Immobilisation of α-amylase on activated amidrazone acrylic fabric: a new approach for the enhancement of enzyme stability and reusability

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 treatments 9 . 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 again 6,10,11 . 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 used 12 . Acrylic fabrics are commonly used as enzyme supports, such as Eupergit C, which has been successfully used in industry for covalent attachment to many enzymes 13 .
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 , Bottomley 15 , Schmidt 16 and Heaton 17 . 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 Miller 18 . A 1 cm 2 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 100 = × 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 7600).
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 use.
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 K m value.
www.nature.com/scientificreports www.nature.com/scientificreports/ Influence of metal ions. The impact of metal ions on the activity of free and immobilized αamylase 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 αamylase in the absence of metal ion.

Effect of metal chelators and inhibitors.
The activity of free and immobilised αamylase 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 αamylase 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 samples [19][20][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 www.nature.com/scientificreports www.nature.com/scientificreports/ 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 NH 2 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 activity 22,23 . 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 αamylase 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 Ahmed 24 . These results indicate that the immobilised α-amylase has sufficient stability and reusability. The reduction of the enzyme activity upon repeated www.nature.com/scientificreports www.nature.com/scientificreports/ 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 activity 25,26 .
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 studies 5,27 . 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 stability 28 . The results presented in Fig. 7a show that the immobilised α-amylase has improved thermal stability compared to that of the free enzyme 29,30 . 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 αamylase 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  www.nature.com/scientificreports www.nature.com/scientificreports/ protein 31 . 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 immobilisation 32 . 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 enzyme 30,32 . 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 K m for the free and immobilised α-amylase was found to be 3.7 and 2.6 mg starch/mL, respectively. The decrease in K m 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 substrate 34 . The apparent V max values for the free and immobilised α-amylase were found to be 0.83 and 0.65 µmol maltose/mL, respectively. The fall in V max seen after immobilisation is in line with the mass transfer restriction of the diffusion layer that surrounds the enzyme particles 31 , 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 enzyme 35 . The observed changes to the catalytic properties could also result from modifications to the three-dimensional protein conformation caused by the enzyme  www.nature.com/scientificreports www.nature.com/scientificreports/ binding to the support. Similar results were reported by Eslamipour and Hejazi 30 and Homaei and Saberi 36 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 Ca 2+ , Ni 2+ and Zn 2+ , retaining 120, 123 and 108% of its activity, respectively. The other metal ions tested had a partial inhibitory effect

Metals ions
Free α-amylase Immobilised α-amylase  Table 2. Effect of metal ions (5 mM) on the activity of free and immobilised α-amylase. Each entry is the mean of three experiments ± S.E.   Table 3. Effect of inhibitors (2 mM) on the activities of the free and immobilised α-amylase. Each entry is the mean of three experiments ± S.E.