Immobilization of peroxidase enzyme onto the porous silicon structure for enhancing its activity and stability
© Sahare et al.; licensee Springer. 2014
Received: 19 May 2014
Accepted: 8 July 2014
Published: 21 August 2014
In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine.
Among microelectronic materials, silicon (Si) has the most mature and low-cost technology; hence, several research groups are approaching Si-compatible technology as an innovative platform for biosensors. Porous silicon has been intensively investigated for a variety of applications such as chemical and biological sensors, medical diagnostics, optical band pass filters, microchemical reactors, and microfuel cells. Moreover, Si-based matrixes have been proved to be a very useful support for the immobilization of enzymes thanks to their capability of retaining biological activity. Silicon (Si) received a lot of attention due to its specific semiconductor properties and furthermore because it allows the development of a broad range of micropatterning processes in order to achieve functional features for future integration in complex systems.
Furthermore, the Si-H and Si-OH groups on porous silicon surface can be easily modified by many reactive reagents and derivatives with receptors, thus enabling the identification of ligands. Microreactors are miniaturized reaction systems fabricated by microtechnology and precision engineering. The microreactors work with micro and nanoliter volumes of reaction media and ensure high efficiency and reproducibility of biocatalytic processes. Enzymatic microreactors have been already proposed as integrated components termed lab-on-a-chip for analytical applications in micrototal analysis systems (MTAS). There, the immobilization strategies to graft different chemical substances on the surface of a microreactor, a support, are used for a design of necessary conditions within the microreactor spaces. Surface modification by silanization is a very common method for particle functionalization. High density of free amino groups (-NH2) lying outwards the particle surface provides an excellent media for further chemical surface modification such as enzyme cross-linking with glutaraldehyde. The immobilization of enzymes in microreactors is mostly carried out in a covalent way. The main advantage of covalent immobilization is the retention of the enzyme during the whole biocatalytic process. Actually, immobilization is a well-established approach in a wide range of industrial applications. Both synthetic and natural inorganic materials such as clay, glass beads, silice-based materials, and celite have been used to immobilize enzymes, the natural catalysts for many biological processes. Among them, mesoporous silicates are the most interesting due to their attractive properties, availability, and simple preparation. Peroxidase immobilization on inorganic mesoporous silicates has proven to be an interesting alternative to improve enzyme functionality. The large regular repeating structures of photonic porous silicon structure offer the possibility of adsorbing or entrapping large biomolecules within their pores, providing a suitable microenvironment to stabilize the enzyme.
Peroxidases (EC 220.127.116.11, etc.) belong to a large family of enzymes that participate in a large number of natural processes developed in living organisms. They are ubiquitous in fungi, plants, and vertebrates. Their principal active sites contain a heme prosthetic group or, alternately, residues of redox-active cysteine or seleno-cysteine groups that are able to oxidize a large number of organic compounds initiated by one electron oxidation step. For all peroxidases, the natural substrate is hydrogen peroxide, but the oxidative process can be performed with many other organic hydro-peroxides such as lipid peroxides. In the oxidation of phenols or aromatic amines, peroxidases produce free radicals that may dimerise or polymerize and thus, in general, form products that are much less soluble in water. This property might be used in removing carcinogenic aromatic amines and phenols from industrial aqueous effluents. Enzymes are also involved in degradation of aromatic compounds and other xenobiotics, including pesticides, polycyclic aromatic hydrocarbons, and dioxins, and thus can be used for removal of aromatic pollutants[12, 13] as antioxidant, as indicators for food processing, in bioelectrodes and in the synthesis of conducting materials. Peroxidases could also be used in the synthesis of fine chemicals and optically and biologically active oxide. 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, 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 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 and its stability under different operational conditions has been analyzed.
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.
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 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. This method presents high sensitivity to small changes in the average refractive index of the porous thin film, allowing for direct and real-time monitoring of the binding of different species to the pore walls[19–22]. Reflective interferometric Fourier transform spectroscopy RIFTS analysis was performed on the specular reflectivity spectra of the PS measured with UV-VIS-NIR spectrophotometer (PerkinElmer Lambda 950, Waltham, MA, USA). As gravimetric measurement is the most direct method of determining the porosity of porous silicon[23–25], the measured porosity of the sample is found to be approximately 80%.
The surface and cross section image of mesoporous silicon was obtained by scanning electron microscope (SEM). Fourier transform infrared (FTIR) spectroscopy was used to identify and characterize the functional groups on the porous silicon surface. The FTIR spectra were collected at a resolution of 2 cm-1 on a Cary 640/660 FTIR Spectrometer - with an ATR accessory (Agilent Technologies, Mexico, Federal District, Mexico).
Steady-state measurements for peroxidase activity were carried out spectrophotometrically using guaiacol as electron donor substrate. Peroxidase activity was measured in 1 mL reaction solution containing 60 mM sodium phosphate buffer pH 6.0 at 25 to 28°C using 3 mM guaiacol, 1 mM hydrogen peroxide as the substrates and by monitoring the absorbance changes at λ = 470 nm using molar extinction coefficient value of 26.6 mM-1 cm-1 for the product tetra-guaiacol formed by the enzymatic reaction. One unit of peroxidase activity was defined as the amount of enzyme that caused the formation of micromoles of tetraguaiacol per min. The protein content was determined by Bradford method with the BioRad protein reagent.
Specific and non-specific immobilization
In an effort to compare the specific and non-specific immobilization of the enzyme load onto the microreactors, three different microreactors has been designed, (1) oxidized support immobilized with enzyme, (2) oxidized and ADPES treated then enzyme immobilization, and (3) oxidized, ADPES, and glutaraldehyde-activated surface incubated with the enzyme. The peroxidase activity of the anchored enzymes onto the pores of microreactors was detected by absorption spectroscopy using guaiacol as substrate at 470 nm.
Three different stabilities were tested for soluble and immobilized peroxidase preparations: Thermostability by incubating at 50°C, stability to organic solvent by incubating in 50% acetronitrile, and against inactivation in the presence of hydrogen peroxide (1 mM). In all cases, aliquots of each sample were withdrawn at different times and assayed for enzymatic activity under the standard condition. The data were adjusted to first-order rate model in order to calculate inactivation rate constants under each condition.
Results and discussion
Preparation of porous silicon substrates
Reflective interferometric Fourier transform spectroscopy
Specific and non-specific immobilization
Effect of immobilization chemistry on the enzyme loading onto PS support
Enzyme activity (U)
Oxidized + enzyme
Oxidized + ADPES + enzyme
Oxidized + ADPES + GTA + enzyme
Effect of PS layer thickness on the enzymatic activity
Effect of PS layer thickness (Si wafer) on the enzymatic activity
Thickness of the porous layer
No detectable activity
Thermal stability of immobilized peroxidase enzyme
Stability of peroxidase in aqueous-organic solvent mixture
Stability of peroxidase in the presence of hydrogen peroxide
This work is focused on porous silicon surface functionalization through the covalent attachment of the peroxidase enzyme with the PS support. The immobilization of the enzyme onto the porous silicon support has been confirmed from the RIFTS and FTIR studies. The study of thickness of the porous layer onto the availability of enzyme showed that higher thickness hinders the passage of substrate into the pores, which results in lower activity. The immobilized support showed lower thermo-stability with respect to soluble/free enzyme due to the major heat transfer through silicon support. The inactivation profile of peroxidase in the presence of acetonitrile indicates that the immobilized peroxidase is protected from acetonitrile deactivation; thus, acetonitrile has been revealed to be a very promising solvent to perform biocatalysis with peroxidase in organic media. While the deactivation of the enzyme in the presence of H2O2 in immobilized support is almost similar as compared to the soluble enzyme, these results conclude that a commercial peroxidase enzyme immobilized onto the porous silicon nanostructure confers more stability against organic solvents for potential industrial applications.
P.S. is a third year PG student at CIICAp, UAEM. RVD is a senior scientist in Biotechnology Institute (IBT) of National Autonomous University of Mexico (UNAM) working in the field of nano-biotechnology and bio-catalysis. MA is a scientist in IBT UNAM. VA is a senior scientist working in Research Centre for Engineering and Applied Sciences in the field of porous silicon and its applications.
The work was financially supported by CONACyT project: Ciencias Basicas #128953.
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