Enzyme structure and catalytic properties affected by the surface functional groups of mesoporous silica

Abstract

The enzyme subtilisin from Bacillus licheniformis (4.1 nm  7.8 nm  3.7 nm) was easily immobilized onto a mesoporous silica (MPS) surface by a direct one-step method and the amount of subtilisin immobilized on each functionalized MPS surface was similar (approximately 0.30 mg of enzyme/mg of MPS support). The catalytic performance (hydrolytic activity and enantioselectivity) of the immobilized subtilisin was found to depend on the properties of the organofunctional group on the MPS surface. In particular, the hydrolytic activity of enzyme immobilized on ethyl-group-modified MPS increased relative to the behavior of free subtilisin (relative activity 143%). The activity of subtilisin immobilized on the modified MPS was improved by facilitation of contact between enzyme and hydrophobic substrate by increase in hydrophobicity with an immobilized carrier. On the other hand, the enantioselectivity of subtilisin immobilized on 3-mercaptopropyl-group-modified MPS significantly decreased (enantioselectivity of 2.6 compared to 4.3 for free subtilisin). This decrease in enantioselectivity indicated that the mercapto group on the MPS surface was changed in the secondary structure of enzyme by interacting between enzyme and immobilized support. The denaturation temperature of subtilisin immobilized on no-substituted MPS increased (65 1C compared with 57 1C for free subtilisin). The denaturation temperature of immobilized subtilisin was dependent on the absorbed fraction of thermal energy by functional groups on the MPS surface.

Enzymes immobilized in mesoporous silica: A physical–chemical perspective

“…Similarly, enzyme inactivation has been explained by a loss of secondary structure [46]. CD has also been used to confirm a maintained structure of peptides within pores at low temperatures (4 ºC) [100] and to prove an increased stability in the pores at elevated temperatures compared to free enzymes in the bulk [57,101,102]. In special cases, CD can be useful in the visible wavelength regions for proteins containing non-amino acid…”

“…For measurements with enzymes inside particles the intensity of the signal may be reduced but the spectral shifts are virtually unaffected by the presence of scattering particles. Tryptophan fluorescence spectra have been used to prove a stabilizing effect of enzymes immobilized in MPS compared to free enzyme in solution during exposure to urea or elevated temperatures [99,101,102]. The technique has also been used to detect the reverse behavior: partial unfolding after immobilization into MPS [46,53].…”

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…A relatively large number of enzyme immobilizates obtained with different methods have been analyzed using CD [21,22,94,108,[111][112][113][114]. Alterations in protein secondary structure after immobilization were noted [21,22,94,108,112,[115][116][117][118], and efforts were made with limited success to relate these to changes in enzyme activity [108] and stability [94,116].…”

Upgrading of Biomass Monosaccharides by Immobilized Glucose Dehydrogenase and Xylose Dehydrogenase

“…A possible approach to overcome these limitations and at the same time increase the biocatalytic productivity via maximizing enzyme “reuse” is enzyme immobilization ,. Enzyme immobilization may moreover reduce the complexity of enzyme separation from the products after reaction ,. Various techniques and methods of immobilization such as adsorption, covalent binding, entrapment or encapsulation have been described previously .…”