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

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.

[1]  J. Fréchet,et al.  Design of reactive porous polymer supports for high throughput bioreactors: poly(2-vinyl-4,4-dimethylazlactone-co-acrylamide- co-ethylene dimethacrylate) monoliths. , 1999, Biotechnology and bioengineering.

[2]  Jyh-Ping Chen.  Immobilization of α‐chymotrypsin to a temperature‐responsive reversibly soluble–insoluble oligomer based on N‐isopropylacrylamide , 1998 .

[3]  M. Bayhan,et al.  Uniform poly(isopropylacrylamide) gel beads for immobilization of α-chymotrypsin , 1998 .

[4]  S. Aksoy,et al.  Stability of α-amylase immobilized on poly(methyl methacrylate-acrylic acid) microspheres , 1998 .

[5]  Jyh-Ping Chen,et al.  Preparations and properties of temperature-sensitive poly(N-isopropylacrylamide) -chymotrypsin conjugates , 1997 .

[6]  H. Ayhan,et al.  Modified PMMA monosize microbeads for glucose oxidase immobilization , 1997 .

[7]  Peter Dunnill,et al.  Non-porous magnetic chelator supports for protein recovery by immobilised metal affinity adsorption , 1996 .

[8]  M. Akashi,et al.  Graft copolymers having hydrophobic backbone and hydrophilic branches. XI. Preparation and thermosensitive properties of polystyrene microspheres having poly (N‐isopropylacrylamide) branches on their surfaces , 1996 .

[9]  R. Jerome,et al.  Enzyme immobilization in reactive nanoparticles produced by inverse microemulsion polymerization , 1996 .

[10]  H. Kawaguchi,et al.  Modification and functionalization of hydrogel microspheres , 1996 .

[11]  Keiji Fujimoto,et al.  Enzyme immobilization on thermosensitive hydrogel microspheres , 1995 .

[12]  A. Kondo,et al.  Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G , 1995, Biotechnology and bioengineering.

[13]  G. Tao,et al.  Synthesis of Porous Polymer Carrier and Immobilization of α-Chymotrypsin , 1995 .

[14]  R. Jerome,et al.  Enzyme Immobilization in Nanoparticles Produced by Inverse Microemulsion Polymerization , 1994 .

[15]  A. Kondo,et al.  Development and application of thermo‐sensitive immunomicrospheres for antibody purification , 1994, Biotechnology and bioengineering.

[16]  Y. Ikada,et al.  Lipoprotein lipase immobilization onto porous polyvinyl alcohol beads , 1993 .

[17]  Toshio Hayashi,et al.  Papain immobilization onto porous poly(λ‐methyl L‐glutamate) beads , 1992 .

[18]  V. Goetz,et al.  A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing , 1991, Biotechnology and bioengineering.

[19]  Y. Ikada,et al.  Protease immobilization onto polyacrolein microspheres , 1990, Biotechnology and bioengineering.

[20]  S. Kamei,et al.  Adsorption of trypsin onto poly(2-hydroxyethyl methacrylate)/polystyrene composite microspheres and its enzymatic activity† , 1987 .

[21]  H. Kawaguchi,et al.  N-Substituted Acrylamide-Styrene Copolymer Latices II. Polymerization Behavior and Thermosensitive Stability of Latices , 1987 .

[22]  D. Clark,et al.  Enzyme immobilization on grafted polymeric microspheres , 1984 .

[23]  M. Wilchek,et al.  A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters. , 1982, Analytical biochemistry.

[24]  M. Lilly,et al.  Nonporous magnetic materials as enzyme supports: Studies with immobilized chymotrypsin , 1977, Biotechnology and bioengineering.

[25]  M. Lilly,et al.  An ultrafiltration‐reactor system using a soluble immobilized enzyme , 1971 .

[26]  M. Heskins,et al.  Solution Properties of Poly(N-isopropylacrylamide) , 1968 .

[27]  S. Słomkowski,et al.  Polypyrrole core/polyacrolein shell latex for protein immobilization , 1995 .

[28]  H. Honda,et al.  PREPARATION OF FINE MAGNETIC PARTICLES AND APPLICATION FOR ENZYME IMMOBILIZATION , 1991 .

[29]  M. A. D. Silva,et al.  Immobilization of α-chymotrypsin onto hydrolyzed poly(ethylene)-g-co-hydroxyethyl methacrylate , 1990 .

[30]  A S Hoffman,et al.  Immobilization and characterization of beta-galactosidase in thermally reversible hydrogel beads. , 1990, Journal of biomedical materials research.