家蚕墨蝶呤还原酶基因的体外表达及酶活性研究

In vitro Expression and Enzymatic Properties Research on Sepiapterin Reductase from the Silkworm,Bombyx mori

家蚕(Bombyx mori)黄体色突变体(lem)的幼虫体壁富含墨蝶呤(SP),SP经墨蝶呤还原酶(SPR)的催化作用合成四氢生物蝶呤(BH4)。作为芳香族氨基酸羟化酶的重要辅酶,BH4的缺乏会导致多种神经性代谢综合症。前期研究已克隆获得家蚕SPR基因(BmSpr),确定了BmSpr为lem突变体的遗传本质。本实验将重组质粒pET-24b-BmSpr转化至E.coli不同菌株的感受态细胞,对BmSpr的体外表达条件进行了优化。SDSPAGE和Western Blot的检测结果表明BmSPR融合蛋白能够在原核表达系统中得到稳定表达,酶活性分析结果显示体外表达的重组BmSPR对其底物SP有较好的催化活性。本研究为进一步以从家蚕lem突变体资源大量提纯的SP为底物,利用原核表达BmSPR,开展体外合成BH4的应用基础研究奠定了实验基础。

Sepiapterin （SP）is contained in the integument of silkworm Bombyx mori mutant lemon （lem）in high con-centration.Sepiapterin reductase （SPR）is a key enzyme to catalyze SP to synthesize tetrahydrobiopterin （BH4）.BH4 is an essential cofactor for aromatic acid hydroxylases,deficiency of which has been associated with many neuropsycho-logical disorders.We have cloned the SPR gene from B.mori （BmSpr）and made it known that BmSpr was responsible for the lem mutant phenotype.In this study,the recombinant plasmid pET-24b-BmSpr was transformed into different E. coli strains and the expression conditions of BmSpr were optimized in vitro.SDS-PAGE and western blot analyses showed that BmSpr could be expressed steadily in the optimized prokaryotic expression system.The results of enzymatic analyses indicated that BmSpr exhibited great activity to the substrate of SP.Findings in this research help to apply the expressed BmSpr to synthesize BH4 in vitro by using extracted SP from the lem mutant as substrate in the future.