Purification And Characterization Of Pyridoxal-5’-Phosphate Hydrolase From Tobacco

Vitamin B6is a group of water-soluble vitamin, including the free forms ofPyridoxine（PN）, Pyridoxamine（PM）, Pyridoxal（PL） and the phosphate forms ofPyridoxine-5’-phosphate（PNP）, Pyridoxamine-5’-phosphate（PMP）, Pyridoxal-5’-ph-osphate（PLP）. Pyridoxal-5’-phosphate acts as the essential coenzyme in many metabolictransition of amino acids and plays an important role in a variety of synthetic andmetabolic reactions. In the mutual transformation between compounds of VB6,Phosphatase plays a significant role and catalyzes dephosphorylation of phosphate formsof VB6to form the free forms.Tobacco is an important economic crop, which plays an important role in nationaleconomic development. Simultaneously, tobacco is also as a model plant is widely usedin plant physiological and biochemical research. Pyridoxal-5’-phosphate hydrolase playsan significant role in the interconversion of VB6from the tobacco.Pyridoxal-5’-phosphate hydrolase was purified from Tobacco plants by ammoniumsulfate, DEAE Sepharose Fast Flow ion exchange chromatography, Sephadex G-100gelfiltration, SP Sephadex C-25ion exchange chromatography. The results showed that theenzyme was purified approximately119.6-fold, specific activity of1110.09U/mg, therecovery of28.49%activity, and the purified protein was a single electrophoretic band inSDS-PAGE with subunit molecular weight of25kDa.Pyridoxal-5’-phosphate hydrolase had an optimal temperature and pH at50℃and5.5, respectively. Under the conditions of50℃, the enzyme was very unstable, however,the activity had a much loss in a short time, while in the conditions of30℃⁴0℃, theenzyme can be better stable.The activity of Pyridoxal-5’-phosphate hydrolase was enhanced by Mg²⁺、Ca²⁺andMn²⁺, yet inhibited by chelating agent EDTA, which inhibited effect was relieved afteradded Mg²⁺. The rate of catalysis was Mg²⁺、Ca²⁺、Mn²⁺、Zn²⁺、Cu²⁺, respectively.Product （trisodium phosphate, PL） had a strong inhibition of enzyme activity, andsubstrate competitive inhibitors of ATP and4-Nitrophenyl phosphate disodium salthexahydrate （pNPP） also were strong inhibitory effects.Under optimal conditions, the Km values for Pyridoxal-5’-phosphate（PLP） andPyridoxamine-5’-phosphate（PMP） were0.23mmol/L and0.56mmol/L, respectively, whichsuggeststhat PLPhadthe better affinitythan PMPin VB6metabolic processes.