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  

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

作　　者：胡斌斌[1,2] 吴萍[2] 刘晓青[2] 丁伟[1] 伍宁丰[2] 

机构地区：[1]甘肃农业大学生命科学技术学院,兰州730070 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2015年第2期64-71,共8页Journal of Agricultural Science and Technology

基　　金：国家863计划项目(2013AA102804)资助

摘　　要：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 widely exists in soil and has potential industrial value in degradation of organic pesticide residues and lignin. In this study, a laccase gene, lacl3H9, 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 lacl3H9 heterologously expressed in Escherichia coli Transseta （DE3） and purified by the affinity chromatography on a Ni-NTA column. The maximal activity of laeease 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 Fe^2＋ could promote laccase enzyme activity, while high concentration could inhibit enzyme activity. Fine enzymatic property of Lac13H9 has laid foundation for its application.

关 键 词：土壤宏基因组 漆酶 基因克隆 酶学性质 

分 类 号：Q78[生物学—分子生物学]