (RSM), a second-order polynomial equation was obtained by multiple regression analysis.Based on response surface, the optimum concentrations of the critical components were obtained as follows:temperature 43.66℃, pH8.38, substrate concentration 5.08 g/L, cell concentration 30.41 g/L and rotating speed 169.74 r/min. With the obtained biotransformation conditions, the maximal a-ketoisocaproate production reached 1264 mg/L...