Ali Riahi-Madvar

This study aimed to improve the functionality and stability of Lepidium draba peroxidase (LDP) for use in glucose biosensing. Following enzyme purification, covalent binding was optimized on chitosan-coated magnetic nanoparticles (nFe₃O₄-CS). Under the optimal conditions of 0.05 g nFe₃O₄-CS, 2.5% glutaraldehyde, and an 18 h coupling duration at 25 °C, enzyme immobilization efficiency reached 30%. The formation of the immobilized enzyme (IE) was confirmed using FT-IR, XRD, and FE-SEM. Upon immobilization, the enzyme’s affinity and catalytic efficiency for TMB were elevated by 1.7 and 11.0 times, respectively, in comparison to the free enzyme. The time course of stability against heat (at 50 °C) and storage stability (after 60 days at 4 °C) of the IE exhibited an increase of more than 225% and 230% as compared with the free enzyme. Moreover, the reusability of the IE was repeated up to 11 times, retaining 40% residual activity. Finally, the applicability of the IE was compared with that of the free enzyme; data showed linearity of the glucose detection range increased 10-fold compared to the free enzyme, and the assay time decreased 3 times relative to the free one. Overall, the improved stability of the immobilized LDP supports its potential use as a suitable enzyme for biomolecule detection in clinical practice and in various industrial applications.

There was an update made to the original publication

The main advantages of the dried enzymes are the lower cost of storage and longer time of preservation for industrial applications. In this study, the spouted bed dryer was utilized for drying the garden radish (Raphanus sativus L.) root extract as a cost-effective source of the peroxidase enzyme. The response surface methodology (RSM) was used to evaluate the individual and interactive effects of main parameters (the inlet air temperature (T) and the ratio of air flow rate to the minimum spouting air flow rate (Q)) on the residual enzyme activity (REA). The maximum REA of 38.7% was obtained at T = 50 °C and Q = 1.4. To investigate the drying effect on the catalytic activity, the optimum reaction conditions (pH and temperature), as well as kinetic parameters, were investigated for the fresh and dried enzyme extracts (FEE and DEE). The obtained results showed that the optimum pH of DEE was decreased by 12.3% compared to FEE, while the optimum temperature of DEE compared to FEE increased by a factor of 85.7%. Moreover, kinetic parameters, thermal-stability, and shelf life of the enzyme were considerably improved after drying by the spouted bed. Overall, the results confirmed that a spouted bed reactor can be used as a promising method for drying heat-sensitive materials such as peroxidase enzyme.

The improvement in the enzyme activity of Aspergillus flavus urate oxidase (Uox) was attained by immobilizing it on the surface of a Ni-based magnetic metal–organic framework (NimMOF) nanomaterial; physicochemical properties of NimMOF and its application as an enzyme stabilizing support were evaluated, which revealed a significant improvement in its stability upon immobilization on NimMOF (Uox@NimMOF). It was affirmed that while the free Uox enzyme lost almost all of its activity at ~40–45 °C, the immobilized Uox@NimMOF retained around 60% of its original activity, even retaining significant activity at 70 °C. The activation energy (Ea) of the enzyme was calculated to be ~58.81 kJ mol⁻¹ after stabilization, which is approximately half of the naked Uox enzyme. Furthermore, the external spectroscopy showed that the MOF nanomaterials can be coated by hydrophobic areas of the Uox enzyme, and the immobilized enzyme was active over a broad range of pH and temperatures, which bodes well for the thermal and long-term stability of the immobilized Uox on NimMOF.

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.