家蚕丝素固定化葡萄糖异构酶的制备方法及其理化性质

Preparation and Properties of Immobilization of Glucose Isomerase on Silk Fibroin

脱胶蚕丝用稀碱溶液处理后制成多孔的碱化丝素 ,分别经物理吸附和戊二醛交联方法制得固定化酶Ⅰ和Ⅱ。每克固定化酶Ⅰ的总活力为 791 0 4U ,活力回收率为 5 2 0 6 % ,活力表现率为 72 2 7% ;每克固定化酶Ⅱ的总活力为 811 39U ,活力回收率为 5 3 6 7% ,活力表现率为 72 97%。蚕丝经高浓度氯化钙溶液溶解、脱盐等处理后制成丝素粉末 ,经吸附后用戊二醛交联固定了葡萄糖异构酶 ,制成固定化酶Ⅲ。每克固定化酶Ⅲ的总活力为 84 4U ,活力回收率为 5 5 2 7% ,活力表现率为 72 4 2 %。经对固定化酶性质的研究表明 :碱化丝素和丝素粉末均能较好地固定葡萄糖异构酶 ;最适温度比游离酶升高了 15℃ ;最适pH没有变化 ,有较强的抗蛋白质变性剂 (8mol/L尿素溶液中的活力在 80 %以上 )能力 ;各固定化酶的Km值在 4× 10 -2 ～ 5× 10 -2 mol/L范围内。实验还发现Co2 + 和Mg2 +离子为固定化酶的激活剂 ,Fe3 + 和Ca2 + 离子为抑制剂。

Basified fibrin was prepared from degumed silk which pretreated with dilute alkali solution and served as supporter for immobilization of glucose isomerase to form the basified fibroin immolbilized glucose isomerase,BFIGI I.The BFIGI I has a total activity of 791.04 U/g,a recovery rate of 52.06%,and an activity express ratio of 72.27%.BFIGI Ⅱ was prepared by appending glutaraldehyde as bridging agent.The activity,the activity recovery ratio and the activity express ratio of BFIGI Ⅱ were 811.39 U/g,53.67% and 72.97% respectively.Fibroin powder prepared by dissolving fibroin in CaCl 2 solution,desalting and subsegment treatment,was used as supporter for immobilization of glucose isomerase,the so called fibroin powder immobilized glucose isomerase FPIGI,when the adsorbed glucose isomerase was immobilized by the bridging agent glutaraldehyde.The activity,the activity recovery ratio and the activity express ratio of FPIGI were 844 U/g,55.27% and 72.42% respectively.Our research showed that both basified fibroin and fibroin powder immobilize glucose isomerase satisfactorily,their optimum temperature being 15 ℃ higher than the free enzyme,and optimum pH was same as the free glucose isomerase.Experiment showed that the immobilized enzymes had high assistance to the action of resisting protein denaturing agent(over 80% of enzyme activity was retained in 8 mol/L urea solution) and a narrow range of Km value (4×10 -2 ～5×10 -2 mol/L).It was found that the Co 2+ and Mg 2+ ions were activating agents for immobilized enzymes and the Fe 3+ and Ca 2+ ions were depressor.