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 nanostruc-
tured materials has recently been demonstrated. The
materials used for this purpose, such as nanoparticles,
nanofibers, nanotubes, nanoporous media, nanocompo-
sites, 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 oppor-
tunities for nanoscale materials in enzyme biotechnology
and highlight recent developments in biofuel production
using more advanced material supports for enzyme immo-
bilization 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 super-
ior 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 chemi-
cal 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, nano-
materials (materials with a length in the order of nano-
meters) 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 selec-
tive, 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 demonstrat-
ed their potential to revolutionize the preparation and use
of biocatalysts. Surface modifications of nanomaterials,
such as silanization, carbodiimide activation, and polyeth-
ylene 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. (munish.puri@deakin.edu.au).
benefits arising from superior volume ratios, due to which
nanoscale biocatalyst systems exhibit unique behavior
that distinguishes them from traditional immobilized sys-
tems. The Brownian motion of nanoparticles, confining
effect of nanopores, and self-assembling behavior of dis-
crete 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 monodisper-
sity, aggregation, precipitation, and thermodynamic sta-
bility [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 great-
ly assists us in better understanding the structure and
function of nanomaterial-bound enzymes. Characteriza-
tion using transmission electron microscopy (TEM), scan-
ning electron microscopy (SEM), FTIR, circular dichroism
(CD), UV-Vis spectroscopy, Raman spectroscopy, and AFM
has revealed the presence of individually dispersed en-
zyme-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 hydro-
lysis 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 pro-
duction has been studied using silica [10], and polymeric
nanoparticles [11]. Simultaneous co-immobilization of
three affinity-tagged cellulase enzymes on gold-doped mag-
netic silicon nanoparticles has successfully achieved sin-
gle-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 hy-
pothesized in Figure 1) for hydrolyzing real biomass (rich
in lignocellulose content) beg urgent investigation.
215
Letters
Fermentaon
Immobilized
enzyme ( )
Pretreated
lignocellulosic/
triglyceride/
non-food
biomass
(sugars)
Bioreactor
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 esteri-
fication reactions can be increased by using nanomater-
ials, 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 pro-
cedures can be better understood and mitigated. The use of
carbon nanotube or graphene oxide nanosheets for immo-
bilizing lipases/cellulases for biofuel production also war-
rants further investigation.
Acknowledgments
The authors are grateful to Prof. Peter Hodgson, Director, Institute
for Frontier Materials, Deakin University for supporting research
work.
References
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Trends in Biotechnology April 2013, Vol. 31, No. 4
Enzymes bound to nanomaterial
NM
Biofuel: ethanol,
butanol, hydrogen
By-products
separaon
Biodiesel
(transesterificaon)
TRENDS in Biotechnology
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