The fatty acid chain length of lipids plays a pivotal role in determining their nutritional value and industrial applicability. However, the synthesis of functional lipids with defined fatty acid compositions is often limited by the insufficient chain-length specificity of conventional lipases. In this study, a chain-length-selective lipase targeting palmitic (C16) acyl chains was engineered using an integrated computational-experimental strategy, and its performance was evaluated at a two-phase microfluidic interface. The engineered variants exhibited a 0.79 to 28.48-fold enhancement in hydrolytic activity toward p-nitrophenyl palmitate (p-NP C16:0), along with improved thermal stability (t1/2 of mutant I291Y increased from 0.40 to 2.04 h). Notably, the dominant mutant I291Y showed markedly enhanced selectivity, with hydrolytic activity toward p-NP C16:0 being 41.65-fold that toward p-nitrophenyl oleate (p-NP C18:1). Consistently, the Vmax of I291Y for p-NP C16:0 was 284.19% higher than that for p-NP C18:1. Molecular docking and molecular dynamics simulations lead to a mechanistic hypothesis in which the substitution of isoleucine 291 with tyrosine is suggested to strengthen the affinity of the substrate binding tunnel for C16 acyl chains while generating steric hindrance against oleic (C18) acyl chains, thereby potentially conferring C16 selectivity. This structural modification also enabled I291Y to selectively hydrolyze C16 acyl chains in glyceryl tripalmitate rather than the C18 acyl chains in glycerol trioleate at the microfluidic two-phase interface. This work demonstrates a computationally guided engineering approach for developing a lipase with enhanced C16 preference, which may facilitate targeted lipid modification and the tailored production of nutritional lipids.

To address the issues of low γ-aminobutyric acid (GABA) synthesis efficiency and excessive ethanol residue in the preparation of cereal-based functional fermented beverages using single-strain fermentation, we utilized a composite fermentation substrate of Cordyceps militaris and germinated brown rice to establish a three-strain sequential fermentation strategy. We screened three functional strains compatible with the composite substrate: Saccharomyces (S.) cerevisiae Fresco, Levilactobacillus (L.) brevis GH1, and Acetobacter (A.) pasteurianus CICC20001. The optimal sequential fermentation process was optimized: first inoculating S. cerevisiae and fermenting at 37°C for 24 h, then cofermenting with L. brevis at 34.4°C for 72 h, and finally inoculating A. pasteurianus and fermenting at 30°C for 48 h. Under these conditions, GABA production reached 24.29 g/L, ethanol content decreased to 4.86 g/L, organic acid components in the beverage were optimized, and flavour quality significantly improved. Additionally, by controlling the fermentation pH using lactic acid, GABA production was further increased to 30.03 g/L. During the co-fermentation phase, S. cerevisiae metabolizes acids to reduce the microenvironmental pH, thereby activating glutamate decarboxylase (GAD) and utilizing nucleotide metabolism to provide precursor substances for the synthesis of pyridoxal phosphate (PLP) coenzymes. L. brevis maintained nitrogen balance through glutamine and γ-glutamyl dipeptide metabolism, stabilizing GAD activity and promoting GABA synthesis. Our results clarify the optimal strategy for three-strain sequential fermentation, elucidate the GABA enrichment mechanisms, and develop a novel low-alcohol, GABA-rich, medicinal and edible cereal-based fermented beverage.

(3-Glucanases are extensively utilized in biomass conversion processes. However, their inadequate catalytic performance under industrial high temperatures limits their application in biorefinery. In this study, a highly active 1,3-1,4-(3-glucanase from Polychaeton citri (PcGlu16B) was identified through protein database screening and heterologously express. To optimize its thermal stability, directed evolution was employed, leading to the selection of three beneficial variants (N11Y, S209Q, and G228P). The optimal combined mutant, N11Y/S209Q (M1), exhibited significantly improved properties compared to the wild-type (WT) with thermal half-life at 50 degrees C extended by 157 min, catalytic efficiency increased by 1.29-fold (4000 vs. 3100 mL s- 1 mg- 1), and enhanced resistance to proteases and chemical reagents. Structural analysis revealed that increased hydrogen bonding, salt bridges, and rigid secondary elements conferred greater rigidity and thermal stability. Meanwhile, enhanced long-range negative interactions modulated key residues within the catalytic channel, improving the catalytic efficiency. Synergistically, M1 with xylanase under alkaline pretreatment exhibited the most effective corn cob degradation, yielding 5.7 mmol/g fermentable sugars with a maximum synergy degree of 2.1. These results demonstrate the database-oriented variant M1 as a thermostable and promising candidate for industrial biomass conversion and provide a strategic framework for enzyme engineering in biorefinery applications.

Chiral alcohols derived from "difficult-to-reduce" ketones are key precursors in pharmaceutical intermediates and high-value fine chemicals. Despite advances in alcohol dehydrogenases (ADHs), the stereoselective reduction of "difficult-to-reduce" ketones remains a major challenge. In this study, we identified 10 short-chain dehydrogenases/reductases (SDRs) through gene mining using tetrahydrofuran-3-one and tetrahydrothiophene-3-one as substrates. Structure-guided mutagenesis combined with docking and molecular dynamics simulations revealed key residues regulating enantiomer selectivity. The A3-E145W mutation enhanced S-selectivity for tetrahydrofuran-3-one by optimizing electrostatic and aromatic stabilization, whereas the A3-A94N/E145S mutant achieved >99% ee (R) for tetrahydrothiophene-3-one through strengthened hydrogen bonding and steric gating. Taken together, these results reveal a general mechanism by which electrostatic tuning and pocket confinement synergistically influence SDR stereoselectivity, providing a versatile strategy for engineering biocatalysts for challenging ketone reduction reactions.

Bioreactors play a crucial role in biocatalysis, particularly where energy efficiency and catalytic stability remain critical challenges. This study reports a continuous-flow photothermal bioreactor based on a MXene/covalent organic framework (MCOF) composite, synthesized in one pot with high surface area and excellent photothermal biocatalytic performance. Multilayer MCOF membranes with nanoscale confinement effects were constructed, which enabled highly efficient enzyme immobilization and enhanced mass transfer. Under near-infrared light irradiation, the MCOF membrane rapidly converted light into heat, facilitating efficient thermal management of enzymes. Compared to the conventional heat conduction heating method, this system reduced energy consumption by more than 86%, requiring only 2.14 Wh of energy per hour. Additionally, the confined structure of the MCOF membrane promoted substrate-enzyme interactions and continuous-flow catalysis. To achieve the same product-conversion effect, the confined structure required only 45% of the free enzyme reaction time and 55% of the dispersed MCOF powder reaction time. After six cycles, the MCOF bioreactor retained over 80% catalytic activity and maintained high conversion rates under various flow conditions. This work integrates photothermal management and confinement-enhanced catalysis to establish a new strategy for developing efficient, reusable, and low-energy bioreactors suitable for scalable green biomanufacturing.

The silkworm, Bombyx mori, is an economically important insect resource in China. With the transformation and upgrading of the sericulture industry, genetically engineered silkworms have emerged as efficient animal bioreactors for producing bioactive proteins and functional compounds. However, the diversity of available transgenic silkworm bioreactor resources remains limited and requires further expansion. Herein, we focused on human lactoferrin (hLF), which exhibits potent antibacterial activity due to its strong iron-chelating capacity, and established an efficient recombinant hLF (rhLF) expression system in the posterior silk glands (PSGs) of transgenic silkworms using piggyBac technology. The rhLF production reached 45.13 ± 2.46 mg/g cocoon shell weight, with N-glycosylation modifications consistent with those of native hLF. Functionally, rhLF exhibited robust antibacterial activity, achieving inhibition rates of 31.64% ± 4.66% against Escherichia coli and 74.35% ± 0.87% against Staphylococcus aureus. Additionally, rhLF significantly promoted cell migration and accelerated wound healing, with a migration rate of 17.06% ± 4.05%. The resulting chimeric fibroin fibers containing rhLF demonstrated enhanced antibacterial activity while maintaining cytocompatibility. Collectively, we constructed a novel transgenic silkworm strain enabling high-yield rhLF production and achieved the biomanufacturing of functional silk fibers. These findings provide a cost-effective strategy for generating high-value products within conventional agricultural systems.

The column purification process plays a critical role in the production of ginkgo ketone ester tablets, and monitoring the concentrations of key compounds is essential for ensuring final product quality. Herein, a quality‑by‑artificial‑intelligence concept integrating real‑time spectral monitoring with predictive modeling was proposed, utilizing portable Raman and near‑infrared spectrometers (NIRS) to acquire spectral data from 217 eluate samples. UPLC‑MS/MS was employed to quantify seven key compounds, including four terpene lactones (bilobalide, ginkgolides A-C) and three flavonol glycosides (quercetin, kaempferol, isorhamnetin). Subsequently, seven quantitative calibration models were constructed using seven different artificial intelligence algorithms. Notably, the ResNet50‑based model exhibited optimal performance. For Raman spectral data analysis, the correlation coefficients (RP2) between predicted and reference values for the seven active components were 0.9944, 0.9953, 0.9945, 0.9899, 0.9961, 0.9953, and 0.9931, with corresponding root mean square error of prediction (RMSEP) values of 0.0172, 0.0199, 0.0184, 0.0265, 0.0181, 0.0176, and 0.0152, respectively. For NIRS spectral data, the R²ₚ values were determined to be 0.9712, 0.9865, 0.9826, 0.9820, 0.9851, 0.9664, and 0.9773, with RMSEP values of 0.0359, 0.0315, 0.0329, 0.0311, 0.0361, 0.0553, and 0.0300, respectively. Notably, Raman spectroscopy provided richer feature information for quantifying these compounds in column chromatography eluate, whereas NIRS spectroscopy yielded more limited discriminative bands. Furthermore, the Grad‑CAM algorithm was applied to visualize and interpret the optimal ResNet50 models, effectively identifying the most influential spectral features for each analyte. Collectively, these results demonstrate the feasibility of integrating portable Raman and NIRS spectrometers with the ResNet50 quantitative calibration model for real‑time monitoring during the production of ginkgo ketone ester tablets.

Combined use of compound enzymes and low-oxygen conditions is a promising approach for tobacco fermentation. Nevertheless, the regulatory mechanism of this co-fermentation system on flavor formation in cigar tobacco leaves remains unclear. This work aims to explore the effects of combined fermentation treatment on sensory properties and metabolic regulation of cigar tobacco leaves. Air-cured ‘Dexue 1’ cigar tobacco leaves were selected as experimental materials. Five treatments were set up: unfermented control (CK), conventional fermentation (T1), low-oxygen fermentation (T2), compound enzyme fermentation (T3), and combined compound enzyme and low-oxygen co-fermentation (T4). A combination of electronic nose analysis, volatile metabolomics and proteomics was used to identify key regulatory proteins and aroma components in fermented tobacco leaves. The optimal dosages were determined as 78 U/g of neutral protease, 82 U/g of glucoamylase and 59 U/g of polyphenol oxidase. All treatments enhanced the sensory quality of tobacco leaves. T4 obtained the highest overall sensory score, as it maintained an optimal balance between enriched desirable aromas and decreased irritating substances. These key proteins Nta23g19450, Nta20g06770, Nta18g02140 and Nta17g02240 modulated phenylpropanoid biosynthesis and tyrosine metabolism, thereby altering the accumulation of caramelized-sweet, woody, floral, and fruity aroma volatiles such as β-damascenone, 2-ethyl-3,5-dimethylpyrazine and geranylacetone. These changes shape the unique flavor profile of cigar tobacco leaves. This study provides a theoretical basis for promoting the fermentation quality of cigar tobacco leaves via compound enzyme and low-oxygen co-fermentation.

Drug molecules used to treat complex diseases often contain cyclic chiral moieties. Specifically, the synthesis of fused-ring alcohols (e.g., chiral β-tetralol and its derivatives) by chemical methods is challenging. Medium-chain alcohol dehydrogenase (MDR) are widely used in the asymmetric reduction of potential chiral ketones to chiral alcohols, and can be used to synthesize chiral fused-ring alcohols. In this study, we integrated enzyme sequence and structural information in an ancestral sequence reconstruction method. This process identified ancestral medium-chain alcohol dehydrogenase N42, with average conversion and selectivity rates exceeding 98% towards 19 ketone substrates (classified into fused-ring, heterocyclic, and aryl groups), and enhanced thermostability (melting temperature 9.9 °C higher than that of its descendant). Computational analysis explained the origin of the N42 activity and revealed a unique set of functional regulatory sites in the MDR family. This ancestral reconstruction strategy combines sequence and structural information provides novel insights into enzyme discovery and functional regulation, might prove useful for studying enzyme functional evolution in families with deep structural differentiation.

Endocrine-disrupting chemicals (EDCs) are considered a highly heterogeneous and persistent class. However, the biodegradation of such compounds is limited by poor stability, low substrate mass transfer efficiency, and low reusability. This study explores a novel engineered biofilm catalyst that integrates bacterial cell-surface display to efficiently degrade EDCs. Under the optimized catalytic conditions, the degradation efficiency of estrone (E1), bisphenol A (BPA), and 2,4-dichlorophenol (2,4-DCP) reached 93.2%, 91.8%, and 81.7%, respectively, within 30 min. Compared to the surface-displayed laccase whole cell catalyst, the catalytic efficiency of the surface-displayed laccase biofilm increased by 22.5%, 15.8%, and 23.7%. The biofilm catalyst showed excellent stability, maintaining over 40% activity across a pH range of 3-7, 97% activity at 60 °C, and retaining 50% efficiency after 7 cycles. These findings demonstrate that the biofilm catalyst is an effective approach to enhancing degradation efficiency, providing a scalable, environmentally benign strategy for advanced wastewater treatment.

Glucose oxidase (GOX) is widely recognized for its potential in food preservation, yet its practical application is often limited by insufficient stability and catalytic efficiency under processing conditions. In this study, a GOX from Aspergillus luchuensis (AlGoxA) was engineered through directed evolution, yielding the combinatorial mutant M1 (A179 V/T302L). The variant exhibited a 10.8-fold increase in catalytic efficiency and an 81.1% reduction in Km compared to the wild type. M1 retained 81.9% of its activity after 60 min at 60 °C, a remarkable improvement over the wild type's 6.2%. It also demonstrated broad pH tolerance, maintaining over 66% activity under alkaline conditions. When applied to apple juice preservation, M1 effectively suppressed browning, with the polyphenol oxidase inhibition rate reaching 87.1%, increased vitamin C retention by 37.9%, and exhibited direct antifungal activity against common spoilage fungi. This study provides a highly efficient and robust biocatalyst for extending juice shelf life while preserving nutritional quality.

Enzymatically modified isoquercitrin (EMIQ), a natural compound known for its antioxidant, anti-proliferative, and anti-inflammatory properties, exhibits remarkable bioavailability and low toxicity, making it a promising therapeutic agent for diabetic kidney disease. However, conventional chemical synthesis of EMIQ is hampered by inefficiency, high reagent consumption, and substantial by-product formation. In this study, EMIQ was produced from rutin and sucrose using a novel Heteropeptide Self-Assembly-Cascaded Dual-Enzyme (HSC-DE) system, which co-immobilized α-L-rhamnosidase and amylosucrase via SpyTag/SpyCatcher pairs. This system significantly enhances reaction efficiency by adding the substrate and gradient temperature, achieving a sucrose conversion rate of 98.3% within 24 h, which was increased by 5.7% compared to previous (92.6%). Notably, the yield of quercetin-3-O-tetraglucoside, the form with optimal bioavailability, reached 62.1%, an increase of 12% over previous methods. This study presents a novel, sustainable, and highly effective bioengineering strategy for the synthesis of EMIQ, highlighting its potential for scalable and environmentally friendly production.

Collagen, a crucial structural protein in the extracellular matrix (ECM), exhibits exceptional biocompatibility with broad applications in biomedicine and cosmetics, of which type III collagen exhibits ubiquitous distribution across crucial tissues including vasculature and skin, where it performs essential physiological functions. However, high-efficiency production of bioactive recombinant human type III collagen (rhCOLIII) remains challenging. This study developed an effective strategy for synthesizing functional rhCOLIII in genetically engineered silkworms, which was driven by fibroin heavy chain (FibH) expression system, making rhCOLIII specifically expressed in the posterior silk gland (PSG) of silkworms. Moreover, rhCOLIII was successfully secreted into the cocoons at the yield of 7.8 mg/g cocoon shell weight. RNA-seq analysis revealed that differentially expressed genes (DEGs) enriched predominantly in endoplasmic reticulum protein processing pathways. Notably, the purified rhCOLIII protein exhibited excellent cytocompatibility and significantly promoted cell proliferation and migration of NIH/3T3, indicating its potential for accelerating wound healing. Additionally, the commercially important traits of the silkworms also remained unchanged, while the existence of rhCOLIII significantly improved the extensibility and toughness of silk fibers. These findings establish the silkworm bioreactor as a viable platform for high-efficiency production of bioactive rhCOLIII and provide a strategy for synthesizing other functional recombinant proteins.

ABSTRACT The Fe(II)/α‐ketoglutarate‐dependent dioxygenase (αKGD) superfamily enables various C−H functionalization supported by their active pocket architectures composed of one or more loop elements. However, their conserved architectural features and broader functional roles across the catalytic cycle remain incompletely defined, although such loops have been implicated in substrate recognition or proton transfer in specific catalytic processes. Here, we applied protein structuromics analysis to the PF10014 family within the αKGD superfamily, which contains a single long active‐pocket loop and therefore provides a tractable model for dissecting loop‐associated structure–function relationships. This analysis identified a conserved structural motif termed the half‐open active pocket, which is proposed as a hallmark feature of the αKGD superfamily. Furthermore, enhanced sampling simulations and mutagenesis experiments targeting isoleucine dioxygenase, a representative member of the PF10014 family, revealed the multifaceted role of the loop that constitutes the half‐open active pocket architecture throughout the catalytic cycle, encompassing the modulation of half‐open active pocket conformation, substrate recognition and anchoring, as well as participation in molecular transport. These findings provide a broader structure–function landscape in which the flexible loop within this family‐conserved half‐open active pocket serves as a key catalytic element with multiple functions.

Melanoidins are brown macromolecular products of Maillard reaction formed through thermal processing, which provide unique flavor and bioactive function of food. This study aims to reveal the changes of structure and bioactive characteristics in beer melanoidin-rich fraction (BM) during different chemical treatments and in vitro digestion and fermentation. The results showed that BM had colloidal properties with 24.21-99.28 nm and negative charge, which mainly contained carbohydrates. In addition, microscopic morphology showed surface cracks after acid treatment, blocky particles after alkali treatment, and some porous small particles after salt treatment. These might be caused by the disruption of polysaccharide, protein, and the skeleton structure through spectra. Moreover, during simulated in vitro digestion stage, Mw of BM was slightly decreased, which might be due to the breakage of glycosidic bonds. Microscopic morphology presented a relatively complete block structure. However, the contents of total phenolics and flavonoids and the production of short-chain fatty acids were significantly increased during colonic fermentation process. Phloroglucinol was the main phenolic compound and its content also increased during colonic fermentation. Collectively, the results indicated that BM had relative stability during the digestion stage, and released bioactive compounds during colonic fermentation. These findings suggest that BM, as a novel colloidal prebiotic, possesses potential role in intestinal health.

Biofilms catalysis has broad application prospects in the biocatalysis due to its high cellular stability, strong reusability, and operational simplicity. However, the naturally limited biofilm-forming ability of Escherichia coli restricts its performance in catalytic processes. In this study, we developed a rational engineering strategy to enhance E. coli-based biofilms catalysis by overexpressing adhesion-related genes in combination with carrier materials. Specifically, adhesin gene overexpression enhances cell adhesion and biofilm formation while decreasing planktonic cell number. Furthermore, biofilm biomass in the ecpA- and proQ-overexpressing strains increased by 563.88% and 550.36%, respectively (p < 0.001). In addition, after the addition of hydrophilic polyurethane fiber carriers, rutin conversion rate of 97.81% and isoquercitrin yield of 90.11% were achieved using biofilms catalysts formed by the ecpA-overexpressing strain (p < 0.05). These findings demonstrate that adhesin gene overexpression combined with hydrophilic polyurethane fibers as carriers is an effective strategy for constructing robust E. coli biofilms catalysts and enhancing biofilm-mediated biocatalysis.

Immobilized enzyme catalytic reactions reduce pollution and enable enzyme reuse, making them suitable for industrial biocatalysis. However, immobilized enzymes often face mass-transfer limitations and restricted substrate access to active sites, which lowers catalytic efficiency. To address these challenges, this study developed a Marangoni-poly(N-isopropylacrylamide) (Marangoni–PNIPAM) hydrogel rotor bioreactor (MPR) through compartmentalized photopolymerization and covalent organic framework (COF)-based immobilization of β-glucosidase. The MPR reached a maximum rotation speed of 4,319 rpm, sustained rotation for 22 min, and exhibited a maximum reaction rate (Vmax) of 1.35 mM·min-1. It converted 87.2% of the substrate within 30 min, demonstrating high catalytic efficiency. Performance decreased markedly upon scale-up. By integrating multiple rotors into a spin-array configuration, the arrayed MPR (AMPR) achieved a conversion rate of 97.3%. Furthermore, it maintained 90% efficiency in scaled-up systems—a level more than four times higher than that of the single-rotor MPR and representing a 197% improvement over COF@β-G. Simulation analyses showed that the AMPR generates vortices through both rotor self-rotation and inter-rotor interactions, promoting fluid movement that equalizes substrate concentration and accelerates transport to the carrier surface. In addition, the induced fluid disturbances enhance substrate penetration through intricate pore channels, allowing access to enzymatic active sites, thereby accelerating conversion.

Continuous-flow adsorption utilizing macroporous cryogels is pivotal for the industrial-scale remediation of dye-containing wastewater. However, fabricating robust polysaccharide-based cryogels remains challenging due to the kinetic conflict between ice crystal growth and polymer crosslinking. Herein, we report a freeze-induced chemical crosslinking strategy for constructing robust quaternized polysaccharide-based cryogels. Using bis(vinylsulphonyl)methane (BVSM) as a crosslinker and microfibrillated cellulose (MFC) as a reinforcer, we successfully engineered a robust, macroporous quaternized agarose (QA) cryogel. Crosslinking experiments combined with density functional theory (DFT) calculations suggest that the β-hydroxyl group adjacent to the quaternary ammonium moiety provides a unique reactive site that lowers the activation barrier for Michael addition to BVSM, thereby enabling efficient covalent crosslinking under cryo-concentrated conditions. The resulting QA/MFC cryogel exhibits a highly interconnected macroporous structure with an average pore diameter of 30 ± 9 μm, a porosity of 88%, superhydrophilicity, and exceptional elasticity. Owing to the dominant contributions of electrostatic attraction and hydrogen bonding, the cryogel presents a high Congo red (CR) adsorption capacity of 1475 mg/g. Fixed-bed experiments further demonstrate efficient continuous-flow removal of CR and favorable fitting by the Thomas and Yoon-Nelson models. This work establishes a versatile strategy for preparing polysaccharide-based cryogels and provides mechanistic insight into freeze-induced chemical crosslinking of quaternized polysaccharides for water purification.

Achieving simultaneous improvements in activity, substrate specificity, stereoselectivity, and thermal stability remains a central challenge in laboratory enzyme evolution. Enzymes with industrial properties require synergistic development of various functions. This study developed a computational pipeline integrating sequence-structure information to regulate the activity, thermal stability, and stereoselectivity of carbonyl reductases. Our framework employs an unsupervised epistasis model to map residue interdependencies across the entire protein structure, combined with Delta Delta G fold calculations and conservation analysis, enabling global evolutionary engineering. This strategy dramatically reduced 6,720 potential mutations to 27 prioritized candidates. Greedy combinatorial strategy generated optimized mutants with enhanced activity (up to 28-fold), high stereoselectivity toward 22 structurally diverse substrates, and improved thermal stability (Delta T m up to 5.8 degrees C). For the substrate 2-acetylpyridine (H1), I51L/Y61F/D147E (M3) is the dehydrogenase with the highest activity reported so far. Systematic analysis of crystal structures and molecular dynamics simulations revealed that distal mutations reorganized interdomain communication networks, increasing the active population of prereaction state conformations. The introduction of distal mutations balanced overall protein fluctuations by redistributing flexibility across different regions, contributing to the simultaneous improvement of catalytic activity and thermal stability. This work demonstrates the efficiency of a computer-aided protein design approach for synergistically enhancing multifunctional compatibility, offering a transformative strategy for advancing biomanufacturing of high-value chiral compounds.