Latex Particles with Thermo‐Flocculation and Magnetic Properties for Immobilization of α‐Chymotrypsin

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Abstract
Core‐shell‐type latex particles composed of styrene, N‐isopropylacrylamide (NIPAAm), and N‐acryloxysuccinimide (NAS) were synthesized by surfactant‐free emulsion polymerization. The latex particles show thermo‐flocculation behavior due to the presence of temperature‐sensitive monomer NIPAAm and could be used for immobilization of α‐chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo‐flocculated latex particles in 20 min at 30 °C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature‐sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo‐flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one‐half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid‐liquid separation rate after thermo‐flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.