Abstract

In our previous study, we firstly cloned the gene of a novel ReNHase (NHase from Rhodococcus erythropolis CCM2595), and the strain was shown to degrade only phenol, hydroxybenzoate, p-chlorophenol, aniline and other aromatic compounds. Here in, we further purified the ReNHase from R. erythropolis CCM2595 and detected its properties of biodegradation. We constructed a plasmid with the gene of ReNHase with His-tag. The encoding ReNHase was cloned and overexpressed in recombinant Escherihia coli and confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The substrate scope of the new heterologous ReNHase was tested and the efficiency of degradation of nitriles by the ReNHase was also investigated by high efficiency liquid chromatography. We also studied the effect of temperature and pH on the catalysis of adiponitrile by the purified ReNHase. The recombinant E.coli showed high catalytic regioselectivity with high substrate affinity towards dinitriles (especially for adiponitrile) whereas lower affinity towards mononitriles. Compared to whole-cell catalysis, the catalytic time was shortened significantly with the purified ReNHase. The enzyme activity of crude recombinant E.coli was 635 U g⁻¹ (DCW), while the specific activity of purified ReNHase was 63.107 U mg⁻¹. The apparent Km value for the purified ReNHase is 6.6252 mmol L⁻¹, which revealed the good affinity between the purified ReNHase and adiponitrile. The reaction Kcat is 82.77 s⁻¹ and Kcat/Km is 1.249 × 10⁴ (mol⁻¹ L s⁻¹), which showed high catalytic activity towards adiponitrile. We propose that this purified ReNHase may be applied for the industry and sewage treatment for environmental protection.

摘要

在我们之前的研究中，我们首先克隆了一种新型 ReNHase（来自红球菌 erythropolis CCM2595 的 NHase）的基因，该菌株仅能降解苯酚、羟基苯甲酸酯、对氯苯酚、苯胺和其他芳香族化合物。在此，我们进一步纯化了来自R. erythropolis CCM2595 的 ReNHase 并检测了其生物降解特性。我们用带有His标签的ReNHase基因构建了一个质粒。在重组大肠杆菌中克隆并过表达编码 ReNHase并经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）证实。测试了新异源 ReNHase 的底物范围，并通过高效液相色谱研究了 ReNHase 降解腈的效率。我们还研究了温度和 pH 值对纯化的 ReNHase 催化己二腈的影响。重组大肠杆菌表现出高催化区域选择性，对二腈（尤其是己二腈）的底物亲和力高，而对单腈的亲和力较低。与全细胞催化相比，纯化的 ReNHase 的催化时间显着缩短。粗重组大肠杆菌酶活为635 U g ^-1(DCW)，而纯化的 ReNHase 的比活性为 63.107 U mg ^-1。纯化的 ReNHase的表观Km值为 6.6252 mmol L ^-1，表明纯化的 ReNHase 与己二腈之间具有良好的亲和力。反应Kcat为82.77  s ^-1，Kcat/Km为1.249 × 10 ⁴ (mol ^-1 L s ^-1 )，对己二腈显示出高催化活性。我们建议这种纯化的 ReNHase 可用于工业和污水处理以实现环保。