疏水-亲水型硅胶固定化葡萄糖氧化酶的研究

目的　评价疏水 亲水型硅胶作为载体固定化葡萄糖氧化酶的性能 ,以期制备出能用于临床体液中葡萄糖浓度测定的生物传感器。方法　以甲基三甲氧基硅 (MTMOS)和γ 氨丙基三甲氧基硅烷 (APTMOS)为原料 ,利用溶胶 凝胶技术制成了一种疏水 亲水型硅胶 ,以 1 乙基 3 (3 二甲基氨丙基 ) 碳二亚胺·盐酸 (EDC)和N 羟基琥珀酰亚胺(NHS)为催化剂 ,利用葡萄糖氧化酶 (GOD)分子中的羧基基团与硅胶中氨基基团间的碳二亚胺偶联反应 ,实现了GOD在硅胶上的固定化 ,考察了固定化GOD的基本性质。结果　固定化GOD活力最优时的条件如下 :APTMOS的体积分数为 70 %、给酶量为 16 80 0U、最适温度和最适pH值分别为 35℃和 5 .5 ,固定化GOD具有很好的操作稳定性和贮存稳定性 ,其米氏常数Km为 9.1mmol·L-1。结论　疏水 亲水型硅胶是一种新型的酶固定化载体 ,可用于多种酶的固定化。

The study of immobilization of glucose oxidase on hydrophilic-hydropholic silica gel

Objective To evaluate the performances of glucose oxidase immobilized on hydrophilic-hydrophobic silica gel, which may be employed to prepare glucose sensor for the determination of glucose in body fluids. Methods The silica gel was prepared from precursors γ-aminopropyltrimethoxysilane (APTMOS) and methyltrimethoxysilane (MTMOS) by sol-gel technique. Glucose oxidase (GOD) was covalently attached to the silica gel via carbodiimide coupling reaction between a carboxylic acid group on enzyme and an amine group of the silica gel under the participation of the linking reagents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide. The performances of immobilized GOD were explored. Results The optimum conditions were obtained as follows: volume fraction of APTMOS 70%, enzyme content given 16 800 U, the temperature of 35 ℃ and buffer pH 5.5. The decrement in the activity of immobilized GOD for the first 2 weeks was less than 10% of its original activity, and the activity of immobilized GOD retained more than 75% of its original activity after 1 month of testing. Six independently prepared immobilized GOD on the silica gel resulted in an average bioactivity of 1 290.9 μmol·min -1·g -1 with an R.S.D. of 3.4%. The Michaelis constant (K m) of immobilized GOD was 9.1 mmol·L -1. Conclusion Immobilizing GOD on the silica gel via the formation of peptide bonds is an outstanding enzyme immobilization method.