REVIEW ARTICLE


Immobilization of Cholesterol Oxidase: An Overview



Shubhrima Ghosh, Razi Ahmad, Sunil Kumar Khare*
Enzyme and Microbial Biochemistry Laboratory, Department of Chemistry, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi-110016, India


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© 2018 Ghosh et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Enzyme and Microbial Biochemistry Laboratory, Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India; Tel: +911126596533; Fax +911126581102; E-mails: skkhare@chemistry.iitd.ac.in, skhare@rocketmail.com


Abstract

Background:

Cholesterol oxidases are bacterial oxidases widely used commercially for their application in the detection of cholesterol in blood serum, clinical or food samples. Additionally, these enzymes find potential applications as an insecticide, synthesis of anti-fungal antibiotics and a biocatalyst to transform a number of sterol and non-sterol compounds. However, the soluble form of cholesterol oxidases are found to be less stable when applied at higher temperatures, broader pH range, and incur higher costs. These disadvantages can be overcome by immobilization on carrier matrices.

Methods:

This review focuses on the immobilization of cholesterol oxidases on various macro/micro matrices as well as nanoparticles and their potential applications. Selection of appropriate support matrix in enzyme immobilization is of extreme importance. Recently, nanomaterials have been used as a matrix for immobilization of enzyme due to their large surface area and small size. The bio-compatible length scales and surface chemistry of nanoparticles provide reusability, stability and enhanced performance characteristics for the enzyme-nanoconjugates.

Conclusion:

In this review, immobilization of cholesterol oxidase on nanomaterials and other matrices are discussed. Immobilization on nanomatrices has been observed to increase the stability and activity of enzymes. This enhances the applicability of cholesterol oxidases for various industrial and clinical applications such as in biosensors.

Keywords: Cholesterol oxidase, Immobililization, Nanoparticles, Enzyme, Biocatalysis, Biosensors.



1. INTRODUCTION

Enzymes are macromolecular biocatalysts, which accelerate biochemical reactions with high specificity and efficiency [1-3]. In recent years, enzymes have found immense applications in industrial bioprocesses, environmental remediation, biofuel production, pharmaceuticals and diagnostics [4]. However, many such applications of free enzymes face the drawback of poor stability, high cost and difficulty in reuse. The activity and stability of enzymes for the above applications have been improved by various methods [5]. Immobilization is one of the important techniques which have been exploited to improve enzyme characteristics. The macro/micro surfaces used in immobilization play significant role in enabling the use of enzymes in non-native applications such as in environmental remediation, biosensors, bioreactors and other applied biotechnology fields [6, 7]. The interaction of enzymes with material surfaces are of concern for many applications such as biosensors, biotransformations, in food industry, pharmaceutical transformations, imaging and drug delivery technologies. After immobilization on the matrix surfaces, the observed activity and specificity of the enzyme may undergo changes. Mostly, the enzyme properties are found to be enhanced in nature. However in some cases, enzymes lose their activity or undergo distortion in structure due to the physicochemical properties of the enzyme, nature of the material support and their mutual interactions [8, 9]. The multiple factors responsible for the limited functioning of the enzymes after attachment to the support include alteration of the native protein configuration, slower diffusion rates of the substrate towards the bulk-attached enzyme and steric hindrance [9-12]. Recently, many researchers have utilized Nanoparticles (NPs) as enzyme carriers for eliminating these negative effects of enzyme immobilization on macro/micro carriers.

Nanomaterials (particles with length scales < 100 nm), due to their high surface area to volume ratios possess unique characteristics that enhance biocatalysis. They also exhibit surface chemistry well-suited for bioconjugation, and length scales that integrate well with metabolic processes and gene expression [13]. These inherent capabilities make nanomaterials valuable for a wide variety of biotechnological applications including biosensing, drug delivery, bioremediation, biofuel production, antibacterial activity and disease diagnostics [14-24]. Nanomaterials have the ability to enhance the performance and activity of the immobilized enzyme [25, 26], as they influence the factors that determine the efficiency of biocatalyst, such as surface area to volume ratio, mass transfer resistance and effective enzyme loading [3, 27-35].

Cholesterol oxidase is a bacterial flavo-enzyme which finds great commercial value in the determination of cholesterol in food and clinical samples, as a catalyst for bioconversion of sterol compounds and as an insecticide [36]. The current review summarizes the different matrices and techniques for nanoimmobilisation, followed by discussion on some typical macro/micro and nanocarriers used for conjugating cholesterol oxidase and describes the applicability of such nano-preparations.

2. CHOLESTEROL OXIDASE

Cholesterol oxidase (EC 1.1.3.6) is a monomeric FAD-dependent oxido-reductase which catalyzes steroid substrates having a hydroxyl group at the 3-β position of the steroid ring. These enzymes are strictly of bacterial origin, and not found in plant or animal systems. First reported in Rhodococcus sp. [37, 38], cholesterol oxidase has been found to be produced by Pseudomonas sp. [39, 40], Burkholderia sp. [41], Mycobacterium sp. [42], Chromobacterium DS-1 [43] and Streptomyces sp. [44, 45]. among others. In fact, actinomycetes are the largest class of cholesterol oxidase producers. The enzyme exists both as membrane-bound and extra-cellular forms in bacteria. Based on their bonding to the cofactor FAD, cholesterol oxidases can be broadly divided into two classes:

Class-I cholesterol oxidases have non covalently-bound FAD, the covalent attachment found to consist of the imidazole ND1 atom of a histidine residue (His121) [46]. Rhodococcus and Brevibacterium cholesterol oxidase belong to this kind.

Class–II cholesterol oxidases have covalently bound FAD. The attachment to the protein, occurs through a bond linking the polypeptide chain to the 8-methyl group of the isoalloxazine moiety [47]. Cholesterol oxidase from Burkholderia and Chromobacterium belong to this kind.

2.1. Structure of Cholesterol Oxidase

The general structure of cholesterol oxidase consists of a substrate-binding domain and a FAD-binding domain, with the single protein chain meandering back and forth between the regions that provide the binding features for the cofactor and the substrate [47]. In most cholesterol oxidases, eight-stranded mixed beta-pleated sheet and six alpha-helices form the substrate binding domain. This domain is positioned over the isoalloxazine ring system of the FAD cofactor allowing oxidation of cholesterol and subsequent isomerisation to cholest-4-en-3-one [48]. The active site contains a hydrophobic pocket, sealed off from the outer environment by flexible loops [47].

In most flavin-containing enzymes, there exists a consensus sequence of repeating glycine residues (GXGXXG) followed by an Asp/Glu 20 residues further in the primary sequence, this motif forming the nucleotide binding domain [49]. The amino acid sequence, structure and redox properties vary a lot between the two different classes of enzymes. The noncovalent form of cholesterol oxidase exhibits a consensus sequence of glycines (G17-X-G19-X-G21-G⁄ A22) followed by a glutamate Glu40, indicating a nucleotide-binding fold. In the case of the covalently bound cholesterol oxidase, this consensus is notably absent [47]. In the non-covalent form, the FAD is bound to the protein by non-covalent interactions, which can be released only under denaturing conditions such as heating at 90°C.

2.2. Biological Functions of Cholesterol Oxidase

Cholesterol oxidase is found to play myriad roles in the bacterial metabolism. In cholesterol assimilating bacteria, it is involved in the first step of cholesterol metabolism, converting cholesterol to 4-cholesten-3-one [50, 51]. Bacteria such as Mycobacterium and Rhodococcus equi secrete cholesterol oxidase enzyme to damage the cell-membrane of the hosts, hence considered responsible for the pathogenticity of these bacteria [42, 52]. Additionally, product of the gene pimE coding for cholesterol oxidase in Streptomyces Natalensis, acts as a signaling protein for the production of pimaricin [53].

2.3. Applications of Cholesterol Oxidase

An important application of cholesterol oxidases is as a diagnostic tool to determine cholesterol levels in serum [54], HDL, LDL [55], on the cell membrane of erythrocytes [56], in human bile and in gall stones [57], in atherosclerotic diseases and other lipid disorders. In most cases, the enzyme is accompanied by cholesterol esterase, EC 3.1.1.13 (which frees esterified cholesterol present in blood serum) and peroxidase EC 1.11.1.7 (which reacts with hydrogen peroxide and a dye to give a colored end product) to aid in detection. This reaction forms the primary basis for most cholesterol biosensors having immobilized forms of cholesterol oxidase.

Cholesterol oxidase proves to be an effective biocatalyst for the transformation of cholesterol and other sterol compounds to form pharmaceutically important products such as 4-cholesten-3-one, androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione [58]. It could also be used in the bioconversion of cholesterol into bile acids, where cholesterol oxidase is used to convert 3β, 7α-cholest-5-ene-3,7-diol to 7α-hydroxycholest-4-en-3-one [59]. Cholesterol oxidase from Rhodococcus erythropolis was employed for the preparative oxidation of cyclic allylic, bicyclic and tricyclic alcohols [60]. These enzyme mediated processes are eco-friendly, cost effective and operate under milder reaction conditions.

In the early 1990s, Monsanto Co. (St Louis, MO, USA) discovered a highly efficient insecticidal protein effective against the boll weevil (Anthonomus grandis Boheman) larvae and other lepidopterans [61]. This was later found to be cholesterol oxidase with an insecticidal activity at 50% concentration (LC50). When cholesterol oxidase is included in the diet of the larvae, it induces lysis in the midgut epithelium. However, adult boll weevils are insensitive to cholesterol oxidase. Transgenic leaf tissues expressing ChOx have also been found to exert insecticidal activity against boll weevil larvae [62].

Cholesterol oxidase provides an interesting target for bacterial infections from species such as Rhodococcus equi, which resides in the macrophages of hosts and acts as an opportunistic pathogen in immune-compromised individuals. Mycobacterium tuberculosis and Mycobacterium leprae have also been found to produce cholesterol oxidase [63]. The bacteria are found to induce membrane lysis facilitated by the induction of extracellular cholesterol oxidase, thus can be used to target the virulence [52].

3. IMMOBILIZATION OF ENZYMES

3.1. Immobilization Methods

Generally, immobilization of enzymes can be carried out by physical as well as chemical methods. In physical methods, the enzyme is bound to the matrix by weak interactions, while in chemical methods, covalent bond is formed between the matrix and the enzyme. Recently, the advances in organic chemistry and molecular biology for protein immobilization to support resulted in the development of some very potent, efficient and site-specific applications such as functional protein microarrays, biosensors, and continuous flow reactor systems [2, 30, 64-66]. Broadly, enzyme immobilization methods can be divided into three categories, direct binding to a support, entrapment and cross-linking as summarized in Fig. (1). In each case, researchers try to maintain the catalytic activity while achieving the expected technological advantages through immobilization protocols. However, the stress developed by the immobilization procedures may induce conformational changes and partial loss of the enzyme activity [67].

Fig. (1). Schematic diagram of immobilization of enzymes.

Binding to a carrier can occur through adsorption or covalent attachment processes [66]. The adsorption through the physical method generally involves multipoint protein adsorption between the protein molecule and binding sites on the immobilization surface through ionic and hydrogen bonding, van der Waals forces and hydrophobic interactions [68, 69]. In these methods, the enzyme is easily desorbed from the immobilization surfaces by fluctuations of pH and temperature [70]. A few advantages of the adsorption methods are that they are easy to carry out, no reagents are involved, minimum activation step is required, are comparatively cheap, and are less disruptive to protein structure than most chemical methods. Covalent coupling is the most frequently used approach to immobilization in which covalent bonds are formed between surface amino acids of the enzyme and the matrix. The advantages of the covalent bonding method include strong linkage of enzyme to the support (due to which no leakage or desorption occurs), a comparatively simple operation, availability of a variety of supports with different functional groups, and wide applicability. The disadvantages of the covalent bonding method include chemical modification of the enzyme leading to functional conformational loss and enzyme inactivation by a change in the conformation of the active site. This can be overcome through immobilization with the substrate or a competitive inhibitor of the enzyme.

Entrapment of enzymes involves the inclusion of the protein in a polymer matrix, such as silica sol–gel and polyacrylamide or artificial membranes like microcapsule and hollow fiber. However, the enzyme leakage cannot be entirely prevented due the weak nature of the physical bonds, therefore implicating additional covalent attachment. For efficient entrapment, the synthesis of the polymeric matrix must take place in the presence of the enzyme. Thermo reverse polymerization has been used to entrap enzymes in natural polymers such as agar, agarose and gelatin, while ionotropic gelation has been used for alginate and carrageenan matrices [71]. Additionally synthetic polymers like polyvinyl alcohol hydrogel [72], polyacrylamide [73] have also been explored for their application in enzyme entrapment. The advantages of the entrapment method are that it is fast, inexpensive, milder conditions are required, and there is less chance of conformational change in the enzyme. The disadvantages include leakage of the enzyme, pore diffusion limitation, and chance of microbial contamination. Immobilization of cholesterol oxidase by entrapment methods have been done by many researchers [74, 75].

Cross-linking of enzyme aggregates is obtained by binding of enzymes to each other by bi- or multifunctional reagents or ligands to prepare carrierless macroparticles [71]. Cross-linking is a simple immobilization process as it does not involve any support matrix. In this method, covalent bond is formed between the biocatalyst, because of which the biocatalyst may undergo conformational changes, resulting in activity loss. Glutaraldehyde is a bifunctional agent generally used for cross-linking. It has aldehyde groups at both ends to react with the free amino groups of enzymes. Thus, carrier-free immobilized enzymes, such as Cross-Linked Enzyme Crystals (CLECs), and Cross-Linked Enzyme Aggregates (CLEAs) are finding increasing interest in the industries [76, 77]. The advantage of the cross-linking method is that it is issued mostly as a means of stabilizing the adsorbed enzyme and also for preventing leakage. The disadvantage of this method is that it may cause significant change in the active site of the enzyme which may lead to a loss of activity.

The factors that determine the category of immobilization to be employed depend on the type of enzyme and the matrix used. Whenever a new immobilization protocol is developed, importance should be given to enzyme recovery percentage, selectivity, operational stability, and reduction in inhibition by the products or any other component of the media. The interaction between the matrix and the enzyme should be controlled and its catalytic properties maintained by proper orientation of the protein. Most importantly, use of toxic and highly unstable reagents during the immobilization processes should be kept to a minimum, making it eco-friendly as compared to other established technologies.

3.2. Choice of Support for Immobilization

Support material is one of the major components for the immobilization of enzymes. Thus, it should be possessed with some important characteristics like large surface area, suitable shape and particle size, high rigidity, hydrophilic character, permeability, insolubility, chemical, mechanical and thermal stability, resistance to microbial attack, and regenerability [78]. Support materials can be classified based on morphology (porous and nonporous support) and their chemical nature (organic and inorganic). Organic support materials are further classified as natural and synthetic. Natural organic supports include polysaccharides and proteins [79, 80]. The surface of most inorganic supports is mainly composed of oxide and hydroxyl groups, such as silanol group in glass, and provides a mild reactive surface for activation and protein binding [71, 81, 82].

Properties of the immobilized enzyme inevitably depend on the choice of the matrix. Nanomaterials possess the ideal size as immobilization matrices as they occupy lesser volume in bioreactors as compared to conventional matrices while providing high surface/volume ratio. Nanoscale materials also have additional advantage of low cost, rapid reaction, mild conversion conditions, robust activities, mobility, high loading, operational stability and minimum diffusional limitation [83]. Moreover, the small size and multifunctionality of nanoparticles (of metals such as Au, Fe, Zn and Ag) make them suitable to interact and operate at biomolecular levels finding applications in biosensors, tumor location analysis and drug delivery [84].

4. IMMOBILIZATION OF CHOLESTEROL OXIDASE

Cholesterol oxidases from bacterial sources have been immobilized to improve their enzymatic properties, reusability and operational stability. The current section summarises some of the established instances of cholesterol oxidase immobilization onto various macro/micro and nanocarriers.

4.1. Immobilization on Macro/ Micro Carriers

To improve its catalytic properties, various macro/micro carriers have been employed to immobilize cholesterol oxidase. Cholesterol oxidase from Rhodococcus sp. NCIM 2891, immobilized on chitosan beads was found to have increased stability in organic solvents, and was reusable upto 12 successive cycles of operation. This immobilized preparation was used for the biotransformation of cholesterol to 4-cholesten-3-one with around 88% millimolar yield [85]. Two important reports by the same group describe the co-immobilization of Cholesterol Oxidase (ChOx) with Horseradish Peroxidase (HRP) and Cholesterol Esterase (ChEt) respectively [74, 86]. While horseradish peroxidase aids in colorimetric detection of cholesterol, cholesterol esterase breaks down the esterified cholesterol present in blood serum into free cholesterol for easy detection. The co-immobilization was carried out on Tetraethyl Orthosilicate (TEOS) derived sol-gel films, by physisorption, physically entrapped sandwich and microencapsulation with ChOx/HRP while covalent modification was carried out for the preparation with ChOx/ChEt. The sol-gel/ChOx/ChEt complex reveal high thermal stability upto 55°C and detection limit of 12 mg dL−1 and sensitivity of 5.4×10−5 Abs. mg−1 dL−1 for cholesterol [74]. All the three techniques used for the immobilization of ChOx/HRP showed linearity for 2-10 mM cholesterol [86]. A similar co-immobilization of ChOx, ChEt and HRP was carried out on alkylamine glass beads and PVC [87, 88]. N-ethyl-N’-3-dimethylaminopropyl carbodiimide activated sepharose is another popular matrix for immobilization of cholesterol oxidase. The immobilized preparation retained 90% of activity at 4 and 9 pH, but did not possess commendable storage stability [89]. Conducting polymers such as thiophene-3-boronic acid and 3-aminophenyl boronic acid increase the interaction between the enzyme and the electrode and have been used to immobilize Brevibacterium sp. cholesterol oxidase for biosensing applications [90]. Cholesterol oxidase has also been conjugated to polymers such as electrochemically-polymerized polyaniline with Triton X-100 and functionalized poly(methyl methacrylate-co-glycidyl methacrylate) and poly(acrylamide-co-acrylicacid)/polyethyleneimine supports [91, 92].

4.2. Immobilization on Magnetic Nanoparticles

Even though a variety of matrices have been used to immobilize cholesterol oxidase, reports on magnetic nanoparticles have been scarce. Many such studies employ co-precipitation of magnetic Fe3O4 nanoparticles with other matrices. For application as a biosensor, nanoparticles synthesized by the co-precipitation of Fe2+ and Fe3+ and functionalization with poly(styrene-co-acrylic acid) have been used to conjugate cholesterol oxidase followed by deposition on the planar platinum polyaniline (PANi) modified electrode [93]. An attempt at the immobilization on magnetic fluorescent nanoparticles, was done through APTES mediated covalent coupling [94]. The maximal catalytic activity was obtained at pH 7.0 and 50 °C. The immobilized preparation retained 80% of its activity at 50ºC for 5h and it was reusable for 7 consecutive operations. Interestingly, the immobilized enzyme was optically sensitive to oxygen in solution, thus rendering it applicable for fibre optic based biosensors. A similar study on immobilization of cholesterol oxidase from various bacterial sources was carried out on APTES modified iron (II, III) oxide nanoparticles followed by glutaraldehyde crosslinking. These nanoconjugates increased the thermal and pH stability and were further used for the production of 4-cholesten-3-one and 4-cholesten-3,5-dione [95]. Cholesterol oxidase has also been attached to magnetic nanoparticles (Fe3O4) synthesized by thermal co-precipitation of Fe2+ and Fe3+ chlorides, through carbodiimide linkage. Such conjugation of cholesterol oxidase resulted in almost 98-100% binding with enhanced pH and temperature stability [96]. Functionalized silica-coated magnetic nanoparticles have also been used for cholesterol oxidase immobilization. Synthesis of maghemite (γ-Fe2O3) nanoparticles was done by thermal co-precipitation of iron ions in alkaline ammonia solution, followed by deposition of silica. Secondary functionalization was done using organosilane and cross-linking by glutaraldehyde [97]. Cholesterol oxidase immobilized on the silica-coated maghemite has been found to retain around 60% of its activity [98]. Cholesterol biosensors have also been developed by co-immobilizing cholesterol oxidase with Fe2O3 micro-pine shaped hierarchical structures with very high and reproducible sensitivity and low detection limit [99].

4.3. Immobilization on Metal Nanoparticles

Majority of the cholesterol oxidase biosensors comprise of gold as the electrode. Thus, gold (and other metal) nanoparticles aid in the deposition of cholesterol oxidase on the electrode surface. In one such report, gold electrode was modified with 1,6-hexanedithiol, followed by deposition of gold nanoparticles. The surface of the nanoparticles was functionalized with carboxyl groups using 11-mercaptoundecanoic acid and cholesterol oxidase was immobilized on the surface of the gold nanoparticle film by covalent bonding using the N-ethyl-N’-(3-dimethylaminopropyl carbodimide) and N-hydroxysuccinimide ligand chemistry [100]. A novel biosensor for quantitatively estimating cholesterol in pork liver and egg yolk samples was made by a glassy carbon electrode with nano-composite mixture consisting of Molybdenum Disulfide (MoS2) and gold nanoparticles (AuNPs), over which cholesterol oxidase was deposited [101]. Another interesting take on the cholesterol oxidase nanoimmobilization was the synthesis of aggregates of enzymes in the nanometer scales. The aggregates were achieved by cross-linking of cholesterol oxidase and cholesterol esterase with glutaraldehyde followed by deposition on Au electrode for manufacturing the biosensor. These ENPs retained 50% of initial activity during its regular use over a period of 60 days when stored at 4°C [102].

4.4. Immobilization on Other Nanoparticles

In addition to conventional nanomatrices, various novel materials have been explored for their capacity to immobilize cholesterol oxidase. Successful immobilization of cholesterol oxidase has been carried out on Mg2AlCO3 Hydrotalcite Layered Double Hydroxide nanomaterials for biosensor applications [103]. This group used extracts of Acacia salicina leaves in order to scavenge the free radicals such as H2O2 liberated during the enzymatic reaction. Polymers such as Poly-Ethylene Glycol (PEG), decorated polystyrene (PS) nanoparticles along with Congo red have also been used to immobilize cholesterol oxidase [104]. APTES modified and glutaraldehyde cross-linked silica nanoparticles were used to conjugate cholesterol oxidase, subsequently used as a biosensor with 200s response time and detection limit of 4.2 mg/dL [105].

Immobilization of enzyme to improve upon their biochemical properties, stability and reusability has been explored for a variety of industrial and clinically important enzymes. Apart from cholesterol oxidase, enzymes successfully conjugated onto macro/micro carriers as well as nanoparticles include cellulose [30, 106] keratinase [107, 108], lipase [109, 110] laccase [111, 112] and α-amylase [113, 114]. From the biosensing point of view, enzymes which has been immobilised apart from cholesterol oxidase include glucose oxidase [115], horseradish peroxidase [116], glucose dehydrogenase [117] and lactate dehydrogenase [118]. In fact, glucose oxidase forms one the largest class of oxidase enzymes immobilised for clinical applications.

5. APPLICATIONS OF IMMOBILIZED CHOLESTEROL OXIDASE

Immobilization of cholesterol oxidase renders it useful for varied industrial and medical applications. The majority of immobilized forms have been used as biosensors for the detection of cholesterol in food samples, blood serum and other clinical samples. Other applications include bioconversions for the production of steroid compounds. The general enzymatic cholesterol biosensors comprise of cholesterol esterase (which converts cholesterol esters present in blood to free cholesterol), cholesterol oxidase (which converts the free cholesterol to 4-cholesten-3-one) and horseradish peroxidase (it reacts with the liberated H2O2 to result in the colored compound which is detected). However, a limitation of this specific cholesterol detection scheme is the high cost of enzyme involved. Cholesterol oxidase based biosensors are considered superior as compared to traditional detection methods such as HPLC or GC, owing to their simpler working protocols, higher reusability, higher specificity and low cost.

Ordinary fabricated biosensors are limited by inadequate performance in terms of detection limit, sensitivity and stability. These challenges can be overcome by the use of nanomaterials, which provide catalytic, optical and electronic properties. Incorporating nanomaterials in biosensors enhance the surface chemistry, electroconductivity and compatible length scales for bioconjugation. Some of the nanomatrices used for the immobilization of cholesterol oxidase for biosensing applications are discussed here.

The physicochemical properties of cholesterol oxidase biosensors are generally studied through electrochemical impedance spectroscopy and cyclic voltammetry. In one report, the linear range of the biosensor made by electrodepositing cholesterol oxidase nanoparticles was 10-700 mg/dL for cholesterol. It optimally responded at pH 5.5 and 40°C within 5s when polarized at +0.25 V versus Ag/AgCl [101]. A gold based biosensor was created by covalently conjugating cholesterol oxidase to gold nanoparticles, which in turn were deposited on a gold electrode [100]. This biosensor exhibited a linear response to cholesterol in the range of 0.04-0.22mM with a detection limit of 34.6µM, apparent Km of 0.062mM with cholesterol and a high sensitivity of 9.02µA mM−1. Polymeric biosensors created by cholesterol oxidase immobilization onto Polystyrene/PEG/Congo red particles yielded linear response in the cholesterol concentration range of 100 mg dL−1 to 300 mg dL−1 [104].

Immobilization of cholesterol oxidase has proved to be a versatile technology for medical and food applications. For an enzyme like cholesterol oxidase with varied clinical applications, it becomes imperative that the substrate specificity is maintained after immobilization. However, the effect of immobilization on substrate specificity need to be studied further.

CONCLUSION

The science of immobilization of enzymes and other biological molecules on different supports is now well established and developed earlier. Selection of appropriate support matrix in enzyme immobilization is of extreme importance. Recently, nanomaterials have been used as a matrix for immobilization of enzyme due to their large surface area and small size. In this review, immobilization of cholesterol oxidase on nanomaterials and other matrices were discussed. Immobilization on nanomatrices has been observed to increase the stability and activity of enzymes. This enhances the applicability of cholesterol oxidases for various industrial and clinical applications such as in biosensors.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Shubhrima Ghosh is grateful to the Indian Institute of Technology Delhi for Senior Research Fellowship. Razi Ahmad is thankful to DST-SERB [PDF/2016/000090] for providing national postdoctoral fellowship.

REFERENCES

[1] Radzicka A, Wolfenden R. A proficient enzyme. Science 1995; 267(5194): 90-3.
[2] Ahmad R, Sardar M. Enzyme immobilization: An overview on nanoparticles as immobilization matrix. Biochem & Anal Biochem 2015; 4(3): 1-8.
[3] Ahmad R, Mishra A, Sardar M. Peroxidase-TiO2 nanobioconjugates for the removal of phenols and dyes from aqueous solutions. Adv Sci Eng Med 2013; 5(10): 1020-5.
[4] Kirk O, Borchert TV, Fuglsang CC. Industrial enzyme applications. Curr Opin Biotechnol 2002; 13(4): 345-51.
[5] Koeller KM, Wong CH. Enzymes for chemical synthesis. Nature 2001; 409(6817): 232-40.
[6] Palmer T, Bonner PL. Enzymes: Biochemistry, biotechnology, clinical chemistry 2007.
[7] Ansari SA, Husain Q. Potential applications of enzymes immobilized on/in nano materials: A review. Biotechnol Adv 2012; 30(3): 512-23.
[8] Rodrigues RC, Ortiz C, Berenguer-Murcia Á, Torres R, Fernández-Lafuente R. Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev 2013; 42(15): 6290-307.
[9] Talbert JN, Goddard JM. Enzymes on material surfaces. Colloids Surf B Biointerfaces 2012; 93: 8-19.
[10] Bryjak J, Kolarz BN. Immobilisation of trypsin on acrylic copolymers. Process Biochem 1998; 33(4): 409-17.
[11] Janssen MHA, van Langen LM, Pereira SRM, van Rantwijk F, Sheldon RA. Evaluation of the performance of immobilized penicillin G acylase using active-site titration. Biotechnol Bioeng 2002; 78(4): 425-32.
[12] Fernandez-Lafuente R. Stabilization of multimeric enzymes: Strategies to prevent subunit dissociation. Enzyme Microb Technol 2009; 45(6): 405-18.
[13] Albanese A, Tang PS, Chan WCW. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu Rev Biomed Eng 2012; 14: 1-16.
[14] Hwang ET, Gu MB. Enzyme stabilization by nano/microsized hybrid materials. Eng Life Sci 2013; 13(1): 49-61.
[15] Sotiropoulou S, Chaniotakis NA. Carbon nanotube array-based biosensor. Anal Bioanal Chem 2003; 375(1): 103-5.
[16] Mishra A, Ahmad R, Perwez M, Sardar M. Reusable green synthesized biomimetic magnetic nanoparticles for glucose and H2O2 detection. Bionanoscience 2016; 6(2): 93-102.
[17] Mishra A, Ahmad R, Sardar M. Biosynthesized Iron Oxide nanoparticles mimicking peroxidase activity: Application for biocatalysis and biosensing. J Nanoeng Nanomanuf 2015; 5(1): 37-42.
[18] Bellino MG, Soler-Illia GJAA. Nano-designed enzyme-functionalized hierarchical metal-oxide mesoporous thin films: En route to versatile biofuel cells. Small 2014; 10(14): 2834-9.
[19] Dizge N, Keskinler Bl. Enzymatic production of biodiesel from canola oil using immobilized lipase. Biomass Bioenergy 2008; 32(12): 1274-8.
[20] Tao Y, Ning M, Dou H. A novel therapeutic system for malignant glioma: Nanoformulation, pharmacokinetic, and anticancer properties of cell-nano-drug delivery. Nanomedicine (Lond) 2013; 9(2): 222-32.
[21] Ahmad R, Khatoon N, Sardar M. Antibacterial effect of green synthesized TiO2 nanoparticles. Adv Sci Lett 2014; 20(7-9): 1616-20.
[22] Ahmad R, Mohsin M, Ahmad T, Sardar M. Alpha amylase assisted synthesis of TiO2 nanoparticles: Structural characterization and application as antibacterial agents. J Hazard Mater 2015; 283: 171-7.
[23] Khatoon N, Ahmad R, Sardar M. Robust and fluorescent silver nanoparticles using Artemisia annua: Biosynthesis, characterization and antibacterial activity. Biochem Eng J 2015; 102: 91-7.
[24] Liu J, Lu C-Y, Zhou H, Xu J-J, Chen H-Y. Flexible gold electrode array for multiplexed immunoelectrochemical measurement of three protein biomarkers for prostate cancer. ACS Appl Mater Interfaces 2014; 6(22): 20137-43.
[25] Johnson BJ, Algar WR, Malanoski AP, Ancona MG, Medintz IL. Understanding enzymatic acceleration at nanoparticle interfaces: Approaches and challenges. Nano Today 2014; 9(1): 102-31.
[26] Sinha R, Khare SK. Immobilization of halophilic Bacillus sp. EMB9 protease on functionalized silica nanoparticles and application in whey protein hydrolysis. Bioprocess Biosyst Eng 2015; 38(4): 739-48.
[27] Ahmad R, Khatoon N, Sardar M. Biosynthesis, characterization and application of TiO2 nanoparticles in biocatalysis and protein folding. J Proteins & Proteomics 2013; 4(2): 115-21.
[28] Ahmad R, Mishra A, Sardar M. Simultaneous immobilization and refolding of heat treated enzymes on TiO2 nanoparticles. Adv Sci Eng Med 2014; 6(12): 1264-8.
[29] Ahmad R, Sardar M. Immobilization of cellulase on TiO2 nanoparticles by physical and covalent methods: A comparative study. Indian J Biochem Biophys 2014; 51(4): 314-20.
[30] Grewal J, Ahmad R, Khare SK. Development of cellulase-nanoconjugates with enhanced ionic liquid and thermal stability for in situ lignocellulose saccharification. Bioresour Technol 2017; 242: 236-43.
[31] Perwez M, Ahmad R, Sardar M. A reusable multipurpose magnetic nanobiocatalyst for industrial applications. Int J Biol Macromol 2017; 103: 16-24.
[32] Jia H, Zhu G, Wang P. Catalytic behaviors of enzymes attached to nanoparticles: The effect of particle mobility. Biotechnol Bioeng 2003; 84(4): 406-14.
[33] Khoshnevisan K, Bordbar A-K, Zare D, et al. Immobilization of cellulase enzyme on superparamagnetic nanoparticles and determination of its activity and stability. Chem Eng J 2011; 171(2): 669-73.
[34] Wang L, Wei L, Chen Y, Jiang R. Specific and reversible immobilization of NADH oxidase on functionalized carbon nanotubes. J Biotechnol 2010; 150(1): 57-63.
[35] Zhang B, Xing Y, Li Z, Zhou H, Mu Q, Yan B. Functionalized carbon nanotubes specifically bind to α-chymotrypsin’s catalytic site and regulate its enzymatic function. Nano Lett 2009; 9(6): 2280-4.
[36] Doukyu N. Characteristics and biotechnological applications of microbial cholesterol oxidases. Appl Microbiol Biotechnol 2009; 83(5): 825-37.
[37] Turfitt GE. The microbiological degradation of steroids: 4. Fission of the steroid molecule. Biochem J 1948; 42(3): 376-83.
[38] Ghosh S, Khare SK. Biodegradation of 7-ketocholesterol by Rhodococcus erythropolis MTCC 3951: Process optimization and enzymatic insights. Chem Phys Lipids 2017; 207(Pt B): 253-9.
[39] Doukyu N, Aono R. Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomonas sp. strain ST-200. Appl Environ Microbiol 1998; 64(5): 1929-32.
[40] Ghosh S, Khare SK. Biodegradation of cytotoxic 7-Ketocholesterol by Pseudomonas aeruginosa PseA. Bioresour Technol 2016; 213: 44-9.
[41] Doukyu N, Aono R. Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200. Appl Microbiol Biotechnol 2001; 57(1-2): 146-52.
[42] Brzostek A, Dziadek B, Rumijowska-Galewicz A, Pawelczyk J, Dziadek J. Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis. FEMS Microbiol Lett 2007; 275(1): 106-12.
[43] Doukyu N, Shibata K, Ogino H, Sagermann M. Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance. Appl Microbiol Biotechnol 2008; 80(1): 59-70.
[44] Gadda G, Wels G, Pollegioni L, et al. Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum. Eur J Biochem 1997; 250(2): 369-76.
[45] Yazdi MT, Zahraei M, Aghaepour K, Kamranpour N. Purification and partial characterization of a cholesterol oxidase from Streptomyces fradiae. Enzyme Microb Technol 2001; 28(4-5): 410-4.
[46] Lim L, Molla G, Guinn N, Ghisla S, Pollegioni L, Vrielink A. Structural and kinetic analyses of the H121A mutant of cholesterol oxidase. Biochem J 2006; 400(1): 13-22.
[47] Vrielink A, Ghisla S. Cholesterol oxidase: Biochemistry and structural features. FEBS J 2009; 276(23): 6826-43.
[48] Coulombe R, Yue KQ, Ghisla S, Vrielink A. Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair. J Biol Chem 2001; 276(32): 30435-41.
[49] Eventoff W, Rossmann MG. The evolution of dehydrogenases and kinases. CRC Crit Rev Biochem 1975; 3(2): 111-40.
[50] García JL, Uhía I, Galán B. Catabolism and biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol 2012; 5(6): 679-99.
[51] Drzyzga O, Fernández de las Heras L, Morales V, Navarro Llorens JM, Perera J. Cholesterol degradation by Gordonia cholesterolivorans. Appl Environ Microbiol 2011; 77(14): 4802-10.
[52] Navas J, González-Zorn B, Ladrón N, Garrido P, Vázquez-Boland JA. Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi. J Bacteriol 2001; 183(16): 4796-805.
[53] Mendes MV, Recio E, Antón N, et al. Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin. Chem Biol 2007; 14(3): 279-90.
[54] Singh S, Solanki PR, Pandey MK, Malhotra BD. Cholesterol biosensor based on cholesterol esterase, cholesterol oxidase and peroxidase immobilized onto conducting polyaniline films. Sens Actuators B Chem 2006; 115(1): 534-41.
[55] Assmann G, Jabs HU, Kohnert U, Nolte W, Schriewer H. LDL-cholesterol determination in blood serum following precipitation of LDL with polyvinylsulfate. Clin Chim Acta 1984; 140(1): 77-83.
[56] Ott P, Binggeli Y, Brodbeck U. A rapid and sensitive assay for determination of cholesterol in membrane lipid extracts. Biochim Biophys Acta 1982; 685(2): 211-3.
[57] Roda A, Festi D, Sama C, et al. Enzymatic determination of cholesterol in bile. Clin Chim Acta 1975; 64(3): 337-41.
[58] Ahmad S, Roy PK, Khan AW, Basu SK, Johri BN. Microbial transformation of sterols to C19-steroids by Rhodococcus equi. World J Microbiol Biotechnol 1991; 7(5): 557-61.
[59] Alexander DL, Fisher JF. A convenient synthesis of 7 α-hydroxycholest-4-en-3-one by the hydroxypropyl-β-cyclodextrin-facilitated cholesterol oxidase oxidation of 3 β,7 α-cholest-5-ene-3,7-diol. Steroids 1995; 60(3): 290-4.
[60] Biellmann JF. Resolution of alcohols by cholesterol oxidase from Rhodococcus erythropolis: Lack of enantiospecificity for the steroids. Chirality 2001; 13(1): 34-9.
[61] Purcell JP, Greenplate JT, Jennings MG, et al. Cholesterol oxidase: A potent insecticidal protein active against boll weevil larvae. Biochem Biophys Res Commun 1993; 196(3): 1406-13.
[62] Pollegioni L, Piubelli L, Molla G. Cholesterol oxidase: Biotechnological applications. FEBS J 2009; 276(23): 6857-70.
[63] Kumari L, Kanwar SS. Cholesterol oxidase and its applications. Adv Microbiol 2012; 2(02): 49-65.
[64] Rodrigues RC, Berenguer-Murcia A, Fernandez-Lafuente R. Coupling chemical modification and immobilization to improve the catalytic performance of enzymes. Adv Synth Catal 2011; 353(13): 2216-38.
[65] Garcia-Galan C, Berenguer-Murcia A, Fernandez-Lafuente R, Rodrigues RC. Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal 2011; 353(16): 2885-904.
[66] Sheldon RA, van Pelt S. Enzyme immobilisation in biocatalysis: Why, what and how. Chem Soc Rev 2013; 42(15): 6223-35.
[67] Petry I, Ganesan A, Pitt A, Moore BD, Halling PJ. Proteomic methods applied to the analysis of immobilized biocatalysts. Biotechnol Bioeng 2006; 95(5): 984-91.
[68] Spahn C, Minteer SD. Enzyme immobilization in biotechnology. Recent Pat Eng 2008; 2(3): 195-200.
[69] Johnson RD, Wang ZG, Arnold FH. Surface site heterogeneity and lateral interactions in multipoint protein adsorption. J Phys Chem 1996; 100(12): 5134-9.
[70] Rao SV, Anderson KW, Bachas LG. Oriented immobilization of proteins. Mikrochim Acta 1998; 128(3): 127-43.
[71] Datta S, Christena LR, Rajaram YRS. Enzyme immobilization: An overview on techniques and support materials. 3 Biotech 2013; 3(1): 1-9.
[72] Grosová Z, Rosenberg M, Rebros M, Sipocz M, Sedlácková B. Entrapment of beta-galactosidase in polyvinylalcohol hydrogel. Biotechnol Lett 2008; 30(4): 763-7.
[73] Deshpande A, D’Souza SF, Nadkarni GB. Coimmobilization of D-amino acid oxidase and catalase by entrapment of Trigonopsis variabilis in radiation polymerised polyacrylamide beads. J Biosci 1987; 11(1): 137-44.
[74] Singh S, Singhal R, Malhotra BD. Immobilization of cholesterol esterase and cholesterol oxidase onto sol-gel films for application to cholesterol biosensor. Anal Chim Acta 2007; 582(2): 335-43.
[75] Murai K, Kato K. Development of cholesterol biosensor with high sensitivity using dual-enzyme immobilization into the mesoporous silica materials. Appl Surf Sci 2013; 258(5): 1725-32.
[76] Jegan Roy J, Emilia Abraham T. Strategies in making cross-linked enzyme crystals. Chem Rev 2004; 104(9): 3705-22.
[77] Velasco-Lozano S, Lopez-Gallego F, Mateos-Diaz JC, Favela-Torres E. Cross-Linked Enzyme Aggregates (CLEA) in enzyme improvement-a review. Biocatalysis 2014; 1(1): 166-77.
[78] Brena BM, Batista-Viera F. Immobilization of enzymes: A literature survey. Immobilization of enzymes and cells 2006; 15-31.
[79] Yong Y, Bai Y, Li Y, Lin L, Cui Y, Xia C. Preparation and application of polymer-grafted magnetic nanoparticles for lipase immobilization. J Magn Magn Mater 2008; 320(19): 2350-5.
[80] Bryjak J, Trochimczuk AW. Immobilization of lipase and penicillin acylase on hydrophobic acrylic carriers. Enzyme Microb Technol 2006; 39(4): 573-8.
[81] Hartmann M, Kostrov X. Immobilization of enzymes on porous silicas-benefits and challenges. Chem Soc Rev 2013; 42(15): 6277-89.
[82] Hudson S, Cooney J, Magner E. Proteins in mesoporous silicates. Angew Chem Int Ed Engl 2008; 47(45): 8582-94.
[83] Betancor L, Luckarift HR. Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol 2008; 26(10): 566-72.
[84] Sardar M, Mishra A, Ahmad R. Biosynthesis of metal nanoparticles and their applications 2014; 239-66.
[85] Ahmad S, Goswami P. Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production. Process Biochem 2014; 49(12): 2149-57.
[86] Kumar A, Malhotra R, Malhotra BD, Grover SK. Co-immobilization of cholesterol oxidase and horseradish peroxidase in a sol-gel film. Anal Chim Acta 2000; 414(1): 43-50.
[87] Pundir CS. Co-immobilization of cholesterol esterase, cholesterol oxidase and peroxidase onto alkylamine glass beads for measurement of total cholesterol in serum. Curr Appl Phys 2003; 3(2): 129-33.
[88] Chauhan N, Pundir CS. Co-immobilization of cholesterol esterase, cholesterol oxidase and peroxidase on PVC strip for serum cholesterol determination. Anal Methods 2011; 3(6): 1360-5.
[89] Chen Y, Xin Y, Yang H, et al. Immobilization and stabilization of cholesterol oxidase on modified sepharose particles. Int J Biol Macromol 2013; 56: 6-13.
[90] Dervisevic M, Çevik E, Şenel M, Nergiz C, Abasiyanik MF. Amperometric cholesterol biosensor based on reconstituted cholesterol oxidase on boronic acid functional conducting polymers. J Electroanal Chem 2016; 776: 18-24.
[91] Khan R, Kaushik A, Mishra AP. Immobilization of cholesterol oxidase onto electrochemically polymerized film of biocompatible polyaniline-Triton X-100. Mater Sci Eng C 2009; 29(4): 1399-403.
[92] Akkaya B, Sahin F, Demirel G, Tumturk H. Functional polymeric supports for immobilization of cholesterol oxidase. Biochem Eng J 2009; 43(3): 333-7.
[93] Le Huy N, Thuy NTM, Binh NH, et al. Covalent immobilization of cholesterol oxidase and poly (styrene-co-acrylic acid) magnetic microspheres on polyaniline films for amperometric cholesterol biosensing. Anal Methods 2013; 5(6): 1392-8.
[94] Huang J, Liu H, Zhang P, Zhang P, Li M, Ding L. Immobilization of cholesterol oxidase on magnetic fluorescent core-shell-structured nanoparticles. Mater Sci Eng C 2015; 57: 31-7.
[95] Ghosh S, Ahmad R, Gautam VK, Khare SK. Cholesterol-oxidase-magnetic nanobioconjugates for the production of 4-cholesten-3-one and 4-cholesten-3, 7-dione. Bioresour Technol 2018; 254: 91-6.
[96] Kouassi GK, Irudayaraj J, McCarty G. Examination of Cholesterol oxidase attachment to magnetic nanoparticles. J Nanobiotechnology 2005; 3(1): 1.
[97] Šulek F, Drofenik M, Habulin M, Knez Ž. Surface functionalization of silica-coated magnetic nanoparticles for covalent attachment of cholesterol oxidase. J Magn Magn Mater 2010; 322(2): 179-85.
[98] Šulek F, Knez Ž, Habulin M. Immobilization of cholesterol oxidase to finely dispersed silica-coated maghemite nanoparticles based magnetic fluid. Appl Surf Sci 2010; 256(14): 4596-600.
[99] Umar A, Ahmad R, Hwang SW, Kim SH, Al-Hajry A, Hahn YB. Development of highly sensitive and selective cholesterol biosensor based on cholesterol oxidase co-immobilized with α-Fe2O3 micro-pine shaped hierarchical structures. Electrochim Acta 2014; 135: 396-403.
[100] Saxena U, Chakraborty M, Goswami P. Covalent immobilization of cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive amperometric detection of cholesterol in real samples. Biosens Bioelectron 2011; 26(6): 3037-43.
[101] Lin X, Ni Y, Kokot S. Electrochemical cholesterol sensor based on cholesterol oxidase and MoS2-AuNPs modified glassy carbon electrode. Sens Actuators B Chem 2016; 233: 100-6.
[102] Aggarwal V, Malik J, Prashant A, Jaiwal PK, Pundir CS. Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase. Anal Biochem 2016; 500: 6-11.
[103] Temani M, Baccar ZrM, Mansour HB. Activity of cholesterol oxidase immobilized on layered double hydroxide nanomaterials for biosensor application: Acacia salicina scavenging power of hypercholesterolemia therapy. Microelectron Eng 2014; 126: 165-8.
[104] Silva RA, Carmona-Ribeiro AM, Petri DFS. Enzymatic activity of cholesterol oxidase immobilized onto polymer nanoparticles mediated by Congo red. Colloids Surf B Biointerfaces 2013; 110: 347-55.
[105] Li M, Huang J, Zhang P, Zhang P, Ding L, Eds. Immobilization of cholesterol oxidase on SiO2 nanoparticles and its application in fiber optic cholesterol sensor. Asia-Pacific Optical Sensors Conference 2016.
[106] Ahmad R, Khare SK. Immobilization of Aspergillus niger cellulase on multiwall carbon nanotubes for cellulose hydrolysis. Bioresour Technol 2018; 252: 72-5.
[107] Konwarh R, Karak N, Rai SK, Mukherjee AK. Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase. Nanotechnol 2009; 20(22): 225107.
[108] Kim J-D. Immobilization of keratinase from Aspergillus flavus K-03 for degradation of feather keratin. Mycobiology 2005; 33(2): 121-3.
[109] Huang SH, Liao MH, Chen DH. Direct binding and characterization of lipase onto magnetic nanoparticles. Biotechnol Prog 2003; 19(3): 1095-100.
[110] Chiou S-H, Wu W-T. Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups. Biomater 2004; 25(2): 197-204.
[111] Das A, Singh J, Yogalakshmi KN. Laccase immobilized magnetic iron nanoparticles: Fabrication and its performance evaluation in chlorpyrifos degradation. Int Biodeterior Biodegradation 2017; 117: 183-9.
[112] Kunamneni A, Ghazi I, Camarero S, Ballesteros A, Plou FJ, Alcalde M. Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers. Process Biochem 2008; 43(2): 169-78.
[113] Namdeo M, Bajpai SK. Immobilization of α-amylase onto Cellulose-Coated Magnetite (CCM) nanoparticles and preliminary starch degradation study. J Mol Catal B Enzym 2009; 59(1): 134-9.
[114] Dey G, Bhupinder S, Banerjee R. Immobilization of alpha-amylase produced by Bacillus circulans GRS 313. Braz Arch Biol Technol 2003; 46(2): 167-76.
[115] Kim D-M, Kim MY, Reddy SS, et al. Electron-transfer mediator for a NAD-glucose dehydrogenase-based glucose sensor. Anal Chem 2013; 85(23): 11643-9.
[116] Wang H-S, Pan Q-X, Wang G-X. A biosensor based on immobilization of horseradish peroxidase in chitosan matrix cross-linked with glyoxal for amperometric determination of hydrogen peroxide. Sensors (Basel) 2005; 5(4): 266-76.
[117] Li G, Xu H, Huang W, Wang Y, Wu Y, Parajuli R. A pyrrole quinoline quinone glucose dehydrogenase biosensor based on screen-printed carbon paste electrodes modified by carbon nanotubes. Meas Sci Technol 2008; 19(6): 065203.
[118] Chaubey A, Gerard M, Singhal R, Singh VS, Malhotra BD. Immobilization of lactate dehydrogenase on electrochemically prepared polypyrrole-polyvinylsulphonate composite films for application to lactate biosensors. Electrochim Acta 2001; 46(5): 723-9.