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

doi:10.13560/j.cnki.biotech.bull.1985.2017-0839

摘要/Abstract

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1 390 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10 d活性没有下降，在70℃保温10 h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6 h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。

关键词: 细菌漆酶, 短小芽孢杆菌, 胞外表达, 毕赤酵母, 酶学性质

Abstract: 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.

Key words: bacterial laccase, Bacillus pumilus, extracellular expression, Pichia pastoris, enzymatic property

李成名, 王佳懿, 卢磊,. 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[J]. 生物技术通报, 2018, 34(3): 185-193.

LI Cheng-ming, WANG Jia-yi, LU Lei. Extracellular Expression of Laccase Gene from Bacillus pumilus LC01 in Pichia pastoris and Characterization of the Recombinant Laccase[J]. Biotechnology Bulletin, 2018, 34(3): 185-193.

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