Abstract:

In order to provide valuable evidences for the in-depth understanding and application of glycogen phosphorylase from Thermoanaerobacter tengcongensis MB4T (Tte-GlgP), the substrate spectrum of Tte-GlgP was determined by ESI-MS method. When soluble starch and maltoheptaose were respectively used as the substrates, the production of alpha-D-glucose- 1-phosphate (G-1-P) was clearly revealed in the ESI-MS chromatogram. When glycogen, maltodextrin and maltotriose were subjected to the same reaction, the production of G-1-P was also clearly detected. These results suggested Tte-GlgP had a relative wide substrate spectrum. To evaluate the relative activity of Tte-GlgP towards these substrates, a modified coupledenzyme method was used to measure the production of G-1-P in these reactions. When 0.25% soluble starch, glycogen, maltodextrin, maltoheptaose, maltopentaose, and maltotriose were used as substrate, respectively, it was established that the transformation efficiency represented by the production of G-1-P (μmol/L), was relatively higher for maltooligosaccharides (maltoheptaose and maltopentaose, with production of 86.83 μmol/L and 85.79 μmol/L G-1-P, respectively), maltodextrin (82.9 μmol/L G-1-P) and soluble starch (69.68 μmol/L G-1-P), but lower for glycogen and maltotriose (45.81μmol/L and 43.60 μmol/L G-1-P, respectively) at the same reaction condition. These results were consistent with those of ESI-MS analysis. Both suggested that Tte-GlgP could transform a wide variety of glucans into G-1-P, with maltooligosaccharides, maltodextrin and starch as the optimum substrates. For the determination of optimum conditions for reaction of Tte-GlgP with soluble starch as the substrate, 50 mmol/L potassium phosphate buffers with pH ranging from 5.0 to 9.0 were used to prepare the reaction mixtures. The reactions were performed respectively at 50, 60 ℃ and 80 ℃ for 30 min, and the highest activity of Tte-GlgP was achieved at pH 8.0 in all these assays, suggesting that the optimum pH for Tte-GlgP was pH 8.0. Then with this optimum pH (8.0), the reactions were performed at different temperature from 37 ℃ to 80 ℃ for 30 min to detect the optimum temperature, which was revealed to be 60 ℃. To determine the thermostability, Tte-GlgP was incubated at different temperatures from 50℃ to 80℃ for 0－21 h, and the residual activity was measured by the modified coupled-enzyme method (0.25% soluble starch was used as substrate). Results suggested that there were still 90% and 64% residual activity after treatment for 6 h at 60 ℃ and 70 ℃, respectively. Thus the Tte-GlgP is indeed a thermostable enzyme as expected.

Key words: Tte-GlgP, substrate spectrum, optimum pH, optimum temperature, thermostability