固定化E.coliBL21(pTrc-gsh)细胞催化合成谷胱甘肽

Enzymatic Synthesis of Glutathione by Immobilized Cell of E.coli BL21 (pTrc gsh)

分别以卡拉胶、明胶、海藻酸钠包埋 E.coli BL 2 1 ( p Trc- gsh)细胞催化合成谷胱甘肽( GSH)。从酶活收率及机械强度方面进行比较 ,选择卡拉胶为包埋载体 ,其最适 p H为 7.0 ,最适温度为 40°C。相关物质对 GSH的合成均有影响 :半胱氨酸、甘氨酸的最适浓度为 2 0 mmol/ L,谷氨酸的最适浓度为 60 mmol/ L;Mg2 + / ATP为 1～ 5 ( V/ V)较合适 ;腺苷二磷酸 ( ADP)浓度为 5 mmol/ L时对酶活的抑制为 2 0 %。优化条件下罐式反应器中 GSH的产量为 0 .84g/ L,操作稳定性较好 ;延迟加入甘氨酸时 GSH产量可提高 1 7.5 % ;与酵母生产 ATP体系相耦联的共固定化体系在填充床中反应 ,GSH合成量达 1 .2 4 g/ L,收率比直接加入 ATP提高 2 4 .2 %

Recombinant E.coli BL21(pTrc gsh) cells were embedded in carrageenan gel, sodium alginate, and gelatin respectively for the synthesis of GSH. The carrageenan gel was selected for the better enzyme recovery and mechanical strength. The optimal pH and temperature were 7.0 and 40 °C respectively. Higher concentrations of glutamate than that of cysteine and glycine facilitated the synthesis of GSH. Mg 2+ and ATP were necessary in the enzymatic synthesis process, ADP, however, was an inhibitor in this process. Under the optimal conditions, the yield of GSH was 0.84g/L for 1g of immobilized cells in tank reactor. The addition of glycine later after glutamate and cysteine made the yield rising up to 17.5%. When the co immobilized recombinant E.coli cells and S. cerevisiae cells which produced ATP from adenosine were employed in a packed bed, the yield could be reached to 1.24g/L, which was 24.2% higher than that of GSH produced by the ATP adding system.