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Current Biotechnology, 2014, 3, 207-217 207

Immobilized Enzymes: Strategies for Overcoming the Substrate Diffusion-
Limitation Problem
M.S. Mohy Eldin
*,1
and D.G. Mita
2,3

1
Polymer Materials Research Department, Advanced Technology and New Materials Research Institute, SRTAC, New
Boarg El-Arab City 21934, Alexandria, Egypt
2
Institute of Genetics and Biophysics, CNR, Via Pietro Castellino, 111, 80131 Naples, Italy
3
Interuniversity Consortium Biostructures and Biosystems, Viale Medaglie dOro, 305, Rome, Italy
Abstract: Immobilized enzymes offer many advantages over the free counterparts. These advantages include, but are not
limited to, easy separation, reuse, induced stabilities and potential of continuous operation. However, the immobilization
process at the same time causes some drawbacks including loss of catalytic activity of the immobilized enzymes. Many
interpretations have been given to explain the reasons behind such drawback. Some of them are related to the enzymes
themselves, such as deactivation of enzymes and miss-orientation. Others refer to diffusion limitation of substrates and/or
to inhibitory effects of the products. Substrate diffusion limitation presents the most serious one.
In this review some strategies for overcoming this drawback are reported. Among these strategies are the ones related to
enzyme immobilization on: a) soluble-insoluble matrices, b) thermally reversible hydrogels, c) pressure-sensitive gels, d)
on catalytic hydrophobic membranes operating under non-isothermal conditions, and finally, on carriers' surface, are
presented and discussed.
Keywords: Activity decay, diffusion limitation, easy separation, enzymes immobilization, inhibition, non-isothermal
bioreactors, pressure-sensitive gels, products, reuse ability, soluble-insoluble matrices, stabilities, substrates, surface
immobilization, thermally reversible hydrogels.
INTRODUCTION
Enzyme immobilization using insoluble materials as
supports offers several advantages over free enzymes,
including easy recovery, potential of continuous operation,
simplified downstream processing, and enhanced stability.
This technique has found widespread applications in many
industrial processes.
However, when the enzymes are used in an insoluble
form, effective enzyme reaction can be hampered by solid-
liquid heterogeneous reaction and diffusion limitation of
substrate and/or product in carriers, especially when the
product is an inhibitor of the enzyme reaction. On the other
hand, it is impossible to use the insoluble enzyme in enzyme
reactions using a solid substrate or solid product.
The different strategies adopted to overcome the problem
of an insoluble enzyme reaction system are reported in this
review article.
Immobilization of Enzymes onto Soluble-Insoluble
Matrices
This type of enzyme immobilization shows a reversible
soluble and insoluble state. That means the immobilized


*Address correspondence to this author at the Polymer Materials Research
Department, Advanced Technology and New Materials Research Institute,
SRTAC, New Boarg El-Arab City 21934, Alexandria, Egypt; Tel/Fax:
00203 459 3414; E-mail: m.mohyeldin@mucsat.sci.eg
enzymes are in soluble form during the enzymatic reaction,
and can be recovered in their insoluble form by changing the
pH, ionic strength, temperature, and/or salt concentration of
the reaction medium after completion of the reaction.
Immobilization of Enzymes in Thermally Reversible
Hydrogels
Hydrogels exhibiting a lower critical solution
temperature (LCST) shrink and Deswell when warmed up to
their LCST. Above the LCST, the gel collapses. Reversibly,
the gel expands and reswells when it is cooled below the
LCST. The thermal cycling acts like a 'hydraulic pump'
which enhances mass transfer of the substrate into and of the
product out of the gel, minimizing the diffusion limitation
and product inhibition problems.
Immobilization of Enzymes in Pressure-Sensitive Gels
These gels swell or collapse sharply upon the rise or fall
of environments pressure. Under pressure cycling operation,
the gel behaves analogously to a cylinder and a piston, such
as in a 'micropump'. The piston pushes up and draws back,
corresponding to the swelling and collapsing of the gel. In
addition to the diffusive flow, a convective flow occurs
enhancing in this way the mass transfer within the gel and
reducing diffusion limitation and product inhibition.
2211-551X/14 $58.00+.00 2014 Bentham Science Publishers
208 Current Biotechnology, 2014, Volume 3, No. 3 Eldin and Mita
Processing Enzymatic Reactions Under Non-Isothermal
Conditions
The reaction can be carried out under non-isothermal
conditions in a membrane bioreactor. The temperature
difference across a catalytic and hydrophobic porous
membrane causes thermal transport of both water and
solutes, including the substrate, which is known as Thermo
dialysis process. This effect brings the substrate in and the
product out through the membrane, thus minimizing both the
diffusion-limitation and the product-inhibition problems. The
net result is the increase of the enzyme reaction rate,
proportional to the transmembrane temperature difference.
Immobilization of Enzymes on Carriers' Surface
The immobilization of enzymes on the surface of a
carrier by physical absorption or covalent binding, makes
them more available for the substrate molecules and avoids
the traffic jam of the substrate-products inside the carriers'
pores. Non-porous carriers are one option. Inducing affinity
groups in the carriers structure, which act as a protecting
agent to protect the enzyme active site from engaging in any
kind of bonding during the immobilization process, is
another option.
A discussion of these strategies, accompanied by
examples, will follow below.
IMMOBILIZATION OF ENZYMES ONTO SOLUBLE-
INSOLUBLE MATRICES
PH-Sensitive Soluble-Insoluble Matrices
The great strength of enzymes immobilized onto soluble-
insoluble carriers is apparent when dealing with soluble or
poorly soluble substrate of high molecular weight. Here
water insoluble carriers suffer from poor contact between a
high molecular weight or insoluble substrate and the
immobilized enzyme. Furthermore separation of the
immobilized enzyme from unreacted solid-substrate residues
will be difficult. The hydrolysis of cellulose represents a
good example of this problem. Taniguchi et al. [1]
immobilized cellulase covalently to a meth acrylic
acid/methylmethacrylate copolymer, which is reversibly
soluble above pH 5.0 and insoluble below pH 3.9. The
immobilized cellulase was repeatedly used to hydrolyze
microcrystalline cellulose. Experiments confirmed that 100%
of the immobilized enzyme activity can be recovered by
precipitation and by dissolving it again by alternately
changing the pH. Comparison of the specific activity of the
immobilized cellulase towards different substrates with that
of native cellulase revealed that 'immobilized' cellulase had
very close kinetic values to those of the free counterpart,
indicating the absence of diffusion limitation.
Margolin et al. [2] immobilized penicillin amidase on
polyelectrolyte complexes. The enzyme was covalently
attached to the polycation part of the complex by using cyan
uric chloride as a coupling agent. The enzyme-
polyelectrolyte complex was found to revert to the insoluble
state either by a slight change in pH from 6.0 to 5.8 or by an
increase of the ionic strength up to 0.3M NaCl. This
concentration can be reduced to 0.01 M when using bivalent
cations, because they effectively bind to carboxylate anions.
The mechanism for transition from the soluble to the
insoluble state and the reverse is shown in Fig. (1).
The authors found that all activity was recovered during
repeated precipitation and re-dissolution cycles. In
comparison to the kinetic parameters of the native enzyme,
the Km value of the immobilized enzyme for benzyl
penicillin was not increased showing the absence of
diffusion limitation. Interestingly, the inhibition constant of
the product, phenyl acetic acid, increased from 2*10
-5
to
1*10
-3
M. This result showed the remarkable effect of the
negatively charged shell of the polymethacrylic acid which
causes repulsion with the negative charges on phenyl acetic
acid, removing it away from the enzyme active site. A
unique character of this enzyme derivative comes from its
high sensitivity to changes in ionic strength of the reaction
medium. Since the reaction was carried out at a constant pH
by adding KOH to neutralize the liberated phenyl acetic acid,
the ionic strength increased by time. The former leads to
precipitation of the enzyme matrix thus stopping the
reaction. By varying the initial ionic strength of the solution,
the reaction can be stopped at any degree of conversion.
Other examples of enzymes immobilized onto different
soluble-insoluble pH sensitive supports to eliminate the
diffusion limitation problem are listed in Table 1 [1-8].
Temperature-Sensitive Soluble-Insoluble Matrix
Hoshino et al. [9] immobilized an amylase onto a novel
thermo-responsive polymer. The polymer was prepared by
copolymerization of methacrylic acid (MAA) and N-
isopropyl acrylamide (NIAM). The enzymes were covalently
attached to the carboxylic groups of MAA by using soluble
carbodiimide, producing a good soluble complex below 32
C and insoluble above 42 C. The response to temperature
change was sharper in the presence of NaCl. The
immobilized enzyme showed a high specific activity as
compared to that of the free enzyme and superiority to other
immobilized enzyme preparations especially when using
uncooked starch as substrate. The Km and the inhibition
constant for glucose of the immobilized enzyme were found
to be 50 mM and 65%, respectively, less than the values of
the free enzyme showing no indications of the presence of
diffusion limitation. The reusability test showed that after
thirty batches of soluble starch hydrolysis, the immobilized
enzyme only lost 20% of its specific activity. This decrease
in activity was explained by the incomplete recovery of the
immobilized enzyme after each reaction cycle and not by
enzyme inactivation. A possible degradation/loss of 3D
enzyme structure resulting from keeping the enzyme at high
temperature for long time is a reasonable hypothesis. To
prove this possibility, following up changes in Km after
thirty batches was necessary. Unfortunately these results are
not available.
Selection of the precipitation temperature of the support
was done by copolymerization of the N-isopropyl
acrylamide with a suitable hydrophilic or hydrophobic co
monomer. Polymers with an adjustable precipitation
temperature in the range of 25C - 53 C were obtained [10-
12], which is an added advantage for these kinds of supports.
Table 2 represents different enzymes immobilized on
different supports sensitive to temperature changes which
Immobilized Enzymes Current Biotechnology, 2014, Volume 3, No. 3 209
confirmed the positive effect of using such supports on the
elimination of the diffusion-limitation problem.
Salt- Sensitive Soluble-Insoluble Matrices
One of the main components of bovine milk proteins, ccsl
- casein, precipitates as calcium caseinate in the presence of
an appropriate concentration of calcium ions. The
precipitated casein can be reversibly solubilized by trapping
the calcium ions with a chelating agent such as EDTA.
Therefore, it is worth attempting to use this property of ccsl -
casein for the construction of soluble-insoluble interconvert-
ible enzymes complexes. Chiba and collaborators
immobilized different proteins on casein to derive soluble-






















Fig. (1). Influence of pH and ionic strength on the reversible transition between the soluble and insoluble forms of penicillin amidase
immobilized in polyelectrolyte (adapted from reference 2).
Table 1. Different enzymes immobilized on soluble-insoluble supports.

Support Name Enzyme Soluble pH Insoluble pH Reaction Ref.
Meth acrylic acid-methyl acrylate-methyl methacrylate (MPM-06)
Papain
Chymotrypsin
> 5.8 < 4.8
Hydrolysis
Synthesis
[5]
Meth acrylic acid-methyl methacrylate (Eudragit L100) Cellulase > 5.0 < 3.7 Hydrolysis [1]
Hydroxy propyl methyl cellulose acetate succinate (AS-L) Cellulase > 5.0 < 3.8 Hydrolysis [6]
Hydroxy propyl methyl cellulose acetate succinate (AS-L) Chitinase > 5.2 < 4.5 Hydrolysis [7]
Hydroxy propyl methyl cellulose acetate succinate (AS-L) Lysozyme > 6.2 < 4.6 Hydrolysis [8]
Polyelectrolyte complex of poly(4-vinyl-N-ethylpyridinium bromide) and
poly(meth acrylic acid)
Penicillin
amidase
> 6.0
< 0.2
M NaCl
< 5.8
> 0.2 - 0.4 M
NaCl
Hydrolysis [2]
Polyelectrolyte complex of poly(4-vinyl-N-ethylpyridinium bromide) and
poly(meth acrylic acid)
-Chymotrypsin
Urase
> 6.1 < 5.7 Hydrolysis [4]
Polyelectrolyte complex of poly(4-vinyl-N-ethylpyridinium bromide) and
poly(meth acrylic acid)
Alcohol
dehydrogenase
> 5.9 < 5.7 Hydrolysis [3]
210 Current Biotechnology, 2014, Volume 3, No. 3 Eldin and Mita
insoluble protein preparations [17-18]. They first prepared an
enzyme-asl-casein conjugate using a hetero bifunctional cross
linking reagent. However, the enzyme-ccsl-casein conjugate
did not show sufficient calcium-dependent precipitation.
Modification of the immobilization method by performing
the polymerization of the enzymes-casein conjugate by
transglutaminase enhanced the enzyme-preparation and the
response to the CaCl2 concentration. Almost complete
precipitation was obtained in the presence of over 50 mM
CaCl2. Comparison of the activity of the immobilized
enzymes with that of the free ones revealed that the overall
reactions in both catalytic systems proceeded at exactly the
same rate. This result indicated that the immobilized
enzymes were working without diffusion limitations.
Phosphoglyceromutase, enolase, and peroxidase were also
used in that study. It is worthy to mention that this technique
is not suitable for those enzymes that require metal as
catalytic cofactors where EDTA acts to extract the catalytic
cofactor and affects negatively on the catalytic activity of the
immobilized enzymes. Affinity of EDTA towards metal
cofactors must be determined. Those metal cofactors of less
affinity to EDTA than calcium will be less affected with this
immobilization technique.
IMMOBILIZATION OF ENZYMES IN THERMALLY
REVERSIBLE HYDRO GELS
Thermally reversible hydro gels, exhibiting a lower
critical solution temperature (LCST), Deswell and collapse
when warmed up to and over their LCST. Reversibly, the gel
expands and reswells when it is cooled below the LCST. A
schematic diagram of the process is shown in Fig. (2). The
thermal cycling acts like a 'hydraulic pump' which enhances
mass transfer of the substrate into and the product out of the
gel, thereby increasing the conversion dramatically relative
to isothermal operation at either the upper or lower
temperature. The increased conversion can also be the result
of reduced product inhibition. Park et al. [19] entrapped -
galactosidase in a thermally reversible hydrogen prepared
from copolymers of N-isopropyl acrylamide (NISAAm) and
acrylamide (AAm) cross linked by N, N' methylene bis-
acrylamide. The enzyme was entrapped in the formed
copolymer beads during inverse suspension polymerization.
In Fig. (3) a comparison is given of the conversion of O-nitro
phenol (3-D-galactopyranoside (ONPG) by the immobilized
-galactosidase operated under thermal cycling between 30
and 35 C and isothermal conversion at 30 and 35C. The
Figure clearly showed that the conversion increased by about
60% due to the effect of the temperature cycling. Other
enzymes and cells entrapped in the same matrix are
presented in Table 3 showing the same behavior with
temperature cycling.






























Fig. (2). Schematic diagram of the water and pore structure in a
swollen and collapsed thermally reversible (LCST) hydro gel
containing an immobilized enzyme (adapted from reference 19).
Table 2. Some thermally sensitive supports for enzyme immobilization.

Support Name Enzyme Soluble-Insoluble Temp. Remaining Activity/No. Cycles Reference
N-isopropyl acrylamide-co-meth acrylic acid Amylase < 32
0
C - > 42
0
C 80%/30 cycles [9]
N-isopropyl acrylamide-co-N-acryloxysuccinimide Amylase < 34
0
C - > 34.7
0
C 89%/12 cycles [13]
N-isopropyl acrylamide-co-glycidyl methacrylate
Alkaline
phosphatase
< 34
0
C - > 34
0
C NA [14]
N-isopropyl acrylamide-co-glycidyl methacrylate Amylase < 32
0
C - > 44
0
C NA [15]
N-isopropyl acrylamide-co-2-hydroxyethyl methacylate Trypsin < 32
0
C - > 34
0
C NA [16]
Immobilized Enzymes Current Biotechnology, 2014, Volume 3, No. 3 211













Fig. (3). Conversion of O-nitrophenol P-D-galactopyranoside by -
galactosidase as a function of time in the packed bed reactor
operated isothermally at 30 and 35 C or cycled between 30 and 35
C. Conversion is the ratio of outlet product (molar) concentration
to inlet substrate (molar) concentration (adapted from reference 19).
IMMOBILIZATION OF ENZYMES IN PRESSURE
SENSITIVE GELS
Wang et al. [23] entrapped -galactosidase by inverse
suspension polymerization in poly (N-isopropyl acrylamide)
(NIPA); a pressure sensitive gel. In a normal reaction system
under isobaric operation, the gel keeps the enzyme from
leaking out and allows substrate and reaction product to
respectively enter and exit by diffusion (Fig. 4). A noticeable
increase in the conversion rate was achieved by the pressure-
cycling operation (Figs. 4, 5). The improvement in
conversion rate ranged between 27 and 58%. The authors
proved that pressure cycling and not the high pressure was








Fig. (4). The micro-pump characteristics of pressure-cycling
operation as compared to isobaric operation (adapted from
reference 23).












Fig. (5). Conversion of O-nitro phenol p-D-galactopyranoside by
immobilized -galactosidase under pressure-cycling and isobaric
operations. Symbols: () = 1 x 10
5
Pa, () = 60 x 10
5
Pa, (o) = 1 x
10
5
- 60 x 10
5
Pa (cycle) (adapted from reference 23).
responsible for the conversion-rate improvement, since
pressure was found not to have effect on the activity of the
free enzyme up to 120*10
5
Pa. They optimized the
operational conditions and found that increasing the
pressure-cycling amplitude (pressure difference), increased
the conversion, whereas the pressure-cycling range had no
effect. The pressure- cycling period (time necessary for
completion of one cycle) also strongly affected the
conversion increment. The increase in the conversion could
be explained by the enhancement of mass transfer inside the
gel beads during the pressure-cycling operation in which the
gel swelled or shrinked sharply upon the fall or rise of the
operational pressure, respectively.
PROCESSING THE ENZYMATIC REACTIONS
UNDER NON-ISOTHERMAL CONDITIONS
Recently, Mita and his collaborators [24] discovered that
the activity of immobilized enzymes increased significantly
when the biocatalytic reaction proceeded under non-
isothermal conditions in a membrane reactor. Their
biocatalytic system normally consisted of a catalytic porous
and hydrophobic membrane separating two substrate
solutions kept at different temperatures. The first experiment
was carried out with two components: an immobilization
matrix, gelatin, in which the biocatalyst was entrapped, faced
to a hydrophobic porous membrane to ensure complete
hydraulic separation between cold and warm substrate
solutions. The apparatus usually employed in later
experiments is shown in Fig. (6). In the first publication
about this effect, the authors investigated the behavior of the
Table 3. Biocatalysts entrapped in N-isopropyl acrylamide-co-acrylamide thermally reversible hydro gel.

Enzyme or Cells Reaction Swell Temp.
0
C Deswell Temp.
0
C References
-galactosidase ONPG hydrolysis < 32
0
C > 33
0
C [19]
Conjugate asparaginase Aspargine hydrolysis < 30-35
0
C > 35-45
0
C [20]
Arthrobacter simplex cells Dehydrogenation of steroid < 27
0
C > 32
0
C [21, 22]
212 Current Biotechnology, 2014, Volume 3, No. 3 Eldin and Mita
invertase catalytic activity [24]. The enzyme was entrapped
in a cross linked gelatin membrane. A Teflon 200 membrane
was used as hydrophobic membrane to induce the thermo
dialysis process. They found that the activity under non-
isothermal conditions was higher than under isothermal
conditions (Fig. 7). The temperature of the system under the
non-isothermal conditions was taken as average temperature,
assuming a uniform enzyme distribution and a linear
temperature change over the membrane thickness.
The percentage of activity increment under non-isothermal
conditions increased with the increase in temperature difference
across the membrane. The percentage of increment ranged
between 100 and 300 %, being proportional to temperature
differences between 10 and 30C, respectively.
To have real benefits from applying this technique, the
activity of the system under non isothermal conditions at a
specific average temperature must be higher than the activity
of the system in isothermal conditions at the higher
temperature, i.e. the temperature of the warm side in the non-
isothermal experiment. For example, the activity measured at
average temperature (Tav) = 30C with applied temperatures
of 20C and 40C for the cold side and warm side,
respectively (A2o/4o); should be higher than the activity of
the system at 40C under isothermal conditions (A40/40). Mita
et al. showed that this was indeed the case. The increased
product removal from the enzyme complex by means of a
process of matter transport associated to the flux of thermal
energy is indicated as the way by which the temperature
gradient affects the enzyme activity.
-Galactosidase immobilized by the same method to
another hydrophobic membrane showed the same behavior,
but with a lower percentage of activity increment [25]. Vice
versa, when enzymes were covalently immobilized on
hydrophilic membrane operating under non isothermal
conditions did not change the activity compared to that of the
entrapped enzyme at the average temperature [25-29]. In
different experiments the authors compared the kinetic
parameters of -galactosidase, immobilized covalently onto a
nylon membrane, under isothermal and non-isothermal
conditions. The results showed that using lactose as
substrate, the maximum activity under non-isothermal
conditions resulted higher than under the higher isothermal
temperature. The decrease of the Km value when the reaction
proceeded under non-isothermal conditions was noteworthy
in comparison to the Km value of the immobilized enzyme
operating under isothermal conditions, so suggesting a
reduction of diffusion limitation. Mita et al. explained the
effect of the non-isothermal conditions on the activity by two
theories. The first one concerns the higher rate of product
removal and substrate enrichment as a result of thermo
dialysis while the second one hypothesizes conformational
changes of the enzyme structure when the immobilized
enzyme is crossed by the heath flux. They believe that both
effects occur as a result of the flux of thermal energy. To
confirm the effect of temperature difference on the substrate
and transmembrane movement, they measured these fluxes
under non-isothermal conditions with an enzyme-free
membrane system. What they found was that the flux of the
products transported away from the membrane was higher
than the flux of the substrate available. This indeed may be
decisive for the increase of the apparent reaction rate of the
enzyme.
A simple and interesting experiment was designed to
make the case clearer. The cold half-cell of the apparatus
was filled with a buffer solution containing lactose at a
concentration of 300mM, and the warm half-cell was filled
with a substrate-free buffer solution. In this way the

















Fig. (6). Schematic representation (not to scale) of a thermo dialysis experimental unit: (A) warm working volume, (B) cold working
volume, (C and D) thermostating jackets; (R) supporting nets, (th) thermocouples (Adapted from reference 24).
Immobilized Enzymes Current Biotechnology, 2014, Volume 3, No. 3 213
immobilized enzyme was able to interact with the substrate
by diffusion through the nylon membrane when the system
was isothermal, or by thermal diffusion and thermo dialysis
when the system was non-isothermal. The results for the
isothermal and non-isothermal conditions are shown in Fig.
(8a, b), respectively. Comparison of the two figures shows
that the substitution of purely diffusive by thermo diffusive
transport increases the apparent velocity of the enzyme
reaction by a full order of magnitude. More recently, the
authors tried, by grafting technique, to simplify the
biocatalytic membrane system by using a membrane that was
both hydrophobic and catalytic. Table 4 presents the
different membranes used, i.e. Teflon or Nylon [24-26], with
the maximum activity increment obtained.













Fig. (7). Isothermal and non-isothermal activity as a function of
average temperature (adapted from reference 24).
IMMOBILIZATION OF ENZYMES ON CARRIERS'
SURFACE
A simple and new strategy has been recently investigated
concerning the immobilization of enzyme on the surface of
nonporous carriers. Recently reported work in this area has
revealed the great potential for the use of nonporous [30-36],
nanofibrous [31-37], and nanoparticles [32, 38-41] materials
as a new class of carriers for biocatalysts. The effective
enzyme loading on nanomaterials can be considerably high
(e.g., it can reach over 10 wt % with particles smaller than
100 nm), and a large surface area per unit mass is also
provided to facilitate reaction kinetics. The considerable
specific surface area afforded by nanoparticles is not the only
size-dependent feature important to the performance of
attached enzymes. It is believed that immobilized enzymes
onto nanoparticles have no problems with soluble or
insoluble substrate as long as the reaction will be carried out
in aqueous medium. However, concerns about the separation
and/or recovery of immobilized nano-biocatlysts from the
reaction medium are raised.
Alexey et al. [42] used lysozyme as a model enzyme, and
demonstrated that the structure and activity of an enzyme are
strongly dependent upon the size of the carrier. Less
perturbation of lysozymes secondary structure is observed
when the protein is adsorbed onto smaller nanoparticles
under similar attachment conditions. The structural
information strictly correlates with activity recovery for
adsorption on the smaller nanoparticles. In short, their
pioneering experiment suggested that smaller nanoparticles,
because of their greater surface curvature, promote the
retention of more native-like protein structure and function
better as compared to their larger (and hence less curved)
particle counterparts.
The mobility of catalyst in solution is another crucial
factor in determining the enzyme activity and this
circumstance also provides an explanation for the higher
activities usually observed in enzymes attached to
nanoparticles rather than when attached to larger particles
[43]. Jia et al. [44] used chymotrypsin as an enzyme model,
demonstrated that the mobile state of the immobilized
enzymes is a key factor in their activity recovery. They
believed that unlike solid materials of large size,
nanoparticles dispersed in a solution are mobile and show
Brownian motion. In that sense, the enzymes attached to the
nanoparticles can be considered not immobilized and thus












Fig. (8). Apparent activity of the system -galactosidase-nylon as a function of the average temperature, under the effect of substrate
transport by diffusion (a) or by thermo dialysis (b) (adapted from reference 25).
214 Current Biotechnology, 2014, Volume 3, No. 3 Eldin and Mita
are different from traditional concept of immobilization.
Such solution behavior may point to a transitional region
between homogeneous catalysis with free native enzymes
and a heterogeneous one with immobilized enzymes.
According to the Stokes-Einstein equation, owing to their
relatively larger size, the mobility and diffusivity of the
nanoparticles should be smaller than those of native enzymes
[43, 44]. Both theoretical modeling and early experimental
measurements demonstrated the mobility-activity relation. It
was also believed that the deterioration in the intrinsic
activities of tethered enzymes would cause loss of catalyst
mobility, in addition to other factors such as conformation
changes of protein.
It was found that the reactions catalyzed by enzymes can
also affect the motion of nanoparticles [45-47]. This may be
referred to the attachment of enzymes to nanoparticles
functioned as nanomotors [43].
Mohy Eldin et al. [48] immobilized Penicillin Acylase
(PA) on nylon particles grafted at 8% with poly-
diethylenegly-dimethacrylate (PDGDA). After optimization
of the factors affecting the activity of the immobilized
enzyme, i.e. both the conditions of the amino alkylation- and
the immobilization process, 12% of the activity was retained.
The probable reasons behind the loss of enzyme activity
could be, binding in the wrong orientation of the
immobilized enzyme, or multi-point attachment to the
matrix, or binding with groups in the active site. It was found
that the increase of the amine-groups concentration on the
matrix surface had a negative influence on the activity. By
protecting the active site of the PA through the
immobilization process using phenyl acetic acid (PAA) as
active-site protecting agent, both the activity and the
retention of activity were improved by a factor of 2. The
latter was reaching 30%.
Mohy Eldin et al. [49] prepared a new matrix for
enzymes covalent immobilization by the activation of
alginates OH groups using p-Benzoquinone. The
immobilization of -galactosidase onto the surface of -
Benzoquinone activated alginate beads has been presented as
a strategy to overcome the diffusion limitation problem of
the substrate. Factors affecting the activation
process and their impact on the activity of immobilized
enzyme were studied. Changes in the temperature and pH
profile for the immobilized form in comparison with the free
form were observed. Moreover, the effect of the
immobilization on the kinetic parameters of the immobilized
enzyme has been monitored. The effects of introducing
ethylene diamine spacer on the catalytic activity and on the
kinetic parameters of immobilized -galactosidase onto -
Benzoquinone activated alginate beads surface were also
studied. Different factors affecting the process such as
concentration of ethylene diamine and reaction time were
considered. Shift of the optimum temperature towards lower
values and of the optimum pH towards acidic region in case
of the immobilized enzyme as compared with free enzyme
were determined. No signs of diffusion limitation were
detected considering the kinetic characteristics of the
immobilized enzyme forms [50].
-Benzoquinone activated alginate beads were also
prepared by Mohy Eldin et al. [51-52] as a new carrier for
affinity covalent immobilization of glucoamylase enzyme.
The alginate played a double role here as a "substrate like"
and as a "carrier" for the enzyme. The immobilized
glucoamylase was found to keep almost 80% of its native
activity giving proof of non-significant substrate (starch)
diffusion limitation. The proposed affinity covalent
immobilizing technique would rank among the potential
strategies for efficient immobilization of glucoamylase
enzyme.
Mohy Eldin et al. [53-54] covalently immobilized -
galactosidase from Aspergillus oryzae onto amino-
functionalized polyvinyl chloride (PVC) microspheres
surfaces after activation with glutaraldehyde. PVC
microspheres were functionalized by introducing terminal
amine groups via amination process with ethylene diamine
(EDA). Furthermore, verifications of amination process were
obtained using Thermal Gravimetric Analysis (TGA), FT-IR,
and SEM analysis. Different factors affecting the amination
process were investigated and their impact on the activity
and the retention of immobilized enzymes activity was
monitored. Concentration of ethylene diamine, amination
temperature, and time were found for a determined effect.
Reaction with EDA (0.025%) at 30 C for 40 minutes was
found for optimum conditions. Variation of PVC: EDA mass
ratio over 1: 1 was found with neglect able effect. Thermal
stability of immobilized enzyme was recognized where the
immobilized form kept 80% of its original activity compared
with 20% for the free form. Denaturation tolerance against
pH was observed where immobilized form kept 60% of its
original activity after 300 minutes incubation at pH 7.0 in the
absence of substrate, while the free form kept only 15% of
its activity under the same conditions.
Moreover, immobilized forms showed storage stabilities
where immobilized form kept 40% of its original activity
after four weeks while the free form kept only 25% of its
initial activity. Under optimum conditions for enzyme
immobilization, 1 kg of immobilized enzyme retains 87% of
its native activity and has 11,000 activity Units. Effect of
immobilization conditions was investigated. The maximum
obtained immobilization yield is 27 Units g
-1
while the
maximum expressed activity is 12mole min
-1
g
-1
. The
optimum pH of the free enzyme was observed at 5.2, while
Table 4. Percentage activity increment of different enzymes in non-isothermal bioreactors.

Grafted Support Name Enzyme Grafting Technique Maximum Activity Increment % Reference
Teflon-g-methylmethacrylate -galactosidase -radiation 47% [27]
[Teflon-g-meth acrylic acid]-g-2-hydroxy ethyl
methacrylate
-galactosidase -radiation 70% [28]
Nylon-g-methyl methacrylate penicillin G acylase -radiation 100% [29]
Immobilized Enzymes Current Biotechnology, 2014, Volume 3, No. 3 215
the optimum of the immobilized one was shifted to 4.4. The
optimum temperature for both the free and immobilized
forms was recognized at 60

C. A change of the temperature

profile of the immobilized enzyme below 60

C was observed
where response to elevation temperature was less than the
free counterpart. The K
m
and V
max
values of the free and
immobilized -galactosidase were found to be 52.03 and
123.1 mM, and 6.12 and 9.98 mol/min., respectively.
Lactose hydrolysis, 500 mL of 100 mM concentration, was
studied using 400 Units of either free or immobilized
enzymes at pH 6.0 and 60
o
C. Almost 80% hydrolysis
percentage has been obtained after 6 hours hydrolysis time
with immobilize enzyme compared with 63% after two hours
for the free counterpart. Investigation of the operational
stability at 40

C shows that the immobilized enzyme kept its

activity for five hours over ten successive lactose hydrolysis
cycles so providing potentials for practical application in
milk and whey hydrolysis.
Mohy Eldin et al. [55-56] studied also the covalent
immobilization of penicillin G acylase (PGA) onto the
surface of NH
2
-poly (vinyl chloride) (PVC) membranes.
PGA was chosen because it plays a relevant role in the
pharmaceutical industry, catalyzing the production of an
important intermediate for the industrial production of semi
synthetic penicillin and cephalosporin. Because PVC has no
functional groups in its structure attention was focused on
the functionalization of PVC with primary amine functional
groups for the covalent immobilization of PGA. This goal
was achieved through an amino alkylation process of the
surface of the PVC membranes with ethylene diamine
followed by activation with glutaraldehyde to finally
immobilize the enzyme. Different factors affecting the
modification and activation processes were studied and their
impacts on the catalytic activity of the immobilized PGA
were followed. The functionalized membranes were
characterized with Fourier transform infrared spectroscopy,
thermo gravimetric analysis and scanning electron
microscopy to verify the modification process. In addition,
the changes resulting from the modification in physical
characteristics, such as surface roughness, water uptake, and
mechanical properties were monitored [55]. Parameters
affecting the immobilization process, and hence the catalytic
activity of the immobilized enzyme, such as enzyme
concentration, immobilization time and temperature were
investigated. Enzyme concentration and immobilization time
were found of fundamental relevance. Higher activity was
obtained through performing enzyme immobilization at
room temperature. Both optimum temperature (35
o
C) and pH
(8.0) of immobilized enzyme have not been altered upon
immobilization.
However, immobilized enzyme acquires stability against
changes in the pH and temperature of reaction medium
values especially at the higher temperature region and lower
pH region. The residual relative activities after incubation at
60C were more than 75% compared to 45% for free enzyme
and above 50% compared to 20% for free enzyme after
incubation at pH 4.5. The apparent kinetic parameters K
M

and V
M
were determined. KM of the immobilized PGA (125.8
mM) was higher than that of the free enzyme (5.4 mM)
indicating a lower substrate affinity of the immobilized
PGA. Operational stability for immobilized PGA was
monitored over 21 repeated reuse cycles. The catalytic
membranes were retained up to 40% of their initial activity
after 10.5 working hours [56]. In conclusion, the
immobilization of PGA enzyme affecting its characters and
has a positive effect on the stabilities parameters. This
presents and advantage for the immobilized enzyme under
the operational conditions for producing 6 amino penicillin
acid (6-APA) which represents an intermediate product in
the antibiotics production process.
CONCLUDING REMARKS
Different strategies for overcoming diffusion limitation
in immobilized-enzyme systems have been devised.
In the case of a high molecular weight, soluble or poorly
soluble substrate, the solution to immobilize the enzymes
onto a support should have the ability to remain soluble
under the reaction conditions and to precipitate by changing
the pH, ionic strength, temperature, and/or salt
concentration.
However, not all the supports are suitable for application
on industrial scale.
For example, casein and polyelectrolyte complexes
precipitate in the presence of calcium and /or monovalent
cation such as sodium or potassium ions, which usually are
part of enzymatic reaction mixture, limiting their use.
Enzymes immobilized on N-isopropyl acrylamide
polymer or its copolymers lost their activity due to
incomplete recovery of all enzyme matrices when repeatedly
going through precipitation and dissolution as a result of
temperature cycling. Also, the temperature-change cycles
affected the stability of the immobilized enzyme.
More or less the same can be said about the polymers
which are sensitive to pH changes of the solution.
Furthermore, with enzyme soluble-insoluble carriers, there is
the problem of product stability under conditions of
insolubilization. In addition, most of these carriers need
centrifugation to separate them from the reaction medium,
which means additional energy costs.
Indeed, some carriers have the properties of auto-
precipitation, but what remains is the problem of incomplete
recovery of the carriers. For instance, 80% of the carrier
could be recovered after auto-precipitation [6].
Other solutions have been found for soluble substrates
apart from enzymes immobilization onto soluble-insoluble
carriers. These solutions depend on the ways of facilitating
the removal of the products from and the supply of the
substrate into the carriers. This goal has been achieved by the
following techniques:
1. Immobilization of the enzymes on environmentally
(pressure and thermal) sensitive hydro gels.
The main disadvantages of this technique are the
additional cost of recycling the temperature and the
pressure on the temperature and pressure-sensitive
hydro gel, respectively.
2. Carrying out the enzymatic reaction under non-
isothermal conditions:
216 Current Biotechnology, 2014, Volume 3, No. 3 Eldin and Mita
The main disadvantage of non-isothermal bioreactors
is the cost of keeping the temperature difference
constant when using large volume bioreactors.
3. Immobilization of the enzymes on carriers' surface.
The main disadvantages of this technique are the stability
deterioration due to the exposure of the immobilized enzyme
molecules directly to the medium and the mechanical
stability of the carriers especially the hydro gels.
The balance between the additional costs and stability
from one side and the obtained benefits from applying these
techniques on the other side is obviously the decisive factor.
Among the different techniques that have been found to
overcome the diffusion limitation problem not one of them is
suitable for all enzymes. It is essential, therefore, that a
careful choice of strategy must be searched for each enzyme
type. Combining the benefits from more than one technique
probably is the best solution to have more advantages and
fewer disadvantages. For example, the use of magnetic nano-
carriers with enzymes immobilized onto their surfaces solves
the problem of separating the nano-biocatalysts and offers
the advantages of high surface area and loading capacity of
the carriers. Analogously, developing integrated processes
providing the separation of products from the reaction
medium at the time of their production is another direction to
increase the efficiency of the catalytic process using
immobilized enzymes onto soluble-insoluble carriers. The
same we can say about the non-isothermal bioreactors which
will shorten the reaction time by increasing the membrane
life time, reducing, and even preventing, the possible fouling
drawbacks.
CONFLICT OF INTEREST
The authors declare no commercial or financial conflict
of interest.
ACKNOWLEDGEMENTS
Declared none.
REFERENCES
[1] Taniguchi M, Kobayashi M, Fuji M. Properties of a reversible
soluble-insoluble cellulase and its application to repeated
hydrolysis of crystalline cellulose. Biotechnol Bioeng 1989; 34:
1092-1097.
[2] Margolin AL, Izumrudov VA, Svedas VK, Zezin AB, Kabanov
VA, Berezin IV. Preparation and properties of penicillin amidase
immobilized in polyelectrolyte complexes. Biochimica et
Biophysica Acta 1981; 660: 359-365.
[3] Margolin AL, Izumrudov VA, Svedas VK, Zezin AB. Soluble-
insoluble immobilized enzymes Biotechnol Bioeng 1982; 24: 237-
240.
[4] Margolin AL, Sherstyuk SF, Izumrudov VA, Zezin AB, Kabanov
VA. Enzymes in polyelectrolyte complexes. Eur J Biochem 1985;
146: 625-632.
[5] Fujimura M, Mori T, Tosa T. Preparation and properties of soluble-
insoluble immobilized proteases. Biotechnol Bioeng1987; 29: 747-
752.
[6] Taniguchi M, Hoshino K, Watanabe K, Sugai K, Fujii M.
Production of soluble sugar from cellulosic materials by repeated
use of a reversibly soluble-autoprecipitating cellulose. Biotechnol
Bioeng 1992; 39: 287-292.
[7] Chen J-P, Chang K-C. Immobilization of chitinase on a reversibly
soluble-insoluble polymer for chitin hydrolysis. J Chem Tech
Biotechnol 1994; 60: 133-140.
[8] Chen J-P, Chen Y-C. Improvement of cell lysis activity of
immobilized lysozyme with reversibly soluble-insoluble carriers.
Biotechnol Tech 1996; 10: 749-754.
[9] Hoshino K, Taniguchi M, Sasakura T, Katagiri M, Fujii M.
Hydrolysis of starch materials by repeated use of amylase
immobilized on a novel thermo-responsive polymer J Ferm Bioeng
1994; 77: 407-412.
[10] Steinke K, Vorlop KD. Proc. 4th European Congress on
Biotechnology. 1987; 2: 185-188.
[11] Vorlop KD, Steinke K. Homogene Biokatalyse mit durch
Temperaturnderung ausfllbaren Polymer-Enzym-Konjugaten.
Chemi Ing Tech 1988; 60: 790-791.
[12] Auditore-Hargreaves K, Houghton RL, Monji N, Priest JH,
Hoffman AS, Noviski RC. Phase-separation immunoassays. Clin
Chem 1987; 33: 1509-1516.
[13] Chen J-P, Chu D-H, Sun Y-M. Immobilization of -amylase to
temperature responsive polymers by single or multi point
attachments. J Chem Tech Biotechnol 1997; 69: 421-428.
[14] Nguyen AL, Loung JHT. synthesis and application of water soluble
reactive polymers for purification and immobilization of
biomolecules. Biotechnol Bioeng 1989; 34: 1186-1190.
[15] Hoshino K, Taniguchi M, Katagiri M, Fujii M. Properties of
amylase immobilized on a new reversibly soluble-insoluble
polymer and its application to repeated hydrolysis of soluble starch.
J Chem Eng 1992; 25: 569-574.
[16] Steinke K, Vorlop KD. Precipitable enzyme-polymer conjugates
with high affinity. DECHEMA Biotechnol Conf 1990; 4: 889-892.
[17] Yoshikawa M, Goto M, Ikura K, Sasaki R, Chiba H.
Transglutaminase-catalysed formation of coenzymatically active
NAD
+
Analogcasein conjugates .Agri Biol Chem 1982: 46: 207-
213.
[18] Okumura K, Ikura K, Yoshikawa M, Sasaki R, Chiba H.
Preparation of Soluble-insoluble Interconvertible Enzymes :
EnzymePolymerized s1-Casein Conjugates. Agri Biol Chem
1984; 48: 2435-2440.
[19] Park TG, Hoffman AS. Effect of temperature cycling on the
activity and productivity of immobilized beta-galactosidase in a
thermally reversible hydrogel bead reactor. Appl Biochem
Biotechnol 1988; 19: 1-9.
[20] Dong LC, Hoffman AS. Thermally reversible hydrogels: III.
Immobilization of enzymes for feedback reaction control. J Contr
Rel 1986; 4: 223-227.
[21] Park TG, Hoffman AS. Immobilization of Arthrobacter simplex in
a thermally reversible hydrogel: Effect of temperature cycling on
steroid conversion. Biotechnol Bioeng 1990; 35: 152-159.
[22] Park TG, Hoffman AS. Immobilization of Arthrobacter simplex in
thermally reversible hydrogels: comparative effects of organic
solvent and polymeric surfactant on steroid conversion. Biotechnol
Lett 1989; 11: 17-22.
[23] Wang Y, Zhong X, Wang S. Pressure Cycling to Enhance an
Immobilized Enzymic Reaction by Enzyme Entrapment in a
Pressure-Sensitive Gel. J Chem Tech Biotechnol 1996; 67: 243-
247.
[24] Mita DG, Pecorella MA, Russo P, et al. Effect of temperature
gradients on the activity of enzymes immobilized on natural
polymeric matrices. J Membrane Sci 1993; 78: 69-81.
[25] Mita DG, Portaccio M, Russo P, et al. Immobilized enzyme
activity in isothermal and non-isothermal conditions: studies on B -
galactosidase as a model enzyme. Biotechnol Applied Biochem
1995; 22: 281-294.
[26] Portaccio M, Stellato S, Rossi S, et al. Efficiency increase of a
bioreactor employing catalytic membranes under non isothermal
conditions. Biotechnol Appl Biochem 1996; 24: 25-31.
[27] Eldin MS, De Maio A, De Martino S, et al. Non-isothermal
bioreactors utilizing catalytic Teflon membranes grafted by-
radiation. J Membrane Sci 1998; 146: 237-248.
[28] Eldin M S, De Maio A, Di Martino S, et al. Isothermal and non-
isothermal lactose hydrolysis by means of -galactosidase
immobilized on single (double grafted) Teflon membrane.
Membrane Sci 2000; 168: 143-158.
[29] Eldin MS, Santucci M, Rossi S, et al. Non-isothermal cephalexin
hydrolysis by penicillin G acylase immobilized on grafted nylon
membranes. J Mol Catal B Enzymatic 2000; 8: 221-232.
Immobilized Enzymes Current Biotechnology, 2014, Volume 3, No. 3 217
[30] Balcao VM, Paiva AL, Malcata FX. Bioreactors with immobilized
lipases: state of the art. Enzyme Microb Technol 1996; 18: 392-
416.
[31] Kennedy JF, Melo EHM, Jumel K. Immobilized enzymes and cells.
Chem Eng Prog 1990; 86: 81-89.
[32] Tischer W, Wedekind F. Immobilized Enzymes: Methods and
Applications. Top Curr Chem 1999; 200: 95-126.
[33] Haupt B, Neumann TH, Wittemann A, Ballauff M. Activity of
enzymes immobilized in colloidal spherical polyelectrolyte brus
hes. Biomacromolecules 2005; 6: 948-955.
[34] Fernandez-Lorente G, Palomo JM, Mateo C, et al. Glutaraldehyde
Cross-Linking of Lipases Adsorbed onAminated Supports in the
Presence of Detergents Leads toImproved Performance.
Biomacromolecules 2006; 7: 2610-2615.
[35] Jia H, Zu G, Vugrinovich B, Katapinan W, Reneker DH, Wang P.
Enzyme-carrying polymeric nanofibers prepared via
electrospinning for use as unique biocatalysts. Biotechnol Prog
2002; 18: 1027-1032.
[36] Jia K, Zhu G, Wang P. Catalytic behaviors of enzymes attached to
nanoparticles: the effect of particle mobility. Biotechnol Bioeng
2003; 84: 406-414.
[37] Gole A, Dash C, Soman C. On the preparation, characterization,
and enzymatic activity of fungal protease-gold colloid bioconju-
gates. Bioconjugate Chem 2001; 12: 684-690.
[38] Caruso F, Schuler C. Enzyme multilayers on colloid particles:
Assembly, stability, and enzymatic activity. Langmuir 2000; 16:
9595-9603.
[39] Daubresse C, Grandfils C, Jerome R, Teyssie P. Enzyme
immobilization in reactive nanoparticles produced by inverse
microemulsion polymerization. Colloid Polym Sci 1996; 274: 482-
489.
[40] Liao MH, Chen DH. Immobilization of yeast alcohol
deshydrogenase on magnetic nanoparticles. Biotechnol Lett 2001;
23: 1723-1727.
[41] Martins FB, Simoes ID, Cruz EM, Gaspar R. Development of
enzyme-loaded nanoparti-cles: Effect of pH. J Mater Sci Mater
Med 1996; 7: 413 - 414.
[42] Vertegel A, Siegel R, Dordick JS. Silica Nanoparticle Size
Influences the Structure and Enzymatic Activity of Adsorbed
Lysozyme. Langmuir 2004; 20: 6800-6807.
[43] Wang P. Nanoscale biocatalyst systems. Curr Opin Biotechnol
2006; 17: 574-579.
[44] Jia H, Zhu G, Wang P. Catalytic behaviors of enzymes attached to
nanoparticles: The effect of particle mobility. Biotechnol Bioeng
2003; 84: 406-414.
[45] Lee B, Lee S, Holliday L. Biochemistry of Mechanoenzymes:
Biological Motors for Nanotechnology. Biomed Microdevices
2003; 5: 269-280.
[46] Blum D, Ko Y, Hong S, Rini D, Pedersen P. ATP Synthase Motor
Components: Proposal and Animation of Two Dynamic Models for
Stator Function. Biochem Biophys Res Commun 2001; 287: 801-
807.
[47] Wada Y, Sambongi Y, Futai M. Biological nano motor, ATP
synthase F(o)F(1): from catalysis to gammaepsilonc(10-12) subunit
assembly rotation. Biochim Biophys Acta 2000; 1459: 499-505.
[48] Eldin MS, Schroen PH, Janssen EM, Mita DG, Tramper J.
Immobilization of Penicillin G acylase onto chemically grafted
nylon particles. J. Mol Catal B: Enzym 2000; 10: 445-451.
[49] Eldin MS, Hassan EA, Elaassar MR. -galactosidase covalent
immobilization on the surface of alginate beads and its application
in lactose Hydrolysis. Deutsch lebensmittel-Rundschau 2005;101:
255-259.
[50] Eldin MS. -galactosidase covalent immobilization on the surface
of alginate beads and its application in lactose hydrolysis: Effect of
introducing ethylene diamine spacer. Deutsch lebensmittel-
Rundschau 2005; 101: 309-314.
[51] Eldin MS, Serour EI, Nasr M, Teama H. Affinity covalent
immobilization of glucoamylase onto chemically modified alginate
beads for starch hydrolysis application. I. Beads modification. J
Appl Biochem Biotechnol 2011; 164: 10-22.
[52] Eldin MS, Serour EI, Nasr M, Teama H. Affinity covalent
immobilization of glucoamylase onto chemically modified alginate
beads for starch hydrolysis application. II. Enzyme immobilization
and characterization. J Appl Biochem Biotechnol 2011; 164: 45-57.
[53] Eldin MS, El Zatahry AA, Hassan EA, Elaassar MR. Covalent
immobilization of -galactosidase onto functionalized PVC
microspheres. J Appl Polymer Sci 2012; 125: 1724-1735.
[54] Eldin MS, El-Aassar MR, El Zatahry AA, Al-Sabah MB. Covalent
Immobilization of -galactosidase onto amino functionalized
polyvinyl Chloride (PVC) microspheres: Enzyme immobilization
and characterization. Adv Polym Technol 2014; 33: 21379-21389.
[55] Eldin MS, Hassan EA, Enshasy HA, Haroun BM, Hassan MA.
Covalent immobilization of penicillin G acylase onto chemically
activated surface of PVC membrane for 6-APA production from
penicillin Hydrolysis Process: - Optimization of surface
modification and its characterization. J Appl Polym Sci Special
Issue: Mem 2012; 124: E27-E36.
[56] Eldin MS, El Enshasy HA, Hassan ME, Haroun B, Hassan EA.
Covalent immobilization of penicillin G acylase onto amine
functionalized PVC membranes for 6-APA production from
penicillin hydrolysis Process. . Enzyme immobilization and
characterization. J Appl Polym Sci 2012; 125: 3820-3828.


Received: November 25, 2013 Revised: March 22, 2014 Accepted: April 1, 2014