CONTROL OF OPTIMUM pH FOR IMMOBILIZED ENZYMES

为了控制固定化酶的最佳pH值，在载体上引入酸性和碱性基团（羧基和氨基）的影响进行了研究。以平均粒径为0.2μm的磁铁矿颗粒为载体，从磁铁矿颗粒表面引发丙烯酸和丙烯酰胺共聚反应，将丙烯酸-丙烯酰胺共聚物接枝到磁铁矿颗粒表面。葡萄糖氧化酶（GOx）固定在磁铁矿颗粒通过缩合与接枝共聚物的羧基。固定在共聚物接枝的磁铁矿上的GOx的最佳pH值被观察到介于天然GOx（pH 4-5）和固定在聚（丙烯酸）接枝的磁铁矿上的GOx（pH 7）之间。另一方面，尽管尝试了乙二胺或N，N-二甲基乙二胺与磁铁矿表面上的羧基的缩合，但由于某些未知原因，引入到表面上的氨基没有被清楚地证实。相反，作为参考实验，将苯基硼酸和胺同时引入到磁铁矿的表面上，以检查共存的氨基对表面上的苯基硼酸的酸度指数pKa的影响。结果表明，虽然固定化苯硼酸的pKa值大于游离苯硼酸，但氨基的存在使其pKa值降低。这表明，固定化酶的最佳pH值可以通过载体上的碱性基团控制在酸性一侧。本研究结果为控制酸性或碱性基团共存的固体载体上固定化酶的最适pH提供了一种实用的技术。该技术已应用于生物传感器酶电极的制作。

In order to control the optimum pH for immobilized enzymes on solid supports, effects of acidic and basic groups(carboxyl and amino groups) introduced onto the supports were investigated. Magnetite particles (average particle size : 0.2μm) were used as the support, and copolymerization of acrylic acid and acrylamide was initiated from the surface of the magnetite particles to graft acrylic acid-acrylamide copolymers onto the surface. Glucose oxidase(GOx) was immobilized on the magnetite particles by condensation with carboxyl groups of the grafted copolymer. An optimum value of pH for the GOx immobilized on the copolymer-grafted magnetite was observed between that for native GOx (pH 4-5) and that for the GOx immobilized on poly(acrylic acid)-grafted magnetite (pH 7). On the other hand, although condensation of ethylenediamine or N,N-dimethylethylenediamine with the carboxyl groups on the surface of the magnetite was attempted, the amino groups introduced onto the surface were not confirmed clearly for some unknown reason. Instead, as a reference experiment, phenylboronic acid and amine were introduced simultaneously onto the surface of the magnetite to examine the effect of coexisting amino groups on the acidity index pKa of phenylboronic acid on the surface. It was found that the value of pKa was decreased by coexistent amino groups though the pKa value of the immobilized phenylboronic acid was larger than that of free one. This suggests that the optimum pH for immobilized enzymes can be controlled toward an acidic side by basic groups on the supports. The results obtained in this research present a practical technique to control the optimum pH for the immobilized enzymes on solid supports with coexistent acidic or basic groups. This technique has been applied to fabrication of enzyme-electrodes for biosensors.