短小芽孢杆菌LC01漆酶基因在毕赤酵母中的胞外表达及重组漆酶酶学性质研究

摘要

为了获得表达量高、稳定性好及染料脱色效率高的细菌漆酶,通过PCR扩增出短小芽孢杆菌LC01的漆酶基因并构建重组表达载体pPICZαA-lac,转化毕赤酵母菌株SMD1168H后利用甲醇诱导培养重组菌获得重组漆酶,纯化并分析了重组漆酶的性质.重组菌株产漆酶活性在第7天达到最高,为1390 U/L.纯化的重组漆酶分子量为65 kD,以丁香醛连氮为底物的最适反应温度和pH分别为70℃和6.8.在pH 9.0放置10d活性没有下降,在70℃保温10h后仍保留36％的酶活.Al3+、Fe3+和Mn2+完全抑制漆酶活性.在介体乙酰丁香酮参与下该漆酶能够有效脱色RB亮蓝、活性黑5和靛红,在pH 9.0时6h的脱色率达到了90％以上,表明该重组漆酶能有效应用于染料废水的脱色处理.%To obtain high-yield bacterial laccase with fine stability and efficient decolorization ability,the laccase gene from Bacillus pumilus LC01 was amplified by PCR and cloned into the expression vector pPICZαA.The constructed vector pPICZαA-lac was transformed into Pichia pastoris SMD1168H.The positive P.pastoris strain was induced by methanol to produce the recombinant laccase.Then the recombinant laccase was purified and characterized.The highest laccase activity reached 1 390 U/L at 7 days after cultivation.The purified recombinant laccase had a molecular weight of 65 kD.The optimal pH and temperature for syringaldazine oxidation were 6.8 and 70℃,respectively.The initial enzyme activity was totally retained after 10 d storing at pH 9.0 and 36％ of activity remained upon 10 h incubation at 70℃.The purified laccase was found to be totally inhibited by Al3+,Fe3+ and Mn2+.The purified laccase efficiently decolorized remazol brilliant blue R,reactive black 5 and indigo carmine in the presence of acetosyringone.More than 90％ of the tested dyes were decolorized within 6 h at pH 9.0,indicating the recombinant laccase is an ideal candidate for the decolorization of dye effluents.