Biotechnology Advances 30 (2012) 512–523

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Potential applications of enzymes immobilized on/in nano materials: A review

Shakeel Ahmed Ansari a, Qayyum Husain b,⁎
a
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India
b
Faculty of Applied Medical Sciences, Jazan University, Jazan, Post Box 2092, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Several new types of carriers and technologies have been implemented in the recent past to improve traditional
Received 23 May 2011 enzyme immobilization which aimed to enhance enzyme loading, activity and stability to decrease the enzyme
Received in revised form 26 August 2011 biocatalyst cost in industrial biotechnology. These include cross-linked enzyme aggregates, microwave-assisted
Accepted 12 September 2011
immobilization, click chemistry technology, mesoporous supports and most recently nanoparticle-based immo-
Available online 17 September 2011
bilization of enzymes. The union of the specific physical, chemical, optical and electrical properties of nanoparti-
Keywords:
cles with the specific recognition or catalytic properties of biomolecules has led to their appearance in myriad
Nanoparticles novel biotechnological applications. They have been applied time and again for immobilization of industrially
Immobilization important enzymes with improved characteristics. The high surface-to-volume ratio offered by nanoparticles
Biosensors resulted in the concentration of the immobilized entity being considerably higher than that afforded by experi-
Biomedical applications mental protocols based on immobilization on planar 2-D surfaces. Enzymes immobilized on nanoparticles
showed a broader working pH and temperature range and higher thermal stability than the native enzymes.
Compared with the conventional immobilization methods, nanoparticle based immobilization served three im-
portant features; (i) nano-enzyme particles are easy to synthesize in high solid content without using surfactants
and toxic reagents, (ii) homogeneous and well defined core–shell nanoparticles with a thick enzyme shell can be
obtained, and (iii) particle size can be conveniently tailored within utility limits. In addition, with the growing
attention paid to cascade enzymatic reaction and in vitro synthetic biology, it is possible that co-immobilization
of multi-enzymes could be achieved on these nanoparticles.
© 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
2. Magnetic nanoparticles in enzyme immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
3. Enzyme immobilization on novel nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
4. Enzyme immobilization on nonmagnetic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
5. Nanoparticles-based enzymes in biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
6. Nanobiocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
7. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521

Abbreviations: GOX, glucose oxidase; NSP, nickel-impregnated silica paramagnetic
particles; EI-NSP, enzyme immobilized on NSP; HRP, horseradish peroxidase; ITO, indi-
um tin oxide; GOS, galacto-oligosaccharides; CGTase, cyclodextrin glycosyl transferase;
SiMNs, silica-coated magnetic nanoparticles; PAN-co-MMA, poly-acrylonitrile-co-
methyl-methacrylate; PEG, polyethylene glycol; PEI, polyethyleneimine; PAZO, poly
[1-[4-(3-carboxy-4-hydroxyphenylazo) benzenesulfonamido]-1,2-ethanediyl, sodium
salt].
⁎ Corresponding author. Tel.: + 966 595021577(Mobile).
E-mail address: qayyumbiochem@gmail.com (Q. Husain).
1. Introduction
Recent interest in nanotechnology has provided a wealth of diverse
nanoscaffolds that could potentially support enzyme immobilization
due to their potential applications in biotechnology, immunosensing
and biomedical areas. Immobilization of enzymes is advantageous for
commercial application due to convenience in handling, ease of separa-
tion of enzymes from the reaction mixture and reuse, low product cost
and a possible increase in thermal and pH stability (Husain, 2010; Lei et

0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.09.005
 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523 513

al., 2002; Tischer and Wedekind, 1999; Wang, 2006). An important re-
quirement for protein immobilization is that the matrix should provide
a biocompatible and inert environment, i.e. it should not interfere with
the native structure of the protein, which thereby could compromise its
biological activity (Mitchell et al., 2002).
Enzymes immobilized to nanosized scaffolds such as spheres, fibers
and tubes have recently been reported (Kim et al., 2006a, b; Martin
and Kohli, 2003; Yim et al., 2003). Consequently, a number of processes
for enzyme immobilization in silica nanotubes (Mitchell et al., 2002),
within phospholipid bilayers (Hamachi et al., 1994), on self-assembled
monolayers (Hung et al., 2006), in Langmuir–Blodgett films (Troitsky
et al., 2003), within a polymer matrix (Kim et al., 2005a; Kim et al.,
2005b), galleries of R-zirconium phosphate (Kumar and McLendon,
1997), mesoporous materials (Lei et al., 2006), on polystyrene latex par-
ticles (Caruso and Mohwald, 1999), gold nanoparticles assembled on
polymers and zeolite (Bhat et al., 2003; Phadtare et al., 2003) and in ther-
mally evaporated fatty lipid films (Sastry et al., 2002) have been devel-
oped, each with its characteristic pros and cons. Moreover, polymer
microspheres and various metal nanoparticles have also been successful-
ly conjugated with proteins and enzymes (Schuler and Caruso, 2000;
Willner et al., 2006). The surface modification of nanoparticles via en-
zyme molecules and biomembrane probes provided unique biofunction-
ality to them. Qhobosheane et al. (2001) have immobilized lactate
dehydrogenase (LDH) and glutamate dehydrogenase onto the silica
nanoparticles in order to modify them biochemically. The immobilized
preparations showed excellent enzymatic activities and detection capa-
bility. They used dye-doped silica nanoparticles as biomarkers for cell
staining to demonstrate their bioapplicability in several biochemical
and biotechnological applications. Thus, these approaches demonstrated
the feasibility of utilizing such nanoparticles in biosensing and biomark-
ing applications.
The premise of using nanoscale structures for immobilization is to
reduce diffusion limitations and maximize the functional surface area
to increase enzyme loading (Xie et al., 2009). In addition, the physical
characteristics of nanoparticles such as enhanced diffusion and parti-
cle mobility can impact inherent catalytic activity of attached en-
zymes (Jia et al., 2003). Thermal stability, increased surface area and
irradiation resistance are the other advantages that expedite their po-
tential applications in photodetectors, solar cells, biosensors, nano-
generators and ceramics (Moghaddam et al., 2009). In the last few
years, several types of nanoparticles have been extensively employed
in the production of various nanostructures like nanorods, nanotubes,
nanowires, nanorings etc. (Ali and Winterer, 2010; Ni et al., 2007).
Amongst all, biosystems and its components have a remarkable im-
pact as biotechnology deals with nanometer size molecules such as
protein and nucleic acids. Several bio-nano processes have been de-
veloped using fabricated nanoscale structures with biomolecules as
nanoblocks (Yamashita, 2001).
2. Magnetic nanoparticles in enzyme immobilization
Magnetic fields have been utilized in support systems to study
enzyme immobilization (Bayramoglu et al., 2008; Gupta and
Gupta, 2005; Kuroiwa et al., 2008; Pimentel et al., 2007; Selim et
al., 2007). Several magnetic particles and magnetic supports such
as microspheres of various biomaterials encapsulating the magnetic
particles and copolymers with magnetic particles have been used
with good results (Dyal et al., 2003; Koneracka et al., 1999; Kouassi
et al., 2005; Saiyed et al., 2003). The high surface to volume ratio pro-
vided by the magnetic nanoparticles favors high binding capacity and
high catalytic specificity of the conjugated enzyme (Johnson et al.,
2008; Konwarh et al., 2009). In addition, magnetic field susceptibility

Table 1
Enzymes immobilized on magnetic nanoparticles and their biotechnological applications.

Enzyme Nanoparticle utilized Applications Kinetic parameters References

Cholesterol oxidase Fe3O4 Analysis of total Free enzyme: Ea = Kouassi et al., 2005
nanoparticles cholesterol 13.6 kJ/mol;
in serum immobilized
enzyme: Ea = 9.3
kJ/mol
Haloalkane dehalogenase Silica coated Production of – Johnson et al., 2008
iron oxide fusion proteins
nanoparticle containing
dehalogenase
sequences
Laccase Chitosan-magnetic nanoparticle Bioremediation Free enzyme: Km Kalkan et al., 2009
of environmental (10−2 mM) = 5.69,
pollutants Vmax (10−2 mM min−1) = 7.70;
immobilized enzyme:
Km (10−2 mM) = 10.69, Vmax
(10−2 mM min−1) = 14.00
Keratinase Fe3O4 Synthesis of keratin Soluble enzyme: Konwarh et al., 2009
nanoparticle specific activity = 37 U mg− 1;
immobilized enzyme:
specific activity = 128.97 U mg− 1
α-Amylase Cellulose-coated magnetite nanoparticles Starch degradation Immobilized enzyme: Namdeo and Bajpai, 2009
Km = 7.5 × 10− 7 μmol ml− 1, rmax
(maximum degradation rate)
= 0.04 × 10− 6 μmol− 1 g− 1 min− 1
β Galactosidase Con A layered ZnO nanoparticles Lactose hydrolysis Free enzyme: Ansari and Husain, 2011a
Km = 2.38 mM,
Vmax = 0.520 mM min− 1;
immobilized enzyme:
Km = 5.88 mM,
Vmax = 0.460 mM min−1
Lipase Fe3O4 nanoparticles Hydrolysis of pNPP Immobilized enzyme: Huang et al., 2003
Ea = 6.4 kJ mol−1,
specific activity = 1.07 μmol min−1
mg, Km = 0.4 mM
514 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523
revealed a mechanism for efficient recovery of the enzyme complex
thereby preventing the enzyme contamination of the final product. En-
zyme stability is maximized with nanoscaled support with the addition-
al advantages of possible modulation of the catalytic specificity, lower
transfer resistance to solve the diffusion problem and lower operational
cost (Bruno et al., 2005). Table 1 lists various magnetic nanoparticles
used for the immobilization of enzymes and their biotechnological
applications.
Entrapment of enzymes has also been performed by using iron–silica
composites because of its better biocompatibility (Besanger et al., 2003).
Prakasham et al. (2010) utilized nickel-impregnated silica paramagnetic
particles (NSP) as biocatalyst immobilization matrices. A greater catalytic
behavior of enzyme immobilized on NSP (EI-NSP) compared to its solu-
ble counterpart revealed that immobilization of an enzyme on these NSP
protects its deactivation at higher and lower pH. Moreover, the improved
enzyme activity might be attributed to the fact that enzyme configura-
tion was altered under an immobilized environment. Such a variation
of improved activity under immobilized conditions has been observed
with other microbial systems and enzymes (Ramkrishna and Prakasham,
1999; Srinivasulu et al., 2002). In addition, immobilization of diastase on
NSP was evaluated for starch hydrolysis at different temperatures rang-
ing from 30 to 60 °C at pH 5.0. EI-NSP showed improved starch hydroly-
sis activity compared to a free enzyme at all investigated temperatures
and it showed enhanced thermostability as a result of immobilization
(Prakasham et al., 2007).
Kim et al. (2009) coated magnetic particles (10 nm) with silica (di-
ameter of 40 nm) and charged them with Cu 2+ ions via a multidentate
ligand, imino-diacetic acid for the immobilization of His-tagged Bacillus
stearothermopilus L1 lipase. Microporous silica gel with a mean particle
diameter of 115 μm was used as a comparative support material. The
molar ratio of Cu2+ to imino-diacetic acid was found to be 1:1.14 and
1:1.99 in the silica gel and the silica-coated magnetic nanoparticles
(SiMNs), respectively. The specific activity of the immobilized enzyme
exhibited the following order: Cu2+-charged SiMN N SiMNN Cu2+-
charged silica gel N silica gel. Lipase immobilized on Cu2+charged
SiMNs retained 70% activity after its 5th repeated use while other
immobilized preparations lost considerably very high activity under
similar experimental conditions. Similarly, keratinase from Bacillus
subtilis immobilized onto polyethylene glycol-supported Fe3O4 super-
paramagnetic nanoparticles showed a marked increase in stability
against various types of physical and chemical denaturants as compared
to its soluble counterpart (Konwarh et al., 2009).
Levison et al. (1998) prepared Fe2O3 nanoparticles by sonication
of Fe (CO) in decalin and then annealed amorphous Fe2O3 nanoparti-
cles for their subsequent use in immobilizing industrial enzymes.
Several investigators have earlier reported an improvement in the
stability of Candida rugosa lipase when immobilized on Fe2O3 mag-
netic nanoparticles. This system offered a relatively simple technique
for separating and reusing enzymes over a longer period than that for
free enzymes alone and for enzymes immobilized by physisorption.
The separation of an immobilized enzyme was facilitated by using a
magnet where either the substrate solution was removed while the
immobilized enzyme was held in place with a magnetic field or vice
versa (Dyal et al., 2003; Gardimalla et al., 2005). In another study, C.
rugosa lipase (CRL) was immobilized on novel nanosupport, phos-
phorous-containing polyurethanes. Pre-treatment of the support
with polar or non-polar solvents had a negative impact on lipase
loading. Non-ionic surfactants increased the immobilization yield of
CRL by 30–40%. Immobilization has improved the thermal stability
of enzyme and exhibited a half-life of 70 h at 55 °C. The highest syn-
thetic activity of CRL-polyurethane preparations were observed at
aw = 0.33. The biocatalyst retained 80% activity after fifteen subse-
quent five-hour cycles when utilized in esterification of palmitic
acid with cetyl alcohol (Guncheva et al., 2011).
Johnson et al. (2008) demonstrated the feasibility of specifically
attaching haloalkane dehalogenase to silica-coated or uncoated iron
oxide superparamagnetic nanoparticles using affinity peptides. The
enzyme was cloned from Xanthobacter autotrophicus strain GJ10
into Escherichia coli to produce fusion proteins containing dehalogen-
ase sequences with C-terminal polypeptide repeats that have a specif-
ic affinity for either silica or iron oxide. Researchers are employing
current technologies and developing new applications that utilize en-
zymes immobilized on nanoparticles. Many enzymes currently used
in biotechnology, including glucose oxidase and peroxidase, have
been covalently immobilized to magnetic nanoparticles (MNPs)
using several different ligands. Glucose oxidase that was used to mea-
sure blood glucose was covalently immobilized on MNPs. It was
found to be stable over a wide range of pH and temperature range
and retained significant activity for 3 months (Rossi et al., 2004). Li-
pase, which is important for processing lipids in food and pharmaceu-
tical industries, has been covalently immobilized on nanoparticles by
a carbodiimide linkage and exhibited significant activity after
1 month of storage (Dyal et al., 2003). Trypsin and chymotrypsin
have also been stabilized against denaturation and self-digestion at
the air–water interface by immobilization on iron and gold nanopar-
ticles (Hansen et al., 2006; Jordan et al., 2006). Streptokinase, which
has been used as a therapeutic agent, was covalently immobilized to
MNPs by carbodiimide linkage for use in localized lysis of blood
clots in vivo. The magnetic properties of the particle-streptokinase
congener would allow focusing on treatment at the exact location
where the clot is present, thus reducing the amount of enzyme re-
quired and in turn decreasing the risk of eliciting an immune re-
sponse (Fernandes et al., 2006; Koneracka et al., 2002).
Kouassi et al. (2005) successfully bound cholesterol oxidase to
Fe3O4 nanoparticles via carbodiimide activation and confirmed its
binding by FT-IR spectroscopy. The binding efficiency was between
98% and 100% irrespective of the amount of particles used. Kinetic
studies revealed that the stability and activity of the enzyme was sig-
nificantly improved against pH, temperature and substrate concen-
tration upon its binding to nanoparticles in comparison to the free
enzyme. The activation energy for free and bound cholesterol oxidase
was 13.6 and 9.3 kJ mol −1, respectively. The study demonstrated that
the stability and activity of this enzyme could be enhanced via attach-
ment to the magnetic nanoparticles, which could henceforth contrib-
ute to the better use of enzymes in various biological and clinical
applications.
Pseudomonas cepacia lipase immobilized onto magnetic nanopar-
ticles via carbodiimide activation prepared by co-precipitating ferric
and ferrous chlorides in alkaline solution under hydrothermal condi-
tions showed a binding efficiency of 90% and the corresponding activ-
ity recovery of 70% when the weight ratio of lipase bound to magnetic
nanoparticles was 0.09. The thermal stability was significantly im-
proved after immobilization thereby attracting its use in the produc-
tion of biodiesel (Mak et al., 2009). Nevertheless, organic–inorganic
complex technology was exploited to prepare zinc tetra-amino-
phthalocyanine-Fe3O4 nanoparticle composites. Laccase bound to
such support via glutaraldehyde by the active amino groups of mag-
netic carriers showed immobilization yields and Km value of 25%
and 20.1 μM, respectively. Immobilized laccase had good thermal,
storage and operational stability, which could be used as the sensing
biocomponent for the fiber optic biosensor based on enzyme catalysis
(Huang et al., 2007).
A novel and efficient immobilization of β galactosidase from
Aspergillus oryzae was recently developed by using magnetic Fe3O4-
chitosan nanoparticles as support. The magnetic Fe3O4-chitosan
nanoparticles were prepared by electrostatic adsorption of chitosan
onto the surface of Fe3O4 nanoparticles made through co-precipita-
tion of Fe 2+ and Fe 3+. β Galactosidase was covalently immobilized
onto the nanocomposites using glutaraldehyde as activating agent.
The immobilization process was optimized by examining immobi-
lized time, crosslinking time, enzyme concentration, glutaraldehyde
concentration, and initial pH values of glutaraldehyde and enzyme
 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523
solution. As a result, the immobilized enzyme presented a higher
storage, pH and thermal stability than the soluble enzyme. Galacto-
oligosaccharide (GOS) was formed with lactose as substrate by
using the immobilized enzyme as biocatalyst with a maximum yield
of 15.5% (w/v) obtained when 50% lactose was hydrolyzed (Pan et
al., 2009).
Recently, cellulosic ethanol has received a lot of attention as an al-
ternative transportation fuel to reduce global dependence on oil.
However, due to high cost of enzymes, the process is not currently
economically feasible. Cellulase multi-enzyme mixtures along with
extra β glucosidase are the key enzymes for biomass degradation.
Glucose oxidase (GOX) was selected as a model enzyme to demon-
strate the immobilization strategies. Three different size magnetic
nanoparticles (5 nm, 25 nm and 50 nm) were fabricated to explore
the effect of various particle sizes on diffusion efficiency. Two differ-
ent methods, co-precipitation and oxidation of Fe (OH)2 were used
to fabricate different sizes of magnetic nanoparticles functionalized
with amine groups by 3-(amino propyl) triethoxysilane. Glutaralde-
hyde was used as a crosslinking agent between functionalized mag-
netic nanoparticles and GOX. It was found that activity of GOX
immobilized on different sized magnetic nanoparticles retained
marked activity after 40 day storage at various temperatures. Trans-
mission electron micrograph (TEM) showed that the morphology of
magnetic nanoparticles was spherical and the sizes agreed with re-
sults obtained from those of a Brunauer, Emmett, Teller method.
The magnetic strength of nanoparticles was analyzed via a physical
property measurement system. X-ray photoelectron spectroscopy con-
firmed each step of the magnetic nanoparticle surface modification and
successful enzyme immobilization. Recycling stability studies showed
20% loss in activity during 10 consecutive recycles for large (50 nm)
and medium (25 nm) size enzyme magnetic nanoparticles.
Laccase is a potentially attractive catalyst for bioremediation of envi-
ronmental pollutants especially organic substances such as chlorophe-
nols and aromatic hydrocarbons of petrochemical industry, toxins of
olive oil, mill wastewater, de-lignification in paper industry and dye de-
colorization in textile industry. Laccase from Trametes versicolor immo-
bilized onto chitosan coated magnetite nanoparticles by using reversed-
phase suspension adsorption retained about 71% of its initial activity at
the end of its 30th repeated use (Kalkan et al., 2009).
α Amylase immobilized on Fe2O3 nanoparticle had significantly
greater thermal, storage and operational stability as compared to its
free counterpart. Both the soluble and immobilized enzymes were
characterized by Fourier transform infrared spectroscopy (FT-IR),
atomic force microscopy (AFM), thermogravimetric analysis (TGA)
and differential thermal analysis (DTA). The results for the continu-
ous hydrolysis of starch in a batch process suggested that α amylase
immobilized on Fe2O3 nanoparticle might be a good candidate for
the hydrolysis of starch and other polysaccharides used in food indus-
tries (Khan et al., Communicated). Recently, catalytic removal of
bisphenol A was investigated with hemoglobin immobilized on
amino-modified magnetic nanoparticles (Tang et al., 2011). The
amino-modified magnetite nanoparticles were firstly prepared by
the co-precipitation of Fe 2+ and Fe 3+ with NH3·H2O and then modi-
fied by 3-aminopropyltriethoxysilane. It was observed that immobi-
lized hemoglobin could remove bisphenol A up to 80.3%. The
immobilization had a beneficial effect on the stability of hemoglobin
and conversions of bisphenol A.
Rapid immobilization with one-pot purification of galactitol dehy-
drogenase (GatDH) and formate dehydrogenase (FDH) was achieved
by using iminodiacetic acid with chelated Co2+ modified magnetic
nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombi-
nant E. coli and FDH commencing Candida methylica were used as an
auxiliary enzyme for the regeneration of NADH/NAD +. The affinity
magnetic nanoparticles were characterized by scanning electron mi-
croscopy (SEM) and FTIR while the purity of GatDH and FDH was
assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
515
(SDS-PAGE). An immobilized two-enzyme system showed optimal activ-
ity at pH 7.0 and 8.0 for (S)-1,2-propanediol and L-tagatose production,
respectively. It retained 70% activity after 1 week of repeated use. The
use of affinity magnetic nanoparticles offers the advantage of a one-pot
purification of His(6)-tagged GatDH and FDH followed by the production
of rare sugar and chiral diol (Demira et al., 2011).
3. Enzyme immobilization on novel nanoparticles
Gold nanoparticles serve as excellent biocompatible surfaces for
the immobilization of enzymes and proteins since the interaction be-
tween amino and cysteine groups of proteins with gold nanoparticles
is as strong as that of the commonly used thiols. Thus, amino acids
and proteins may be directly immobilized on gold nanoparticles
without any modification (Crespilho et al., 2006; Lia et al., 2010; Xu
et al., 2007). Gold nanoparticles can be easily and efficiently functio-
nalized with thiolated molecules and carboxylic groups, which in
turn, are conjugated with amino groups of the proteins (Jun et al.,
2001; Vertegel et al., 2004). Enzyme immobilized on polyurethane
microsphere–gold nanoparticles has shown enhanced storage and
thermal stability (Kumar et al., 2003). Phadtare et al. (2004) have in-
vestigated the assembly of gold nanoparticles on the surface of the
amine-functionalized zeolite microspheres for the formation of zeo-
lite-gold nanoparticle “core–shell” structures and its use in immobi-
lizing fungal protease. The assembly of gold nanoparticles on the
zeolite surface occurs through the amino groups present in 3-amino-
propyltrimethoxysilane. The fungal proteases bound to the massive
“core–shell” structures were easily separated from the reaction medium
by mild centrifugation and exhibited excellent reuse characteristics. The
biocatalytic activity of fungal protease in bioconjugate was marginally
enhanced relative to the free enzyme in solution. It has also shown an
enhancement in pH and temperature stability and a shift in the temper-
ature-optima.
Yang et al. (2006) have studied multilayer films of GOX on Au-
nanoparticle electrode surface using cysteamine as a covalent attach-
ment cross-linker. However, the main disadvantage of this method
was the addition of a redox mediator to the reaction medium which
was less efficient than using an immobilized redox mediator. Moreover,
submicron-sized poly (N-isopropyl acrylamide)/polyethyleneimine
core–shell microgels were prepared in aqueous media by using tert-
butyl hydroperoxide as an initiator and then the gold nanoparticles
(8 nm) were formed on the surface of microgels. The amino groups on
the polyethyleneimine (PEI) chains act as the binder for the assembly
of gold nanoparticles/microgel complex. The microgels were highly sta-
ble with gold nanoparticles on their extended PEI chains in aqueous
media, and this multi-scale nanoparticle complex could be recovered
from water and redispersed in water. The nanogold/microgel particles
were further conjugated with the enzymes; horseradish peroxidase
(HRP) and urease. It was found that under identical assay conditions,
the enzyme/nanogold/microgel systems exhibited enhanced biocata-
lytic activity over free enzymes in solution, especially at lower enzyme
concentrations. Moreover, HRP/nanogold/microgel systems showed
higher activity at various pH, temperatures and storage stability as com-
pared to free enzyme (Xu et al., 2007). The interaction of colloidal gold
particles with proteins/enzymes have been employed for the immobili-
zation of pepsin and xanthine oxidase (Gole et al., 2001; Zhao et al.,
1996). The high surface area of host gold nanoparticles renders the
immobilized enzyme “quasi free” and at the same time retained the ad-
vantages of immobilization such as ease in reusing, an enhanced tempo-
ral and thermal stability, etc.
Crespilho et al. (2009) demonstrated an extremely promising ap-
proach for constructing enzyme biosensors. They immobilized urease
on electroactive nanostructured membranes made up of poly-aniline
and silver nanoparticles (AgNP) stabilized in polyvinyl alcohol
(PAni/PVA-AgNP). Fabrication of this modified electrode comprised
chemical deposition of poly-aniline followed by drop-coating of
516 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523
PVA-AgNP and urease, resulting in a final ITO/PAni/PVA-AgNP/urease
electrode configuration. Investigation on the performance of this
electrode toward urea hydrolysis via amperometric measurements
revealed a fast increase in cathodic current with a well-defined peak
upon addition of urea to the electrolytic solution. A modified elec-
trode with an immobilized enzyme promoted an efficient conversion
of urea into ammonium and bicarbonate ions. Km app
of 2.7 mM L − 1 in-
dicated that the electrode architecture employed might be advanta-
geous for fabrication of enzymatic devices with improved biocatalytic
properties. This applied potential ensures minimization of interference
effects when the biosensor was used in real and complex matrices such
as biological media, food and beverages. The measurements carried out
with H2O2 and glucose demonstrated that this new approach was ex-
tremely promising for the construction of enzyme biosensors.
GOX was covalently immobilized on thiolated gold nanoparticles
via N-ethyl-N′-(3-dimethyl-aminopropyl) carbodiimide and N-
hydroxysuccinimide. Its binding was confirmed by UV–visible, FT-
IR, CD and TEM studies. The immobilized enzyme exhibited a re-
sponse time of 30 s, a shelf life of more than 6 months and improved
tolerance to both pH and temperature. Lower Km (3.74 mg dL −1) of
immobilized GOX showed enhanced activity which indicated a fa-
vorable conformational change in the GOX structure upon attach-
ment to thiolated gold nanoparticles. These GOX thiolated gold
nanoparticles were used for estimating glucose up to 300 mg dL -1.
The result showed that these biocompatible thiol-modified gold
nanoparticles might serve as a promising matrix for the immobiliza-
tion of other enzymes and proteins with enhanced stability and ac-
tivity for biosensor applications (Pandey et al., 2007). Recently,
gold nanoparticles were used to immobilize acetylcholinesterase.
AFM demonstrated that gold nanoparticles enhanced interaction of
the enzyme with the gold electrode. The modified electrode was
exploited for electrochemical detection of thiocholine at the gold
surface after hydrolyzing acetylthiocholine by the immobilized enzyme.
The sensor response to acetylthiocholine was significantly reduced and
the utility of the electrode was limited in the absence of nanoparticle
layer. The ability of the nanoparticle-based sensor to reliably measure
concentrations of the organophosphate pesticide carbofuran at lesser
concentrations was demonstrated by monitoring the inhibition of hy-
drolysis of acetylthiocholine.
Mahmoud et al. (2009) have prepared a novel nanocomposite
consisting of cellulose nanocrystals functionalized with gold nanopar-
ticles. Cyclodextrin glycosyl transferase (CGTase) and alcohol oxidase
conjugated on an activated cellulose nanocrystal/gold nanoparticle
matrix exhibited a significant biocatalytic activity with excellent en-
zyme stability and without apparent original activity loss. The recov-
ered specific activities were 70% and 95% for CGTase and alcohol
oxidase, respectively. Similarly, bioconjugates of an enzyme and
gold nanorods by fabrication via electrostatic interactions between
anionic GOX and positively charged gold nanorods were constructed.
The assembled processes were monitored by UV–vis spectra, zeta po-
tential measurements and transmission electron microscopy (TEM).
The enzyme activity of the obtained bioconjugates displayed relative-
ly enhanced thermostability behavior in contrast with that of free en-
zyme. Free GOX in solution had lost 78% of its relative activity at 90 °C
while enzyme immobilized on gold nanorods retained about 39.3%
activity under similar incubation conditions (Ma and Ding, 2009).
Nanosized dispersions of platinum have gained considerable at-
tention because of their size-dependent electrical, chemical and opti-
cal properties. The catalytic and electrochemical activity of highly
dispersed platinum nanoparticles has been the focus of keen interest
in the fabrication of amperometric biosensors (Genies et al., 1998; Li
et al., 2007; Park and Cheon, 2001; You et al., 2003). Platinum nano-
particle-doped sol–gel solution was used as a binder for multi-walled
carbon nanotubes (CNT) for the fabrication of electrochemical sen-
sors (Yang et al., 2006). The resulting CNT-silicate material brought
new capabilities for electrochemical devices by using synergistic
action of electrocatalytic activity of platinum nanoparticles and CNT.
The combined electrocatalytic activity permitted low-potential detec-
tion of H2O2 with remarkably improved sensitivity. Platinum-CNT
based biosensor responded more sensitively to glucose than CNT-
based biosensor. The sensitivity of platinum-CNT based biosensor
was four times greater than that of CNT-based biosensor. Similarly,
Zhao et al. (2007) have demonstrated a sensitive amperometric glu-
cose biosensor based on platinum nanoparticles and CNTs electrode.
Platinum-nanoparticles enhanced the electrocatalytic activity of elec-
trode for electro-oxidating H2O2. GOX electrode exhibited good re-
producibility and excellent response performance to glucose with
linear range from 1 × 10 −5 to 7 × 10 −3 mol L −1 and fast response
time within 5 s.
4. Enzyme immobilization on nonmagnetic nanoparticles
Nanoscaled materials provide upper limits in balancing the key
factors that determine the efficiency of biocatalysts including surface
area, mass transfer resistance and effective enzyme loading. The ex-
ploitation of various nanomaterials such as nanoparticles, nanofibers,
nanotubes and nanoporous matrices for biotechnological applications
has been excellently reviewed by Wang (2006). Beyond their high
surface area to volume ratios, nanoscale biocatalyst systems exhibit
unique behaviors that distinguish them from traditional immobilized
systems and extend their remarkable applications in engineering of
biomolecular catalysis (Jia et al., 2011; Kim et al., 2011).
Several workers have developed a one-step method for preparing
cellulase-immobilized on nanoparticles that consisted of well-defined
poly-methyl methacrylate (PMMA) cores and cellulase shells (Ho et
al., 2008; Kin et al., 2008). Immobilized cellulase showed improved
thermostability and retained significantly very high activity at
broader pH range as compared to soluble counterpart. These nano-
particles have thick and evenly distributed enzyme shells and this
preparation can be produced in high concentrations of up to 18%
(w/w) solid content. Thus, this method might provide a new com-
mercially viable route to the immobilization of thermally stable en-
zyme to form nanoparticle enzyme complexes (Ho et al., 2008).
Table 2 illustrates some common nanoparticles that have been
employed in the recent past for immobilizing enzymes of major in-
dustrial applications.
Eldin et al. (2010) prepared copolymers of poly-acrylonitrile-
co-methyl methacrylate (PAN-co-MMA) nanospheres using a
precipitation–polymerization technique and ethylenediamine by
covalent attachment with carbonyl groups of PAN-co-MMA nano-
particles. This preparation was used for the immobilization of
β galactosidase using glutaraldehyde as a coupling agent. The catalytic
activity of the immobilized β galactosidase was 22.35 μM min−1 g−1
of nanospheres. Operational, thermal and storage stabilities were
found to increase upon immobilization. Different factors affecting the
modification and activation processes were studied and the resultant
activated nanospheres were characterized by FT-IR, TGA SEM. Polyure-
thane powder from hexamethylene diisocyanate and butanediol has
also been exploited to immobilize Penicillium canescens β galactosidase
by covalent binding between the amino groups of enzyme and the iso-
cyanate groups in polyurethane powder (Budriene et al., 2005). Candida
antarctica lipase B is one of the most recognized biocatalysts used in a
broad range of synthetic applications of industrial importance including
kinetic resolutions, aminolysis, esterification and trans-esterification
(Anderson et al., 1998; Ghanem, 2008; Schmid and Verger, 1998).
Several workers have reported increased utilization of immobilized
enzyme in chemical transformations and polymerization reactions
(Kumar et al., 2000; Mahapatro et al., 2004). Miletic et al. (2010) have
immobilized lipase on polystyrene nanoparticles and found that the
activity of immobilized enzyme was remarkably improved as compared
to the free enzyme.
 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523
Table 2
Enzymes immobilized on non magnetic nanoparticles and their applications.
Enzyme Nanoparticle used
Glucose oxidase Thiolated gold nanoparticle
Lipase Polystyrene nanoparticle
α-Chymotrypsin Polystyrene nanoparticles
β galactosidase POS-PVA
CGTase + alcohol oxidase Activated cellulose nanocrystal/gold
nanoparticle matrix
Diastase Silica coated nickel nanoparticle
α-Chymotrypsin and lipase were immobilized in hierarchically-
ordered mesocellular mesoporous silica (HMMS) in a simple and ef-
fective way by enzyme adsorption followed by glutaraldehyde cross-
linking. It resulted in the formation of nanometer scale crosslinked
enzyme aggregates (CLEAs) entrapped in the mesocellular pores of
HMMS (37 nm) which did not leach out of HMMS through narrow
mesoporous channels (13 nm). CLEA of α-chymotrypsin (CLEA-CT)
in HMMS showed high enzyme loading capacity and significantly in-
creased enzyme stability even after 2 weeks under a rigorously shak-
ing condition, while the free enzyme demonstrated a rapid
inactivation due to the enzyme leaching. Moreover, CLEA of lipase
in HMMS retained 30% specific activity with greatly enhanced stabil-
ity (Kim et al., 2007).
Immobilization of enzymes and proteins on activated supports
permitted simplification of the reactor design and may be used to im-
prove some enzyme properties (Gaberc-Porekar and Menart, 2001;
Iyer and Ananthnarayan, 2008; Mateo et al., 2007). Supports contain-
ing epoxy groups proved useful to generate intense multipoint cova-
lent attachment with different nucleophiles placed on the surface of
enzyme molecules (e.g., amino, thiol, hydroxyl groups). However,
the intermolecular reaction between epoxy groups and soluble en-
zymes was extremely slow. Here, enzyme immobilization was per-
formed via a two-step process: (i) an initial physical or chemical
intermolecular interaction of the enzyme surface with the new func-
tional groups introduced on the support surface, and (ii) a subsequent
intense intramolecular multipoint covalent reaction between the nu-
cleophiles of the immobilized enzyme and the epoxy groups of the
supports. The first immobilization might involve different enzyme re-
gions which were further rigidified by multipoint covalent attach-
ment (Mateo et al., 2007).
Liu et al. (2009) reported covalent attachment of NAD(H) to silica
nanoparticles and found successful coordination of particle-immobi-
lized enzymes which enabled multistep biotransformation. Moreover,
silica nanoparticle-attached glutamate dehydrogenase, LDH and
NAD(H) were prepared and applied to catalyze the coupled reactions
for production of α-ketoglutarate and lactate with the cofactor regener-
ated within the reaction cycle. The use of particle-attached cofactor
promises a new biochemical processing strategy for cofactor-depen-
dent biotransformation.
In another study, multistep reactions catalyzed by a covalently
immobilized enzyme−cofactor−enzyme system were achieved (El-
Zahab et al., 2004). LDH, glucose dehydrogenase (GDH) and cofactor
517
Applications Kinetic parameters References
Estimation of glucose level Immobilized enzyme: Pandey et al., 2007
up to 300 mg mL−1 Km = 3.74 mM, soluble
enzyme = 5.85 mM
Aminolysis, esterification, – Miletic et al., 2010
trans-esterification
Proteolysis (cleaves peptide Immobilized enzyme: Jia et al., 2003
amide bonds) Km = 31.7 μM, kcat = 20.0 s−1;
soluble enzyme:
Km = 47.8 μM, kcat = 17.8 s−1
GOS synthesis Immobilized enzyme: Neri et al., 2009
k1 = 1.41 h−1;
soluble enzyme: k1 = 1.16 h−1
Enhanced enzyme _ Mahmoud et al., 2009
loading and stability
Starch hydrolysis Immobilized enzyme: Prakasham et al.,
Km = 8,414 mM, 2007
Vmax = 4.92 μM min−1 mg−1;
soluble enzyme:
Km = 10,176 mM,
Vmax = 2.71 μM min−1 mg−1
NADH were incorporated into two porous silica glass supports
(30 nm and 100 nm pore size) to obtain effective shuttling of cova-
lently bound NADH between LDH and GDH. It was observed that
the glass of 30 nm pore size afforded enzyme activities which were
two-folds as observed for the glass of 100 nm pore size thereby indi-
cating that the former provided better enzyme−cofactor integration.
A similar multienzyme system immobilized on nonporous polysty-
rene particles of 500 nm diameter was only 2% active as the glass-
supported system. It is believed that nanoporous structure of the
glass supports enhanced molecular interactions among the immobi-
lized enzymes and cofactor, thus improving the catalytic efficiency
of the system. Applications of such catalysts in industrial chemical
processing may provide efficient in situ cofactor regeneration, easy
catalyst/cofactor reuse and simple product purification.
Synthesis of GOS by A. oryzae β galactosidase immobilized on POS-
PVA was reported by Neri et al. (2008). A maximum GOS concentration
of 26% (w/v) of total sugars was achieved at 55% lactose conversion
from 50% (w/v) lactose solution at pH 4.5 and 40 °C. Trisaccharides
accounted for more than 81% of the total GOS produced. GOS formation
was not considerably affected by pH and temperature. Its formation was
unaffected by enzyme immobilization in the POS-PVA matrix, thus indi-
cating the absence of diffusional limitations in the enzyme carrier. Fur-
thermore, this immobilized derivative could be reused 10 times with
the retainment of nearly 84% of the initial activity. Wan et al. (2007)
have exploited electrospun nanofibers bearing porphyrin pendants to
covalently immobilize catalase thereby improving the activity and sta-
bility of the immobilized enzyme. The facilitation of electron transfer
between the immobilized enzyme and the ability of porphyrin pen-
dants to retain the active conformation of the catalase might be respon-
sible for the improvement in its activity and stability.
Hamlin et al. (2007) have reported a successful immobilization and
improved stabilization of β galactosidase on polyelectrolyte surfaces. β
Galactosidase was covalently immobilized in a channel of a borosilicate
glass microchip by using channel specific coating technology (Xiong
and Regneir, 2001). Immobilization of β galactosidase onto polyelectro-
lyte multilayer assemblies of the polyanion, poly [1-[4-(3-carboxy-4-
hydroxyphenylazo) benzenesulfonamido]-1,2-ethanediyl, sodium salt]
(PAZO) and the polycation, PEI constructed by electrostatic self-assem-
bly has been monitored. A single layer of β galactosidase was deposited
over a precursor film comprising up to five bilayers of the PEI/PAZO
polyelectrolyte pair. The enzyme was deposited on both PEI and PAZO
surfaces.
518 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523
Quartz crystal microbalance with dissipation monitoring, single-
wavelength ellipsometry and UV–visible absorption spectroscopy
revealed differences in both the amount of β galactosidase incorpo-
rated in each of the multilayer assemblies and the resulting enzyme
packing density in the films. The enzymatic films were immersed in
a reaction solution containing o-nitrophenyl β-D-galactopyranoside
and absorbance measurements were used to monitor the concentra-
tion of o-nitrophenol. Although the data indicated that a comparable
amount of β galactosidase was incorporated onto both surfaces, enzy-
matic activity was substantially inhibited when β galactosidase was
immobilized on the polyanionic surface compared to the enzyme on
the polycationic surface. The difference in catalytic activities reflects
the different abilities of two polyelectrolytes to screen the protein ac-
tive site from the substrate environment. In both assemblies, the pro-
tein interpenetrated the PEI/PAZO multilayer, disrupting the J-
aggregated state of the PAZO chromophores. This work demonstrated
that the charge, conformation and composition of underlying poly-
electrolyte cushions have a significant effect on the structure and
function of an immobilized protein within functional nanoassemblies
(Hamlin et al., 2007). Chitosan, modified by linolenic acid and 1-
ethyl-3-(3-dimethylaminopropyyl)-carbodiimide coupling displayed
improved enzyme loading capacity (0.04 to 37.57%) with increasing
BSA concentration (Liu et al., 2005).
Daubresse et al. (1996) reported immobilization of alkaline phos-
phatase by physical entrapment within colloidal particles produced
by inverse micro-emulsion polymerization. Functionality was inde-
pendently imparted to the nanoparticle surface by copolymerization
of acrylamide, N, N′ methylene-bis-acrylamide with N-acryloyl-1,6-
diaminohexane and acrylic acid. They studied the effect of functional
co-monomers on the size and zeta potential of the reactive latexes.
Integrity of the immobilized enzyme was ascertained from its catalyt-
ic activity towards hydrolysis of p-nitrophenylphosphate. This meth-
od also conferred the immobilized enzyme with greater stability
against environmental changes such as pH and temperature, as well
as higher selectivity toward the substrates. Kim et al. (2006a, b)
have demonstrated successful immobilization of M. javanicus lipase
on silica nanoparticles. The attachment of glutaraldehyde and 1,4
phenylene diisothiocyanate onto the ethylene diamine-activated sili-
ca nanoparticles resulted in a remarkable increase in the enzyme
loading capacity and activity. Immobilized lipase retained a high
level of activity over a wider range of pH than the free form. Thermal
stability was considerably enhanced by the immobilization. Since this
nanoparticle-based enzyme immobilization preserves high enzyme
loading and activity with enhanced stability, it can be applied to var-
ious enzymatic applications, such as biosensors and bioremediations.
In the past, Li et al. (2003a, b) have succeeded in coupling pepsin to
alumina in an orientation-specific manner. A comparative analysis
revealed that conjugated pepsin retained higher enzymatic activity
upon immobilization mainly because of the lack of diffusion limita-
tions of the substrate. Additionally, upon attachment to the alumina
nanoparticles, the thermal stability of pepsin was enhanced. Mao et
al. (2006) developed novel core–shell nanoparticles consisting of
poly(methyl-methacrylate) cores coated with synthetic polymer
and other biopolymers shells via direct graft copolymerization of
methyl methacrylate. Much greater enzymatic yield (90%) was
achieved by this novel procedure of enzyme immobilizations. Naik
et al. (2004) demonstrated the entrapment of cobalt–platinum and
cadmium–selenium and zinc sulfide nanoparticles within the silica
matrix. Both free and entrapped cadmium–selenium and zinc sulfide
nanoparticles exhibited similar emission profiles. It was observed that
addition of magnetic nanoparticles to the entrapped enzyme–silica
matrix facilitated easy separation of the entrapped enzyme from the
product. Iron oxide nanoparticles were entrapped along with the en-
zyme in a silica matrix using the bio-silicification reaction which
resulted in the reusability of entrapped enzyme after magnetic separa-
tion. Biomolecules entrapped using this method is widely applicable
in biocatalytic and biosensing applications. Similarly, chitosan nano-
particles were prepared and used as carriers for immobilizing enzyme
(Tang et al., 2007). They choose various methods for preparing chitosan
nanoparticles and the effect of several factors such as the molecular
weight of chitosan, concentration of chitosan, TPP concentration and so-
lution pH on the size of chitosan nanoparticles were studied. The results
showed that solution pH, TPP concentration and chitosan concentration
affected the size of chitosan nanoparticles significantly. The adequacy of
the model equation for predicting the orders of the size of chitosan
nanoparticles was verified effectively by the validation data. The mini-
mum particle size was about 42± 5 nm. The optimal conditions of im-
mobilization were as follows: 1 mg of neutral proteinase was
immobilized on chitosan nanoparticles for about 15 min at 40 °C and
the obtained enzyme activity yield was 84.3%.
A successful immobilization of GOX on the surface of silica nano-
particles (SNPs) was done via physical entrapment within photo-po-
lymerized hydrogels prepared from two different molecular weights
(575 and 8000 Da) of polyethylene glycol (PEG) (Jang et al., 2010).
The hydrogel entrapment resulted in a decrease in reaction rate and
an increase in apparent Km of silica nanoparticle-immobilized GOX,
but these negative effects were minimized by using hydrogel with a
higher MW PEG, which provided higher water content and larger
mesh size. The catalytic rate of the PEG 8000 hydrogel was about
ten times faster than that of the PEG 575 hydrogel because of en-
hanced mass transfer. A long-term stability test demonstrated that
SNP-immobilized GOX entrapped within the hydrogel maintained
more than 60% of its initial activity after a week, whereas non-entrapped
SNP-immobilized GOX and entrapped GOX without SNP immobilization
retained less than 20% of their initial activity. Incorporation of SNPs into
hydrogel enhanced the mechanical strength of the hydrogel six-fold rel-
ative to bare hydrogels. Moreover, a hydrogel microarray entrapping
SNP-immobilized GOX was fabricated using photolithography and suc-
cessfully used for quantitative glucose detection. Nattokinase purified
from Bacillus subtilis using ionic exchange chromatography was success-
fully immobilized upon polyhydroxybutyrate nanoparticles (Deepak et
al., 2009). Immobilization resulted in the enhanced stability with a 20%
increase in enzyme activity. Moreover, the activity was completely
retained for 25 days upon storage at 4 °C. β Galactosidase from Kluyvero-
myces lactis was covalently immobilized onto a mPOS-PVA using glutar-
aldehyde as activating agent. A soluble and immobilized enzyme
displayed the same pH-optimum (6.5) and temperature-optimum
(50 °C). However, the immobilized enzyme showed a marked increase
in stability against various physical and chemical denaturants. The appar-
app
ent Km and activation energy for both soluble and immobilized β galac-
tosidases were evaluated. Immobilized β galactosidase presented higher
operational, greater lactose hydrolyzing activity and thermal stability
than the soluble enzyme.
In the author's laboratory, ZnO nanoparticles (ZnO-NP) were
exploited for the immobilization of A. oryzae β galactosidase by sim-
ple bioaffinity based adsorption. Ansari and Husain (2011a, b) dem-
onstrated a novel and an efficient method for the immobilization of
A. oryzae β galactosidase by using concanavalin A (Con A) layered
zinc oxide nanoparticles as bioaffinity support. AFM analysis showed
that the prepared matrix has an advantageous microenvironment and
large surface area available for binding a significant amount of en-
zyme. Con A layered ZnO-NP retained 84% of the enzyme activity
upon immobilization. Soluble and immobilized β galactosidase exhib-
ited the same pH-optimum at pH 4.5 while the temperature-opti-
mum was increased from 50 °C to 60 °C for the immobilized
enzyme. The free enzyme lost 81% activity when it was exposed at
60 °C for 2 h whereas an immobilized enzyme retained 60% activity
under similar experimental conditions. Michaelis constant, Km was
2.38 mM and 5.88 mM for free and immobilized β galactosidase, re-
spectively. Vmax for the soluble and the immobilized enzyme was
0.520 mM/min and 0.500 mM/min, respectively. The functional
groups present in native and parent compounds were monitored by
 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523
FTIR. Immobilized enzyme retained more than 80% activity even after
its 6th repeated use. In another study, comparative stability studies of
β galactosidase immobilized on native ZnO and ZnO-NP were investi-
gated. The activity yield of immobilization was 85% for the enzyme
adsorbed on ZnO-NP as compared to the enzyme adsorbed on native
ZnO, which retained only 60% β galactosidase upon immobilization.
ZnO-NP adsorbed β galactosidase exhibited greater thermal, storage
and operational stability as compared to ZnO adsorbed β galactosi-
dase. In addition, ZnO-NP adsorbed β galactosidase hydrolyzed a
greater amount of lactose from milk and whey as compared to simply
ZnO adsorbed β galactosidase (Husain et al., 2011).
In the recent past, Al2O3-NPs has been exploited as a potential candi-
date in targeted drug delivery and biosensor application (Frey et al., 1997;
Heilmann et al., 2003) but no study has been carried out which could
strengthen its importance in immobilizing industrially important en-
zymes. Thus, a high yield immobilized preparation was obtained by spe-
cific adsorption of K. lactis β galactosidase on a highly efficient and
selective bioaffinity support, Con A layered Al2O3-NPs. TGA of bioaffinity
support revealed 6% loss in weight at 600 °C whereas its thermal decom-
position was observed at 350 °C by DTA. No significant change was no-
ticed in the band intensity of pUC19 plasmid upon its treatment with
Con A layered Al2O3-NPs. Comet assay further exhibited negligible change
in tail length of comet after treating the lymphocytes by bioaffinity ma-
trix. AFM revealed a large surface area of Con A layered Al2O3-NPs for
binding higher amounts of the enzyme. Moreover, FTIR has shown bind-
ing of β galactosidase on bioaffinity support by exhibiting a broadening in
peaks at 3220.61 cm−1 and 3447.27 cm−1. Soluble and immobilized β ga-
lactosidase exhibited the same pH-optima at pH 7.0. However, the immo-
bilized enzyme exhibited enhanced pH stability and a broad spectrum
temperature optimum. Immobilized β galactosidase was found to be
highly stable against product inhibition by galactose and retained 85% ac-
tivity after its sixth repeated use. Due to easy production, excellent tem-
perature stability, non-toxic nature and greater specificity of the
bioaffinity support, it might serve as a powerful biorecognition probe in
biosensor applications (Ansari and Husain, 2011a, b).
5. Nanoparticles-based enzymes in biosensors
Special attention has been given to bioassay applications using
nanomaterials to develop biosensors, biomedical devices, biofuel
cells and bioreactors (Akella and Mitra, 2007; Cui and Gao, 2003;
Penn et al., 2003; Ramanavicius et al., 2005). DNA, antibodies and en-
zymes have been immobilized to nanostructure materials by cova-
lent binding, adsorption and entrapment to develop sensitive
biosensors and bioassay methods (Li et al., 2003a, b; Liu and Ju,
2002). Recently, a large number of enzyme biosensors based on
nanoparticles (Besteman et al., 2003; Xiao et al., 2003) or nanotubes
have been developed. The ability to tailor the properties of nanoma-
terials offers excellent prospects for enhancing the performance of
enzyme-based biocatalytic sensors.
Biosensors has many advantages such as high sensitivity, fast re-
sponse, low cost, immunity from electrical and magnetic disturbances,
geometrical versatility, remote sensing capability, small size and light-
weight, and can provide an alternative to laboratory-based measure-
ment methods (D'souza, 2001; Kishen et al., 2003). Salimi et al.
(2007) have successfully co-deposited GOX on nickel-oxide (NiO)
nanoparticles at a glassy carbon electrode. Cyclic voltametry was used
for electro-deposition of NiO nanoparticle and GOX immobilization.
The direct electron transfer of immobilized GOX displayed a pair of
well defined and nearly reversible redox peaks with a formal potential
(E0′) of −0.420 V in pH 7.0 phosphate buffer and the response showed
a surface controlled electrode process. The surface coverage and hetero-
geneous electron transfer rate constant of GOX immobilized on NiO film
glassy carbon electrode were 9.45 × 10−13 mol cm− 2 and 25.2±0.5 s −1
which indicated a high enzyme loading ability of the NiO nanoparticles
and great facilitation of the electron transfer between GOX and NiO
519
nanoparticles. The biosensor showed excellent electro-catalytical re-
sponse to the oxidation of glucose when ferrocene-methanol was
used as an artificial redox mediator. It exhibited good stability, repro-
ducibility and long life time. Furthermore, Kiapp of 2.7 mM for GOX on
the nickel oxide nanoparticles exhibited excellent bio-electrocatalytic
activity of immobilized enzyme toward glucose oxidation. In addition,
this glucose biosensor showed fast amperometric response (3 s) with
a sensitivity of 446.2 nA mM−1, detection limit of 24 μM and wide con-
centration range of 30 μM to 5 mM.
A novel disposable biosensor for rapid determination of H2O2 was
prepared by entrapping HRP in a colloidal gold nanoparticle modified
chitosan membrane (Au-chitosan) to modify the ITO electrode. This
membrane was very efficient for retaining the enzyme activity and
preventing its leakage out of the membrane. The biosensor was char-
acterized by SEM, AFM and electrochemical methods. Parameters af-
fecting the performance of the biosensor, including concentrations
of o-phenylenediamine and pH of substrate solution were optimized
and the linear calibration range was obtained from 0.01 to 0.5 mM
with a correlation coefficient of 0.997 (n = 8). The amperometric re-
sponse of the biosensor did not show an obvious decrease after the
substrates were injected continuously 34 times into the flow cell.
The prepared biosensor was economic due to the low-cost of ITO
film electrode obtained from industrial mass production. It enabled
a simple, sensitive and rapid determination for H2O2 and thus, pro-
vided a useful platform for the development of biosensors. The bio-
sensor possesses good detection precision, acceptable accuracy and
storage stability for fabrication in batch (Lin et al., 2007). The devel-
oped sensor showed rapid electrocatalytic response for monitoring
H2O2 and provided a promising platform for the development of bio-
sensors, affinity supports and immobilized enzyme reactors.
Glucose oxidase was immobilized on tin oxide using a membrane
entrapment method which provided a simple process for fabricating
enzyme electrode. The immobilized glucose oxidase showed a rela-
tively large current response as compared to other glucose biosen-
sors. It exhibited good reproducibility and stability. The sensitivity
of the biosensor was 19.55 μA mM −1 and a linear response was ob-
served between 0 and 3 mM glucose. Such an amperometric glucose
biosensor can be used for detecting glucose in blood, urine, beverages
and fermentation systems (Park et al., 2008).
β Galactosidase was covalently immobilized onto a gold-coated
magneto-elastic film via a self assembled monolayer (SAM) of ω-car-
boxylic acid alkylthiol containing terminal carboxylic group. The en-
zyme was then attached to the monolayer via carbodiimide/N-
hydroxysuccinimide-mediated coupling (Ball et al., 2003). The immo-
bilized enzyme exhibited a Km of 1.2 mM by using indolyl galactopyr-
anoside as substrate. Kinetic parameters for non-precipitating and
precipitating enzymatic substrates were examined by competitive in-
hibition. Moreover, this method could also be employed for screening
enzyme inhibitors or determining concentrations of enzyme sub-
strates or inhibitors based on wireless, magnetoelastic transduction.
Li et al. (2003a, b) prepared a novel method for immobilizing an-
tibodies (antigens) based on magnetic nanoparticles for piezoelectric
immunoassay. The goat-anti-IgG antibody as the model analyte was
first covalently immobilized to magnetic nanoparticles which were
surface modified with amino-groups. The magnetic bionanoparticles
thus formed were attached to the surfaces of quartz crystal with the
help of a permanent magnet. The detection of IgG was performed
with the prepared sensor. The piezoelectric immunosensor can deter-
mine IgG in the range of 0.6–4.9 μg ml –1 with a detection limit of
0.36 μg ml –1. The magnetic bionanoparticles and immunocomplex
layer could be removed easily by taking away the magnetic field,
making the regeneration of a piezoelectric sensor easier and more
efficient.
A mediator less biosensor was fabricated with a double-sided micro-
porous gold electrode by successively immobilizing a mixed self-assem-
bled monolayer comprising of carboxylic acid and thiol-terminated
520 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523

thiolate (DL-thiorphan and 1,8-octanedithiol, GOX) and finally a gold With an increasing H2O2 concentration the amperometric response of
nanoparticle (Au-NP) on one working side. The double-sided micropo- the sensor increased. The linear response range of the sensor to H2O2
rous gold electrodes were formed by plasma sputtering of gold on a po- concentration was from 1.7 × 10−6 to 5.3 × 10 −4 mol L −1 with a corre-
rous nylon substrate, yielding a face-to-face type two-electrode lation coefficient of 0.998 (n = 12) and a detection limit of
electrochemical cell. While the straight chain molecule 1,8-octane- 9.2 × 10−7 mol L −1 at a signal-to-noise ratio of 3. The apparent Michae-
dithiol forms a dense insulating monolayer, the side armed DL-thior- lis–Menten constant was determined to be 0.31 mM L −1. The storage
phan forms a low density layer for the diffusion of redox couples to stability of the sensor was examined by amperometric response at
the electrode surface. The mixed self-assembled monolayer not only H2O2 concentration of 106 μM L −1. After 60 days of storage and inter-
provided the linking functional groups for both enzyme and gold nano- mittently measuring every 5 days, the results showed that the response
particles but also resulted in an appropriately spaced monolayer for di- of the sensor still maintain about 80% of the original values (Zhang et al.,
rect electron transfer process from the center of the redox enzyme to 2004a, b).
the electrode surface. GOX was covalently immobilized onto carboxyl- Recently, single enzyme nanoparticles have been used for various
ic-acid terminated monolayer gold nanoparticles by simply dispensing applications in bioscience research from biosensors to new drug de-
it in colloidal gold solution. It was observed that the resulting ampero- velopment, food, environmental monitoring, proteomics, biomarker
metric biosensor exhibited quantitatively the same response to glucose analysis and in virus detection. Biological signal transducers are wide-
in the presence and of dissolved oxygen, which provides evidence that ly used for the manufacture of nanobiosensors (Bhinder and Dadra,
the gold nanoparticles immobilized on and around the GOX promote di- 2009). Kishen et al. (2003) have developed a fiber optic biosensor in
rect electron transfer from the enzyme to the electrode, assuming the the recent past to monitor Streptococci activity in human saliva. Wu
role of a common redox mediator (Zhang et al., 2006). The steady- et al. (2009a, b) presented a novel bionanocomposite film consisting
state response of H2O2 biosensor was measured by using selenium of glucose oxidase/platinum/functional grapheme sheets/chitosan
nanoparticles. It achieved 95% of steady-state currents within 10 s. for glucose sensing. The biosensor exhibited good sensitivity with a
detection limit of 0.6 μM glucose and better responses due to its
large surface area and fast electron transfer of graphene and Pt nano-
Table 3
Nanoparticles exploited to synthesize enzyme based biosensors.
particles. It had good reproducibility and long-term stability. The in-
terfering signals from ascorbic and uric acids were negligible. This
Enzyme Type of nanoparticle Application References work showed that the hybrid GOD/Pt/FGS/chitosan nanocomposite-
immobilized
based biosensor has great potential for clinical utility and home care
Glucose Selenium H2O2 biosensor, Zhang et al., for rapid monitoring of glucose. The graphene-based biosensor fabri-
oxidase nanoparticle determination of free 2004a, b
cation method demonstrated in this work was readily applicable to
glucose in body fluids,
food and agricultural the fabrication of other biosensors based on oxidases such as biosen-
products sors for cholesterol, alcohol, lactate, acetylcholine, hypoxanthine and
Uricase ZnO nanoparticle Uric acid biosensor, Zhang et al., xanthine (Wang and Lin, 2008). Several other nanoparticle based en-
evaluation of serum 2004a, b
zymes that have been utilized to develop biosensors of industrial im-
uric acid
Glucose Gold nanoparticle Biosensors, catalytic Yang et al., 2006;
portance and clinical applications have been summed up in Table 3.
oxidase nanodevice to Li et al., 2007 A reagentless uric acid biosensor based on uricase immobilized on
construct nanoreactors ZnO nanorods was developed by Zhang et al. (2004a, b). ZnO nanorods
Laccase Zinc-tetra-amino- Fiber optic biosensor, Huang et al., derived electrode retained enzyme activity and could enhance the elec-
phthalocyanine- catechol biosensor and 2007; Zhang et
tron transfer between the enzyme and the electrode. This sensor showed
Fe3O4, laccase- in degrading bisphenol al., 2007;
modified silica A Galliker et al., a high thermal stability up to 85 °C and an electrocatalytic activity to the
nanoparticles, 2010 oxidation of uric acid without the presence of an electron mediator. The
magnetic core–shell electrocatalytic response showed a linear dependence on the uric acid
(Fe3O4–SiO2) concentration ranging from 5.0×10−6 to 1.0×10−3 mol L−1 at 3σ. The
nanoparticles
Glucose Nickel oxide Amperometric Salimi et al.,
biosensor developed for phenol detection on ZnO nanorods exhibited
oxidase nanoparticle biosensor and 2007 good performance showing response time less than 5 s upon adding phe-
electrochemical nol to the buffer solution. The sensitivity and detection limit were im-
investigation of glucose proved to 103.08 μA mM−1 and 0.623 μM due to the pretreatment of
oxidase
the gold electrode (Gu et al., 2009).
Glucose Platinum Biosensors, naked eye Zhao et al., 2007;
oxidase nanoparticle, gold detection of glucose in Radhakumary
nanoparticles urine and Sreenivasan, 6. Nanobiocatalysis
2011
Urease Silver nanoparticle, Biosensors, analysis of Crespilho et al., Driven by the recent advances in molecular biotechnology and
phosphonate grafted urea content in blood, 2009, Sahoo et
iron oxide urine, alcoholic al., 2011
nanoscale science and engineering, development of nanobiocatalysts
nanoparticle beverages, natural has shown a multiscale trend that includes the optimization of protein
water and molecular structures, manipulation of nanoscale environments of the
environmental enzymes, microscale properties of materials and fluids and operational
wastewaters
factors of macroscale reactors (Wang, 2009). Compared with enzymes
Peroxidase Gold-chitosan Rapid deterioration of Lin et al., 2007
nanoparticle H2O2, water treatment, immobilized on micrometric supports, a nanobiocatalyst could achieve
pharmaceutical and a much higher enzyme loading capacity and significantly enhanced
biomedical applications mass transfer efficiency. Recent advances of nanobiocatalytic ap-
Glucose Pt/FGS/chitosan, Glucose biosensor, Wu et al., 2009a, proaches have improved the performance of protein digestion by
oxidase silica nanoparticle quantitative estimation b, Jang et al.,
of glucose 2010
using various nanomaterials such as nanoporous materials, magnetic
Cyclodextrin Cellulose-silver Biosensors, Ma and Ding, nanoparticles and polymer nanofibers. Trypsin stabilization was
glycosyl nanoparticle determination of 2009; Mahmoud achieved in the form of trypsin-coated nanofibers which showed negli-
transferase, methanol and methyl et al., 2009; Ling gible decrease in activity after repeated uses for 1 year and exhibited
alcohol ester content of pectin and Heng, 2010
greater resistance to proteolysis, therefore it can be employed in the de-
oxidase
velopment of automated, high-throughput and on-line digestion
 S.A. Ansari, Q. Husain / Biotechnology Advances 30 (2012) 512–523
systems. This novel method implicates improved performance of pro-
tein digestion in speed, detection sensitivity, recyclability and trypsin
stability (Kim et al., 2010). A simple and unique method was developed
to prepare bioactive materials through a spin coating process (Tong et
al., 2008). These workers have investigated the preparation of protease,
subtilisin carlsberg-coated plastic films and examined their activity for
hydrolysis of chicken egg albumin (CEA). This novel method produced
enzymatic coatings with a typical loading of 13 μg cm−2 and retained
46% chicken egg albumin activity in aqueous solutions and exhibited re-
markably improved thermal stability.
Wang et al. (2009) have developed an attractive approach for fab-
ricating a nanobiocatalyst by immobilizing chloroperoxidase on iron
oxide MNPs which were cheap and biocompatible with a core con-
taining multiple iron oxide MNPs and a thick polymer shell. Several
enzymes such as hydrolase, glucose oxidase and alcohol dehydroge-
nase have been immobilized on such MNPs in the recent past with
great success (Blakemore, 1975; Kim et al., 2008; Petros and DeSi-
mone, 2010).
Ginet et al. (2011) have functionalizing lipid-coated nano-magnets
with phosphohydrolase and successfully engineered an easy-to-purify,
robust and recyclable biocatalyst to degrade ethyl-paraoxon, a commonly
used pesticide. They genetically fused the opd gene from Flavobacterium
sp. ATCC 27551 encoding a paraoxonase tomamC, an abundant protein
of the magnetosome membrane in Magnetospirillum magneticum AMB-
1. The MamC protein acts as an anchor for the paraoxonase to the magne-
tosome surface, thus producing magnetic nanoparticles displaying phos-
phohydrolase activity. Magnetosomes functionalized with Opd were
easily recovered from genetically modified AMB-1 cells. The catalytic
properties of the immobilized Opd which were measured by the hydroly-
sis of ethyl-paraoxon exhibited Km (and kcat) values of 58 μM (and
178 s− 1) and 43 μM (and 314 s− 1) for the immobilized and purified en-
zyme, respectively. The immobilized enzyme was found to be quite stable
over repeated uses for pesticide degradation and thus it makes it applica-
ble for pesticide bioremediation in contaminated effluents.
Magnetosomes are nano-sized particles of magnetite (Fe3O4) or
greigite (Fe3S4) that are biomineralized by a phylogenetically diverse
group of prokaryotes, the magnetotactic bacteria. They cover a broad
range of applications such as magnetic resonance imaging, hyperther-
mia cancer treatment or molecules and cell sorting. Functional soluble
proteins (luciferase) have been grafted on magnetosome surfaces by
chemical coupling of specific ligands to membrane lipids or proteins,
or displayed by gene fusion using as an anchor proteins specifically tar-
geted to the magnetosome membrane (Lang et al., 2007). The latter ap-
proach circumvents the purification steps, as the enzyme is genetically
targeted to the membrane surrounding the crystal: subsequently, the
functionalized magnetite particles can be magnetically recovered after
a simple disruption of the cells. The production of magnetotactic bacte-
ria can be achieved with a reasonable yield under controlled conditions
in bioreactors. In another study, single-step production of a reusable
magnetic biocatalyst was carried out for bioremediation of pollutants
from soil and water by utilizing in vivo immobilization of a bacterial
phosphotriesterase. This dimeric zinc metalloenzyme was able to hy-
drolyze organophosphorous neurotoxins such as pesticides (i.e. para-
oxon) or some nerve agents (Van Dyk and Pletschke, 2011).
7. Concluding remarks
Nanocrystalline and nanoporous metal oxide surfaces are serving
as novel matrices for enzyme immobilization. Recently, the use of
nanoparticles has emerged as a versatile tool for generating excellent
supports for enzyme stabilization due to their small size and large
surface area. Nanoparticles strongly influence the mechanical proper-
ties of the material like stiffness and elasticity and provide biocom-
patible environments for enzyme immobilization. The availability of
pure enzymes is another key factor in preparing very active immobi-
lized biocatalysts for the development of biosensors since it facilitated
521
full occupation of the surface of the support by the enzyme. More-
over, the use of pure enzymes avoids undesired side reactions cata-
lyzed by contaminant enzymes of crude preparations. To overcome
the problems associated with enzyme purification like time-consum-
ing, high cost, enzyme inactivation, etc., one suitable solution would
be to exploit nanoparticles for immobilization by highly selective im-
mobilization procedures. The utility of such highly efficient matrices
can be further extended in the development of therapeutic agents
as novel targeted drug delivery for treating cancer patients across
the globe.
Acknowledgments
The authors are thankful to the Council of Science and Technology,
Lucknow, Uttar Pradesh for funding the project entitled “Immobiliza-
tion of plant and fungal β galactosidase by using bioaffinity supports
—its application in the hydrolysis of lactose in whey and milk.” The
authors extend their thanks to Dr. Humaira Ashraf and Dr. Rukhsana
Satar for their critical reading and providing valuable suggestion in
preparing this manuscript.
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