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The organophosphate (OP)-hydrolyzing enzyme phosphotriesterase (PTE, variant L7ep-3a) immobilized within a partially oxidized mesoporous silicon nanoparticle cage is synthesized and the catalytic performance of the enzyme@nanoparticle construct for hydrolysis of a simulant, dimethyl p-nitrophenyl phosphate (DMNP), and the live nerve agent VX is benchmarked against the free enzyme. In a neutral aqueous buffer, the optimized construct shows a ≈2-fold increase in the rate of DMNP turnover relative to the free enzyme. Enzyme@nanoparticles with more hydrophobic surface chemistry in the interior of the pores show lower catalytic activity, suggesting the importance of hydration of the pore interior on performance. The enzyme@nanoparticle construct is readily separated from the neutralized agent; the nanoparticle is found to retain DMNP hydrolysis activity through seven decontamination/recovery cycles. The nanoparticle cage stabilizes the enzyme against thermal denaturing and enzymatic (trypsin) degradation conditions relative to free enzyme. When incorporated into a topical gel formulation, the PTE-loaded nanoparticles show high activity toward the nerve agent VX in an ex vivo rabbit skin model. In vitro acetylcholinesterase (AChE) assays in human blood show that the enzyme@nanoparticle construct decontaminates VX, preserving the biological function of AChE when exposed to an otherwise incapacitating dose.

Prolyl oligopeptidases from psychrophilic, mesophilic, and thermophilic organisms found in a range of natural environments are studied using a combination of protein structure prediction, atomistic molecular dynamics, and trajectory analysis to determine how the S9 protease family adapts to extreme thermal conditions. We compare our results with hypotheses from the literature regarding structural adaptations that allow proteins to maintain structure and function at extreme temperatures, and we find that, in the case of prolyl oligopeptidases, only a subset of proposed adaptations are employed for maintaining stability. The catalytic and propeller domains are highly structured, limiting the range of mutations that can be made to enhance hydrophobicity or form disulfide bonds without disrupting the formation of necessary secondary structure. Rather, we observe a pattern in which overall prevalence of bound interactions (salt bridges and hydrogen bonds) is conserved by using increasing numbers of increasingly short-lived interactions as temperature increases. This suggests a role for an entropic rather than energetic strategy for thermal adaptation in this protein family.

AbstractFungal lipases are the leading industrial biocatalyst due to their broad applications, but high cost limits their commercial usage. The low-cost agri-residues substrates can reduce the cost of lipase production. However, the compatibility of agri-residue with fungal species, recovery process of lipase and stability of the enzyme are crucial steps. The aim of the present work was optimization of lipase production from a suitable combination of fungal culture with a locally available vegetable oilseed cake (mustard/groundnut/almond/cottonseed) in solid-state fermentation process and its direct immobilization. The enzyme produced using selected combination of Rhizopus oryzae and mustard oilseed cake was optimized by Plackett–Burman design, one-factor-at-a-time and central composite design (CCD). The highest enzyme activity of 25.08 U/gds was obtained by CCD at urea 2.11% w/w, inoculum size 1.18% v/w, and moisture content 69.99% w/w. The crude enzyme from the extract was immobilized on functionalized magnetic nanoparticles with the results of protein loading 68.88 ± 3.54 µg/mg of MNPs and activity recovery of 60.33 ± 3.03%. This study can be helpful to explore the suitability of locally available agri-residue for production of lipase and utilization of enzyme in different industrial applications. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic compounds (PACs) are pervasive environmental contaminants derived from diverse sources including pyrogenic (e.g., combustion processes), petrogenic (e.g., crude oil), and biological origins. They are commonly found in gasoline, coal, and crude oil, reflecting their prevalence and varied origins in natural and anthropogenic activities. The aim of this study is to use Bacillus tropicus which is a spore-forming, gram-positive and facultative anaerobic bacteria, containing a gene for PACs degradtion. In this study bacterial sample was collected from women's vaginal discharge through streaking and spreading techniques. The DNA was extracted from bacterial culture and then the bacterium was identified through 16S rRNA which appeared to be B.tropicus. Then the computational analysis was conducted where the sequence similarity and functional analysis of alcohol dehydrogenase EutG protein from B.tropicus was analyzed through PSI-BLAT and SMART tool, respectively. The PSI-BLAST showed 100% query coverage score and 9 domains of alcohol dehydrogenase EutG protein were predicted through SMART tool. The quality of the protein was also assessed through ProQ server with a predicted LQ score of 8.091, a Maxsub score of − 0.350 and a z score of − 10.76. Then the phylogentic analysis was conducted to know the evolutionary relationship and closely related taxa. The 3D structure of the protein was predicted through SWISS MODEL and its quality was predicted through ERRAT with overall qauality factor of 98.708. The Ramachandran plot also predicted its quality and showed that 93.8% residues were in the most favored region. After this, 3D stucture of PACs were obtained from PubChem and molecular docking of the protein was performed with each of the compound. The lowest energy of − 10.3 was obtained with Indeno[1,2,3-cd] pyrene and the best docked complex was visulaized through discover studio to analyze its binding residues. Lastly, in-silico site-directed mutagenesis studies were performed which showed that the EutG gene (codes for alcoholic dehydrogenase) obtained from B. tropicus, will not get altered or have any decreasing effect on the enzyme's stability if it goes through any mutations. This suggests that B. tropicus can act as an efficient, non-virulent, and reliable candidate for the eco-friendly and cost-effective bioremediation of PACs. [ABSTRACT FROM AUTHOR]

Glucokinase (GK), an enzyme critical to glucose metabolism, exhibits thermal instability, which can affect its enzymatic activity under physiological and pathological conditions. This study aims to mathematically model the thermal denaturation kinetics of GK and empirically validate the model using experimental data. To establish a mathematical model on thermal denaturation of glucokinase (E.C.2.7.1.2) and its experimental validation, the enzyme glucokinase was investigated in a 0.075M Tris HCl buffer with pH 9.0 at 30∘C and 0.6M MgCl2. A first-order kinetic model was developed to describe the enzyme’s denaturation, incorporating temperature-dependent reaction rates based on the Arrehenius equation. Empirical data were collected through Spectrophotometer across a temperature range of 20∘–60∘C. Experimental validation revealed that GK undergoes irreversible denaturation above 60∘ with a significant reduction as temperature increases. Moreover, the thermal denaturation of GK in the presence of osmolyte Urea is a critical process affecting enzyme stability and function. This study also aims to mathematically model and empirically validate the impact of Urea on GK’s thermal denaturation behavior. Results demonstrated that Urea significantly reduces the thermal stability of GK, lowering its denaturation temperature. The results are simulated graphically using the Wolfram MATHEMATICA software. The mathematical predictions closely matched experimental data, confirming the model’s accuracy. [ABSTRACT FROM AUTHOR]

Hypoxia, high ROS levels and chronic inflammation are the main factors that hinder the healing of diabetic wounds. Long-term exposed wounds are prone to bacterial infection, especially MRSA infection, which exacerbates the complex wound microenvironment of diabetes and threatens patients' lives. Here, we developed a ROS nanopurifier (CSVNP), which was prepared by loading superoxide dismutase (SOD), catalase (CAT) and vancomycin into nanogels through in-situ polymerization. CSVNP can effectively increase enzyme loading and stability, and improve cascade reaction efficiency between enzymes through nanosize effect, so that CSVNP can use a variety of ROS (H 2 O 2 and ·O 2 -) as oxygen sources to generate much oxygen in situ, which can effectively alleviate the hypoxic environment and inflammatory response of diabetic tissues, theraby promoting cell migration and angiogenesis, and accelerating wound healing. In addition, the generated oxygen can further promote the transformation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages and reduce pro-inflammatory factors (TNF-α, IL-6, and IL-1β) release. CSVNP can also effectively eradicate MRSA by releasing vancomycin, preventing bacterial infection from exacerbating the deterioration of diabetic wounds. This multifunctional ROS nanopurifier with antiphlogosis, antibacterial and in-situ oxygen supply, provides a new strategy with universal and translational prospects for clinical diabetic tissue damage. Methicillin-resistant staphylococcus aureus (MRSA)-infected diabetic wounds face significant challenges in clinical care, characterized by high ROS levels, acute inflammation, vascular lesions, and hypoxia, which impede healing and risk severe complications. Here, we originally developed a reactive oxygen species (ROS) nanopurifier prepared by in-situ polymerization of superoxide dismutase (SOD), catalase (CAT), and vancomycin. It uses SOD and CAT to continuously convert ROS (H 2 O 2 and ·O 2 -) into O 2 in diabetic tissues, effectively improving hypoxia and chronic inflammation, thereby promoting angiogenesis and cell proliferation and migration, and accelerating diabetic wound healing. Vancomycin can effectively kill MRSA bacteria, avoid bacterial infection spread, and reduce complications risk. This safe, efficient and easy-to-prepare ROS nanopurifier provides a general strategy for repairing MRSA-infected diabetic tissue damage. [Display omitted] [ABSTRACT FROM AUTHOR]

Maximizing saccharification efficiency of lignocellulose and minimizing the production costs associated with enzyme requirements are crucial for sustainable biofuel production. This study presents a novel semi-fed-batch saccharification method that uses a co-culture of Clostridium thermocellum and Thermobrachium celere strain A9 to efficiently break down high solid-loading lignocellulosic biomass without the need for any external enzymes. This method optimizes saccharification efficiency and enhances glucose production from alkaline-treated rice straw, a representative lignocellulosic biomass. Initially, a co-culture of C. thermocellum and T. celere strain A9 was established with a treated rice straw loading of 150 g/l, supplemented with Tween 20, which enhanced enzymes stability and prevented unproductive binding to lignin, achieving a remarkable glucose concentration of up to 90.8 g/l. Subsequently, an additional 100 g/l of treated rice straw was introduced, resulting in a total glucose concentration of up to 140 g/l, representing 70.1% of the theoretical glucose yield from the 250 g/l treated rice straw load. In contrast, batch saccharification using an initial substrate concentration of 250 g/l of alkaline-treated rice straw without Tween 20 resulted in a glucose concentration of 55.5 g/l, with a theoretical glucose yield of only 27.7%. These results suggest that the semi-fed-batch saccharification method using co-cultivation of C. thermocellum and T. celere strain A9, supplemented with Tween 20 is an efficient microbial method for saccharifying high-concentration biomass. Moreover, this approach effectively manages high solids loading, optimizes efficiency, and reduces the need for external enzymes, thus lowering production costs and simplifying the process for industrial applications. [ABSTRACT FROM AUTHOR]

The cultivation of opium poppy is the only commercially viable source of most morphinan alkaloids. Bioproduction of morphinan alkaloids in recombinant whole-cell systems provides a promising alternate source of these valuable compounds. The enzyme codeine 3-O-demethylase can transform morphinan alkaloids by O-demethylation and has been applied in single step biotransformation reactions or as part of larger biosynthetic cascade, however, the productivity for these reactions remains low and suboptimal enzyme properties could be improved. This mutagenesis study targeted non-conserved N-and C-terminal residues, which were replaced with the equivalent residues from enzyme thebaine 6-O-demethylase. Whole cell biotransformation performance was significantly improved in Escherichia coli expressing codeine 3-O-demethylase mutants, with a ~ 2.8-fold increase in the production of oripavine from thebaine and ~ 1.3-fold increase in the production of morphine from codeine. Statistical analysis of biotransformation yield, enzyme expression and stability, predicted using changes in Gibbs free energy (ΔΔG) with deep-learning-based model DDmut, suggested that altered enzyme stability and/or expression of soluble protein may contribute to the observed improvements in biotransformation. This approach could be beneficial for screening future codeine 3-O-demethylase mutations and for other enzymes. [ABSTRACT FROM AUTHOR]

Climate change and the energy crisis, driven by excessive CO2 emissions, have emerged as pressing global challenges. The conversion of CO2 into high-value chemicals not only mitigates atmospheric CO2 levels but also optimizes carbon resource utilization. Enzyme-catalyzed carbon technology offers a green and efficient approach to CO2 conversion. However, free enzymes are prone to inactivation and denaturation under reaction conditions, which limit their practical applications. Metal–organic frameworks (MOFs) serve as effective carriers for enzyme immobilization, offering porous crystalline structures that enhance enzyme stability. Moreover, their high specific surface area facilitates strong gas adsorption, making enzyme@MOF composites particularly advantageous for CO2 catalytic conversion. In this paper, we review the synthesis technologies and the application of enzyme@MOFs in CO2 catalytic conversion. Furthermore, the strategies, including the enhancement of CO2 utilization, coenzyme regeneration efficiency, and substrate mass transfer efficiency, are also discussed to further improve the efficiency of enzyme@MOFs in CO2 conversion. The aim of this review is to present innovative ideas for future research and to highlight the potential applications of enzyme@MOFs in achieving efficient CO2 conversion. [ABSTRACT FROM AUTHOR]

This study explores various methods for the covalent immobilization of cysteine proteases (ficin, papain, and bromelain). Covalent immobilization involves the formation of covalent bonds between the enzyme and a carrier or between enzyme molecules themselves without a carrier using a crosslinking agent. This process enhances the stability of the enzyme and allows for the creation of preparations with specific and controlled properties. The objective of this study is to evaluate the impact of covalent immobilization under different conditions on the proteolytic activity of the enzymes. The most favorable results were achieved by immobilizing ficin and bromelain through covalent bonding to medium and high molecular weight chitosans, using 5 and 3.33% glutaraldehyde solutions, respectively. For papain, 5 and 6.67% glutaraldehyde solutions proved to be more effective as crosslinking agents. These findings indicate that covalent immobilization can enhance the performance of these enzymes as biocatalysts, with potential applications in various biotechnological fields. [ABSTRACT FROM AUTHOR]

The cytochrome P450 family of enzymes have been shown to be powerful biocatalysts for a wide range of selective transformations. However, the industrial uptake of P450 enzymes has been low due to issues with enzyme stability and the requirement for exogenous cofactors to drive the reaction. Herein we describe a facile and scalable method for the stabilisation and solubilisation of a P450 decarboxylase enzyme in ionic liquids. The utilisation of ionic liquids allowed for solubilisation of the relatively water-insoluble fatty acid substrate of the enzyme and the modified enzyme was found to be significantly more thermally stable in ionic liquids relative to the enzyme in aqueous media. The shift to non-aqueous solvent allowed for the enzyme to operate in the Vmax region, which when coupled with the improved thermal stability, ultimately resulted in a 1000-fold increase in the process intensity of fatty acid decarboxylation. A novel photochemical method for driving the reaction was also discovered which removed the requirement for exogenous H2O2 to be added to the reaction. These results highlight the potential of this strategy as it facilitates a holistic process of biocatalysis engineering where by solvent consideration and increased thermal stability significantly broadens the capability of the enzyme, crucial for the wider realization of industrial biocatalysis. [ABSTRACT FROM AUTHOR]

The pursuit of obtaining enzymes with high activity and stability remains a grail in enzyme evolution due to the stability-activity trade-off. Here, we develop an isothermal compressibility-assisted dynamic squeezing index perturbation engineering (iCASE) strategy to construct hierarchical modular networks for enzymes of varying complexity. Molecular mechanism analysis elucidates that the peak of adaptive evolution is reached through a structural response mechanism among variants. Furthermore, this dynamic response predictive model using structure-based supervised machine learning is established to predict enzyme function and fitness, demonstrating robust performance across different datasets and reliable prediction for epistasis. The universality of the iCASE strategy is validated by four sorts of enzymes with different structures and catalytic types. This machine learning-based iCASE strategy provides guidance for future research on the fitness evolution of enzymes. The authors design an isothermal compressibility-assisted dynamic squeezing index perturbation (iCASE) methodology to improve enzyme stability and efficacy, which is combined with machine learning predictive models to advance enzyme optimization. [ABSTRACT FROM AUTHOR]

DNA aptamers have emerged as a promising class of probes for the development of biosensors. However, the only viable strategy thus far for adjustment of probe densities is tuning DNA concentrations. Herein, we constructed a class of Supra-EAB sensors to introduce localized high probe densities and achieved significantly improved stability against enzymes. [ABSTRACT FROM AUTHOR]

Enzyme immobilization in the microfluidic chip channel to improve enzyme activity and stability has become a powerful strategy to enhance biocatalysis and biomass conversion. Here, a miniaturized glucose biosensor in which glucose oxidase (GOx) was efficiently immobilized in the PDMS chip microchannel by layer-by-layer self-assembly was developed and used successfully for amperometric determination of glucose. After the surface of PDMS microfluidic chip was treated with 365 nm ultraviolet light, the methacrylic acid monomer was grafted onto it using poly dimethyl diallyl ammonium chloride as linker, then GOx was electrostatically adsorbed on the inner wall of the PDMS chip microchannel to construct multilayer GOx. According to the results of confocal laser scanning microscopy, immobilized enzyme activity, and GOx loading, the maximum quantity of enzyme immobilized on 4 layers of GOx was observed within the PDMS microchannels. The amperometric response of glucose with the biosensor under the optimal conditions exhibited linear relationship in the range of 0.4 to 2.0 mM with correlation coefficient 0.9973, and the limit of detection was 84 μM. In addition, the microfluidic system greatly reduced the consumption of samples during tests and showed excellent accuracy, stability and reproducibility. [ABSTRACT FROM AUTHOR]

In recent years, research on farmland soil stability has gained attention due to climate change. Studying the thermal stability of soil enzymes at key crop growth stages in response to increased CO2, drought, and warming is critical for evaluating climate change impacts on crop production and soil ecosystem stability. Despite its importance, research on the thermal stability of soil nutrient cycling enzymes remains limited. A pot experiment was conducted using the soil of winter wheat (Triticum aestivum L.), one of China's main grain crops, as the research object. An artificial climate chamber was used to simulate four growth stages of winter wheat (jointing stage, flowering stage, grain filling stage, and maturity stage). Different levels of CO2 concentration (400 and 800 μmol mol−1), temperature conditions (current temperature and 4 °C higher), and water conditions (80% and 60% of field water capacity) were set, and their interactions were examined. By analyzing the temperature sensitivity (Q10) of soil enzyme activities related to soil carbon (C), nitrogen (N), and phosphorous (P) cycles in response to different treatments, the results showed that doubling CO2 concentration decreased soil C cycle enzyme Q10 and increased soil N and P cycle enzyme Q10 significantly. Additionally, soil C cycle enzyme Q10 decreased with increasing temperature, while other enzymes showed inconsistent responses. Mild drought significantly decreased the soil N-cycling enzyme Q10 in the early growth stage of winter wheat and the soil P-cycling enzyme Q10 in each growth stage, but significantly increased the soil N-cycling enzyme Q10 in the mature stage. The interaction between CO2 concentration doubling and warming exhibited a single-factor superimposed effect in reducing soil C cycle enzyme Q10. Moreover, doubling CO2 concentration offset the effect of mild drought stress on soil P cycle enzyme Q10. Above-ground biomass, soil total dissolved nitrogen, and nitrate nitrogen were identified as the primary factors influencing soil C, N, and P cycling enzyme Q10. This study is of great significance in exploring the effects of global warming on food production and the mechanism of soil ecosystem functional stability under future climate change. [ABSTRACT FROM AUTHOR]

Feruloyl esterases (FAEs) play critical roles in industrial applications such as food processing, pharmaceuticals, and paper production by breaking down plant cell walls and releasing ferulic acid. However, most bacterial FAEs function optimally in acidic environments, limiting their use in alkaline industrial processes. Additionally, FAEs with alkaline activity often lack the thermal stability required for demanding industrial conditions. In this study, an alkaline feruloyl esterase, PhFAE, from Pandoraea horticolens was identified that exhibits high catalytic activity but suffers from thermal instability, restricting its broader industrial applications. To address this limitation, molecular dynamics simulations were used to analyze enzyme stability, and FireProt, an automated computational tool, was employed to design stabilizing mutations. The engineered S155F mutant demonstrated a 7.8-fold increase in half-life at 60 °C and a 1.72-fold improvement in catalytic efficiency (Kcat/Km), corresponding to 680% and 72% enhancements, respectively, compared to the wild-type enzyme. Molecular docking and dynamics simulations revealed that these enhancements were likely due to increased hydrophobic interactions and altered surface charge, which stabilized the enzyme's structure. This study provides an effective strategy for improving the functional properties of FAEs and other industrial enzymes, broadening their applicability in diverse industrial processes. [ABSTRACT FROM AUTHOR]

In recent years, lignin derived from lignocellulosic biomass has emerged as a critical component in modern biorefinery systems. The production yield and reactivity of lignin are critical factors for advancing the research and development of lignin-derived biochemicals. The recovery of high-purity lignin, along with carbohydrates, is accomplished through the application of various advanced pretreatment techniques. However, biological pretreatment using lignin-degrading enzymes to facilitate lignin depolymerization is an environmentally benign method for the sustainable production of valuable products that occurs under mild conditions with high substrate specificity. The current review presents the role of biocatalysis in lignin valorization, focusing on lignin-degrading enzymes that facilitate different bond cleavage in the lignocellulosic biomass. The review also highlights the recent advancements in enzyme engineering that have enabled the enhancement of enzyme stability and catalytic efficiency for improving lignin valorization processes. Furthermore, the integration of omics technologies that provide valuable insights into the microbial and enzymatic pathways involved in lignin degradation is presented. The challenges and future prospects in this emerging field of study for a biorefinery concept are also outlined for improving lignin depolymerization efficiency. [ABSTRACT FROM AUTHOR]

The GM2 gangliosidoses, Tay-Sachs disease and Sandhoff disease, are devastating neurodegenerative disorders caused by β-hexosaminidase A (HexA) deficiency. In the Sandhoff disease mouse model, rescue potential was severely reduced when HexA was introduced after disease onset. Here, we assess the effect of recombinant HexA and HexD3, a newly engineered mimetic of HexA optimized for the treatment of Tay-Sachs disease and Sandhoff disease. Enzyme replacement therapy was administered by repeat intracerebroventricular injections in Sandhoff disease model mice with dosing beginning before and after signs of neurodegeneration. As previously observed, HexA effectively increased the lifespan of Sandhoff disease mice by 3.5-fold only when treatment was started before onset of neurodegeneration. In contrast, HexD3 halted motor decline and ameliorated late-stage disease severity even when dosing began late, after neurodegeneration onset. Additionally, HexD3 had advantages over HexA in enzyme stability, distribution potential, and homodimer activity. Overall, our data indicate that advanced therapeutics may widen the treatment window for neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Thermophilic Geobacillus kaustophilus HTA426 genome possesses a monoacylglycerol lipase (MAGL) gene. MAGLs can synthesize emulsifiers for use in the food and pharmaceutical industries from fatty acids and glycerol. They can also be used to analyze monoacylglycerol (MAG) levels in serum and food. The MAGL gene from strain HTA426 was artificially synthesized and heterologously expressed in Escherichia coli BL21(DE3). The recombinant His-tag fused MAGL (GkMAGL) was purified using a Ni2+-affinity column. The purified enzyme showed a temperature optimum at 65 °C and was stable up to 75 °C after 30 min incubation. In addition, the enzyme exhibited a pH optimum of 7.5 and was stable from pH 5.0 to 11.0. The enzyme hydrolyzed monoacylglycerols and showed the highest activity toward 1-monolauroylglycerol. The enzyme was stable in the presence of various organic solvents and detergents. The addition of Triton X-100 significantly increased GkMAGL activity. The thermal stability of the enzyme was higher than that of thermostable MAGL from Geobacillus sp. 12AMOR1 (12AMOR1_MAGL). Circular dichroism spectral analysis showed that the conformational stability of the GkMAGL was higher than that of 12AMOR1_MAGL at higher temperatures. These results indicate that the GkMAGL has useful features that can be used for various biotechnological applications. [ABSTRACT FROM AUTHOR]

The immobilization of enzymes in polyamide-based polymeric materials through covalent bonding is an established technique to stabilize and reuse biocatalysts in industrial processes. Traditionally, enzymes are immobilized using crosslinking agents that activate functional groups on both the support and the enzyme, creating strong bonds that securely anchor the enzyme to the surface. While effective for maintaining enzyme activity over multiple cycles, this method can reduce catalytic efficiency due to rigid binding and involves complex activation steps. Recently, in situ immobilization approaches have emerged as promising alternatives. In this method, enzymes are directly entrapped within the polymer matrix during the synthesis of the polyamide support, such as nylon, simplifying the process and offering enhanced control over enzyme distribution. For instance, studies have demonstrated that in situ immobilization can improve enzyme stability by protecting it within the polymeric network, while reducing production costs and waste. This review explores the ability of polyamide as a support material for immobilization of enzymes, analyzing key techniques, performance across applications, and future strategies to optimize polymer-enzyme interactions for industrial use. [ABSTRACT FROM AUTHOR]

Background: The emergence of multidrug-resistant hypervirulent Klebsiella pneumoniae (hvKp) has made it difficult to treat and control infections caused by this bacterium. Previously, the therapeutic effectiveness of phage-encoded depolymerase Dep_kpv74 in a mouse model of K. pneumoniae-induced thigh soft tissue infection was reported. In this study, the effect of Dep_kpv74 on blood parameters in mice, the proliferation and subpopulation composition of spleen lymphocytes, and the activity and stability of the enzyme at different pH and temperatures were further explored. Results: The stability tests showed that Dep_kpv74 remained active in the temperature range from 8 °C to 55 °C. The optimal pH value for maintaining the activity of Dep_kpv74 ranged from 5.0 to 9.0. The depolymerase was detected in the blood, spleen, and lungs of mice 10 min after intraperitoneal administration, reaching maximum activity values after 1–3 h and maintaining activity a day after administration. The introduction of Dep_kpv74 at the therapeutic dose (10 μg/mouse) or at a 10-fold higher dose did not lead to reliable changes in bloodstream cell content compared with the reference values of intact mice. The biochemical results of the studies indicated that Dep_kpv74 did not exert any toxic effects on liver and kidney functions. The results of the analysis of lymphocyte proliferative activity demonstrated that Dep_kpv74 depolymerase has a mild immunomodulatory effect. Conclusions: Thus, the results of this study provide one more confirmation that depolymerase Dep_kpv74 is a potential candidate for the treatment of infections caused by hvKp expressing K2 capsular polysaccharides. [ABSTRACT FROM AUTHOR]

Antioxidant enzymes are considered to be the most direct and safe candidates to effectively resist oxidative stress and treat senile osteoporosis. However, due to their size, existing enzyme delivery carriers are inevitably endocytosed by cells and subsequently enter endosomes/lysosomes with low pH and rich in acid hydrolase, resulting in the limited therapeutic effects. In this work, an ultrasmall superoxide dismutase (SOD) nanoformulation reinforced with ultrathin ZIF‐8 coating (utZIF‐SOD) via in situ coordination‐mediated self‐assembly strategy is constructed. Notably, utZIF‐SOD achieves direct, efficient cellular uptake mediated by small size effect, thereby avoiding lysosomal degradation. In particular, ultrathin ZIF‐8 coating strengthened the stability of enzyme. Even in aged cells with high oxidative stress levels, utZIF‐SOD maintained excellent stability, and its efficiency in scavenging excess intracellular reactive oxygen species (ROS) is ≈1.5 times that of native SOD, better promoting the osteogenic differentiation of aged bone marrow mesenchymal stem cells. In a senile osteoporosis mouse model, the bone mineral density increment after treatment with utZIF‐SOD is ≈2 times that of native SOD, achieving the reversal of senile osteoporosis. This work demonstrates the great promise in the synthesis of ultrasmall nanoformulations with ultrathin metal‐organic framework coating and opens new avenues for efficient enzyme delivery. [ABSTRACT FROM AUTHOR]

AbstractFish viscera, a fish processing by-product, is currently underutilized, despite its complex composition of high value-added components, such as proteins, lipids, and polysaccharides. This work explores the potential for revalorizing fish viscera as a sustainable source of proteins, lipids and polysaccharides in the food industry. Furthermore, their potential food applications and future perspectives are discussed. Fish viscera, constituting 12–18% of the total fish weight, is abundant in proteins, lipids, and polysaccharides. Protein hydrolysates derived from fish viscera exhibit diverse bioactivities, such as antioxidant, ACE-inhibitory, and antibacterial activities. Various enzymes with high stability properties can be extracted from fish viscera. Moreover, fish viscera-derived lipids are abundant in saturated/unsaturated fatty acids and phospholipids, which can exhibit excellent bioactivities such as immune regulatory, anti-inflammatory, lipid metabolism-regulatory and anti-tumor activities. Additionally, polysaccharides present in fish viscera display anticoagulant and antithrombotic activities. Overall, fish viscera have great potential as a good source of proteins, lipids and polysaccharides. [ABSTRACT FROM AUTHOR]

Phospholipase D3 (PLD3) has emerged as an important 5′‐exonuclease in charge of removing single‐stranded DNA in lysosomes. Rare genetic variants of the gene encoding PLD3 have been implicated in late‐onset Alzheimer's disease (AD). Ishii et al. have produced the soluble domain of human PLD3 with the aim of determining its three‐dimensional structure using X‐ray crystallography. The high‐resolution structure (2.3 Å) provides new insights into the biochemical properties of the enzyme and paves the way to a deeper understanding of amino acid replacements affecting the stability and activity of the enzyme. [ABSTRACT FROM AUTHOR]

Mesoporous metal‐organic frameworks (MOFs) are promising supports for the immobilization of enzymes, yet their applications are often limited by small pore apertures that constrain the size of encapsulated enzymes to below 5 nm. In this study, we introduced labile linkers (4,4′,4′′‐(2,4,6‐boroxintriyl)‐tribenzoate, TBTB) with dynamic boroxine bonds into mesoporous PCN‐333, resulting in PCN‐333‐TBTB with enhanced enzyme loading and protection capabilities. The selective breaking of B−O bonds creates defects in PCN‐333, which effectively expands both window and cavity sizes, thereby unlocking hidden mesopores for enzyme encapsulation. Consequently, this strategy not only increases the adsorption kinetics of small enzymes (<5 nm) such as cytochrome c (Cyt C) and horseradish peroxidase (HRP), but also enables the immobilization of various large‐sized enzymes (>5 nm), such as glycoenzymes. The glycoenzymes@PCN‐333‐TBTB platform was successfully applied to synthesize thirteen complex oligosaccharides and polysaccharides, demonstrating high activity and enhanced enzyme stability. The dynamic linker‐mediated enzyme encapsulation strategy enables the immobilization of enzymes exceeding the inherent pore size of MOFs, thus broadening the scope of enzymatic catalytic reactions achievable with MOF materials. [ABSTRACT FROM AUTHOR]

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Introduction: The demand for gluconic acid (GA) has risen recently, driven by its extensive applications in the food, healthcare, and construction industries. The biocatalysis of gluconic acid, facilitated by glucose oxidase and catalase, hinges on enzyme stability, significantly influencing catalytic efficiency. Nonetheless, catalase requires enhancements in thermal stability and activity to meet the requirements of practical applications. Methods: We evaluated ten catalases expressed in Aspergillus niger , ultimately selecting the catalase from the thermophilic fungus Thermoascus aurantiacus , labeled as TaCat, for its superior thermal stability and operational performance. We further characterized the enzymatic properties of the recombinant catalase, focusing on its thermostability. Simultaneously, we used AlphaFold2 for structural predictions and conducted in-depth analyses via accelerated molecular dynamics simulations. Results and discussion: We successfully obtained a strain with the highest catalase activity by optimizing signal peptides and overexpressing the crucial heme synthesis enzyme. Enzyme production reached an impressive 321,779.5 U/mL in a 50-L fermenter. Our application studies confirmed the considerable advantages of TaCat in terms of GA production. In conclusion, TaCat, distinguished by its remarkable thermal stability and high activity, holds substantial potential for GA production. [ABSTRACT FROM AUTHOR]

Phenolic contamination is one of the crucial concerns for the safety of drinking water. Enzymatic degradation is a green and efficient manner for phenolic compounds removal from water. However, enzymatic degradation of phenolic pollutants in water is limited as a result of the low activity, stability and reusability of the enzyme. Herein, we propose a novel strategy to degrade phenolic pollutants in water by using an enzyme‐metal hybrid catalyst constructed by in situ formation of ultrafine Cu nanoparticles on the cross‐linked Laccase aggregates. The designed Cu/Lac CLEAs showed excellent performance on phenolic pollutants removal due to the cooperative catalysis between Lac CLEA and Cu NPs and the enrichment of phenolic pollutants in hybrid catalyst. The degradation efficiency of 2,4,6‐trichlorophenol catalyzed by Cu/Lac CLEAs was improved by 33 % compared to the Lac CLEAs, while Cu NPs barely catalyzed the degradation process of phenolic pollutants. The Cu/Lac CLEAs hybrid catalyst exhibit high catalytic activity at room temperature in a wide pH range of 5–8, making the degradation of phenolic pollutants more practically operational. In other words, this study develops a novel hybrid catalyst for the efficient removal of pollutants from water. [ABSTRACT FROM AUTHOR]

Copper oxide (CuO) nanostructures have gained popularity in glucose biosensor development due to their excellent electrochemical properties, affordability and ease of fabrication. This review examines the progress of glucose biosensors based on CuO nanostructures and shows differences between enzyme-based and enzyme-dependent systems. Enzyme-based glucose oxidase sensors offer high specificity but are hampered by difficulties associated with enzyme stability at different temperatures, pH and interfering inhibitors. Unlike nonenzyme-based sensors, those using glucose oxidation directly offer high sensitivity and long working life but are struggling with specificity. The main drivers of these sensors are nanostructure morphology, synthesis techniques, surface modifications and electrode materials. The switch from more conventional CuO to sophisticated nanostructures significantly improved the sensitivity and durability of the sensor. This research focuses on optimizing these variables to address existing limitations and to advance CuO nanostructures as leads for next-generation glucose biosensing technologies. [ABSTRACT FROM AUTHOR]

The immobilization of enzymes increases their stability and allows their reuse, and bacterial cellulose (BC) is a material that can be used in this technique. This work aims to produce an enzymatic product to degrade oils and fats using BC as a matrix. Komagataeibacter hansenii bacteria produce BC membranes, and lipase is immobilized on the membranes by ex situ method. Then, the surface of the membranes is modified with zein, a hydrophobic corn protein. The membranes are characterized by TGA, FTIR, scanning electron microscopy (SEM) analysis and oil degradation test. TGA demonstrates higher stability for the membranes with lipase and zein. The FTIR spectrum of pure BC membrane and zein‐modified membrane are very similar because of the high zein coating. SEM analysis shows that zein‐modified membranes with lipase present smaller amounts of pores. Finally, using soy oil, lipase degrades oil even after immobilization during the degradation test. [ABSTRACT FROM AUTHOR]

This study focuses on the immobilization of horseradish peroxidase (HRP) on modified acrylic fabrics incorporating gold and silver nanoparticles. The process involves treating the acrylic fabrics with hydroxylamine hydrochloride and then coating them with silver and gold nanoparticles. Both obtained materials, treated acrylic fabrics-coated with silver nanoparticles (AgNPs@TA-HAC) and treated acrylic fabrics-coated with gold nanoparticles (AuNPs@TA-HAC), were utilized as supporters for HRP. The physicochemical properties of modified acrylic fabrics were investigated using FTIR, SEM, and zeta potential. HRP immobilized on AgNPs@TA-HAC displayed an activity of 69 units/g support with a specific activity of 4.55 units/mg protein, whereas HRP immobilized on AuNPs@TA-HAC demonstrated an activity of 76 units/g support with a specific activity of 4.75 units/mg protein. After the 15 repetitive cycles, the immobilized HRP on AuNPs@TA-HAC and AgNPs@TA-HAC retained 75 and 59% of their enzymatic activity, respectively. The immobilized HRP on both material supporters retained its activity better compared to the free HRP when exposed to all tested organic solvents. The Michaelis constant (Km) for free HRP and HRP immobilized on AuNPs@TA-HAC and AgNPs@TA-HAC were determined to be 6.23, 8.65, and 9.11 mM, respectively. The maximum reaction rates (Vmax) for the immobilized HRP on both supports were slightly reduced at 0.71 and 0.69 U/mL, compared to 0.74 U/mL for free HRP. This approach of utilizing acrylic fabrics and metal nanoparticles provides a promising method for enzyme immobilization, with potential applications in various industries. [ABSTRACT FROM AUTHOR]

Amylases are highly needful product in food, beverage, and detergent industry. With the objective of isolating α-amylases producing strains, samples were collected from local bakery chimneys and kitchens, Chennai, Tamil Nadu, India. The potential α-amylase producers were isolated and screened by Starch-Iodine plate assay method. The optimized parameters such as 1.5% of starch concentration in the media with 7.2-pH at 35 °C for 36 h enhanced the production of α-amylase. The strain showing high potential, identified by 16s rRNA sequencing was found to be Bacillus subtilis. The Bacillus subtilis strain produced extracellular α-amylase with activity of 1330.14 U/mL. Further partial purification by ammonium sulphate precipitation followed by dialysis and column chromatography enhanced the specific activity to 1695.306 U/mg. The molecular weight of enzymes was found to be around 36 kDa. The enzyme stability and activity characterization revealed that the enzyme was thermotolerant which was able to retain more than 70% activity at 70 °C, 80 °C and 90 °C. The purified enzyme was also found to be Ca2+ independent. The hydrolysis of raw starch from rice, wheat and corn flour using the purified α-amylase from Bacillus subtilis showed significant activity of 72.8%, 76.1% and 65.2% with 4 h incubation respectively. The potentiality of the enzyme as an additive with commercial detergent was also determined to be effective. This study favourably attracts the attention towards industrial applications. [ABSTRACT FROM AUTHOR]

The method of enzyme immobilization can ameliorate the overall stability and restoration of enzymes, hence facilitating their broader application in several sectors. This investigation utilized cellulase as a hydrolytic enzyme. In order to enhance the stability and performance of the cellulase enzyme, the research employed immobilization technology to secure the Cellic Ctec2 cellulase to the synthesized Cs@Fe3O4 nanocomposites. Fe3O4 nanoparticles (NPs) were coated with chitosan obtained from co-precipitation method that served as enzyme carrier. The NPs (Cs@ Fe3O4) were observed under XRD; VSM (vibrating-sample magnetometer) shows saturation magnetizations (Ms), UV–vis, field emission scanning electron microscopy (FESEM), and Fourier transform infrared spectroscopy (FT-IR). Response surface approach was applied to optimize the conditions for immobilization of cellulase. The optimum immobilization of cellulase reaches to 99.1% of loading efficiency and 69.7% of recovery activity with 2.5% of glutaraldehyde concentration. Furthermore, under ideal circumstances the immobilized enzyme's thermostability, pH stability, temperature tolerance and reusability, were studied with respect to free cellulase. Higher relative activity of cellulase enzyme was observed at pH 5 with 50 °C temperature than free enzyme. One percent CMC hydrolysis is considered for reusability of free and immobilized enzyme and releases 222 mg glucose/g substrate at 24 h, showing great quiescence in cellulosic biomass conversion. Immobilized cellulase demonstrated high reusability by retaining almost 61.2% up to the 5th cycles and 51.2% of activity-maintained 10th cycle of hydrolysis. Reusability of cellulase enzyme can attain a gradual decrease in relative activity as number of repeats of the cycle increases to 10 during hydrolysis and increases in glucose yield after hydrolysis. [ABSTRACT FROM AUTHOR]

Wuyi rock tea residues, generated after brewing or aroma extraction, are rich in nutrients. This study aimed to prepare ACE inhibitory peptides from the protein hydrolysate of Wuyi rock tea residues and investigate their stability. The optimal proteolysis conditions for neutral protease were optimized as: a substrate concentration of 2% (w/v), pH 7.0, hydrolysis temperature of 45°C, enzymatic digestion time of 3 h, and enzyme dosage of 6800 U/g. Under these conditions, the ACE inhibition rate of protein hydrolysate of Wuyi rock tea residues reached 70.47 ± 0.29% at a concentration of 0.5 mg/mL. Additionally, the prepared ACE inhibitory peptides demonstrated excellent stability against acid, base, heat, and certain metal ions. These findings suggest that the proteins extracted from Wuyi rock tea residues can serve as a promising source to produce highly active ACE inhibitory peptides. These peptides could be utilized in the development of nutraceuticals, functional foods, and pharmaceuticals. [ABSTRACT FROM AUTHOR]

Xylooligosaccharides (XOS) are potential prebiotic ingredients for food industries, mainly obtained after xylan hydrolysis by endoxylanases. Enzyme immobilization offers opportunities for recovery and reuse, while also enhancing its physical and chemical characteristics, such as stability and catalytic efficiency. This work aimed to immobilize the SM2 xylanase derived from the xynA gene from Orpinomyces sp. PC-2 and to evaluate its potential for XOS production. For this, SM2 xylanase was immobilized using the cross-linking methodology. The free and immobilized enzymes were characterized regarding the effect of pH, temperature, and thermostability. The cross-linked enzyme aggregate was evaluated for reuse and storage conditions and used for xylooligosaccharide production. Both free and immobilized SM2 xylanase showed maximal activity at 60 °C. The immobilized enzyme was more active at acidic and neutral conditions, and the free enzyme showed greater activity at basic conditions. The half-life of the free and immobilized xylanase was 30 and 216 h, respectively. In reuse tests, enzymatic activity increased with each cycle, and there was no statistical difference in the activity of SM2 xylanase aggregate stored at 4 and 25 °C. After saccharification, xylobiose (0.895 g/L), xylotriose (0.489 g/L), and xylohexose (0.809 g/L) were detected. As a result, immobilization enhanced thermostability, shifted the pH of maximum activity to 5, facilitated reuse, and eliminated the need for refrigerated packaging. Finally, the xylooligosaccharides produced by the SM2 xylanase are known for their prebiotic role, providing potential application of the immobilized enzyme in the food industry. [ABSTRACT FROM AUTHOR]

Coordinated by intricate crosstalk between various phytohormones, petal senescence characterizes an extraordinary stage of flower development, involving precisely regulated biochemical and physiological reprogramming. The process involves a gradual deterioration of physiological processes and structural integrity, eventually leading to the death of the flowers. Polyamines are small organic compounds that play crucial roles in plant growth, development and stress. These compounds are involved in various physiological processes, including cell division, flowering, fruit set and maturation. Given this, the current study elucidates the potential role of polyamines in regulating the senescence of cut spikes of Antirrhinum majus. Spikes, harvested with a minimum of two buds at one day before the anthesis stage, were treated with different polyamines such as Putrescine (Put), Spermidine (Spd) and Spermine (Spm) at 0.25 and 0.75 mM concentrations. A separate set of flower spikes was maintained in distilled water, which served as the control. Our results indicated that polyamines delayed the senescence of A. majus significantly leading to an extended postharvest lifespan of flower spikes. Among these treatments, 0.75 mM Put was the most effective treatment in enhancing the longevity of A. majus. On the contrary, the control spikes showed early senescence. The study showed that polyamines accentuated the activity of antioxidant enzymes and enhanced membrane stability, besides reducing lipoxygenase activity and ethylene content in flowers. Moreover, polyamines significantly increased the content of soluble proteins, total phenols and sugar fractions in the floral tissues. Also, polyamines reduced bacterial growth and improved solution uptake in flowers. The investigation elucidated that the polyamines regulate the flower senescence of A. majus by orchestrating various physio-biochemical processes. [ABSTRACT FROM AUTHOR]

The species Yarrowia lipolytica is an aerobic yeast that produces different lipase isoforms, including extracellular, intracellular, and membrane-bound ones. The immobilization of lipases, such as those from Y. lipolytica, increases enzyme stability and lowers operational costs, through its reuse. The characterization of those biocatalysts is highly important to orientate their technological applications. The present work aims to obtain different Y. lipolytica lipases, through fermentation and immobilization techniques, and to evaluate the ester synthesis and hydrolysis activity of these biocatalysts in comparison to a commercial lipase produced by Candida rugosa and test them for phytosterol ester production. High immobilization yield was achieved by microencapsulating Y. lipolytica lipase extract on magnetic nanoparticles (>99.7%). However, immobilization significantly reduced their activity (more than 90%). Lipases from Y. lipolytica showed greater 4-nitrophenyl laurate synthesis in relation to the lipase from C. rugosa. However, C. rugosa lipase was still the best biocatalyst for β-sitosterol oleate synthesis, with a conversion of more than 99%. Y. lipolytica lipases can be good catalysts for ester hydrolysis reactions, even for ester synthesis, but are not good catalysts specifically for phytosterol esters synthesis. [ABSTRACT FROM AUTHOR]

A biocatalyst has been developed for application in the simultaneous isomerization and fermentation (SIF) of xylose, which could enable operation in repeated batches and the use of xylose from biomass hemicellulose for the production of second-generation (2G) ethanol. To this end, the enzyme xylose isomerase (XI) was immobilized on eleven different supports (based on chitosan, modified silica, agarose and magnetic supports) to obtain a derivative that is stable under process conditions and easy to recover from the fermented medium for future industrial application in biorefineries. Immobilization was performed with 5 mg/gsupport, with a support-to-suspension ratio of 1:20. Phosphate (pH 7.0) and carbonate–bicarbonate (pH 10.05) buffer were used for uni-point and multi-point immobilization, respectively. Among the immobilized enzymes, the magnetic microparticle Captura N exhibited the best immobilization parameters (67% recovered activity and half-life of 10 h at 80 °C), in addition to its magnetic properties, which facilitates purification. The SIF of crude sugarcane straw acid hydrolysate was carried out in repeated batches using XI-chitosan and XI-Captura N. Although economically promising, chitosan-based supports did not enhance enzyme stability. Therefore, magnetic microparticles are a promising option as XI immobilization supports for biorefinery applications. [ABSTRACT FROM AUTHOR]

Phosphatases are enzymes that catalyze the hydrolysis of phosphate esters. They play critical roles in diverse biological processes such as extracellular nucleotide homeostasis, transport of molecules across membranes, intracellular signaling pathways, or vertebrate mineralization. Among them, tissue-nonspecific alkaline phosphatase (TNAP) is today increasingly studied, due to its ubiquitous expression and its ability to dephosphorylate a very broad range of substrates and participate in several different biological functions. For instance, TNAP hydrolyzes inorganic pyrophosphate (PPi) to allow skeletal and dental mineralization. Additionally, TNAP hydrolyzes pyridoxal phosphate to allow cellular pyridoxal uptake, and stimulate vitamin B6-dependent reactions. Furthermore, TNAP has been identified as a key enzyme in non-shivering adaptive thermogenesis, by dephosphorylating phosphocreatine in the mitochondrial creatine futile cycle. This latter recent discovery and others suggest that the list of substrates and functions of TNAP may be much longer than previously thought. In the present review, we sought to examine TNAP within the alkaline phosphatase (AP) superfamily, comparing its sequence, structure, and evolutionary trajectory. The AP superfamily, characterized by a conserved central folding motif of a mixed beta-sheet flanked by alpha-helices, includes six subfamilies: AP, arylsulfatases (ARS), ectonucleotide pyrophosphatases/phosphodiesterases (ENPP), phosphoglycerate mutases (PGM), phosphonoacetate hydrolases, and phosphopentomutases. Interestingly, TNAP and several ENPP family members appear to participate in the same metabolic pathways and functions. For instance, extra-skeletal mineralization in vertebrates is inhibited by ENPP1-mediated ATP hydrolysis into the mineralization inhibitor PPi, which is hydrolyzed by TNAP expressed in the skeleton. Better understanding how TNAP and other AP family members differ structurally will be very useful to clarify their complementary functions. Structurally, TNAP shares the conserved catalytic core with other AP superfamily members but has unique features affecting substrate specificity and activity. The review also aims to highlight the importance of oligomerization in enzyme stability and function, and the role of conserved metal ion coordination, particularly magnesium, in APs. By exploring the structural and functional diversity within the AP superfamily, and discussing to which extent its members exert redundant, complementary, or specific functions, this review illuminates the evolutionary pressures shaping these enzymes and their broad physiological roles, offering insights into TNAP's multifunctionality and its implications for health and disease. [ABSTRACT FROM AUTHOR]

The cross-linking process of collagen is one of the more important ways to improve the mineralization ability of collagen. However, the regulatory effect of dynamic cross-linking on biomineralization in vitro remains unclear. Dynamic-cross-linked mineralized collagen under different cross-linking processes, according to the process of cross-linking and mineralization of natural bone, was prepared in this study. Mineralization was performed for 12 h at 4, 8, and 12 h of collagen cross-linking. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed the characteristics of dynamic-cross-linked mineralization in terms of morphological transformation and distribution. Fourier transform infrared spectroscopy (FTIR) analysis showed the crystallinity characteristics of the hydroxyapatite (HA) crystal formation. Pre-cross-linked dynamic-cross-linked mineralization refers to the process of cross-linking for a period of time and then side cross-linked mineralization. The mineral content, enzyme stability, and mechanical properties of mineralized collagen were improved through a dynamic cross-linking process of pre-cross-linking. The swelling performance was reduced through the dynamic cross-linking process of pre-cross-linking. This study suggests that the dynamic cross-linking process through pre-cross-linking could make it easier for minerals to permeate and deposit between collagen fibers, improve mineralization efficiency, and, thus, enhance the mechanical strength of biomineralization. This study can provide new ideas and a theoretical basis for designing mineralized collagen scaffolds with better bone repair ability. [ABSTRACT FROM AUTHOR]

Enzymatic reactions play an important role in numerous industrial processes, e.g., in food production, pharmaceuticals and the production of biofuels. However, a major challenge when using enzymes in industrial applications is maintaining their stability and activity, especially under harsh operating conditions. To solve this problem, enzyme immobilization techniques have been developed. Immobilization involves fixing the enzymes on solid supports, which increases their stability, enables their reusability and facilitates the easy separation of reaction mixtures. In addition, immobilized enzymes are ideal for continuous flow systems such as millireactors, where they allow better control of reaction conditions, improving efficiency and product consistency. Glucose dehydrogenase is an important enzyme in biotechnology, particularly in biosensors and the production of biofuels, as it catalyzes the oxidation of glucose to gluconolactone, reducing NAD+ to NADH. However, like many other enzymes, it tends to lose activity over time. The immobilization of glucose dehydrogenase in a millireactor provides a controlled environment that increases the stability and activity of the enzyme. The aim of this study was to investigate the effects of different immobilization strategies on the performance of glucose dehydrogenase in a 3D printed millireactor. The enzyme was immobilized in alginate gel in three immobilization strategies: as beads, on the bottom surface, and on both the top and bottom surfaces of the millireactor. The results showed that the application of the enzyme on both surfaces improved the glucose conversion two-fold compared to immobilization in beads and four-fold compared to immobilization only on the bottom surface. The dual-surface enzyme immobilization strategy showed the highest efficiency, achieving the highest conversion of 95.76 ± 1.01% (τ = 131 min) and NADH productivity of 0.166 ± 0.01 mmol/(L·min) (τ = 7.11 min) combined with operational stability over five days. Effective diffusion rates comparable to those of aqueous solutions confirmed the suitability of alginate gels for biocatalysis. These advancements highlight the potential of this modular and scalable platform for various biotechnological applications. [ABSTRACT FROM AUTHOR]

This study evaluates the feasibility and effectiveness of using immobilized pectinase enzymes for juice processing to reduce cloudiness while preserving nutritional and bioactive properties. The research is driven by the increasing demand for innovative food products that offer enhanced functionality and health benefits. It focuses on the development and application of immobilized biocatalysts in bioprocessing, specifically using pectinase encapsulated in a sol–gel matrix. Reaction parameters for the interaction between immobilized pectinase and its primary substrate, pectin, were optimized through systematic experimentation. Optimal conditions were established, achieving enhanced enzyme activity and stability with 0.15 g of lens-shaped capsules containing 10.0 mg/mL pectinase in 24-well microplates as microreactors. Kinetic studies indicated improved substrate affinity after immobilization (Km = 0.115 mg/mL), particularly when magnetized (Kmi = 0.041 mg/mL). Operational stability and reusability assessments demonstrated potential for extended use with magnetized pectinase capsules retaining higher residual activity after a fourth reuse cycle (155% > 75%). The application of immobilized pectinase in processing peach nectar successfully reduced cloudiness and increased the release of bioactive compounds, enhancing antioxidant and anti-inflammatory activities, as evaluated by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay and the albumin method, respectively. In vitro digestion studies revealed dynamic activity profile changes, highlighting the impact of juice bioprocessing on bioavailability. [ABSTRACT FROM AUTHOR]

Trypsin and chymotrypsin were purified from the Kamori goat pancreas by affinity and ion exchange chromatography, and their activity was checked. The activity and stability of extracted enzymes under various conditions, including temperature, pH, and substrates, were studied. Extracted enzyme's activity and stability studies demonstrated that both enzymes possessed excellent activity at pH 8 and reached maximum activity after 8 hours. Purity was increased to 22.1-and 12.8-fold, with 29.2% and 25.3% yields for trypsin and chymotrypsin, respectively. According to SDS-PAGE, their molecular weights were approximately 24 and 25.5 kDa, respectively. Trypsin and chymotrypsin displayed maximum activity at 50°C, pH 8, using BAPNA (Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride) and BTEE (N benzoyl-L-tyrosine ethyl ester) as substrates. Both enzymes were stabilized in the presence of Ca++ ions. The enzymes activity declined continuously as the concentration of NaCl increased. Enzymes were effectively inhibited by SBTI and PMSF but not by pepstatin A. Kcat of trypsin and chymotrypsin was 16.8 and 36.7 S−1, and Km was 0.91 and 0.17 mm, respectively. Results suggest that Kamori goat pancreas is a promising source of trypsin and chymotrypsin due to their excellent activity and stability. [ABSTRACT FROM AUTHOR]