表面偶极离子亲水磁珠固定化异柠檬酸裂解酶及其表征

Immobilization of Isocitrate Lyase on Amphoteric Ion-Rich Hydrophilic Magnetic Beads and Its Characterization

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6×His-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 6×His-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.