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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Mn~(2+)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.Mn~(2+)和Cu~(2+)可显著提高MnPs的活性,但Zn~(2+),Fe~(2+),EDTA和半胱氨酸则会不同程度地抑制其活性,Hg~(2+)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) were prepared from wild and mutant strains of Coriolus versicolor IBL-04 and purified by ammonium sulfate precipitation, dialysis, ion exchange and gel permeation chromatography etc. The purified enzyme was immobilized on twelve SDS-PAGE showed a single band in the 43 kDa region, and its suitable pH value and temperature were 5.0 and 40 ° C. The mutant MnPs showed a higher frequency than the wild-type MnPs A wider range of active pH values and higher thermal stability.The purified MnPs from the selected mutants showed almost the same electrophoretic properties, steady-state kinetics, metal ion and EDC degradation efficiency as the wild-type MnP. The enzyme catalyzed with Mn 2+ had the fastest reaction rate, but the highest affinity corresponded to ABTS, methoxylolglycol, 4-aminophenol and reactive dyes.Mn 2+ And Cu 2+ significantly increased the activity of MnPs. However, Zn 2+, Fe 2+, EDTA and cysteine inhibited the activity of MnPs to some extent, Is the strongest active inhibitor.MnPs from all sources can effectively catalyze the degradation of EDCs, nonylphenol and triclosan, which can be removed more than 80% after 3 h of treatment and can enter into MnPs-medium system Step to 90%. In summary, PSK MnPs enzyme has higher thermal stability and applicability pH unique Michaelis-Menten kinetic parameters and a high removal efficiency characteristics EDCs, which is expected to industrial applications