葡萄糖氧化酶的有机相共价固定化  

Covalent immobilization of glucose oxidase within organic media

作　　者：周涛[1,2] 朱雄军[1] 苏建华[1] 姚冬生[1,2] 刘大岭[1] 

机构地区：[1]暨南大学生命科学技术学院微生物技术研究所,广东广州510632 [2]广东省生物工程药物重点实验室,广东广州510632

出　　处：《生物工程学报》2012年第4期476-487,共12页Chinese Journal of Biotechnology

基　　金：广东省科技攻关项目(No.2005B20401004)资助~~

摘　　要：将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。Activity losing during the covalent immobilization of enzyme is a serious problem. Here we studied organicphase immobilization by using glucose oxidase （GOD） as a model. After lyophilized at optimum pH, GOD is covalently immobilized onto glutaraldhyde-activated chitosan microsphere carrier under the condition of water, 1, 4-dioxane, ether and ethanol separately. The special activities, enzyme characterization and kinetic parameters are determined. Results show that all of the organic phase immobilized GODs have higher special activities and larger gcat than that of aqueous phase. Under the conditions of 0.1% of glutaraldehyde, l.6% moisture content with 80 mg of GOD added to per gram of carrier, 2.9-fold of the special activity and 3-fold of the effective activity recovery ratio were obtained, and 3-fold of the residue activity was demonstrated after 7 runs when comparesl, 4-dioxane phase immobilized GOD with water phase immobilized one. In addition, kinetic study shows that 1,4-dioxane immobilized GOD （KraapP=5.63 mmol/L, Vmax=1.70 μmol/（min·mg GOD）, Kcat=0.304 s-1） was superior to water immobilized GOD （KmaPP=7.33 mmol/L, Vmax=1.02 μmol/（min-mg GOD）, Kcat=0.221 s-l）. All above indicated GOD immobilized in proper organic media presented a better activity with improved catalytic performance. Organic phase immobilization might be one of the ways to overcome the conformational denature of enzyme protein during covalent modification.

关 键 词：葡萄糖氧化酶 共价固定化 有机介质 酶比活 

分 类 号：Q814.2[生物学—生物工程]