阅读说明：本技术 一种锰过氧化物酶的共交联固定化方法 (Co-crosslinking immobilization method of manganese peroxidase ) 是由 吴嘉沁 张瑞丰 李艳 肖通虎 龙能兵 于 2019-05-07 设计创作，主要内容包括：本发明是关于一种锰过氧化物酶的共交联固定化方法。使用油溶性的异氰脲酸三丙烯酸酯作为交联剂,水相中的反应物为含有氨基的锰过氧化物酶以及胺化环氧树脂与β-环糊精形成的超分子复合物,利用双键与氨基的迈克尔加成反应,在较低的温度下发生共交联聚合反应,制备出不同负载量的固定化锰过氧化物酶。通过控制交联程度,提高分散性,改善其内部的传质微环境,该固定化酶具有较高的催化活性,负载量在78mg酶/g载体时具有最高的比活性,达到游离酶的94％。(The invention relates to a co-crosslinking immobilization method of manganese peroxidase, which uses oil-soluble isocyanuric acid triacrylate as a crosslinking agent, reactants in a water phase are manganese peroxidase containing amino and a supermolecular complex formed by aminated epoxy resin and β -cyclodextrin, and the co-crosslinking polymerization reaction is carried out at a lower temperature by utilizing the Michael addition reaction of double bonds and amino to prepare immobilized manganese peroxidase with different loading amounts.)

1. A manganese peroxidase co-crosslinking immobilization method is characterized in that a water/oil two-phase reaction system is used, the oil phase is isocyanuric acid triacrylate as a crosslinking agent, and the structure is as follows:

the reactant in the water phase is manganese peroxidase and a molecular compound with the following structure:

the manganese peroxidase co-crosslinking immobilization method comprises the following steps:

1) mixing bisphenol A epoxy resin with the number average molecular weight of 454, methanol and diethylenetriamine according to the mass ratio of 2: 1, stirring and reacting for 4-5 hours at the temperature of 25-35 ℃, pouring the mixture into water, repeatedly washing precipitates with water to remove methanol and a small amount of amine, and then putting the precipitates into a vacuum oven to dry at normal temperature to obtain an epoxy resin amide;

2) adding the epoxy resin aminated substance and β -cyclodextrin into water according to the molar ratio of 1: 2.1-1: 2.3, heating and stirring until the epoxy resin aminated substance is completely converted into a molecular compound and dissolved in the water, and keeping the total mass concentration of the aqueous solution within the range of 5-6 wt%;

3) dissolving manganese peroxidase in a sodium phosphate buffer solution with the pH value of 7.5, keeping the concentration of the enzyme within the range of 1.0-7.0 mg/mL, and mixing manganese peroxidase solutions with different concentrations with the molecular complex aqueous solution according to the ratio of 55mL to 20 mL;

4) adding 1.2g of isocyanuric acid triacrylate into the mixed aqueous solution under stirring, keeping the reaction temperature within the range of 25-30 ℃, forming white gel particles after 10-15 minutes, stopping stirring to allow the reaction system to stand for 6-7 hours, and filtering to obtain manganese peroxidase immobilized products with different loading amounts.