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Starch hydrolysis to maltose by nano-magnetic combined cross-linked enzyme aggregates of α-amylase and maltogenic amylase (NM-Combi-CLEAs) is an important step to open new perspectives for special food and pharmaceutic production. Improvement of mass transfer, thermostability, functional specificity, and reusability of combined enzymes was performed. The obtained results exhibited that, 1:9 ratio of α-amylase/maltogenic amylase, use of tert-butanol as precipitant, 2 mM glutardialdehyde, 1:0.75 ratios of combined enzymes to lysine, 20 h crosslinking at 3-4 °C are well-suited conditions. The dynamic light scattering (DLS) results implied that the nanomagnetites diameter was about 81.9-88.9 nm, with polydispersity index (PDI) of 0.242 and a Ȥ-potential of -21 mV. Moreover, the particle size, PDI, and Ȥ-potential of NM-Combi-CLEAs were around 99.6 nm, 0.088, and -32 mV respectively. The NM-Combi-CLEAs kept 80.4% of its original activity after 10 cycles, its Km value exhibited about 1.5 folds reduction with about 1.5 times enhance in thermostability at 95 °C than free one. Immobilization activity yield revealed about 84% of activity retaining by NM-Combi-CLEAs strategy. Accordingly, this efficacious nanobiocatalyst with high thermostability and reusability recommended for starch conversion to maltose.

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In this work, the structural thermostabilization of the characterized nanomagnetic cross-linked enzyme aggregates of naringinase have been considered. Comparisons have been made between free and immobilized enzyme by the determination of temperature-dependent half-lives (t1/2), energy barriers of thermal inactivation (Ea(in)) process, and thermodynamic parameters (ΔH*, ΔG*, and ΔS*) in a storage thermostability approach. Samples of NM-NGase-CLEAs were treated at different temperatures in the range of 40-80 °C for 90 min. The Km values of immobilized enzyme was reduced about 10.7 folds compared to the free one. The catalytic efficiency (kcat/Km) was raised about 10.5 folds after immobilization. Enzyme half-life (t1/2) of NM-NGase-CLEAs increased from 18.7 to 52.9 min (about 3 folds) at 80 °C. The thermodynamics study indicated that Ea(in) of the free enzyme increased from 38.51 to 49.14 (KJ·mol-1) and ΔH* increased from 35.57 to 46.20 (KJ·mol-1) after immobilization, which indicates an increase in the thermostability of this multimeric enzyme after nanomagnetic CLEAs fabrication. The NM-CLEAs of naringinase preserved 73% of its original activity after 10 cycles, which implies strong operational stability. Thus, the developed method for nanomagnetic CLEAs preparation has provided an efficient and simple approach for the productive and reusable nanobiocatalyst together with ease in enzyme handling.

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Magnetic nanoparticles (NPs) were functionalised with soy protein isolate (SPI) and bovine serum albumin (BSA) for inulinase immobilisation. The results revealed the nanomagnetite size of about 50 nm with a polydispersity index (PDI) of 0.242. The average size of the SPI NPs prepared by using acetone was 80-90 nm (PDI, 0.277), and SPI-BSA NPs was 80-90 nm (PDI, 0.233), and their zeta potential was around -34 mV. The mean diameter of fabricated Fe3O4@SPI-BSA NPs was <120 nm (PDI, 0.187). Inulinase was covalently immobilised successfully through glutaraldehyde on Fe3O4@SPI-BSA NPs with 80% enzyme loading. Fourier transform infrared spectra, field emission scanning electron microscopy, and transmission electron microscopy images provided sufficient proof for enzyme immobilisation on the NPs. The immobilised inulinase showed maximal activity at 45°C, which was 5°C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilised enzyme was shifted from 6 to 5.5. Thermal stability of the enzyme was considerably increased to about 43% at 75°C, and Km value was reduced to 25.4% after immobilisation. The half-life of the enzyme increased about 5.13-fold at 75°C as compared with the free form. Immobilised inulinase retained over 80% of its activity after ten cycles.

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In this research, the preparation and characterization of a novel biocatalyst comprising nano-magnetic cross-linked enzyme aggregates of naringinase (NM-NGase-CLEAs), which was covalently bounded to lysine-assisted magnetic nanoparticles, were studied. The Schiff base formed between ɛ-amino groups of the lysine residues and aldehyde groups of glutaraldehyde was reduced by ascorbic acid. Among the six different precipitants, tert-butanol performed the best for naringin hydrolysis. The optimal conditions for the immobilization process required 10 mM glutaraldehyde, 1:10 ratio of lysine/enzyme, and 3 h crosslinking at 3-4 °C. The morphology of the NM-NGase-CLEAs implied a non-uniform, semi-pyramid and semi-cubic rods. The dynamic light scattering (DLS) results showed that the nanomagnetite particle size was around 81.9-96.5 nm, with a polydispersity index (PDI) of 0.238. After NM-NGase-CLEAS formation, the particle size was reduced to around 13.2-15.3 nm, with PDI of 0.177, respectively. Moreover, the Ȥ-potential of -28 mV also confirms the improvement of CLEAs stability. The NM-NGase-CLEAs kept 73% of its original activity after 10 cycles, which proposes strong operational stability. In conclusion, the NM-NGase-CLEAs are thermo-stable, reusable, and efficient nanobiocatalyst for debittering of citrus juices.

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Inulinase can produce a high amount of fructose syrup from inulin in a one-step enzymatic process. Inulinase from Aspergillus niger was immobilized covalently on Fe3O4 magnetic nanoparticles functionalized with wheat gluten hydrolysates (WGHs). Wheat gluten was enzymatically hydrolyzed by two endopeptidases Alcalase and Neutrase and related nanoparticles were prepared by desolvation method. Magnetite nanoparticles were coated with WGHs nanoparticles and then inulinase was immobilized onto it using glutaraldehyde as crosslinking agent. Parallel studies employing differential scanning calorimetry and field emmision scanning electron microscopy were carried out to observe functional and structural variations in free inulinase during immobilization. Optimum temperature of immobilized inulinase was increased, while, pH and Km values were decreased compared to free enzyme. Overall, a 12.3 folds rise was detected in enzyme half-life value after Immobilization at 75 °C and enzyme preserved 70% of its initial activity after 12 cycles of hydrolysis with 75% of enzyme loading.

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The structural and storage and functional thermostabilization of endo-inulinase (EC 3.2.1.7) through semi-rational modification of surface accessible lysine residues by pyridoxal-5'-phosphate (PLP) and ascorbate reduction have been explored. Improved stability was observed on modifications in the absence or presence of inulin, which indicates storage or functional thermostabilization, respectively. Comparisons have been made between non-modified and modified enzyme by the determination of Tm as an indicator of structural stability, temperature-dependent half-lives (t1/2), energy barrier of the inactivation process, and thermodynamic parameters (ΔH, ΔG, and ΔS) in a storage thermostability approach. These parameters coincided well with the observed stabilization of the engineered enzyme. Moreover, relative activities with sucrose and inulin were determined for non-modified and modified endo-inulinases at different temperatures. A comparison of the sucrose-to-inulin ratios of the initial rate of hydrolysis as an indicator of substrate specificity revealed about twofold improvement in inulinase versus sucrose activity by enzyme modification. Molecular dynamics simulations and molecular docking approaches were employed to explain the observed structural and functional thermostabilization of endo-inulinase upon modification. We hypothesize the establishment of intramolecular interactions between the covalently attached PLP-Lys381 and Arg526 and Ser376 residues as a representative of modification-originated intramolecular contacts in the modified enzyme.

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