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

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.