聚丙烯腈膜固定化海藻糖合成酶的研究

Research on Immobilization of Trehalose Synthase on Polyacrylonitrile

以聚丙烯腈膜(PAN)为载体,采用吸附法固定化海藻糖合成酶粗酶液。通过单因素法探讨最佳固定化条件,并分析固定化酶酶学性质。结果表明,最佳固定化条件为:pH 7.6、30℃、加酶量0.1 mg/cm^2条件下震荡吸附3 h;该条件下制备的固定化酶最适反应条件为:温度40℃、pH 7.4、初始麦芽糖底物浓度200 g/L。与游离酶相比,固定化酶热稳定性提高10℃、酸碱稳定性由pH 6.6～7.4扩展至pH 5.4～8.0;反应达到平衡时,反应液中海藻糖含量为50.9%,副产物葡萄糖含量为9.1%,较游离酶降低6个百分点,在目前已报道的固定化海藻糖合成酶催化反应中副产物含量最低;在40℃、pH 7.4、麦芽糖底物浓度200 g/L条件下,重复使用累计72 h后,固定化酶活仍保留在初始酶活的64.4%。

The crude trehalose synthase is immobilized on polyacrylonitrile membrane by adsorbtion. The optimal conditions of immobilization is research and the properties of immobilized enzyme by single factor method is analyzied. The results show that the optimum immobilization conditions are:p H 7.6,30 ℃ for 3 h with 0.1 mg trehalose synthase per square centimeter PAN membrane. The optimal reaction conditions of the immobilized trehalose synthase are 7.4,40 ℃ and initial substrate concentration 200 g/L. Compared with the free trehalose synthase,the thermal stability increases by 10 ℃,pH stability expande from pH 6.6 ~ 7.4 to pH 5.4 ~ 8.0. The max trehalose content is 50.9% when the reaction reached equilibrium,the by-product of glucose content is 9.1%,which reduced by 6 percentage points compared with the free enzyme and is the lowest byproduct content of the reported immobilization trehalose synthase. Under the optimum reaction conditions of p H 7.4,40 ℃,substrate concentration 200 g/L,the immobilized enzyme remains64.4% of the initial enzyme activity after repeating used for 72 h.