从野生与突变株云芝IBL-04提纯锰过氧化物酶及其表征

doi:10.1016/S1872-2067(15)61044-0

摘要/Abstract

摘要：

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 ℃. 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.

关键词: 锰过氧化物酶, 云芝IBL-04, 提纯, 表征, 热稳定性, 生物降解

Abstract:

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 ℃, respectively. 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⁺, ethylenediaminetetraacetic 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 thermal stability, as well as unique Michaelis-Menten kinetic parameters and high EDC elimination efficiency, render them promising candidates for industrial exploitation.

Key words: Manganese peroxidase, Trametes versicolor IBL-04, Purification, Characterization, Thermo-stability, Biodegradation

Muhammad Asgher, Muhammad Ramzan, Muhammad Bilal. 从野生与突变株云芝IBL-04提纯锰过氧化物酶及其表征[J]. 催化学报, 2016, 37(4): 561-570.

Muhammad Asgher, Muhammad Ramzan, Muhammad Bilal. Purification and characterization of manganese peroxidases from native and mutant Trametes versicolor IBL-04[J]. Chinese Journal of Catalysis, 2016, 37(4): 561-570.

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