The use of isocyanide-based multicomponent reaction for covalent immobilization of: Rhizomucor miehei lipase on multiwall carbon nanotubes and graphene nanosheets

Article abstract of DOI:10.1039/c6ra14142k

We describe here a novel and simple method for making bioconjugation and immobilization of Rhizomucor miehei lipase (RML) on carboxylated multiwall carbon nanotubes (MWCNT-COOH) and carboxylated graphene nanosheets (Gr-COOH) by using an isocyanide-based f

Full text of DOI:10.1039/c6ra14142k

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The use of isocyanide-based multicomponent reaction for covalent immobilization of
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Rhizomucor miehei lipase on multiwall carbon nanotubes and graphene nanosheets
Mehdi Mohammadi*a, Maryam Ashjaria, Maryam Garmroodib, Maryam Yousefi**c, Ali
Asghar Karkhaned
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bBioprocess Engineering Department, Institute of Industrial and Environmental
Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB),
P.O.Box:14965/161 Tehran, Iran.
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bDepartment of Chemistry, University of Zanjan, Zanjan, Iran.
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cNanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
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dSystems Biotechnology Department, Institute of Industrial and Environmental
Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Iran,
Tehran, Iran
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* Corresponding author
Phone: (+98) 21 44580461
Fax: (+98) 21 44580399
E-mail: M.mohammadi@nigeb.ac.ir
**Co-corresponding author
Phone: +98 2 122 432 020
Fax: +98 2 122 432 021
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E-mail: m.yousefi@avicenna.ac.ir
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Abstract
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We describe here a novel and simple method for making bioconjugate and immobilization of
Rhizomucor miehei lipase (RML) on carboxylated multiwall carbon nanotubes (MWCNT-
COOH) and carboxylated graphene nanosheets (Gr-COOH) by using an isocyanide based
four-component reaction. In this approach, the enzyme supplies amino groups, the support
supplies carboxylic acid groups and the missing components (isocyanide and aldehyde) are
added to the reaction medium. This coupling reaction was carried out in water at 25°C, in
which rapid and high enzyme loading were achieved. The maximum loading capacity of
530mg and 680mg was obtained for Gr-COOH and MWCNT-COOH, respectively. A variety
of techniques including FTIR, Raman spectroscopy, XRD, SEM and TGA were employed to
characterize the immobilized derivatives of RML. The immobilized preparations showed
significantly increased thermal stability and co-solvent stability as compared to the soluble
enzyme. Kinetic parameters and optimum pH activity of RML and its immobilized
preparations were also determined. The Km values of 0.44, 0.23 and 0.18mM and the
maximum reaction rates (Vmax) of 0.09, 0.1 and 0.08 mM min−1 were obtained for
MWCNTs-RML, Gr-RML and free RML, respectively. This approach may provide a general
and efficient method to attach biomolecules on a variety of carboxylated solid surfaces at
ambient conditions.
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Keywords
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Covalent immobilization; Graphene nanosheets; Multiwall carbon nanotubes; Rhizomucor
miehei lipase
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1. Introduction
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Enzymes possess efficient properties in terms of high catalytic activity, operations under mild
conditions, exclusion of undesirable side reactions and having different types of selectivities
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in chemical reactions . Therefore application of enzymes is increased in several industrial
demands 1. Among them, lipases; with the natural catalytic function of hydrolyzing fatty acid
ester bonds are suitable for organic syntheses because of accepting a wide range of non-
natural substrates without requiring cofactors, stability and activity in organic solvents and
availability from several (micro) organisms 2, 3. Application of enzymes in free form are
limited by enzyme inactivation under harsh condition and difficulty for recovering and
reusing the native enzyme from the reaction medium 4, 5. Therefore utilizing an economically
attractive process that allows improving the operational stability of enzyme, preserving its
catalytic properties is a challenge of great interest 6, 7. A great number of materials, both
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inorganic and organic have been used as carrier for enzyme immobilization
. Combine
with recent progress in nanotechnology, nanoscale materials with large surface-to-volume
ratio such as metal nanoparticles, nanomesopourus materials and carbon nanotubes are in
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spotlight as the immobilization support
11. In particular carbon-based nano materials
(CNMs) are used widely for the immobilization of bio-macromolecules due to their
mechanical, thermal and electrical properties and general biocompatibilities 12, 13. Especially,
when the support has a porous structure, highly stable biocatalyst and high immobilization
yield can be obtained 14. In fact immobilization of biomolecules on these materials is often a
key step in their biotechnological and biological applications. Both covalent and none-
covalent attachment can be used for immobilization of proteins on the surface of CNMs 12, 15
.
Although non-covalent immobilization preserves the structure of both the nanomaterial and
the protein well, leaching of the protein from the CNMs cannot be excluded 16, 17. On the
other hand the best stability and selectivity will be achieved via covalent bonding because of
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its ability to rigidify the location of the immobilized protein 18. However covalent attachment
in many cases reduces enzyme activity 19. Moreover the surface of CNMs must be modified
with proper functional groups to bind with proteins via covalent attachment. Therefore,
except electrochemical methods, there is only small number of protocols for covalent linkage
of proteins onto CNMs and are generally limited to 1. direct coupling of a protein using
diimide-activated amidation procedure and 2. chemical amination of the surface of the
support and subsequent immobilization of a protein by using a proper linker or a cross-linker
reagent 20. For example, covalent attachment of bovine serum albumin (BSA) by direct
coupling of carboxylic acid groups of carbon nanotubes with surface-exposed amino groups
of the protein using N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride
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(EDAC) or N,N’- dicyclohexylcarbodiimide (DCC) as coupling agents has been reported .
The other universal method on covalent immobilization of proteins on these materials is
based on further modification of carboxylic acid groups to produce other electrophilic
moieties on the surface of the support 20. The immobilization of enzymes onto CNMs has also
been performed using linker molecules such as N-succinimidyl 1-pyrenebutanoate, 1-
aminopyrene, polyethylene glycol and glutaraldehyde 20. Although the amino functionalized
CNMs are a well-known support for enzyme immobilization, the support modifications is
normally time consuming and requires more chemical reagents 22.
Shortening the number of steps required for functionalization of the support will decrease the
total cost of the process. Therefore, it remains a big demand for the discovery of simple
protocols for functionalization of CNMs and subsequent immobilization of biomolecules on
it. Recently we have reported the results of using the Ugi four-component reaction for
covalent attachment of enzymes on aldehyde functionalized supports 23. This simple and
efficient procedure offers great flexibility and can accommodate different chemistries as well
as surfaces of choice. In fact by utilizing this method a large number of supports and
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functional groups can be used for immobilization of biomolecules. The Ugi four-component
reaction is a condensation reaction between an aldehyde, an amine, a carboxylic acid and an
isocyanide (Scheme 1).
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Scheme 1. The Ugi four-component reaction
We describe here using the Ugi four-component reaction for one-pot immobilization of
biomolecules on carboxylic acid-functionalized multiwall carbon nanotubes (MWCNTs-
COOH) and graphene nanosheets (Gr-COOH) for the first time. For this, Rhizomucor miehei
lipase was selected as a model enzyme in order to assess the potential applicability of CNMs
as carrier for immobilization of biomolecules. The lipase from Rhizomucor miehei is a
commercially available enzyme in both soluble and immobilized form. The enzyme is
described as a single polypeptide chain of 269 residues with molecular size of 31,600 Da and
a pI of 3.8 24. RML is a quite active and stable lipase and presents more advantages in
industry compared to other lipases 25. The results of the present protocol are promising to be
used not only for immobilization of biomolecules but also for chemical functionalization of
CNMs.
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2. Exprimental
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2.1. Materials
The graphite used for preparing graphene nanosheets was expandable graphite, supplied by
Shandong Qingdao Graphite Company (China). Carboxylated multiwalled carbon nanotubes,
(MWNTs-COOH, D: 8-15nm, length: 50nm, surface area per unit mass:
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233m2/g, Purity>95%) was obtained from Neutrino Port. Co., (Cheap Tubes Company,
USA). The lipase from Rhizomucor miehei, p-nitrophenyl butyrate (p-NPB), cyclohexyl
isocyanide, and acetaldehyde were from Sigma. Dioxane, 1-propanol, 2-propanol, THF,
DMSO, acetonitrile, sulfuric acid and nitric acid were purchased from Merck. Other reagents
and solvents used were of analytical grade. Fourier transform infrared spectra (FT-IR) were
recorded on a Bomen FT-IR-MB-series instrument with a KBr pellet technique.
Thermogravimetry (TGA) and differential thermal analysis (DTA) were carried out from 10
°C to 800 °C at a heating rate of 20°C/min in air atmosphere using a STA 503M system from
Bähr GmbH, Germany.
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2.2. Methods
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2.2.1. Preparation of graphene nonosheets
The expandable graphite was heat treated at 950 10°C for 30s to obtain expanded graphite.
The expanded graphite was immersed in a 75% of aqueous alcohol solution in an ultrasonic
bath. The mixture was sonicated for 10h, and then filtered and washed with enough distilled
water and ethyl alcohol. The obtained graphene nanosheets were dried under vacuum at
ambient temperature for 12h and stored in room temperature.
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2.2.2. Carboxylation of graphene nanosheets
Carboxylation was carried out by reacting of graphene nanosheets with strong acids oxidizing
solution. Graphene nanosheets (1g) were added to a 3:1 mixture of concentrated H2SO4,
HNO3 (120: 40mL). The reaction was sonicated in an ultrasonic bath for 1h and then stirred
for 24h at room temperature. The mixture was centrifuged at 6000 rpm for 1h and the solid
materials were washed with 300 mL of deionized water (repeatedly till no acid was detected
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in the supernatant) and then with ethanol (2 x 300mL), followed by vacuum drying. The
obtained carboxylated graphene nanosheets were stored in room temperature.
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2.2.3. Enzyme immobilization on multi-walled carbon nanotubes and graphene nanosheets
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An amount (5mg) of the MWNTs-COOH (or Gr-COOH) was added to 2mL of distilled water
(pH 7.0) containing a certain amount of RML and acetaldehyde (20µL). Immobilization of
RML was started by addition of 10µL of cyclohexyl isocyanide to the solution under gently
stirring at 25°C. Periodically, samples of the supernatant were withdrawn and analyzed by
activity assay and the Bradford’s method26 for determination of the protein concentration.
Finally the immobilized RML derivatives were washed by distilled water and stored at 4°C
for further characterization (section 3.1).
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2.2.4. Enzymatic activity assay
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Two methods were used for activity measurement of the derivatives. In the first method the
activities of the soluble lipase and its immobilized preparations were analyzed
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spectrophotometrically by measuring the increment in absorbance at 348nm (∈
= 5150M-1cm-
1) . The increase in absorbance produced by the release of p-nitrophenol in the hydrolysis of
p-NPB in 25mM sodium phosphate buffer at pH 8.0 and 25°C. Briefly, 0.05-0.2mL of the
lipase suspension or solution (blank or supernatant without further dilution) was added to
2.5mL of substrate solution (0.8mM) under magnetic stirring 27. Spontaneous hydrolysis of
p-NPB was measured using 2.5ml of substrate solution (0.8mM) in the absence of RML as
control. Enzyme specific activity is given as 1µmol of p-nitrophenol released per minute per
mg of the enzyme (IU) under the conditions described above. In the second method the
activity of the derivatives was assayed in a pH-stat system (Metrohm, Switzerland) by using
20ml substrate (1% of each substrate, 2% gum arabic, mixed and ultrasonic for 5 min, pH 8.0
and 25°C). In this way a certain amount of each derivative was titrated automatically with
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0.05M NaOH. Substrate specificity of the lipase was determined by measuring activity in the
presence of C8, C12 and C18 as substrate. Enzyme specific activity is defined as the amount
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of lipase that release 1ꢀmol of fatty acid from the substrate in 1 min at 25°C.
2.2.5. Determination of the amount of RML bound to the carriers
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The concentration of RML in the solutions was determined by the Bradford’s method. The
reported yields of immobilization were calculated as the ratio of protein bound to the support
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to the initial amount of protein. Yields were expressed as percentage
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2.2.6. Thermal inactivation of RML immobilized preparations
Free enzyme and immobilized preparations of RML were incubated in 25mM sodium
phosphate at pH 7.0 at different temperatures. The suspension of each sample was withdrawn
periodically and their activities were measured using the p-NPB assay.
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2.2.7. Stability of RML immobilized preparations in presence of organic solvents
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Free enzyme and immobilized preparations were incubated in a total volume of 1 mL solution
containing 25mM sodium phosphate buffer pH 7.0 and 10% or 20% of dioxane, 1-propanol,
2-propanol, methanol, acetaldehyde, DMSO, and THF at 25°C. The activity of each sample
was measured using the p-NPB assay.
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2.2.8. Leaching experiment
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Different preparations of MWNTs-RML and Gr-RML (5mg) were incubated in a solution
containing 1M of NaCl with vigorous magnetic stirring for 24h. Then the concentration of
RML in the supernatant was measured by using enzyme activity assay and the Bradford’s
method.
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2.2.9. Determination of kinetic parameters
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Kinetic parameters of the free and immobilized preparations of RML were determined using
different p-NPB concentrations in the range of 0.063-1.25mM in 25mM sodium phosphate
buffer (pH 8.0). Km and Vmax values of the free and immobilized lipase were calculated via
non-linear regression fitting of the of the Michaelis-Menten equation using Prism 6
(Graphpad Software).
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3. Results and discussion
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3.1. Covalent immobilization of RML on graphene nanosheets
The choice of the reactive functional group on carbon-based materials was simply made,
keeping in mind that one of the most common functional groups which can be easily
introduced on the surface of CNMs is carboxylic acid groups. To demonstrate the utility of
the proposed method, RML was immobilized on carboxylated graphene nanosheets. This
novel approach is based on the use of the Ugi multi-component reaction to covalent
attachment of enzymes. In this four-component reaction the support supplies carboxylic acid
groups, the enzyme supplies amino groups and the two missing components (isocyanide and
aldehyde) are added to the reaction medium. Extremely mild condition (water and 25°C) was
used to perform this coupling reaction. Similar to our previous report 23, negligible
immobilization of RML on both supports was observed in sodium phosphate buffer solution
even at low ionic strength (5mM) after long time of incubation. Further investigation is
needed to find the reasons of negative effect of impurities on enzyme immobilization in this
procedure. Carboxylated graphene nanosheets have a unique planar structure and intriguing
mechanical properties that have attracted intensive interests. To investigate the effect of the
amount of offered enzyme on immobilization yield and final activity of the immobilized
derivatives, different amounts of RML (400, 500, 800 and 1000mg) were offered to
immobilize on 1g of Gr-COOH under gently stirring for 5h. Further incubation of the enzyme
for long time (24h) had negligible effect on the immobilization yield in all cases. However
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complete immobilization of up to 100mg of the enzyme was performed shortly after 30
minutes of incubation. Table 1 shows the parameters of immobilization of RML on Gr-
COOH using various enzyme-to-support weight ratios. Immobilization of 400mg of RML on
Gr-COOH produced 86% of immobilization yield after 5h of incubation. It means that 340mg
of the offered enzyme is immobilized on the support at this condition. Increasing the enzyme
to 500mg caused to decrease the immobilization yield to 73%. However the amount of the
immobilized enzyme improved to 365mg on 1g of the support. With further increasing the
amount of offered enzyme to 800 and 1000mg, lower immobilization yields were obtained.
This investigation also showed that the maximum loading capacity of 1gr of Gr-COOH is
about 530mg of the enzyme. Up to date different loading capacities for carboxylated
graphene nanosheets has been reported in literature. To the best of our knowledge this is the
highest reported capacity of immobilization of an enzyme on this support via covalent
linkage. However physical adsorption of 800mg/g of acid pectinase onto graphene nanosheets
has been achieved previously 28.
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Table 1. Immobilization of Rhizomucor miehei lipase on multiwall carbon nanotubes and
graphene nanosheets
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Offered
enzyme
(mg/g)
Specific activityc
(U/mg RML)
Yielda (%)
MWCNTs
mg enzyme/gr support
Desorptionb (mg)
Gr
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73
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53
Gr
MWCNTs
368
Gr
12
17
13
21
MWCNTs
Gr
5.7
5.8
5.5
5.3
MWCNTs
400
500
800
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90
80
68
340
365
448
530
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19
5.8
5.6
5.5
5.1
450
640
1000
680
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bYield is defined as the percentage of the soluble enzyme that becomes attached to the supports after 5h.
bDesorption was performed in a solution containing 1M NaCl.
cSpecific activity (U/mg lipase) is expressed as micromole of substrate hydrolyzed per minute per mg of RML.
The specific activity of the soluble RML was 5.9.
Desorption experiment was performed by incubation of the immobilized derivatives in a
solution containing 1M NaCl under vigorous stirring in order to ensure if the immobilization
is covalent. After 24h, the solid was separated from the supernatant by centrifugation. The
Bradford assay showed releasing of only 12-21 (3.5-4%) of the immobilized enzyme in
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solution clearly proving that by using the multicomponent approach, immobilization is
performed via covalent binding. The results also showed no detectable immobilization in
directed attachment of RML on Gr-COOH without using isocyanide and aldehyde.
The specific activity of the immobilized preparations of RML on graphene nanosheets (Gr-
RML) was also investigated as a function of the amount of loaded RML. The enzymatic
activity was characterized by measuring the hydrolysis rate of p-NPB, as a model substrate.
The catalytic hydrolysis of p-NPB produces equimoles of p-nitrophenol which is determined
spectrophotometrically. The results showed a little decrease (90-98%) in specific activity of
the immobilized derivatives compared to specific activity of free RML, presenting
remarkable efficiency of the used method for enzyme immobilization. The results also
showed that with increasing the amount of enzyme, the specific activity of the final derivative
decreases. Decrease in enzyme specific activity is most likely because of diffusion limitation
of substrate/product after immobilization of the enzyme 18.
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3.2. Covalent immobilization of RML on carboxylated multiwall carbon nanotubes
(MWCNTs-COOH)
Covalent immobilization of RML on carboxylated multiwall carbon nanotubes was examined
using the Ugi four-component approach. Various amounts of the enzyme were offered to the
support. Immobilization of up to 100mg of the enzyme was completed quickly after 15
minutes of incubation while for the higher amounts of RML the longer times were needed. As
Table 1 shows that both immobilization yields and loading capacities were greatly improved
compared to Gr-COOH, suggesting that MWCNTs-COOH is more efficient immobilization
matrix for proteins. For example with 400mg of RML in the initial solution, 92% of
immobilization yield was obtained. It means that 368mg of the enzyme is immobilized on the
support after 5h of incubation. With increasing the amount of offered enzyme to 500, 800 and
1000mg, immobilization yields decreased to 90, 80 and 68% respectively. Despite of these
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low immobilization yields, the loading amount of RML on the support dramatically
improved. The higher capacity of carboxylated multiwall carbon nanotubes compared to Gr-
COOH can be explained by its three dimensional structure which enables higher enzyme
loading 29. The maximum loading capacity of MWCNTs was determined as 680mg which
was much higher than those reported previously 30, 31. The results also showed that in higher
immobilization yields, the specific activity of the obtained derivatives slightly decreased. For
example with immobilization of 680 mg of the enzyme, specific activity of the derivative
decreased to 5.1 which it is about 86% of the specific activity of the free enzyme. In fact in a
high enzyme loading, diffusion of the substrate to the enzyme and of the product away from
the enzyme becomes limited. To confirm the covalent nature of the enzyme attachment,
desorption experiment was performed. The results showed that only 3% of the immobilized
enzyme has physical interactions and leached out from the support, indicating that the
enzyme was irreversibly bound to the support. Desorption experiment for the derivative
obtained from direct immobilization of RML on MWCNTs-COOH without using coupling
agents (isocyanide and aldehyde) was also performed at the same condition. More than 98%
of the adsorbed enzyme was released to the solution, confirming that physical adsorption is
the main mechanism of immobilization in the used condition.
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3.3. Characterization of the immobilized derivative of MWCNTs-COOH
Characterization of the MWCNTs-COOH before and after immobilization of RML was
carried out by using scanning electron microscopy (SEM), infrared spectroscopy (IR),
thermal gravimetric analysis (TGA), X-ray Diffraction analysis (XRD) and Raman
spectroscopy.
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Fig. 1. Scanning electron micrographs of a) MWCNTs-COOH, b) MWCNTs-RML, c) Gr-
COOH and d) Gr-RML.
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Fig.
1
shows the high-resolution SEM images of the carboxyl-functionalized
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MWCNTs/graphene nanosheets and their RML immobilized derivatives. The surface of the
MWCNTs-COOH has uniform and smooth layers and a clear rode-like shape, whereas its
aggregates structure in the image of MWCNTs-RML was clear evidence for successful
immobilization of the enzyme. It also shows that the immobilized preparation become thicker
due to attachment of RML on the surface of MWCNTs. In other words, the diameter of
MWCNTs-RML is slightly increased as compared to that of MWCNTs-COOH. These
morphological characteristics are similar to the images reported in the previous study of
enzyme organophosphate hydrolase on functionalized carbon nanotubes 32.
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Fig.2. FT-IR spectra of a) MWCNTs-COOH, b) MWCNTs-RML, c) Gr-COOH and d) Gr-
RML.
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In the FTIR spectrum of MWCNTs-COOH (Fig. 2 (a)), the peaks at 1764 and 3291cm-1 are
due to the C=O and O–H stretching vibrations of the carboxylic groups. For the MWCNTs-
RML (Fig. 2 (b)) the C=O stretching frequencies of the amide bond formed by the
functionalization reaction appeared at 1666 and 1538cm-1 which clearly indicate the
successful attachment of RML on MWCNTs. Furthermore the bands at 2928 and 2863cm-1
can be clearly assigned to the C–H stretching vibrations of the attached RML on MWCNTs.
The successful immobilization of RML on MWCNTs-COOH was further confirmed by
thermogravimetric analysis (Fig. 3 (a, b)). While the MWCNTs-COOH only showed a minor
mass loss at 492°C, the mass loss of 29% in sample MWCNTs-RML at the same temperature
corresponds to removal of the enzyme molecules bond to the support surface. Similar
observation has reported by Shi et al. 33 for MWCNTs–lipase preparations.
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a
b
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c
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Fig. 3. TG analysis of a) MWCNTs-COOH, b) MWCNTs-RML, c) Gr-COOH and d) Gr-
RML.
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Fig. 4 (a, b) shows typical XRD patterns of MWCNTs-COOH and MWCNTs-RML. The first
diffraction peak for the samples at the angle (2θ) of 25.70◦ can be indexed as the C (0 0 2)
reflection of the hexagonal structure 34, 35. The other characteristic diffraction peaks at 2θ of
about 44◦, 64.48◦ and 78.01◦ are associated with C (1 0 0), C (0 0 4) and C (1 1 0)
diffractions, respectively 34. There is no significant difference between the two patterns of
MWCNTs-COOH and MWCNTs-RML, indicating that the carbon crystallite structure of
MWCNTs was not affected by the enzyme loading in immobilization process.
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333
Fig. 4. XRD analysis of a) MWCNTs-COOH, b) MWCNTs-RML, c) Gr-COOH and d) Gr-
RML.
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Raman scattering was used for investigation of disorder in the carbon skeleton of MWCNTs-
COOH before and after immobilization of RML (Fig. 5 (a, b)). The most characteristic peaks
at 1338, 1565 and 2700cm-1, in the Raman spectra of both samples, correspond to graphite D-
band, G-band and 2D band, respectively. The G-band at 1338 cm-1 can be attributed to sp2-
hybridized carbons of Raman-active E2g mode of graphite while the D-band at 1338cm-1 is
attributed to either sp3-hybridized carbons or to structural defect sites of the sp2-hydridized
carbon network 36. The relative intensity of D to G band (ID/IG) can be used to determine the
degree of disorder in the carbon network. The profiles of Raman spectra of both MWCNTs-
COOH (1.06) and MWCNTs-RML (0.98) are almost the same, suggesting that MWCNTs
retains its basic structural properties during the immobilization of RML.
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346
Fig. 5. Raman spectra of a) MWCNTs-COOH, b) MWCNTs-RML, c) Gr-COOH and d) Gr-
RML.
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3.4. Characterization of the immobilized derivative of Gr-COOH
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Functionalized graphene nanosheets before and after immobilization was also systematically
characterized using SEM, IR, TGA, XRD and Raman spectroscopy. Fig. 1 (c, d) shows the
SEM image of carboxylated-graphene nanosheets which confirms that there is no damage
effect on the graphene nanosheets even under the strongest oxidation treatment of using
mixtures of nitric acid sulfuric acid. The SEM micrograph of the obtained Gr-COOH also
shows irregular particles with smooth surfaces. The changes in the surface morphology of the
Gr-COOH after covalent immobilization of the enzyme are also observed.
In the IR spectrum of Gr-COOH (Fig. 2 (c)), the peak at 3312cm−1 can be attributed to the –
OH vibration stretching. Besides, it also shows other bands at 1662cm−1 for carboxyl groups
and 1419cm-1 for C–O. In the immobilized derivative, the strong bands at 1667 and 1540cm−1
are according to N–H in-plane stretching of the amide I and amide II presented 37. In addition,
appearance of C–N stretching frequency at 1247cm−1 and aliphatic C–H stretch band at 2855-
2940cm−1 clearly demonstrated successful functionalization of the support by RML (Fig. 2
(d)).
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The successful immobilization of RML on graphene nanosheets can also be concluded from
TGA curves. As shown in Fig. 3 (c, d), comparing with Gr-COOH, Gr-RML shows much
lower thermal stability. Its main mass loss takes place around 231–478°C, which can be
attributed to the removal of the enzyme from the surface of the support. The observed small
mass loss over the temperature range between 478 and 705°C can also be assigned to the
removal of more stable functional groups.
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The XRD pattern of Gr-COOH gives a distinguishable (002) graphite peak at 26° together
with three other peaks at 44.22°, 54.41° and 64.43° with low intensities. As Fig. 4 (d) shows,
after immobilization of the enzyme, the XRD profile is not changed, suggesting that the
crystalline structure of the support remained almost unchanged after the enzyme attachment.
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Raman spectroscopy was also used as an effective tool to investigate the structural
characteristics and properties of Gr-RML. Fig. 5 (c, d), shows a moderate D-band (~1348cm-
1), a moderate G-band (~1570)cm-1 and a strong 2D band (~2714cm-1) in the Raman spectrum
of Gr-COOH. On the other hand three Raman bands are observed for Gr-RML: a band with
relative intensity at 1452cm-1 (D-band), a band at 1509cm-1 (G-band) and a band with high
intensity in 2428cm-1 region. Comparing with Gr-COOH (1.19), the D/G intensity of Gr–
RML (1.02) is almost similar; implying that immobilization of RML by the Ugi four-
component reaction has no significant effect on the graphite structure of Gr-COOH.
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3.5. Thermal and co-solvent stability of the free and immobilized derivatives
382
Organic solvents can cause conformational changes in enzyme structure, thus changing its
catalytic properties. This is due to the fact that a few amounts of water molecules are required
for enzymatic function. Organic solvents, particularly those having log P values below 2,
strongly distort this essential water-enzyme interaction, thereby inactivating or denaturing the
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enzyme structure. In order to investigate the effect of immobilization procedure on the
stability of RML, seven water-miscible solvents (10% and 20% of 1-propanol, 2-propanol,
dioxane, THF, Acetonitrile, DMSO and methanol) with log P < 2 were used. Both
immobilized derivatives showed improved stability compared to the soluble enzyme,
retaining 100% of their initial activities after 24h of incubation in the presence of 10% of the
seven organic solvents, while the soluble enzyme retained 68-80% of its initial activity at this
condition (results not shown).
387
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393
120
120
45
45
50
80
80
50
55
40
0
55
60
40
0
60
0
10
20
0
10
20
time (h)
time (h)
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395
a
b
80
40
0
Gr-RML
Free RML
MWCNTs-RML
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c
Fig. 6. Thermal (a, b) and Co-solvent (c) stability of free RML and immobilized preparations.
398
Further investigation was performed using 20% of all solvents in order to determine the effect
of increasing the organic solvents percentage on stability of both free enzyme and the
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immobilized derivatives. Fig. 6 (c) shows the results of the enzyme inactivation in the
presence of 20% of seven chosen organic solvents. Both immobilized derivatives
(MWCNTs-RML and Gr-RML) retain 100% of their initial activities after 24h of
incubation in the presence of 20% of methanol, while the free enzyme loses 80% of its
activity at the same condition. However increasing the percentage of methanol to 50% leads
to almost 75% and 90% decrease in activity of MWCNTs-RML and Gr-RML, respectively.
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Incubation of the soluble RML in the presence of dioxane 20% leads to 72% decrease in
initial activity of RML, while immobilized derivatives are stable (more than 95%) at the
same condition. On the other hand, while Gr-RML is quite stable in presence of 20%
acetonitrile, MWCNTs-RML and free RML keep 75% and 20% of their activities after 24h of
incubation, respectively. The most promising result obtained from investigation on co-solvent
stability of MWCNTs-RML. This derivative retains 71-100% of its initial activity after 24h
incubation in the presence of 20% of the seven organic solvents.
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Thermal stability of free RML and its immobilized derivatives (MWNTs-RML and Gr-RML)
was also investigated in various temperatures (Fig. 6 (a, b)). Complete profile of thermal
stability of the free RML has been reported in our previous study 23. The soluble RML is
relatively unstable and loses 80% of initial activity at 50°C after 6h of incubation. Rapid
inactivation of RML was also observed by increasing the temperature to 55°C after 2.5h of
incubation. As results show immobilization of RML on MWNTs-COOH leads to significant
improvement in thermal stability of the immobilized derivative (Fig. 6(a)). MWNTs-RML is
quite stable and retains 100% of its initial activity after 24h of incubation at 45°C.
Furthermore, the results reveal retaining about 93% and 80% of initial activity MWNTs-
RML during 24h of incubation at 50°C and 55°C, respectively. By increasing the temperature
to 60°C only 24% of activity of MWNTs-RML disappears during 8h of incubation which
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clearly confirms the positive effect of the immobilization procedure on stability of RML. The
improved thermal stability is also observed for RML immobilized on functionalized Gr-
COOH. As can be seen from Fig. 6b, Gr-RML has great thermal resistance by retaining 100%
of its activity after 24h of incubation at 45-50°C. This derivative also shows about 94% of its
activity after 24h of incubation at 55°C. Furthermore, covalent immobilization of RML
increases the stability of the obtained derivative with retaining 65% activity after 8h
incubation at 60°C. Complete inactivation of the derivative is observed after 24h incubation
of the derivative at 60°C. In comparision, the thermal stability of MWNTs-RML is more
improved compared to immobilized preparation obtained from immobilization of RML on
Gr, most likely becouse of three dimentional structure of MWNTs which keeps away the
enzyme structure from distorting agents. Another experiment was performed in order to study
thermal stability of the derivatives at 45°C for a period of 5 days. The results showed that
free RML, Gr-RML and MWNTs-RML retain their initial activities in the first four days at
this condition. In the fifth day of incubation 69, 94 and100% of activities were observed for
free RML, Gr-RML and MWNTs-RML, respectively. These results were further confirmed
the positive effect of both immobilization and the structure of the carrier on stability of the
immobilized enzyme.
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3.6. Substrate specificity of free and immobilized RML
442
Three different substrates with acyl chain lengths of C8, C12 and C18 were also used to
investigate the activity and substrate specificity of free and immobilized preparations of RML
by using a pH-stat system at 25°C and pH 8.0 (Fig. 7). The results show that with increasing
the chain length, activity of RML and its immobilized derivatives decreases. The maximum
specific activity of 24890, 17662 and 14970 U/mg was observed for free RML while using
C8, C12 and C18 triacylglycerides as substrate, respectively. Decrease in enzyme activity
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with increasing the chain length was also observed for the immobilized preparations.
Furthermore the results show that with a same triacylglyceride, the specific activity of RML
decreases after immobilization on both supports. The maximum decrease in enzyme activity
(43%) of the immobilized preparations is observed for MWCNs-RML in hydrolyzing C18
triacylglyceride compared to the free RML.
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450
451
452
453
30000
Free RML
20000
Gr-RML
10000
MWCNTs-RML
0
C8
C12
C18
Substrate
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Fig. 7. The activity and substrate specificity of free and immobilized preparations of RML
toward substrates with three different acyl chain lengths
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3.7. Kinetic study and evaluation of optimal pH activity of free and immobilized preparations
of RML
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The optimum pH for free RML is at pH 7.5, whereas the optimum pH of the Gr-RML and
MWCNTs-RML are shifted by 1 and 1.5 units towards the alkaline side, respectively (Fig. 8).
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120
80
40
0
Free RML
Gr-RML
MWCNTs-RML
4
5
6
p7H
8
9
10
461
462
Fig. 8. Optimum pH activity of RML and immobilized preparations
This increase in optimum pH can be explained by the effect of immobilization on alteration
of the microenvironment of the enzyme. A similar increase in the optimum pH value after
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enzyme immobilization has been previously documented 23, 38
.
Kinetic parameters of RML and its immobilized derivatives were determined by
spectrophotometrically measuring the initial reaction rates for each form with varying
amounts of substrate (p-NPB) (Fig. 9).
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Fig. 9. Michaelis-Menten plot for the free and immobilized preparations of RML
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471
Km values for MWCNTs-RML and Gr-RML were 0.44 and 0.23mM, respectively which
were slightly more than that of the free RML (0.18mM). The higher values of Km represent
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lower affinity between enzymes and substrates. The effect of immobilization in increasing the
Km values can be explained by slightly decreased access of the substrate to the active site of
enzyme in the immobilized form 39. The maximum reaction rates (Vmax) of the free RML,
MWCNTs-RML and Gr-RML were 0.08, 0.09 and 0.1mM min−1, respectively. The observed
results are in accordance with the values reported by Zhang et al. 40.
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4. Conclusion
478
Covalent immobilization of RML on carboxylated multiwall carbon nanotubes and
carboxylated graphene nanosheets was performed via a four-component reaction in an
extremely mild condition. This immobilization procedure was performed at room temperature
in water and for the small amounts of offered protein (up to 100mg) was accomplished in a
very short time (15-30min) without significant decrease in specific activity of the enzyme.
This short immobilization time will maximize the survival rate of particularly unstable
proteins. The results showed that the applied approach can be used as a simple, rapid, useful
and versatile method for covalent linkage of biomolecules onto carbon-based solid materials.
This protocol can also be easily used to conjugate any other useful molecules with amine
groups, such as nucleic acids, antibodies and even chemical compounds on carboxylated solid
supports. The leaching experiment confirmed the immobilization of RML on the supports via
irreversible covalent linkage. The results showed strongly increased capacity of MWCNTs-
COOH and Gr-COOH for covalent immobilization of enzymes. The immobilized derivatives
also showed greatly improved enzyme stability compared to the free enzyme.
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Acknowledgment
493
This work was financially supported by the National Institute of Genetic Engineering and
Biotechnology for which the authors are thankful.
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Table of Contents:
One-pot immobilization of RML on carbon-based nanomaterials was performed by using the
Ugi four component reaction in an extremely mild condition