Isolation, Dentification, Phylogenetic Analysis, Optimization Of Enzyme Producing Conditions, Cell Immobilization And The Industrial Fermentation Of Rhodococcus Sp. Strain M1 Producing Cis-epoxysuccinate Hydrolase

Strain Ml, capable of producing cis-epoxysuccinate hydrolase (ESH) with high yield, was isolated from the soil on campus of Zhejiang University, and identified as Rhodococcus ruber based on physiological experiment , Biolog carbon source utilization experiment and phylogenetic analysis of 16S rDNA sequence. The conditions for enzyme formation of strain Ml were optimized. The optimum conditions were 1.2% of glucose as carbon source, 0.6% of (NH4)2SO4 as nitrogen source, and 0.5% of yeast-extract, the time and quantity of adding disodium cis-epoxysuccinate at 32h of incubation and 3.58%, respectively. The ESH activity was about 750U/g wet cells when the organism was cultured in the 250mL-flask.The different scale of magnifications in 80L fermentor, 8001 fermentor and 5000L fermentor were performanced. The highest enzyme activity about 300U/g was obtained with about 53h of fermentation time, and the pH value 8.5 of fermentation liquid in 80L fermentor. The highest enzyme activity about 540U/g wet cell did with about 40h of fermentation, the pH value 7.3 of the fermentation liquid in 800L fermentor fermentation. And about 550U/g wet cell did about 63h of fermentation, the fermentation time is, the pH value 7.5 of the fermentation liquid in 5000L fermentor.The effect on the concentration of the thalli, the pH value and the enzyme activity of different time after inducer addition was observed. There appeared some differences among different scale of fermentor. The second addition of inducer and some organic nitrogen source was helpful to resolve the problem of which the pH value enhanced slowly and lower enzyme activity in 5 000L fermentor.The immobilization of Rhodococcus ruber strain Ml cells producing cis-epoxysuccinate hydrolase (ESH) firstly using Carrageenan-Koniak polysaccharide- carboxymethyl cellulose sodium gel was used to biotransform disodium cis-epoxysuccinate to L(+)-tartaric acid. The conditions for immobilization of strain Ml were opitimized, resulting in Carrageenan added with 1.2%, Koniak polysaccharide added with 0.2%, carboxymethyl cellulose sodium added with 0.3%, Glutaraldehyde and KC1 , with 0.5% andl%, the treatment time 30min, respectively. The scanning electron micrograph (SEM graph) showed that the shape of strain Ml cells was not changed after immobilization. Meanwhile, the gel without cells of strain Ml contained many tiny holes that were helpful for cells immobilization. The enzyme activity of strain Ml was stable after immobilization. In shaking flask, the transformation stability of immobilized cells was satisfactory for 30 batches of transformation of disodium cw-epoxysuccinate to L (+)-tartaric acid, resulting in a satisfactory transformation in a 4 000L reactor.