| 引用本文: | 马娅萍, 黄龙全, 张剑韵.烟草磷酸吡哆醛水解酶的分离纯化与表征[J].广西植物,2012,(5):710-713.[点击复制] |
| MA Ya-Ping, HUANG Long-Quan, ZHANG Jian-Yun.Purification and characterization of pyridoxal-5'-phosphate hydrolase from tobacco[J].Guihaia,2012,(5):710-713.[点击复制] |
|
| 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Km值分别为0.23 mmol/L和0.56 mmol/L。 |
| 关键词: 烟草 磷酸吡哆醛 水解酶 纯化 酶性质 |
| DOI：10.3969/j.issn.1000-3142.2012.05.027 |
| 分类号:Q945.14 |
| 基金项目: |
|
| Purification and characterization of pyridoxal-5'-phosphate hydrolase from tobacco |
|
MA Ya-Ping1, HUANG Long-Quan1, ZHANG Jian-Yun2*
|
|
1.College of Tea &2.Food Sciences, Anhui Agricultural University, Heifei 230036, China;3.2.College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
|
| 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 Km values for pyridoxal-5'-phosphate(PLP)and pyridoxamine-5'-phos-phate(PMP)were 0.23 mmol/L,0.56 mmol/L,respectively. |
| Key words: tobacco pyridoxal-5'-phosphate hydrolase purification enzymological properties |
