A Comparison of Covalent Immobilization and Physical Adsorption of a Cellulase Enzyme Mixture

Abstract

This paper reports the first use of a linker-free covalent approach for immobilizing an enzyme mixture. Adsorption from a mixture is difficult to control due to varying kinetics of adsorption, variations in the degree of unfolding and competitive binding effects. We show that surface activation by plasma immersion ion implantation (PIII) produces a mildly hydrophilic surface that covalently couples to protein molecules and avoids these issues, allowing the attachment of a uniform monolayer from a cellulase enzyme mixture. Atomic force microscopy (AFM) showed that the surface layer of the physically adsorbed cellulase layer on the mildly hydrophobic surface (without PIII) consisted of aggregated enzymes that changed conformation with incubation time. The evolution observed is consistent with the existence of transient complexes previously postulated to explain the long time constants for competitive displacement effects in adsorption from enzyme mixtures. AFM indicated that the covalently coupled bound layer to the PIII-treated surface consisted of a stable monolayer without enzyme aggregates, and became a double layer at longer incubation times. Light scattering analysis showed no indication of aggregates in the solution at room temperature, which indicates that the surface without PIII-treatment induced enzyme aggregation. A model for the attachment process of a protein mixture that includes the adsorption kinetics for both surfaces is presented.

Immobilization of Cellulase on a Functional Inorganic–Organic Hybrid Support: Stability and Kinetic Study

“…This is confirmed by the findings of Hirsh et al, who used polystyrene film after surface activation by plasma immersion ion implantation. They reported that, when cellulase was immobilized via covalent bonds, desorption was strongly reduced and the enzyme retained its catalytic properties [30]. Moreover, changes in the relative activity of immobilized cellulase after treatment with NaCl solutions at various concentrations indicated that the ionic strength of the solutions affected the catalytic activity of the immobilized cellulase.…”

Evaluation of nanoparticle‐immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

“…Endoglucanase from Thermomonospora fusca or T. reesei immobilized to polystyrene showed a 20% decrease in enzyme activity when the adsorption time was increased from 30 min to 24 h (Kongruang et al, 2003). A comparison between crude cellulase physisorbed or chemisorbed to polystyrene showed that plasma immersion ion implantation produced a distributed layer of enzyme that retained its native conformation; whereas, physisorbed cellulase unfolded and tended to form aggregates on the untreated polystyrene (Hirsh et al, 2010).…”

“…Materials that have been used as solid supports for enzyme immobilization include: polystyrene, Eupergit 1 , Sepharose, nylon, iron oxide, and others (Calsavara et al, 2001;Chim-Anage et al, 1986;Hirsh et al, 2010;Ho et al, 2008;Jain and Wilkins, 1987;Karagulyan et al, 2008;Kongruang et al, 2003;Sundstrom et al, 1981;Suvajittanont et al, 2000a,b;Tebeka et al, 2009;Woodward et al, 1984). It has been shown that the support used to immobilize the enzyme, as well as the immobilization method, will affect the function of the protein (Cao, 2005;Engasser and Horvath, 1976;Haynes and Norde, 1994;Norde, 2003).…”

Ion-implanted polytetrafluoroethylene enhances Saccharomyces cerevisiae biofilm formation for improved immobilization

“…From this, we can conclude that proteins on the untreated PTFE were physically adsorbed, while those on PIII-treated surfaces were covalently bonded. The use of this SDS washing protocol as a test of covalency has been demonstrated in literature over a range of surfaces of different surface energies [16,29,30].…”