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

Paper ID Volume ID Publish Year Pages File Format Full-Text
17092 42640 2013 8 PDF Available
Title
Catalytic activity and thermostability of enzymes immobilized on silanized surface: Influence of the crosslinking agent
Abstract

•Optimization of multimeric enzymes immobilization on silanized silicon surface using a variety of bifunctional crosslinkers.•Influence of crosslinkers to preserve catalytic activity and thermo stability.•Comparison of the different crosslinkers in terms of the number of covalent bonds and the stability of the links formed upon grafting.•For multimeric enzymes, steric hindrance could be beneficial to stabilize dimers.

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).These effects can be explained by a multipoint attachment model, in which a higher number of anchoring points stabilizes the three-dimensional structure and especially the binding of the two subunits in the active dimer, at the expense of a greater rigidity which is detrimental to the absolute activity. The differences observed with the crosslinkers are mainly due to steric hindrance at the interface which seems to be greatly influenced by the structure and the reactivity of the linkers.

Graphical abstractModifying the linker influences thermal and operational stabilities of enzymes immobilized on a silanized surface.Figure optionsDownload full-size imageDownload as PowerPoint slide

Keywords
Crosslinkers; Glucose-6-phosphate dehydrogenase (G6PDH); Multimeric enzyme; Enzyme immobilization; Silane; Thermostability
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Catalytic activity and thermostability of enzymes immobilized on silanized surface: Influence of the crosslinking agent
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 52, Issues 6–7, 10 May 2013, Pages 336–343
Authors
, , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us