Immobilisation of α-amylase on activated amidrazone acrylic fabric: a new approach for the enhancement of enzyme stability and reusability
immobilisation of α-amylase on activated amidrazone acrylic fabric: a new approach for the enhancement of enzyme stability and reusability
Ahmed R. Al-najada
Yaaser Q. Almulaiky
Reda M. el-Shishtawy
Saleh A. Mohamed
Wesam H. Abdulaal
Sami A. Al-Harbi
Published: xx xx xxxx in this study, amidrazone acrylic fabric was applied as an immobilising support for α-amylase. the immobilised α-amylase was characterised by fourier transform infrared spectroscopy and scanning electron microscopy. Furthermore, the optimum conditions for immobilisation efficiency, immobilisation time, reusability, kinetic parameters and pH, for the immobilisation process were examined. The study demonstrated that with 4% cyanuric chloride, and a pH of 7.0, the highest immobilization efficiency of 81% was obtained. Around 65% of the initial activity was maintained after storage at 4 °C for 8 weeks. The immobilised enzyme retained 53% of its original activity after being reused 15 times and exhibited improved stability compared with the free enzyme in relation to heavy metal ions, pH, temperature and inhibitors. the immobilised enzyme presented kinetic parameters of 2.6 mg starch and 0.65 µmol maltose/mL for Km and Vmax respectively, compared with 3.7 mg starch and 0.83 µmol maltose/ mL for the free enzyme. the improvements in the enzyme's catalytic properties, stability and reusability obtained from immobilisation make amidrazone acrylic fabric support a good promising candidate for bio-industrial applications.
Therefore, for the large-scale industerial use of amylases, their stability needs to be improved via immobilisation
by different physiochemical treatments9. Immobilisation defined as attaching enzymes to a support so that a
reduction or loss of mobility maybe achieved with catalytic activity retention, thereby allowing continuous use
of the enzyme. This approach allows for improved stability of the biocatalyst, easier separation of substrates and
products, and recovery of the enzyme so that it can be used again6,10,11.
Support materials can be categorised as either organic or inorganic, depending on their chemical composition.
The best support materials are inert towards the supported enzymes, low cost, physically resistant to compression,
resistant to microbial attack and hydrophilic with an appropriate surface area, alongside minimal restriction of
substrate diffusion. Immobilising support plays a substantial role in the immobilised enzyme’s performance. If
immobilisation is to be achieved through the use of a surface binding technique, the bonding strength is strongly
dependent on the physical link or chemical type used12. Acrylic fabrics are commonly used as enzyme supports,
such as Eupergit C, which has been successfully used in industry for covalent attachment to many enzymes13.
Hydrazine is a ligand that can be used to create complexes. It is an area of increasing research interest and
has been studied by Audrieth and Ogg 14, Bottomley15, Schmidt16 and Heaton17. Hydrazine is also of great
academic interest because of its versatility, which is attributed to the existence of two free electron pairs and four
replaceable hydrogen atoms. Its difunctional nature allows for many uses, including dibasic ligand reactions,
which gives polymeric amides that can be drawn into fabrics. It can also be used in polymeric materials because
of its cross-linking properties. In this study, amidrazone acrylic fabric activated with different concentrations of
cyanuric chloride is used as a support for α-amylase by cross-linking methods. Moreover, the immobilised and
free enzymes are characterised and compared. This is the first investigation of the cross-linking immobilisation of
α-amylase on amidrazone acrylic fabric.
Materials and Methods
α-Amylase. α-Amylase from Bacillus subtilis, and other materials were purchase from Sigma-Aldrich and
then used as obtained.
Acrylic fabrics. A 1/1 woven acrylic acquired from El-Mehalla Co. in Egypt was used in this work. The fabric
was washed with ethanol and then dried at ambient temperature.
α-Amylase assay. To determine the activity of the immobilised and free enzymes, we opted for a technique
outlined by Miller18. A 1 cm2 piece of activated acrylic fabric was used for the routine assay of the activity of
immobilised enzyme. Immobilised α-amylase was removed after 30 minutes’ incubation with 1% starch (1 mL) at
37 °C and 1 mL of dinitrosalicylic acid (DNS) was included to help develop colour. The tube containing this
mixture was then incubated for 10 minutes in a boiling water bath before cooling then the absorbance was recorded
at 560 nm.
preparation of support. Acrylic fabric of a certain weight was pretreated with 1% hydrazine hydrochloride
in the presence of 2% ammonium acetate at a liquor ratio of 50:1 at 85 °C for 60 minutes with shaking. Then, the
modified acrylic fabric was completely rinsed with water and air-dried. The pretreated fabric was then activated
with cyanuric chloride at different levels (2–6% w/w) in an acetone/water mixture (50% v/v) at pH 4.0 and 0 °C
for 2 hours. Subsequently, the fabric support was thoroughly rinsed with cold water and, acetone then dried in a
ventilated refrigerator prior to α-amylase immobilisation.
immobilisation procedure. The treated fabric was used as a support for the immobilisation of α–amylase
dissolved in 50 mM Tris–HCl buffer at pH 8.0, or 7.0, or 50 mM sodium acetate buffer at pH 4.0. The reaction was
carried out at room temperature overnight. The fabric was then allowed to dry at room temperature. To confirm
the progression of the immobilization, the relative activity (%) was determined by using the following equation:
Relative enzyme activity% =
Activity of immobilized enzyme
Initial activity of enzyme
Morphological characterisation. The attenuated total reflectance Fourier transform infrared (ATR –
FTIR) spectra of the immobilised enzyme were obtained using a PerkinElmer Spectrum 100 FTIR spectrometer.
The morphology of the immobilised enzyme was examined with a scanning electron microscope (FESEM, JEOL
Reusability of immobilised enzyme. The reusability was assessed over 15 cycles of repeated usage. The
original activity was assumed as the 100% control so that the percentage activity could be gauged after repeated
Storage stability. The free and immobilised enzymes were stored for 60 days at 4°C. The residual activity
was assayed under the optimum conditions described above.
physicochemical characterisation. Temperatures from 35 to 85 °C and pHs from 4.0 to 8.5 were
investigated to determine the optimal temperature and pH for the free and immobilised α-amylase. Thermal stability
was investigated by assessing the relative activity of free and immobilised α-amylase after 30 minutes of
incubation at temperatures from 35 to 85 °C before the addition of the substrate. The percentage relative activity versus
temperatures was plotted. A Lineweaver-Burk plot with different concentrations of the substrate starch was used
to gauge the Km value.
Ethanol, refulx HN
Acrylic f abric
Influence of metal ions. The impact of metal ions on the activity of free and immobilized α- amylase was
determined through incubation with 5 mM metal ion for 15 minutes before the addition of the starch to the
mixture. The control (100%) was set as the activity of α- amylase in the absence of metal ion.
Effect of metal chelators and inhibitors. The activity of free and immobilised α- amylase was
determined under the influence of Ethylenediaminetetraacetic acid(EDTA), sodium oxalate, sodium citrate and metal
chelators, 1,10 phenanthroline monohydrate, inhibitors 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and
phenylmethylsulfonyl fluoride(PMSF)at a concentration of 2 mM. The enzyme activity without the addition of these
compounds was taken as 100% and the % relative activity in their presence was determined.
Results and Discussion
The existence of surface functional groups and the bonding of α- amylase to the amidrazone acrylic fabric
activated with cyanuric chloride were confirmed by ATR-FTIR spectroscopy and scanning electron microscopy
(SEM). Figure 1 illustrates the method used for the amidrazone activation of the acrylic fabric for the
immobilisation of α-amylase. The treated fabric was user-friendly and had numerous easily derivatised functional groups,
good mechanical and chemical stability, and good capacity for enzyme immobilisation.
The ATR-FTIR spectra of the acrylic, hydrazine-treated, cyanuric-activated and amylase-immobilised samples
are presented in Fig. 2. The first three samples exhibit similar absorption peaks with slight differences, whereas
the spectrum of the amylase-immobilised sample is remarkably different. Characteristic broad bands owing to
overlapped OH and NH groups present in the amylase enzyme are evident at 3279 cm−1 in the immobilised
enzyme sample, while CH stretching vibration mode is observed around 2934 cm−1 in all of the samples19–21.
Additionally, amide bands are also observed for the immobilised enzyme sample at 1646 cm−1 (CO stretch, amide
I), 1553 cm−1 (NH bend, amide II) and at 1224 cm−1 (weak band, CN stretch, amide III). The success of the
hydrazine treatment is clearly indicated by the decrease in the intensity of the nitrile absorption peak in the spectrum
of the hydrazine-treated sample compared with the untreated acrylic fabric as the nitrile group is partially
converted to amidrazone and/or cross-linked amidine (Fig. 1). Furthermore, a clear CN stretching vibration peak is
observed at 969 cm−1 in the hydrazine-treated sample. The presence of carboxylate groups in the acrylic fabric is
also indicated by the C=O peak at 1732 cm−1 and the C–O–C stretching vibration at 1071 cm−1. These peaks were
either shifted or, their intensity changed upon hydrazination, cyanuric activation and α-amylase immobilisation.
Cyanuric activation takes place via nucleophilic substitution reaction with NH and/or NH2 groups present in the
treated samples (Fig. 1), and thus the broad peak centred at 3370 cm−1 would change upon reaction of the treated
sample with cyanuric chloride, as shown in Fig. 2. In addition, the vibrations observed at 850 and 1530 cm−1 for
the C–Cl bond and the triazinyl ring, respectively, could indicate the presence of a dichlorotriazinyl ring. Overall,
the peak changes and the appearance of amylase peaks in the FTIR spectra indicate that the amylase
immobilization was successful.
The morphology of the surface of the untreated acrylic, hydrazine-treated, cyanuric-activated and
amylaseimmobilised samples was studied by SEM (Fig. 3). As can be seen in Fig. 3a, the untreated acrylic fabric exhibited
a dermis-like structure on the surface. Significant differences were observed on the surface of the acrylic fabric
after treatment with hydrazine hydrochloride, as shown in Fig. 3b. The surface structure of the hydrazine-treated
acrylic appears to be more compact and exhibits a smooth surface. Moreover, after treatment with cyanuric
chloride, the morphology of the acrylic fabric was still retained but minor changes could be observed, such as small
cracks and defects, as shown in Fig. 3c. After immobilisation of the enzyme (Fig. 3d), it can be seen that the
enzyme was dispersed on the surface of the acrylic fabric in different locations, indicating the successful
immobilisation of the enzyme.
α-Amylase immobilisation on amidrazone acrylic fabric activated with varying concentrations of cyanuric
chloride (2, 4, 6%) at immobilisation pHsof4.0, 7.0 and 8.0 was evaluated. The maximum immobilisation
efficiency of α-amylase was 81% for the sample activated with 4% cyanuric chloride at an immobilisation pH of7.0
(Table 1). The immobilisation efficiencies decreased in the order: 4%> 2% > 6% cyanuric chloride concentration.
At a high concentration of cyanuric chloride, one would expect that excess enzyme attachment sites, might reduce
the activity of the enzyme owing to changes to its stereochemical configuration. This result is in agreement with
previous reports regarding the effects of cyanuric chloride on enzyme activity22,23. The effect of immobilisation
time on the relative activity of the immobilised enzyme is presented in Fig. 4a. Clearly, the immobilised enzyme’s
activity initially experienced a rapid increase with immobilisation time, reaching a plateau after 8 hours as a result
of saturation. An important factor in industrial use is enzyme reusability. α- Amylase that has been immobilised
on amidrazone acrylic fabric can be separated from the reaction mixture very easily making it possible to reuse
the enzyme multiple times for starch hydrolysis. The activity of the immobilised α- amylase was examined over
15 reaction cycles to determine the reusability (Fig. 4b). As shown in the figure, 53% of the initial enzyme activity
was retained after 15 reuses, which is superior to the 21.4% obtained by Ahmed24. These results indicate that the
immobilised α-amylase has sufficient stability and reusability. The reduction of the enzyme activity upon repeated
usage could be attributed primarily to two factors: the change in the stereochemical structure of the enzyme and
leaching of the enzyme during the recycling process. Moreover, the frequent interaction of the substrate with the
active site of the immobilised enzyme causes distortion of the active site, leading to loss of activity25,26.
To evaluate the storage stability, the activities of the immobilised and free enzymes were evaluated after being
stored at 4 °C for a period of 60 days. The catalytic activity was measured every 15 days. The results showed
that the enzyme’s storage stability was enhanced by the immobilisation (Fig. 5) with the immobilised α-amylase
retaining 65% of its original activity after 8 weeks, while the free enzyme retained 39% of its original activity. Thus,
the immobilisation significantly prevented enzyme deactivation and enhanced the enzyme’s storage stability.
The immobilised and free enzymes exhibited their maximum activity at pH 6.5 and 5.5, respectively (Fig. 6),
in agreement with the results reported from other studies5,27. The change of the optimal pH may be attributed to
immobilized method, support structure and conformation change of enzyme after bounded. The immobilised
α-amylase retained 95 to 54% of its activity at pH range 7.5 to 8.5. For industrial application of α-amylase, it is
important that the immobilised enzyme has good thermal stability28. The results presented in Fig. 7a show that
the immobilised α-amylase has improved thermal stability compared to that of the free enzyme29,30. Free
αamylase exhibited the highest activity at 45 °C, while immobilisation caused this temperature to rise to 55 °C. The
results show that amidrazone acrylic fabric has a protective effect on α- amylase activity at higher temperatures.
The development of many covalent bonds between the support and the enzyme reduces conformational
flexibility, thermal vibrations, and enzyme mobility, while also preventing aggregation and unfolding of the enzyme
protein31. The reduction of the relative activity of the unbound enzyme was reduced by thermal denaturation
at higher temperatures, whereas the immobilised enzyme’s activity reduced more slowly above 55 °C. This is
because the three-dimensional structure of the protein is protected from thermal denaturation by the restriction
of the enzyme mobility upon immobilisation32. Upon varying the temperature in the range of 50–80 °C, the free
enzyme was observed to be more temperature-sensitive than the immobilised one, indicating the usefulness of
immobilisation (Fig. 7b). Similarly, Pereira et al.5 and Mohamed et al.33 reported the optimum temperature for
immobilised α-amylase to be 50 °C. This increase in thermal stability and optimum working temperature for
the immobilised enzyme might be attributed to changes in the stereochemical configuration of the enzyme30,32.
The relation between the rate of the enzymatic reaction and the substrate concentration can be displayed using a
Lineweaver–Burk plot (Fig. 8). The apparent Km for the free and immobilised α-amylase was found to be 3.7 and
2.6 mg starch/mL, respectively. The decrease in Km for the immobilised enzyme shows that the enzyme’s affinity
for starch increased by 1.4-fold as a result of immobilisation, and thus the activity also increased. The α-amylase
molecule might be extended over the supporting surface with an improved orientation, leading to more
available active sites and a higher affinity for the substrate34. The apparent Vmax values for the free and immobilised
α-amylase were found to be 0.83 and 0.65 µ mol maltose/mL, respectively. The fall in Vmax seen after
immobilisation is in line with the mass transfer restriction of the diffusion layer that surrounds the enzyme particles31,
which limits the diffusion of the starch towards amidrazone acrylic fabric-immobilised α-amylase. It can also be
explained through the consistent orientation of the enzyme molecules on the amidrazone acrylic fabric surface,
which leads to a considerable fall in the affinity of the enzyme35. The observed changes to the catalytic
properties could also result from modifications to the three-dimensional protein conformation caused by the enzyme
binding to the support. Similar results were reported by Eslamipour and Hejazi30 and Homaei and Saberi36 for
free and immobilised α-amylase.
Table 2 illustrates the influence of metal ions on the activity of free and immobilised α-amylase. As can be
seen from this table, the immobilised α-amylase is highly stable in the presence of Ca2+, Ni2+ and Zn2+,
retaining 120, 123 and 108% of its activity, respectively. The other metal ions tested had a partial inhibitory effect
This report describes the immobilisation of α-amylase by cross-linking and covalent bonding on acrylic fabric
with amidrazone that has been activated using cyanuric chloride as a new organic support. The immobilisation
of the enzyme on the carrier improved the thermal stability, pH levels and affinity for starch. Furthermore, the
enzyme’s storage stability and reusability were also improved by immobilisation. The immobilised enzyme can be
separated with great ease from the reaction mixture, after which it is can be used again in a fresh enzymatic
reaction. Confirmation that the α-amylase was attached to the treated acrylic fabric was obtained by FTIR and SEM.
In conclusion, amidrazone acrylic fabric has been demonstrated to be an excellent carrier for industrial enzymes
and the authors recommend its use for enzyme immobilisation. It should prove useful for industrial applications
involving biopharmaceuticals, biocatalysts and biotechnology.
Y.Q.A., R.M.E., M.A. and S.A.M. designed the experiment. Y.Q.A., A.R.N., M.A., M.B., A.A., S.A.A. and W.H.A.
performed the experiments and carried out the analysis. All authors read and approved the final manuscript.
Competing Interests: The authors declare no competing interests.
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