聚甲基丙烯酸缩水甘油酯固定化青霉素酰化酶性质的研究

Studies on Some Properties of the Immobilized Penicillin G Amidase by Beaded Glycidyl Methacrylate Copolymer

应用反相悬浮技术合成了珠状甲基丙烯酸缩水甘油酯 - N ,N′-亚甲基双 (丙烯酰胺 )共聚物 ,并将巨大芽孢杆菌 ( Bacillus megaterium)青霉素酰化酶共价偶联到甲基丙烯酸缩水甘油酯共聚物载体上 ,制成固定化青霉素酰化酶 ,其表观活性为 3 71 U/g(干重 ) ,水解青霉素 G钾盐的最适温度为 47°C,最适 p H为 8.5 ,在 p H4.5～ 9.0 ,温度 40°C以下时酶的活性稳定 ,表观米氏常数 Km为 1 .3 3× 1 0 -2 mol/L ,最大反应速度 vmax为 1 .2 7× 1 0 -5mol/min,固定化酶在 4°C冰箱保存 3 5 d,再水解 w=0 .0 2的青霉素 G钾盐溶液 ,重复使用 3 0次 ,保留酶活性 88.1 %。

Reactive, macroporous and beaded glycidyl methacrylate copolymer bearing oxirane groups and amide groups was synthesized by inverse suspension polymerization and used as the support for the immobilization of penicillin G amidase. The apparent activity of the enzyme was 371U/g (dry weight). The optimal pH and temperature were 8.5 and 47 °C for hydrolysis of penicillin G respectively. The immobilized enzyme was stable in the range of pH 4.5～9.0, and at the temperature below 40 °C. The apparent Michaelis constant K_m and v_ max of the immobilized enzyme were 1.33×10 -2mol/L and 1.27×10 -5mol/min, respectively. Being applied for 30 times, the remained activity of the immobilized enzyme kept in 4 °C refrigerator for 35 day was about 88.1%.