- Email: [email protected]
Enzyme immobilization on nanomaterials for biofuel production
Letters
Enzyme immobilization on nanomaterials for biofuel production Munish Puri, Colin J. Barrow, and Madan L. Verma Bioprocessing Laboratory, Centre for Chemistry and Biotechnology (CCB), Geelong Technology Precinct, Waurn Ponds, Deakin University, Victoria 3217, Australia
The efficient immobilization of enzymes using nanostructured materials has recently been demonstrated. The materials used for this purpose, such as nanoparticles, nanofibers, nanotubes, nanoporous media, nanocomposites, and graphene all possess large surface areas that improve biocatalytic efficiency for industrial applications by increasing enzyme loading and facilitating reaction kinetics [1]. In this report, we present the research opportunities for nanoscale materials in enzyme biotechnology and highlight recent developments in biofuel production using more advanced material supports for enzyme immobilization and stabilization. Immobilized enzymes (IMEs) are used commercially for an array of large-scale biocatalyst applications, because their applications can yield both improved product quality and lower processing costs [2]. Typically, IMEs have superior thermal and operational stability through a range of pH values and ionic concentrations and are more resistant to denaturation than their native soluble forms. IMEs can additionally be recycled by utilizing the physical or chemical properties of the matrix or carrier. Substantial research efforts have aimed to optimize the structure of carrier materials for better catalytic efficiency. To this end, nanomaterials (materials with a length in the order of nanometers) represent the upper limit in terms of several key factors that determine the efficiency of biocatalysts, such as high surface areas for greater enzyme loading, lower mass transfer resistance, reduced fouling effect, and selective, nonchemical separation from the reaction mixture by application of a magnetic field. Currently, there is considerable interest in the use of nanoscale material and process development, which is aimed at exploiting the unique phenomena associated with these small length materials to improve their function. Advances in fabrication have given researchers access to a variety of nanomaterials that possesses unique optical, electronic, magnetic, mechanical, and chemical properties. Various nanomaterials, such as nanoparticles, nanofibers, nanotubes, and nanoporous matrices have all demonstrated their potential to revolutionize the preparation and use of biocatalysts. Surface modifications of nanomaterials, such as silanization, carbodiimide activation, and polyethylene glycol (PEG) or polyvinyl alcohol (PVA) spacing, aid in the binding of single or multienzyme systems to the nanoparticles (Figure 1 inset), whereas crosslinking using glutaraldehyde can also stabilize the attached enzymes [2]. Beyond the high surface areas of nanomaterials, we see Corresponding author: Puri, M. ([email protected]).
benefits arising from superior volume ratios, due to which nanoscale biocatalyst systems exhibit unique behavior that distinguishes them from traditional immobilized systems. The Brownian motion of nanoparticles, confining effect of nanopores, and self-assembling behavior of discrete nanostructures also represent exciting opportunities in this field. There are however specific disadvantages with regard to the handling of nanomaterials, chiefly represented by health and environmental concerns [3]. Difficulties with regard to nanomaterial preparations such as monodispersity, aggregation, precipitation, and thermodynamic stability [4] are expected to be addressed in the near future. The physical and chemical approaches to nanomaterial synthesis are well documented. Understanding protein interactions with nanomaterials at the structural and functional level is important for improving the application of these interesting hybrid materials [5]. Enzyme kinetic studies in conjunction with atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy greatly assists us in better understanding the structure and function of nanomaterial-bound enzymes. Characterization using transmission electron microscopy (TEM), scanning electron microscopy (SEM), FTIR, circular dichroism (CD), UV-Vis spectroscopy, Raman spectroscopy, and AFM has revealed the presence of individually dispersed enzyme-bound nanomaterials in solution. Two types of enzymes, namely cellulases and lipases [6], are the primary candidates for large-scale implementation of enzymatic biofuel production [7]. Enzyme-based hydrolysis of biomass (an eco-friendly route) [8] can be improved economically by increasing thermal stability, efficiency, and reusability of enzymes, all of which can be assisted by the immobilization of enzymes on support matrixes [9]. Immobilization of cellulases for application in biofuel production has been studied using silica [10], and polymeric nanoparticles [11]. Simultaneous co-immobilization of three affinity-tagged cellulase enzymes on gold-doped magnetic silicon nanoparticles has successfully achieved single-step hydrolysis of cellulose [12]. In these various applications of nanomaterial-bound enzymes, biocatalytic efficiency in biofuel production has been observed to have improved; a crucial step towards future application in biofuel production. Despite the ongoing academic interest, these bench-scale technologies currently lack verified applications on an industrial scale, and studies using nanomaterial-bound cellulase or enzyme cocktails (as hypothesized in Figure 1) for hydrolyzing real biomass (rich in lignocellulose content) beg urgent investigation. 215
Letters
Trends in Biotechnology April 2013, Vol. 31, No. 4
Enzymes bound to nanomaterial
NM Fermentaon
Biofuel: ethanol, butanol, hydrogen Immobilized enzyme ( ) By-products separaon Pretreated lignocellulosic/ triglyceride/ non-food biomass (sugars)
Biodiesel (transesterificaon)
Bioreactor TRENDS in Biotechnology
Figure 1. Flow diagram representing the use of nanomaterial-bound enzyme in a bioreactor for biofuel production using various types of raw materials. Inset shows binding of large quantities of 3D structure of enzymes on a nanomaterial (NM; presenting high surface area).
To conclude, nanomaterial-bound enzyme-catalyzed biofuel production processes are still in their infancy. Recent studies have established that the activity and stability of immobilized enzymes in hydrolysis and esterification reactions can be increased by using nanomaterials, due to the protection from denaturation and increased activation of enzymes that they afford. It is possible that co-immobilization of multienzymes could be achieved on these nanomaterials, thus facilitating application of various enzymes in hydrolyzing complex substrates for biofuel production. However, significantly more research is required before technical bottlenecks such as biocompatibility issues, restricted mass transfer, enzyme leaching upon reuse, and the complexity and expense inherent to current nanomaterial synthesis procedures can be better understood and mitigated. The use of carbon nanotube or graphene oxide nanosheets for immobilizing lipases/cellulases for biofuel production also warrants further investigation. Acknowledgments The authors are grateful to Prof. Peter Hodgson, Director, Institute for Frontier Materials, Deakin University for supporting research work.
References 1 Ansari, S.A. and Husain, Q. (2012) Potential applications of enzymes immobilized on/in nanomaterials. Biotechnol. Adv. 30, 512–523
216
2 Johnson, P.A. et al. (2011) Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization. Methods Mol. Biol. 679, 183–191 3 Shu, W.H. et al. (2009) Multifunctional nanosystems at the interface of physical and life sciences. Nano Today 4, 27–36 4 Krumov, N. et al. (2009) Production of inorganic nanoparticles by microorganism. Chem. Eng. Technol. 32, 1026–1035 5 Li, L. et al. (2010) Analytical strategies for detecting nanoparticleprotein interactions. Analyst 135, 1519–1530 6 Trans, D.T. et al. (2012) Immobilization of lipase on a ferric silica nanocomposite for biodiesel production. J. Biotechnol. 158, 112–119 7 Verma, M.L. et al. (2013) Immobilization of glucosidase on a magnetic nanoparticle improves thermostability: application in cellobiose hydrolysis. Biores. Technol. http://dx.doi.org/10.1016/j.biortech.2013. 01.047 8 Puri, M. et al. (2012) Biofuel production: prospects, challenges and feedstock in Australia. Renew. Energy Res. Rev. 16, 6022–6031 9 Zhang, Z. et al. (2012) Advancements and future directions in enzyme technology for biomass conversion. Biotechnol. Adv. 30, 913–919 10 Chang, R.H.Y. et al. (2011) Cellulase immobilized mesoporous silica nanocatalysts for efficient cellulose to glucose conversion. Green Chem. 13, 2844–2850 11 Lupoi, J.S. and Smith, E.A. (2011) Evaluation of nanoparticle-immobilized cellulase for improved yield in simultaneous saccharification and fermentation reactions. Biotechnol. Bioeng. 108, 2835–2843 12 Cho, E.J. et al. (2012) Co-immobilization of three cellulases on Audoped magnetic silica nanoparticles for the degradation of cellulose. Chem. Commun. 48, 886–888 0167-7799/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2013.01.002 Trends in Biotechnology, April 2013, Vol. 31, No. 4
Enzyme immobilization on nanomaterials for biofuel production Munish Puri, Colin J. Barrow, and Madan L. Verma Bioprocessing Laboratory, Centre for Chemistry and Biotechnology (CCB), Geelong Technology Precinct, Waurn Ponds, Deakin University, Victoria 3217, Australia
The efficient immobilization of enzymes using nanostructured materials has recently been demonstrated. The materials used for this purpose, such as nanoparticles, nanofibers, nanotubes, nanoporous media, nanocomposites, and graphene all possess large surface areas that improve biocatalytic efficiency for industrial applications by increasing enzyme loading and facilitating reaction kinetics [1]. In this report, we present the research opportunities for nanoscale materials in enzyme biotechnology and highlight recent developments in biofuel production using more advanced material supports for enzyme immobilization and stabilization. Immobilized enzymes (IMEs) are used commercially for an array of large-scale biocatalyst applications, because their applications can yield both improved product quality and lower processing costs [2]. Typically, IMEs have superior thermal and operational stability through a range of pH values and ionic concentrations and are more resistant to denaturation than their native soluble forms. IMEs can additionally be recycled by utilizing the physical or chemical properties of the matrix or carrier. Substantial research efforts have aimed to optimize the structure of carrier materials for better catalytic efficiency. To this end, nanomaterials (materials with a length in the order of nanometers) represent the upper limit in terms of several key factors that determine the efficiency of biocatalysts, such as high surface areas for greater enzyme loading, lower mass transfer resistance, reduced fouling effect, and selective, nonchemical separation from the reaction mixture by application of a magnetic field. Currently, there is considerable interest in the use of nanoscale material and process development, which is aimed at exploiting the unique phenomena associated with these small length materials to improve their function. Advances in fabrication have given researchers access to a variety of nanomaterials that possesses unique optical, electronic, magnetic, mechanical, and chemical properties. Various nanomaterials, such as nanoparticles, nanofibers, nanotubes, and nanoporous matrices have all demonstrated their potential to revolutionize the preparation and use of biocatalysts. Surface modifications of nanomaterials, such as silanization, carbodiimide activation, and polyethylene glycol (PEG) or polyvinyl alcohol (PVA) spacing, aid in the binding of single or multienzyme systems to the nanoparticles (Figure 1 inset), whereas crosslinking using glutaraldehyde can also stabilize the attached enzymes [2]. Beyond the high surface areas of nanomaterials, we see Corresponding author: Puri, M. ([email protected]).
benefits arising from superior volume ratios, due to which nanoscale biocatalyst systems exhibit unique behavior that distinguishes them from traditional immobilized systems. The Brownian motion of nanoparticles, confining effect of nanopores, and self-assembling behavior of discrete nanostructures also represent exciting opportunities in this field. There are however specific disadvantages with regard to the handling of nanomaterials, chiefly represented by health and environmental concerns [3]. Difficulties with regard to nanomaterial preparations such as monodispersity, aggregation, precipitation, and thermodynamic stability [4] are expected to be addressed in the near future. The physical and chemical approaches to nanomaterial synthesis are well documented. Understanding protein interactions with nanomaterials at the structural and functional level is important for improving the application of these interesting hybrid materials [5]. Enzyme kinetic studies in conjunction with atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy greatly assists us in better understanding the structure and function of nanomaterial-bound enzymes. Characterization using transmission electron microscopy (TEM), scanning electron microscopy (SEM), FTIR, circular dichroism (CD), UV-Vis spectroscopy, Raman spectroscopy, and AFM has revealed the presence of individually dispersed enzyme-bound nanomaterials in solution. Two types of enzymes, namely cellulases and lipases [6], are the primary candidates for large-scale implementation of enzymatic biofuel production [7]. Enzyme-based hydrolysis of biomass (an eco-friendly route) [8] can be improved economically by increasing thermal stability, efficiency, and reusability of enzymes, all of which can be assisted by the immobilization of enzymes on support matrixes [9]. Immobilization of cellulases for application in biofuel production has been studied using silica [10], and polymeric nanoparticles [11]. Simultaneous co-immobilization of three affinity-tagged cellulase enzymes on gold-doped magnetic silicon nanoparticles has successfully achieved single-step hydrolysis of cellulose [12]. In these various applications of nanomaterial-bound enzymes, biocatalytic efficiency in biofuel production has been observed to have improved; a crucial step towards future application in biofuel production. Despite the ongoing academic interest, these bench-scale technologies currently lack verified applications on an industrial scale, and studies using nanomaterial-bound cellulase or enzyme cocktails (as hypothesized in Figure 1) for hydrolyzing real biomass (rich in lignocellulose content) beg urgent investigation. 215
Letters
Trends in Biotechnology April 2013, Vol. 31, No. 4
Enzymes bound to nanomaterial
NM Fermentaon
Biofuel: ethanol, butanol, hydrogen Immobilized enzyme ( ) By-products separaon Pretreated lignocellulosic/ triglyceride/ non-food biomass (sugars)
Biodiesel (transesterificaon)
Bioreactor TRENDS in Biotechnology
Figure 1. Flow diagram representing the use of nanomaterial-bound enzyme in a bioreactor for biofuel production using various types of raw materials. Inset shows binding of large quantities of 3D structure of enzymes on a nanomaterial (NM; presenting high surface area).
To conclude, nanomaterial-bound enzyme-catalyzed biofuel production processes are still in their infancy. Recent studies have established that the activity and stability of immobilized enzymes in hydrolysis and esterification reactions can be increased by using nanomaterials, due to the protection from denaturation and increased activation of enzymes that they afford. It is possible that co-immobilization of multienzymes could be achieved on these nanomaterials, thus facilitating application of various enzymes in hydrolyzing complex substrates for biofuel production. However, significantly more research is required before technical bottlenecks such as biocompatibility issues, restricted mass transfer, enzyme leaching upon reuse, and the complexity and expense inherent to current nanomaterial synthesis procedures can be better understood and mitigated. The use of carbon nanotube or graphene oxide nanosheets for immobilizing lipases/cellulases for biofuel production also warrants further investigation. Acknowledgments The authors are grateful to Prof. Peter Hodgson, Director, Institute for Frontier Materials, Deakin University for supporting research work.
References 1 Ansari, S.A. and Husain, Q. (2012) Potential applications of enzymes immobilized on/in nanomaterials. Biotechnol. Adv. 30, 512–523
216
2 Johnson, P.A. et al. (2011) Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization. Methods Mol. Biol. 679, 183–191 3 Shu, W.H. et al. (2009) Multifunctional nanosystems at the interface of physical and life sciences. Nano Today 4, 27–36 4 Krumov, N. et al. (2009) Production of inorganic nanoparticles by microorganism. Chem. Eng. Technol. 32, 1026–1035 5 Li, L. et al. (2010) Analytical strategies for detecting nanoparticleprotein interactions. Analyst 135, 1519–1530 6 Trans, D.T. et al. (2012) Immobilization of lipase on a ferric silica nanocomposite for biodiesel production. J. Biotechnol. 158, 112–119 7 Verma, M.L. et al. (2013) Immobilization of glucosidase on a magnetic nanoparticle improves thermostability: application in cellobiose hydrolysis. Biores. Technol. http://dx.doi.org/10.1016/j.biortech.2013. 01.047 8 Puri, M. et al. (2012) Biofuel production: prospects, challenges and feedstock in Australia. Renew. Energy Res. Rev. 16, 6022–6031 9 Zhang, Z. et al. (2012) Advancements and future directions in enzyme technology for biomass conversion. Biotechnol. Adv. 30, 913–919 10 Chang, R.H.Y. et al. (2011) Cellulase immobilized mesoporous silica nanocatalysts for efficient cellulose to glucose conversion. Green Chem. 13, 2844–2850 11 Lupoi, J.S. and Smith, E.A. (2011) Evaluation of nanoparticle-immobilized cellulase for improved yield in simultaneous saccharification and fermentation reactions. Biotechnol. Bioeng. 108, 2835–2843 12 Cho, E.J. et al. (2012) Co-immobilization of three cellulases on Audoped magnetic silica nanoparticles for the degradation of cellulose. Chem. Commun. 48, 886–888 0167-7799/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2013.01.002 Trends in Biotechnology, April 2013, Vol. 31, No. 4