Functional characterization of a novel terpene synthase GaTPS1 involved in (E)-α-bergamotene biosynthesis in Gossypium arboreum.

Terpenoids in plants are mainly synthesized by terpene synthases (TPSs), which play an important role in plant-environment interactions. Gossypium arboreum is one of the important cotton cultivars with excellent pest resistance, however, the biosynthesis of most terpenoids in this plant remains unknown. In this study, we performed a comparative transcriptome analysis of leaves from intact and Helicoverpa armigera-infested cotton plants. The results showed that the H. armigera infestation mainly induced the JA signaling pathway, ten TPS genes were differentially expressed in G. arboreum leaves. Among them, a novel terpene synthase, GaTPS1, was heterologously expressed and functionally characterized in vitro. The enzymatic reaction indicated that recombinant GaTPS1 was primarily responsible for the production of (E)-α-bergamotene. Moreover, molecular docking and site-directed mutagenesis analysis demonstrated that two amino acid residues, A412L and Y535F, distinctly influenced the catalytic activities and product specificity of GaTPS1. The mutants GaTPS1-A412L and GaTPS1-Y535F resulted in a decrease in the proportion of products (E)-α-bergamotene and D-limonene, while an increase in the proportion of products (E)-ß-farnesene, α-pinene and ß-myrcene. Our findings provide valuable insights into understanding the molecular basis of terpenoid diversity in G. arboreum, with potential applications in plant metabolism regulation and the improvement of resistant cotton cultivars.

Molecular Identification and Engineering a Salt-Tolerant GH11 Xylanase for Efficient Xylooligosaccharides Production.

This study identified a salt-tolerant GH11 xylanase, Xynst, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xynst and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xynst in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (Km = 0.30 mg mL-1 for Xynst and 0.18 mg mL-1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (Kcat/Km = 15483.33 mL mg-1 s-1). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xynst did. The conversion efficiencies of Xynst and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.

A disulfide bond mutant of Pseudoalteromonas porphyrae κ-carrageenase conferred improved thermostability and catalytic activity and facilitated its utilization in κ-carrageenan industrial waste residues recycling.

In this study, Discovery Studio was employed to predict the potential disulfide bond mutants of the catalytic domain of Pseudoalteromonas porphyrae κ-carrageenase to improve the catalytic activity and thermal stability. The mutant N205C-G239C was identified with significantly increased catalytic activity toward κ-carrageenan substrate, with activity 4.28 times that of WT. The optimal temperature of N205C-G239C was 55 °C, 15 °C higher than that of WT. For N205C-G239C, the t1/2 value at 50 °C was 52 min, 1.41 times that of WT. The microstructural analysis revealed that the introduced disulfide bond N205C-G239C could create a unique catalytic environment by promoting favorable interactions with κ-neocarratetraose. This interaction impacted various aspects such as product release, water molecule network, thermodynamic equilibrium, and tunnel size. Molecular dynamics simulations demonstrated that the introduced disulfide bond enhanced the overall structure rigidity of N205C-G239C. The results of substrate tunnel analysis showed that the mutation led to the widening of the substrate tunnel. The above structure changes could be the possible reasons responsible for the simultaneous enhancement of the catalytic activity and thermal stability of mutant N205C-G239C. Finally, N205C-G239C exhibited the effective hydrolysis of the κ-carrageenan industrial waste residues, contributing to the recycling of the oligosaccharides and perlite.

Hyperglycosylation impairs the inhibitory activity of rCdtPLI2, the first recombinant beta-phospholipase A2 inhibitor.

Crotoxin, a phospholipase A2 (PLA2) complex and the major Crotalus venom component, is responsible for the main symptoms described in crotalic snakebite envenomings and a key target for PLA2 inhibitors (PLIs). PLIs comprise the alpha, beta and gamma families, and, due to a lack of reports on beta-PLIs, this study aimed to heterologously express CdtPLI2 from Crotalus durissus terrificus venom gland to improve the knowledge of the neglected beta-PLI family. Thereby, recombinant CdtPLI2 (rCdtPLI2) was produced in the eukaryotic Pichia pastoris system to keep some native post-translational modifications. rCdtPLI2 (~41 kDa) presents both N- and O-linked glycans. Alpha-mannosidase digested-rCdtPLI2 (1 mol) strongly inhibited (73%) CB-Cdc catalytic activity (5 moles), demonstrating that glycosylations performed by P. pastoris affect rCdtPLI2 action. Digested-rCdtPLI2 also inhibited PLA2s from diverse Brazilian snake venoms. Furthermore, rCdtPLI2 (1 mol) abolished the catalytic activity of Lmr-PLA2 (5 moles) and reduced the CTx-Cdc (5 moles) enzyme activity by 65%, suppressing basic and acidic snake venom PLA2s. Additionally, crotalic antivenom did not recognize rCdtPLI2, suggesting a lack of neutralization by antivenom antibodies. These findings demonstrate that studying snake venom components may reveal interesting novel molecules to be studied in the snakebite treatment and help to understand these underexplored inhibitors.

Bioinspired synergy strategy based on the integration of nanozyme into a molecularly imprinted polymer for improved enzyme catalytic mimicry and selective biosensing.

Nanozymes offer many advantages such as good stability and high catalytic activity, but their selectivity is lower than that of enzymes. This is because most of enzymes have a protein component (apoenzyme) for substrate affinity to enhance selectivity and a non-protein element (coenzyme) for catalytic activity to improve sensitivity. The synergy between molecularly imprinted polymers (MIPs) and nanozymes can mimic natural enzymes, with MIP acting as the apoenzyme and nanozyme as the coenzyme. Despite researchers' attempts to associate MIPs with nanozymes, the full potential of this combination remains not well explored. This study addresses this gap by integrating Fe3O4-Lys-Cu nanozymes with peroxidase-like catalytic activities within appropriate MIPs for L-DOPA and dopamine. The catalytic performance of the nanozyme was improved by the presence of Cu in Fe3O4-Lys-Cu and further enhanced by MIP. Indeed, the exploration of the pre-concentration property of MIP has increased twenty-fold the catalytic activity of the nanozyme. Moreover, this synergistic combination facilitated the template removal process during MIP production by reducing the extraction time from several hours to just 1 min thanks to the addition of co-substrates which trigger the reaction with nanozyme and release the template. Overall, the synergistic combination of MIPs and nanozymes offers a promising avenue for the design of artificial enzymes.

Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis.

The L-isoleucine-4-dioxygenase converts L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C-45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.

Combined Liposome-Gold Nanoparticles from Honey: The Catalytic Effect of Cassyopea® Gold on the Thermal Isomerization of a Resonance-Activated Azobenzene.

Gold nanoparticles (AuNPs) have been synthesized directly inside liposomes using honey as a reducing agent. The obtained aggregates, named Cassyopea® Gold due to the method used for their preparation, show remarkable properties as reactors and carriers of the investigated AuNPs. A mean size of about 150 nm and negative surface charge of -46 mV were measured for Cassyopea® Gold through dynamic light scattering and zeta potential measurements, respectively. The formation of the investigated gold nanoparticles into Cassyopea® liposomes was spectroscopically confirmed by the presence of their typical absorption band at 516 nm. The catalytic activity of the combined liposome-AuNP nanocomposites was tested via the thermal cis-trans isomerization of resonance-activated 4-methoxyazobenzene (MeO-AB). The kinetic rate constants (kobs) determined at 25 °C in the AuNP aqueous solution and in the Cassyopea® Gold samples were one thousand times higher than the values obtained when performing MeO-AB cis-trans conversion in the presence of pure Cassyopea®. The results reported herein are unprecedented and point to the high versatility of Cassyopea® as a reactor and carrier of metal nanoparticles in chemical, biological, and technological applications.

Oxidative stress-modifying effects of TiO2nanoparticles with varying content of Ti3+(Ti2+) ions.

Nanoparticles (NPs) with reactive oxygen species (ROS)-regulating ability have recently attracted great attention as promising agents for nanomedicine. In the present study, we have analyzed the effects of TiO2defect structure related to the presence of stoichiometric (Ti4+) and non-stoichiometric (Ti3+and Ti2+) titanium ions in the crystal lattice and TiO2NPs aggregation ability on H2O2- and tert-butyl hydroperoxide (tBOOH)-induced ROS production in L929 cells. Synthesized TiO2-A, TiO2-B, and TiO2-C NPs with varying Ti3+(Ti2+) content were characterized by x-ray powder diffraction, transmission electron microscopy, small-angle x-ray scattering, x-ray photoelectron spectroscopy, and optical spectroscopy methods. Given the role of ROS-mediated toxicity for metal oxide NPs, L929 cell viability and changes in the intracellular ROS levels in H2O2- and tBOOH-treated L929 cells incubated with TiO2NPs have been evaluated. Our research shows that both the amount of non-stoichiometric Ti3+and Ti2+ions in the crystal lattice of TiO2NPs and NPs aggregative behavior affect their catalytic activity, in particular, H2O2decomposition and, consequently, the efficiency of aggravating H2O2- and tBOOH-induced oxidative damage to L929 cells. TiO2-A NPs reveal the strongest H2O2decomposition activity aligning with their less pronounced additional effects on H2O2-treated L929 cells due to the highest amount of Ti3+(Ti2+) ions. TiO2-C NPs with smaller amounts of Ti3+ions and a tendency to aggregate in water solutions show lower antioxidant activity and, consequently, some elevation of the level of ROS in H2O2/tBOOH-treated L929 cells. Our findings suggest that synthesized TiO2NPs capable of enhancing ROS generation at concentrations non-toxic for normal cells, which should be further investigated to assess their possible application in nanomedicine as ROS-regulating pharmaceutical agents.

Engineering of Substrate-Binding Domain to Improve Catalytic Activity of Chondroitin B Lyase with Semi-Rational Design.

Dermatan sulfate and chondroitin sulfate are dietary supplements that can be utilized as prophylactics against thrombus formation. Low-molecular-weight dermatan sulfate (LMWDS) is particularly advantageous due to its high absorbability. The enzymatic synthesis of low-molecular-weight dermatan sulfates (LMWDSs) using chondroitin B lyase is a sustainable and environmentally friendly approach to manufacturing. However, the industrial application of chondroitin B lyases is severely hampered by their low catalytic activity. To improve the activity, a semi-rational design strategy of engineering the substrate-binding domain of chondroitin B lyase was performed based on the structure. The binding domain was subjected to screening of critical residues for modification using multiple sequence alignments and molecular docking. A total of thirteen single-point mutants were constructed and analyzed to assess their catalytic characteristics. Out of these, S90T, N103C, H134Y, and R159K exhibited noteworthy enhancements in activity. This study also examined combinatorial mutagenesis and found that the mutant H134Y/R159K exhibited a substantially enhanced catalytic activity of 1266.74 U/mg, which was 3.21-fold that of the wild-type one. Molecular docking revealed that the enhanced activity of the mutant could be attributed to the formation of new hydrogen bonds and hydrophobic interactions with the substrate as well as neighbor residues. The highly active mutant would benefit the utilization of chondroitin B lyase in pharmaceuticals and functional foods.

Correlative Effects of Carbon Support Structures and Surface Properties on ORR Catalytic Activities of Loaded Catalysts.

As a complex three-phase heterogeneous catalyst, the oxygen reduction reaction (ORR) catalyst activity is determined by the interfacial and surface structures and chemical state of the catalyst support. As a typical biomass carbon-based support, rice husk-based porous carbon (RHPC) has natural unique hierarchical porous structures, which easily regulate the microstructure and surface properties. This study explored the correlative effects of RHPC structure and surface properties on ORR catalytic activity through the typical modification methods, namely, alkali etching, high temperature, oxidation, and ball milling. The various factors for the joint effects are defined as the specific surface area, oxygen-containing functional groups, graphite edge defects, resistivity, and contact angle. The analysis of such joint influences is difficult to quantitatively evaluate due to the large number of experimental factors and small sample sizes. Partial least-squares (PLS) can better deal with such problems. Therefore, a PLS regression model was established to evaluate the relative weight of each factor on the catalytic activity for the RHPC-based support catalysts. The results reveal that the regression coefficients of four factors yield similar magnitude for the effect of the half-wave potential (E1/2). However, graphite edge defects had a more significant impact on the limiting diffusion current density (J) and electron transfer number (n). Furthermore, an optimal support named BM-RHPC-3 was prepared with more defects and oxygen-containing functional groups, which prepared Fe-NS/BM-RHPC-3 presenting the best ORR catalytic activity (E1/2 = 0.880 V, J of 5.15 mA cm-2), superior to Pt/C (E1/2 = 0.844 V, J of 4.99 mA cm-2). The statistical regression model is validated with a relative error of less than 5% between predicted and true values for analyzing RHPC-based ORR catalysts' catalytic performance. It shows the feasibility of experiment-informed learning for data-driven material discovery and design.

Crucial amino acids identified in Δ12 fatty acid desaturases related to linoleic acid production in Perilla frutescens.

Perilla oil from the medicinal crop Perilla frutescens possess a wide range of biological activities and is generally used as an edible oil in many countries. The molecular basis for its formation is of particular relevance to perilla and its breeders. Here in the present study, four PfFAD2 genes were identified in different perilla cultivars, PF40 and PF70, with distinct oil content levels, respectively. Their function was characterized in engineered yeast strain, and among them, PfFAD2-1PF40, PfFAD2-1PF70 had no LA biosynthesis ability, while PfFAD2-2PF40 in cultivar with high oil content levels possessed higher catalytic activity than PfFAD2-2PF70. Key amino acid residues responsible for the enhanced catalytic activity of PfFAD2-2PF40 was identified as residue R221 through sequence alignment, molecular docking, and site-directed mutation studies. Moreover, another four amino acid residues influencing PfFAD2 catalytic activity were discovered through random mutation analysis. This study lays a theoretical foundation for the genetic improvement of high-oil-content perilla cultivars and the biosynthesis of LA and its derivatives.

Zinc Tellurium with Anionic Vacancies Anchored on Ordered Macroporous Carbon Skeleton Enabling Accelerated Polysulfide Conversion for Lithium-Sulfur Batteries.

Lithium-sulfur batteries (LSBs) showcase great promise for large-scale energy storage systems, however, their practical commercialization is seriously hindered by the sluggish redox reaction kinetics and detrimental shuttle effect of soluble polysulfides. Herein, small ZnTe1- x nanoparticles with anionic vacancies firmly anchored on 3D ordered macroporous N-doped carbon skeleton (3DOM-ZnTe1- x@NC) are elaborately constructed as a high-efficiency electrocatalyst for LSBs. The ordered macroporous carbon skeleton not only greatly increases the external surface area to expose sufficient active sites but also facilitates the electrolyte penetration. Additionally, the experimental studies combined with theoretical calculations confirm the presence of Te vacancies optimizes the electronic structure to enhance the intrinsic catalytic activity and chemical absorption. Consequently, LSBs assembled with the 3DOM-ZnTe1- x@NC modified separators exhibit high specific discharge capacity, as well as superior rate performance and good long-term cycling stability. Even under a high sulfur loading of 6.5 mg cm-2 and lean electrolyte, an impressive areal capacity of 5.28 mAh cm-2 is achieved at 0.1 C after 100 cycles. More significantly, the 3DOM-ZnTe1- x@NC based pouch cells are also fabricated to demonstrate its potential for practical applications. This work highlights that the rational combination of 3DOM architecture and vacancy engineering is important for designing advanced Li-S electrocatalysts.

Role of pretty nanoflowers as novel versatile analytical tools for sensing in biomedical and bioanalytical applications.

In recent years, an encouraging breakthrough in the synthesis of immobilized enzymes in flower-shaped called "organic-inorganic hybrid nanoflowers (hNFs)" with greatly enhanced catalytic activity and stability were reported. Although, these hNFs were discovered by accident, the enzymes exhibited highly enhanced catalytic activities and stabilities in the hNFs compared with the free and conventionally immobilized enzymes. Herein, we rationally utilized the catalytic activity of the hNFs for analytical applications. In this comprehensive review, we covered the design and use of the hNFs as novel versatile sensors for electrochemical, colorimetric/optical and immunosensors-based detection strategies in analytical perspective.

P-doped RuSe2 on Porous N-doped Carbon Matrix as Catalysts for Accelerated Sulfur Redox Reactions.

Monocomponent catalysts exhibit the limited catalytic conversion of polysulfides due to their intrinsic electronic structure, but their catalytic activity can be improved by introducing heteroatoms to regulate its electronic structure. However, the rational selection principles of doping elements remain unclear. Here, we are guided by theoretical calculations to select the suitable doping elements based on the balanced relationship between the adsorption strength of lithium polysulfides (LiPSs) and catalytic activity of lithium sulfide. We apply the screening method to develop a new catalyst of phosphorus doped RuSe2, manifesting the further enhanced conductivity compared with original RuSe2, facilitating charge transfer and further modulating the d-band center of RuSe2, thereby augmenting its effectiveness in interacting with LiPSs. Consequently, the assembled cell exhibits an areal capacity of 7.7 mAh cm-2, even under high sulfur loading of 8.0 mg cm-2 and a lean electrolyte condition (5.0 µL mg-1). This rational screening strategy offers a robust solution for the design of advanced catalysts in the field of lithium-sulfur batteries and potentially other domains as well.

Manipulating Orbital Hybridization of CoSe2 by S Doping for the Highly Active Catalytic Effect of Lithium-Sulfur Batteries.

In recent years, various transition metal compounds have been extensively studied to deal with the problems of slow reaction kinetics and the shuttle effect of lithium-sulfur (Li-S) batteries. Nevertheless, their catalytic performance still needs to be further improved by enhancing intrinsic catalytic activity and enriching active sites. Doping is an effective means to boost the catalytic performance through adjusting the electron structure of the catalysts. Herein, the electron structure of CoSe2 is adjusted by doping P, S with different p electron numbers and electronegativity. After S doping (S-CoSe2), the content of Co2+ increases, and charge is redistributed. Furthermore, more electrons are transferred between Li2S4/Li2S and S-CoSe2, and optimal Co-S bonds are formed between them with optimized d-p orbital hybridization, making the bonds of Li2S4/Li2S the longest and easy to break and decompose. Consequently, the Li-S batteries with the S-CoSe2-modified separator achieve improved rate performance and cycling performance, benefiting from the better bidirectional catalytic activity. This work will provide reference for the selection of the anion doping element to enhance the catalytic effect of transition metal compounds.

Enhanced catalytic performance of Candida rugosa lipase through immobilization on zirconium-2-methylimidazole: A novel biocatalyst approach.

Immobilization of enzymes on suitable supports is a critical approach for enhancing enzyme stability, reusability, and overall catalytic efficiency. This study explores the immobilization of Candida rugosa lipase on zirconium-based 2-methylimidazole (ZrMI) nanoparticles, aiming to develop a stable and reusable biocatalyst. The ZrMI was produced via a solvothermal technique and analyzed using various characterization methods. Candida rugose lipase was immobilized using cross-linking agents, achieving an 87 % immobilization efficiency. The immobilized enzyme exhibited significantly enhanced thermal stability, broader pH tolerance, and increased catalytic efficiency compared to free C. rugose lipase. The ZrMI@lipase retained 69 % of its enzymatic activity following a 60-day storage period at 4 °C. Notably, it displayed significant reusability, maintaining 65 % of its original activity after undergoing 15 catalytic cycles. Examination of the kinetics revealed that the immobilized enzyme possessed a heightened substrate affinity (Km of 4.1 mM) and maximal reaction rate (Vmax of 85.7 µmol/ml/min) in comparison to the free enzyme (Km of 5.4 mM and Vmax of 69 µmol/ml/min), indicating enhanced catalytic efficiency. Validation through zeta potential and hydrodynamic size assessments verified the successful binding of the enzyme and the consistent colloidal characteristics. These results suggest that ZrMI is a promising support for C. rugose lipase immobilization, offering improved stability and reusability for various industrial applications. The study highlights the potential of ZrMI@lipase as an efficient and durable biocatalyst, contributing to advancements in enzyme immobilization technology and sustainable industrial processes.

Nanozymes and their biomolecular conjugates as next-generation antibacterial agents: A comprehensive review.

Antimicrobial resistance (AMR), the ability of bacterial species to develop resistance against exposed antibiotics, has gained immense global attention in the past few years. Bacterial infections are serious health concerns affecting millions of people annually worldwide. Therefore, developing novel antibacterial agents that are highly effective and avoid resistance development is imperative. Among various strategies, recent developments in nanozyme technology have shown promising results as antibacterials in several antibiotic-sensitive and resistant bacterial species. Nanozymes offer several advantages over corresponding natural enzymes, such as inexpensive, stable, multifunctional, tunable catalytic properties, etc. Although the use of nanozymes as antibacterial agents has provided promising results, the specific biomolecule-conjugated nanozymes have shown further improvement in catalytic performance and associated antibacterial efficacy. The exclusive design of functional nanozymes with theranostic potential is found to simultaneously inhibit the growth and image of AMR bacterial species. This review comprehensively summarizes the history of nanozymes, their classification, biomolecules conjugated nanozyme, and their mechanism of enzyme-mimetic activity and associated antibacterial activity in antibiotic-sensitive and resistant species. The futureneeds to effectively engineer the existing or new nanozymes to curb AMR have also been discussed.

Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor.

BACKGROUND: D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants. RESULTS: The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle. CONCLUSION: All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

Oxygen vacancy-engineered cerium oxide mediated by copper-platinum exhibit enhanced SOD/CAT-mimicking activities to regulate the microenvironment for osteoarthritis therapy.

Cerium oxide (CeO2) nanospheres have limited enzymatic activity that hinders further application in catalytic therapy, but they have an "oxidation switch" to enhance their catalytic activity by increasing oxygen vacancies. In this study, according to the defect-engineering strategy, we developed PtCuOX/CeO2-X nanozymes as highly efficient SOD/CAT mimics by introducing bimetallic copper (Cu) and platinum (Pt) into CeO2 nanospheres to enhance the oxygen vacancies, in an attempt to combine near-infrared (NIR) irradiation to regulate microenvironment for osteoarthritis (OA) therapy. As expected, the Cu and Pt increased the Ce3+/Ce4+ ratio of CeO2 to significantly enhance the oxygen vacancies, and simultaneously CeO2 (111) facilitated the uniform dispersion of Cu and Pt. The strong metal-carrier interaction synergy endowed the PtCuOX/CeO2-X nanozymes with highly efficient SOD/CAT-like activity by the decreased formation energy of oxygen vacancy, promoted electron transfer, the increased adsorption energy of intermediates, and the decreased reaction activation energy. Besides, the nanozymes have excellent photothermal conversion efficiency (55.41%). Further, the PtCuOX/CeO2-X antioxidant system effectively scavenged intracellular ROS and RNS, protected mitochondrial function, and inhibited the inflammatory factors, thus reducing chondrocyte apoptosis. In vivo, experiments demonstrated the biosafety of PtCuOX/CeO2-X and its potent effect on OA suppression. In particular, NIR radiation further enhanced the effects. Mechanistically, PtCuOX/CeO2-X nanozymes reduced ras-related C3 botulinum toxin substrate 1 (Rac-1) and p-p65 protein expression, as well as ROS levels to remodel the inflammatory microenvironment by inhibiting the ROS/Rac-1/nuclear factor kappa-B (NF-κB) signaling pathway. This study introduces new clinical concepts and perspectives that can be applied to inflammatory diseases.

How carbohydrate-binding module affects the catalytic properties of endoglucanase.

The important role of Carbohydrate-binding module (CBM) in the cellulases catalytic activity has been widely studied. CBM3 showed highest affinity for cellulose substrate with 84.69 % adsorption rate among CBM1, CBM2, CBM3, and CBM4 in this study. How CBM affect the catalytic properties of GH5 endoglucanase III from Trichoderma viride (TvEG3) was systematically explored from two perspectives: the deletion of its own CBM(TvEG3dc) and the replacement of high substrate affinity CBM3 (TvEG3dcCBM3). Compared with TvEG3, TvEG3dc lost its binding ability on Avicel and filter paper, but its catalytic activity did not change significantly. The binding ability and catalytic activity of TvEG3dcCBM3 to Avicel increased 348.3 % and 372.51 % than that of TvEG3, respectively. The binding ability and catalytic activity of TvEG3dcCBM3 to filter paper decreased 51.7 % and 33.33 % than that of TvEG3, respectively. Further structural analysis of TvEG3, TvEG3dc, and TvEG3dcCBM3 revealed no changes in the positions and secondary structures of the key amino acids. These results demonstrated that its own CBM1 of TvEG3 did not affect its catalytic activity center, so it had no effect on its catalytic activity. But CBM3 changed the adsorption affinity for different substrates, which resulted in a change in the catalytic activity of the substrate.