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漆酶广泛存在于土壤中,在有机农药残留和木质素的降解方面具有潜在的工业价值。从土壤宏基因组中得到了一个全长为1 389 bp漆酶基因lac13H9,编码462个氨基酸,预测理论分子量为50 k Da,含有一个17个氨基酸组成的信号肽,与NCBI蛋白数据库比对结果表明是一个新的漆酶基因。将lac13H9转入到大肠杆菌Transseta(DE3)中通过微好氧发酵进行异源表达,并通过Ni柱亲和层析纯化。获得的重组漆酶Lac13H9具有良好的酶学性质,其最适温度为40℃,最适p H为6.0,且具有较好的热稳定性,在50℃下保温90 min还有60%的剩余酶活力,同时发现低浓度的Fe2+促进漆酶酶活,高浓度则抑制酶活。良好的酶学性质为该酶的应用奠定了基础。
Laccase is widespread in soil and has potential industrial value in the degradation of organic pesticide residues and lignin. A lac13H9 full-length cDNA of lac13H9 encoding a polypeptide of 462 amino acids with a predicted molecular mass of 50 kDa and a 17 amino acid sequence was obtained from the soil metagenome. The results of comparison with the NCBI protein database Is a new laccase gene. The lac13H9 was transformed into E. coli Transseta (DE3) for heterologous expression by microaerobic fermentation and purified by Ni column affinity chromatography. The obtained laccase laccase Lac13H9 has good enzymatic properties, the optimum temperature is 40 ℃, the optimum p H is 6.0, and has better thermal stability. After incubation at 50 ℃ for 90 min and 60% Enzyme activity, at the same time found that low concentrations of Fe2 + promote laccase activity, high concentrations inhibit enzyme activity. Good enzymatic properties laid the foundation for the application of the enzyme.