过氧化物酶的凝胶化包埋及对酚类物质的降解

Encapsulation of enzymes into nanogel and associated degradation of phenolic compounds

过氧化物酶催化活性强,是氧化降解酚类污染物的有效手段,但其环境耐受性差,难以在污水处理中广泛应用.为增强辣根过氧化物酶(horseradish peroxidase,HRP)的环境耐受性并用于酚类物质降解,将修饰后的HRP进行凝胶化包埋,通过HRP表面的氨基或羧基进行丙烯酰化修饰,与丙烯酰胺(acrylamide,AAm)发生自由基聚合反应,分别制备纳米凝胶酶n-HRP@PAM和c-HRP@PAM.质谱表征显示,HRP羧基能接枝双键多达9个,多于氨基接枝的双键数;激光光散射显示,纳米凝胶酶均匀分散在水溶液中.借助于四甲基联苯胺的显色反应,探讨纳米凝胶酶的酶活性和热稳定性;以多种酚类化合物的降解率为性能指标,研究HRP浓度和双氧水添加量对酚类降解率的影响,对比天然HRP和纳米凝胶酶的环境耐受性及回收再利用效果.结果表明,相比天然HRP和n-HRP@PAM,c-HRP@PAM具有更紧密的高分子凝胶网络、更高的热稳定性和环境耐受性,对酚类废水的降解效果也最佳.研究证明,对生物酶表面羧基丙烯酰化并原位制备纳米凝胶是一种提升生物酶环境耐受性的有效手段.

As an effective means of degradation of phenolic pollutants,peroxidase exhibits high catalytic activity but poor environmental tolerance,and it is difficult to be widely used in sewage treatment.In order to enhance the environmental tolerance of horseradish peroxidase(HRP)and be used for phenol degradation,the modified horseradish peroxidase(HRP)was gelatinized and embedded in gel and acryloylated through the amino or carboxyl groups on the surface of HRP,and two encapsulated enzymes(n-HRP@PAM,c-HRP@PAM)were prepared by in situ free radical polymerization reaction with acrylamide(AAm).Mass spectrometry revealed that up to nine double bonds could be grafted on the carboxyl groups of HRP,and more than the number of double bonds grafted on its amino groups.Dynamic laser scattering showed that the two encapsulated enzymes were uniformly dispersed in aqueous solution.Furthermore,the enzymatic activity and thermal stability of two encapsulated enzymes were explored with the colorimetric reaction of tetramethylbenzidine,and the influence of HRP concentration and hydrogen peroxide on the degradation of phenolic compounds was studied.The environmental tolerance and recycling performance of natural HRP and two encapsulated enzymes were compared.Results show that,compared with natural HRP and n-HRP@PAM,c-HRP@PAM has a denser polymeric network,higher thermal stability and environmental tolerance,and the best degradation effect on phenolic wastewater.Overall,this study proves that acrylation of carboxyl groups on the peroxidase surface and the in situ preparation of nanogel are an effective way to improve the environmental tolerance of enzyme.