凤尾菇(Pleurotus sajor-caju)漆酶Lac4基因在黑曲霉中表达研究

研究将凤尾菇漆酶基因Lac4(Gene Bank登录号AJ507327.1)cDNA克隆到表达载体pSZHG10-6-2上,通过农杆菌介导法导入黑曲霉CICC2462中,筛选得到糖化酶基因glaA位点发生同源重组的黑曲霉转化子。摇瓶发酵后取上清液作SDS-PAGE检测、酶活测定及酶学性质和酶稳定性分析,研究重组漆酶对染料脱色影响。结果表明,成功分泌表达重组漆酶r Lac4,其活性在第9天时达酶活最高峰1 211 U·L~(-1);酶学性质研究发现,其最适反应温度为60℃、pH为4.5,且在10~30℃及pH 5.0~6.0稳定性较好。最适反应条件下,重组漆酶对ABTS的Km为0.142 mmol·L~(-1),最大反应速率Vm为0.693 mmol·L~(-1)·min~(-1)·mg~(-1);通过重组漆酶研究4大类染料脱色能力,发现ABTS为介体时该漆酶对三苯基甲烷类孔雀绿脱色可达44%,杂环类中性红、偶氮类甲基橙脱色率分别13%和11%,而对蒽醌类活性亮蓝(RBBR)降解作用不明显,重组漆酶对孔雀绿的脱色效率具有较大应用价值潜力,对含三苯基甲烷染料废水处理应用前景良好。

Expression of Pleurotus sajor-caju laccase Lac4 in Aspergillus niger

In the study,the laccase Lac4 gene (GeneBank login AJ507327.1) cDNA from Pleurotus sajor-caju had be cloned to expression vector pSZHG10-6-2,and through the method of agrobacterium mediated import Aspergillus niger CICC2462,screening by homologous recombination in glaA loci homozygous transformation.After the fermentation was carried out,the supematant was subjected to SDS-PAGE,enzymatic activity and enzymatic properties and enzyme stability.The effects of recombinant laccase on the decolorization of the dye were investigated.The results showed that the recombinant laccase rLac4 was successfully secreted and its activity reached the highest peak of 1 211 U· L-1 on the 9th day.The optimal temperature was 60 ℃C,the optimum pH was 4.5,and the enzyme had good stability at 10-30 ℃C and pH 5.0-6.0.Under the optimum reaction conditions,the Km and Vm of recombinant protein laccase with ABTS as the substrate were 0.142 mmol· L-1 and 0.693 mmol· L-1· min-1· mg-1;In addition,the decolorization ability of four kinds of dyes by recombinant laccase was studied.It was found that when the ABTS was mediator,the lignase could decolorize 44％ of the triphenylmethane peach green,the neutral red and azo methyl Orange decolorization rates of methyl orange were 13％ and 11％,respectively.The degradation effect of anthraquinone active blue (RBBR) was not obvious,indicating that the recombinant laccase had great potential for the decolorization efficiency of malachite green.The wastewater treatment of methane dyes has a good application prospect.