家蚕磷酸吡哆醇氧化酶在E.coli中的表达及酶学特征研究  

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

作　　者：王振[1] 张剑韵[2] 黄龙全[1] 

机构地区：[1]安徽农业大学茶与食品科技学院,合肥230036 [2]安徽农业大学生命科学学院,合肥230036

出　　处：《蚕业科学》2010年第3期503-506,共4页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.30870338)

摘　　要：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。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 Ni^2＋ 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 opti- mum 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.

关 键 词：家蚕 磷酸吡哆醇氧化酶 原核表达 分离纯化 酶学性质 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]