Nanoscale roughness affects the activity of enzymes adsorbed on cluster-assembled titania films.
In this study, we investigated how the adsorption properties governed by the nanometer-scale surface morphology of cluster-assembled titanium oxide films influence the catalytic activity of immobilized serine-protease trypsin. We developed an activity assay for the parallel detection of physisorbed enzyme activity and mass density of the adsorbed proteins in microarray format. The method combines a microarray-based technique and advanced quantitative confocal microscopy approaches based on fluorescent labeling of enzymes and covalent labeling of active sites of surface-bound enzymes. The observed diminishing trypsin binding affinity with increasing roughness, as opposed to the steep rise in its saturation uptake, was interpreted as heterogeneous nucleation-driven adsorption of trypsin at the rough nanoporous titania surface. The increase in relative activity of adsorbed trypsin is proportional to the fractional saturation of titania surfaces, expressed as percentage of saturation uptake. In turn, the specific activity, that is, the ratio of active proteins to the absolute number of adsorbed proteins, drops with growing saturation uptake and surface roughness, witnessing a reduction in the accessibility of enzyme active sites. Both geometrical constraints of titania nanopores and the clusterwise adsorption of trypsin were identified as the key factors underpinning the steric hindrance of the immobilized enzyme. These findings are relevant for the optimization of rough nanoporous surfaces as carriers of immobilized enzymes. The proposed activity assay is particularly advantageous in the screening of candidate materials for enzyme immobilization.
European School of Molecular Medicine (SEMM), IFOM-IEO, Via Adamello 16, 20139 Milano, Italy.
Lasma
L
Gailite
Pasquale E
PE
Scopelliti
Vimal K
VK
Sharma
Marco
M
Indrieri
Alessandro
A
Podestà
Gabriella
G
Tedeschi
Paolo
P
Milani
D004800:Enzymes, Immobilized / Q000737:chemistry*; D008567:Membranes, Artificial; D040081:Protein Array Analysis; D013499:Surface Properties; D014025:Titanium / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; C009495:titanium dioxide; D014025:Titanium; D014357:Trypsin