有机磷降解酶在重组大肠杆菌中的表达及一步纯化固定化  

Expression of organophosphohydrolase in recombinant Escherichia coli and its purification and immobilization by one-step method

作　　者：周丽亚[1] 欧阳亚萍 薛赛光 姜艳军[1] ZHOU Li-Ya;OUYANG Ya-Ping;XUE Sai-Guang;JIANG Yan-Jun(School of Chemical Engineering and Technology,Hebei University of Technology,Tianjin 300130,China)

机构地区：[1]河北工业大学化工学院,天津300130

出　　处：《微生物学通报》2020年第7期2094-2105,共12页Microbiology China

基　　金：国家自然科学基金(21878068);河北省自然科学基金(B2017202056);河北省高校百名优秀创新人才项目(SLRC2017029);河北省高层次人才保障计划(A2016002027)。

摘　　要：【背景】有机磷化合物作为一类广谱杀虫剂,因其用量大、毒性强且不易降解,在自然界中的残留已对环境造成了严重污染。【目的】有机磷降解酶(organophosphohydrolase,OpdA)可降解多种有机磷化合物,探究其被固定到NiCo2O4载体上后用于有机磷化合物的降解效果。【方法】构建含组氨酸标签(histidine tag,His-tag)的OpdA,以pET-28a(+)为载体,Escherichia coli Rosetta(DE3)为宿主细胞,在终浓度为1.0 mmol/L的IPTG诱导下表达His-tagged OpdA。采用一步纯化固定化方法,实现固定化酶(OpdA@NiCo2O4)的制备。【结果】采用水热处理和煅烧制备了含过渡金属离子的NiCo2O4,利用过渡金属离子对酶分子表面组氨酸咪唑基的配位作用,实现了发酵粗酶液中His-taggedOpdA的一步纯化固定化,在优化条件下获得了高稳定性的OpdA@NiCo2O4;然后将其用于有机磷化合物的降解,在NaBH4存在条件下,通过级联反应和降解条件优化,实现了有机磷化合物的高效降解。【结论】该研究不但实现了重组酶的一步分离纯化和固定化,也为有机磷化合物的降解提供了一条安全、高效、环保的新途径。[Background]As a kind of broad-spectrum insecticides,organophosphorus compounds have caused serious environmental pollution due to their high dosage,strong toxicity and non-degradability.[Objective]To degrade the organophosphorus compounds,organophosphohydrolase(OpdA)was immobilized on NiCo2O4 and the properties of the catalyst in degrading organophosphorus compounds were investigated.[Methods]Specifically,histidine tag(His-tag)was introduced into OpdA to construct His-tagged OpdA,and the His-tagged OpdA was efficiently expressed in Escherichia coli Rosetta(DE3)using pET-28 a(+)as vector under the induction of 1.0 mmol/L isopropyl-β-D-thiogalactopyranoside.The immobilized OpdA(OpdA@NiCo2O4)was prepared by one-step purification and immobilization method.[Results]NiCo2O4 was prepared by hydrothermal treatment and calcination.Based on the coordination between the transition metal ions on the surface of NiCo2O4 and the imidazole group of histidine on the surface of enzyme,one-step purification and immobilization of His-tagged OpdA were achieved.OpdA@NiCo2O4 with high stability was obtained under optimized conditions,and then it was applied for organophosphorus compounds degradation.The high degradation efficiency of organophosphorus compounds was realized by cascade reaction in the presence of NaBH4 under the optimal degradation conditions.[Conclusion]In conclusion,one-step purification and immobilization of recombinant enzyme were achieved,and it’s also proved that this study provides a safe,efficient and environmental new way for the degradation of organophosphorus compounds.

关 键 词：有机磷降解酶 重组表达 组氨酸标签 一步纯化固定化 有机磷化合物 

分 类 号：X592[环境科学与工程—环境工程] X172