家蚕磷酸吡哆醇氧化酶在E.coli中的表达及酶学特征研究

磷酸吡哆醇氧化酶(pyridoxine-5′-phosphate oxidase,PNPO)是维生素B6(VB6)代谢的关键酶。采用重组质粒pET32a(+)-PNPO原核表达家蚕(Bombyx mori)PNPO,经Ni2+亲和层析纯化后对其基本酶学性质进行分析。结果表明,纯化后的家蚕重组PNPO经SDS-PAGE鉴定为单一条带,比活力为529.81nmol/(min·mg)蛋白,纯化倍数为7.1倍;该酶的最适反应温度为40℃,在50℃以下稳定;在pH8.0～9.0之间酶活力最高,pH6.0～10.0之间活力保持稳定。该结果有助于进一步开展家蚕PNPO的催化作用和表达调控机制的研究。

Expression of Bombyx mori Pyridoxine-5'-phosphate Oxidase in E. coli and Assay of Enzymological Characters

Pyridoxine-5′-phosphate oxidase (PNPO) is the key enzyme in VB6 metabolism. Prokaryotic expression of the recombinant Bombyx mori PNPO was induced with isopropyl-β-D-thiogalactoside (IPTG) by using plasmid pET32a(+ )PNPO. The target protein was purified with Ni2+ affinity chromatography and the enzymological properties of PNPO were assayed. The results indicated that the purified Bombyx mori recombinant PNPO displayed only one band on SDS-PAGE electrophoretogram. Its specific activity was 529. 81 nmol/(min·mg) protein and enrichment times was 7. 1. The optimum temperature and pH of the enzyme were 40 ℃ and 8. 0 to 9. 0,respectively. The enzyme was stable in pH value ranging from 6. 0 to 10. 0 and at temperature below 50 ℃. These results laid a good foundation for further studies on Bombyx mori PNPO’s catalytic and expressional regulatory mechanisms.