CONTROL OF OPTIMUM pH FOR IMMOBILIZED ENZYMES

为了控制固定化酶在固体载体上的最适pH，考察了载体上引入的酸性基团和碱性基团(羧基和氨基)的影响。以平均粒径为0.2μm的磁铁矿粒子为载体，在磁铁矿粒子表面引发丙烯酸和丙烯酰胺的共聚反应，将丙烯酸-丙烯酰胺共聚物接枝到磁铁矿粒子表面。通过与接枝共聚物的羧基缩合，将葡萄糖氧化酶(GOx)固定在磁铁矿颗粒上。接枝共聚物的磁铁矿固载GOx的最佳pH值介于天然GOx(pH 4~5)和聚丙烯酸接枝磁铁矿(PH 7)之间。另一方面，虽然乙二胺或N，N-二甲基乙二胺与磁铁矿表面的羧基进行了缩合反应，但由于未知的原因，引入到磁铁矿表面的氨基并没有得到明确的确认。相反，作为参考实验，将苯基硼酸和胺同时引入到磁铁矿表面，以考察共存的氨基对苯基硼酸表面酸性指数pKa的影响。研究发现，虽然固定化苯基硼酸的pKa值大于游离的pKa值，但共存氨基的pKa值有所降低。这表明固定化酶的最适pH可以通过载体上的碱性基团控制在酸性一侧。本研究提供了一种实用的方法来控制固定化酶在酸性或碱性基团共存的固体载体上的最适pH。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.