Protein Immobilization on Epoxy-Activated Thin Polymer Films: Effect of Surface Wettability and Enzyme Loading

Abstract

A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

Lipase B from Candida antarctica binds to hydrophobic substrate–water interfaces via hydrophobic anchors surrounding the active site entrance

“…2B, thick plane) which analyses atomic solvation parameters, interfacial polarity profiles, and ionization energies of charged residues. The predicted plane is also in agreement with the experimentally observed height of 1.64 nm for CALB monomers upon binding to a hydrophobic surface as determined by atomic force microscopy [39]. Helix ␣5 and ␣10 which are predicted to interact with the hydrophobic substrate layer have previously been intensively analysed by simulation of CALB in free solution [40].…”

Solid−Solid Interface Adsorption of Proteins and Enzymes in Nanophase-Separated Amphiphilic Conetworks

“…In fact, CaLB is known to form aggregates on surfaces, presumably by interprotein interactions between hydrophobic patches. 9 Conetwork Composition. Variations in the PDMS content in amphiphilic PHEA-l-PDMS conetworks greatly influence the morphology of the APCNs, as reported earlier.…”

Immobilization of biocatalysts for enzymatic polymerizations: Possibilities, advantages, applications

“…Variation in comonomer composition of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared, in order to explore the relationships between surface wettability and CALB binding to surfaces (Chen et al, 2008).…”