碳酸酐酶在多孔玻璃上的固定化及性质研究

Evaluation and Characterization of Immobilized Bovine Carbonic Anhydrase on Controlled Pore Glass

将牛碳酸酐酶共价固定到多孔玻璃CPG上,拟应用于烟道气中CO2的强化吸收。多孔玻璃经过烷基化和戊二醛活化后,与酶共价结合。以p-硝基苯乙酸酯水解活力来表示碳酸酐酶的活性,在最优固定化pH=8.0下,固定化酶的活力为1.32U/g CPG,固定化酶载量为14.7mg/g CPG,固定化效率因子为0.334。测得固定化酶的米氏动力学参数k2和Km的值分别是258min-1和259×10-4 mol/L,分别是游离酶的61.7%和2.23倍。以pH=7.85和40℃时的酶活作为比较标准,当pH>7.85和T>40℃时,固定化酶的性质优于游离酶。在pH=10和40℃下,固定化酶的活性似乎不稳定,在7天内下降为原来的37%。然而,以压力法测得的CO2水合活力表示酶的活性,在上述条件下,固定化酶却很稳定,活性在21天内几乎没有变化,表明酯酶活力法测定酶活不能用来评估CPG-固定化碳酸酐酶的稳定性。

The bovine carbonic anhydrase （CA） was covalently immobilized on the controlled pore glass （CPG） for the potential use in the post-combustion CO2 absorption. CPG was activated by γ-aminopropyl triethoxysilane and glutaldehyde, then coupled with CA enzyme. Based on p-nitrophenyl acetate （p-NPA） hydrolysis activity for CA, the activity of the immobilized enzyme （CPG-CA） and the immobilization factor （IF） was 1.32 U/g CPG （dry） and 0. 334 respectively. The enzyme loading was 14. 7 mg/g CPG. The Michaelis-Menten kinetic parameters k2 and Km for CPG- CA was 258 min^-1 and 259×10^-4 mol/L, which was 61.7% and 223% of those for the free CA respectively. Being pH=7.85 and 40 ℃ as the standard of comparison, CPG-CA had better performance than the free CA at pH〉7.85 and T〉40 ℃. At pH= 10 and 40 ℃, the CPG-CA seemed unstable because its activity decreased to 37% of the original in 7 days. However, based on CO2 hydration activity, the real CA activity measured by the manometric method, the CPG-CA was stable because its activity was almost unchanged in 21 days. That meant that the p-NPA hydrolysis activity couldn＇t be applied for evaluating the stability of the CPG-CA.