重组血红密孔菌漆酶的纯化、酶学性质及染料脱色研究

Purification,characterization and dye decolorization of a recombinant Pycnoporus sanguineus laccase

为了研究血红密孔菌漆酶重组表达后的酶学性质和染料脱色效果,利用超滤、阴离子交换层析和凝胶层析从毕赤酵母发酵液中纯化出重组漆酶。结果表明:重组漆酶分子质量为62.8kD,氧化底物2,2′-连氮-二(3-乙基苯并噻唑-6-磺酸)(ABTS)、丁香醛连氮和2,6-二甲氧基苯酚的最适pH分别为2.2、4.2和4.0,动力学常数Km分别为20.73、48.48和1252.39μmol/L;该漆酶在有机溶剂中的活性稳定性:甲醇>乙醇>丙酮>二甲基亚砜;在铜离子和铝离子中酶活比较稳定,汞离子对酶活有较强的抑制作用;纯化的重组漆酶在无介体时可有效脱色RB亮蓝,但对靛红和结晶紫的脱色较为缓慢;当添加介体ABTS或紫脲酸时,可在短时间内实现对靛红和结晶紫的脱色,显示出该重组漆酶在染料废水处理中具有较好的应用前景。

To study the characterization and dye decolorization ability of a recombinant Pycnoporus sanguineus laccase, the enzyme was purified from the fermentation liquid of Pichia pastoris using uhrafihration, anion-exchange chromatography and gel filtration. The results showed that the recombinant laccase had a molecular weight of around 62.8 kD. It oxidized the substrates 2,2＇-azino-bis（3-ethylbenzothiazoline-6- sulfonic acid） （ABTS） , syringaldazine and 2,6-dimethoxyphenol at optimum pH of 2.2, 4.2 and 4.0, with Km values of 20. 73, 48.48 and 1 252.39 μmol/L, respectively. The stability of lacease activity in organic solutions was methanol 〉 ethanol 〉 acetone 〉 dimethyl sulfoxide. High laccase activity was found in the presence of Cu^2＋ and Al^3＋ , while Hg^2＋ showed a strong inhibition effect. Remazol brilliant blue R could be efficiently decolorized by the purified laccase in the absence of mediator, while the decolorization of indigo carmine and crystal violet proceeded slowly. When ABTS or violuric acid was added as redox mediators, the enzyme could rapidly decolorize indigo carmine and crystal violet. The results demonstrate the potential application of recombinant laccase in the treatment of dye effluents.