Search Results (76)

Traditional enzyme immobilization strategies often rely on chemical crosslinkers or solid carriers, thereby increasing processing complexity and potentially compromising catalytic efficiency. Here, we present a carrier-free approach for generating stable biocatalytic particles by exploiting the intrinsic aggregation behavior of four phylogenetically distinct AA10 LPMO homologues (Kpap_p40, Kari_p40, Alip_p40, and Psuf_p40) as scaffolds for catalytically active inclusion bodies (CatIBs) in Escherichia coli. Each AA10 variant was genetically fused to either mCherry or a thermostable Bacillus α-amylase (BacAmy) and expressed in E. coli BL21(DE3), resulting in the predominant formation of insoluble protein inclusion bodies (IBs). Protein partitioning was quantified by SDS–PAGE densitometry, intracellular localization by confocal microscopy, particle size and morphology by dynamic light scattering and FESEM, and secondary structure by FTIR spectroscopy. All variants assembled into submicron, structured aggregates with hydrodynamic diameters ranging from 620 to 824 nm and were enriched in α-helical and β-sheet secondary structure, consistent with the formation of structured aggregates rather than extensive amorphous misfolding. mCherry IBs retained fluorescence and displayed polar localization in vivo, while BacAmy CatIBs exhibited maximal catalytic activity at 80 °C, maintained substantial activity up to 95 °C, and demonstrated broad pH tolerance with pronounced pH stability from a slightly acidic to a mild alkaline range. FTIR analysis showed that BacAmy CatIBs contained 47–54% α-helical structure, while mCherry IBs contained 42–45% α-helical structure, indicating the preservation of partially native protein conformations within the aggregated state. Differences among variants influenced particle size, dispersity, and aggregate morphology. These findings demonstrate the potential of AA10 LPMO domains as versatile structural modules for engineering thermostable, carrier-free biocatalysts and provide a foundation for expanding their application beyond oxidative polysaccharide cleavage toward sustainable enzyme material design. Full article

The accelerating global demand for sustainable energy, driven by population growth, industrialization, and environmental concerns, has intensified the search for renewable alternatives to fossil fuels. Biofuels, including bioethanol, biodiesel, biogas, and biohydrogen, offer a viable and practical pathway to reducing net carbon dioxide (CO₂) emissions. Yet, their large-scale production remains constrained by biomass recalcitrance, high pretreatment costs, and the enzyme-intensive nature of conversion processes. Recent advances in enzyme immobilization using magnetic nanoparticles (MNPs), covalent organic frameworks, metal–organic frameworks, and biochar have significantly improved enzyme stability, recyclability, and catalytic efficiency. Complementary strategies such as cross-linked enzyme aggregates, carrier-free immobilization, and site-specific attachment further reduce enzyme leaching and operational costs, particularly in lipase-mediated biodiesel synthesis. In addition to biocatalysis, nanozymes—nanomaterials exhibiting enzyme-like activity—are emerging as robust co-catalysts for biomass degradation and upgrading, although challenges in selectivity and environmental safety persist. Green synthesis approaches employing plant extracts, microbes, and agro-industrial wastes are increasingly adopted to produce eco-friendly nanomaterials and bio-derived supports aligned with circular economy principles. These functionalized materials have demonstrated promising performance in esterification, transesterification, and catalytic routes for biohydrogen generation. Technoeconomic and lifecycle assessments emphasize the need to balance catalyst complexity with environmental and economic sustainability. Multifunctional catalysts, process intensification strategies, and engineered thermostable enzymes are improving productivity. Looking forward, pilot-scale validation of green-synthesized nano- and biomaterials, coupled with appropriate regulatory frameworks, will be critical for real-world deployment. Full article

With the booming development of natural anticancer agents, icaritin has been widely used in clinical liver cancer treatment due to the smaller number of side effects. Among them, enzymes as catalysts for producing icaritin have attracted considerable attention. Industrial production remains in its infancy due to the poor reusability of free enzymes, despite enzymes possessing favorable efficiency and green catalytic effects. The present study investigated two immobilization methods, including cross-linked enzyme aggregates (CLEAs) and magnetic cross-linked enzyme aggregates (MCLEAs). The optimal temperature and pH for the two hydrolases were determined, followed by characterization using VSM, XRD, FT-IR, and TEM techniques. The experiments have demonstrated that MCLEAs eradicate the need for ultrafiltration; MCLEAs are beneficial for enhancing fixation efficiency. Additionally, MCLEAs exhibited significantly higher catalytic activity, which raised the catalytic activity by approximately 30% compared with CLEAs. Moreover, after 10 consecutive reuse cycles, the catalytic activity of MCLEAs remained above 70%, maintaining a conversion rate of epimedin C at 61.59%. To summarize, MCLEAs offer an efficient strategy for enzyme immobilization. MCLEAs not only significantly enhanced both the enzyme’s ability to catalyze and resist but also eliminated the necessity for ultrafiltration as well as enabled rapid product separation. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) represents a multifactorial condition requiring multi-target therapeutic strategies beyond traditional single-marker approaches. In this work, we present a fully in silico nutraceutical screening pipeline that integrates molecular prediction, systemic aggregation, and technological design. A curated panel of ten MASLD-relevant targets, spanning nuclear receptors (FXR, PPAR-α/γ, THR-β), lipogenic and cholesterogenic enzymes (ACC1, FASN, DGAT2, HMGCR), and transport/regulatory proteins (LIPG, FABP4), was assembled from proteomic evidence. Bioactivity records were extracted from ChEMBL, structurally standardized, and converted into RDKit descriptors. Predictive modeling employed a stacked ensemble of Random Forest, XGBoost, and CatBoost with isotonic calibration, yielding robust performance (mean cross-validated ROC-AUC 0.834; independent test ROC-AUC 0.840). Calibrated probabilities were aggregated into total activity (TA) and weighted TA metrics, combined with structural clustering (six structural clusters, twelve MOA clusters) to ensure chemical diversity. We used physiologically based pharmacokinetic (PBPK) modeling to translate probabilistic profiles into minimum simulated doses (MSDs) and chrono-specific exposure (%T>IC50) for three prototype concepts: HepatoBlend (morning powder), LiverGuard Tea (evening aqueous form), and HDL-Chews (postprandial chew). Integration of physicochemical descriptors (MW, logP, TPSA) guided carrier and encapsulation choices, addressing stability and sensory constraints. The results demonstrate that a computationally integrated pipeline can rationally generate multi-target nutraceutical formulations, linking molecular predictions with systemic coverage and practical formulation specifications, and thus provides a transferable framework for MASLD and related metabolic conditions. Full article

The valorization of agricultural by-products represents a sustainable strategy to reduce waste and create high-value biotechnological products. This review highlights Colombian plant-derived peroxidases (PODs) obtained from Guinea grass, royal palm, African oil palm, lemongrass, sleepy plant, and sweet potato. These enzymes catalyze oxidative reactions and show potential in biosensing, polymer synthesis, environmental remediation, and health monitoring. We summarize extraction and purification strategies while addressing current challenges such as operational stability, scalability, and cost. Special emphasis is given to applications like cross-linked enzymatic aggregates (CLEAs) and electrochemical biosensors, where Colombian PODs demonstrate superior stability and sensitivity compared to horseradish peroxidase (HRP). This review frames these advances within the circular bioeconomy, presenting insights into waste reduction and CO₂ savings. By integrating local biodiversity into innovative processes, Colombian PODs can drive sustainable technologies and provide industrial and environmental solutions. Full article

Enzymes, as nature’s precision biocatalysts, hold transformative potential across industrial, environmental, and biomedical sectors. However, their instability, solvent sensitivity, and limited reusability in their free form necessitate advanced immobilization strategies to enhance their robustness and scalability. This review critically examines cutting-edge advancements in enzyme immobilization, focusing on the integration of artificial intelligence (AI), novel nanomaterials, and dynamic carrier systems to overcome the traditional limitations of mass transfer, enzyme leakage, and cost inefficiency. Key innovations such as metal–organic frameworks (MOFs), magnetic nanoparticles, self-healing hydrogels, and 3D-printed scaffolds are highlighted for their ability to optimize enzyme orientation, stability, and catalytic efficiency under extreme conditions. Moreover, AI-driven predictive modeling and machine learning emerge as pivotal tools for rationalizing nanomaterial synthesis, multi-enzyme cascade design, and toxicity assessment, while microfluidic systems enable precise biocatalyst fabrication. This review also explores emerging carrier-free strategies, including cross-linked enzyme aggregates (CLEAs) and DNA-directed immobilization, which minimize diffusion barriers and enhance substrate affinity. Despite progress, challenges persist in regards to eco-friendly nanomaterial production, industrial scalability, and real-world application viability. Future directions emphasize sustainable hybrid material design, AI-aided lifecycle assessments, and interdisciplinary synergies between synthetic biology, nanotechnology, and data analytics. By connecting laboratory innovation with industrial needs, this work provides a forward-thinking framework to harness immobilized enzymes for achieving global sustainability goals, particularly in bioremediation, bioenergy, and precision medicine. Full article

The immobilization of a laccase from Pycnoporus sanguineus UEM-20 via the formation of cross-linked enzyme aggregates (CLEAs) was optimized through a central composite design (CCD) of response surface methodology (RSM). Both free and immobilized enzymes were investigated for their physico-chemical characteristics, and their adequacy in removing bisphenol A (BPA) and decolorizing malachite green dye in solution was evaluated. The immobilization caused only minor differences in thermostability. Upon immobilization, the enzyme experienced some changes in its kinetic properties. The V_max decreased by a factor of 1.1, and the K_M increased by a factor of 1.89. These kinetic changes did not modify in any remarkable way the capacity of the immobilized enzyme in degrading BPA and decolorizing malachite green dye. Its sensitivity to NaCl was also minimally affected by immobilization. However, its sensitivity to sodium sulfate was substantially decreased. After 1 month’s conservation, the activity of the free form had suffered a drastic drop. The immobilized form, by contrast, remained 100% active after 6 months. All these findings predict that the immobilized laccase from P. sanguineus UEM-20 may be useful in the enzymatic bioremediation of pollutants such as endocrine disruptors and synthetic dyes. Full article

The growing demand for plant-based protein alternatives has driven interest in protein modifications to enhance their functional properties in food applications. Enzymatic cross-linking using laccases derived from Rhus vernicifera (LR) and transglutaminase (TG) offers a promising strategy to enhance protein solubility, emulsifying properties, and foaming properties of food proteins. This study varied the enzymatic reaction conditions, including enzyme concentration, pH, temperature, incubation time, and ferulic acid addition, for the most effective cross-linking between proteins in lupin flour (LF) and soy protein isolate (SPI), resulting in changes in physicochemical and functional properties of the cross-linked proteins. LR-induced cross-linking in lupin and soy proteins was most favourable at 142.5 U/100 mg protein, pH 6, and 20 °C, where ferulic acid enhanced cross-linking efficiency with prolonged incubation (20 h). TG-induced cross-linking in lupin and soy proteins was most favourable at 1.25 U/100 mg protein, pH 6 and 30 °C, where high-molecular-weight aggregates were observed. Cross-linking modified protein surface characteristics, increasing ζ-potential and particle size due to protein aggregation, while ferulic acid further enhanced polymerisation. Morphological analysis revealed a porous powder structure across all samples with increased porosity in cross-linked samples as evidenced by the predominance of small fragments within the particles. Prolonged incubation led to partial disaggregation in LR-treated samples unless they were stabilised by ferulic acid. Under mild conditions (1 h, pH 6, 20 °C), LR and ferulic acid-added samples showed minor and significant improvements in protein solubility and foaming stability, respectively. Additionally, a significant increase in foaming ability was observed in ferulic acid-added LR samples after prolonged incubation (20 h), compared to the corresponding control. In contrast, prolonged incubation (20 h) or TG treatment had a lower foaming stability compared to the mild LR treatment. Emulsifying ability and emulsion stability showed limited variation across treatments. These findings suggest that cross-linking conditions influence specific functional properties, highlighting the need for further optimisation to achieve desired protein functionality in food applications. Full article

Gelsemine, a naturally occurring indole alkaloid derived from plants of the Gelsemium species of the Gelsemiaceae family, has been extensively investigated for its neuroprotective and anti-inflammatory properties. Recent studies have demonstrated that gelsemine exerts neuroprotective effects against beta-amyloid (Aβ) oligomers, a key neurotoxic peptide implicated in the pathogenesis of Alzheimer’s disease (AD). However, despite these beneficial effects, the precise molecular targets underlying gelsemine’s neuroprotective actions in AD remain unidentified. Here, we employed a combination of bioinformatic, biochemical, and functional assays in neuronal models to investigate the mechanism of gelsemine’s action in AD cellular models. Our findings indicate that gelsemine inhibits the activity of transglutaminase 2 (TG2), an enzyme involved in protein cross-linking with emerging roles in Aβ aggregation and neurotoxicity. Molecular modeling and biochemical analyses reveal that gelsemine interacts with the TG2 catalytic site, leading to its inhibition. Furthermore, gelsemine modulates the TG2-mediated Aβ aggregation process, thereby attenuating Aβ-induced neurotoxicity and preserving neuronal function. These findings establish TG2 as a previously unrecognized molecular target of gelsemine and underscore the potential of Gelsemium-derived alkaloids as neuroprotective agents. The modulation of TG2 activity by natural alkaloids may provide a novel therapeutic approach for mitigating Aβ toxicity and preserving neuronal function in AD. Full article

Efficient hydrolysis of cellulose in agricultural waste (e.g., coconut fiber) is critical for biorefining processes such as second-generation bioethanol (2G ethanol) production. However, free cellulases suffer from low thermal stability and challenges in recovery. To address this, we developed cross-linked enzyme aggregates (CLEAs) combined with magnetic nanoparticles (magnetic CLEAs, m-CLEAs) to enhance enzyme stability and reusability. In this context, solutions of ethanol, acetone, and ammonium sulfate were used to prepare enzymatic aggregates, with subsequent use of glutaraldehyde and magnetic nanoparticles to obtain the biocatalysts. The addition of bovine serum albumin (BSA) protein was also tested to improve immobilization. Biocatalysts with ethanol and acetone performed better. Acetone (AC) and BSA yielded the highest enzymatic activities (287.27 ± 42.59 U/g for carboxymethyl cellulase (CMCase) with Celluclast; 425.37 ± 48.11 U/g for CMCase with Cellic CTec2). Magnetic nanoparticles were incorporated to expand the industrial applicability, producing m-CLEAs with excellent thermal stability and high catalytic activities. The m-CLEA–Celluclast–AC–BSA–GA 5% maintained 58% of its activity after 72 h at 70 °C. The m-CLEA–Celluclast-AC–BSA–GA 2.5% proved effective in hydrolyzing coconut fiber and isolated cellulose, producing up to 0.91 ± 0.01 g/L of glucose and 2.7 ± 0.15 g/L of glucose, respectively, after 72 h. Therefore, this approach supports sustainability by using coconut fiber, which is often discarded into the environment. Full article

This study aims to evaluate the therapeutic potential of phloretin–chitosan nanoparticles (Ph-ChNPs), alone and in combination with the anticancer drug tamoxifen, in modulating breast cancer markers and improving in vivo treatment outcomes. Ph-ChNPs were prepared by ionic gelation in the presence of Tripolyphosphate (TPP) solution as a crosslinker agent. The nanoparticles were characterized using DLS, TEM, UV-VIS and FT-IR spectroscopy. In vitro cytotoxic assay of Ph-ChNPs on MCF-7 breast cancer cell lines revealed anticancer activity with an IC₅₀ value of 32.12 ± 1.63 µg/mL. In vivo studies were carried out on mice, treated with DMBA to induce breast cancer and followed the effect of the prepared nanoparticle, either alone or with combination with tamoxifen, on mice health. The biochemical parameters measured after treatment with Ph-ChNPs alone showed an improvement in lipid profile with decreased total cholesterol (TC) and Triglyceride (TG) levels and increased HDL-c levels. Ph-ChNPs significantly reduced IL-6 and cyclin D1 levels, with a slight increase in cyclin E2 levels. Antioxidant enzyme levels were improved, and oxidative stress markers were reduced. The combination treatment showed a synergistic effect in reducing inflammation and cell proliferation. DMBA-injected mice had substantially increased BRCA1 and BRCA2 gene expression. Ph-ChNP-treated mice showed well-organized mammary gland structures, while DMBA-injected mice displayed dense tumor cell aggregations. Ph-ChNPs and tamoxifen treatments improved histopathological variations, with the combination treatment showing significant apoptosis of tumor cells. This study demonstrates the significant potential of Ph-ChNPs combined with tamoxifen in breast cancer treatment. The combination therapy effectively reduces tumor growth, induces apoptosis and modulates critical breast cancer markers, offering a promising therapeutic strategy. Full article

The formation of cross-linked enzyme aggregates (CLEAs) using macromolecular cross-linkers improves substrate accessibility and enhances enzyme retention. However, there have been few studies exploring the use of macromolecular cross-linkers due to the challenges related to cross-linker screening. In compliance with our previous computational and experimental screening, dextran is the optimal macromolecular cross-linker to develop CLEAs of endolevanase from Bacillus lehensis G1 (rlevblg1-dex-CLEA) for levan-type-fructooligosaccharides (L-FOS) production. In this study, rlevblg1-dex-CLEAs was optimized, and the activity recovery continued to increase and reached 90.5%. Subsequently, the rlevblg1-dex-CLEAs were characterized and they displayed higher thermal stability after 1 h of incubation in comparison to the free enzyme. Moreover, the rlevblg1-dex-CLEAs were reusable for five cycles and exhibited greater storage stability over 180 days at 4 °C (60.9%) than that of free rlevblg1. In addition, the rlevblg1-dex-CLEAs demonstrated similar catalytic efficiency as the free enzyme and generated a substantial amount of L-FOS with a longer degree of polymerization, which is more beneficial for industrial use. Full article

We present the synthesis of a cross-linking enzyme aggregate (CLEAS) of a peroxidase from Megathyrsus maximus (Guinea Grass) (GGP). The biocatalyst was produced using 50%v/v ethanol and 0.88%w/v glutaraldehyde for 1 h under stirring. The immobilization yield was 93.74% and the specific activity was 36.75 U mg⁻¹. The biocatalyst surpassed by 61% the free enzyme activity at the optimal pH value (pH 6 for both preparations), becoming this increase in activity almost 10-fold at pH 9. GGP-CLEAS exhibited a higher thermal stability (2–4 folds) and was more stable towards hydrogen peroxide than the free enzyme (2–3 folds). GGP-CLEAS removes over 80% of 0.05 mM indigo carmine at pH 5, in the presence of 0.55 mM H₂O₂ after 60 min of reaction, a much higher value than when using the free enzyme. The operational stability showed a decrease of enzyme activity (over 60% in 4 cycles), very likely related to suicide inhibition. Full article

Isomaltulose is a novel sweetener and is considered healthier than the common sugars, such as sucrose or glucose. It has been internationally recognized as a safe food product and holds vast potential in pharmaceutical and food industries. Sucrose isomerase is commonly used to produce isomaltulose from the substrate sucrose in vitro and in vivo. However, free cells/enzymes were often mixed with the product, making recycling difficult and leading to a significant increase in production costs. Immobilized cells/enzymes have the following advantages including easy separation from products, high stability, and reusability, which can significantly reduce production costs. They are more suitable than free ones for industrial production. Recently, immobilized cells/enzymes have been encapsulated using composite materials to enhance their mechanical strength and reusability and reduce leakage. This review summarizes the advancements made in immobilized cells/enzymes for isomaltulose production in terms of refining traditional approaches and innovating in materials and methods. Moreover, innovations in immobilized enzyme methods include cross-linked enzyme aggregates, nanoflowers, inclusion bodies, and directed affinity immobilization. Material innovations involve nanomaterials, graphene oxide, and so on. These innovations circumvent challenges like the utilization of toxic cross-linking agents and enzyme leakage encountered in traditional methods, thus contributing to enhanced enzyme stability. Full article

The modern global trend toward sustainable processes that meet the requirements of “green chemistry” provides new opportunities for the broad application of highly active, selective, and specific enzymatic reactions. However, the effective application of enzymes in industrial processes requires the development of systems for the remote regulation of their activity triggered by external physical stimuli, one of which is a low-frequency magnetic field (LFMF). Magnetic nanoparticles (MNPs) transform the energy of an LFMF into mechanical forces and deformations applied to enzyme molecules on the surfaces of MNPs. Here, we demonstrate the up- and down-regulation of two biotechnologically important enzymes, yeast alcohol dehydrogenase (YADH) and soybean formate dehydrogenase (FDH), in aggregates with gold-covered magnetic nanoparticles (GCMNPs) triggered by an LFMF. Two types of aggregates, “dimeric” (with the enzyme attached to several GCMNPs simultaneously), with YADH or FDH, and “monomeric” (the enzyme attached to only one GCMNP), with FDH, were synthesized. Depending on the aggregate type (“dimeric” or “monomeric”), LFMF treatment led to a decrease (down-regulation) or an increase (up-regulation) in enzyme activity. For “dimeric” aggregates, we observed 67 ± 9% and 47 ± 7% decreases in enzyme activity under LFMF exposure for YADH and FDH, respectively. Moreover, in the case of YADH, varying the enzyme or the cross-linking agent concentration led to different magnitudes of the LFMF effect, which was more significant at lower enzyme and higher cross-linking agent concentrations. Different responses to LFMF exposure depending on cofactor presence were also demonstrated. This effect might result from a varying cofactor binding efficiency to enzymes. For the “monomeric” aggregates with FDH, the LFMF treatment caused a significant increase in enzyme activity; the magnitude of this effect depended on the cofactor type: we observed up to 40% enzyme up-regulation in the case of NADP⁺, while almost no effect was observed in the case of NAD⁺. Full article