Purification And Enzymatic Properties Of Pyridoxamine-Pyridoxal Conversion Enzymes From Bean Sprouts

Vitamin B6is the general term for a kind of chemical compounds, including the free forms of Pyridoxine(PN), Pyridoxamine(PM), Pyridoxal(PL) and the phosphate forms of Pyridoxine-5’-phosphate(PNP), Pyridoxamine-5’-phosphate(PMP), Pyridoxal-5’-phosph-ate(PLP). Pyridoxal-5’-phosphate acts as the essential coenzyme in many metabolic transi-tion of amino acids and plays an important role in a variety of synthetic and metabolic rea-ctions.PPAT can catalyze conversion between PM and PL, it with PM and pyruvate as the substrate generated PL and L-alanine which have been confirmed in microbiology meanwhile GOT in Pig heart can catalytic PM and PL transformation only on the condition of taking of the PLP. The study found that the plants also exist PM and PL transformation, but the enzymology properties and enzyme is not clear, which could be ordinary aminotransferase or the PPAT specificity.First of all, GOT was purified from bean sprouts by twice ammonium sulfate, twice Sephadex G-100gel filtration,twice DEAE-Sepharose Fast Flow anion exchange chromatography. The apparent purity was examined by SDS-PAGE.The conclusion this enzyme was purified approximately200.8-fold. Through the analysis of the substrate specificity of purified enzyme, found that glutamic-oxaloacetic holotransaminase can not catalytic PM and pyruvate generated PL and alanine, only under the condition of taking off the PLP.After GOT purified and analysed from bean sprouts, Pyridoxamine-pyruvate aminotransferase was purified from bean sprouts by twice ammonium sulfate, twice Sephadex G-100gel filtration,twice DEAE-Sepharose Fast Flow anion exchange chromatography. The apparent purity was examined by SDS-PAGE. By further study of Pyridoxamine-pyruvate aminotransferase, lead to the conclusion that this enzyme was purified approximately207.56-fold, the recovery of7.84%activity; In PM and pyruvic acid as substrate, the PPAT showed a maximum activity; under optimal conditions, the Km values for Pyridoxine and Pyruvate were1.7765mmol/L and5.3335mmol/L, respectively.