根癌农杆菌介导脂肪酶在无孢黑曲霉中的高效表达

Effective Expression of Lipase in Non-Spore Aspergillus niger via Agrobacterium tumefaciens-Mediated Transformation

黑曲霉(Aspergillus niger)具有高效的蛋白表达和分泌能力.为实现异源蛋白在无孢黑曲霉中的高效重组表达,在优化遗传筛选标记的基础上,建立了根癌农杆菌介导的、以潮霉素抗性基因为筛选标记的黑曲霉转化体系.利用该体系介导米黑根毛霉脂肪酶(RML)转化黑曲霉SH-1,通过PCR鉴定、酶活检测、镍柱亲和层析及SDS-PAGE电泳检测等确定成功获得了RML的黑曲霉转化子,并通过发酵条件优化实现了RML的高效表达,对硝基苯酚比色法测得转化子酶活最高可达15 U/mL,是其在酵母中表达水平的10倍以上.

Aspergillus niger（A.niger） owns effective capabilities of protein expression and secretion.In order to implement the effective recombinant expression of heterologous protein in non-spore A.niger,several selection mar-kers were evaluated and hygromycin B resistance gene was selected to establish an Agrobacterium tumefaciens（A.tumefaciens）-mediated transformation system of A.niger.Then,Rhizomucor miehei lipase（RML） was transformed into A.niger SH-1 via the A.tumefaciens-mediated transformation and was verified by means of PCR identification,enzyme activity assay,Ni affinity chromatography and SDS-PAGE.Finally,RML transformants effectively expres-sing RML protein were obtained after the fermentation optimization.The p-nitrophenol colorimetry results show that the transformants possesses a lipase activity of 15 U/mL,which is more than ten times that expressed in yeast.