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

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 alpha-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 degrees 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.

Enabling multienzyme biocatalysis using nanoporous materials

“…Since the nonporous particles do not suffer internal mass transfer limitations, usually much higher reaction rates can be expected than porous materials. (Chen and Su, 2001;Daubresse et al, 1996;Jia et al, 2003). This observation particularly demonstrated that the nanopores afforded efficient integration of the enzymes and cofactor, and thus fostered faster reactions.…”

“…Individual enzymes attached to submicron particles have demonstrated high enzyme activities that were close to those of free enzymes (Chen and Su, 2001;Daubresse et al, 1996); however, no work has been reported on the use of these particles for multienzyme systems. In this work, the triad catalytic system was coimmobilized onto the outer surface of 500-nm (diameter) polystyrene particles (with PEG 10,000 as the spacer) prepared according to a procedure reported previously (Jia et al, 2003), with final loadings as 3.65 mg NADH and 0.38 mg enzyme per gram of particles (molar ratio of cofactor to enzyme: 416, similar to those of nanoporous glass).…”

Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility

“…The enzyme was covalently attached onto polymeric particles via a coupling reaction between the succinimide ester group of NAS and amino groups of enzyme (Chen and Su, 2001). Typically, the precleaned nanoparticles were first dispersed into 0.1M pH 6.0 phosphate buffer at a concentration of f50 mg/mL, and then 30 mg of CT was dissolved in 2 mL of this particle-containing buffer in a 20-mL vial.…”

“…Recently reported work in this area has revealed the great potential for the use of nanoporous (Wang et al, 2001), nanofibrous (Jia et al, 2002), and nanoparticle (Caruso and Schuler, 2000;Daubresse et al, 1996;Liao and Chen, 2001;Martins et al, 1996) materials as a new class of carriers for biocatalysts. The effective enzyme loading on nanomaterials can be very high (for example, it can reach over 10 wt% with particles smaller than 100 nm (Chen and Su, 2001)), and a large surface area per unit mass is also provided to facilitate reaction kinetics.…”

Design of novel nano-carriers for multi-enzyme co-localization

“…It has been reported that up to 10 wt.% effective enzyme loading can be achieved on nanoparticles (Chen and Su, 2001). Attachment of enzymes on nanoparticle surfaces also significantly avoids the internal diffusion resistances observed with porous materials.…”