Magnetic nanoparticles: a versatile carrier for enzymes in bio‐processing sectors

Abstract

Many industrial processes experience the advantages of enzymes which evolved the demand for enzymatic technologies. The enzyme immobilisation technology using different carriers has trustworthy applications in industrial biotechnology as these techniques encompass varied advantages such as enhanced stability, activity along with reusability. Immobilisation onto nanomaterial is highly favourable as it includes almost all aspects of science. Among the various techniques of immobilisation, the uses of nanoparticles are remarkably well perceived as these possess high‐specific surface area leading to high enzyme loadings. The magnetic nanoparticles (MNPs) are burgeoning in the field of immobilisation as it possess some of the unique properties such as high surface area to volume ratio, uniform particle size, biocompatibility and particularly the recovery of enzymes with the application of an external magnetic field. Immobilisation of industrially important enzymes onto nanoparticles offers overall combined benefits. In this review, the authors here focus on the current scenario in synthesis and functionalisation of MNPs which makes it more compatible for the enzyme immobilisation and its application in the biotechnological industries.

1 Introduction

The biotransformation reactions catalysed by the enzymes; the most versatile catalyst in living systems have been used in diverse bioprocess applications largely due to their ability to catalyse the reactions under very mild conditions with a varying degree of substrate specificity. The recovery, reuse, and stability of the enzymes play a vital role for large‐scale applications besides having excellent catalytic properties [1]. The problem of enzyme in making any biocatalysts can be easily solved by their immobilising them [2]. Several approaches including chemical re‐engineering [3], genetic modification of proteins [4], and site‐directed mutagenesis have been reported to improve the enzyme properties [5, 6]. The immobilisation of enzymes onto the solid support improves the stability and recovery which greatly reduces the cost of the process ultimately [7]. The enzyme and the support material have on direct impact on the properties of immobilised enzyme, and their interaction influences the specific chemical, biochemical, and kinetic properties [8].

The conventional immobilisation processes such as covalent and ionic binding, adsorption, entrapment, and encapsulation and offers several disadvantages like poor mass transfer limitation of the reactants, poor dispersibility of the products, limited loading capacity, and minimal recovery of the active enzyme fractions [9]. The advent of nanotechnology and its application in the field of biotechnology has drawn greater attention during the past few decades [10]. The nanobiotechnology involves engineering, construction fabrication, and manipulation of molecules in the 1–10 nm range different approaches for the benefit of biological systems. The enzymes have been immobilised using nanoparticles, encapsulated into hyper branched polymers/dendrimers, and single enzyme nanoparticles [11, 12, 13, 14]. Here in this review we focus on the different methods available for the synthesis and functionalisation techniques involved during the preparation of MNPs which makes it more suitable for the enzyme immobilisation. The application of MNPs for the enhanced the enzymes activity and their applications in the bioprocess industries have been discussed.

1.1 Importance of enzymes and their role in industries

Enzymes have been used since ancient time's traditional biotechnological approaches indirectly yeasts and bacteria for the manufacture of cheese, beer, wine, and vinegar [15]. The isolated‐free enzyme laid its first application in the year 1914 and their proteinaceous nature was proved in 1926 followed by their large‐scale production using microbes in 1960s. Due to the advancement in production technologies, properties of engineered enzyme and their application in various fields have led to the steady growth of industrial enzyme sector. The global market of these industrial enzymes is increasing constantly and it is now worth >2 billion dollars per annum. The production of these enzymes has been improved by the application of biotechnology [16]. The enzyme catalysis is extensively used in pharmaceutical, textile, food, and beverage industries. The versatility of enzymes has attracted more researchers in making benign industrial manufacturing processes [17]. Currently, the application of enzymes in analytical techniques as a biosensor is burgeoning in the field of medical sciences. Industrially produced enzymes are used in the leading industries such as detergents (17%), leather and paper (17%), textiles (8%), and pharmaceuticals (41%) [16, 18] as shown in Table 1.

Table 1.

Applications of various industrially important enzymes

Industries	Enzymes employed	Purpose
enzymes for detergency and personal care	proteases, carboxypeptidases amylases, lactases, sucrases maltases, lipases	modification of starch, tissue and fibre stickies and pitch control, removal of stains deinking, drainage improvement
textile	alkaline pectinase, cellulases catalases, proteases, lipases	bio scouring of cotton, bio blasting of cotton fabric, treatment and processing of silk and wool
paper and pulp industry	amylases, xylanases, cellulases, lipases, esterases	bleaching of paper pulp
pharmaceutical	nitrite hydrolase, penicillin G acylase, thermolysin, lipase	organic synthesis of pharmaceutical proteins
food	glucoamylase glucose oxidase, lipase, lipoxygenase	fruit juice processing, wine making, enhancement of flavours, processing of sugar, production of sweeteners, production of glucose syrups, cheese ripening and increase in yield

1.2 Enzyme immobilisation and stabilisation of enzymes

‘ Enzymes’ have its own excellent properties such as higher activity, selectivity and specificity. On comparison with the conventional catalysts, enzyme biocatalysts increase the reaction rate without affecting the reaction thermodynamics. Thus, by the use of these properties, the most complex chemical processes may be performed under favourable environmental conditions. However, most of these enzymes are relatively unstable in spite of the production and purification of enzymes is still being expensive. The difficulty of using these enzymes efficiently lies in the recovery of active enzyme and reuse of the enzymes greatly reduces the cost of the enzymes [1, 19]. Moreover, the application of enzymes as biocatalysts has not yet reached a significant level due to their high costs, poor recovery, reusability, less stability over extended pH and temperature, substrate and product inhibition nature [20, 21, 22, 23]. In addition, when compared with the conventional heterogeneous catalysis, the enzyme catalysis is performed under homogeneous conditions. Thus, there is a chance for the enzyme to contaminate the product if it is not be recovered in the active form from the reaction mixtures for reuse. To overcome the above said technical difficulties, immobilisation is the most vibrant technique to increase the success possibilities [24]. An economical industrial process can be achieved by the efficient reutilising of the biocatalyst [2]. The immobilisation capacity and activity recovery are the two important criteria for an efficient immobilisation [25]. Different approaches have been proposed to overcome the limitations conferred by the free enzymes, including chemical re‐engineering [3], genetic modification of proteins [4], and site‐directed mutagenesis which have been reported to improve the enzyme properties [5, 6]. The enzyme immobilisation is considered to be the most successful method to improve the enzyme properties [26, 27, 28]. By means of immobilisation, enzymes are retained either on the surface or on the support matrix [29].

Through immobilisation we can form the heterogeneous enzyme catalyst system by binding with or within the solid supports. Any suitable method should consider the following factors for enzyme immobilisation such as enzyme topology, active site orientation, reaction medium compatibility, operational cost and stability as shown in Fig. 1. The enzyme immobilisation onto the insoluble organic or inorganic carriers has become a vital area in enzyme technology [21]. The immobilisation of enzymes on a matrix is the most frequently used method for the improvement of enzyme activity [7]. The immobilised enzymes are those which are physically confined or localised in a certain defined region of space with retention to their catalytic activities and which can also be recycled efficiently. The most important attribute of an immobilised enzyme is to finally reduce the cost of the process especially for large‐scale applications. The properties of an immobilised enzyme are governed by both the enzyme and the support material used for the immobilisation and this interaction provides specific chemical, biochemical, and kinetic properties [8].

Fig. 1.

Factors considered for the successful immobilisation of enzymes on to support

Several methods have been successfully used for the production of immobilised enzymes as shown in Fig. 2. This may be broadly classified as physical method (weak interactions exist between the support and the enzyme) and chemical method (covalent bonds are formed with the enzyme). Basically, there are four principle techniques for the enzyme immobilisation such as covalent binding, entrapment, and adsorption cross‐linking [9]. Among all the techniques, the industrial applications of biocatalysts are highly supported by the covalent binding [30].

Fig. 2.

Depiction of different methods of immobilisation

(a) Covalent binding, (b) Entrapment, (c) Encapsulation, (d) Adsorption, (e) Ionic binding, (f) Affinity binding, (g) Metal ion binding, E ‐enzyme M‐metal

1.3 Enzyme immobilisation using nanomaterials

The use of nanomaterials as a carrier for enzyme immobilisation has received more attention during the recent years, because of its large specific surface area offering higher enzyme‐loading capacity [31], decreased mass transfer resistance, and increased mechanical resistance [32]. The enzymes have been immobilised using different matrices, encapsulated into hyper branched polymers/dendrimers and single enzyme nanoparticles [11]. The recent review by Ge et al. [3] describes well about the enhancement of enzyme stabilisation by fabricating the nanobiocatalyst using recently developed methods. To meet the advantages, the different nanoparticles have been reported by several researchers for the bioprocess applications such as gold [33, 34, 35], alumina [36], silica [37, 38], cadmium sulphide [39], iron oxide [40, 41].

Crespilho et al. [42] developed a strategy for enzyme immobilisation on layer‐by‐layer dendrimer‐gold nanoparticle electrocatalytic membrane‐incorporating redox mediator to produce nanostructured electrocatalytic membranes using a combination of the three methods. The layer‐by‐layer technique employs poly vinylsulfonic acid layers on indium tin oxide electrodes which were used alternatively for the poly amidoamine dendrimers with cobalt hexacyanoferrates‐modified gold nanoparticles. The film is then used for the immobilisation of glucose oxidase using glutaraldehyde as the cross‐linker and used for biosensing applications. Glucose oxidase monolayer was covalently immobilised on the surface of gold nanoparticles for the fabrication of bio‐conjugate complex. The bio‐conjugates of glucose oxidase/gold nanoparticles can be considered as catalytic nanodevices to construct nanoreactor based on glucose oxidation for the biotechnological purposes. The immobilisation of glucose oxidase onto gold nanoparticles which had better thermostability [43]. Bai et al. [44] developed a glucose biosensor by the immobilisation of IO₄ ⁻ ‐oxidised‐glucose oxidase on gold nanoparticles‐mesoporus silica composite modified gold electrode with 2‐aminoethanethiol as a cross‐linker. The biosensor exhibited faster response time of <7 s with high sensitivity and conductivity. A novel approach of fabricating gold nanowire for the immobilisation in biosensor applications have also been studied [45]. The gold nanowires were used to immobilise glucose oxidase and the detection of glucose was performed with phosphate buffer and exhibited short response time of 8 s.

Crespilho et al. [46] defined a simple strategy to obtain an efficient bio‐electrochemical enzymatic device in which urease was immobilised on indium tin oxide electrodes. In this, the electrodes were previously modified with an electro active nanostructured membrane containing polyaniline and silver nanoparticles stabilised by polyvinyl alcohol. Chen et al. [47] grafted various carboxylic acids with different alkyl chain lengths on to zirconia nanoparticles for immobilising Pseudomonas cepacia lipase

Cruz et al. [48] described a wide range of surface coverage which allows the detection of optimum conditions for the preparations of immobilisates. It has also been discussed on the impact of structure modifiers on the conformational stability conditions and thereby on immobilisation of enzymes on fumed silica for organic catalysis. Liu et al. [49] reported the covalent attachment of nicotinamide adenine dinucleotide to silica nanoparticles for multistep biotransformation. Moreover, silica nanoparticle‐attached glutamate dehydrogenase, lactate dehydrogenase and nicotinamide adenine dinucleotide were prepared and applied to catalyse the coupled reactions for production of α‐ketoglutarate and lactate. The use of nano particle‐attached cofactor promises a new biochemical processing strategy for cofactor‐dependent biotransformation.

1.4 MNPs as a versatile carrier for enzymes in bioprocess applications

In the past few decades, extensive research has been carried on MNPs and has drawn greater attention because of their unique properties. The use of MNPs for immobilisation is extensively studied which allows the easier recovery of the enzymes and retains their residual activity after many cycles. The MNPs are a class of engineered materials that can be manipulated under the influence of an external magnetic field. Also, among various nanoparticles, MNPs have been considered as suitable for immobilisation of enzyme due to their super‐paramagnetic behaviour and low toxicity [50]. Also, MNPs are not completely porous supports which have unique advantage of no external diffusion [51]. They display the property of super‐paramagnetism that offering the advantage of reducing the aggregation of particles. They possess diversified biological applications in the fields of medicine, detection of bacteria, immobilisation of enzymes, hyperthermia, and magnetic resonance imaging. The controlled size of MNPs and the ability to manipulate by an external magnetic field and their super‐paramagnetic properties makes them a valuable tool for many biomedical and biotechnological applications [52, 53, 54]. The enzyme immobilisation with MNPs is burgeoning in the field of enzyme technology [55, 56, 57] and has multifarious industrial applications. Cao et al. [58] elaborated the exploitation of enzyme immobilised the MNPs for the purification of proteins, analyse of food samples, and food engineering. The enhancement of the stability of the enzymes, possibility for modulation of catalytic activity of the enzymes, lower mass transfer resistance, higher enzyme‐loading capacity, and easier recovery of the enzymes with the application of an external magnetic field are some of the excellent features provided by the MNPs when compared with the traditional methods of immobilisation. Koneracká et al. [59] immobilised several clinically significant proteins and enzymes onto MNPs and the immobilised MNPs with the enzymes exhibited no modification or loss in the enzyme activities.

Several materials have been reported for the synthesis of MNPs including cobalt, iron, and nickel. Among these materials, iron oxide including maghemite (γ ‐Fe₂ O₃) and magnetite (Fe₃ O₄) nanoparticles are promising for their biocompatibilities, low cost, as well as relatively simple synthetic procedures compared with other magnetic materials. Conventionally, the synthesis of MNPs could be classified into five categories: (i) co‐precipitation, (ii) micro‐emulsion, (iii) thermal decomposition, (iv) hydro thermal, and (v) sol–gel methods [60]. Since it is difficult to immobilise enzymes directly onto the MNPs, suitable functionalisations of MNPs are essential for extending them to suitable applications. Several approaches have been employed to modify these MNPs offering other advantages like protecting particle from undesired aggregation and increasing the stability of the particles more stable. The MNPs with small size possess unique features; like single domain limit, large surface area and enduing the particles with magnetic properties. For applications in bio‐related areas, especially in vivo, in vitro applications, biocompatible MNPs are favourable. which could help to maintain the original bimolecular features of the tissues, cells, and proteins [61, 62].

MNPs are more prefered support matrices for enzymes rendering many advantages such as (i) increased enzyme‐loading capacity and (ii) biocompatibility. The super‐paramagnetic properties enable easier and faster recovery of the enzymes by the application of the external magnetic field thus making the process simpler, inexpensive, and enhanced purity [63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75].

Immobilisation of pectinase was carried out in MNPs and surface‐modified MNPs [13, 14, 76], the comparative study of kinetic parameters revealed that MNPs were the best support for pectinase immobilisation. On comparing various supports (Con A‐Seralose 4B, Alginate beads, Chitosan tethered silica, Composite membranes, Amphiphilic PS‐b‐PAA diblock Copolymers, Silica‐coated chitosan particles), lower K _m and higher V _max was achieved for the MNPs. It was evident that the increase in V _max due to negligible steric hindrance and less diffusional resistance. The decrease in K _m value was due to greater affinity of immobilised pectinase to pectin. The synthesised MNPs were extended for real applications such as clarification of Malus domestica juice [14] and enhanced degradation of a textile recalcitrant [76].

1.5 Synthesis and functionalisation of MNPs

Diverse methods have been constantly reported by the researchers for the synthesis of MNPs involving various phases. In general, the physicochemical characteristics of any nanoparticle such as size, dispersity, crystal structure, and morphology are greatly influenced by the mode of synthesis involved [77]. The MNPs produced by the materials in either gas or solid phase with high‐energy requirement are referred to as physical methods (chemical vapour deposition, arc discharge, and annealing) and the synthesis of particles in solution under moderate temperatures are called chemical methods co‐precipitation, thermal decomposition, micro‐emulsion, and hydrothermal synthesis [78, 79].

Chemical vapour deposition is based on the addition of the precursors for layer deposition in the gaseous phase. The layer composition is determined by the parameters of the activating plasma and most notably, the types and the proportions of the gases. The desirable size and morphology of the nanopowders can be achieved by the heat treatment with high purity gas streams [80, 81].

Arc vaporisation works on the principle of gas phase synthesis in which a metal precursor is packed inside a graphite electrode [82, 83, 84]. In this method, the examination of size‐dependent properties of MNPs enabled natural separation at different depths of water [85]. This is not a convenient method for handling large quantities of nanoparticles for coating. Annealing is the solid‐phase synthesis which employs heating at low temperatures to yield. Nanoparticles with the size of 8 nm embedded on amorphous carbon matrix [86].

Micro‐emulsion is a chemical method employing the dispersion of two immiscible liquids under thermodynamically stable isotropic conditions. One or more liquids which consist of nanosized domains are stabilised by interfacial film phenomena using surface‐active compounds [87]. This method is used to synthesise the uniform‐sized MNPs with narrow size distribution. The micro‐emulsion can be prepared as either water‐in‐oil or oil‐in‐water based on which it forms the continuous and dispersed phase. The water‐in‐oil formulation is predominantly used for the synthesis of many nanoparticles and is prepared by mixing water and oil with an amphiphilic surfactant. Some compounds such as Triton X‐100, Igepal CO‐520 and Brij‐97 were also used for the preparation of micro‐emulsions with the base source of ammonium hydroxide, sodium hydroxide and with some added solvents such as acetone and ethanol [88, 89, 90, 91, 92].

By the thermal decomposition method, the MNPs with uniform particle size distribution are synthesised successfully by thermal decomposition method. The precursors include diverse metal‐containing compounds such as organo‐metallic compounds and are more preferable for the synthesis of MNPs. The precursors are allowed to decompose on heating to high temperature or UV irradiation, while the reagents ratios, time, and ageing have been considered to control the morphology and the size of the MNPs [45, 93].

The broad range hydrothermal principle of nanosized particles can be synthesised through. Here, the reaction is carried out in aqueous media under high pressure 2000 psi at 200°C. Recently, some other solvents are also employed for better performances. For instance, nickel ferrite nanoparticles were synthesised using ethylene glycol as a solvent. Continuous hydrothermal and microwave‐hydrothermal are the advanced methods through which specific size, rapid synthesis of MNPs are achieved under low temperature [94, 95, 96, 97, 98, 99, 100].

Chemical co‐precipitation method is an easy and convenient way to synthesise MNPs. The reaction is carried out with the aqueous ferrous (Fe²⁺) and ferric (Fe³⁺) salt solution by the addition of a base under inert atmosphere at the elevated temperature. The size distribution, shape, and composition depend on the type of salts used (e.g. chlorides, sulphates, and nitrates), ratio of Fe²⁺ /Fe³⁺, reaction temperature, pH value, and ionic strength of the media [45, 101, 102].

$$\mathrm{F}{\mathrm{e}}^{2+}+2\mathrm{F}{\mathrm{e}}^{3+}+8\mathrm{O}{\mathrm{H}}^{-}\to \mathrm{F}{\mathrm{e}}_3{\mathrm{O}}_4+4{\mathrm{H}}_2\mathrm{O}$$ (1)

$$\mathrm{F}{\mathrm{e}}_3{\mathrm{O}}_4+0.25{\mathrm{O}}_2+4.5{\mathrm{H}}_2\mathrm{O}\to 3\mathrm{Fe}{\left(\mathrm{OH}\right)}_3$$ (2)

$$\mathrm{F}{\mathrm{e}}_3{\mathrm{O}}_4+2{\mathrm{H}}^{+}\to \gamma -\mathrm{F}{\mathrm{e}}_2{\mathrm{O}}_3+\mathrm{F}{\mathrm{e}}^{2+}+{\mathrm{H}}_2\mathrm{O}$$ (3)

The details about the synthesis of MNPs through different methods and its analysis are shown in Table 2 [45]. In terms of simplicity in synthesis, co‐precipitation is widely preferred. Considering size and morphology control of the MNPs, thermal decomposition seems to be the best method developed till date. As an alternative, micro‐emulsions can also be used to synthesise mono‐dispersed nanoparticles with various morphologies. However, this method requires large quantities of solvent. Hydrothermal synthesis is relatively less explored method for the preparation of high‐quality MNPs. MNPs prepared from co‐precipitation and thermal decomposition are the best‐studied methods and can be prepared on a large scale. Thus, in order to prevent the above inconveniences, the MNPs are usually functionalised by coating the surface of the particles with the organic compounds [45].

Table 2.

Comparative summary of chemical methods for MNPs synthesis

Name of synthesis	Reaction condition	Temperature, °C	Time period	Nature of solvent	Capping agents	Size distribution	Shape control	Yield
co‐precipitation	very simple, ambient conditions	20–90	min	water	needed, added during or after reaction	relatively narrow	not good	high/scalable
thermal decomposition	complicated, inert atmosphere	100–320	h–d	organic compound	needed, added during or after reaction	very narrow	very good	high/scalable
micro‐emulsion	complicated, ambient conditions	20–50	H	organic compound	needed, added during or after reaction	relatively narrow	good	low
hydrothermal synthesis	simple, high pressure	220	h–d	water‐ethanol	needed, added during or after reaction	very narrow	very good	medium

The MNPs generally includes γ ‐Fe₂ O₃ and Fe₃ O₄ which are predominantly used for the biological applications as they are biocompatible, non‐toxic than the other MNPs. Although a variety of physical and chemical methods involving various phases have been employed for the synthesis of MNPs, the chemical co‐precipitation method is more advantageous in terms of simplicity and easier surface modification for the immobilisation of enzymes [103]. Hu et al. [104] synthesised six different kinds of ferrites, MeFe₂ O₄ (Me = Mn, Co, Cu, Mg, Zn, Ni) in nanoscale by co‐precipitation method and analysed for the adsorption–desorption of hexavalent chromium from synthetic wastewater. Furthermore, the effect of magnetic properties on the removal of hexavalent chromium was investigated. A new method for the synthesis of environmental friendly MNPs by ultrasonic chemical co‐precipitation is described by Wu et al. [105]. The resultant nanoparticles were in the range of 15 nm and exhibited super‐paramagnetic effect and hence it can be used for biological applications.

2 Surface modification of MNPs

The surface properties of the synthesised naked MNPs are a major obstacle which greatly influences the amount of immobilised enzymes. The naked MNPs possess hydrophobic surfaces with large surface area lead to the agglomeration and finally results in the formation of clusters. This hinders the enzyme immobilisation and their catalytic activities [106]. The agglomeration of MNPs also has an influence in the loss of super‐paramagnetic behaviour and in the trigger of opsonisation process [107]. Hence, in order to functionalise and modify the porous surface of the MNPs, surface coating and functionalisation of MNPs have been carried out using the compounds such as aminosilanes, chitosan, and carbodiimide. The functionalisation governs the stability, particle size control, and ideal immobilisation of the enzyme over the surface of the MNPs [108, 109, 110]. In situ synthesis methods [111] are present which skips the separate step of functionalisation. However, it is also to be noted that in situ methods produce functionalised nanoparticles of uncontrollable less purity though prepared in a single step.

The enzymes were bound to the matrix through various methods such as surface adsorption, covalent binding, cross‐linking with bifunctional agents such as glutaraldehyde or inclusion in gel phase or encapsulation. When the enzymes bound on to the material's surface, the interaction between them plays a significant role to have a stable and active immobilised enzyme. The nature and linkage between the enzymes and the support matrix are the important factors contributing to the loss of enzyme activity. Hence, many researchers have reported constantly to improve the current state of the art of immobilised systems [112].

The success of immobilisation lies in the availability of diverse functional groups provided by the support material for the attachment of enzymes. The surface of the MNPs is modified to acquire various functional groups for the enzyme immobilisation. This includes several methodologies, of which silanisation, coating with polymers such as chitosan and carbodiimide activation are more popular [113]. Immobilisation of enzyme is also influenced by the porous nature of the MNPs. The porous carriers can support high enzyme loading but it also has high steric hindrances reducing the activity of the immobilised enzyme, whereas the non‐porous carriers involve low enzyme loading but increased activity can be achieved because of less steric hindrances [13, 14].

2.1 Aminosilane coating

The chemical surface modification of MNPs results from the formation of covalent bond that occurs as a result of the reaction of one functional group with another. Some of the compounds form active intermediates with other functional groups rather than being cross‐linked or modified compounds [114, 115]. A second molecule that contains the correct chemical constituent is subsequently coupled with the active ingredient to allow the bond formation to occur. 3‐Aminopropyl triethoxysilane (APTES) is one of such intermediates involved in the surface modification of MNPs. The coating of these silane‐coupling agents on the surface of MNPs offers the high density of functional groups and makes the operation simple [105]. The functional groups designed for specific tasks are anchored as an organic molecule such as APTES as a core shell nanoparticle [116].

Silanes have the general chemical formula as Y–(CH₂) _n –Si–R₃, with Y representing the head group functionality, (CH₂) _n an alkane chain, and Si–R₃ the anchor group by which the silane will be grafted to the oxide surface are bifunctional molecules. The amino group is usually preferred for the binding of different biological groups and this is efficiently provided by the APTES when it is used as the coating material. It is used for the loading of drugs for targeted drug delivery [117]. Recent reports reveal the capability of the aminosilane‐coated MNPs to immobilise the recombinant bacterial enzymes covalently [118].

The hydroxyl groups present on the synthesised MNPs attacks and displaces the ethoxy group (–OC₂ H₅) on APTES and forms a covalent (–Si–O–) bond. This silanisation covers the surface of the MNPs with the functional amino group (–NH₂) forming AMNPS and thus making them available for immobilisation with pectinase [12]. Further, the glutaraldehyde and AMNPs undergoes dehydration to form activated AMNPs, i.e. the reaction between the aldehyde group (–CHO) of glutaraldehyde and the exposed terminal amino group in the functionalised AMNPs. In a similar way, covalent bond is formed between the amino and aldehyde groups of enzyme and glutaraldehyde, respectively, and the mechanisms are shown in Fig. 3.

Fig. 3.

Schematic representation of enzyme immobilisation onto carboxyl group activated AMNPs

2.2 Chitosan coating of MNPs

Chitosan is a cationic polysaccharide formed by the deacetylation of the naturally occurring chitin. Chitin is an unelastic and nitrogenated polysaccharide, found on the walls of the fungi and outer skeleton of arthropodes such as insects, crustaceans and beetles. The natural presence of amino groups in chitosan makes it a cationic polyelectrolyte (pK _a ∼6.5) [119, 120]. The chitosan‐based materials have been successfully employed for enzyme immobilisation. They exist in the form of beads, spheres, or membranes because of their high biological activity, antimicrobial property and stability [66, 121]. Due to the strong magnetic dipole–dipole attractions between particles, the MNPs tend to aggregate in liquid media, modify, and increase their stability. Some biocompatible and biodegradable polymers with specific functional groups have been used as stabilisers. Chitosan, known as 2‐amino‐2‐deoxy‐(1 → 4)‐β ‐d ‐glucopyranan, is one of the most widely distributed biopolymers. Moreover, chitosan is a cationic, non‐toxic, biodegradable, and biocompatible polyelectrolyte. Hence, it has been extensively used in the food, cosmetics, pharmaceutical and biotechnological fields for potential applications [115, 122]. Chitosan is considered to be an ideal support material for enzyme immobilisation because of its varied characteristics such as improved mechanical strength potential anti microbial activity, resistance to chemical degradation, reduced interaction between the metal ions and the enzyme [110, 123]. The magnetic carrier is more significant for the preparation of immobilised enzymes and chitosan. Chitosan can be used as a base material for magnetic carriers as it provides the functional groups for the attachment of MNPs.

Biro et al. [124] synthesised macro, micro, and nanosized chitosan particles by different routes and used for the immobilisation of β‐galactosidase that recorded the highest activity with the nanoparticles and exhibited excellent storage stability with macro and micro spheres. The native chitosan membranes and the membranes cross‐linked with glutaraldehyde were also investigated for the immobilisation of lipase. These supports were characterised for the presence of enzymes and have shown lesser enzyme loaded on the membrane compared with the enzyme added [125]. The chitosan nanoparticles prepared by micro‐emulsion using 2.0% acetic acid and 30% tri‐n ‐octylamine as a solvent and precipitant, respectively, yielded 7 nm sized particles with156 mg/g enzyme‐loading capacity. The immobilised lipase was also able to retain 91% activity after five cycles [126]. Hence, the chitosan was also used as the reducing agent for the synthesis of nanoparticles and it also serves as the surface capping agent. The chitosan is used for the MNPs stabilisation to form a hybrid nanocomposite with excellent hydrophilicity and biocompatibility.

Jiang et al. [127] reported the preparation of magnetic chitosan microspheres by reverse phase suspension methodology. Glutaraldehyde was used as a cross‐linking agent for the immobilisation of enzymes which resulted size distribution. The immobilised laccase exhibited its maximal activity at pH 3.with maximum K_m and V _max than those of free laccase. It also showed an improvement in stability to various parameters such as temperature, reuse, and storage time. A simple and efficient method for the preparation of Fe₃ O₄ ‐chitosan nanoparticles by co‐precipitation and immobilisation via glutaraldehyde cross‐linking was investigated for lipase immobilisation [128].

The laccase from Pycnoporous anguineus was immobilised on the microspheres by adsorption with glutaraldehyde showed an enhancement in the operational stability. Candida rugosa lipase was immobilised on Fe₃ O₄ ‐chitosan nanoparticles via carbodiimide activation in which the optimum conditions for the immobilisation and the factors involved in it were derived by response surface methodology [66, 129].

The mechanism of the enzyme attachment onto the chitosan‐coated MNPswere elucidated and the hydroxyl groups on the MNPs and chitosan reacts to form a hydrogen bond and is accompanied by the release of water molecule. The chitosan coat on the MNPs exposes the amino group (NH₂) on the surface. This amino activated MNPs reacts with the aldehyde group (–CHO) of glutaraldehyde to form activated CMNP_S. The other free aldehyde group in glutaraldehyde forms covalent bonds with amino group of pectinase and thereby immobilisation occurs which is represented in Fig. 4.

Fig. 4.

Schematic representation of enzyme immobilisation onto carboxyl group activated CMNPs

2.3 Silica coating of MNPs

Silica, a chemically inert material, is the most prominent substance for coating without affecting the redox reaction at the core surface. Thus, the silica's surface can easily be functionalised; making the nanoparticles adaptable to many applications. Moreover, the silica coating increases the chemical stability and prevents aggregation of MNPs [130]. The coating material does not disturb the redox reaction at the surface due to the selectivity on the chemical inertness. The schematic representation involved the fabrication of the nanobiocatalyst with the silica functionalised MNPs are shown in Fig. 5. Especially, in silica coating, the reaction between a silanol group and various coupling agents provides a covalent attachment with specific ligands no the surfaces of the MNPs [131]. The silica matrix also acts an anti‐sintering agent and stabilises the maghemite particles. The MNPs with the silica coating renders good stability against organic solvents, anti‐bacterial property, ease in separation and recovery. The aforementioned properties make the surface‐modified MNPs as an ideal carrier for enzyme immobilisation and they also exhibit excellent biocompatibility and hydrophilicity properties [110, 132].

Fig. 5.

Schematic representation of enzyme immobilisation onto carboxyl group activated Amino functionalised silica coated magnetic nanoparticles (ASMNPs)

Wang et al. [133] fabricated cuprous ion‐chelated mesoporous silica MNPs for the immobilisation of laccase where tetraethoxyorthosilicate (TEOS) as the silica source was employed and laccase was adsorbed onto the fabricated nanoparticles with 86.6% residual activity after ten successive cycles of reaction. APTES‐functionalised MNPs were used as the coupling agents for protease for digestion of rape seed meals using 1,4‐phenylenediisothiocyanate as the binding agent [134]. Wang et al. [135] studied the immobilisation of glucose oxidase on novel CoFe₂ O₄ /SiO₂ nanoparticles via cross‐linking with glutaraldehyde. The mono‐dispersed CoFe₂ O₄ /SiO₂ nanoparticles were prepared by a facile route and functionalised CoFe₂ O₄ /SiO₂ nanoparticles with reactive amino groups were synthesised by one‐step method using precursors TEOS and APTES. The immobilised glucose oxidase retained 87% of its initial activity after 7 cycles with the K_m value of 14.6 mM. [136]. The silane‐functionalised nanoparticles were used for the immobilisation of glucose oxidase with an enzyme‐loading capacity of 95 mg/g support and were reported to retain 98% activity after 45 d. More excitingly, the immobilised glucose oxidase showed stability at an elevated temperature of 80°C. Recently, the nanosized amine‐functionalised magnetite nanoparticles were synthesised using APTES as a precursor and was used to immobilise Mucor javonicus lipase using glutaraldehyde as a cross‐linking agent [137]. These nanosized magnetite nanoparticles conjugated with lipase were used to catalyse the solvent‐free synthesis of 1,3‐diacylglycerols from various fatty acids. A ten‐fold increase in specific activity and yield >90% has been achieved by using immobilised lipase [138].

3 Surface activation of MNPs for immobilisation of enzymes

3.1 Carbodiimide activation

Due to its simplicity and high efficiency, a method involving the binding through carbodiimide activation has increased in popularity. 1‐Ethyl‐3‐[3‐dimethylaminopropyl] carbodiimide hydrochloride (EDC) is a water‐soluble cross‐linking agent used to couple carboxyl groups to the primary amines. The formation of amine bonds is facilitated by the cross‐linker which activates the carboxyl groups for spontaneous reaction with primary amines (Fig. 6). The cross‐linking of the carbodiimide can be performed in physiological solutions without adding organic solvents as it is water soluble. However, the excess reagent and the cross‐linking by products can be easily removed by washing with water or dilute acids [139]. The coupling of enzymes onto MNPs through carbodiimide has been constantly reported by researchers for immobilising various enzymes due to its simplicity and high efficiency. Talbert and Goddard [112] utilised the carbodiimide chemistry for the covalent attachment of lactase onto carboxylic acid‐functionalised MNPs of 18 nm particle size. The conjugated enzyme showed excellent catalytic activity with K_m 2.8 × 10³ mol⁻¹ s⁻¹ compared with 2.5 × 10³ mol⁻¹ s⁻¹ for free enzyme that also improved recycle efficiency. The immobilised enzyme retained 78% activity after five repeated cycles. Here the activation of the MNPs takes place in two stages. In the first stage, the carboxylic acid groups were modified to create an amino reactive intermediate followed by the addition of mercaptoethanol. It quenches the unreacted EDC that can lead to protein cross‐linking after the subsequent addition of the enzyme. In the second stage, the lactase was covalently conjugated to the nanoparticles through reactive amine groups associated with the enzyme.

Fig. 6.

Schematic representation of carbodiimide activation

The cellulase which has a significant role in ethanol industry has been covalently immobilised on Fe₃ O₄ via. carbodiimide chemistry to attain the maximum enzyme‐loading capacity and specific activity of 62 IU/mg. This maximum efficiency for enzyme‐to‐support binding was verified at low enzyme loading and the saturation point was confirmed at a weight ratio of 0.02. The immobilised cellulase has also shown a marginal increase in stability over a wider range of temperatures when compared with that of free enzyme and it was also capable of retaining residual activity after six cycles [140].

Sui et al. [141] demonstrated the use of gluconic acid‐functionalised super‐paramagnetic nanoparticles for lipase immobilisation by carbodiimide as the coupling agent. The immobilised enzyme showed an enhancement in the storage stability, pH, and thermal stability than the free lipase. This is mainly due to the carbodiimide which increased the binding efficiency of lipase. The lipase immobilised on MNPs revealed excellent storage stability after 1 month of storage [38]. Kouassi et al. [142] demonstrated the binding of cholesterol oxidase through carbodiimide and achieved 98% binding efficiency.

3.2 Glutaraldehyde activation

Glutaraldehyde is a highly versatile cross‐linking agent widely used in immobilisation (as shown in Figs. 3, 4, 5 [12, 13, 14, 143]). The intermolecular cross‐linking of enzymes by means of bifunctional or multifunctional reagents such as glutaraldehyde, bisadiazobenzidine, or hexamethylenediisocyanate plays a vital role in the immobilisation of enzymes to solid support: This is primarily based on the formation of bonds between enzymes and the matrix through covalent linkage. Using glutaraldehyde for activation enables the nanoparticles to obtain distinct number of reactive arms on the surface [144]. This interaction improves the uniformity, density, and distribution of the immobilised proteins and thereby confers the reproducibility of the surfaces. Ibrahim et al. [145] demonstrated the immobilisation of cyclodextrin glucano transferase from Amphibacillus sp., NPST‐10 onto aminopropyl‐functionalised silica‐coated super‐paramagnetic nanoparticles.

The glutaraldehyde is used along with the chitosan which is used for the functionalisation of MNPs. The amino group on the chitosan reacts with the carboxyl group of glutaraldehyde. Thus, the surface of the activated particles has the carboxyl group of glutaraldehyde. The linkage is formed due to the polymerisation of glutaraldehyde and a bifunctional reagent with several other functional groups and successfully used for the immobilisation of enzymes [146]. This is exceptionally stable at extreme pH and temperatures that facilitate the multipoint covalent attachment [147, 148, 149].

Glutaraldehyde was used for the activation of amino‐functionalised silica‐coated MNPs_. The free amino group of amino‐functionalised silica‐coated MNPs reacts with the terminal aldehyde group of glutaraldehyde to form a Schiff base linkage and provides a free terminal aldehyde which can be then condensed with the free amino groups in the enzyme to form a second Schiff base [150, 151]. The increment in the glutaraldehyde concentration has shown a net significant increase of 94.5% in the enzyme binding efficiency with 5% glutaraldehyde. Li et al. [43] has reported a novel route for the immobilisation of trypsin with glutaraldehyde as the cross‐linking agent. In addition, glutaraldehyde extends its advantage in providing atleast five atom spaces between the enzyme and support which has great importance in catalysis [152]. The immobilisation is mediated by the interaction of the aldehyde groups of glutaraldehyde in functionalised MNPs with the amino groups of trypsin. The immobilised enzymes were conveniently applied for the hydrolysis of proteins and complete protein digestion has been achieved in short time (5 min).

3.3 Polymer‐encapsulated MNPs

The polymers are widely used in nanotechnology for the synthesis of organic and inorganic nanoparticles. The polymer‐assisted synthesis of nanoparticles is a very promising technology, as it considerably decreases the reaction temperature and effectively controls the particle size by preventing agglomeration. The polymers also provide a simple, eco‐friendly, cost‐effective process for the production of multifunctional nanoparticles.

Phillipova et al. [153] reviewed the use of magnetic polymer beads and their unique properties. Since the beads have high magnetic susceptibility to an external magnetic field, they can be easily separated from other components of the mixture with the help of magnets. Attachment of specific functional groups or ligands to the shell of beads can make the separation highly selective and hence it can be used in many biological applications. Yong et al. [154] prepared MNPs by the chemical co‐precipitation of Fe³⁺ and Fe²⁺ ionsand modified the nanoparticles directly by vinyltriethoxysilicane to introduce reactive groups onto the surface of the particles. Glycidyl methacrylate and methacryloxy ethyltrimethyl ammonium chloride, which were then grafted onto the modified nanoparticles by surface‐initiated radical polymerisation. The functionalised MNPs were used for immobilising lipase by electrostatic adsorption and covalent binding. The properties of the immobilised lipase, such as activity recovery, thermal stability, and reusability were investigated. Further, the applications of polymer‐grafted MNPs were used in the immobilisation of lipase. Brahim et al. [155] studied the kinetics of glucose oxidase immobilised in poly hydroxyl methyl methaacrylate‐hydrogel microspheres in a packed‐bed bioreactor. These hydrogel microspheres are pH responsive thereby influencing the action mechanism of immobilised enzymes. The results thus obtained were further analysed in the form of kinetic equations. Liao et al. [156] immobilised and synthesised a novel MNP functionalised with poly vinyl alcohol for the immobilisation of cellulase by micro‐emulsion technique. The immobilised enzyme is applied to improve the hydrolytic efficiency of microcrystalline cellulose combined with ball millingfor the degradation of cellulose.

4 Immobilisation of industrially important enzymes using MNPs

MNPs have been extensively synthesised and functionalised by different methods and are used for commercial applications in industries. The superparamagnetic particles conjugated with therapeutic enzymes have profound applications in the healthcare industries as diagnostic kits [157]. Table 3 summarises the application of different enzymes immobilised onto the MNPs with the kinetic parameters. The immobilisation of some of the useful enzymes onto the MNPs and its applications are discussed here.

Table 3.

Applications of MNPs for immobilisation of enzymes employed in various bioprocessing sectors

Enzyme	Nanoparticles used	Kinetic parameters	Applications	Reference
Food industry
Mucor javonicus lipase	APTES‐glutaraldehyde‐modified MNPs prepared by chemical co‐precipitation method	specific activity (U/mg protein) free enzyme: 0.133 immobilised enzyme:1.42 retained 90% activity after 10 cycles.	conversion of fatty acids to diacyl‐glycerol	[152]
α –amylase	MNPs modified with gum Acacia as a steric stabiliser	Km (µmol/mL/min) free enzyme: 3.4 immobilised enzyme: 5.3 retained 70% activity after 6 cycles	hydrolysis of starch	[153]
lactase	carboxylic acid‐functionalised MNPs by carbodiimide chemistry using EDC and N ‐hydroxysuccimide as coupling agents	K cat (mol−1 s−1) free enzyme: 2.8 × 103 immobilised enzyme: 2.5 × 103 retained 78% activity after 5 cycles	analysis of lactose	[108]
pectinase	docusate sodium salt‐modified MNPs	specific activity (U/mg protein) immobilised enzyme:1.98 retained 80% activity after 6 cycles	hydrolysis of pectin	[154]
amyloglucosidase	cross‐linked enzyme‐aggregate MNPs	Km (µmol/min) Free enzyme: 0.148 immobilised enzyme: 0.141	production of glucose	[155]
β ‐galactosidase	con A layered MNPs	Km (mmol) free enzyme: 2.38 immobilised enzyme: 5.8	degradation of starch	[156]
porcine pancreatic lipase	sodium dodecyl sulphate‐capped MNPs synthesised by chemical co‐precipitation method	specific activity (U/mg protein) free enzyme: 4.75 immobilised enzyme: 9.87 retained 90% activity after 5 cycles	hydrolysis of oils (olive oil)	[157]
Chemical industries
Thermomyces lanuginose lipase	Fe3 O4 /zinc oxide core shell nanoparticles prepared by chemical co‐precipitation method using APTES and TEOS	retained 96% activity after 5 cycles no loss in activity after 15 d	production of chalcone derivatives	[158]
GOD	MNPs by chemical co‐precipitation method	Km (U/g) free enzyme: 640 immobilised enzyme: 780 retained 86% activity after 30 cycles	deoxygenation of water	[102]
GOD	amino‐modified silica‐encapsulated MNPs by microencapsulation method	Km (mmol) free enzyme: 4.8 × 10−2 immobilised enzyme: 1.6 × 10−1 retained 90% activity after 12 cycles	production of quinonenmine dye	[132]
Candida rugosa lipase	chitosan/MNPs prepared by chemical co‐precipitation methods using EDC and N ‐hydroxysuccimide as coupling agents	specific activity (U/g protein) immobilised enzyme: 19.96	hydrolysis of p–nitrophenylpalmitate	[125]
Pharmaceutical industry
Candida rugosa lipase	chitosan‐capped MNPs by carbodiimide activation by coupling with EDC	78% activity retained after five cycles	kinetic resolution of Ibuprofen	[51]
penicillin G acylase	silica‐coated MNPs functionalised by ionic liquids containing ethyoxyl groups	specific activity (U/g protein) immobilised enzyme: 261 retained 62% activity after 10 cycles	production of β ‐lactam antibiotics	[159]
Sensor applications
GOD	single enzyme MNPs functionalised with poly (pyrrole N ‐sulphonic acid) by chemical co‐precipitation method	response time: 4 s detection limit: 0.2 µmol/L shelf life: 30 d	glucose biosensor	[108]
tyrosinase and xanthine oxidase	cyclodextrin‐capped APTES‐coated super‐paramagnetic nanoparticles	detection limit xanthine oxidase: 22 nmol tyrosinase: 2 µmol	electrochemical biosensor for detection of cathecol and xanthine	[160]
cholesterol oxidase	MNPs	E a (kJ/mol) free enzyme: 13.6 immobilised enzyme: 9.3	analysis of total cholesterol in serum	[136]

Enzyme	Nanoparticles used	Kinetic parameters	Applications	Reference
Bioremediation of waste water
Trametes laccase	Cu2+ ‐chelated magnetised mesoporous MNPs functionalised with 3‐chloro propyl trimethoxysilane and ABTS	V max (mmol/L min) free enzyme: 0.103 immobilised enzyme: 0.095 retained 72.6% activity after 10 cycles	degradation of phenol in coking waste water	[161]
horse radish peroxidase	Fe3 O4 /chemically reduced graphene oxide nanocomposite	Km (mmol/L) free enzyme: 10.25 immobilised enzyme: 8.81 retained 70% activity after 10 cycles	removal of phenolic compounds	[162]
Trametes versicolor laccase	bimodal carbon‐based mesoporous magnetic nanocomposite	residual activity free enzyme: 20% immobilised enzyme: 20%	degradation of phenols	[61]
laccase	poly 4(vinyl pyridine)‐grafted MNPS	Km (mmol) free enzyme: 0.32 immobilised enzyme: 0.41 retained 59% activity after 5 weeks	degradation of textile dyes	[163]
laccase	chitosan–MNPs	Km (mmol) free enzyme: 5.69 immobilised enzyme: 10.69	bioremediation of environmental pollutants	[164]

4.1 Lipase

Lipase is a ubiquitous enzyme of considerable importance and industrial potential. They offer as a biocatalyst in a wide range of industrial applications such as in oleochemical, detergent, organic industry, leather industry, environmental management, cosmetic and perfume industries, and many other biomedical applications [165]. It has also been now used in the food industry as a flavour and aroma constituent [166]. The application of lipase as a free enzyme is not favourable for industrial application because of its difficulty to recover and reuse. Also, its poor stability in free form hinders its possibility to withstand many catalytic cycles [167]. This can be overcome by immobilisation of lipase on to various supports [168]. The MNPs has been reported for the immobilisation of lipases from different sources which offer improved stability and recovery [53, 169].

Sui et al. [141] immobilised lipase onto the surface‐modified MNPs by chemical co‐precipitation method through carbodiimide activation. The pH and thermal stability of the lipase was greatly improved than that of the free enzyme. 70% of the initial activity is retained by the MNPs after 30 days. A novel and efficient lipase immobilisation to form chemical cross‐linked enzyme aggregates (CLEAs) by chemical co‐precipitation method using APTES and glutaraldehyde has been reported for the synthesis of 1,3‐diacyl‐glycerol from fatty acids which play a major role in food industry [157].

4.2 Laccase

Laccase, a copper‐containing enzyme, catalyses the oxidation of phenolic and non‐phenolic substrates and other highly recalcitrant environmental pollutants. They have been employed for various bioprocess applications especially in the industrial wastewater treatment [169, 170, 171, 172]. Laccase has been immobilised in various supports including polymers, sepabeads [173], polypyrrole [174], and manganese dioxide nanoparticles decorated with MWCNTS/PANI composite. A novel metal chelating fibrous polymer; poly vinyl pyrrolidone and Cu II were grafted onto the magnetic beads and used for the reversible immobilisation of laccase and used for the degradation of textile dyes at a higher rate [175]. Recent study revealed that laccase immobilisation onto the magnetic nanoparticles (MNPs) was found to be highly supported in the acidic pHs [176].

4.3 Pectinase

Pectinase is one of the most important enzyme which catalyses several important industrial processes such as plant fibre processing industry, coffee and tea extraction, extraction of oils and also in waste water treatment [159]. The treatments of waste in industries become difficult because of the degradation‐resistance and high viscous nature of pectin [177]. It also plays a major role in fruit juice processing industries to degrade the pectic substances imparting turbidity and undesirable suspension. A novel method of impregnating pectinase onto the co‐precipitated silica‐coated surface‐modified MNPs using covalently bound amino and carboxyl groups as coupling agents has greatly improved the enzyme activity and storage stability [13, 143].

4.4 Other industrially important enzymes

The immobilisation of lactase onto MNPs is of considerable importance in the biosensor and ingredient processing applications. Talbert and Goddard [112] has covalently immobilised lactase isolated from A. oryzae by carbodiimide activation to functionalise the MNPs. This enzyme conjugated MNPs open a new horizon to produce a simple and effective conjugated lactase system that achieving both particle and enzyme specificity.

The higher immobilisation rate was observed for amylase immobilisation onto acacia gum used as a stabilising agent for catalysing the hydrolysis of starch [161]. The bound amylase demonstrated high catalytic activity and retained its activity after six cycles of reuse which is extremely advantageous to the industry. MNP‐gold core shell nanoparticles has been synthesised and used for the attachment of trypsin and glucose oxidase. The enzymes immobilised by forming CLEAs onto MNPs are also reported [13], amyloglucosidase (AMG) was covalently attached to the MNPs to form a monolayer and was followed by forming CLEAs with free AMG which greatly improved the enzyme activity and stability with respect to temperature and reusability. Recently, the large‐scale production of superparamagnetic iron oxide nanoparticles has been studied for clinical uses [177].

5 Conclusions

MNPs‐mediated immobilisation exhibits remarkable enzyme stability for industrially important bioprocesses, product development and also possess properties like reusability and recyclability as they can be subjected to magnetic decantation. By analysing various nano carriers, MNPs have been considered as a suitable carrier for immobilisation of enzyme due to their super‐paramagnetic behaviour, low toxicity and has no external diffusion. The applicability towards easy surface modification, activation and as a carrier for wide range of industrially important enzymes attracts many researchers towards further research. The detailed investigation from the above discussion about the interactions between MNPs and enzyme through various surface activation and modification would pave a limelight to the development of enhanced enzymatic activity.