Top Catal (2012) 55:1231–1246
DOI 10.1007/s11244-012-9891-2
REVIEW ARTICLE
Cellulase Immobilized Nanostructured Supports for Efficient
Saccharification of Cellulosic Substrates
Ankush A. Gokhale • Ilsoon Lee
Published online: 9 October 2012
Ó Springer Science+Business Media New York 2012
Abstract Functionalized nanomaterials are promising
candidates for enzyme immobilization to develop efficient
industrial biocatalysts with tailor-made catalytic properties.
Cellulase, a saccharifying hydrolase, can be immobilized
on various nanostructured supports using different types of
binding chemistries. This review examines prior cellulase
immobilization strategies and promising future techniques
to integrate nanotechnology with biocatalysis.
Keywords Nanobiotechnology
Cellulase

Immobilization
Nanosupports
Biocatalysis
1 Introduction
In recent years, with the search for cleaner and greener
routes for bulk synthesis of industrially relevant products
intensifying, enzyme technology has emerged as a viable
alternative. Enzymes are rightly called ‘biocatalysts’ due to
their innate capability of aiding complex transformations in
a more subtle way as nature originally conceived it to be
[1]. Often regarded as the ‘third wave’ following the suc-
cessful implementation of biotechnology in agricultural
and pharmaceutical applications [2, 3], new developments
in industrial enzyme technology are credited to developing
highly substrate specific catalytic mechanisms to produce
enantiomerically pure products. Examples include the
conversion of b-tetralone to the desired amine by the
S-selective transaminase with an enantiomeric excess (ee)
of around 80–94 % [4] as well as bacterial lipase
A. A. Gokhale
I. Lee (&)
Department of Chemical Engineering and Materials Science,
Michigan State University, East Lansing, MI 48824-1226, USA
e-mail: leeil@egr.msu.edu
developed from Pseudomonas aeruginosa with an ee of
over 90 % as opposed to 2 % for wild-type lipase [5].
Biocatalytic processes have often been used to produce
higher end products usually in the range of US $20–30 per
kg [6]. However, enzyme technology has received a con-
siderable boost in the past decade because of cheaper
downstream processing, better understanding of genomics
and a plethora of information provided by bioinformatics
and high throughput screening studies [7]. This has enabled
increased production of lower end goods at cost effective
price. The use of nitrile hydratases as a biocatalyst to
convert acrylonitrile to acrylamide, a well know com-
modity chemical shows that enzyme technology can be
easily scaled up [8, 9]. Case studies have also shown that
the use of enzyme technology helps cut the industrial
operating costs by 10–50 % by bringing down the energy
and raw material costs [10].
The key to improved biocatalysis is to develop engi-
neered enzymes suitable for industrial processes by making
suitable changes in the proposed biocatalyst design as well
as the microenvironment in which they operate. Site-
directed mutagenesis and directed evolution are two com-
monly used tools in protein engineering to modify the
catalytic potential of the enzymes. Site-directed mutagen-
esis provides invaluable insights regarding the relative
importance of specific residues in the overall catalytic
mechanism. Typically, site-directed mutagenesis involves
substitution of specific amino acid residues followed by
expression of the protein. Holloway et al. [11] studied the
effect of site-directed mutagenesis techniques on improv-
ing the range of substrates that can be dechlorinated by X.
autotrophicus haloalkane dehalogenase GJ10. Similarly,
Igarashi et al. [12] have discussed the substitution of cer-
tain key amino acid residues near the active site to improve
the substrate specificity of pyrroloquinoline quinone
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(PQQ)-harboring water-soluble glucose dehydrogenase.
Directed evolution on the other hand aims to create mul-
tiple mutant libraries using recombining or non-recom-
bining mutagenesis methods. This is followed by screening
and selection based on desired parameters such as catalytic
improvements or substrate specificity to accumulate a large
number of beneficial mutant genes [13]. The usefulness of
directed evolution techniques in enzyme biocatalysis has
been demonstrated by several studies. Desantis et.al. [14]
showed the preparation of highly enantioselective nitrilase
using the gene site saturation mutagenesis (GSSM) evo-
lution strategy. Bornscheuer et al. [15] studied the esterase
catalyzed hydrolysis of the 3-hydroxy esters and showed a
25 % ee after using the directed evolution techniques.
The emergence of nanobiotechnology as a new inte-
grated discipline has helped diminish rigid boundaries
between materials science, chemistry and biology. New
techniques to synthesize size-controlled multifunctional
nanoparticles developed by material scientists, novel
physico-chemical binding mechanisms contributed by
chemists and fundamental understanding of cellular sys-
tems provided by microbiologists have helped shape the
contours of this multidisciplinary field. In the past decade,
the use of nanostructured platforms to incorporate bioac-
tive components has generated considerable interest espe-
cially in the fields of pharmacology, and biosensor industry
[16–25]. In certain specialized pharmacological cases,
nanobiotechnology has even made it possible to carry as
well as release drug molecules to a specific organ of
interest [26, 27]. Enzymes such as glucose oxidase have
been successfully immobilized on graphene based
nanomaterials to yield a fast response and improved sen-
sitivity [28, 29]. The widespread use of nanobiotechnology
in pharmaceutical and biosensor applications has led to an
increased effort to replicate its success in other areas of
interest such as bioenergy. The success of nanobiotech-
nology would however depend on the availability of
mature technologies that can reasonably integrate and
support miniaturization of biological functions.
The use of functionalized nanosized materials as sup-
ports for enzyme immobilization has been gaining ground
in recent years. Bare nanostructured supports are suscep-
tible to aggregation. Hence these supports are usually
modified by surface functionalization techniques. The type
of surface chemistry used during modification is highly
specific to the nanoparticle-enzyme system under consid-
eration. Lee and co-workers [16, 17, 21–23] at MSU have
utilized functionalized nanostructured lipid membranes
tethered on electrodes to immobilize proteins such as the
catalytically active esterase domains of neuropathy target
esterase (NTE) by maintaining the activity of membrane
proteins for the development of highly sensitive nanobio-
sensors. In their work, they utilized self-assembly, soft
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Top Catal (2012) 55:1231–1246
lithography, layer-by-layer assembly, and molecular inter-
actions at the interfaces [18, 30–45]. Nanosized materials
usually have high surface area to volume ratio providing
ample opportunities to anchor a number of bioactive
materials [46, 47]. Bioconjugated nanostructured supports
offer several advantages ranging from high loading,
improved dispersability and reduced mass-transfer limita-
tion [48–50]. Proteins undergo conformational changes as
they bind to nanosized supports. Lundqvist et al. [51]
linked the perturbations in the protein secondary structures
to the size of the nanoparticles on which they are immo-
bilized. Smaller nanoparticles with higher surface curva-
ture allow proteins to retain their native structure.
Nanoparticles because of their small sizes, often display
Brownian motion. An interesting study by Jia et al. [52]
used a simple model based on collision theory and Stokes-
equation to predict the enzymatic activity of a-Chymo-
trypsin on polymeric nanoparticles with different sizes.
Particle size of the supports, enzyme mobility and protein
conformational changes are some of the important factors
that govern the ability of immobilized enzyme systems to
deliver improved biocatalytic performance.
There has been growing interest in cellulosic ethanol as
the next generation fuel in recent years. Depleting fossil
fuels, concerns about energy security as well as magnani-
mous government incentives in the form of subsidies and
tax-breaks have positioned cellulosic ethanol to be a strong
contender as an alternative energy source. With several
governments around the world recommending the use of
bioethanol blended fuel to meet the ever increasing energy
demands, there has been considerable effort to find a
practical and economically feasible solution to production
of ethanol from renewable resources. Conversion of cel-
lulosic biomass into reducing sugars has been historically
accomplished by acid-based techniques and enzymatic
processes. According to some estimates, besides being
environmental friendly, enzyme based processes present
roughly the same projected costs as compared to acid-
based processes [53]. In a typical hydrolysis process, cel-
lulose is hydrolyzed to reducing sugars by a bunch of
catalytic and noncatalytic enzyme modules secreted by
cellulolytic microorganisms. These modules are collec-
tively referred to as cellulases. Cellulases comprise of a
number of enzyme systems such as endo, exo glucanase
and b-glucosidase. While endo-exo glucanases are
responsible for disrupting the cellulosic matrix, b-gluco-
sidase converts the cellobiose, an intermediate product
generated during endo-exo synergism to reducing sugars
[54]. The large scale use of cellulase as a biocatalyst for the
production of cellulosic ethanol would entail careful opti-
mization of the specific ratios in which the enzymes need to
be expressed as well as the specific activities of individual
enzymes. Moreover, it would also need developing
Top Catal (2012) 55:1231–1246
effective strategies to ensure the complete use of the bio-
catalytic potential of the added cellulase. According to
some studies, cellulases account for as much as 50 % of the
total hydrolysis costs [55, 56]. A truly cost efficient process
would therefore demand evolving sustainable routes to
ensure recovery of cellulase followed by its reuse. How-
ever, recovery and reuse is complicated due to the fact that
during hydrolysis cellulase tends to get redistributed over
two heterogeneous phases: the solid substrate and the
liquid supernatant [56, 57]. An alternative strategy to pre-
vent this redistribution and facilitate reuse is the technique
of immobilization. The cellulase technology is still in the
nascent stage of development and it is likely that newer
breed of microbes producing highly specialized enzymes
would be designed in the near future. However, the sheer
robustness of the immobilization technique would allow
ready assimilation of these new enzymes within the pre-
existing architecture with or without minor modifications.
In addition, for any industrial process stability of catalyst
over a relatively wide range of process parameters is nec-
essary. Immobilization of enzymes on supports in general
helps to improve the operational stability by increasing the
resilience of enzymes to variation in pH and temperature
[58, 59].
In this review, we discuss past and current developments
in the field of cellulase immobilization on nanostructured
supports for effective conversion of cellulosic substrates to
reducing sugars. Before that, it is important to understand
the structure of cellulase and the mechanism by which it
operates in order to design effective immobilization
strategies.
2 Cellulases
2.1 Background
The use of the term ‘cellulase’ for the class of enzymes that
degrade cellulosic materials goes back to the beginning of
the 20th century [60, 61]. Major impetus to enzyme tech-
nology including cellulase biochemistry was provided
during the Second World War when new protocols for
effective protein separation started emerging [60]. Cellu-
lase was initially conceived to be a consortium of several
enzymes with one set of enzymes termed ‘C1’ exclusively
meant for de-crystallization of the cellulose whereas
another set called ‘Cx’ responsible for the hydrolytic action
[62]. In recent years, there is a common agreement that the
action of cellulase in degrading cellulose to reducing sug-
ars is a result of three major classes of enzymes namely
endoglucanase (EG), exoglucanase and b-glucosidase [63,
64]. With the bioethanol industry growing steadily over the
past few years, the market potential for the cellulase
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consumption is estimated to reach about US $400 million
per year [8]. And this is assuming that cellulase would be
used for hydrolyzing the corn-stover produced in the
Midwest alone. The actual requirement would be consid-
erably higher if the traditional applications of cellulase
such as in detergent, textile, pulp and paper industries are
all factored in. Certain studies were conducted in the past
to examine the efficiency of cellulase from fungal sources
by estimating the enzyme turn over number (kcat). Sinnot
[65] showed that the kcat for cellulase produced from
Trichoderma reesei acting on b-cellobiosyl fluoride was
two orders of magnitude lower than that of Aspergillus
glucoamylase acting on a-glucosyl fluoride. It is hypothe-
sized that cellulase utilize the energy produced from the
cleavage of glycosyl bonds for its own functions as
opposed to improving the process of hydrolysis. Lower
catalytic efficiency means higher cellulase requirement for
cellulose to glucose conversion. This drives up the cost of
the hydrolysis process making commercialization difficult.
Recent development in cellulase technology through col-
laboration between industry and Department of Energy
(DOE) has helped lower the enzyme cost from a high of US
$5.40 per gallon of ethanol to approximately 20 cents per
gallon of ethanol [66]. However, for the process to be truly
competitive, the cost of cellulase needs to drop to less than
7 cents per gallon of ethanol [60, 67]. Further reduction in
the cost of cellulase through genetic engineering or
downstream processing could be very challenging [56].
Thus cellulase recovery and reuse facilitated through
immobilization on supports can help offset the cost of the
enzymes and make the process economically viable.
2.2 Structure
The modular structure of most cellulases (EG, exoglucan-
ase and b-glucosidase) is conceived to exhibit at least two
functional domains: a catalytic domain (CD) that partici-
pates in the cleavage of glycosidic bonds and the cellulose
binding domain (CBD) which as the name suggests binds
to the substrate [68, 69]. A peptide sequence links the two
domains together. Figure 1 shows a typical schematic
diagram of the various modules in a generic cellulolytic
enzyme.
Various experimental techniques such as X-ray crystal-
lography, small angle X-ray scattering and nuclear mag-
netic resonance (NMR) have been used in the past to
examine the 3D architecture of cellulase [71–73]. Studies
have confirmed the existence of two domain proteins in
case of T. reesei cellobiohydrolase (CBH) enzymes—the
larger one corresponding to CD whereas the smaller one
representing CBD [74, 75]. Kraulis et al. [71] showed
through NMR studies that terminal CBD in case of CBH 1
has two sides one hydrophobic and the other hydrophilic.
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Fig. 1 Simplified scheme
showing different functional
domains of cellobiohydrolase,
one of the exo-cellulolytic
enzymes: The CBD anchors the
enzyme to the substrate whereas
the CD attacks the glycosidic
bonds. The polypeptide linker
joins the two domains.
Reproduced with permission
from [70]
The hydrophilic side is rich in tyrosine residues. Since
tyrosine plays a significant role in cellulose binding, it has
been hypothesized that the hydrophilic surface is primarily
responsible for attachment of the CBD to the cellulosic
substrate [76]. The exact role played by the CBD is rela-
tively unclear. Some studies claim that the sole function of
CBD is to anchor the enzyme on the cellulosic substrate
[76] whereas others hypothesize that the CBDs are actually
responsible for releasing individual cellulosic chains from
the substrate of interest [77]. The role of the polypeptide
linker joining the two domains is related to the function of
the CBD. If CBD is assumed to play the mere role of
binding on to the cellulose, the linker is probably just a
spacer connecting the two domains [76]. On the other hand,
if CBD plays a more prominent role in cellulose hydrolysis
as claimed above, the linker can be responsible for the
degree of penetration of the CBD into the substrate and in
controlling the flow of cellulosic chains to the active sites
[78]. Some studies have also concluded that the length of
the linker peptide joining the CD and CBD domain is of
great significance [79–81]. In case of endoglucanse A
derived from Cellulomonas fimi, the deletion of the linker
chain resulted in decrease in catalytic ability, though there
was little effect on the ability to adsorb on microcrystalline
cellulose [80]. On the other hand, in case of Cellobiohy-
drolase I (CBH I) derived from T. reesei, the removal of the
first one-third part of the linker reduced the binding
capacity but did not affect the catalytic ability [81]. Inspite
of several studies, it is difficult to validate the exact
structure of cellulase mainly because the architecture tends
to vary depending on the family of cellulolytic microbes
used for expressing the enzyme. Hence, cellulase is clas-
sified into different types depending on the type and nature
of CDs.
2.3 Synergistic Mechanism of Cellulases
The synergistic action of the various enzyme sub-systems
within cellulase is well-known. While EG attacks the
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internal b-1,4 bonds, exoglucanase cleave the terminal
ends of the cellulose chain. Exoglucanase usually exists in
two types: CBH I and cellobiohydrolase II (CBH II). CBH
I and CBH II are processive in nature because they tend to
attack the reducing and non-reducing ends as they progress
along the cellulose chain. The main product from endo-exo
catalysis is cellobiose. b-Glucosidase, another enzyme sub-
system converts cellobiose to reducing sugar. Industrial
strains of T. reesei can produce up to five types of EGs and
two types of CBHs (CBH I and CBH II) [82]. EGs possess
a higher catalytic ability when it comes to cleaving the
internal glycosidic bonds. EGs have the active site situated
on an open cleft unlike CBH whose catalytic center is
located in a tunnel shaped region [83]. The mechanism of
how EGs and CBHs act cooperatively is still a debatable
question. Various synergistic mechanisms such as exo–exo
or endo-exo have been suggested. The exo–exo synergism
has been suggested on the basis of experimental proof
shown by Fagerstam and Pettersson [84]. The more com-
monly used endo-exo synergism explains the formation of
free cellulosic ends by EGs followed by action of CBHs on
both reducing and non-reducing ends to produce cellobiose
[83]. Figure 2 shows the cleavage of the amorphous cel-
lulosic chains by EGs followed by the processive action of
the CBHs. Glucose is produced from cellobiose units by
b-glucosidase.
The extent of synergism between EG and CBH also
depends on the nature of substrate. Very little or no syn-
ergism was found to exist between the two enzymes when
soluble substrates such as Carboxymethyl Cellulose (CMC)
were used [87]. For crystalline substrates like commercial
avicel, the effect of CBH is more pronounced because the
internal b-1,4 bonds are relatively inaccessible to EGs [88].
However, most crystalline substrates have some amor-
phous regions which remain accessible to EGs. So the
synergistic effect cannot be completely ruled out in this
case. The degree of synergy, an important parameter used
to evaluate the cooperative behavior among different cel-
lulase sub-sets, is a ratio of the effect observed in presence
Top Catal (2012) 55:1231–1246
Fig. 2 The endo-exo
synergistic action of the
cellulolytic enzymes: EGs
cleave the internal b-1,4 bonds.
CBHs attack the reducing/non-
reducing ends. b-Glucosidase
produces glucose from
cellobiose units. Adapted with
permission from [85, 86]
of all enzymes to the sum of effects observed in presence of
individual enzymes. Karlsson et al. [83] carried out a
number of studies to study the effect of synergism on
cellulase systems using steam treated willow as the sub-
strate. One of the prominent observations made in this
study is that presence of b-glucosidase plays a significant
role in cellulolytic hydrolysis. Conversion was noticeably
lower when no b-glucosidase was added. However this was
not the case when EGs were used. This behavior can be
explained on the basis of functions that each enzyme car-
ries out. CBH is primarily responsible to convert the
reducing and non-reducing ends of individual cellulosic
chains into cellobiose. The products of cellulolytic hydro-
lysis can cause significant inhibition. The presence of b-
glucosidase helps eliminate accumulation of cellobiose by
facilitating its conversion to glucose. As per Karlsson et al.
[83], when plotted as a function of the adsorbed enzyme,
EGs show almost similar conversions in presence or
absence of b-glucosidase. These studies also conclude that
if used independently, at lower conversions, the absolute
quantity of sugars produced by EGs per unit of enzyme
used is comparable to that produced by CBHs. At higher
conversions, CBH is more efficient. EGs have a special
preference for disorganized amorphous regions of the
substrate. Once these regions are cleaved by EGs, further
conversion of the remaining crystalline parts of the sub-
strate proceeds at a slower pace. If EG and CBH are used
together, results show that the combined effect of the two
enzymes acting synergistically is quite high as compared to
the sum of their individual effects. Also it was observed
that the degree of synergy was quite high in the initial
duration of hydrolysis. As the time of hydrolysis increased
the value decreased and finally leveled off. The high syn-
ergistic effect could be because of EGs rapidly attacking
the easily hydrolyzable areas of the substrate and gener-
ating several loose ends for the CBH to work with. As the
concentration of these easily hydrolyzable areas goes down
with passage of time, the EGs have to compete with the
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CBH for finding appropriate substrate sites. Glucose is
another product which has been extensively studied for its
inhibitory effect [18, 89]. Accumulation of glucose can
limit the b-glucosidase activity leading to build up of
unutilized cellobiose. This indirectly affects the perfor-
mance of EGs and CBHs. Some reports claim that the rate
limiting step in the hydrolysis of crystalline cellulose is
probably the binding of enzyme to the cellulosic substrate
followed by channelization of individual cellulosic chains
towards the active sites [90]. A number of mechanisms
have been suggested to explain the conversion of cellulose
to reducing sugars. Prominent mechanisms include reten-
tion or inversion of anomeric configurations. Identification
of stereoisomers using p-NMR studies can help identify the
possible mechanism. It has been hypothesized that cellu-
lases having similar active sites tend to produce stereo-
chemically similar isomers [91]. While is most cellulases,
the active sites are located in two carboxylic acid residues
[92], one of which acts as a proton donor and the other as a
nucleophile. The structural topography of the active centers
is such that in retaining enzymes, the distance between the
proton donor and nucleophile is about 5.5 Å which is
enlarged to 10 Å in case of inversion type enzymes [93].
While inversion is a single step process, retaining enzymes
form a substrate-enzyme intermediate which is then
transformed into the product [92].
2.4 Immobilization of Cellulases on Nanostructured
Supports
Immobilization of cellulases on nanomaterials has been
achieved using a diverse range of methodologies. These
methods have been classified on the basis of the interaction
between the cellulase and the support used for immobili-
zation. Table 1 references prior work in this area by pro-
viding information about the binding chemistries used for
cellulase immobilization, nanosupports used for binding
and the types of substrates used for cellulosic hydrolysis.
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Table 1 Immobilization of cellulases on various types of nanostructured supports using different binding chemistries
Immobilization Nanosupport used for Substrate used for hydrolysis Enzyme or Reference
technique immobilization enzyme sub-type

Adsorption on surface of Nanoclay materials p-Nitrophenyl-b-D-glucopyranoside b-glucosidase [94]

nanostructured Soil colloidal particles p-Nitrophenyl-b-D-glucoside b-glucosidase [95]
materials
Hydrophobic teflon silica Laminarin Endo-b-1,3- [96]
nanoparticles glucanase
Activated carbon Methylcellulose solution Cellulase from A. [97]
niger
Superparamagnetic iron-oxide CMC Cellulase from [98]
nanoparticles Trichoderma
viride
Carbon nanotubes 4-Nitrophenyl-b–D-glucopyranoside b-glucosidase [99]
Latex colloidal particles 2-Nitrophenyl-b-D-glucopyranoside b-glucosidase [100]
Covalent binding of PMMA cores-shell particles CMC Cellulase from [101]
cellulase on Aspergillus Sp.
nanomaterials Nanofibrous PAN membrane CMC Cellulases from [102]
A. niger
PVA membrane CMC Cellulase, Source: [103]
–
Non-porous silica nanoparticles CMC Cellulase from T. [104]
reesei
Gold nanoparticles CMC EG from [105]
Fusarium Sp.
Liposomes CMC, Cellulose powder CC31 Cellulase from [106]
Trichoderma
viride
Immobilization within Alginate and polyacrylamide 4-nitrophenyl-b–D-glucopyranoside b-glucosidase [107]
nanopores gels, pore size: – isolated from A.
niger
SBA-15 pore size: 5.4–11 nm Microcrystalline cellulose Cellulase from [108]
Trichoderma
viride
FDU-12 (entrance about: CMC Cellulase from T. [109]
9–10.8 nm and cavity about reesei
19–28 nm)
Small pore 2–4 nm mesoporous Cellulose power pretreated with ionic liquid Cellulase from T. [110]
silica nanoparticles (SPMSN) 1-butyl-3-methyl-imidazolium chloride reesei
Large pore 20–40 nm (BMIM)Cl
mesoporous silica
nanoparticles (LPMSN)

2.5 Adsorption of Cellulase on the Surface
of Nanostructured Supports
Physical adsorption of cellulase on solid supports is per-
haps the most straightforward technique to achieve
immobilization. Lower costs and relatively non-toxic mode
of attachment are some of the major advantages of using
this technique [111]. Vander–Waal forces of attraction,
hydrogen bonding and hydrophobic interactions are some
of the common modes of attachment of the protein mole-
cules on the supports [112]. Some studies have also
claimed that non-specific adsorption of enzymes in general
reduces the chances of internal mass transfer diffusion, a
phenomenon seen in some other modes of immobilization
[111]. A detailed study regarding sorption and desorption
of cellulase on silicate clay materials has been shown
elsewhere [113]. This study claims that organic matter with
higher C/N ratio showed higher affinity for cellulase
sorption. In another recent study, b-glucosidase, one of the
three cellulolytic enzymes, was immobilized on nanoclay
materials [94] as well as fine/coarse soil colloidal particles
[95] and its effect on enzyme activity was monitored. In
case of fine soil colloidal particles, greater enzyme loading
was reported mainly due to higher surface area available
for immobilization. The retention of enzyme activity on
fine colloids was also reported to be higher. Soil colloids in

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general contain a large number of metallic residues. The
catalytic potential of the immobilized b-glucosidase
improved because of the formation of enzyme-metal
complexes which are reported to facilitate better substrate
binding.
Site-directed immobilization of enzymes can ensure that
the CDs of the enzyme are always accessible to the sub-
strate. However, despite several advantages, adsorption of
enzymes on supports remains highly non-specific. More-
over, cellulolytic enzymes bound to solid supports are
likely to desorb and instead re-adsorb on the cellulosic
substrate. As explained in the previous section, cellulases
share a strong affinity for cellulosic substrates because of
the presence of CBDs. The forces responsible for physical
adsorption of cellulase on solid supports are inherently
weak and in the presence of cellulose, a substrate with
which cellulases enjoy natural chemistry, these interactions
may not be strong enough to stop enzyme leaching. With
reference to the glucosidase adsorption on soil colloids as
shown earlier [95], studies have shown that desorption of
b-glucosidase from colloidal soil particles ranged from 17
to 20 % for fine colloids and around 28 % for coarse col-
loids. And this was even before the immobilized enzymes
were subjected to hydrolysis. The extent of enzyme
leaching during and after the hydrolysis cycle should be
comparatively higher.
In another study LamA, a type of endo-b-1,3-glucanase,
was adsorbed on hydrophobic Teflon particles (215 nm) as
well as hydrophilic silica nanoparticles (13 nm) [96].
Based on spectroscopic data and Km values, this study
claims very little change occurred in the conformational
entropy during the immobilization process making its
contribution to the entropy of adsorption minimal. Surface
coverage for Teflon and silica particles was reported to be
78 and 34 %, respectively. Hydrophobic interactions
between the apolar Teflon and protein segments of LamA
ensure significantly higher adsorption as compared to
electrostatic forces acting at the interface of silica particles.
Specific activity of immobilized enzymes was around 50 %
for both Teflon and silica particles as compared to free
enzymes in solution with the loss in catalytic potential
being attributed to random orientation of the enzyme
molecule on the surface of the support.
A more quantitative study of the adsorption process is
described elsewhere [97]. In this study, cellulase from
Aspergillus niger was immobilized on activated carbon.
Immobilization occurs both on the surface as well as within
the micropores. Thermodynamic parameters were esti-
mated to understand the interplay of temperature, equilib-
rium enzyme concentration, the distribution coefficient and
their combined effect on cellulase adsorption. Negative
value for change in Gibb’s free energy, positive change in
enthalpy and entropy reinforce the view that adsorption of
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cellulose on activated carbon is a spontaneous process. A
small positive value for entropy represents relatively lower
randomness in the system which indicates that very little
conformational changes occurred during the immobiliza-
tion process. Enzyme activity tests revealed higher glucose
production when immobilized enzymes with higher
enzyme loadings were used to hydrolyze the substrate. In
addition, the immobilized enzymes display good biocata-
lytic potential (around 70 %) when subjected to five cycles
of reuse.
The nature of surface charge (whether positive or neg-
ative) and the extent of charge distribution on the surface of
proteins are strong functions of solution pH [112]. Figure 3
illustrates how enzyme molecules can exhibit different
types of charges depending on the charge of individual
protein segments. Hence, in such cases, a detailed exami-
nation of ionization states of the enzyme and the charge on
the support/carrier needs to be carried out.
Electrostatic adsorption is a versatile approach that can
be easily extended to a broad spectrum of nanomaterials
such super paramagnetic particles [98] as well as carbon
nanotubes [99]. The benign nature of iron-oxide nanopar-
ticles towards biological applications allows these particles
to be used in several cellular and drug delivery applications
with or without functionalization. Besides this, magnetic
nanoparticles provide a facile route to recycle and reuse the
immobilized enzyme over multiple cycles. Higher pH
stability was reported when cellulase was electrostatically
immobilized on magnetic nanoparticles [98]. However,
after the cellulase attachment the size of the nanoparticles
recorded more than a ten-fold increase indicating some
degree of aggregation.
Another example of electrostatic interaction used for
immobilization of cellulolytic enzymes is the attachment of
b-glucosidase to carbon nanotubes [99]. Carbon nanotube
with its high surface area is another nanoscale material that
can serve as a very good candidate for anchoring a host of
biomolecules. Negatively charged carbon nanotubes were
prepared by pretreatment with nitric acid prior to enzyme
immobilization [99]. Under acidic pH, b-glucosidase has
positively charged protein segments which can be elec-
trostatically assembled on the anionic carbon nanotubes.
However, activity reported after immobilization was about
20 % as compared to free enzymes. The possibility of
formation of amide bond between the amino groups of the
proteins and the carboxylic groups on the acid pretreated
carbon nanotubes leading to conformational changes in the
immobilized enzyme structure cannot be completely ruled
out.
The usefulness of long-chained polyelectrolyte
brushes to immobilize biomolecules on inorganic or poly-
meric supports has been demonstrated by Caruso et al.
[114, 115]. The use of polyelectrolytes creates a benign
123
1238
Fig. 3 Selection of suitable support depending on the charge of the
protein segments on the surface of the enzyme molecule. Reproduced
with permission from [112]
microenvironment for the enzymes to operate on the sub-
strate of interest by reducing support-protein steric hin-
drance. In a related study, b-glucosidase was immobilized
on latex beads using quenched and annealed brushes [100].
The general procedure adopted by the authors for immo-
bilization is depicted in Fig. 4. The robustness of this
approach allows immobilization of enzymes with different
shapes and conformations such as glucoamylase (dumbbell
shaped with flexible linker) as well as b-glucosidase (rigid
globular structure).
The b-glucosidase activity after immobilization was
well preserved because the spatial structure of the enzyme
Fig. 4 Immobilization of
enzymes (b-glucosidase and
glucoamylase) on
polyelectrolyte brush
functionalized latex
nanoparticles. Reproduced with
permission from [100]
123
Top Catal (2012) 55:1231–1246
was largely retained in the brush matrix. The long-chained
polymer segments of the brushes ensure minimum protein–
protein interaction thus ensuring no aggregation within the
brush layers. Another proof of the benign nature of poly-
electrolytes is that the Michaelis–Menten parameters Km
and kcat for the immobilized glucosidase are very close to
those of free enzymes. No remarkable increase was
observed in the value of Km after immobilization indicating
that the active sites of b-glucosidase are still readily
accessible to the substrate [116].
Another significant aspect that needs careful monitoring
during cellulase adsorption is the isoelectric point (pI) of
the enzyme. Proteins in general, show better binding
capability when the pH is close to pI. Charged enzymes
molecules experience mutual repulsion between different
protein segments when the solution pH is far away from the
pI. Near the pI, the net charge on the enzyme molecule is
almost zero because of minimum intramolecular electro-
static repulsion. This facilitates higher protein binding.
Previous reports have confirmed the veracity of this theory
[104].
2.6 Covalent Binding of Cellulase to Nanomaterials
The covalent immobilization of enzymes on solid supports
is often considered as a safe route to minimize protein
desorption. In order to extract the complete biocatalytic
potential of the immobilized enzymes, most industrially
relevant processes require protein leaching to be as low as
possible. Immobilization of cellulases on solid supports can
be carried out using different types of covalent chemistries.
Commonly used binding agents include glutaraldehyde and
carbodiimide derivatives such as1-ethyl-3-[3-dimethyl-
aminopropyl]carbodiimide hydrochloride (EDAC) [117].
However, certain reactive groups are used up during the
Top Catal (2012) 55:1231–1246
formation of the covalent bond. The identification and
location of these groups is of primary importance. Groups
which form a part of the active site of the enzyme should
be generally avoided for bond formation. The general
mechanism for formation of EDAC mediated covalent
bond [118] and glutaraldehyde cross-linking [119] is
depicted in Fig. 5a, b, respectively.
Covalent binding is usually preceded by surface modi-
fication of nanomaterials. Ho et al. [101] used a unique
approach to synthesize PMMA-cellulase core–shell na-
noenzymes. In this study, well defined poly(methyl meth-
acrylate) (PMMA) cores covalently bound to cellulase
were synthesized from mixture of methyl methacrylate
(MMA) and cellulase by direct graft polymerization. The
formation of these novel structures was guided by initiation
of amine groups on the enzyme. Highly uniform and thick
enzyme shells, good pH and temperature stability, and
single step process to synthesize nanometer scale particles
with narrow size distribution are some attractive features of
this approach. Certain issues such as lower activity, high
temperature involved in synthesis (80 °C) and development
of a facile method to recycle and reuse the immobilized
enzymes once addressed would make these core–shell
nanoenzymes truly attractive candidates for cellulase
immobilization.
Cellulase immobilized on electrospun nanofibrous
polyacrylonitrile (PAN) membranes has been reported
recently [102]. Previous studies focused on complicated
surface functionalization of PAN membranes followed by
enzyme immobilization. However, Hung et al. [102] sug-
gested a more convenient direct activation of nitrile groups
on PAN membranes by amidination reaction as per pro-
cedure described elsewhere [120–122]. The activation time
can influence the number of active sites on the PAN
membrane to which the cellulase can covalently bind to.
An activation time of 7.5 min was found optimum for
cellulase immobilization especially since the structural
stability of the PAN membrane is doubtful at longer acti-
vation times [120]. Individual cellulase molecules tend to
occupy the active sites of the PAN membrane rather than
stack over each other, thus ensuring that the catalytic
centers of the enzyme enjoy continued access to the cel-
lulosic substrate [102]. Besides using PAN membranes, use
of polyvinyl alcohol (PVA) nanofibers as scaffolds for
cellulase immobilization has been also reported in litera-
ture [103, 123]. Glutaraldehyde can covalently cross-link
the –OH groups of PVA fibers with the amino groups of the
proteins. Just like the activation time plays an important
role in opening up active sites on PAN membrane, a similar
comparison can be drawn regarding the glutaraldehyde
cross-linking. Longer cross-linking time can reduce the
biocatalytic activity of immobilized cellulase because then
the various protein segments within the enzyme undergo a
1239
high degree of cross-linking blocking the active sites from
accessing the substrate. Also, prolonged cross-linking can
cause –OH groups of the PVA fibers to cross-link among
themselves thus reducing the protein-PVA interaction.
The use of gold nanoparticles for bioconjugation is quite
common. Reliable techniques to synthesize gold nanopar-
ticles starting from 2 to 250 nm, fairly high degree of
colloidal stability over long times and a wide range of
surface functionalization approaches available in literature
have made these particles promising candidates for protein
immobilization [124]. Gole et al. [105] showed the cova-
lent immobilization of endoglucanse on gold nanoparticles
with mean size of around 5 nm. A combination of thiolate
linkages through cysteine residues as well as amine binding
to gold nanoparticles is predicted to be the possible cause
for cellulase attachment. The catalytic behavior of EG
conjugated gold nanoparticles is very much similar to free
enzymes with the optimum of almost 100 % recorded at a
pH of 7 and a temperature of 60 °C. Cellulase has been
covalently bound to nonporous silica nanoparticles [104] as
well as iron-oxide nanoparticles [125, 126]. Using both
physical adsorption as well as covalent cross-linking, Af-
sahi et al. [104] showed a quantitative comparison of both
the methods by immobilizing cellulase on nonporous silica
particles with an average particle size of 14.8 nm and
surface area of 25 m2/gm. These results show for the given
system under consideration, covalent cross-linking with
glutaraldehyde provided better results as compared to
physical adsorption. However enzyme activity in general
was subdued for both the cases (not more than 35–40 %)
indicating that the immobilization strategies used for cel-
lulase attachment on silica particles may be responsible for
constraining the mobility of the attached enzyme. To
overcome the problem of enzyme mobility, Garcia et al.
[126] suggested the use of long-chained spacer molecules
to serve as a bridge between the cellulase enzyme and the
support. Use of high molecular weight ligands such PVA or
polyethylene glycol (PEG) helped to improve the biocata-
lytic activity of the immobilized cellulase. Another inter-
esting approach suggested by Yoshimoto et al. [106] is
using liposomes of various diameters to act as spacers
between the chitosan beads and cellulase. Liposomes with
mean diameters of 50, 100 and 200 nm with aldehyde
terminal groups were used in this study. Protein loading
was size dependent, with 50 nm liposomes showing least
protein loading while 200 nm liposomes showing highest
loading. However, 50 nm liposome showed the highest
specific activity thus indicating that the size of the spacer
and the orientation of the active sites of the enzyme during
immobilization via a spacer can play a significant role in
the biocatalytic activity. The same study also concluded
that cellulase immobilized using 50 nm liposomes were
best suited for multiple cycles of reuse since these
123
1240 Top Catal (2012) 55:1231–1246

Fig. 5 Mechanisms for

formation of covalent bonds
a EDAC mediated amide bond
formation proceeds through
formation of an unstable
o-acylisourea ester (adapted
with permission from [118],
b Glutaraldehyde crosslinking
of proteins occurs through
formation of intermediate
imine. Adapted with permission
from [119]

liposomes were least susceptible to disruption during the
course of the reaction.
2.7 Immobilization of Enzymes Within the Nanopores
Another immobilization technique suggested in literature is
entrapment or encapsulation of the enzyme within an
inorganic or polymeric matrix. Enzyme immobilization
within porous materials can provide the same degree of
stability to the immobilized enzymes as compared to
covalent binding or physical adsorption [127]. Entrapment
exploits the size difference between the substrate or the
product molecule and the enzyme. The entrapping medium
is so selected that it allows free flow of substrate from the
bulk medium to the active site of the enzyme [128].
However, enzyme leaching is a common problem with this

123
Top Catal (2012) 55:1231–1246
Fig. 6 Enzyme immobilization within nanopores: hybrid bimodal
mesoporous silica spheres used for enzyme encapsulation. Repro-
duced with permission from [129]
process since the mode of attachment is more physical than
covalent. Ortega et al. [107] immobilized b-glucosidase
from A. niger into alginate and polyacrylamide gels. The
size of the pores as well as the charge on the gel matrix
plays an important role in deciding the extent of immobi-
lization followed by retention of activity. Wang and Caruso
[129] reported the use of polyelectrolyte modified hybrid
bimodal mesoporous silica spheres to immobilize a host of
proteins and enzymes. Though not specially used for cel-
lulase immobilization, the versatility of this approach as
illustrated in Fig. 6 clearly shows that this method can be
easily extended to cellulase immobilization.
In order to study the effect of pore size on cellulase
immobilization, Takimoto et al. [108] used mesoporous
Santa Barbara amorphous silica-15 (SBA-15) to immobi-
lize cellulase. The pore size of the silica was varied from
5.4 to 11 nm. The authors conclude that the protein loading
on silica particles not only depends on the relative surface
area available for immobilization but also the pore size of
the particles. The authors claim that the enzyme activity for
silica materials with pore size 8.9 nm is highest because
this size closely matches the dimensions of the cellulase
enzyme used in this study. At this pore size the enzyme is
immobilized at the very opening of the pore allowing better
accessibility to the substrate molecules. Nitrogen adsorp-
tion studies confirm this hypothesis. In another study, both
functionalized and unfunctionalized porous face centered
cubic mesoporous silica materials called FDU-12 produced
using a technique developed at the Fudan University, China
1241
were used to immobilize cellulase [109]. The immobilized
cellulase activity was not just the function of the pore
dimensions but also the type of organosilane used for the
synthesis of the mesoporous silica. Organo-functionalized
silica showed lower enzyme leaching as compared to non-
functionalized ones thus enhancing the operational stabil-
ity. It has been noted that the extent of cellulase adsorption
on SBA-15 is limited because of its 2D hexagonal shape
[110]. Amine functionalized FDU-12, on the other hand,
can immobilize a significantly higher quantity of cellulase
among the FDU class supports yet showed lower catalytic
activity [110]. This is because cellulases typically have
their active centers located in aspartic and glutamate acid
residues [130]. Hence, the formation of a covalent bond
between the amine groups of the aminopropyltriethox-
ysilane (APTES) functionalized silica materials with the
carboxylic groups of the catalytic centers can lead to sub-
dued enzyme activity. Vinyl functionalized FDU is another
type of support that has been investigated for cellulase
immobilization. The highest activity achieved using SBA
supports tends to be closer to 70 % [108] whereas when
using the vinyl functionalized FDU supports, it is 71–90 %
[109]. However, the two results are not comparable
because of the difference in the nature of substrates used.
The activity of cellulase immobilized on SBA-15 supports
was tested using microcrystalline cellulose which is an
insoluble substrate. On the other hand, cellulase immobi-
lized on FDU-12 supports was used to hydrolyze CMC, a
semi-soluble form of cellulose. Insoluble substrates
encounter greater difficulty in diffusing through the porous
structure thus severely limiting enzyme-substrate interac-
tions. Soluble substrates on the other hand, can freely
access the enzyme entrapped in the porous silica support.
In addition to the diffusion effects which are inherent in
these systems, cellulase immobilization within a porous
matrix also depends on the nature of interaction between
the enzyme and the immobilizing support. The result is a
synergistic effect where a strong interplay of diffusion,
mode of attachment, resultant stability and enzyme activity
is commonly observed. A recent study tries to understand
this combinational effect by immobilizing cellulase on
porous silica nanoparticles with varying pore dimensions
and by using different types of bonding chemistries [110].
Cellulase was physically adsorbed on both small pore
mesoporous silica nanoparticles (SPMSN) and large pore
mesoporous silica nanoparticles (LPMSN). The different
methods used in the synthesis of these materials give rise to
different functional groups on the surface. As a result,
SPMSN carry a net negative charge whereas LPMSN carry
zero to mildly positive charge. The net negative charge on
cellulase at the immobilization pH along with the small
size of the pores (2–4 nm) inhibits the electrostatic
adsorption of proteins. LPMSN, however, performs better
123
1242
in terms of adsorption because of lower level of mutual
repulsion between the support and the adsorbed proteins.
The study also reveals that covalent functionalized LPMSN
yielded activities upward of 80 % even though the same
type of chemistry described elsewhere showed subdued
cellulase [109]. Better characterization techniques are
therefore needed to identify whether the functional groups
of cellulase participating in the immobilization process
belong to the CD or the CBD region of the enzyme in order
to explain the deviation in the results. Similarly, ensuring
substrate compatibility would also help in a more robust
comparison.
2.8 New Advances in Cellulase Technology and Its
Implication on Immobilization
2.8.1 Cellulosome
Unlike their aerobic counterparts that secrete the regular
cellulase or hemicellulase, anaerobic microbes (celluloso-
mal microbes) during the course of their evolution devel-
oped novel enzyme architecture integrating multiple
enzymatic sub-units to degrade highly crystalline cellulosic
plant cells in a more efficient way [131]. The presence of
dockerin modules and attachment of CBDs to the scaffol-
din sub-unit are some of the key features of cellulosomal
enzymes. The scaffoldin sub-units act as vehicles for effi-
cient assembly of various enzyme modules with different
sub-units joined by flexible linkers. Thus, while in regular
noncellulosomal enzymes, each enzyme sub-unit usually
comes with a CBD to bind to the substrate, cellulosomes
frequently incorporate multiple CBDs within one scaffol-
din sub-unit [131, 132]. Instead of individual enzymes
binding to the substrate, all the enzyme modules on the
same scaffoldin sub-unit are bound to the cellulosic sub-
strate through the common scaffoldin sub-unit. In addition
few enzyme modules may have separate CBDs as well. The
difference between the non-cellulosomal enzymes and
cellulosomal enzymes can be understood from Figs. 1 and
6. Figure 1 shows how each enzyme unit (CBH, EG and
b-glucosidase) participate in the cellulosic hydrolysis by
binding and attacking substrates/intermediate products
whereas Fig. 7 shows the modular structure of cellulo-
somes with multiple CDs attached to the scaffoldin sub-
unit through cohesion-dockerin interactions.
The binding of CBD on cellulosic substrate has been an
area of intense research. Studies have identified the
mechanism and the aromatic residues responsible for the
CBD binding on the cellulosic substrate [134, 135]. Plant
biomass displays a heterogeneous structure with some
portions being highly crystalline while some others show-
ing with high degree of soluble glycan chains [131, 136].
Likewise, the CBDs also display preferential binding
123
Top Catal (2012) 55:1231–1246
ability in response to the difference in the nature of plant
cell-wall. After the scaffoldin is bound to the plant biomass
depending on the preferential affinity of the CBDs, it is
hypothesized that cellulosome undergoes conformational
changes, which includes reorientation of its CDs to
accommodate for the substrate specific degradation. The
highly specialized cohesion-dockerin interaction, one of
the strongest protein–protein interlocking forces, is another
key feature of cellulosomes [137, 138]. The unique plug-
socket design of the cohesion-dockerin assembly ensures
that the CDs of the enzyme remain fused to the main
scaffoldin sub-unit.
Cellulosomes are nature’s answer to improved cellulose
degradation. The ability to blend several enzyme-sub units
together using the same scaffoldin is a powerful tool to
achieve synergy at the nanometer scale. Looking in terms
of enzyme immobilization, each scaffolding sub-unit rep-
resents a ‘nanostructured support’ on which several
enzyme units are ‘immobilized’ using the cohesin-dockerin
arrangement. In the previous sections, the significance of
the use of spacer arms such as polyelectrolyte brushes and
high molecular weight polymer chains to reduce steric
hindrance and improve enzyme-substrate accessibility has
been highlighted. The cohesin-dockerin assembly also
serves the same purpose. Moreover, most of the scaffoldin
units come with their CBDs which enable efficient
adsorption of the cellulosome on to the cellulosic substrate.
2.8.2 Artificial or ‘Designer’ Cellulosomes
The use of cellulosomes as nanostructured scaffolds has
found support within the scientific community in recent
years. Efforts are being made to engineer ‘designer cellu-
losomes’ to accommodate the various types of dockerin-
cohesin modules as well as CDs and CBDs belonging to
different families on the scaffoldin units [139, 140]. In yet
another case, the CDs and the CBDs were assembled on
streptavidin and streptavidin functionalized inorganic
nanoparticles [141]. Enzymes immobilized on curved
supports are observed to have a higher biocatalytic activity
[142, 143]. Thus by attaching CDs and CBDs belonging to
different protein families on cadmium selenide (CdSe)
nanoparticles, Kim et al. [141] effectively used the curved
surface area of the CdSe nanoparticle for bioconjuagation.
The illustration of the same is provided in Fig. 8.
Highly clustered CBDs on CdSe nanoparticles helped
achieve more than seven fold increase in the biocatalytic
activity as compared to free native enzymes. In case of CD
and CBD assembled on streptavidin, improved results were
obtained when an insoluble amorphous substrate was used
in place of soluble substrate. Biotin-avidin interactions, one
of the most commonly used noncovalent forces in binding
biomolecules were utilized for effective clustering of CDs
Top Catal (2012) 55:1231–1246
Fig. 7 Simplified scheme showing different components of a cellu-
losome: scaffoldin sub-unit includes multiple cohesion modules and a
C-terminal divergent dockerin for increased interactions between the
plant cell-wall and bacterial cell envelope. CDs and CBDs are
anchored on scaffoldin through cohesion-dockerin interactions.
Reproduced with permission from [133]
Fig. 8 Artificial/‘Designer’ cellulosomes: catalytic/CBD modules
expressed by different families of cellulolytic microbes assembled
on streptavidin and functionalized nanoparticles to form hybrid
cellulosomes. Reproduced with permission from [141]
and CBD modules on streptavidin and CdSe nanoparticles.
An important observation made in this study was that the
mere homoclustering of CDs may not always provide a
1243
significant improvement in the enzyme activity. Hetero-
clustering of both CDs as well as CBDs helped improve the
catalytic efficiency for amorphous and microcrystalline
substrates. From the Lineweaver–Burke plot, the authors
observed that the clustering of CDs and CBDs on inorganic
nanoparticles is responsible for enhanced substrate-enzyme
interactions. On the other hand, the immobilization of CDs
and CBDs on streptavidin improves the kinetic rate of the
process. Thus the support used for immobilization of
enzymatic modules plays a significant role in the mecha-
nism of activity enhancement.
3 Conclusion
Nanobiotechnology has a unique potential to engineer a
plethora of bioconjugated nanoscale materials with novel
functional properties. In order to make optimum use of
available resources in the most compact manner, the world
is moving rapidly towards developing hybrid technologies
that support miniaturization in a big way. Nanobiotech-
nology is well positioned to deliver these technologies and
is already making significant inroads in certain specialized
industries such as in production of fine chemicals, drug
delivery and many more. The application of cellulase
immobilization on nanostructured supports is yet another
example. Here, in this review various traditional aspects of
cellulase immobilization were examined including the
nature, shape and size of support used the different types of
interactions, cross-linking agents, incubation time and their
collective effect on the biocatalytic activity of the enzyme.
Inspite of attractive features such as remarkable dispers-
ability, high aspect ratio, relative ease of functionalization,
high degree of robustness, easy application to various
forms of reactor configuration and opportunities to recycle,
the use of nanomaterials as scaffolds for cellulase immo-
bilization is not commonly seen in the bioethanol industry.
Two typical reasons are preventing the ascent of this
technology. Firstly, concerns regarding the toxicological
effects of nanomaterials and a perception that large scale,
cost effective production of these materials may not be
feasible has led to a considerable delay. Secondly, with the
focus shifting to engineering improved varieties of cellu-
lolytic microbes using the newly developed techniques of
proteomics, investment in immobilization techniques has
relatively gone down. While concerns regarding handling
of nanomaterials in an industrial setting are not unfounded,
it is hard to ignore the obvious advantages. Similarly,
immobilization should not be treated as a rival technology
to the developing field of cellulase engineering. Instead
both technologies are complementary. An excellent
example of assimilation of both immobilization and pro-
teomics presented in this review is the development of
123
1244
artificial cellulosomes, more specifically the assembly of
various enzymatic modules on streptavidin and CdSe
nanoparticles. With cellulases projected to soon become
the second largest industrially used enzymes, the prospect
of such examples incorporating fusion of these technolo-
gies appear bright.
Acknowledgments The funding from the Michigan University
Research Corridor and the Michigan Initiative for Innovation and
Entrepreneurship and in part from the National Science Foundation
(0609164, 0832730) to support this research is greatly appreciated.
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