现代食品科技2024，Vol.40Issue(3)：65-73,9.DOI:10.13982/j.mfst.1673-9078.2024.3.0350
腺苷酸环化酶在大肠杆菌中的表达及初步应用
Expression and Preliminary Application of Adenylate Cyclase in Escherichia coli
摘要
Abstract
Adenylate cyclase(AC)is essential for the enzymatic synthesis of cyclic adenosine monophosphate(cAMP).In fact,AC catalyzes the synthesis of cAMP and pyrophosphates(PPi)by adenosine triphosphate(ATP).In this study,Thermomonospora echinospora-derived AC(Te AC)was heterologously expressed in Escherichia coli.Following purification of the protein via affinity chromatography,its enzymatic properties were analyzed.Thereafter,the protein was utilized for the catalytic synthesis of cAMP.After inducing the expression of recombinant TeAC in E.coli at 16℃,TeAC was purified via affinity chromatography on a Ni column.Based on SDS-PAGE,the band of the protein of interest corresponded to 40 ku,which aligns with the expected protein size.The optimal temperature and pH of recombinant TeAC enzyme were 50℃and 10.5,respectively.Enzyme kinetic analysis revealed Km,Vmax,and Kcat values of 115.1 mmol/L,64.52 μmol/(mg·min),and 8.13 s-1,respectively,for enzymatic catalysis using the ATP substrate.After 11 h of the reaction catalyzed by TeAC,the yield of cAMP was 19.1 g/L.Overall,TeAC with efficient catalytic performance was successfully expressed,and its application in the catalytic synthesis of cAMP led to increased yield and production efficiency.This study lays the foundation for the enzymatic synthesis of cAMP.关键词
腺苷酸环化酶/环单磷酸腺苷/大肠杆菌Key words
adenylate cyclase/cyclic adenosine monophosphate/Escherichia coli引用本文复制引用
宋捷,郑穗平..腺苷酸环化酶在大肠杆菌中的表达及初步应用[J].现代食品科技,2024,40(3):65-73,9.基金项目
国家重点研发计划项目(2018YFA0901700) （2018YFA0901700）
