由野生及突变株云芝IBL-04制得细胞外锰过氧化物酶(MnPs)，并经过硫酸铵沉淀、透析、离子交换和凝胶渗透层析法等步骤提纯.纯化的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)上于43 kDa区域呈现单一谱带，它适宜的pH值和温度分别为5.0和40°C.突变株MnPs表现出比野生株MnPs更宽的活性pH值范围和更高的热稳定性.从所选突变株所得纯化的MnPs表现出与野生株MnP几乎相同的电泳性质、稳态动力学、金属离子和EDCs降解效率.该生物酶与Mn2+一起催化的反应速率最快，但最高的亲和性对应于ABTS、甲氧基羟苯基乙二醇、4-氨基苯酚和活性染料. Mn2+和Cu2+可显著提高MnPs的活性，但Zn2+， Fe2+， EDTA和半胱氨酸则会不同程度地抑制其活性， Hg2+是最强的活性抑制剂.所有来源的MnPs均可有效催化EDCs、壬基苯酚和二氯苯氧氯酚降解，处理3 h可除去80%以上，在MnPs-介质体系中可进一步提高到90%.综上，云芝MnPs生物酶具有较高的pH适用性和热稳定性、独特的Michaelis-Menten动力学参数和高的EDCs去除效率等特点，因而有望工业化应用.%Extracellular manganese peroxidases (MnPs) produced by native and mutant strains of Trametes versicolor IBL‐04 (EB‐60, EMS‐90) were purified by ammonium sulphate precipitation and dialysis, followed by ion‐exchange and gel‐permeation chromatography. The purified enzymes elucidated a single band in the 43‐kDa region on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. The optimum pH and temperature of the purified enzymes were found to be 5.0 and 40 °C, respec‐tively. Mutant strain MnPs exhibited a broader active pH range and higher thermal stability than native MnP. Purified MnPs from selected mutants showed almost identical properties to native MnP in electrophoresis, steady‐state kinetics, and metal ion and endocrine‐disrupting compound (EDC) degradation efficiency. Although the fastest reaction rates occurred with Mn2+, MnPs displayed the highest affinity for ABTS, methoxyhydroquinone, 4‐aminophenol and reactive dyes. MnP activity was significantly enhanced by Mn2+and Cu2+, and inhibited in the presence of Zn2+, Fe2+, ethylene‐diaminetetraacetic acid and cysteine to various extents, with Hg2+ as the most potent inhibitory agent. MnPs from all sources efficiently catalyzed the degradation of the EDCs, nonylphenol and triclosan, removing over 80%after 3 h of treatment, which was further increased up to 90%in the presence of MnP‐mediator system. The properties of T. versicolor MnPs, such as high pH and ther‐mal stability, as well as unique Michaelis‐Menten kinetic parameters and high EDC elimination effi‐ciency, render them promising candidates for industrial exploitation.
展开▼
