Study Of Dual-enzymatic Synthesis Of D-(-)-p-pHydroxypHenylglycine And Mutation Breeding Of High Producing Strains

In this paper,the underlying parametes about growth property of S.strain ,including growth curve, the change of pH in the liquid of fermentation and the morphology of bacteria, was observed. The result indicated that the concentration of S.strain achieved maximum after about 14 hours, that pH in the liquid of fermentation increased gradually with the change of time. So S.strain is presumed as alkali-yielding bacteria, and the thallus is relatively small and connected, with a shape of branch and roll.On the basis of previous work, the underlying parametes of the process of producing D-hydroxypHenylglycine via the bi-catalysis was investigated .By establishing the method of determining substrate and product, namely HPLC, the ability of conversion was observed and the spectific activity of D-Hase and DCase was also determined. The conclusion was that the spectific activity reached maximum during the anaphase of logarithm growth term. Meanwhile, the condition of bio-conversion, including pH of buffer, the ratio of substrate concentration and bacteria weight, was also optimized elementarily. The result showed that pH 7.0 of buffer and appropriately increasing bacteria weight both result in a relatively high yield of conversion.Hydantoinase and Carbamolyase overproduction strains were bred by use of substrate analogue resistant mutation. S.strain was treated by UV, NTG, UV plus NTG and UV plus LiCl successively. Mutant S-8-56 which produced hydantoinase and carbamolyase 2.15 folds higher than that of original strain was obtained from ten thousand strains of mutants with 5-FU resistance by determination of enzyme activity.Optimal conditions for the growth and enzyme activity of S.strain were defined.The results suggested that the optimal carbon source and nitrogen source were glycerol, sucrose and yeast extract. Enzyme activity was inhibited by citric acid and Cu²⁺ ion. Suitable medium consists of 0.5% sucrose, 1.0% glycero, 0.5% yeast extract , 0.3% NaCl, 0.01% MnSO₄·4H₂O, 0.02% CaCl₂·2H₂O,0.01% MgSO₄·7H₂O,0.01% FeSO₄·7H₂O,0.1% K₂HPO₄·3H₂O. The optimal initial pH of the media was 6.5. The highest enzyme activity was obtained at 30℃ incubation for 14 hours.