Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of β-glucosidase from Thermotoga maritima (tm-β-Glu) on magnetic nanoparticles (Mnps) functionalized with chitin (ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-β-Glucosidase thermostable chitinbinding domain (tm-β-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-β-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10 th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of tm-β-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.
SummaryIn an ultrasonic bath and for 30 min in the presence of water as extraction solvent in a ratio of 1:10 (g/mL), effect of extraction temperature on extraction of caffeoylquinic acids (CQAs) from Moringa oleifera leaves (MOL) was investigated. Gradually increasing in extraction temperature caused a growing in mono‐CQA and declining in 3,5‐diCQA. The extract obtained at 80 °C showed the highest antioxidant capacity, and IC50 values for scavenging activities on DPPH radicals and ABTS radicals were 56.9 and 53.6 µg/mL, respectively. Compared with α‐glucosidase and α‐amylase, the extract showed the highest inhibition on pancreatic lipase (IC50 of 0.073 mg/mL). Increasing the extraction temperature improved cytotoxicity of the extract on cancer cell lines of HeLa and HepG2. Compared with gram‐negative Escherichia coli and Salmonella typhimurium, all the extracts showed higher inhibition on the growth of gram‐positive Bacillus cereus and Staphylococcus aureus. Our results suggested that use of higher extraction temperature (80 °C) improved the biological activity of MOL extract.
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