来源于土壤宏基因组中漆酶Lac13H9基因克隆及其酶学性质分析

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。

Cloning of Laccase Lac13H9 Gene from Soil Metagenome and Analysis of its Enzymatic Properties

Laccase widely exists in soil and has potential industrial value in degradation of organic pesticide residues and lignin. In this study, a laccase gene, lac13H9, with total length of 1 389 bp was obtained from the soil macro genome. It encoded 462 amino acids polypeptide with a calculated molecular mass of 50 kDa and contained a signal peptide composed with 17 amino acid residues. The deduced amino acid sequence was a new protein through aligning with available protein sequences held in the NCBI. The recombinant lac13H9 heterologously expressed in Escherichia coli Transseta (DE3) and purified by the affinity chromatography on a Ni-NTA column. The maximal activity of laccase was observed at 40℃, the optimum pH was 6.0, and it had good thermal stability. About 60% enzyme activity was remained under 50℃ for 90 min. At the same time, low concentration of Fe2+ could promote laccase enzyme activity, while high concentration could inhibit enzyme activity. Fine enzymatic property of Lac13H9 has laid foundation for its application.