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

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，EC 5.4.2.2）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1710 bp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63 kDa。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（pH 4~6）及碱性溶液（pH 8.5~10）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684 min-1和0.24 mM-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。

Abstract:

Exopolysaccharides produced by Paenibacillus mucilaginous as flocculants have 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 63 kDa. 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 0.24 mM-1 on G1P, respectively. This work lays a solid foundation for the construction of genetically engineered strains and metabolic engineering research.