Enhanced Reusability of Horseradish Peroxidase Immobilized onto Graphene Oxide/Magnetic Chitosan Beads for Cost Effective Cholesterol Oxidase Assay

Horseradish Peroxidase (HRP) is an important biocatalyst extensively used in enzymatic reactions. Cholesterol oxidase (ChoX) is a commercially valuable enzyme used in the estimation of cholesterol in human serum. ChoX is an oxygen oxidoreductase class of enzyme which catalyzes the oxidation of cholesterol in the presence of O2, liberating hydrogen peroxide H2O2as a by-product. HRP catalyzes the reduction of this H2O2in the presence of a redox dye (chromophore), producing a pink colored Quinoneimine which can be measured spectrophotometrically. The use of soluble HRP makes this assay method expensive for each time use and the recovery of HRP is not possible.Our aim was to prepare the HRP immobilized beads having magnetic properties for the ease of separation and increasing the reusability of HRP for the low cost ChoX assay.In the present work, we prepared magnetic chitosan beads using chitosan-Fe2O3nanoparticle blend coated with Graphene Oxide (GO), and subsequently activated with 2.5% glutaraldehyde (GA). Enzyme loaded beads were characterized by SEM, FTIR, and XRD analysis.The immobilization efficiency was ~80% and the immobilized HRP retained 90% of its initial activity up to 12 times reuse. The pH and temperature optima were shifted from 6.5 and 50°C for soluble HRP to 7.0 and 55°C for the immobilized HRP, respectively. Storage stability of immobilized HRP was 93.72% and 60.97% after 30 and 60 days storage respectively, at 4°C.On the basis of the present study, the HRP loaded magnetic chitosan/graphene oxide beads could be used for low-cost ChoX assay at laboratory scale due to its enhanced reusability and stability.

[1]  S. Shera,et al.  Artificial Neural Network Modeling to Predict the Non-Linearity in Reaction Conditions of Cholesterol Oxidase from Streptomyces olivaceus MTCC 6820 , 2019, Journal of Biosciences and Medicines.

[2]  Qian Liu,et al.  Horseradish peroxidase immobilized on the magnetic composite microspheres for high catalytic ability and operational stability. , 2019, Enzyme and microbial technology.

[3]  W. Zhuang,et al.  Stability and repeatability improvement of horseradish peroxidase by immobilization on amino-functionalized bacterial cellulose , 2018, Process Biochemistry.

[4]  A. Saboury,et al.  Physical Adsorption of Horseradish Peroxidase on Reduced Graphene Oxide Nanosheets Functionalized by Amine: A Good System for Biodegradation of High Phenol Concentration in Wastewater , 2018, International Journal of Environmental Research.

[5]  John M. Woodley,et al.  Role of Biocatalysis in Sustainable Chemistry. , 2017, Chemical reviews.

[6]  Huahua Yu,et al.  Preparation and characterization of controlled-release fertilizers coated with marine polysaccharide derivatives , 2017, Chinese Journal of Oceanology and Limnology.

[7]  Huimin Zhao,et al.  Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates. , 2017, Bioorganic & medicinal chemistry.

[8]  Ramesh N. Patel,et al.  Biocatalysis for synthesis of pharmaceuticals. , 2017, Bioorganic & medicinal chemistry.

[9]  Yu Zhang,et al.  Graphene oxide-based Fe2O3 hybrid enzyme mimetic with enhanced peroxidase and catalase-like activities , 2016 .

[10]  Roswanira Abdul Wahab,et al.  Review; Agriculture and Environmental Biotechnology an Overview of Technologies for Immobilization of Enzymes and Surface Analysis Techniques for Immobilized Enzymes , 2022 .

[11]  Qing Chang,et al.  Immobilization of Horseradish Peroxidase on NH2-Modified Magnetic Fe3O4/SiO2 Particles and Its Application in Removal of 2,4-Dichlorophenol , 2014, Molecules.

[12]  Gurumallesh Prabu Halliah,et al.  Synthesis, characterization of CH-α-Fe2O3 nanocomposite and coating on cotton, silk for antibacterial and UV spectral studies , 2014 .

[13]  M. Basri,et al.  Enhanced Biocatalytic Esterification with Lipase-Immobilized Chitosan/Graphene Oxide Beads , 2014, PloS one.

[14]  A. Elsayed,et al.  Activity and stability of immobilized Candida rugosa lipase on chitosan coated Fe3O4 nanoparticles in aqueous and organic media , 2014 .

[15]  Rafiq Ahmad,et al.  Development of Highly Sensitive and Selective Cholesterol Biosensor Based on Cholesterol Oxidase Co-Immobilized with α-Fe2O3 Micro-Pine Shaped Hierarchical Structures , 2014 .

[16]  Veeresh Juturu,et al.  Microbial cellulases: Engineering, production and applications , 2014 .

[17]  Niranjan Gopal,et al.  Biosensors in clinical chemistry: An overview , 2014, Advanced biomedical research.

[18]  T. Kumosani,et al.  Horseradish peroxidase and chitosan: activation, immobilization and comparative results. , 2013, International journal of biological macromolecules.

[19]  M. Aliahmad,et al.  Synthesis of maghemite (γ-Fe2O3) nanoparticles by thermal-decomposition of magnetite (Fe3O4) nanoparticles , 2013 .

[20]  G. Favero,et al.  Comparison of three immunosensor methods (surface plasmon resonance, screen-printed and classical amperometric immunosensors) for immunoglobulin G determination in human serum and animal or powdered milks. , 2013, Journal of pharmaceutical and biomedical analysis.

[21]  Iqbal Ahmad,et al.  Induction of ROS, mitochondrial damage and autophagy in lung epithelial cancer cells by iron oxide nanoparticles. , 2012, Biomaterials.

[22]  Shenguang Ge,et al.  Paper-based chemiluminescence ELISA: lab-on-paper based on chitosan modified paper device and wax-screen-printing. , 2012, Biosensors & bioelectronics.

[23]  E. Šturdı́k,et al.  Application of Electrochemical Biosensors in Clinical Diagnosis , 2012, Journal of clinical laboratory analysis.

[24]  Eun-Hyung Yoo,et al.  Glucose Biosensors: An Overview of Use in Clinical Practice , 2010, Sensors.

[25]  M. Monier,et al.  Immobilization of horseradish peroxidase on modified chitosan beads. , 2010, International journal of biological macromolecules.

[26]  Hui Liu,et al.  Graphene oxide as a matrix for enzyme immobilization. , 2010, Langmuir : the ACS journal of surfaces and colloids.

[27]  Hanwen Sun,et al.  Photochemical preparation of magnetic chitosan beads for immobilization of pullulanase , 2009 .

[28]  L. Ananthanarayan,et al.  Glucose oxidase--an overview. , 2009, Biotechnology advances.

[29]  Mustafa Yiǧitoǧlu,et al.  Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber , 2009, Bioprocess and biosystems engineering.

[30]  Zhi‐Kang Xu,et al.  Modification of microfiltration polypropylene membranes by allylamine plasma treatment Influence of the attachment route on peroxidase immobilization and enzyme efficiency , 2006 .

[31]  E. Querol,et al.  Analysis of protein conformational characteristics related to thermostability. , 1996, Protein engineering.

[32]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[33]  P. Fu,et al.  Enzymatic determination of total serum cholesterol. , 1974, Clinical chemistry.

[34]  R. Richterich,et al.  [DETERMINATION OF TOTAL SERUM CHOLESTEROL]. , 1964, Das Medizinische Laboratorium.

[35]  T. M. Devlin.  Enzymatic Methods in Analytical Chemistry , 1959 .

[36]  Long Hao,et al.  Self-Healing Epoxy Coatings Based on Nanocontainers for Corrosion Protection of Mild Steel , 2017 .

[37]  Petr Skládal,et al.  Graphene oxide , 2022 .

[38]  Wei Gao,et al.  The Chemistry of Graphene Oxide , 2015 .

[39]  S. Dutz,et al.  The Role of Interactions in Systems of Single Domain Ferrimagnetic Iron Oxide Nanoparticles , 2012 .

[40]  R. Ruoff,et al.  The chemistry of graphene oxide. , 2010, Chemical Society reviews.

[41]  D. Sellmyer,et al.  ADVANCED MAGNETIC NANOSTRUCTURES , 2006 .