摘要
四氢嘧啶羟化酶(EctD)是双加氧酶超家族的重要成员,其底物四氢嘧啶和产物羟基四氢嘧啶的应用广泛,因此EctD在生物制造领域具有重要的应用价值。从来源于新疆盐湖的需盐色盐杆菌(Chromohalobacter salexigens)中获得四氢嘧啶羟化酶基因(ectD),并在E.coli BL21(DE3)中进行表达,对异源表达的EctD酶学性质进行研究。结果表明:EctD的最适温度是30℃、最适pH是7.5,在30℃、pH 6.5~8.0条件下具有较好的稳定性;Fe^(2+)、Ba^(2+)、Mn^(2+)、Mg^(2+)、Fe^(3+)和Ca^(2+)离子可增强EctD的酶活,而CO_(2)+、Zn^(2+)和Cu^(2+)离子明显抑制EctD的酶活;动力学参数为K_(m)=7.63 mmol/L、k_(cat)/K_(m)=1.011 L/(mmol·s)、V_(max)=6.75μmol/(mL·min)。在最适条件下,该酶以70 mmol/L四氢嘧啶为底物,催化反应25 h后,可生成68 mmol/L的羟基四氢嘧啶,底物转化率可达98%。本研究通过对EctD的表达和表征,为酶法合成羟基四氢嘧啶奠定基础。
Ectoine hydroxylase(EctD),a member of the Fe^(2+)andα-dependent dioxygenase superfamily,catalyzes the hydroxylation of the substrate ectoine to the product hydroxyectoine.Due to the popular use of ectoine and hydroxyectoine in industry,the application of EctD is of great significance in the field of biomanufacturing.First,the ectoine hydroxylase gene(ectD)derived from the salt-tolerant bacteria in salt lakes of Xinjiang,Chromohalobacter salexigens,was cloned and expressed in E.coli BL21(DE3)using molecular biological technology.Such heterologously expressed ectoine hydroxylase was subsequently studied for its enzymatic properties.As a result,the optimum temperature of EctD is 30℃,the optimum pH is 7.5,and the enzyme exhibits excellent stability under the conditions of 30℃and pH 6.58.0.In addition,Fe^(2+),Ba^(2+),Mn^(2+),Mg^(2+),Fe^(3+)and Ca^(2+)ions can enhance the enzyme activity of EctD,while CO_(2)+,Zn^(2+)and Cu^(2+)ions display significant inhibition of EctD.The kinetic parameters are K_(m)=7.63 mmol/L,k_(cat)/K_(m)=1.011 L/(mmol·s),V_(max)=6.75μmol/(mL·min).Under the optimal conditions,the hydroxylation of ectoine(70 mmol/L)was catalyzed by EctD to afford 68 mmol/L of hydroxyectoine,with the substrate conversion rate reaching 98%,after 25 h of catalytic reaction.This research work on the expression and characterization of EctD provides a new approach for the complete conversion of ectoine to hydroxyectoine.
作者
曹莹莹
张皓
王鑫沂
徐虹
李莎
CAO Yingying;ZHANG Hao;WANG Xinyi;XU Hong;LI Sha(College of Food Science and Light Industry,Nanjing Tech University,Nanjing 211800,China)
出处
《生物加工过程》
CAS
2023年第3期263-270,共8页
Chinese Journal of Bioprocess Engineering
基金
江苏先进生物制造协同创新中心自主课题(XTD2201)
材料化学工程国家重点实验室开放课题(KL1912)。
关键词
四氢嘧啶羟化酶
需盐色盐杆菌
重组表达
酶学性质
羟基四氢嘧啶
ectoine hydroxylase
halobacterium halodendron
recombinant expression
enzyme properties
hydroxyectoine
