Immobilization of Isocitrate Lyase on Amphoteric Ion-Rich Hydrophilic Magnetic Beads and Its Characterization
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.
tuberculosisisocitrate lyasehydrophilic magnetic beadsenzyme immobilization
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