| D-phenylalanine(D-phe) is an important intermediate in chemical andpharmaceutical industry. With the development of these industry, D-phenylalaninewill be increasingly needed. Hydantoinase method has been a tendency forD-phenylalanine production due to its advantages of high activity and high opticalpurity of product. One enzyme and one acid method, which obtained D-hydantoinasefrom microorganism, converses D,L-benzylhydantoin(D,L-BH) intoN-Carbamoyl-D-phenylalanine(Nc-D-Phe) by enzyme, and then hydrolyzes Nc-D-Pheby acid method, which was speedy reaction for obtain D-Phe. The yield of D-Phe ishigh (above 95%). Consequently, the process of Nc-D-Phe production byD-hydantoinase become the key for the whole process. Compared with traditionalmethod of single batch reaction catalyzed by free cell, D-hydantoinase methodabandons the cells at each time, immobilized cells or enzyme used as biocatalyst hasthe advantage of continuity and stability, easy operation and separation andpurification for production. Thus, it is significant for promoting the industrializationof D-phenylalanine by immobilization method.In this paper, N-carbamoyl-D-phenylalanine was produced by immobilized cellsor immobilized D-hydantoinase, The research involved the selected of carriers forimmobilized cells, the optimal the immobilization condition of PVA gel, the selectedof carrier for immobilized D-hydantoinase and the effect of microwave on productionof N-carbamoyl-D-phenylalanine by immobilized D-hydantoinase.JS-02 cells were embedded in various carriers and utilized for conversion. Aftermultiply compared with each other in the mechanical strengths of prepared gels,recovery of enzyme activity and manufacturing procedure, it was concluded thatPolyvinyl alcohol (PVA) gel was an ideal carrier.The optimal condition was determined by means of immobilizing cells on PVAthrough single factor test as follows: the best concentration of gel as 10%, the optimalcell loading by direct entrapment at 0.28g/mL. concentration of reagent to strengthenthe gel at 1%, concentration of glycerin at 3%,the optimal pH at 9.0, the optimal itemperature at 50℃,the stability of pH, operation and storage of immobilized cellswas enhanced.Resins with various function groups and different immobilization methods weretested. The immobilized D-hydantoinase combined with carrier D92 was consideredvaluable in view of the recovery of enzyme activity, half-life of immobilizedD-hydantoinase and cost of carrier. The optimum conditions were : concentration ofenzyme at 6mg/mL, absorption temperature at 25℃, absorption time at 12 hours. theoptimum temperature of immobilized enzyme at 45C, optimum pH of immobilizationat 8.5, and the Km was 1.8 times as that of free enzyme. Thermal pH, and storagestability of immobilized D-hydantoinase were improved by immobilization. Thehalf-life of immobilized D-hydantoinase at 45℃ was 11 days.The effects of microwave on conversion reaction with D-hydantoinase werestudied. Compared with common heating, the reaction velocity of immobilizedenzyme was accelerated over 20 times by microwave and the native D-hydantoinasewas accelerated about 8 times. The optimum condition was obtained: temperature at33℃, reaction time at 2 min, when the yield was about 80%, and half-life at 16 min.After five times of repeat operation, the activity of immobilized D-hydantoinasebegan to reduce. We explained the phenomena from the relation of proteinstructure and the enzyme activity . |
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