Covalent immobilization of amyloglucosidase on magnetic-silver nanoparticles for rapid and reusable saccharification of cassava peel.

Short-Term Glucose Release from Ultrasound-Assisted Alkali-Pretreated HH Using Free and Magnetic Nanoparticle-Immobilised Cellulase

The enzymatic conversion of lignocellulosic biomass (LB) into fermentable sugars is important for the development of sustainable biorefineries. This study investigated the immobilisation of Trichoderma reesei (T. reesei) cellulase on amine-functionalised magnetic nanoparticles (MNPs) and evaluated the resulting biocatalyst for the hydrolysis of pretreated hemp hurd (HH) biomass. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) provided evidence consistent with cellulase association with the nanoparticles, with the estimated dry-state particle diameter increasing from 22.4 ± 0.4 to 27.8 ± 0.3 nm after immobilisation. The selected immobilised catalyst loading produced approximately 89% of the total filter-paper assay response obtained with the selected free-enzyme loading, although this comparison was not normalised to protein content. During 7 h hydrolysis experiments, glucose production increased progressively for both enzyme forms. Across the tested enzyme dilutions, immobilised cellulase generated approximately 88–91% of the glucose produced by free cellulase. The immobilised enzyme also retained approximately 64% of its initial hydrolysis performance after five reuse cycles. These findings demonstrate the potential of magnetic cellulase nanobiocatalysts for recoverable and reusable hydrolysis of lignocellulosic biomass. However, further studies are required to determine protein-normalised activity, immobilisation efficiency, longer-term stability, process economics and industrial scalability.

Laccase-mediated silver nanoparticles as a nano-biocatalytic pretreatment platform for enhanced saccharification and bioethanol production from sugarcane bagasse

Immobilization of β-Agarase onto Ni-NTA Magnetic Nanoparticles for Efficient Production of Functional Neoagarooligosaccharides

AgaDcat is a promising β-agarase for the production of functional neoagarooligosaccharides (NAOSs), but its limited thermal stability and poor reusability as a free enzyme increase enzyme consumption and production cost during repeated agarose hydrolysis. In this study, β-agarase AgaDcat was immobilized on nickel-nitrilotriacetic acid magnetic nanoparticles (Ni-NTA-MNPs) through His-tag/Ni-NTA affinity interactions. The successful preparation of Ni-NTA-MNPs and the immobilization of AgaDcat were verified by SEM, EDS, VSM, FT-IR, XRD, and TGA. Following optimization, the immobilized enzyme exhibited an activity retention of 80.47%. Thermal stability assays showed that the immobilized enzyme was more thermostable than the free enzyme. After incubation at 50 °C for 60 min, the immobilized enzyme retained 59.4% of its initial activity, whereas the free enzyme retained only 29.1%. Moreover, the immobilized enzyme displayed good reusability, retaining 90.59% and 58.71% of its activity after 3 and 7 cycles, respectively. HPAEC-PAD analysis showed that the immobilized enzyme reached reaction equilibrium within 4–8 h, with neoagarotetraose (NA4) and neoagarohexaose (NA6) identified as the major degradation products, whereas the free enzyme required 20 h, indicating significantly improved catalytic efficiency. These results indicate that IMAC-based immobilization improves the stability, reusability, and catalytic efficiency of β-agarase, providing a promising reusable biocatalytic strategy for the efficient production of functional neoagarooligosaccharides.

A nanofiber-based biocatalytic system: catalase immobilization on an electrospun PVP/GA carrier platform for hydrogen peroxide decomposition

Hydrogen peroxide (H2O2) is widely used in industrial and environmental processes; however, excessive accumulation of H2O2 may cause oxidative damage and environmental hazards. Catalase (CAT) is an efficient enzyme capable of decomposing H2O2 into water and oxygen, but its practical application is limited by low stability and lack of reusability in its free form. In this study, CAT was immobilized onto electrospun polyvinylpyrrolidone/gum arabic (PVP/GA) nanofibers via glutaraldehyde-mediated covalent cross-linking to develop a stable and reusable biocatalytic system. The effects of key immobilization parameters, including CAT concentration, nanofiber amount, cross-linking time, and glutaraldehyde concentration, were systematically optimized, and a maximum immobilization efficiency of 81.2% was achieved under optimal conditions. The immobilized enzyme exhibited improved thermal stability, broader pH tolerance, enhanced pH stability, and superior storage stability compared to the free enzyme. Kinetic analysis revealed an increase in both Km and Vmax values after immobilization, indicating enhanced catalytic capacity despite partial diffusion limitations. Application experiments demonstrated that the immobilized CAT achieved complete degradation of H2O2 at concentrations up to 100 mM within 25 min, while maintaining significant catalytic performance during repeated operational cycles. Moreover, the immobilized enzyme retained 52% of its initial activity after 30 days of storage, highlighting its improved stability. These findings demonstrate that PVP/GA nanofiber supported CAT represents a promising and reusable biocatalytic system for efficient H2O2 detoxification in environmental and industrial applications.

Magnetically Controlled Wall-Loading of CALB-Immobilized Fe3O4 Nanoparticles for Continuous-Flow Transesterification in a Microreactor

The combination of immobilized enzymes and microreactors offers great advantages in green biomanufacturing. However, improving the catalytic loading and reusability of immobilized enzymes remains a challenge. This study developed a magnetically separable immobilized enzyme system for continuous-flow biocatalysis. Candida antarctica lipase B (CALB) was covalently immobilized onto polydopamine-modified Fe3O4 nanoparticles to prepare a magnetic nanoimmobilized enzyme (CALB@PNMNs). Compared to free enzymes, CALB@PNMNs exhibited enhanced stability in organic solvents and storage stability. CALB@PNMNs microreactor was constructed, with CALB@PNMNs immobilized on its inner walls by an external magnetic field. This layer of magnetic particles allows for the convenient recovery and reuse of the catalyst by removing the applied magnetic field. After optimizing the immobilization process, the actual CALB loading per unit area was 0.30 mg/cm2, and the adsorption yield reached 86.89%. The microreactor was applied to the transesterification of n-butanol and ethyl acetate, with the optimal conditions identified as an alcohol–ester ratio of 1:3, a total flow rate of 10 μL/min, and a reaction temperature of 60 °C, yielding an n-butanol conversion of 63.72%. After short-term continuous operation for 6 h, the microreactor retained 87.63% of the enzyme adsorption capacity and CALB@PNMNs maintained a n-butanol conversion above 50% after 8 reuse cycles. This work has established a stable magnetically controlled immobilized enzyme microreactor (IMER) platform, offering application potential for continuous-flow bioprocesses.

Surface-Engineered Hydrated Silica as a Reusable Support for Flavourzyme Immobilization in Food Protein Hydrolysis