The 2,6-pyridinedicarboxylic acid covalently bonded to the silochrome surface: Immobilization and sorption-desorption properties (original) (raw)
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Biotechnology Progress, 2008
Cytochrome c can be readily adsorbed onto mesoporous silicates at high loadings of up to 10 mmol g -1 of silicate. The adsorbed protein retains its peroxidative activity, with no diffusional limitations being observed. The protein can be adsorbed onto the external surface of the silicate or, provided that the pore diameter is sufficiently large, into the channels. In aqueous buffer, the catalytic activity of the adsorbed protein (for the oxidation of ABTS) decreased with increasing temperature, with the decrease being less marked for cytochrome c held within the silicate channels. Similar results were obtained in 95% methanol. Analysis of kinetic data showed that significant increases in k cat /K M occurred in methanol, ethanol, and formamide, with slight decreases occurring in 1-methoxy-2-propanol. The observed increases were primarily a result of substantial increases in k cat , while the results in 1-methoxy-2-propanol can be ascribed to increases in K M . Resonance Raman spectroscopy indicated that the structure of the heme environment of the adsorbed protein was essentially unchanged, in aqueous buffer and in the nonaqueous solvents, methanol, 1-methoxy-2-propanol, and ethanol. In addition, Raman spectra of the lyophilized protein indicated that there were no apparent changes in the heme structure.
Cytochrome c immobilization into mesoporous molecular sieves
Journal of Molecular Catalysis B-enzymatic, 2000
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Chemistry – An Asian Journal, 2019
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2013
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 −NH 2 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).
Effect of a Methyl-Protecting Group on the Adsorption of Pyrrolidine on Si(100)-2 × 1
The room-temperature adsorption of pyrrolidine and its methyl-protected analogue, N-methylpyrrolidine, on the Si(100)-2 × 1 surface has been investigated using multiple internal reflection Fourier transform infrared spectroscopy and ab initio quantum chemistry calculations. For both compounds, initial adsorption occurs by barrierless formation of a dative bond between the nitrogen lone pair and the electrophilic atom of the Si dimer. However, while pyrrolidine proceeds to chemisorb dissociatively through N-H bond cleavage, methylpyrrolidine is shown to be trapped in its dative-bonded precursor state at room temperature due to a substantial barrier for N-CH 3 cleavage. Additionally, the saturation coverage of methylpyrrolidine on Si is seen to be significantly less than that of pyrrolidine, due likely to both steric factors and charge-transfer effects.
Langmuir, 2014
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 eff ect on catalytic activity. To this end, we used a model multimeric enzyme (G6PDH) and several cross-linking molecules with diff erent chemical properties, including the nature of the end-group (-NCO, -NCS, -CHO), the connecting chain (aliphatic vs aromatic), and geometrical constraints (meta vs paradisubstituted aromatics). There did not seem to be radical diff erences 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 infl uence 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 signifi cantly 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 diff erent 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.