烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。

Purification and characterization of pyridoxal-5'-phosphate hydrolase from tobacco

Pyridoxal-5′-phosphate hydrolase was purified from tobacco by ammonium sulfate , DEAE-Sepharose Fast Flow ion exchange chromatography , Sephadex G-100gel filtration , SP Sephadex C-25ion exchange chromatography. Further investigations of pyridoxal-5′-phosphate hydrolase , reported herein , lead to the conclusion that this enzyme was purified approximately 119.6-fold , the recovery of 28.49% activity , Sephadex G-100gel filtration and SDS-PAGE showed that the molecular weight of the enzyme was 49.6kDa , and the molecular weight of subunit was approximately 25kDa ; The enzyme had an optimal temperature and pH at 50°C and 5.5 , respectively.It was enhanced by Mg 2+ , Ca 2+ and Mn 2+ , yet inhibited by chelating agent EDTA , which inhibited effect was relieved after added Mg 2+ ; under optimal conditions , the K m values for pyridoxal-5′-phosphate ( PLP ) and pyridoxamine-5′-phos-phate ( PMP ) were 0.23mmol / L , 0.56mmol / L , respectively.