Purification And Characterization Of The Pyridoxal-5’-phosphate Hydrolase From Bean Sprouts

Vitamin B6is a general term of the pyridine compounds, including the six forms of Pyridoxine(PN), Pyridoxamine(PM), Pyridoxal(PL), Pyridoxine-5’-phosphate (PNP), Pyridoxamine-5’-phosphate(PMP), Pyridoxal-5’-phosphate(PLP), and they are interconvertible compounds. PLP is the catalytically active form of VB6. The high concentration of PLP can inhibit many enzymes in extracellular, therefore the concentration of PLP was maintained at a low level in intracellular. Phospoatase involved in the regulation mechanism of the PLP, which hydrolysis the PLP to PLThe hydrolysis of PLP is an important mechanism for the level of intracellular PLP. It was clear that the PLP in microorganisms and mammals can be hydrolysis by acid phosphatase and alkaline phosphatase, and can be controlled by the specific PLP phosphatase. The study of PLP hydrolysis in the plants is late, it is not clear that the phosphatase play an role in the mechanism.In this study, bean sprouts was taken as experimental material, purification was conducted with the PLP and pNPP as substrate. Analysis of the enzymatic properties are beneficial for the study of the phosphatase of PLP is specific PLP phosphatase or is not.The enzyme purified by taken the PLP as the substrate had an optimal temperature and pH at5.5. The activity can be enhanced by Mg2+、Zn2+and Mn2+, especially for Mg2+. The activity can be inhibited by fluoride and molybdate. Under optimal conditions, the Km values were0.27mmol/L and0.14mmol/L taken the PLP and pNPP as the substrate, it did not exhibit a strong affinity for the PLP.The enzyme purified by taken the pNPP as the substrate had an optimal temperature and pH at5.5. The activity can be enhanced by Mg2+、Zn2+and Mn2+, especially for Mg2+. The activity also can be inhibited by fluoride and molybdate. Under optimal conditions, the Km values were0.18mmol/L and0.16mmol/L taken the PLP and pNPP as the substrate.The result of the enzymatic properties showed that the phosphatase which hydrolysis the PLP was the unspecific phosphatase.