耐过氧化氢的锰过氧化物酶对三苯甲烷类染料的脱色

Triphenylmethane dye decoloration using hydrogen peroxide-resistant manganese peroxidase

摘要:

【目的】从一株白腐菌Trametes sp. SQ01中获得一种新型的锰过氧化物酶, 探讨该酶的底物特异性和对过氧化氢的耐性, 以及其对三苯甲烷类染料的脱色能力。【方法】通过丙酮沉淀和DEAE-cellulose 52柱层析法纯化锰过氧化物酶。利用UV-2010紫外可见分光光度法研究锰过氧化物酶对过氧化氢的耐性, 同时, 用紫外可见分光光度计对三苯甲烷类染料脱色效果进行分析。【结果】通过两步纯化, 获得了均一性的锰过氧化物酶。该酶的最适pH和温度分别是4.5和70 °C, 在pH 3.0?8.0时, 酶活相对稳定。该酶在二价锰离子存在下能够氧化2,6-二甲氧基苯酚、愈创木酚、2,2′-连氮-双-(3-乙基苯并噻唑啉磺酸)和过氧化氢等化合物, 同时也能作用二价锰离子。在与这些底物反应中, 最适底物为过氧化氢(Km为3.7 μmmol/L)。该酶具有抗过氧化氢漂白能力, 锰过氧化物酶与高浓度的过氧化氢(2.5 mmol/L)作用60 min后仍能保持70%的活性。在所测试的染料中, 锰过氧化物酶对结晶紫的脱色率最高达到65.8%。二价锰离子和过氧化氢对锰过氧化物酶脱色能力的影响进行研究, 与孔雀绿相比, 锰离子和过氧化氢对活性艳蓝脱色的影响很小。【结论】Trametes sp. SQ01锰过氧化物酶对过氧化氢的耐受性, 以及对三苯甲烷类染料的高效脱色能力表明该酶在染料脱色降解方面有着广阔的应用前景。

Abstract:

[Objective] We produced a novel manganese-dependent peroxidase (MnP) by Trametes sp. SQ01 and studied its resistance of MnP to hydrogen peroxide, substrate specificity and ability of decolorizing triphenylmethane dyes. [Methods] MnP was purified through acetone precipitation and DEAE-celluose 52 anion-exchange chromatography. The resistance of MnP against H2O2 and dye decolorization were measured with a UV-visible spectrophotometer. [Results] The homogenous MnP has been obtained through two-step purification. The optimum pH and temperature for the purified MnP were 4.5 and 70 °C, respectively. The enzyme was highly stable in the pH range 3.0?8.0. The enzyme oxidizes various substrates in the presence of manganese, such as 2,6-dimethoxyphenol, guaiacol, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and H2O2, simultaneously, the MnP also regards Mn2+ as its substrate. Among all the substrates tested, the optimum was H2O2 (Km, 3.7?μmmol/L). Interestingly, the enzyme was resistant to H2O2 bleaching. The enzyme retains 70% integrity of its heme after incubation with 2.5 mmol/L H2O2 for 60 min. All of the tested dyes could be discolored by MnP, among which Crystal violet was observed with the highest decolorization rate at 65.8%. The effects of Mn2+ and H2O2 on dye decolorization by MnP were also studied, which revealed that both H2O2 and Mn2+ had less influence on the decolorization of Remazol Brilliant Blue R (RBBR) than Malachite Green. [Conclusion] The resistance of MnP to hydrogen peroxide and its ability of decolorizing triphenylmethane dyes demonstrated its potential application on dye decolorization.