家蚕磷酸吡哆醇氧化酶的体外定点突变及其活性鉴定  

Site-directed mutation of pyridoxine 5'-phosphate oxidase from Bombyx mori and activity assay of the mutants in vitro

作　　者：童宁[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院,合肥230036

出　　处：《昆虫学报》2011年第11期1231-1235,共5页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(30870338)

摘　　要：【目的】研究家蚕Bombyx mori磷酸吡哆醇氧化酶(pyridoxine5'-phosphate oxidase,PNPO)个别保守氨基酸残基对PNPO酶活性的影响。【方法】用重叠延伸法把氨基酸残基Lys111(AAA)突变为Glu(GAA),Ser160(AGC)定点突变为Ala(GCC);构建重组表达载体,在大肠杆菌Escherichia coli Rosetta中诱导表达,经亲和纯化后进行酶活鉴定。【结果】重组蛋白的分子量约为45.0kDa。体外酶活分析发现,家蚕氨基酸残基Lys111突变体K111E活性降低了约78.0%,Ser160突变体S160A的活性降低了67.4%。【结论】结果提示氨基酸残基Lys111和Ser160对维持家蚕PNPO的酶活性有重要作用。本研究明确了家蚕磷酸吡哆醇氧化酶个别保守氨基酸残基的酶学功能。[Aim] To study the effects of single conserved amino acid residue of pyridoxine 5＇-phosphate oxidase（PNPO） from Bombyx mori on its activity.[Methods] Lys111（AAA） and Ser160（AGC）,two of the most conserved residues,were mutated to Glu（GAA） and Ala（GCC） by using over-lap extension,respectively.The obtained expression plasmids were over-expressed in Escherichia coli Rosetta by IPTG induction,and then the expressed products were purified with affinity chromatography and the activity of the purified PNPO were determined.[Results] The molecular mass of the target recombinant protein was ~45.0 kDa.Compared with the wild type PNPO,the activities of the mutants K111E and S160A were reduced by 78.0% and 67.4%,respectively.[Conclusion] The results suggest that the residues Lys111 and Ser160 are important to maintain the activity of PNPO.This study confirms the significance of single conserved amino acid residues on the catalytic function of PNPO of B.mori.

关 键 词：家蚕 磷酸吡哆醇氧化酶 定点突变 基因表达 蛋白纯化 酶活性 

分 类 号：Q966[生物学—昆虫学]