Purification Of Phosphorylase And Enzymatic Sythesis Of Glucose-1-phosphate

Carbohydrates, proteins and nucleotides are the three of the most important materials about life. Carbohydrate Research is a rising hotspot and one of the renaissances in science research. The nucleotide sugars were found to act as sugar donors in transfer reactions. As an activated or naturally C1-protected sugar it may serve as an important intermediate or starting material in the synthesis of nucleotide sugars. It is not only the foundation of nucleotide sugars synthesis, but also the base of the oligosaccharides synthesis. This paper studied on the enzymic-sepectrophotometric analysis and biosynthesis ofα-D-glucose-1-phosphate (G-1-P). Potato phosphorylase which can catalyze the reaction, was purified and studied its charcters. A rapid and specific enzymic-sepectrophotometric method was described for determination of G-1-P in fermentation liquid by using phosphoglucomutase (PGluM EC5.4.2.2), glucose-6-phosphate dehydrogenase (G6P-DH EC1.1.1.49) and NADP, respectively. The mass concentration of G-1-P was linear with the change of absorption ofβ-NADPH (â–³A) in the range of 0⁴00 mg/L (r²=0.999 8), and the recovery of this method was 100.88%(RSD=0.52%). Potato phosphorylase can catalyze the reversible phosphorolysis of soluble starch . (1,4-α-D-glucosyl)_n+ P_i←→(1,4-α-D-glucosyl)_n-1+α-D -Glcose-1-phosphate First, to purify potato phosphorylase. The potato juice was purified by the ammonium sulfate fraction. The precipitate was collected by centrifugation between 30%⁸0% saturation, then filtrated through 50,000 membrane. The Aqueous Two-phase System(ATPS) were suitable for industry production. When DEX was added in the enzyme solution with the concentration of 20%, it did little affection on the enzyme. Incubating in 30 ℃for 17 h, 20% specific activity was lost. 20.9%PEG6000/4.2%DEX40,000, pH6.5, almost all the potato phosphorylase was gathered in the bottom phase. Collected the bottom phase and filtrated through 50,000 membrane to condense the enzyme. Purification folds was 455 and specific activity was 308.8U/mg. The character of potato phosphorylase was the follow: the optimum reaction temperature was 40 ℃. When the temperature exceeded 50 ℃,the activity of potato phosphorylase dropped very fast. The activity of potato phosphorylase lost completely above 65 ℃. The enzyme was more stable under 30 ℃. It could be incubated in 30 ℃for 17 h without activity decrease. The optimal pH was 7.0, and pH6.0⁷.5 was the best condition for potato phosphorylase. The K mA for orthophosphate in the phosphorolytic direction of G-1-P production was 11.71 mmol/L and K mB for soluble starch was 3.82 mg/mL. Na+,K+ and Zn2+ had less effect on the potato phosphorylase's stability; Cu2+,Hg2+ and Ag+ inhibited the activity for they are all heavy metals, which were easily inducing the protein degraded. And the nucleotides did not have any effect on the activity of potato phosphorylase. The substrate conditions for the phosphorolytic direction of G-1-P production was 500 mmol/L orthophosphate and 50 g/L soluble starch. pH 7.0, incubate in 40 ℃for 13 h and the percent of conversion was 17%.