Methodology for the immobilization of enzymes onto mesoporous materials.
Cytochrome c and xylanase were adsorbed onto two mesoporous materials, SBA-15 (a pure silicate) and MSE (an organosilicate), with very similar physical properties but differing chemical compositions. A methodical order was developed whereby the influences of surface area, pore size, extent of order, particle size, surface potentials, isoelectric points, pH, and ionic strength on immobilization were explored. In silico studies of cytochrome c and xylanase were conducted before any immobilization experiments were carried out in order to select compatible materials and probe the interactions between the adsorbents and the mesoporous silicates. The stabilities of the mesoporous materials at different pH values and their isoelectric points and zeta potentials were determined. Electrostatic attraction dominated protein interactions with SBA-15, while weaker hydrophobic interactions are more prominent with MSE for both cytochrome c and xylanase. The ability of the immobilized protein/enzyme to withstand leaching was measured, and activity tests and thermostability experiments were conducted. Cytochrome c immobilized onto SBA-15 showed resistance to leaching and an enhanced activity compared to free protein. The immobilized cytochrome c was shown to have higher intrinsic activity but lower thermostability than free cytochrome c. From an extensive characterization of the surface properties of the silicates and proteins, we describe a systematic methodology for the adsorption of proteins onto mesoporous silicates. This approach can be utilized in the design of a solid support for any protein.
Hudson
Sarah
S
Materials and Surface Science Institute and Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland. sarah.hudson@ul.ie
Magner
Edmond
E
Cooney
Jakki
J
Hodnett
B Kieran
BK
D000327:Adsorption; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D045304:Cytochromes c / Q000737:chemistry; D004563:Electrochemistry; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry; D016062:Porosity; D011506:Proteins / Q000737:chemistry; D017640:Silicates / Q000737:chemistry; D013816:Thermodynamics
D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D011506:Proteins; D017640:Silicates; D045304:Cytochromes c; D043364:Endo-1,4-beta Xylanases; D009584:Nitrogen