具有羟基化酶体系活性的重组大肠杆菌的构建

Construction of Recombinant Escherichia Coli with Hydroxylase System Activity

VD3羟化酶(VD3Hydroxylase,VDH)是一种P450单加氧酶,属于CYP107家族,能够催化维生素D3的25位羟基化。铁氧化还原蛋白(Ferredoxin,FDX)和铁氧化还原蛋白还原酶(Ferredoxin reductase,FDR)是两个电子传递链蛋白,参与羟化反应中电子的传递。构建具有羟基化酶体系活性的重组大肠杆菌,为骨化醇类化合物的高效转化提供参考。分别构建重组质粒p ETDuet-1-fdr-fdx及p ACYCDuet-1-vdh,将两个相容性质粒同时转入大肠杆菌BL21(DE3)中。采用IPTG对重组大肠杆菌进行诱导,使其表达目的蛋白,利用SDS-PAGE检测其蛋白大小及可溶性。采用液体传代的方法研究质粒稳定性。酶切鉴定和测序证明重组质粒p ETDuet-1-fdr-fdx及p ACYCDuet-1-vdh构建成功。SDS-PAGE结果显示目的蛋白大小与预期一致。重组大肠杆菌可以将阿法骨化醇和艾地骨化醇中间体(AD-M07)分别转化成骨化三醇和艾地骨化醇中间体(AD-M08),转化率分别为36. 10%和22. 87%,转化时间为12 h,比自养假诺卡氏菌缩短600%。重组大肠杆菌经传代50次,质粒稳定性达到92%以上,说明该重组大肠杆菌菌株稳定性较好。实验成功构建了具有羟基化酶体系活性的重组大肠杆菌,为高效转化阿法骨化醇和艾地骨化醇中间体(AD-M07)奠定了基础。

VD3 Hydroxylase(VDH)is a monooxygenase which is found to be homologous with the P450 belonging to CYP107 family.VDH shows VD325-hydroxylase activity.Ferredoxin(FDX)and Ferredoxin reductase(FDR)are electron transport proteins,which transfer electron for hydroxylation.The recombinant E.coli with hydroxylase activity was constructed to provide reference for the efficient transformation of Alfacalcidol and AD-M07.The compatible recombinant plasmids p ETDuet-1-fdr-fdx and p ACYCDuet-1-vdh were transformed into E.coli BL21(DE3)and induced by IPTG to obtain the target proteins.The molecular weight and solubility were identified by SDS-PAGE.The stability of plasmids was evaluated by subculturing of the recombinant E.coli.Enzyme digestion and DNA sequencing indicated that the recombinant plasmids of p ETDuet-1-fdr-fdx and p ACYCDuet-1-vdh were constructed successfully.SDS-PAGE showed that the expressed protein was consistent with the expected molecular weight.The recombinant E.coli could biotransform Alfacalcidol and AD-M07 to calcitriol and AD-M08 after 12 h with conversion rate of 36.10%and 22.87%respectively.The conversion time of recombinant E.coli was one-sixth of that of Pseudonocardia autotrophica.92%recombinant E.coli was plasmids-harboring cell in 50 th generation which indicated that this recombinant E.coli was stable.The author has constructed a recombinant E.coli with VD3 25-hydroxylase activity,which laid a foundation for more efficient transformation of Alfacalcidol and AD-M07.