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