Materials
Chitosan (Mw 50–90 kDa with DD of 80%) was supplied from Sigma Chemical, Co., St. Louis, USA.
Polyethyleneimine (PEI, 50%, w/v), glutaraldehyde (GA, 50%, w/v), 3,5-dinitrosalicylic acid (DNS98%), and sodium hydroxide 97% (Mw 40 g/mol) were obtained from Sigma-Aldrich, Chemie GmbH, Riedstr. 2, D-89555 Steinheim, Germany. Soluble starch 99% (Mw 342.3 g/mol) and potassium sodium tartrate (Rochelle salt, 99%, Mw 282.23 g/mol) were obtained from WINLAB, UK. Other chemicals were of analytical grade.
Methods
Enzyme production
α-Amylase enzyme was produced under submerged fermentation from isolated strain B. subtilis strain-MK1 as reported in the previous work by Ahmed et al. 2019a, 2019b. The enzyme was partially purified using 60% (v/v) ethanol precipitation; the precipitate was collected by centrifugation, dried, weighed, and used for α-amylase immobilization.
Preparation of chitosan-magnetic nano-particles (Ch-MNP) beads
Magnetic nano-particles (Fe3O4) were prepared according to Mehta et al. (2006) with some modifications. The method depended on mixing ferric and ferrous ions in a 1:2 molar ratio in highly basic solution at elevated temperature. In brief, iron (III) chloride hexahydrate (0.0551 mol) was dissolved in 150 mL of ammoniated water while 0.0275 mol of iron (II) chloride tetrahydrate was dissolved in 150 mL of ammoniated water. Then, the two solutions were mixed in a 500 mL conical flask and placed in a temperature-controlled water bath equipped with a magnetic stirrer. A sodium hydroxide aqueous solution (12.8 g in 120 mL of distilled water) was then added with a flow rate of 10 mL/min while continuously stirring at 80 °C, and the reaction was continued for 60 min under the same conditions. The resulting Fe3O4 particles were washed 3 times repeatedly with 500 mL of distilled water until neutral pH using magnetic field separation and finally kept in 150 mL distilled water in the fridge for further treatment.
Chitosan was dissolved in distilled water to produce 2% (w/v) solution then mixed with a solution of magnetic nano-particles (Fe3O4). After that, the polymer solution was sprayed into cross-linking solution of NaOH 5% (w/v), through a nozzle of 300 μm using the Inotech Encapsulator. The prepared beads were hardened in cross-linking solutions for 3 h. To modify the gel beads for covalent immobilization of enzyme, the gel beads were soaked in a solution of 4% (v/v) polyethylenimine (PEI) at pH 9.5 for 3 h, followed by soaking in glutaraldehyde (GA) 2.5% for 3 h, and after washing, the gel beads was ready for immobilization (Yuan et al. 2016).
Enzyme binding to activated beads
Partially purified α-amylase from B. subtilis strain-MK1 was immobilized by covalent binding on Ch-MNP. This was performed by mixing 2 mL of the partially purified α-amylase (200 U) with 1 g of the activated beads. The mixture was left for 24 h at 4 °C, and then the beads were washed twice with distilled H2O and were used for enzyme assay (Abdel Wahab et al. 2018).
Determination of α-amylase activity
α-Amylase activity was done according to Sajjad and Choudhry (2012) by mixing 0.5 ml of 1% soluble starch in 0.1 M phosphate buffer (pH 7.0) with 0.5 mL of the partially purified enzyme or 0.2 g of immobilized enzyme and was incubated for 30 min at 40 °C. The reaction was stopped by adding 1 mL of dinitrosalicylic acid (DNS) reagent and kept on boiling water bath for 10 min (Miller 1959), and the color absorbance was read at 540 nm. One unit of enzyme activity (U) is defined as the amount of enzyme that liberated 1 μmol of reducing sugar as glucose/min under assay conditions. All the experiments were performed in triplicate, and the results were expressed as mean values. Immobilization yield (IY%) was calculated according to Wang et al. (2015) as following:
$$ \mathrm{IY}\ \left(\%\right)=I/\left(A-B\right)\times 100 $$
where I is the total activity of immobilized enzyme, A is the total activity offered for immobilization, and B is the total activity of unbounded enzyme.
Optimization of beads modifications using statistical design
Response surface methodology (RSM) based on central composite design (CCD) was used to determine the optimum level of four important factors for beads modification of Ch-MNP beads. These factors include PEI percent (%) (W), PEI activation time (X), GA percent (%) (Y), and GA activation time (Z). These factors were tested at three levels as, low (− 1), central (0), and high (+ 1), resulting in experimental design of 25 experiments with respect to mean of IY (%) of α-amylase on Ch-MNP beads as response. The experimental data were analyzed by the response surface regression procedure to fit the second order polynomial of the equation:
$$ \mathrm{IY}={\beta}_0+\varSigma {\beta}_{\mathrm{i}}{X}_{\mathrm{i}}+\varSigma {\beta}_{\mathrm{i}\mathrm{i}}{X_{\mathrm{i}}}^2+\varSigma {\beta}_{\mathrm{i}\mathrm{j}}{X}_{\mathrm{i}}{X}_{\mathrm{j}} $$
where IY represents response, β0 is the interception coefficient, βi is the coefficient of the linear effect, βii is the coefficient of quadratic effect, βij is the coefficient of the interaction effect, and XiXj are the independent variables which influence the response variable (IY). All the experiments were performed in triplicate, and the results were expressed as mean values. The independent variables of the experimental design were optimized and interpreted using the (JMP) statistical software.
Statistical analysis of data
Statistical analysis of the model was carried out according to the analysis of variance (ANOVA). The quality of the fit of the polynomial model equation was assessed by determining the R2 coefficient and the adjusted R2 coefficient. Also, the significance of statistical and regression coefficient were checked with F test and P value, respectively. The validation of the model was checked by the comparison of experimentally obtained data with the predicted values, and the prediction error was calculated. Three-dimensional (3D) surface plots and corresponding contour plots were constructed to explain the effect of the independent variables on the responses (IY).
Fourier transforms infrared (FTIR) spectroscopy analysis
The FTIR absorption spectra of Ch-MNP/PEI/GA (beads), free α-amylase enzyme, and Ch-MNP/PEI/GA/Enz (immobilized enzyme) were measured by FTIR spectroscopy attenuated total reflection (ATR) mode Bruker VERTEX 70/70v model using the KBr disk technique. This test was performed to detect the presence of the new functional group and carbonyl group formed at all different formulas. The reaction began by mixing 2% (w/w) of the sample with dry KBr. The mixture was ground into a fine powder using an agate mortar before it was compressed into a KBr disk under a hydraulic press at 10,000 psi. Each KBr disk was scanned over a wave number range of 400–4000 cm−1, with a resolution of 4 cm−1, and the characteristic peaks were recorded.
Scanning electron microscope (SEM)
These investigations were performed in order to describe the morphological changes on the beads surface before and after conjugation with the enzyme. Morphological examinations on the surface of Ch-MNP and Ch-MNP/PEI/GA/Enz were carried out using scanning electron microscopy (SEM, Quanta 250 FEG, accelerating voltage 200 V–30 kV, FEI Company, Thermo Fisher Scientific).
Application of Ch-MNP/PEI/GA/enzyme in baking process
For studying application of immobilized α-amylase in baking process, dough was prepared by using 50 g wheat flour, 1.0 g baker’s yeast, 1.0 g Ch-MNP/PEI/GA/Enz (200 U), and 5 mL of H2O (Ahmed et al. 2016). Dough-raising was observed carefully after incubation at room temperature (~ 35 °C) for 2 h and was compared with control (dough without enzyme). After each run, the beads were washed with sodium phosphate buffer (0.1 M, pH 8.0) to remove any residual substrate and reused to start a new run.