A comparison of covalent immobilization and physical adsorption of a cellulase enzyme mixture.
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.
Hirsh
S L
SL
Applied and Plasma Physics, School of Physics (A28), The University of Sydney, Sydney, NSW 2006, Australia. s.hirsh@physics.usyd.edu.au
Bilek
M M M
MM
Nosworthy
N J
NJ
Kondyurin
A
A
dos Remedios
C G
CG
McKenzie
D R
DR
D000327:Adsorption; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010316:Particle Size; D013499:Surface Properties
D004800:Enzymes, Immobilized; D002480:Cellulase