烟草磷酸吡哆醛水解酶的分离纯化与表征

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

关键词:  烟草  磷酸吡哆醛  水解酶  纯化  酶性质

Key words:  tobacco  pyridoxal-5'-phosphate  hydrolase  purification  enzymological properties

摘要:

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49.6 kDa,亚基分子量约为25 kDa; 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K_m值分别为0.23 mmol/L和0.56 mmol/L。

Abstract:

Pyridoxal-5'-phosphate hydrolase was purified from tobacco by ammonium sulfate,DEAE-Sepharose Fast Flow ion exchange chromatography,Sephadex G-100 gel filtration,SP Sephadex C-25 ion 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-100 gel filtration and SDS-PAGE showed that the molecular weight of the enzyme was 49.6 kDa,and the molecular weight of subunit was approximately 25 kDa; The enzyme had an optimal temperature and pH at 50°C and 5.5,respectively. It was enhanced by Mg²⁺,Ca²⁺ and Mn²⁺,yet inhibited by chelating agent EDTA,which inhibited effect was relieved after added Mg²⁺; under optimal conditions,the K_m values for pyridoxal-5'-phosphate(PLP)and pyridoxamine-5'-phos-phate(PMP)were 0.23 mmol/L,0.56 mmol/L,respectively.