Despite the obviously practical value
of peroxidases, at present, their commercial uses are limited, primarily due to its low stability in the presence of hydrogen peroxide, their natural substrate. All heme-proteins, Selleckchem MK-2206 including peroxidases, are inactivated in the presence of some concentrations of hydrogen peroxide. This process, described as a suicide inactivation, is especially important in the absence of reducing substrates, but its mechanism has not been yet fully elucidated . Although the interest to peroxidase started several decades ago, their application as biocatalysts in industrial processes is still negligible due to its inherent instability under operational conditions, mainly caused by the inactivation in the presence Pritelivir ic50 of hydrogen peroxide. The development of techniques for enzyme stabilizing can improve a number of biocatalytic industrial processes. In this work, peroxidase enzyme has been immobilized onto porous silicon (PS) supports for the possible prevention from
its self-inactivation buy Doramapimod and its stability under different operational conditions has been analyzed. Methods A commercial peroxidase, Baylase® RP, was kindly donated by Bayer Mexico (Mexico, Federal District, Mexico). Crystalline silicon was a product from Cemat Silicon (Warsaw, Poland). Glutaraldehyde, 3-aminopropyldiethoxysilane, guaiacol, and bovine serum albumin were from Sigma-Aldrich (St. Louis, MO, USA). Bradford reagent was from Bio-Rad (Hercules, CA, USA). All other chemical reagents used in our experiment were of analytical grade
without further purification. Microreactor fabrication Fabrication of porous silicon(PS) <100 > oriented, heavily doped p-type Si wafers with resistivity 0.002 to 0.005 ohm-cm were electrochemically etched with an electrolyte composed of HF/ethanol/glycerol (3:7:1 (v/v)) at a constant current density of 50 mA cm-2 for 170 s to obtain a porous layer of 3,000 ± 60 nm. Functionalization of porous support The porous silicon samples were subjected to thermal oxidation in air at 600°C for 60 min. Silanization process with 3-aminopropyldiethoxysilane (APDES) was performed by immersing the sample in a 5% APDES in toluene for a period of 1 h and annealed at 110°C for 15 min. Glutaraldehyde (GTA, 2.5%) in Obatoclax Mesylate (GX15-070) phosphate buffer pH 6.0 was subsequently coupled to the support for 1 h and finally incubated with peroxidase for 24 h at 4°C. After each step of functionalization, the percent reflectance was measured and the chemical modification of the surface was verified by FTIR. RIFTS, SEM, FTIR, and gravimetric measurement of enzymatic microreactor Reflective interferometric Fourier transform method provides a fast and convenient method of extracting the basic optical parameters modified during the bio-functionalization steps onto of the PS surface.