摘要
比较两种亲水磁珠固定化结核分枝杆菌异柠檬酸裂解酶(Mycobacterium tuberculosis isocitrate lyase,MtICL)并优化,为天然产物中MtICL先导抑制剂的亲和富集筛选提供适用的固定化酶.构建6xHis-pET28a-ICL,经E.coli BL21(DE3)重组表达、Ni2+-NTA柱纯化和酶学性质表征获得MtICL(重组蛋白质).用羧基功能化和Ni2+-NTA功能化制备两种表面偶极离子亲水磁珠固定化MtICL,比较其固载量、酶活力、稳定性及对已知抑制剂的响应.羧基磁珠固定化酶表观保留比活显著高于Ni2+-NTA固定化酶.当羧基磁珠与重组蛋白质加样质量比为60:1时,固定化酶表观保留比活可达游离酶的85％,此时羧基磁珠对MtICL表观固载量为(7.2±0.2)mg/g(以磁珠质量计,n=3).羧基磁珠固定化MtICL对衣康酸的亲和力与游离酶无显著差异(P＞0.05),且稳定性更强.此羧基磁珠固定化MtICL可望作为磁分离、亲和富集筛选天然产物中MtICL抑制剂类抗结核先导化合物.
Abstract
To provide magnetic immobilized Mycobacterium tuberculosis isocitrate lyase(MtICL)suitable for affinity-driven enrichment screening of MtICL lead inhibitors from natural products,two hydrophilic magnetic beads were compared for immobilization of MtICL under the optimized conditions.MtICL was obtained via expression of recombinant 6xHis-pET28a-ICL plasmid in E.coli BL21(DE3)and purification with Ni2+-NTA agarose column followed by characterization of enzymatic properties.Two amphoteric ion-rich hydrophilic magnetic beads,functionalized with carboxyl and Ni2+-NTA,were used to immobilize MtICL,respectively,for comparing their immobilization quantity,enzymatic activity,stability,and affinities for the known inhibitors.The apparent retention specific activity of carboxyl magnetic beads-immobilized MtICL was significantly higher than that of Ni2+-NTA magnetic bead-immobilized MtICL.When the mass ratio of carboxyl magnetic beads to protein was optimized to 60:1,the apparent retention activity of the immobilized enzyme reached 85％of that of the free enzyme.The apparent saturated immobilization capacity of carboxyl magnetic beads for MtICL was(7.2±0.2)mg/g(calculated by magnetic beads weight,n=3).There was no significant difference in the affinity of carboxyl magnetic bead-immobilized MtICL for itaconic acid compared to that of free MtICL(P＞0.05),and the stability was stronger.MtICL immobilized on carboxylic magnetic beads is suitable for the magnetic separation,affinite-driven,enrichment,and screening of MtICL inhibitors as anti-tuberculosis lead ligands from natural products.
基金项目
国家自然科学基金面上项目(82273900)
重庆市自然科学基金面上项目(CSTC2019jcyjmsxmX0166)
维普
万方数据