锰过氧化物酶的耦合固定化及其性质研究

Studies on the Properties and Co-immobilization of Manganese Peroxidase

摘要:

分别采用海藻酸钠、明胶和壳聚糖为载体，并以戊二醛为交联剂，通过包埋-交联和吸附-交联两种耦合固定化方法制备固定化锰过氧化物酶。探讨了酶的不同固定化条件和固定化酶的部分性能。与游离酶相比，制备的3种固定化酶最适反应pH分别由7.0降低到5.0、5.0和3.0，最适反应温度分别由35℃升高到75℃、55℃和75℃。3种固定化酶的耐热性都显著提高，其中用壳聚糖制成的固定化酶在pH 2.2～11的宽范围内表现出很好的酸碱耐受性。30℃连续测定6～9次酶活力，重复使用的3种固定化酶显示出良好的稳定性。将固定化酶应用在偶氮染料的脱色中，用明胶制成的固定化酶在静置和摇床条件下，以及用海藻酸钠制成的固定化酶在摇床条件下，均表现出与游离酶相近的脱色能力，并且在重复进行的摇床实验中，脱色能力未降低，反应前后的酶活力均没有损失。

Abstract:

White-rot fungus manganese peroxidase (MnP) oxidizes a wide range of substrates, rendering it an interesting enzyme for potential applications. The stability of MnP can be improved by immobilization. With sodium alginate, gelatin, or chitosan as a carrier, and glutaraldehyde as the crosslinking agent，MnP was co-immobilized using the embed-crosslinked method and the adsorb-crosslinked method. The immobilization conditions and the partial properties of the three immobilized enzymes were investigated. When compared with the free enzyme, the optimum pH values and the temperatures of the three immobilized MnPs carried by alginate, gelatin, and chitosan were respectively shifted from 7.0 to 5.0, 5.0, 3.0 and from 35℃ to 75℃, 55℃, 75℃.The thermostabilities of the three immobilized MnPs were considerably better than that of the native enzyme. The chitosan-immobilized enzyme was stable in the wide range of pH 2.2 to pH 11. The enzyme activities of the three immobilized MnPs decreased by less than 5% even after repeated use for 6～9 times. The ability of decolorizing azo dyes in static and shaky situation by gelatin-immobilized MnP approached to the free enzyme, and there was no loss of enzyme activity during 2 repeated batch reactions.