疣孢漆斑菌Myrothecium verrucaria GH-01漆酶的纯化和酶学性质研究

Purification and Enzymatic Properties of Laccase from Myrothecium verrucaria GH-01

疣孢漆斑菌具有生长周期短,分泌漆酶酶活高等特点。利用已分离得到的疣孢漆斑菌GH-01(Myrothecium verrucaria GH-01)发酵产生粗酶液。进而通过分级沉淀、透析和层析的方法对漆酶粗酶液进行纯化。SDS-PAGE和Native-PAGE结果表明纯化可获得具有漆酶活性的单体蛋白。酶学性质研究表明,该漆酶催化最适温度为40℃,在底物ABTS存在条件下的最适p H值为4.0,且在低温及碱性条件具有较好的稳定性。此外通过漆酶对4大类染料脱色能力的研究,发现该漆酶对偶氮类的橙黄Ⅰ和蒽醌类的茜素红脱色有较好的脱色效果,反应1 h脱色率达80%以上;对三苯甲烷类的碱性品红脱色能力较弱,脱色率只能达到20%左右;脱色率最差的是杂环类的亚甲基蓝。在含10 U漆酶的体系中,对50 mg/L的染料降解效果相对最佳。

Myrothecium verrucaria has the advantages of short growth cycle and high enzyme activity of secreting laccase. In this study, the crude enzyme was produced by the M. verrucaria GH-01 obtained by separation. Further we purified the crude enzyme by fractional precipitation, dialysis and chromatography. The results of SDS-PAGE and Native-PAGE indicated that the monosome protein with laccase activity was obtained by purification. The study of enzymatic properties of laccase showed that the optimal temperature of catalyzed reaction for laccase was 40℃, and the laccase activity against ABTS showed a maximum at pH4.0. It had the favorable stability under the low temperature and alkaline conditions. Besides, studying the lacease decolorizing of 4 representative dyes demonstrated that the laccase decolorizing rate for orange I of azo dyes and alizarin red of anthraquinone class was the highest with decolorizing rate over 80% by 1 h, lower for fuchsin of triphenylmethane class with decolorizing rate only 20% and lowest for methylene blue of heterocyclic class. In 10 U laccase system, the decolorizing degree of the 50mg/L dye concentration was optimal.