麻疯树脂酶全长基因克隆、表达及其蛋白质结构预测

脂酶(Lipase,EC3.1.1.3)是普遍应用于皮革、饲料及生物柴油工业的工业酶制剂,具有广泛的应用价值。目前对植物来源的脂酶研究较少。本研究用在生物柴油中具有应用前景的油料植物——麻疯树(Jatrophacurcas)作为研究对象,克隆了该物种的脂酶基因(JcLIP)。通过多序列比对并结合物种的亲缘关系设计了具有较高特异性的简并引物,通过使用RT-PCR和RACE技术,最终获得了麻疯树脂酶基因的全长序列并成功地在大肠杆菌中表达,酶活测定结果表明,麻疯树脂酶在大肠杆菌中表达在包涵体中,但是能产生具有活力的蛋白质,酶活约为0.8U.mL-1。结构预测和比较表明,JcLIP蛋白质具有脂酶的结构核心和催化活性中心,而在非核心区具有较毛霉脂酶更多的插入和随机卷曲,这可能是决定二者之间酶活差异的重要原因。

Cloning and Expression of Lipase Gene JcLIP in Jatropha curcas and Its Protein Structure Prediction

Lipase is one of the most widely used enzymes in industries such as leather, food and bio-diesel production. It is valuable for its application to other fields. But research into lipase from plants is less reported than that of other lipases. In this paper, we describe the cloning of a lipase gene from Jatropha curcas, which has great potential in bio-diesel production. Degenerated primers were designed according to multiple align- ments with homologous sequences relative to J. curcas. RT-PCR and random amplification of cDNA ends (RACE) were performed to obtain full-length cDNA of JcLIP. The lipase was expressed in E. coli. The results of enzyme assay showed that the protein was in inclusion body, with the activity of the supernate only 0.8 U. mL-1. Structural prediction and comparison revealed JcLIP protein with the right structural core and catalytic activity site of lipase. The random coils and insertions of the JcLIP protein sequence is partially different in structure from that of lipase of Rhizomucor miehei, which may imply a different activity.