title: Enzyme immobilization on silane-modified surface through short linkers: fate of interfacial phases and impact on catalytic activity.
abstract: We investigated the mechanism of enzyme immobilization on silanized surfaces through coupling agents (cross-linkers) in order to understand the role of these molecules on interfacial processes and their effect on catalytic activity. To this end, we used a model multimeric enzyme (G6PDH) and several cross-linking molecules with different chemical properties, including the nature of the end-group (-NCO, -NCS, -CHO), the connecting chain (aliphatic vs aromatic), and geometrical constraints (meta vs para-disubstituted aromatics). There did not seem to be radical differences in the mechanism of enzyme adsorption according to the linker used as judged from QCM-D, except that in the case of DIC (1,4-phenylene diisocyanate) the adsorption occurred more rapidly. In contrast, the nature of the cross-linker exerted a strong influence on the amount of enzyme immobilized as estimated from XPS, and more unexpectedly on the stability of the underlying silane layer. DIC, PDC (1,4-phenylene diisothiocyanate), or GA (glutaraldehyde) allowed successful enzyme immobilization. When the geometry of the linker was changed from 1,4-phenylene diisothiocyanate to 1,3-phenylene diisothiocyanate (MDC), the silane layer was subjected to degradation, upon enzyme adsorption, and the amount of immobilized molecules was significantly lowered. TE (terephtalaldehyde) and direct enzyme deposition without cross-linker were similar to MDC. The organization of immobilized enzymes also depended on the immobilization procedure, as different degrees of aggregation were observed by AFM. A correlation between the size of the aggregates and the catalytic properties of the enzyme was established, suggesting that aggregation may enhance the thermostability of the multimeric enzyme, probably through a compaction of the 3D structure.
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06 , F-75005, Paris, France.
authors.forename: Nesrine
authors.initials: N
authors.lastname: Aissaoui
authors.forename: Latifa
authors.initials: L
authors.lastname: Bergaoui
authors.forename: Souhir
authors.initials: S
authors.lastname: Boujday
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Lambert
authors.forename: Christophe
authors.initials: C
authors.lastname: Méthivier
authors.forename: Jessem
authors.initials: J
authors.lastname: Landoulsi
mesh_terms: D055162:Biocatalysis; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D010316:Particle Size; D012821:Silanes / Q000737:chemistry / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D012821:Silanes; D005954:Glucosephosphate Dehydrogenase