Support structure predominantly influences immobilization performance. Thus, particle size analysis of magnetite-cornstarch nanoparticles (MCNs) was performed under different conditions, showing the influence of temperature (room temperature, 50oC, 65oC, and 80oC), molar ratios of ferrous and ferric ions (2:1, 1:1, and 2:3), and cornstarch quantity (0.8 g, 1 g, and 1.2 g) by ZetaSizer. The analysis results showed that the smallest particle distribution was obtained at 65oC (Fig. 2A). The smallest particle sizes were obtained at 2:3 molar ratio of iron ions where the size
was less than 100 nm, and also at 1 g cornstarch as 90 nm (Figs. 2B and 2C). The small size of magnetic nanoparticles resulted in higher surface area in enzyme immobilization .
SERS analysis of glutaraldehyde-modified MCNs was carried out using the surface-enhanced raman spectroscopy (SERS) technique, determining the interactions among starch, magnetite, and glutaraldehyde. SERS analysis results showed that the characteristic raman peaks were determined at 478 cm− 1 and 2917 cm− 1 for cornstarch (Fig. 3A), and 668 cm− 1 for magnetite (Fig. 3B). The peaks of MCNs were determined at the specific points of cornstarch and magnetite (Fig. 3C). For glutaraldehyde-modified MCNs, the number of peaks was increased between 0-1000 cm− 1 of raman shifts, indicating that glutaraldehyde was bound to the MCN (Fig. 3D).
Free esterase was heterologously expressed in Escherichia coli BL21 (DE3) and purified before its immobilization. The enzyme purity was displayed by SDS-PAGE (Fig. 4). The immobilization of the thermoalkalophilic recombinant esterase was performed on glutaraldehyde-modified MCNs. The results showed that immobilization yield and entrapment efficiency were found as 74% and 82%, respectively.
The characterization of the immobilized thermoalkalophilic esterase was performed investigating some parameters such as the influence of pH, temperature, various chemicals, thermostability, and operational stability.
The temperature effect for free and immobilized esterase was studied in a broad range of temperature (25 to 90oC). This analysis showed that both free and immobilized esterase in glutaraldehyde-modified MCNs exhibited maximum activity at 65oC (Fig. 5A). Thus, immobilization of the esterase enzyme in glutaraldehyde-modified MCNs did not change the optimal reaction temperature giving the highest catalytic activity. In one previous work, it was found that the same esterase immobilized using entrapment technique in silicate-coated Ca-alginate beads had an optimum working temperature of 70oC, slightly higher than that of in the present study . There have been several studies on various esterases immobilized by magnetic nanoparticles (MNPs) in the literature. Accordingly, a free esterase of Bacillus pumilus exhibited maximum activity at 37oC for free enzyme and 45oC for its immobilized form on silane functionalized superparamagnetic nanoparticles (SNPs) . Another free and immobilized Zunongwangia sp. esterase using Fe3O4 ~ cellulose nano-composite optimally worked at 30oC and 35oC, respectively . Also, a free Mucor miehei esterase and its immobilized form on core-shell magnetic beads through adsorption and covalent binding showed an optimum temperature at 40oC and 50oC, respectively . Similar to the present study, one report has shown that Pseudomonas putida IFO12996 esterase immobilization by MNPs exhibited a similar optimal working temperature compared to its free form . In line with this, having a similar optimum temperature of the immobilized and free esterase has been also shown in some reports using different support materials [45, 46].
The pH effect for free and immobilized esterase was investigated in the interval of pH 4 and 11. The analysis showed that both free and immobilized esterase in glutaraldehyde-modified MCNs showed the highest activity at pH 9, exhibiting a similar pH effect profile (Fig. 5B). A previous study demonstrated that immobilization of the same esterase in silicate-coated Ca-alginate beads reduced the optimal pH by one unit, exhibiting maximum activity at pH 8 . Most of the esterase immobilization works using MNPs did not alter the optimum pH points compared to the free esterases as in the present study [35–37]. Only one study on Mucor miehei esterase immobilization using core-shell magnetic beads enhanced the optimal working pH as much as one unit, relative to the free esterase .
Thermostability of free and immobilized esterase was studied in a temperature range of 40-80oC for one hour of incubation. The results showed that the immobilized esterase possessed a maximum residual activity at 65oC, higher than that in the free form of the enzyme. Also, the residual activity of free esterase dramatically reduced to 60% and 6% at 70oC and 80oC, respectively. Nevertheless, the immobilized form of the esterase highly kept its residual activity, showing 90% at 70oC and 65% at 80oC after 1 h of incubation (Fig. 6). In previous work, the same thermoalkalophilic esterase entrapped by silicate-coated Ca-alginate beads possessed approximately 60% of residual activity at 80oC upon one hour of incubation , showing slightly lower thermal stability compared to the present study. This situation could be associated with esterase position in the immobilization support material. The conformational change of esterase might be constricted by the immobilization matrix without temporarily affecting under denaturant conditions such as high temperatures. Similar to the present study results, three reports have shown that esterase immobilization on different MNP supports including core-shell magnetic beads , silane functionalized superparamagnetic nanoparticles (SNPs)  and Fe3O4 ~ cellulose nano-composite  improved thermal stability relative to their free esterases. Only one study has reported that the esterase immobilization process on MNPs did not change the enzyme thermostability .
The pH stability was investigated in a range of pH (4–12) upon 1 h of incubation for immobilized esterase in the present study. The results showed that the residual activity of the immobilized enzyme was mostly conserved at alkaline pH points (pH 8–12) after 1 h of incubation, whereas it reduced at acidic pH points (pH 4–6) (data not shown).
The influence of various chemicals on free and immobilized esterase was studied under different metal ion conditions (1 mM CaCl2, 1 mM ZnCl2, 1 mM MgCl2, and 1 mM CuSO4), as well as 1% SDS. This analysis demonstrated that ZnCl2, to some extent, enhanced the immobilized esterase activity, while the other metal ions did not change, except CuSO4 reducing its activity by 10% (Fig. 7). In literature, there have been some reports about the effect of metal ions on esterase immobilized by different support materials. Regarding this, the activity of immobilized esterase (Lx-EstBASΔSP) was slightly decreased by Zn2+ and increased by Mg2+ . Also, Cu2+ enhanced and Ca2+ sharply reduced another immobilized hNF-NmSGNH1 esterase on hybrid nanoflowers . The present study demonstrated that SDS inactivated the immobilized esterase activity (Fig. 7). In literature, there have been several reports acquiring similar findings to the present study [36, 47–49].
The operational stability of an immobilized biocatalyst is a significant factor for the enzyme utilization in large-scale processes since it declines the operation price. For this reason, the operational stability of the immobilized esterase was analyzed up to seven biocatalyst reaction cycles in 0.1 M Tris-HCl buffer (pH 8.0) at 90 rpm and 55oC for 5 min using pNPA substrate. The specific activity was determined after each cycle during the biocatalyst reaction. The analysis demonstrated that the immobilized esterase kept its residual activity of 75% after three sequential cycles, suggesting that it possesses favorable operational stability (Fig. 8). In one previous study, the same esterase in silicate-coated Ca-alginate beads kept above 72% of the esterase activity following three subsequent cycles . Similar findings have been reported about immobilized esterases on various support materials including MNPs. They have possessed a residual activity of above 70% next three sequential cycles [35, 47–51].
The surface morphology of MCNs and also esterase immobilized onto MCNs was monitored by scanning electron microscopy (SEM) at magnitudes of 2500x and 40x (Fig. 9). Scanning Electron Microscopy (SEM) micrographs showed that MCNs were spherical shape, uniform, and well dispersed. Esterase immobilized MCNs were displayed similar morphology as free MCNs having compact structures.