Matrices For Enzyme Immobilization Research Articles

Preparation of cobalt nanoparticles from polymorphic bacterial templates: A novel platform for biocatalysis

Nanoparticles have gathered significant research attention as materials for enzyme immobilization due to their advantageous properties such as low diffusion rates, ease of manipulation, and large surface areas. Here, polymorphic cobalt nanoparticles of varied sizes and shapes were prepared using Micrococcus lylae, Bacillus subtilis, Escherichia coli, Paracoccus sp., and Haloarcula vallismortis as bacterial templates. Furthermore, nine lipases/carboxylesterases were successfully immobilized on these cobalt nanoparticles. Especially, immobilized forms of Est-Y29, LmH, and Sm23 were characterized in more detail for potential industrial applications. Immobilization of enzymes onto cobalt oxide nanoparticles prepared from polymorphic bacterial templates may have potential for efficient hydrolysis on an industrial-scale, with several advantages such as high retention of enzymatic activity, increased stability, and strong reusability.

Immobilization of Proteins in Poly-Styrene-Divinylbenzene Matrices: Functional Properties and Applications

Supports based on poly-styrene-divinylbenzene (PSD) are commercially available since a long time ago. However, they are not commonly used as enzyme immobilization matrices. The main reason for this lies in the negative effect of the very hydrophobic surface on enzyme stability that produces the instantaneous enzyme inactivation in many instances. However, they have recently regained some impact in enzyme immobilization. They are easy to modify, and have been prepared with different modifiers. We will pay special attention to the coating of these supports with ionic liquids, which permits to have the ionic liquid phase anchored to the solid and modulate the enzyme properties without risk of losing these expensive and potentially toxic compounds. Thus, this review will present the covalent or physical immobilization of enzymes on PSD supports, submitted to different modifications. Moreover, lipases immobilized via interfacial activation on some naked PSD supports have shown some unexpected improvement in their catalytic properties, with uses in reactions like hydrolysis, esterification or transesterification. Keywords: Hydrophobic supports, enzyme destabilization, interfacial activation of lipases, modulation of lipase properties, supported ionic liquids.

Covalent Immobilization of Catalase onto Regenerated Silk Fibroins via Tyrosinase-Catalyzed Cross-Linking.

Regenerated silk fibroins could be used as medical scaffolds and carrier materials for enzyme immobilization. In the present work, tyrosinase enzyme was used for enzymatic oxidation of silk fibroins, followed by immobilization of catalase onto the fibroin surfaces through physical adsorption and covalent cross-linking as well. Spectrophotometry, SDS-PAGE, and Fourier transform infrared spectroscopy (FTIR) were used to examine the efficiency of enzymatic oxidation and catalase immobilization, respectively. The results indicate that tyrosine residues in silk fibroins could be oxidized and converted to the active o-quinones. Incubating silk fibroins with catalase and tyrosinase led to a noticeable change of molecular weight distribution, indicating the occurrence of the cross-links between silk fibroins and catalase molecules. Two different pathways were proposed for the catalase immobilizations, and the method based on grafting of catalase onto the freeze-dried fibroin membrane is more acceptable. The residual enzyme activity for the immobilized catalase exhibited higher than that of the control after repeated washing cycles. Meanwhile, the thermal stability and alkali resistance were also slightly improved as compared to free catalase. The mechanisms of enzymatic immobilization are also concerned.

Reduced graphene oxide enhances horseradish peroxidase stability by serving as radical scavenger and redox mediator

Graphene-based nanomaterials have been widely studied as high-performance matrices for enzyme immobilization and in the development of biosensors. Surface O-functionalities of graphene induce changes in chemical reactivity and electronic conductivity of nanomaterials and may interfere with enzymatic processes; however, the mechanisms are not fully understood. We compare the effects of three commercially available graphene-based nanomaterials, namely a graphene, a graphene oxide (GO), and a reduced graphene oxide (RGO), on the activity/stability of horseradish peroxidase. Both graphene and GO significantly reduced enzyme stability by altering enzyme conformation, which was evidenced by circular dichroism spectroscopy. However, RGO improved enzyme stability up to 7-fold. This increased stability was attributed to the capability of RGO to quench superoxide radicals, which were primarily responsible for the enzyme deactivation. The basal plane of RGO, mainly through quinone moieties, may act as a redox mediator to facilitate enzymatic turnover. Overall, the radical scavenging plus redox mediating capabilities of RGO suggest the potential for graphene-based nanomaterials to enhance enzyme engineering and enzyme-based sensors.

Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry.

Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions.

Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels

Chitosan-grafted hydrogels were employed for immobilization and controlled released of β-galactosidase. These hydrogels containing immobilized enzymes were employed to simulate the production of lactose-free food and controlled release of β-galactosidase into lactose-intolerant individuals. The degree of swelling, efficiency of immobilization (i.e., fractional uptake of enzyme), and controlled release were studied as a function of pH and temperature. The degrees of swelling decreased in acidic media: 49.4g absorbed water per g hydrogel at pH 7.0, and 8.4g absorbed water per g hydrogel at pH 3.5. The immobilization efficiency was 19%, indicating that chitosan-grafted hydrogels are promising matrices for enzyme adsorption and immobilization. Cyclic experiments reveal that chitosan-grafted hydrogels containing immobilized enzymes can be reused several times without introducing additional enzyme prior to each cycle. There is no significant decrease in the activity of the immobilized enzyme during reutilization studies. All results were conducted in triplicate by considering t-tests at a 95% significance level. Analysis of β-galactosidase activity and controlled release reveals that chitosan-grafted hydrogels containing immobilized enzymes are useful for the production of lactose-free food and controlled enzyme release with high performance.

Enzyme Immobilization: An Overview on Nanoparticles as Immobilization Matrix

Immobilization process is to optimize the operational performance of an enzyme for industrial applications. So far different matrices have been described in the literature to improve the performance of the immobilized enzymes. With the advent of nanotechnology, the nanomaterials because of their unique physico-chemical properties constitute novel and interesting matrices for enzyme immobilization. The nanomaterials possess ideal characteristics to equilibrate principal factors which determine biocatalysts efficiency, including specific surface area, mass transfer resistance and effective enzyme loading. This review presents the current scenario and techniques in enzyme immobilization. An overview of the main methods used to combine proteins/enzymes with nanoparticles is given in the study. The advantages and disadvantages of nanoparticles as immobilization matrix are also discussed.

Rhizopus oryzae lipase immobilized on hierarchical mesoporous silica supports for transesterification of rice bran oil.

The tunable textural properties of self-oriented mesoporous silica were investigated for their suitability as enzyme immobilization matrices to support transesterification of rice bran oil. Different morphologies of mesoporous silica (rod-like, rice-like, and spherical) were synthesized and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and nitrogen adsorption-desorption isotherms. The surface area, pore size, and ordered arrangement of the pores were found to influence the immobilization and activity of the enzyme in the mesopores. The immobilization in rod-like silica was highest with an immobilization efficiency of 63 % and exhibited minimal activity loss after immobilization. Functionalization of the mesoporous surface with ethyl groups further enhanced the enzyme immobilization. The free enzyme lost most of its activity at 50 °C while the immobilized enzyme showed activity even up to 60 °C. Transesterified product yield of nearly 82 % was obtained for 24 h of reaction with enzyme immobilized on ethyl-functionalized SBA-15 at an oil:methanol ratio of 1:3. Fourier transform infrared spectroscopy (FT-IR) and Gas chromatography-mass spectrometry (GC-MS) were used to characterize the transesterified product obtained. The reusability of the immobilized enzyme was studied for 3 cycles.

Characterization of Waste Clay from Palm Oil Mill Effluent and Enzyme Immobilization Study for Cassava Saccharification Process

Waste clay recovered from palm oil mill effluent (POME) was characterized and used as an enzyme-supporting material for the cassava saccharification process. The clay was treated by the Soxhlet extraction method to remove the residual oil and then characterized using a BET surface area analyser, XRF, XRD, FTIR, TGA, and FESEM. The chemical analysis showed that the sample had a high amount of CaO (93%) with a minor content of SiO2 (1.378%) and Al2O3 (0.707%), with a surface area of 1.15 m2/g. The XRD analysis revealed the major mineral presence to be calcite, as confirmed by FESEM analysis. The FTIR results also attested to the presence of a calcite phase and carbonate groups. To study the performance of the waste clay for enzyme immobilization application, the recovered waste clay was further used as an enzyme supporting material for enzyme immobilization in the cassava saccharification process. Results showed that the enzymes were successfully encapsulated and gave the highest immobilization yield of 70% with 2% clay concentration. In addition, the encapsulated enzymes also enhanced the reusability, where the enzyme retained 32% of its activity after seven cycles of saccharification processing.

Enzyme immobilization on polymethacrylate-based monolith fabricated via thermally induced phase separation

Polymer-based monolithic materials with interconnected porous structure have received much attention as high-performance matrices for enzyme immobilization. The present work describes immobilization of horseradish peroxidase (HRP) onto a polymethacrylate-based monolith which was fabricated by thermally induced phase separation (TIPS) method. The poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith was modified with adipic acid dihydrazide (AADH) and ethylenediaminetetraacetic dianhydride (EDTAD) to yield carboxyl group-bearing PGM (PGM-COOH) monolith. This monolith was reacted with N-hydroxysuccinimide (NHS) to activate the carboxyl groups on the monolith and further modified with HRP. The immobilized HRP showed much higher thermal stability than the free one. Furthermore, the immobilized HRP could be reused for at least 6 times with the remaining activity of 52%. The PGM monolith may have a high potential as a solid support for enzyme immobilization.

A comparative study of the effectiveness of β-glucosidase immobilized on CNT-nanoparticles and Ca-alginate beads

Enzymes are extensively used in various industrial, biomedical and biopharmaceutical applications. However, enzymes in their free form are unstable and expensive besides being characteristically susceptible to inhibition by high product concentrations and are highly sensitive to pH and temperature changes. Immobilization technology offers solutions to these challenges besides enhancing operational stability, longevity and ease of separation. β-glucosidase has been widely employed as model enzyme for enzymatic studies. Ca-alginate beads provide a gentle environment for immobilization, but have certain limitations such as low stability, high porosity and limitations in biocompatibility. Carbon nanotubes (CNTs) on the other hand have excellent mechanical, thermal and electrical properties, as well as dimensional and chemical compatibility with biomolecules like DNA and enzymes, suitable for biosensor design. Here, β-glucosidase was immobilized in Ca-alginate gel and multi-walled carbon nanotubes (MWCNT) using standard techniques and their activity was compared with that of free enzyme. The activity was found highest (12.53 U/mL) for the free enzyme and lowest (9.768 U/mL) for the immobilized Ca-alginate. The activity of immobilized MWCNT (12.20 U/mL) was close to the free enzyme activity. The enzyme reaction was found to follow Michaelis-Menten kinetics. The Michaelis constants, Km and Vmax, determined using Langmuir linearized plot are respectively 0.09048 μmol/mL and 0.00989 μmol/mL.min for immobilized Ca-alginate; and 0.0985 μmol/mL and 0.01237 μmol/mL.min for immobilized MWCNT. The corresponding values for the free enzyme are 0.0854 μmol/mL and 0.01263 μmol/mL.min. Thus, MWCNT appears to be a promising support material for enzyme immobilization. Keywords: β-glucosidase, immobilization, Ca-alginate, carbon-nanotube, Michaelis-Menten kinetics

Nanoscale roughness affects the activity of enzymes adsorbed on cluster-assembled titania films.

In this study, we investigated how the adsorption properties governed by the nanometer-scale surface morphology of cluster-assembled titanium oxide films influence the catalytic activity of immobilized serine-protease trypsin. We developed an activity assay for the parallel detection of physisorbed enzyme activity and mass density of the adsorbed proteins in microarray format. The method combines a microarray-based technique and advanced quantitative confocal microscopy approaches based on fluorescent labeling of enzymes and covalent labeling of active sites of surface-bound enzymes. The observed diminishing trypsin binding affinity with increasing roughness, as opposed to the steep rise in its saturation uptake, was interpreted as heterogeneous nucleation-driven adsorption of trypsin at the rough nanoporous titania surface. The increase in relative activity of adsorbed trypsin is proportional to the fractional saturation of titania surfaces, expressed as percentage of saturation uptake. In turn, the specific activity, that is, the ratio of active proteins to the absolute number of adsorbed proteins, drops with growing saturation uptake and surface roughness, witnessing a reduction in the accessibility of enzyme active sites. Both geometrical constraints of titania nanopores and the clusterwise adsorption of trypsin were identified as the key factors underpinning the steric hindrance of the immobilized enzyme. These findings are relevant for the optimization of rough nanoporous surfaces as carriers of immobilized enzymes. The proposed activity assay is particularly advantageous in the screening of candidate materials for enzyme immobilization.

Graphene-based field effect transistor enzymatic glucose biosensor using silk protein for enzyme immobilization and device substrate

A silk fibroin-encapsulated graphene field effect transistor (FET) enzymatic biosensor that utilizes silk protein as both device substrate and enzyme immobilization material was developed for glucose detection. This biosensor detected glucose levels by measuring the differential drain-source current and the Dirac point shift of the graphene transistor as the glucose is oxidized by glucose oxidase that was immobilized in silk fibroin film on the graphene FET. The fabricated biosensors showed 0.1–10mM large linear detection range, which covers the reference range of medical examination for diabetes diagnostics. The detection limit of the fabricated biosensors was approximately 0.1mM (S/N=3) with excellent selectivity, and the average sensitivity was 2.5μA/mM measured at Vds=100mV and Vg=0V. Because this fibroin-encapsulated graphene FET enzymatic biosensor is biocompatible, flexible, and long-term stable, it holds a great promise for portable, wearable, and implantable continuous glucose monitoring applications.

Acetylcholinesterase biosensor for carbaryl detection based on interdigitated array microelectrodes.

In this study, an acetylcholinesterase (AChE) biosensor with superior accuracy and sensitivity was successfully developed based on interdigitated array microelectrodes (IAMs). IAMs have a series of parallel microband electrodes with alternating microbands connected together. Chitosan was used as the enzyme immobilization material, and AChE was used as the model enzyme for carbaryl detection to fabricate AChE biosensor. Electrochemical impedance spectroscopy was used in conjunction with the fabricated biosensor to detect pesticide residues. Based on the inhibition of pesticides on the AChE activity, using carbaryl as model compounds, the biosensor exhibited a wide range, low detection limit, and high stability. Moreover, the biosensor can also be used as a new promising tool for pesticide residue analysis.

Adsorption of microbial esterases on Bacillus subtilis-templated cobalt oxide nanoparticles

Due to low diffusion rates and large surface areas, nanomaterials have received great interest as supporting materials for enzyme immobilization. Here, the preparation of a cobalt oxide nanoparticle using Bacillus subtilis as a biological template and use of the nanostructure for microbial esterase immobilization is described. Morphological features and size distributions were investigated using electron microscopy (EM) and dynamic light scattering (DLS). Catalytic properties of enzyme-coated nanostructures were investigated using 4-methylumbelliferyl acetate and p-nitrophenyl (PNP) acetate as model substrates. Enzyme-coated nanostructures were observed to retain ∼85% of the initial activity after 15 successive reaction cycles, and enzyme immobilization processes could be repeated four times without a loss of immobilization potential. The present work demonstrates that B. subtilis-templated cobalt oxide nanoparticles have the potential to be used as biocompatible immobilization materials, and are promising candidates for the preparation of effective nanobiocatalysts.

Enzymatic interesterification of soybean oil and methyl stearate blends using lipase immobilized on magnetic Fe3O4/SBA-15 composites as a biocatalyst.

The magnetic Fe3O4/SBA-15 composites were prepared, and treated with 3-aminopropyltriethoxysilane as a carrier material for enzyme immobilization. The immobilization of Candida rugosa lipase onto the amino-functionalized Fe3O4/SBA-15 composite was investigated by using glutaraldehyde as a coupling reagent. The immobilized lipase was then employed as a biocatalyst for the interesterification of soybean oil and methyl stearate in a laboratory-scale operation at 45°C. Various techniques, such as Fourier transform infrared spectroscopy (FT-IR), powder X-ray diffraction (XRD), and vibrating sample magnetometry (VSM), were used for the characterization of the immobilized lipase composite. The immobilized lipase behaved superparamagnetic and showed excellent response at applied magnetic field. The obtained results showed that the immobilized lipase could efficiently catalyze the interesterification reaction. Moreover, the interesterification reaction parameters, such as reaction temperature, substrate ratio and reaction time were investigated regarding the stearoyl incorporation into the triacylglycerols. Further, the immobilized lipase proved to be easily separated from the reaction mixture by applying an external magnetic field and to be stable in the repeated use for four cycles.

Palygorskite-poly(o-phenylenediamine) nanocomposite: An enhanced electrochemical platform for glucose biosensing

Palygorskite (Pal) may be a promising material for enzyme immobilization due to its large surface, high biocompatibility and stability. This attractive material combined with a conducting polymer, poly(o-phenylenediamine), was exploited as a platform for the immobilization of glucose oxidase (GOD) using glutaraldehyde as crosslinker, and thus a novel glucose biosensor was obtained. The results of electrochemical impedance spectroscopy (EIS) and SEM indicated the successful entrapment of GOD in the clay polymer nanocomposite (CPN) film. Amperometric detection of glucose was performed by holding the potential at the CPN electrode at 0.6V for the oxidation of H2O2 generated in the enzymatic reaction. The apparent Michaelis–Menten constant (KMapp) was calculated to be 5.25mM, which is close to that of the free enzyme. The proposed biosensor exhibited a wide linear range, a low detection limit, a good reproducibility and accepted stability in the determination of glucose, providing a biocompatible platform for glucose biosensing.

Physico-Chemical and Morphological Changes of Sayong Kaolinite Clay Treated with Sulphuric Acid for Enzyme Immobilization

This study analyzed the effects of sulphuric acid (H2SO4) treatment on pysico-chemical properties and morphological changes of clay obtained from Sg. Sayong, Perak. The clay was ground and sieved to <150μm and treated with different concentrations of H2SO4. The treatment was completed by refluxing the clay with different concentration of H2SO4 (1M, 5M and 10M ) at 100 °C for 4 hours and followed by calcination at 500 °C for 1 hour. The physic-chemical properties and morphological changes of the untreated and treated clay were compared using Surface Area Analyser, X-Ray Diffraction (XRD), Field Emission Scanning Electron Micrograph (FESEM), X-Ray Diffraction (XRD) and Fourier Transformed Infrared Spectroscopy (FTIR). The results showed that acid treatment of 5M increased the surface area from 25 m2/g to 75 m2/g and the pore volume increased from 0.1518 cc/g to 0.3546 cc/g. The nanopore size of the clay decreased from 24.8 nm to 19.4 nm after treated with acid. This can be explained due to the elimination of the exchangeable cations and generation of microporosity. The results of XRF showed SiO2 increased from 58.34% to 74.52% and Al2O3 reduced from 34.6% to 18.31%. The mineral oxides such as Fe2O3, MgO, CaO, K2O and TiO2 also reduced. This concluded that H2SO4 treatment has led to significant removal of octahedral Al3+, Fe3+ cations and other impurities. In conclusion, this study showed the physico-chemical properties and morphology of Sayong clay were improved once treated with H2SO4 and therefore suggests better supporting material for enzyme immobilization.

Selective enzyme immobilization on arrayed microelectrodes for the application of sensing neurotransmitters

The method for selective enzyme immobilization on arrayed microelectrodes has been investigated in order to fabricate a near-real time biosensor for potential ex vivo or in vivo applications (e.g. in fermentation processes, in neuroscience studies, etc.). In this study, chitosan is used as the enzyme immobilization material, permselective polymers (polypyrrole and Nafion®) are used as the exclusion layers for charged interferents, and glutamate oxidase is used as the model enzyme for immobilization. To fabricate a glutamate sensor in the array, glutamate oxidase is selectively immobilized on a microelectrode site which is closely arrayed to other microelectrodes (platinum microelectrodes with ∼100μm separation) with the electrode dimension of ∼30μm×140μm by adsorbing the enzyme on the electrodeposited chitosan matrix on the electrode surface. When testing the glutamate sensor with glutamate at physiological concentrations, it has a linear detection range up to 217μM, a response time ∼1s, a limit of detection 2.5±1.2μM, and a sensitivity 38.1±5.4nA/μMcm2 (n=8); on the other hand, the closely arrayed control sensor without the chitosan film showed no signal upon the addition of glutamate. Little interferent current was observed. The sensor can retain up to 70% of its initial sensitivity for at least 9 days and no observable decrease in sensor sensitivity was detected after 20 times of continuous operations. Successful selective glutamate oxidase immobilization on closely packed microelectrodes for monitoring glutamate is demonstrated.

Activity of catalase adsorbed to carbon nanotubes: Effects of carbon nanotube surface properties

Nanomaterials have been studied widely as the supporting materials for enzyme immobilization. However, the interactions between enzymes and carbon nanotubes (CNT) with different morphologies and surface functionalities may vary, hence influencing activities of the immobilized enzyme. To date how the adsorption mechanisms affect the activities of immobilized enzyme is not well understood. In this study the adsorption of catalase (CAT) on pristine single-walled carbon nanotubes (SWNT), oxidized single-walled carbon nanotubes (O-SWNT), and multi-walled carbon nanotubes (MWNT) was investigated. The adsorbed enzyme activities decreased in the order of O-SWNT>SWNT>MWNT. Fourier transforms infrared spectroscopy (FTIR) and circular dichrois (CD) analyses reveal more significant loss of α-helix and β-sheet of MWNT-adsorbed than SWNT-adsorbed CAT. The difference in enzyme activities between MWNT-adsorbed and SWNT-adsorbed CAT indicates that the curvature of surface plays an important role in the activity of immobilized enzyme. Interestingly, an increase of β-sheet content was observed for CAT adsorbed to O-SWNT. This is likely because as opposed to SWNT and MWNT, O-SWNT binds CAT largely via hydrogen bonding and such interaction allows the CAT molecule to maintain the rigidity of enzyme structure and thus the biological function.