The irreversible change in protein conformation was unavoidable when the protein was co-valently immobilized. This problem was very common and critical in drug modification, protein chip and preparation of biological enzyme electrodes. In the present study, chitosan microsphere was used as a carrier and trypsin as model enzyme to compare the dynamic characteristic of enzymes immobilized with a-queous phase and organic media. This results showed that, the Kmapp of all the organic covalently immobilized trypsin were better than those immobilized with traditional aqueous phase. The Km value of trypsin immobilized with aqueous phase was 3.69 times higher than that immobilized with 1,4-dioxane. Comparing with aqueous phase immobilized enzymes, Kmapp of the organic covalently immobilized trypsin were much closer to its Km value of the corresponding free enzymes. Moreover, the organic operation environment endowed higher Kcat to immobilized trypsin than the water environment. For instance, the Kcat of trypsin immobilized with ethyl acetate was 58. 64 times higher than that immobilized with aqueous phase.Therefore, it is effective to maintain the native conformation of enzymes by immobilized them with organic media, which could be explained by that, the proteins are "rigid" in organic phase, and are not easy to denature than those in aqueous phase during covalent modification.%蛋白质共价固定时,容易发生由于共价化学反应而引起蛋白质构象的不可逆变化而造成蛋白活性丧失的现象,该问题在诸如药物修饰、蛋白质芯片、酶生物电极等制备中十分重要.本研究以壳聚糖为载体,以胰蛋白酶为研究对象,比较了水相与多种有机相条件下的共价固定化酶的动力学特性.结果发现,固定化酶特征常数Km值:所有的有机相条件下共价固定的胰蛋白酶均优于传统的水相中共价固定的该酶,且更接近于游离酶的Km值(水相中固定的胰蛋白酶其Km是1,4-二氧六环中固定的胰蛋白酶的3.69倍),提示有机相中固定的胰蛋白酶比水相中固定的该酶更接近于酶的天然构象；与水相中共价固定的胰蛋白酶相比,所有有机相中共价固定的胰蛋白酶均获得了更高的转换数Kcat(乙酸乙酯中固定的胰蛋白酶其转换数Kcat是水相中固定的该酶的58.64倍),即有机相中共价固定的胰蛋白酶均显示出比传统的水相中共价固定的该酶更高的催化效率.本研究表明,利用酶蛋白在有机相中变得刚硬的特性,在有机相中对某些酶进行共价固定化修饰有利于维持酶蛋白天然构象,一定程度上能够克服传统水相中进行共价固定化过程中造成的酶变性失活现象.
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