三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。

The Study of Immobilizing Trypsin to Triethanolamine-polystyrene Resin by Covalenting Method

Trypsin was coupled to porous triethanolaminepolystyrene (TMPS) resin by CNBr. IR analysis indicated that the reaction mechanism of coupling enzyme to TMPS is similar to that of coupling enzyme to polysaccharide by CNBr. The immobilized trypsin increased with the increase of the CNBr concentration but its specific activity decreased.The amount of immobilized trypsin was appropriate and the specific activity was retained relatively high at coupling pH of about 10. Since trypsin-catalyzed reaction involved the liberation of hydrogen ions, the hydrogen ion concentration in the microenviroment is higher than in the bulk solution. Consequently, the pH optima of immobilized trypsin shifted to basic region and broadened.Trypsin retained only 48% of its enzyme activity after heating at 60℃ for 90 min. In contrast, under the same condition the immobilized trypsin showed enhanced resistance to heat inactivation, retaining 90% of its original activity. Because of low cost and good mechanical strength, TMPS may become a very useful matrix for covalenting enzymes.