http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–12 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.928811

REVIEW ARTICLE

Recent trends in nanomaterials immobilised enzymes for biofuel production Madan L. Verma, Munish Puri, and Colin J. Barrow Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 10/15/14 For personal use only.

Centre for Chemistry and Biotechnology, Geelong Technology Precinct, Waurn Ponds, Deakin University, Victoria, Australia

Abstract

Keywords

Application of nanomaterials as novel supporting materials for enzyme immobilisation has generated incredible interest in the biotechnology community. These robust nanostructured forms, such as nanoparticles, nanofibres, nanotubes, nanoporous, nanosheets, and nanocomposites, possess a high surface area to volume ratios that can cause a high enzyme loading and facilitate reaction kinetics, thus improving biocatalytic efficiency for industrial applications. In this article, we discuss research opportunities of nanoscale materials in enzyme biotechnology and highlight recent developments in biofuel production using advanced material supports for enzyme immobilisation and stabilisation. Synthesis and functionalisation of nanomaterial forms using different methods are highlighted. Various simple and effective strategies designed to result in a stable, as well as functional protein-nanomaterial conjugates are also discussed. Analytical techniques confirming enzyme loading on nanomaterials and assessing post-immobilisation changes are discussed. The current status of versatile nanomaterial support for biofuel production employing cellulases and lipases is described in details. This report concludes with a discussion on the likely outcome that nanomaterials will become an integral part of sustainable bioenergy production.

Bioreactors, biomass, cellulases, immobilisation, lipases, nanomaterials

Introduction Nanotechnology is a rapidly expanding field, broadly encompassing the fabrication and use of nanometre-scale materials (Ansari & Husain, 2012; Jiang et al., 2013; Verma et al., 2013a). Recently, advances in nanofabrication have given researchers access to a variety of nanomaterials that possess unique optical, electronic, magnetic, mechanical and chemical properties (Biswas et al., 2012; Wu et al., 2008). Nanomaterials possess structural components in the nanoscale size range, and of comparable dimensions to biological macromolecules, such as enzymes (proteins) and nucleic acids (Stark, 2011). The resulting intersection between biotechnology and nanotechnology has made possible the development of a new hybrid nanobiocatalytic system, which combines the catalytic and selective recognition properties of biological molecules with unique characteristics that nanomaterials can provide (Chronopoulou et al., 2011). Versatility of nanomaterials applications can easily be engineered by the process of functionalisation (Jiang et al., 2013). Processes such as silanisation, glutaraldehyde/carbodiimide activation, synthetic/natural polymers, aid in the binding of single/multienzyme systems to the nanomaterials (Johnson et al., 2011).

Address for correspondence: Dr. Madan L. Verma, Centre for Chemistry and Biotechnology, Geelong Technology Precinct, Waurn Ponds, Deakin University, Victoria, Australia. Tel: +61 35227 2619. Fax: +61 35227 2170. E-mail: [email protected]

History Received 7 October 2013 Revised 21 February 2014 Accepted 27 February 2014 Published online 11 July 2014

Various nanomaterials, such as nanoparticles, nanofibres, nanotubes, nanoporous, nanosheets and nanocomposites, have shown potential to revolutionise the preparation and the use of biocatalysts (El-Zahab et al., 2004; Hwang & Gu, 2013; Lee et al., 2009; Nair et al., 2007; Shi et al., 2007; Pavlidis et al., 2012a,b; Verma et al., 2013a, b,d). The depletion of global non-renewable fossil fuels continues at an unprecedented rate, a problem brought to light by the rising price of crude oil and its refined products. This phenomenon is a key driver of the growing focus on sustainable sources of alternative energy, having already given rise to a host of potential alternative fuels. Biofuels are one example, offering a potential route towards satisfying future energy requirements by utilising domestically available renewable resources (Pugh et al., 2011; Puri et al., 2012). Biofuel is biodegradable and non-toxic, and is produced from various feedstocks, including vegetable oils and biomass (Kralova & Sjooblom, 2010). Redundant plant biomasses have attracted particular attention as potential renewable sources for the production of alternatives to petroleum based fuels. Enzyme-based processes represent an attractive potential route for biofuel production, as they are environmentally benign, selective, and able to function efficiently at ambient temperatures (Kumari et al., 2009; Du et al., 2005). Two enzymes, namely cellulases and lipases, are the primary candidates for large scale implementation of enzymatic biofuel production. Lipases (EC 3.1.1.3) are widely studied for their ability to catalyse reactions at the

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hydrophobic/hydrophilic interface, such as hydrolysis, and synthesis of fats and oils, with high enantioselectivity. Lipases may therefore be used in an array of applications, ranging from food technology, such as in the hydrolysis of oils and synthesis of structured lipids (Goswami et al., 2013; Kralovec et al., 2010, 2012), to energy, such as in the synthesis of biodiesel (Wang et al., 2011a) to fine chemical production (Adlercreutz, 2013; Kanwar & Verma, 2010; Kanwar et al., 2009; Stergiou et al., 2013; Verma et al., 2008b, 2009, 2011). Cellulases (EC 3.2.1.4) have been used successfully in the saccharification of lignocellulosic materials (i.e. plant biomass) for ethanol production (Abraham et al., 2014; Lee et al., 2010; Margeot et al., 2009; Matano et al., 2013; Puri et al., 2012; Verma et al., 2013b,c). Cellulase is a common collective term for a mixture of three different enzymes, namely endoglucanase, exoglucanase and b-glucosidase. These enzymes work synergistically to produce glucose which is subsequently fermented into ethanol (Chandel et al., 2012; Garvey et al., 2013; Lynd et al., 2002).

Tangible challenges There are several challenges facing commercially viable enzyme catalysed biofuel production. Significant limitations of enzyme assisted biofuel production include: (i) enzyme inactivation by solvents (ethanol, methanol and glycerol), (ii) high enzyme costs, and (iii) barriers to scale up (Shimada et al., 2002; Watanabe et al., 2000). High enzyme costs can be reduced to a significant extent by introducing novel nanoscale matrices that may lead to efficient immobilisation methods. Multiple reuses with easy separation of magnetic nanomaterials as well as nanocomposites supports by an external magnetic field can significantly lower the cost of an enzymatic process. Immobilisation of the biomolecules (whole cells/enzymes) is a well-established technology, with the potential to significantly improve the economic viability of these processes in an industrial setting. Enzyme immobilisation is a fundamental route for the development of bioreactors and biosensors (Song et al., 2012). A number of nanosupports and methods have been applied in enzyme immobilisation with a diverse array of applications (Ansari & Husain, 2012; Li et al., 2011; Lee et al., 2010; Lupoi & Smith, 2011; Verma et al., 2012, 2013a,b). Besides easily separation from the product, immobilised enzymes have the advantage of thermal and operational stability at extreme environments of pH, temperature and organic solvent than their native soluble forms (Ansari & Husain, 2012; Hwang & Gu, 2013; Verma et al., 2013a). They are, therefore, the right candidate to use commercially for large scale biocatalyst applications. Industrial application of immobilised forms can result in both improved product quality and lower processing costs (Johnson et al., 2011; Kim et al., 2008; Mateo et al., 2007; Puri et al., 2013; Verma & Kanwar, 2008; Verma et al., 2008a). A further limitation of free and immobilised enzymes is the shear stress from the stirring in a batch reactor that may disrupt the enzyme carrier through physical agitation. The use of automated packed bed reactors (PBR) can partially overcome this limitation (Wang et al., 2011a). This economic

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consideration makes it desirable to use the enzyme repeatedly in a continuous reactor (Jegannathan et al., 2008). In cellulosic ethanol production, the fermentative ability of yeast reduces during extreme environments, such as high ethanol concentrations, low pH or high temperatures. Binding the cells to novel nanomaterials supports has been shown to improve ethanol tolerance and enhanced thermal stability (Ivanova et al., 2011). The immobilised forms of cellulases and lipases therefore represent good alternatives to free enzymes for catalysis in aqueous/non-aqueous media. The development of nanostructured materials whose unique attributes can make them versatile and invaluable components in biofuel research and development is a major focus of researchers in the nanotechnology field (Table 1). Nanotechnology has the potential to interface with biotechnology in new and powerful ways to further promote the generation of sustainable bioenergy production.

Advantages of nanomaterials as enzyme immobilisation supports Nanomaterials exhibit advantageous features over bulk materials in terms of their role in enzyme immobilisation. The large surface area to volume ratios and subsequent higher enzyme loading and good biocatalytic potential makes nanomaterials far more suitable candidates for enzyme immobilisation than the conventional materials (Gupta et al., 2011; Hwang & Gu, 2013; Verma et al., 2013a). The number of enzyme molecules bound to nanomaterials can easily be estimated based on the physical property of the support and biomolecules. For example, assuming the Fe3O4 nanoparticles were spherical, the density of Fe3O4 is 5.18 g/cm3 and the molecular weight of esterase was 33 000 dalton, it could be calculated that about four esterase molecules were bound to a Fe3O4 nanoparticle (Shaw et al., 2006). The effect of support size on the enzyme loading was investigated using macro and nanoparticles of chitosan (Klein et al., 2012). High protein loading in nanoparticles (204 mg) was achieved than the macroparticle (40 mg) per gram of dry support. High enzyme loading led to maximum enzyme activity in nanoparticle (18 330 U) as compared to macroparticle (210 U) per gram of dry support. The BET specific surface area for the macroparticles (87 m2
g 1) was higher than nanoparticles (29 m2
g 1), as macroparticles present a higher mesoporosity than nanoparticles (Klein et al., 2012). Thus, the surface area to volume ratios rather than the surface area is the best parameter to use as an indicator of a material’s potential for an enzyme immobilisation support. The low mass transfer resistance of nanomaterial bound enzymes as compared to macroscale matrixes further enhances the enzyme activity (Kim et al., 2006, 2008). The immobilised nanomaterial exhibits significant Brownian motion due to stable monodispersion behaviour in aqueous suspension (Wang, 2006). According to the Stokes-Einstein equation, the mobility and diffusivity of nanomaterial must be smaller than those of the macroparticle, owing to relatively larger sizes, and thus introducing size dependence in nanoparticle-enzyme activity. It has been shown that Brownian motion may be responsible for the high activities obtained when enzymes are immobilised on nanoparticles

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Table 1. Nanomaterial bound enzymes used for biofuel production. Enzyme Cellulase from Trichoderma viride Cellulase from T. viride

Nanomaterial

Substrate

Silica nanoparticle

Adsorption

Silica nanoparticle

Adsorption, Covalent Covalent

Microcrystalline cellulose Microcrystalline cellulose Cellulose

Covalent

Exo and Endo-Glucananse, Au-dopped silica Glucosidase nanoparticle b-Glucosidase from Polystyrene Aspergillus niger nanofibre b-Glucosidase from Silicon oxide Agaricus arvensis nanoparticle

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Type of Bonding

Product

Reference

Ethanol

ND

Lupoi & Smith, 2011

Glucose

ND

Chang et al., 2011

ND

ND

Cho et al., 2012

Cellulose

ND

ND

Lee et al., 2010

Covalent

Synthetic substrate

ND

b-Glucosidase from A. niger

Iron oxide nanoparticle

Covalent

Cellobiose

Glucose

b-Glucosidase from Trichoderma reesei

Magnetic nanoparticle

Covalent

Glucose

Lipase from Burkholderia sp.

Ferric-silica nanocomposite

Adsorption

Wheat straw, Eucalyptus globules pulp Olive oil + Methanol

Lipase from Thermomyces lanuginosus, Candida Antarctica Lipase from Rhizopus miehei Lipase from Burkholderia sp. Lipase from Pseudomonas cepacia

Kinetic parameters

Fe3O4 nanoparticle

Covalent

Waste grease + Methanol

Silica nanoparticle

Encapsulation

Magnetic nanoparticle PAN nanofibre

Adsorption

Triolein + Methanol Olive oil + Methanol Soyabean oil + Methanol

Covalent

Lipase from P. cepacia

Fe3O4 nanoparticle

Covalent

Lipase from P. cepacia

Fe3O4 nanoparticle

Covalent

Lipase from P. cepacia

Nanoporus gold

Adsorption

Lipase from P. cepacia

Polyacryonitrile fibre Adsorption

Lipase from Thermomyces lanuginosa Xylanase from Pholiota adiposa

Fe3O4 nanoparticle

Covalent

Silicon oxide nanoparticle

Covalent

Soyabean oil + Methanol Soyabean oil + Methanol Soyabean oil + Methanol Rapeseed oil + Methanol Soyabean oil + Methanol Birchwood xylan

Km: 2.5 and 3.8 mM, Singh et al., 2011 Vmax: 3028 and 3347 Umg 1 for free and immobilised enzyme Km: 3.5 and 4.3 mM, Verma et al., 2013b Vmax: 0.72 and 0.89 Umg 1 for free and immobilised enzyme ND Valenzuela et al., 2014

Biodiesel Km: 0.09 and 3.65 mM, Vmax: 133.3 and 131.4 Umg for free and immobilised lipase Biodiesel ND

Tran et al., 2012 1

Ngo et al., 2013

Biodiesel

ND

Macario et al., 2013

Biodiesel

ND

Liu et al., 2012

Biodiesel Km: 56.7 and 88.4 mM, Vmax: 22.5 and 18.3 Umg for free and immobilised enzyme Biodiesel ND

Li et al., 2011 1

Wang et al., 2009

Biodiesel

ND

Wang et al., 2011a

Biodiesel

ND

Wang et al., 2011b

Biodiesel

ND

Sakai et al., 2010

Biodiesel

ND

Xie and Ma, 2010

ND

Kcat: 2109 and 2183 s 1 for free and immobilised enzyme

Dhiman et al., 2012

ND: Not defined.

(Wang, 2006). Nanomaterials, particularly nanofibres, offer larger flexibility in reactor design as they are easier to handle (Nair et al., 2007; Sakai et al., 2008). An enzyme immobilised fibre bioreactor was developed with continuous steady hydrolysis conversion at a constant flow rate (Huang et al., 2008). Recovery for reuse of magnetic nanoparticles is easily

achieved by using an external magnetic field that avoids high speed centrifugation for as long a time as is required for nonmagnetic nanoparticle separation. Thus, the ease of separation of magnetic nanomaterial bound enzymes consequently lowered the mechanical shearing and process costs, thus

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improving the operational stability of enzyme (Ren et al., 2011; Safarik & Safarikova, 2009; Yiu & Keane, 2012). It is important to understand the advantages and limitations of nanomaterials before proposing their application in bioenergy production. Nanomaterial preparations have some specific technological challenge in regards to formation, monodispersity and thermodynamic stability (Krumov et al., 2009). In some processes, specific reaction conditions (pH and temperature control) are required to avoid aggregation and precipitation of the particles out of solution. Such nanomaterials are stabilised by additional steps involving coating with polymer layers (Cui et al., 2005). Nanomaterials with homogeneous sizes are often isolated by precipitation techniques, resulting in particles with a strongly hydrophobic surface. Nanomaterial stabilisation against aggregation can also be achieved by coating the nanomaterial with a polymer shell because of a large decrease of their surface energy in comparison with pristine nanomaterial (Rozenberga & Tenne, 2008). There are however drawbacks to the handling of nanomaterials, which presents certain health and environmental concerns (Arico et al., 2005). For example, carbon nanotubes are toxic in their pure powder state (Buzea et al., 2007). Currently, there are no specific regulatory requirements for various nanomaterials (Verma et al., 2011). The key aspects of nanomaterial toxicity testing that encompasses screening of physiochemical properties (size, surface chemistry, shape, protein absorption gradient and surface smoothness/roughness), in vitro studies (absorption, cytotoxicity), in vivo models studies (efficacy, imaging studies) and in silico studies, have been reviewed comprehensively (Oberdorster et al., 2005; Sharifi et al., 2012). For safety measures prior using nanomaterial, firstly, there is need of selection of representative for each nanomaterial, in terms of structure, size and property. Secondly, a toxicity testing protocol needs to be developed for specific nanomaterial. Thirdly, manufacturing process, in specific, area of fabrication, handling and storage adhered to good manufacturing practice to ensure the quality and stability of the nanomaterial. Finally, assessing the impact of nanomaterial on the environment as a proactive risk management and detecting/monitoring the exposure level in the workplace, air/waterborne releases, humans and other organisms and environmental media is important (Chan, 2006). In order to safely develop of future nanotechnology, a fundamental understanding of the biological interactions of nanomaterial with cells, proteins, and tissues, is crucial and nanomaterials must be endow with a high degree of biocompatibility, with minimal negative effects on blood components, genetic materials, and cell viability (Sharifi et al., 2012). Thus, academia, industry and regulatory bodies help in elucidating the mechanisms of action, balancing its risk and benefit, thus maximising the utility of these nanomaterials without compromising public health and environmental integrity (Chan, 2006).

Preparation and functionalisation of nanomaterial Top down methods, where bulk materials are fractured into nanoscale size materials, as well as a bottom up approach, where constituent molecules are assembled into a

Crit Rev Biotechnol, Early Online: 1–12

nanostructure entity, were used to synthesise nanomaterials (Biswas et al., 2012). The physical and chemical approaches to nanomaterial synthesis are hitherto well documented (Borlido et al., 2013; Wu et al., 2008). Nanoparticles were prepared by the co-precipitation method (Kalantari et al., 2012; Wu et al., 2008). Briefly, ferric chloride and ferrous chloride were dissolved in deionised water at optimised concentrations and stirred in a nitrogen environment. Ammonium/sodium hydroxide solution was then added drop-wise, resulting in the formation of a black precipitate. The resulting dark brown suspension was collected with a magnet and washed repeatedly with water to remove non-magnetic by products. Nanofibre synthesis was conducted by electrospinning, self-assembly, and phase separation techniques (Bhardwaj & Kundu, 2010). Of these, electrospinning is the most widely used technique for nanofibre synthesis due to its simple and effective process. This technique uses electrostatic forces to produce nanoscale dimension fibres from polymer solutions or melts. The electrospinning system basically comprised three components: a high voltage power supply, a spinneret and a grounded collecting plate and utilises a high voltage source to inject charge of a certain polarity into a polymer solution or melt, which is then accelerated towards a collector of opposite polarity (Sill & Recum, 2008). The electrospinning process thus offers a simplified technique for nanofibre formation. Nanoporous gold with different pore sizes was obtained by simple dealloying and thermal annealing methods. It was made by chemically dealloying AgAu alloy foils (Ag78Au22) in concentrated nitric acid at room temperature (Qiu et al., 2008). A variety of techniques have been documented for the production of high quality carbon nanotubes (Volder et al., 2013). The nanotubes were synthesised by arc discharge, laser ablation and chemical vapour deposition techniques. All three methods require an energy source; electricity is required in arc discharge, heat is required for chemical vapour deposition, and high intensity light is required for laser ablation. A carbon source is also required to produce fragments of groups or single carbon atoms that can recombine to generate the carbon nanotubes (Saifuddin et al., 2013). Nanosheet was synthesised by thermal exfoliation of graphite oxide (Kishore et al., 2012). Graphite powder was reacted with a strong oxidising solution of concentrated sulphuric acid, nitric acid and potassium chlorate at room temperature. Graphite oxide was then thermally exfoliated to synthesise graphene by rapid heating to 1050  C in the presence of argon gas. Thermally exfoliated graphene differed from the brownish graphite oxide, in that it was a light, reflected black powder (Kishore et al., 2012). In addition to laboratory-based nanomaterial syntheses protocols, there are many companies involved in the manufacturing. It is estimated that processing and using applications of the nanotechnology products expected to grow to $3.1 trillion by 2015 (Becker, 2013 and references therein). Recently, a complete list of different world class companies engaged in the carbon nanomaterials manufacturing have been reviewed comprehensively (Volder et al., 2013). Surface functionalisation is a crucial step to improve the efficiency of nanomaterials (Johnson et al., 2011; Pavlidis

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DOI: 10.3109/07388551.2014.928811

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et al., 2012a,b; Verma et al., 2013d; Wu et al., 2008). It involves grafting of desirable functional groups onto nanomaterial’s surface (Shim et al., 2002). This surface modification can affect their dispersability and interactions with enzymes, thus altering the catalytic activity of the immobilised enzyme significantly (Pavlidis et al., 2010). Prior to nanomaterials application, their surface is functionalised to provide stability, biocompatibility and functionality. Currently, functionalisation is achieved with a variety of organic and inorganic materials such as silica, natural polymers (dextran, starch, gelatin, chitosan), synthetic polymers like poly (ethylene glycol), poly (vinyl alcohol), poly (lactide acid), polymethylmethacrylate, polyacrylic acid, biopolymers, dendrimers and small molecules (Jiang et al., 2013). The surface functional groups play several roles in enzyme immobilisation such as: (i) to change the surface charge of nanoporous support for controlling electrostatic interaction with adsorbed enzyme, (ii) to chemically link with the amino acid groups of the targeted enzyme, and (iii) to decrease the size of pore entrance for entrapping the enzymes in the nanochannels (Lee et al., 2009).

Nanocomposites have improved the pristine nanomaterial property by surface coating with desired functional groups. Nanoparticles are non-porous nanomaterials (Sen & Bruce, 2009). They could cause damage to native nanoparticles due to erosion of reacting agents (Lu et al., 2007). In order to avoid this environmental reaction, nanoparticles are covered by the deposition of an inorganic/organic layer such as silica/ silane/oleic acid etc. on nanocores, creating hybrid nanomaterial/nanocomposite. These nanocomposites exhibit more facilitated grafting to functional groups, making them ideal supports for enzyme immobilisation (Deng et al., 2008; Macario et al., 2013; Sen et al., 2010; Tran et al., 2012). The covalent grafting of b-glucosidase enzyme on twice functionalised g-Fe2O3@SiO2 core-shell magnetic nanocomposites was reported (Georgelin et al., 2010). The functionalised nanoparticles provided non-toxic platforms that are stable in high concentrations and further show chemical groups to allow further coupling with enzymes. This approach produces a colloidal suspension of covalently grafted enzymes that remain stable for months and mimics the enzyme-substrate interactions in solution.

Comparable benefits of nanomaterial forms

Strategies for enzyme immobilisation on nanomaterial

Nanomaterial forms such as magnetic/non-magnetic, pristine/ functionalised, powder/suspension and membrane, have been used for enzyme immobilisation (Verma et al., 2013a). Nanoparticles present highly attractive platforms for a diverse array of biological applications. Nanoparticles have a nanoscale diameter, defined as 1–100 nm, and represent the current forefront of nanotechnology. The ability to fabricate and control the structure of nanoparticles allows scientists to influence the resulting properties and, ultimately, to design materials with desired modifications. The potential applications for magnetic nanoparticles are growing in biofuel production (Ngo et al., 2013; Liu et al., 2012; Wang et al., 2009, 2011a; Xie & Ma, 2009). Nanoparticles, such as Fe2O3, Fe3O4, polymeric, gold, and silica, have been used for the immobilisation of cellulases and lipases (Andrade et al., 2010; Lee et al., 2010; Verma et al., 2013a,b; Wang et al., 2011b). Dispersion in reaction solutions and difficulty in subsequent recovery of non-magnetic nanomaterials for reuse are the primary limitations of nanoparticles. Nanofibres have provided a substitute to overcome this problem (Kim et al., 2008). Electrospun nanofibres provide a large surface area and high porosity with well-interconnected pores for the attachment or entrapment of enzymes. Nanofibres are durable and easily separable. In the case of porous nanofibres, they can reduce the diffusional path of the substrate from the reaction medium to the enzyme active sites because of the reduced dimension in size as compared to macroparticles, improving the efficiency of immobilised enzymes (Jia et al., 2002). The electrospinning process can be used for different polymers/polymer blends, and operates at room temperature so that biological compounds can be loaded into the nanofibres without compromising its native biological activity. These attractive features of nanofibres have generated some recognition as potential supports for enzyme immobilisation (Li et al., 2011; Lee et al., 2010; Bhardwaj and Kundu, 2010).

There are considerable advantages to modifying nanomaterial properties through surface functionalisation techniques for effective enzyme immobilisation. Interfacing proteins with nanostructured materials offers the possibility to develop a novel and robust bioconjugates for multiple applications (Chronopoulou et al., 2011). Immobilisation of cellulase and lipase has been extensively studied using magnetic, silica, cellulose, gold, TiO2, and polymeric nanomaterials, among others (Cho et al., 2012; Huang et al., 2011; Pavlidis et al., 2012a,b; Verma et al., 2013a). It has been reported that proteins adsorbed onto nanomaterials are more stable in strongly denaturing environments of high temperatures and organic solvents (Dordick et al., 2012). The development of nanobiocatalytic systems that are highly stable, efficient, or that function as molecular machines to catalyse multiple reactions, is rapidly reshaping our vision of biocatalysis (Johnson et al., 2011; Kim et al., 2008). Lipase immobilisation on nanoparticles by covalent methods using glutaraldehyde as a cross-linker has most commonly been employed (Scheme 1a; Wang et al., 2009). Such a method gave good retention of enzyme activity (70%) and high loading efficiency (25 mg lipase/g particles; Xie & Ma, 2010). Lipase was covalently attached to the carbon nanotubes via carbodiimide activation (Scheme 1b; Ji et al., 2010). Immobilised nanomaterials retained the selectivity of the native lipase and exhibited high stability. The nitrile groups of the polyacrylonitrile (PAN) nanofibres were activated by an amidination reaction followed by reaction with lipase in phosphate buffer solution (Scheme 1c; Li et al., 2011). Activated nanofibre showed high protein loading (43 mg lipase/g fibres) and retained good activity of lipase (80%) compared to free enzyme. Nanofibres were used for enzyme immobilisation by cross-linking additional enzyme molecules and aggregates onto the covalently attached enzyme (Kim et al., 2005). The apparent activity of

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Scheme 1. Schematic representation of enzyme immobilised to nanoparticles (1a; Wang et al., 2009), nanotubes (1b; Ji et al., 2010), nanofibre (1c; Li et al., 2011), nanotubes (1d; Wang and Jiang, 2011), nanocomposites (1e; Georgelin et al., 2010). (a) Amino-functionalised nanomaterial is activated with a cross-linker (glutaraldehyde) to immobilise the enzyme by covalent bonding between amino group of enzyme and amino group of the support. (b) Carbon nanotube is oxidised to carboxylated carbon nanotube. Modified nanotube is reacted with cross-linker to make a covalent bond between enzyme and support. (c) Nanofibre is oxidised to modified nanofibre. Activated nanofibre is reacted with enzyme to make a covalent bond between enzyme and support. (d) Carbon nanotube is oxidised to carboxylated carbon nanotube. Modified nanotube is reacted with a cross-linker in presence of cobalt ions so that His-tagged enzymes bind to the support by affinity method. (e) Magnetic nanoparticle is treatment with a polymer to make ferric-silica nanocomposite. Glutaraldehyde activated nanocomposite was reacted with enzyme to make a covalent binding between enzyme and support.

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DOI: 10.3109/07388551.2014.928811

enzyme coatings on nanofibres was nine times higher than that of nanofibres with just a layer of covalently attached enzyme molecules, and the operational enzyme stability of enzyme-aggregate-coated nanofibres was greatly improved. Peptide nanotubes were employed for the incorporation of CRL, through hydrogen bonding between nanotube amidic groups and complementary groups on the surface on the protein surface (Yu et al., 2005). CRL was immobilised in the internal cavity of the nanotubes that was large enough to allow entry of the substrates. Enzymes can be covalently attached or non-covalently absorbed onto carbon nanotubes (Lee et al., 2010; Shi et al., 2007). The covalent immobilisation of enzymes on pristine nanotubes was performed by allowing the reaction of the free amine groups (on the protein surface) with carboxylic acid groups that are generated by sidewall oxidation of nanotubes with concentrated sulphuric acid and nitric acid, which is facilitated by cross-linker EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) (Saifuddin et al., 2013). The immobilisation of lipase on several kinds of mesoporous silica, such as MCM-41, MCM-48, MCF, SBA-15 has been investigated (Kim et al., 2007). One serious problem with the adsorption method employed for enzyme binding onto the nanoprous support is enzyme leaching. This can be controlled using the covalent method with a cross-linker like glutaraldehyde. Nanoporous support (SBA-15) was used for the lipase adsorption using glutaraldehyde (Gao et al., 2010). Wang and Jiang (2011) studied a detailed site-specific method based on the affinity between His-tagged enzyme and single-walled carbon nanotubes modified with Na,Na-bis(carboxymethyl)-L-lysine hydrate (Scheme 1d). This method overcomes the enzyme leaching, diffusion resistance limitations and provided the right orientation for enzyme active site. The synthesis of coreshell nanoparticles twice functionalised with amino groups and PEG chains has been used for the covalent grafting of b-glucosidase (Scheme 1e; Georgelin et al., 2010). Cellulases have recently been immobilised to gold-doped silica nanoparticles using affinity method, finding application in one step hydrolysis of cellulose to glucose (Cho et al., 2012). Candida rugosa lipase was immobilised on the magnetic nanoparticle via hydrophobic interaction (Wang et al., 2012). The nanoparticles were functionalised with different alkyl chain lengths to modulate their surface hydrophobicity. The performance of nanoparticle immobilised lipase improved with increased degree of alkyl chains hydrophobicity. Most lipases show interfacial activation phenomena. Enzymes contain a lid domain controlling access to the active site. The interaction of the lipase with lipid aggregates induces the displacement of the lid, which makes the active site accessible to substrate molecules and increases catalytic performance (Derewenda & Sharp, 1993). Therefore, the hydrophobic support improved lipase performance via an interfacialactivation phenomenon (Jin et al., 2011). A good understanding of interactions of the enzyme with the functional groups of nanomaterial surface and, more generally, with its nano-environment at the support/solvent interface, could eventually lead to the possibility of directing the self-assembly of the enzyme at the nanometer scale (Pavlidis et al., 2010; 2012a,b; Verma et al., 2013d). This is

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necessary for the development of efficient bioreactors and biosensors-based specific application on the controlled deposition of enzymes at the molecular level (Chronopoulou et al., 2011).

Analytical techniques employed for investigating enzyme-nanomaterial interaction Interfacing nanomaterials and enzymes has led to rapid advances in nanobiotechnology. Understanding enzyme interactions with nanomaterials at the structural level is paramount for improving the application of these interesting hybrid materials (Cruz et al., 2010; Pavlidis et al., 2012a,b). Biophysical characterisation of enzyme post immobilisation changes using TEM (transmission electron microscopy), SEM (scanning electron microscopy), AFM (atomic force microscopy), CD (circular dichroism), UV-Vis spectroscopy, Raman spectroscopy, FTIR (Fourier transform infrared) spectroscopy and XPS (X-ray photoelectron) spectroscopy elucidate the structure of enzymes adsorbed on/in the nanomaterials. Investigation of the conformation stability of these adsorbed enzymes led to the discovery of a novel property of nanomaterials like their ability to enhance protein activity and stability (Andrade et al., 2010; Lee et al., 2009). As proteins adsorb onto the surfaces of nanomaterials, maintenance of an active, tertiary structure depends upon characteristic conformational stability, the properties of the immobilisation support, as well as the intermolecular interactions occurring between the protein and the support (Cruz et al., 2010; Shang et al., 2007). Accordingly, the potential stabilising effect of nanostructured materials remains enzyme-specific. A thorough understanding of the conformational changes that an enzyme undergoes in its immobilised state is restricted by a limited understanding of the structural, chemical, and electrostatic interactions that exist between proteins and nanomaterials (Ganesan et al., 2009). As conventional techniques to study enzyme structure in solution rely upon light scattering phenomena, they are generally rendered ineffective for studying nanomaterial immobilised proteins as a result of background noise from the carrier material and potential for aggregate formation. The size and morphology of nanomaterials before and after enzyme immobilisation were determined by TEM, SEM, and AFM, respectively. The XRD analyses showed that the magnetic nanoparticles had no crystal phase change after lipase was bound to the support (Xie & Ma, 2010). The FTIR analysis of nanomaterial and enzyme bound nanomaterials was performed in order to confirm binding of the enzyme onto the support. The characteristic peaks of the protein were observed in the spectra of free enzyme and enzyme bound nanoparticles, which clearly demonstrated the binding of enzymes to the surface of the nanomaterials. CD spectroscopy was also used to analyse the influence of covalent attachment onto the secondary structure of enzymes (Ganesan et al., 2009; Verma et al., 2013d). Lipase attached to carbon nanotubes retained 62% of the a-helix content of the native lipase revealed from CD spectra analysis (Ji et al., 2010). Application of an enzyme in a given process is controlled by its conformational stability on the surface of the nanomaterial. Such sophisticated analytical techniques are

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used to characterise in situ structural stability of enzymes immobilised on supports and can predict the operational stability of the immobilised biocatalyst (Ganesan et al., 2009). Thus, a good understanding of protein-nanomaterial interactions is useful to redesign the functional biocatalyst system.

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Impact of the nanomaterials immobilised enzymes in improving biofuel production Various nanomaterials forms have been successfully employed in biofuel production. Covalent immobilisation of lipases has been carried out on magnetic Fe3O4 nanoparticles for application in biodiesel production at a reactor scale (Wang et al., 2009). Porcine pancreas lipase (PPL), Candida rugosa lipase (CRL), and Pseudomonas cepacia lipase (PCL) were immobilised onto the amino-functionalised Fe3O4 nanoparticles for 30 min. Consequently, the PCL, being resistant to methanol, has been selected for use in enzymatic transesterification reactions (Wang et al., 2009). Higher conversion of soybean oil to biodiesel production was recorded with the immobilised PCL in a reactor as compared to flask reactions. Complete yield (100%) was observed in the first 3 cycles, being the result of using agitation systems with impellers, which offers better substrate mixing and transferring. These results are comparatively superior to those of other reports, which stated that a conversion rate of only 67% was achieved at the optimal conditions in biodiesel production from soybean oil (Kaieda et al., 2001). The especially high conversion indicated that the immobilised PCL retains sufficiently high stability and recyclability in the transesterification of vegetable oil to warrant use in a reactor. High protein load (85% of original lipase solution) rendered high activities of immobilised enzyme (95%) than the free enzyme (82%). The high biodiesel production was attributed to higher enzyme loading due to the larger surface area of nanoparticles for the attachment of enzymes. A high conversion of biodiesel production (90%) was reported from covalently immobilised Thermomyces lanuginosus lipase to the amino-functionalised magnetic nanoparticles for 12 h (Xie & Ma, 2009, 2010). Transesterification efficiency of immobilised lipase using glutaraldehyde crosslinker was high than immobilised lipase using an EDC crosslinker to achieved 90% biodiesel production. The superiority of glutaraldehyde chemistry of nanomaterial’s activation over EDC as a suitable cross-linker for enzyme immobilisation has been reported. Liu et al. (2012) immobilised lipase sourced from isolated Burkholderia sp. onto hydrophobic magnetic particles (HMP) via an adsorption method for 8 h. The immobilised lipase was used six times for transesterification without recorded loss of activity. The transesterification conditions with the immobilised enzyme were optimised by response surface methodology (RSM) for biodiesel production. The transesterification efficiency achieved by HMP-lipase was also comparable with a commercial lipase Novozyme 435. The conversion of oil to fatty acid methyl esters was ca. 70% within 12 h and the biodiesel production rate was 43.5 g/L/h. High adsorption capacity of HMP for the lipase (4619 U/g) based on the Langmuir adsorption isotherm and RSM optimisation for

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transesterification established the feasibility of using the developed immobilised lipase in practical applications. Pseudomonas cepacia lipase was immobilised via an adsorption method onto the nanopores (35 nm) of a nanoporous gold support (Wang et al., 2011b). The lipasenanoporous conjugate achieved high biodiesel production (90%), whereas, the conversion of soybean oil to biodiesel by free lipase was 74% at 24 h. The performance of an immobilised enzyme also improved during extreme reaction conditions of high temperatures and organic solvents. High catalytic performance of immobilised lipase might be explained by their physical confinement inside the relatively small pores. Specifically, the matching of pore dimension and molecular diameter of lipase and the suitability of gold to function as an immobilisation support are key factors in achieving high enzymatic stability. Expensive gold material (Wolf, 2011) has been used for a wide range of processes due to biocompatibility and chemical stability (Jacoby, 2013). Thus, it is cost-effective to reuse the particles once the enzyme has been exhausted. Lipase was covalently immobilised on a functionalised magnetic nanoparticle for 30 min (Wang et al., 2011a). Immobilised lipase was employed for biodiesel production based on soybean oil methanolysis using continuous PBR (Wang et al., 2011a). Emulsification before methanolysis has improved the reaction rate. Immobilised lipase showed high activity and stability in the single PBR at an optimal flow rate. The conversion rate has been increased with the reaction time. The maximum conversion rate (75%) was recorded at 12 h. In contrast, after nine cycles, the conversion rate remained at only 18.5% in the batch process (Wang et al., 2009). Such a noticeable increase in the conversion rate from that of the batch process was probably a decrease in the deactivation of the immobilised lipase resulting from the high methanol concentration and shearing force. The same researchers developed a four-PBR system for repeated and effective use of immobilised lipase (Wang et al., 2011a). Besides the stability, the conversion rate achieved using the four-PBR was much higher than those achieved using the single PBR. The conversion of biodiesel was maintained at a high rate (88%) for 192 h, and it only slightly dropped to approximately 75% after 240 h of reaction. These results were quite encouraging as compared to other earlier studies (Hama et al., 2007; Thanh et al., 2011). Reasons attributed for the high conversion rate and good stability in the four-PBR was the longer residence time of the reaction mixture in the reactor and reduction in the inhibition of the immobilised lipase by products. From an economic perspective, the cost involved in lipase production would result in an overall increase in the fabrication cost of biodiesel, the main obstacle preventing complete reuse of lipase (Al-Zuhair, 2007; Hama et al., 2007). Thus, the advantages of using an immobilised enzyme could outweigh the increased cost of biodiesel. These studies highlighted the possibility of designing and operating even larger-scale enzymatic systems for biodiesel production. Green and efficient production of biodiesel from waste grease containing high amount of free fatty acid was achieved by using novel magnetic nanoparticle (Ngo et al., 2013). Thermomyces lanuginosus lipase (TLL) and Candida antartica lipase B (CALB) were covalently immobilised to

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magnetic nanoparticles for 4 h, followed by freeze-drying to give high enzyme loading (61 mg TLL or 22 mg CALB per gram particles). Immobilised TLL showed the best performance among immobilised enzymes known thus for the production of biodiesel from grease with methanol, giving high yield (99%) in 12 h. The immobilised lipase retained high productivity (88%) even after 11 cycles. Cellulase was immobilised to silica nanoparticles by the physical adsorption method for 12 h. Immobilised cellulase was used to increase ethanol yields in the simultaneous saccharification and fermentation reaction of cellulose (Lupoi & Smith, 2011). Immobilised enzyme produced more glucose yield as compared to free enzyme. Immobilisation onto silica nanoparticles can stabilise the enzyme and promote activity at non-optimum reaction condition, such as under extreme temperatures and ethanol concentrations. Two mesoporous silica nanoparticles with different pore sizes, and surface area were employed as supports for the immobilisation of cellulase via either covalent or non-covalent binding for 24 h (Chang et al., 2011). Cellulase bound to mesoporous silica nanoparticles via covalent binding achieved an effective yield (80%) of cellulose-to-glucose conversion as compared to cellulase bound to nanoparticle via physical adsorption. Singh et al. (2011) reported the drastic improvement in thermal stability of glucosidase once it was covalently immobilised on functionalised silicon oxide nanoparticles for 36 h. Nanoparticle bound enzymes were stable and retained their full catalytic activity even after usage for 25 cycles at 50  C, demonstrating its commercial application to ethanol production. Recently, b-glucosidase was covalently immobilised to the non-porous magnetic particles for 2 h by using different activation agents (cyanuric chloride, polyglutaraldehyde, carboxyl, tosyl, and long-arm tosyl) (Alftren & Hobley, 2013). Magnetic particles activated with cyanuric chloride (105 U/g) and polyglutaraldehyde (82 U/g) yielded high immobilised enzyme activity. Immobilised enzymes enhanced real complex lignocellulosic substrate (pretreated spruce) hydrolysis in conjunction with free cellulase and retained enzyme activity for four hydrolysis cycles. Batch and continuous reactors were used for butylbiodiesel production by electrospun PAN nanofibre bound PCL lipase (Sakai et al., 2010). The enzyme was immobilised on nanofibres via physical adsorption by incubating the nanofibres in an aqueous solution containing lipase for 11 h. About 94% conversion to biodiesel was achieved in 48 h incubation. The initial reaction rates were 65-fold higher than those detected for commercial immobilised lipase (Novozyme 435) on a total catalyst mass basis. Moreover, the immobilised lipase showed no reduction in catalytic activity during 20 days of operation in a continuous flow-through reactor system. The enhancement of catalytic activity of the lipase on nanofibres may be the result of a conformational change of lipase that enables free access of substrates to the active centres of the enzymes. PAN nanofibre was activated via amidination reaction for covalently immobilisation of PCL enzyme for 30 min (Li et al., 2011). The immobilised enzyme (9 U/mg proteins) showed good activity retention of the free lipase (11 U/mg proteins). The biodiesel production achieved was 90% with only 10% loss of original conversion recorded even after 10

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recycles. Higher biocatalytic efficiency for biodiesel production was attributed to a good immobilisation strategy with only slight changes in enzyme kinetic parameters after covalent binding. Lee et al. (2010) studied high b-glucosidase activity and stability upon binding to magnetic nanofibres using two immobilisation approaches for ca. 3 h. Covalently bound enzyme retained more than 91% of original activity after 20 d of continuous incubation under constant shaking as compared to enzymes coated on the support. Application of lipase bound to ferric silica nanocomposites by simple and effective adsorption method of 24 h were employed for biodiesel production (Tran et al., 2012). Maximum adsorption capacity of lipase (29.45 mg/g particles) from lab isolated Burkholderia sp. on alkyl-functionalised nanocomposites was estimated based on the Langmuir isotherm. The biodiesel production achieved was higher than 90% validating the novel support. This immobilised lipase exhibited high methanol tolerance and reusability as compared to earlier studies (Tran et al., 2012). Cystein-tagged protein cellulases were engineered to enable functional immobilisation on a gold nanoparticle surface. Simultaneous co-immobilisation of three affinitytagged cellulase enzymes on gold doped magnetic silicon nanoparticles for 1 d has successfully achieved one step hydrolysis of cellulose. These results demonstrated the feasibility of ethanol production using enzymes-cascade immobilisation (Cho et al., 2012). Recently, an organicinorganic nanosupport with spherical morphology was synthesised to immobilise Rhizomucor miehei lipase (RML) for 50 h by an encapsulation method (Macario et al., 2013). Lipase was encapsulated to a liposome hybrid nanosphere. The liposomal phase coated with porous inorganic silica for stabilise the internal liposomal phase and, consequently, protected the lipase. Immobilised lipases produced biodiesel by transesterification reaction of triolein with methanol. The immobilised enzyme performed a faster triolein conversion with respect to the lipase encapsulated into mesoporous/ surfactant matrix (Macario et al., 2013). This recent study demonstrated the immobilised lipase retaining its free and stable conformation, due to the biocompatible microenvironment inside the liposome membrane. Total productivity of the immobilised enzyme was superior to that of the equivalent amount of free enzyme. In these various applications of nanomaterials bound enzymes, biocatalytic efficiency in biofuel production has been improved; a crucial step towards, future applications in biofuel production.

Conclusions and future prospective Immobilising enzymes onto a variety of nanostructured materials has led to several benefits when compared to immobilisation on larger materials, or un-immobilised enzymes. Application of nanomaterials in enzyme immobilisation results in higher enzyme loading, multiple recycling and protection from denaturation of enzymes particularly in a packed-bed reactor scale. Some nanomaterials bound enzymes have been employed in biofuel development. Also, the use of carbon nanotube/nanosheets on immobilised lipases/cellulases for exploration of their potential in

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biofuel production needs investigation. With the possibility of co-immobilisation of multi-enzymes on these nanomaterials could facilitate the application of various enzymes in hydrolysing complex substrates for biofuel production (Alper & Stephanopoulous, 2009). However, more studies are required to understand the technical bottlenecks such as sophisticated and expensive synthesis procedures for nanomaterials, toxicity issues and development of safety evaluation guidelines (Sharifi et al., 2012; Verma et al., 2011). With the recent advances in molecular biotechnology and nanotechnology, development of effective biocatalytic systems through the immobilisation of enzymes on functionalised nanomaterials supports appears to be promising. Thus, from the processing of nanomaterials to the engineering of enzymes for biofuel production, nanotechnology has the potential to tremendously impact the biofuel research field. Only two studies of nanomaterial bound enzymes for biodiesel production have been conducted in a packed-bed reactor which needs further investigation. In the future, increasing co-integration of nanotechnology will be important to facilitate the development of sustainable and cost-effective biofuel production. Further studies are required for process improvement and scale-up of nanomaterials as novel matrices to exploit their full potential at the industrial level.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. One of the authors (MLV) thank Deakin University for awarding the Alfred Deakin Post-Doctoral fellowship (Project ID#RM24013).

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