Catalytic activity and thermostability of enzymes immobilized on silanized surface: Influence of the crosslinking agent

In this work, we investigate the influence of crosslinkers on the operational and heat stability of immobilized enzymes on a silanized silicon surface. To this end, glucose-6-phosphate dehydrogenase (G6PDH), a model multimeric enzyme, was attached through bifunctional crosslinkers able to bind covalently the −NH2 in the silane layer and of amine residues in the enzyme. Five bifunctional crosslinkers in the form of “X-spacer-X” were used, differing by the reactive functional groups (X = aldehyde: −CHO, isothiocyanate: −NCS, isocyanate: −NCO), by the nature of the spacer chain (aromatic or aliphatic) or by the geometry (bifunctional groups positioned in meta- or para- on an aromatic ring). A thermostability enhancement has been obtained for enzymes immobilized using 1,4-phenylene diisothiocyanate (PDC) and 1,4-phenylene diisocyanate (DIC). Moreover, using the latter crosslinker, activity was the mostly preserved upon successive uses, thus giving the best operational stability achieved. Changing the geometry of the cross-linker, i.e., 1,4- as compared to 1,3-phenylene diisothiocyanate (PDC and MDC, respectively), has a crucial effect on operational and thermal stabilities. Indeed, among all used crosslinkers, the most important loss was observed for MDC (residual activity after 6 times use is ~16%). Using dialdehyde crosslinkers: glutaraldehyde (GA) and terephtalaldehyde (TE), activity was significantly less well preserved than with DIC and PDC (for GA and TE, a loss of about 50% at 30 °C against no loss for PDC and DIC).