Inulin hydrolysis was performed by inulinase from Aspergillus niger covalently immobilized on magnetite nanoparticles (Fe 3 O 4 ) covered with soy protein isolate (Fe 3 O 4 /SPI) functionalized by bovine serum albumin (Fe 3 O 4 /SPI/BSA) nanoparticles as a new bio-functional carrier. The specific activity and protein content of the immobilized enzyme were 25.99 U/mg and 3.52 mg/mL, respectively, with 80% enzyme loading. The immobilized inulinase showed maximum activity at 45 C, which is 5 C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilized enzyme shifted from 6 to 5.5, which is more acidic compared to that of the free enzyme. The K m value of immobilized inulinase decreased to 2.03 mg/mL. Thermal stability increased considerably at 65 and 75 C, and a 5.13-fold rise was detected in the enzyme half-life at 75 C after immobilization. Moreover, 80% of initial activity of immobilized inulinase remained after 10 cycles of hydrolysis.
Silicon oxide was initially loaded on a Fe3O4 magnetic nanoparticle substrate (Fe3O4@SiO2) and then functionalized with ─NH2 group (Fe3O4@SiO2@NH2) to construct a novel hierarchical magnetic nanocomposite. A sensitive urea biosensor medium involving a dip‐coated hierarchical magnetic nanocomposite on F‐doped SnO2 conducting glass was designed (Fe3O4@SiO2@NH2/SnO2:F) to achieve an excellent platform for urease (Urs) enzyme immobilization via covalent linking to the exposed NH2 groups through glutaraldehyde (Urs/Fe3O4@SiO2@NH2/SnO2:F). The hierarchical magnetic nanocomposite selection criteria were based on enhancement of urea biosensing by Urs immobilization via covalent linking to the exposed NH2 groups, while the SnO2:F selection as substrate was based on its ability to afford high electronic density to the biosensor surface as an electrostatic repulsion layer for the anionic interferents in the biological environment. FE‐SEM, TEM, FTIR, CV, EIS, and I–V techniques established the morphology of the modified electrode's surface and electrochemical behavior of urea on the fabricated Urs/Fe3O4@SiO2@NH2/SnO2:F biosensor. The sensing mechanism can be clarified on the basis of the two reactions, namely (1) catalytic reaction and (2) oxidation or reduction of metal oxides, same as in the case of solid‐state gas sensors. The I–V results display high sensitivity for urea detection of within 5–210 mg/dL and a limit of detection of 3 mg/dL.
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 FeO@SPI-BSA NPs was <120 nm (PDI, 0.187). Inulinase was covalently immobilised successfully through glutaraldehyde on FeO@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 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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