胶质芽孢杆菌葡萄糖磷酸变位酶基因的克隆、表达与酶学性质研究  

Cloning, Expression, and Characterization of a Phosphoglucomutase from Paenibacillus mucilaginosus

作　　者：唐家毅[1] 蓝东明[2] 王永华[2] 杨博[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]华南理工大学轻工与食品学院广东广州510641

出　　处：《现代食品科技》2015年第3期38-42,37,共6页Modern Food Science and Technology

基　　金：广东省自然科学基金(S2012040007734);中央高校基金(2014ZM0062)

摘　　要：胶质芽孢杆菌所产胞外多糖作为絮凝剂在食品工业废水处理方面比传统的工业絮凝剂具有更好应用效果而引起了广泛的关注。葡萄糖磷酸变位酶（PGM,EC5.4.2.2）能将葡萄糖-6-磷酸转变成葡萄糖-1-磷酸而被认为是多糖合成路径上的关键酶之一。本研究克隆获得来源于自胶质芽孢杆菌GIM1.16编码PGM的基因pgm。序列分析表明,该基因包含一个1710bp的读码框。在大肠杆菌中表达了pgm基因并纯化了重组蛋白,SDS-PAGE电泳结果显示重组的PGM分子量约为63kDa。重组的PGM最适反应温度为40℃,但当反应温度超过55℃时,PGM基本丧失活性,PGM在酸性（pH4-6）及碱性溶液（pH8.5-10）中均表现出低活性,其最适合反应pH值为7.5。PGM对底物葡萄糖-1-磷酸的Kcat和Km值分别为684min-1和0.24mM-1。本研究为胶质芽孢杆菌菌株的基因工程改造和代谢工程研究奠定了坚实基础。Exopolysaccharides produced by Paenibacillus mucilaginous as flocculantshave attracted much attention from wastewater treatment in the food industry because of their better performance over traditional industrial flocculants. Phosphoglucomutase（PGM, EC 5.4.2.2）, which is responsible for the transformation of glucose 6-phosphate（G6P） into glucose 1-phosphate（G1P）, was considered to be the key enzyme involved in the biosynthesis of polysaccharides. In this article, a pgm gene encoding PGM from P. mucilaginous GIM1.16 was cloned. Sequence analysis showed that the pgm contained a 1710 bp open reading frame（ORF）. Subsequently, the pgm gene was expressed in Escherichia coli, and SDS-PAGE analysis demonstrated that the molecular weight of the recombined protein was 63k Da. The optimum temperature for the recombined PGM activity was 40 ℃, but the enzyme became inactive once the incubation temperature exceeded 55 ℃. The recombined PGM showed low activity in acidic conditions（pH 4.0-6.0） and alkaline conditions（pH 8.5-10.0）, and the optimum pH for the activity of this enzyme was 7.5. The Kcat and Km values of PGM were 684 min-1 and 0.24m M-1 on G1P, respectively. This work lays a solid foundation for the construction of genetically engineered strains and metabolic engineering research.

关 键 词：胞外多糖 胶质芽孢杆菌 葡萄糖磷酸变位酶 

分 类 号：Q78[生物学—分子生物学] Q55