This study addressed the issue of insufficient activity in CALB lipase during the catalytic synthesis of key chiral intermediates for moxifloxacin. A structure-guided protein engineering strategy was employed to systematically modify its functional domains. Through molecular dynamics simulations of CALB-I189K, multiple regions exhibiting high conformational flexibility were preliminarily identified. Subsequently, by integrating 3D structural alignment with active site pocket distance analysis, the functionally most critical region (143-146) was selected. A site-directed saturation mutation library was constructed specifically targeting this region. Building upon the previously reported CALB-I189K, a mutant I189K/L144R/A146K was ultimately obtained through high-throughput screening combined with chiral HPLC validation. This mutant maintains excellent stereoselectivity (E = 206.52) while enhancing catalytic efficiency (kcat/Kappa m) to 273.73 min-1 & centerdot;mM-1, approximately 4.5-fold that of I189K. At a substrate concentration of 1 M, it achieves 50% conversion within 2.6 h, demonstrating kinetic resolution capabilities approaching industrial standards. Molecular simulation analysis indicates that the L144R and A146K mutations synergistically enhance catalytic performance primarily by optimizing spatial distances between catalytic residues. This study not only provides a high-performance catalyst for the efficient biosynthesis of moxifloxacin chiral intermediates but also offers new insights for enzyme rational design based on dynamic structural information.

Conventional sutures provide passive mechanical closure but lack active therapeutic functions, often resulting in delayed healing and excessive scarring. Here, we report a bioresorbable living electronic suture that integrates mechanically robust spider silk protein (spidroin) with liquid metal, enabling near-infrared-triggered contractile actuation, triboelectric energy harvesting, and microfluidic-guided drug delivery within a single filament. Mechanical training during post-spinning processing enhances tensile strength (>115 MPa) and structural stability, while surface microcolumn imprinting improves interfacial conductivity and programs directional capillary transport. The resulting fiber converts tissue motion into localized electric fields for real-time wound sensing, and surface microstructures enable programmable microfluidic transport and drug delivery. Owing to the intrinsic biodegradability and biocompatibility of spidroin, the suture gradually resorbs during tissue regeneration. In vivo murine studies demonstrate accelerated and higher quality wound repair, with scar area reduced by 58.3% compared with commercial sutures. Collectively, this work establishes a scalable strategy for converting structural protein fibers into multifunctional bioelectronic sutures, advancing next-generation smart wound care.

GH11 xylanases are widely used for hemicellulose depolymerization and xylooligosaccharide (XOS) production, yet their catalytic efficiency often remains suboptimal for practical application. Here, we implemented a gate-neighborhood engineering strategy to enhance catalytic efficiency of GH11 xylanases by reshaping the dynamic ensemble surrounding the conserved aromatic-proline gatekeeper motif. Using the GH11 xylanase XynNTU-CDLs from Paenibacillus campinasensis NTU-11 as a model, docking-coupled MD simulations identified dynamic hotspots adjacent to the W21/P134 gate, which were subsequently subjected to focused saturation mutagenesis. The resulting double variant, S24D/K137S, exhibited a 7.4-fold increase in catalytic efficiency (kcat/Km) toward beechwood xylan, arising from both improved substrate affinity and accelerated turnover, while preserving the characteristic endo-acting product profile. Molecular simulations revealed that this improvement did not arise from simple gate expansion or narrowing, but from a redistribution of loop flexibility that promotes a two-stage “access-then-clamp” mechanism, stabilizes productive substrate positioning, strengthens protein-substrate interactions, and reshapes the free-energy landscape toward a more focused low-energy basin. In contrast, direct disruption of the W-P gate (W21G/P134G) enlarged gate opening but reduced catalytic activity, highlighting the importance of dynamic regulation rather than static geometric opening. Extension of this strategy to two additional GH11 xylanases also identified gate-adjacent variants with improved catalytic activity, supporting gate-neighborhood regulation as a practical route for tuning GH11 xylanase catalysis.

Multienzyme cascades provide an efficient biocatalytic strategy for synthesizing nucleoside analogs from simple sugar donors and nucleobases. In pentose-to-nucleoside cascades, purine nucleoside phosphorylase (PNP) catalyzes the terminal N-glycosylation step and strongly influences product formation and substrate scope. Here, seven putative PNP homologs from microorganisms isolated from low-temperature environments were evaluated under EcRK-EcPPM-PNP cascade conditions. A PNP from Colwellia sp. WH041, designated ColPNP, showed the highest arabinofuranosylation activity and was selected for further characterization. ColPNP displayed optimal activity at 45 °C and exhibited a 12.7-fold higher catalytic efficiency than Escherichia coli PNP at the same temperature. Molecular dynamics simulations supported the favorable catalytic performance of ColPNP under moderate-temperature conditions. After optimization of the EcRK-EcPPM-ColPNP cascade, vidarabine conversion reached 58.2% within 12 h. The optimized system was further applied to synthesize diverse nucleoside analogs with conversions of 45.8%-89.8%, and guanosine analogs were obtained through sequential EcADA-mediated deamination. This study expands the PNP toolbox and demonstrates ColPNP as an efficient module for enzymatic nucleoside analog synthesis.

Efficient multiphase bioprocessing for sustainable lipid valorization and biodiesel synthesis relies on robust solid–liquid biointerfaces. Their rational design is fundamentally constrained by severe reaction–diffusion constraints and the intrinsic activity–stability trade-off. Herein, we report a synergistic “orient-and-lock” strategy on bifunctional mesoporous SBA-15 to decouple these limitations. Hydrophobic domains first drive the oriented adsorption and “lid-opening” hyperactivation of Thermomyces lanuginosus lipase, followed by rapid isocyanide-mediated covalent anchoring to kinetically trap this metastable state without compromising structural flexibility. Molecular dynamics simulations elucidate a critical microenvironmental compartmentalization effect. This spatial partitioning selectively enriches lipophilic substrates while isolating polar inhibitors through localized hydrogen-bond networks and hydrophobic compartments, thereby effectively reducing substrate transfer resistance and minimizing active site blockage. Consequently, the immobilized biocatalyst exhibits an unprecedented 40-fold enhancement in apparent specific activity and a 57-fold increase in catalytic efficiency (kcat/Km) compared to the free enzyme. Its excellent tolerance to methanol enables a highly efficient one-step methanol addition reaction. The system demonstrates exceptional operational robustness, retaining 80% activity over 25 consecutive cycles and achieving a 96.8% biodiesel yield, establishing a synergistic paradigm of conformational engineering and transport regulation for high-performance heterogeneous biocatalysts.

Aliphatic alpha, w-dicarboxylic acids (alpha, w-DCAs) are essential monomers for polyesters and polyamides, yet their production remains dominated by energy-intensive and unsustainable chemical routes. This study reports a selfsustained in vitro biotransformation (ivBT) platform that converts renewable fatty acids into alpha, w-DCAs through an enzyme-driven cascade. This system integrates three catalytic modules: an H2O2-dependent cytochrome P450 enzyme MCC for terminal hydroxylation, a bifunctional alcohol dehydrogenase ADH2 for sequential oxidation of alcohol groups to carboxylic acids, and a flavin-bridged catalyst F4 that enables NAD+/NADH cofactor cycling with in situ H2O2 generation. Through enzyme engineering and reaction optimization, 2.06 g/L dodecanedioic acid was produced via a one-pot shake-flask scale reaction, with broad compatibility across even-numbered C8C16 fatty acids. This self-sustained system provides an economically viable route for sustainable alpha, w-DCAs production, establishing a general framework for developing advanced in vitro multi-enzyme catalytic systems.

Bacterial-related wound infections are usually accompanied by robust inflammation, which delay the rate of healing of traumatic wounds. The hydrogel wound dressings with antibacterial properties are expected to promote wound healing progress. Herein, a multifunctional composite hydrogel (PRLM) was developed via a green freeze-thaw cycling process. The composite hydrogel incorporated with polyvinyl alcohol (PVA), regenerated cellulose (RC), lignin and MXene. The incorporation of RC significantly affected the mechanical strength of composite hydrogels through hydrogen bonding interactions. Lignin and MXene were loaded into the hydrogel to endow it with photothermal antibacterial performance. The inhibition rates of PRLM hydrogel against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) reached 99.1 % and 98.6 %. Moreover, in vitro studies demonstrated that the composite hydrogel exhibited good cytocompatibility with L929 cells. Therefore, this work may provide novel insights into the development of dressings for bacterial-infected wounds.

Background: Inflammatory bowel disease (IBD) involves dysregulated mucosal immunity and compromised intestinal barrier integrity. Prebiotic oligosaccharides have emerged as immunomodulatory candidates, yet their sex-specific effects on piscine mucosal immunity remain poorly understood. Zebrafish (Danio rerio) offers a robust vertebrate model for dissecting intestinal immunopathology due to conserved innate and adaptive immune signaling.Methods: This study employed network pharmacology to predict GOS anti-colitis targets, followed by in vivo validation in a TNBS-induced zebrafish colitis model. Male and female zebrafish were fed 0.4% GOS or inulin for 35 days prior to intraperitoneal TNBS challenge. Intestinal histopathology was assessed by H&E and AB-PAS staining; mRNA expression of IL-1β, IL-6, TNF-α, IFN-γ1, and IL-10 was quantified by RT-qPCR.Results: GOS (96.62% purity) was biosynthesized via β-galactosidase BMG and showed favorable biocompatibility without growth impairment. Post-TNBS challenge, GOS significantly improved survival compared to inulin controls. Notably, sex-specific immunomodulation was observed: female zebrafish exhibited downregulated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ1), whereas males displayed marked IL-10 upregulation. Histologically, GOS restored goblet cell counts, reduced crypt damage, and recovered mucus secretion, indicating reinforced intestinal barrier function.Conclusion: These findings demonstrate that GOS exerts dual anti-colitis mechanisms through sex-biased cytokine regulation and mucosal barrier restoration in zebrafish, supporting its potential as an immunoprotective feed additive in aquaculture and a translational model for human IBD therapeutics.

Methods for accurate and efficient protein quantification are crucial in the fields of biomanufacturing, synthetic biology and protein development for optimizing related bioprocesses. Herein, we developed a fluorescent probe, FL-NO 2 , composed of fluorescein linked to dinitroethylene groups, enabling site-specific covalent labeling of cysteine residues. This probe exhibits high specificity for CPGC-tagged proteins, and the impacts of the location of the inserted dual-Cysteine tag, as well as the introduction of a flexible GGGGS linker, on the protein activity and the labeling efficiency were investigated to extend the practical applicability of this fluorescent tool. Using FL-NO 2 as a fluorescence-guided expression platform for in situ protein detection during bacterial fermentation allowed the key parameter optimization. Moreover, this fluorescence-guided platform enabled high-throughput screening of promoter libraries overcoming the labor-intensive limitations of traditional protein expression analysis. This work provides a platform that facilitates the development of in situ protein quantification and high-throughput protein engineering techniques.

Given the complex relationship between the structural features of carbon nanotube (CNT) membranes and their water permeability, predicting the performance of CNT membranes poses a significant challenge. The Bayesian optimization-based Extreme Gradient Boosting (Bayes-XGBoost) algorithm demonstrates considerable potential in capturing the intricate influences of various feature parameters on water permeability. An experimental dataset comprising 572 sets of data derived from molecular dynamics simulations serves as the characteristic dataset for machine learning, utilizing the Bayes-XGBoost algorithm to elucidate the connection between the structural features of CNT membranes and their filtration performance. The results indicate that, in predicting the permeability of CNT membranes, the Bayes-XGBoost algorithm achieves an impressive prediction accuracy of 97.82%, exhibiting faster convergence speed and higher predictive precision compared to traditional machine learning algorithms. Additionally, the optimal combination of CNT membrane feature parameters was identified through a genetic algorithm, providing robust support for the design and fabrication of high-performance CNT membranes. This highlights the significant potential of the Bayes-XGBoost in the field of material design.

Pretreatment is a key step in the formation of biofuels and has pervasive impacts on other major operation steps in the overall biorefinery. Nevertheless, low bulk density, microbial contamination, inefficient processing and high-temperature or high-pressure operations during pretreatment cause huge problems for biomass logistics and biomass conversion. In this work, we developed a novel, efficient and easy-to-operate pretreatment technology: Densifying lignocellulosic biomass with sodium hydroxide (DLC(sh)) for corn stover (CS). A systematic investigation into the effects of DLC(sh) pretreatment on the biomass logistics, enzymatic hydrolysis, and microbial fermentation performance of lignocellulosic feedstocks will enable a comprehensive evaluation of DLC(sh) pretreatment technology's viability in commercial application. DLC(sh) pretreatment owning high bulk density and uniform shape, greatly facilitates feedstock handling, transportation and storage. The DLC(sh) pretreatment densified CS with low energy consumption, which avoided the use of high-temperature or high-pressure equipment and scaled up for pretreated biomass. DLC(sh)-CS showed highly enzymatic digestibility (glucan and xylan conversion>90 %) and highly fermentability (0.219 g ethanol/g dry corn stover) without detoxification of the pretreated biomass. Hence, these results demonstrated that DLC(sh) pretreatment has great commercial potential to convert corn stover to ethanol.

The use of CO2 as a C1 carbon source for the synthesis of valuable chemicals through biotechnology methods represents an effective strategy to fix carbon dioxide. Phenolic acid decarboxylases possess the capability to introduce a carboxyl group into para-hydroxystyrenes for the regionally selective synthesis of (E)-para-hydroxycinnamic acids, utilizing bicarbonate as a CO2 source. It is difficult to achieve this reaction with traditional chemical methods, and only a few enzymes have been isolated and characterized. Here, we mined which low amino acid sequence shared its identity with those of related decarboxylases and which heterologously expressed phenolic acid decarboxylase PAD_Cs from Clostridium sp. DSM 8431 in E. coli. The recombinant PAD_Cs displayed maximum activity at 50 °C, and pH 5.0. PAD_Cs showed distinct carboxylation ability. The carboxylated substrates have a wide range of substitution modes on aromatic systems, including alkyl and alkoxy groups as well as halogens. Furthermore, the carboxylation conversion rates were impressive: para-hydroxystyrene exceeded 20% and 2-methoxy-4-vinylphenol surpassed 26%. This study indicated that PAD_Cs might serve as a potential enzyme source in biotechnological CO2 fixation.

The fungal cell wall provides the cell with enough strength to withstand turgor pressure and keeps adequate plasticity to extend the cell wall size under turgor pressure for cell growth. The cell walls of apical growing hyphae and budding growth yeast have been studied in detailed which share common components of chitin and β-1,3-glucan in their scaffold structures while other polysaccharide components vary on species. In contrast, the cell walls of elongating growth mushroom stipe remains poorly studied. This study explored that in addition to chitin, β-1,3-glucan with β-1,6-linkage branches, and β-1,6-glucan with β-1,3-linkage branches, the scaffold structure of C. cinerea cell wall also incorporated β-1,4-glucan, a component not previously reported in fungal cell walls. After converting chitin to chitosan, we identified three distinct forms of chitosan (chitin). The first was a free chitosan-β-glucan complex, in which chitosan was covalently linked to low molecular weight β-1,6-, β-1,3-, or β-1,4-glucan, which could be extracted using 10 % acetic acid. The second form consisted of chitosan-β-glucan complexes covalently linked to the β-1,4-glucan matrix polysaccharide via β-1,6-glucan, β-1,3-glucan, or β-1,4-glucan, which could be released from the cell wall by hydrolases hydrolysis. The third form involved the release of insoluble chitosan as chitooligosaccharides by chitosanase.

Processive endoglucanases, which possess both endo- and exoglucanase activities, are considered highly promising catalysts in cellulose degradation. In this study, we employed multiple deep learning models, including MutCompute, DeepSequence, and ESM-1v, to guide the engineering of EG5C-1, a processive endoglucanase derived from Bacillus subtilis BS-5. This enabled a systematic exploration of the enzyme's sequence space. Through a combination of clustering analysis and a greedy algorithm, we optimized combinations of amino acid substitutions and ultimately identified an elite variant, M8 (R23Q/E43Q/K91I/K191P/A198T/Q237D/V240P/S245A), composed entirely of substituted residues. Compared to the wild-type enzyme, M8 exhibited 10-fold and 5-fold improvements in catalytic efficiency (kcat/Km) toward soluble substrate carboxymethyl cellulose-Na (CMC) and insoluble substrate phosphoric acid-swollen cellulose (PASC), respectively, along with enhanced optimal temperature and thermostability. Molecular mechanistic analyses revealed that all distal substituted residues enhanced dynamic coupling and coordination, primarily influencing the conformation of three loops near the substrate pocket. These structural changes modulated substrate binding and product release, thereby contributing to improved catalytic efficiency (kcat/Km). This work not only suggests a feasible strategy to explore the "dark space" within sequences but also provides insights into the practical application of machine learning in experiments.

UDP-glycosyltransferases (UGTs) are pivotal biocatalysts for synthesizing pharmaceutically valuable active components; however, their application is frequently constrained by poor regioselectivity and suboptimal catalytic efficiency. In this study, a tailored, free energy-driven, substrate-binding pocket reshaping strategy is implemented to pinpoint the specific residues in UGTBL1 that control bidirectional regioselective glycosylation of tyrosol, enabling the synthesis of salidroside and icariside D2 without the need for large-scale screening. Additionally, modifications in the tunnel lead to two strictly regioselective mutants with improved catalytic efficiency due to the faster release of the products. Remarkably, while wild-type UGTBL1 exhibits poor regioselectivity toward the alcoholic and phenolic hydroxyl groups of tyrosol, generating an almost equal mixture of products (1:1 ratio), mutant M2 achieved 99.2% regioselectivity toward the alcoholic hydroxyl group of tyrosol, coupled with a 14.8-fold enhancement in catalytic efficiency for salidroside production. Similarly, mutant M2-1 displays 99.1% regioselectivity toward the phenolic hydroxyl group, along with a 3.6-fold improvement in catalytic efficiency for icariside D2 synthesis. Molecular dynamics simulations reveal details about the mechanism for improved regioselectivity and catalytic efficiency. This work provides important insights for protein engineering of UDP-glycosyltransferase with the spacious active pocket in constructing small but smart mutant libraries.

To address the challenges of quantitative immobilization and unavoidable spatial hindrance of multiple enzymes in traditional multi-enzyme cascade immobilization, we developed a novel bienzyme immobilization system based on a hierarchically structured MXene@LDHs@Fe3O4 (MLF) carrier. The MLF composite, with a high theoretical surface area (similar to 300 m(2)/g), adjustable interlayer spacing (10-50 nm), and strong magnetization (20 emu/g), provides an ideal support for high-capacity enzyme loading. The system was fabricated through the assembly of MXene nanosheets with layered double hydroxides (LDHs) and magnetic Fe3O4 nanoparticles, integrating horseradish peroxidase (HRP) on the MLF core, a polydopamine (PDA) interfacial barrier, and surface-immobilized glucose oxidase (GOx) through melamine-genipin mediated dendritic cross-linking. This spatially organized catalytic architecture enables quantitative enzyme loading while minimizing spatial hindrance, thereby enhancing catalytic efficiency. The co-immobilized bienzyme reactor (GOX@PDA@HRP@MLF) achieved loading capacities of 272.46 mg/g for HRP and 414.33 mg/g for GOx, with an activity recovery of 85.3 % during 2,4-dichlorophenol degradation. The immobilized enzymes retained similar to 80 % of their initial activity after five operational cycles. Under optimized conditions, the GOX@PDA@HRP@MLF material achieved a degradation rate of 4.6 for 2,4-dichlorophenol in a continuous flow system, representing a similar to 21-fold increase in spacetime yield compared to random multi-enzyme immobilization on single-layer resin materials. These results demonstrate that the dendritic cross-linking strategy employed in this study can serve as a universal method for multi-enzyme immobilization, with significant potential for industrial applications.

The economic viability of cellulose biotransformation remains constrained by high enzyme costs, with processive endoglucanases emerging as promising candidates due to their dual-function hydrolysis mechanism. However, comprehensive kinetic and synergistic analyses of these enzymes are notably limited. This study investigates the kinetic properties of GH5 processive endoglucanase (M3-1) through various kinetic models. Inverse Michaelis-Menten analysis revealed M3-1's superior substrate recognition capacity, demonstrating 95.5 % productive binding site coverage compared to 48.8 % in non-processive endoglucanases. This enhanced efficiency is attributed to M3-1's distinctive structural features, particularly its open and deep cleft configuration. Pre-steady-state kinetics identified substrate association as the rate-limiting step, providing crucial direction for enzyme engineering efforts. Synergistic studies with cellobiohydrolase (CBH) demonstrated remarkable degradation synergy (DS value up to 8.2 on filter paper) and improved substrate resistance compared to traditional EG/CBH combinations. We propose a novel bidirectional degradation mechanism for the M3-1/CBH system, operating both inside-out and outside-in. The effectiveness of M3-1/CBH combination was further enhanced by up to 320 % through the addition of nonionic surfactants and expansin. These findings advance our understanding of processive endoglucanases and their potential applications in biomass conversion.

In clinical anticoagulant therapy, the drug Bivalirudin (Biva) presents a lower incidence of adverse events and more predictable pharmacokinetics in comparison to heparin. However, its short half‐life of ≈20 min leads to poor patient compliance and increased medical burden. Here, a long‐acting anticoagulant hydrogel based on Biva for antithrombotic treatment is described. The fusion peptide ( d ‐RADA)8‐B that integrates Biva, a D‐type self‐assembly motif, and an activated factor X (FXa)‐responsive motif exhibits both supramolecular reservoir and prodrug‐like properties. After subcutaneous injection, the anticoagulant peptide forms a semi‐solid depot with protease‐degradation resistance and slowly disassembles to release prodrug ( d ‐RADA)8‐B into the bloodstream. The circulating prodrug acts as an inert sentinel, which can be activated to release Biva to inhibit thrombus formation when exposed to the thrombus‐related protease FXa. One week after the administration of ( d ‐RADA)8‐B, significant embolism suppression is observed in animal models of carotid artery thrombosis and pulmonary embolism without increasing hemorrhagic side effects. This study demonstrates a concise strategy to engineer a supramolecular anticoagulant hydrogel with long‐term, high drug loading, and on‐demand antithrombotic activation.

Glucansucrases are extracellular enzymes capable of synthesizing diverse α-glucan polymers and oligosaccharides, including the industrially relevant mutan. The mutI-encoded mutansucrase (MUT-I) from Leuconostoc pseudomesenteroides G29 was biochemically characterized as a robust biocatalyst for α-glucan engineering. Recombinant MUT-I (165.1 kDa) demonstrated optimal activity at pH 5.5/30 °C with a high substrate affinity (Km = 3.46 mM) and catalytic efficiency (kcat = 10.35 s-1). It synthesized water-insoluble mutan (91% α-1,3 linkages; Mw = 644,083 g/mol) exhibiting exceptional thermal stability (degradation onset 287.65 °C) and compact fibrous morphology. The enzyme displayed dual functionality: (1) generating gluco-oligosaccharides through maltose-dependent acceptor reactions and (2) introducing α-1,3 branches into dextrans (74% and 14% branching efficiency for dextran T3 and T70, respectively). The deep learning tool AlphaFold 3 identified conserved catalytic residues (Asp476, Glu514, and Asp587) governing polymerization dynamics. This study establishes MUT-I's potential for sustainable production of structurally defined α-glucans as biodegradable alternatives to synthetic polymers in food and biomedical applications.

The sequence design of artificial amyloid spider silk protein (spidroin) is important for the production and wide application of artificial spidroin. However, such micro/nanoscale sequence designs are rarely translated into attractive macroscopic properties for applications with specific functional materials. Here, a sequence design strategy is proposed for artificial spidroins with amyloid peptides based on different grand averages of hydropathicity (GRAVY). The obtained artificial spidroins are capable of efficient heterologous expression and can form programmable hydrophilic/phobic meta-spidroin dressings through self-assembly. Combined with computational analysis, this study explores the molecular mechanism of its high yield. Notably, macroscopic meta-spidroin films with corresponding hydrophilicity and hydrophobicity are directly obtained by designing the GRAVY of the sequence of spidroins, achieving a connection between microscopic design and macroscopic performance. Furthermore, by combining the gradient microstructure formed by microneedles and microcolumns, meta-spidroin dressings achieve precise control of droplet behavior on the film surface and are used to treat diabetic wounds in mice. Taken together, this study not only provides a reasonable reference for designing spidroin dressings applicable in biomedical applications but also offers an effective solution for the treatment of diabetic wounds.