BIOSYNTHESIS, CHARACTERIZATION AND APPLICATION OF TIO2 NANOPARTICLES IN BIOCATALYSIS AND PROTEIN FOLDING (original) (raw)
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Simultaneous Immobilization and Refolding of Heat Treated Enzymes on TiO2 Nanoparticles
Advanced Science, Engineering and Medicine, 2014
Titanium dioxide (TiO 2 nanoparticles (<25 nm) were found to effectively assist refolding of thermally inactivated trypsin and alpha amylase enzymes. The native enzymes (alpha amylase and trypsin) were adsorbed on TiO 2 nanoparticles separately, by incubating the native enzymes and the TiO 2 nanoparticles for 2.0 hours at 25 C. The enzymes after adsorption on TiO 2 nanoparticles retained 86% of enzyme activity in case of trypsin and 95% activity in the case of alpha amylase enzyme. Both the enzymes after adsorption on TiO 2 nanoparticles were found to be thermally more stable as compared to the free enzymes. When the enzymes were incubated at 60 C for 60 min the free enzyme loses all of its activity whereas the adsorbed enzymes retained 80% of its activity in the case of trypsin and 94% activity in the case of alpha amylase. To study the nanoparticlesprotein interaction, trypsin and alpha amylase enzymes were inactivated by heating at 60 C for 1.0 hour. Thermally inactivated enzymes were adsorbed on TiO 2 nanoparticles in a similar manner as the native enzymes, and the activity in the adsorbed enzymes were determined after regular intervals of time. The thermally inactivated trypsin enzyme regains 90% of its activity after 2.0 hours of incubation on TiO 2 nanoparticles and thermally inactivated alpha amylase regains nearly 100% activity after 1.5 hour. Thermally inactivated trypsin and alpha amylase enzymes were substantially refolded towards their native conformation after adsorption on TiO 2 nanoparticles as evidenced by FTIR spectroscopy.
Simultaneous Immobilization and Refolding of Heat Treated Enzymes on TiO2 Nanoparticles
Advanced Science, Engineering and Medicine, 2015
ABSTRACT Titanium dioxide (TiO2 nanoparticles (&lt;25 nm) were found to effectively assist refolding of thermally inactivated trypsin and alpha amylase enzymes. The native enzymes (alpha amylase and trypsin) were adsorbed on TiO2 nanoparticles separately, by incubating the native enzymes and the TiO2 nanoparticles for 2.0 hours at 25 C. The enzymes after adsorption on TiO2 nanoparticles retained 86% of enzyme activity in case of trypsin and 95% activity in the case of alpha amylase enzyme. Both the enzymes after adsorption on TiO2 nanoparticles were found to be thermally more stable as compared to the free enzymes. When the enzymes were incubated at 60 C for 60 min the free enzyme loses all of its activity whereas the adsorbed enzymes retained 80% of its activity in the case of trypsin and 94% activity in the case of alpha amylase. To study the nanoparticles-protein interaction, trypsin and alpha amylase enzymes were inactivated by heating at 60 C for 1.0 hour. Thermally inactivated enzymes were adsorbed on TiO2 nanoparticles in a similar manner as the native enzymes, and the activity in the adsorbed enzymes were determined after regular intervals of time. The thermally inactivated trypsin enzyme regains 90% of its activity after 2.0 hours of incubation on TiO2 nanoparticles and thermally inactivated alpha amylase regains nearly 100% activity after 1.5 hour. Thermally inactivated trypsin and alpha amylase enzymes were substantially refolded towards their native conformation after adsorption on TiO2 nanoparticles as evidenced by FTIR spectroscopy.
Applicability of Nanomatrices Immobilized α- amylase in Biotechnology
HCTL Open International Journal of Technology Innovations and Research (IJTIR)
Recent advancement in nanotechnology has provided us diverse nanostructured materials for wider applications. Nanomaterials are emerging innovative field that have attracted considerable attention for the enzyme technology. The current demand is to develop and implement new technologies in order to enhance enzyme immobilization. The entrapment of enzymes in suitable matrices facilitates enhanced catalytic activity, stability, catalyst recovery, loading ability and reusability etc. These unique properties are inevitable and cost effective for large scale application in industrial biotechnology. The present review delineates some of these aspects in the field of nanotechnology for enzyme stabilization.
Immobilization of cellulase from Aspergillus niger on TiO2 nanoparticles was studied by two different approaches - physical adsorption and covalent coupling. This strain was selected as it is ubiquitous in nature and has been observed in the broad range of habitat, moreover produces cellulase extracellulary. For covalent methods, TiO2 nanoparticles were modified with aminopropyltriethoxysilane (APTS). The adsorbed enzymes and covalently immobilized showed 76% and 93% activity respectively, as compared to the equivalent free enzyme. The catalytic efficiency Vmax/Km increased from 0.4 to 4.0 after covalent attachment, whereas in adsorption method the increased slightly from 0.4 to 1.2. The covalently immobilized and adsorbed cellulase lost only 25% and 50% of their activity respectively after 60 min of incubation at 75°C. The reusability and operational stability data also showed that covalent coupling increased the stability of the enzyme. The presence of enzyme on TiO2 nanoparticles was confirmed by Fourier transform infrared spectroscopy. The High-resolution transmission electron microscopy and Atomic Force Microscopy studies indicated that there was aggregation of enzyme when adsorbed on TiO2 surface, whereas a monolayer of enzyme is observed in covalently attached enzyme. Thus, it is advisable to exploit fully the various methods and techniques of immobilization to achieve most stable enzyme preparation.
Food chemistry, 2018
Stability of enzymes is an important parameter for their industrial applicability. Here, we report successful immobilization of β-amylase (bamyl) from peanut (Arachis hypogaea) onto Graphene oxide-carbon nanotube composite (GO-CNT), Graphene oxide nanosheets (GO) and Iron oxide nanoparticles (FeO). The Box-Behnken Design of Response Surface Methodology (RSM) was used which optimized parameters affecting immobilization and gave 90%, 88% and 71% immobilization efficiency, respectively, for the above matrices. β-Amylase immobilization onto GO-CNT (bamyl@GO-CNT) and FeO(bamyl@FeO), resulted into approximately 70% retention of activity at 65 °C after 100 min of exposure. We used atomic force microscopy (AFM), scanning and transmission electron microscopy (SEM and TEM), Fourier transformed infrared (FT-IR) spectroscopy and fluorescence microscopy for characterization of free and enzyme bound nanostructures (NS). Due to the non-toxic nature of immobilization matrices and simple but elegant...
Protein aggregation is a problem in biotechnology. High temperature is one of the most important reasons to enhance enzyme inactivation and aggregation in industrial systems. This work focuses on the effect of TiO 2 and SiO 2 nanoparticles on refolding and reactivation of lysozyme. In the presence of TiO 2 and SiO 2 nanoparticles, after enzyme heat treatment at 98 • C for 30 min, not only aggregates were observed, but the amount of those increased. Hence the residual activity of lysozyme (without additives) and even in the presence of TiO 2 and SiO 2 nanoparticles after heat treatment was very low (<5%). T m of the aggregated lysozyme after this heat treatment was decreased with increasing concentrations of TiO 2 and SiO 2 nanoparticles from 0 to 0.02 mg/ml in neutral pH, Whether the T m of natural enzyme was above 373 (K) or 100 • C. these nanoparticles help enzyme denaturation and misfolding in heating.
Enzymes and nanoparticles: Modulation of enzymatic activity via nanoparticles
International Journal of Biological Macromolecules, 2018
Enzymes are biocatalysts that speed up the reactions taking place inside the cell. They are widely used in industries, scientific research and clinical diagnostics. Enzymes are specific for their substrates. They increase the rate of reaction by lowering the activation energy required to convert the substrates into the products. The catalysis by an enzyme is influenced by the nature of medium, substrate, enzyme concentration, temperature, pH, and the presence of activators and inhibitors. Nanoparticles are solid dispersion particulates of size range 10-1000 nm. They cause enhancement of particle mobility, diffusion, thermal stability, storage capacity, greater surface area and also modulate catalytic activity of the attached enzymes. Enzymes can be immobilized on nanoparticles by simple adsorption or via chemical linkages. Immobilization is a commercially applicable and a convenient method because it usually results in enhanced thermal and pH stabilities of the enzyme, lower cost of production, reusability with easy handling and separation. Primary objective of writing this review is to give an overview of the various aspects of enzymology, enzyme catalysis, enzyme immobilization and modulation of enzyme activity with special emphasis on modulation through different types of nanoparticles including their synthesis, characterization and applications.
Use of Nanomaterials for the Immobilization of Industrially Important Enzymes
Immobilization enables enzymes to be held in place so that they can be easily separated from the product when needed and can be used again. Conventional methods of immobilization include adsorption, encapsulation, entrapment, cross-linking and covalent binding. However, conventional methods have several drawbacks including reduced stability, loss of biomolecules, less enzyme loading or activity and limited diffusion. The aim of this study is the evaluation of importance of nanomaterials for the immobilization of industrially important enzymes. Nano materials are now in trend for the immobilization of different enzymes due to their physi-chemical properties. Gold nanoparticles, silver nanoparticles, nano diamonds, graphene, carbon nanotubes and others are used for immobilization. Among covalent and non-covalent immobilization of enzymes involving both single and multiwalled carbon nanotubes, non-covalent immobilization with functionalized carbon nanotubes is superior. There-fore, enzymes immobilized with nanomaterials possess greater stability, retention of catalytic activi-ty and reusability of enzymes.