植物乳杆菌WU146-磷酸-β-葡萄糖苷酶的基因克隆、蛋白表达及生物信息学分析

Cloning,Protein Expression and Bioinformatics Analysis of 6-Phosphor-β-glucosidase Gene in Lactobacillus plantarum WU14

植物乳杆菌是一类兼性异养,兼性厌氧的同型发酵乳酸菌,其在改善食品风味和发酵特性等方面发挥着重要作用,而这种功能特征与其具有糖苷酶活性密切相关。为了挖掘植物乳杆菌糖苷酶的应用价值,以植物乳杆菌WU14为研究对象,利用聚合酶链式反应(PCR)技术成功克隆了8个WU14的糖苷水解酶,生物信息学分析表明均为糖苷水解酶1家族(GH1)的6-磷酸-β-葡萄糖苷酶基因。鉴于植物乳杆菌中该类糖苷酶鲜有报道,采用原核异源表达展开研究。序列分析表明8个酶蛋白氨基酸序列具有GH1家族的6-磷酸-β-葡萄糖苷酶典型的2个保守催化位点,序列一致性在32%~74%之间。经SDS-PAGE,8个蛋白均在大肠杆菌中表达,其中BglAW14、BglCW14、BglFW14为部分可溶性表达,其余为蛋白表达,形成包涵体。生物信息学分析显示:8个基因的编码蛋白都无信号肽和跨膜结构,疏水性较强。由亚细胞定位预测可知,除BglEW14主要存在于分泌结构内和BglHW14主要存在于细胞膜外,其余6个基因都存于细胞质内。对8个6-磷酸-β-葡萄糖苷酶基因的克隆表达及生物学序列分析,为进一步探究植物乳杆菌来源的6-磷酸-β-葡萄糖苷酶的分子机理研究奠定了基础。

Lactobacillus plantarum is a kind of facultative heterotrophic and facultative anaerobic homo-fermenting lactic acid bacteria,which has glycosidase activity and plays an important role in improving food flavor and fermentation characteristics.Using PCR technology,86-phosphor-β-glucosidase genes from the glycoside hydrolase 1 family of L.plantarum WU14 were successfully cloned.It is showed that eight genes contain a complete open reading frame,and the amino acid sequence alignment reveals there are two conservative catalytic sites of the 6-phosphor-β-glucosidase gene in glycoside hydrolase 1 family,and the sequence identity is between 32%-74%.The results of SDS-PAGE shows that all proteins were expressed in E.coli.Among them,BglAW14,BglCW14,and BglFW14 are partially soluble expression.A purified and recovered to single target band was obtained,and the rest are expression of inclusion bodies.Bioinformatics analysis tells that the encoded proteins have weak hydrophobicity,and none of them have signal peptides and transmembrane structures.The prediction of subcellular structure shows that,except for BglEW14 mainly distributes in the secretion and BglHW14 mainly exists in the cell membrane.The remaining six genes are all present in the cytoplasm.The cloning,protein expression and biological sequence analysis of 6-phosphor-β-glucosidase genes laid a theoretical foundation for further exploring the molecular research of 6-phosphor-β-glucosidase derived from Lactobacillus plantarum.