Polycaprolactone (PCL) is a biodegradable polyester that can degrade slowly under ambient environmental conditions. Here, we explored natural fungal sequence diversity to identify cutinase-like enzymes (CLEs) with PCL-degrading activity. Using a previously reported Cryptococcus CLE as the query, bioinformatics analysis identified 14 fungal homologs that were subsequently expressed in the heterologous hostSaccharomyces cerevisiae and screened using a plate-based assay. Nine homologs exhibited detectable PCL degradation, with three producing larger degradation zones than the Cryptococcus reference enzyme. These three homologs exhibited distinct apparent optimal temperatures of 30°C, 37°C, and 40°C, compared with 45°C for the reference enzyme under the tested conditions. Signal peptide replacement and GFP-based analyses suggested that native signal peptide recognition alone was unlikely to account for the five inactive homologs. Collectively, this study provides a practical bioinformatics-to-experiment strategy for screening natural enzyme diversity and prioritizing promising candidates for further biochemical characterization.
Antibiotic therapy for STEC O157:H7 infections induces Shiga toxin release, highlighting the need for alternative, food-compatible antimicrobial strategies. The ethanolic leaf extract of Vitex negundo L. was characterized by UPLC-qTOF-MS/MS, integrated with Feature-Based Molecular Networking (GNPS), which putatively annotated 56 metabolites. Computational screening of all annotated compounds against the Stx1 and Stx2 B-subunits identified compound 8, an iridoid glycoside, as the top-ranked candidate with predicted Stx1-binding potential (ΔG = -6.9 kcal/mol; K i = 8.7 µM). Over 100 ns of molecular dynamics, the Stx1 B-subunit backbone remained conformationally stable (Cα RMSD < 1.5 Å) and 2–3 intermolecular hydrogen bonds with compound 8 were maintained; ADMET prediction returned a gut-restricted profile. A sweet potato starch-based film incorporating the crude extract showed antibacterial activity against E. coli O157:H7 and complete visual disintegration within 28 days of soil burial, indicating preliminary potential as an eco-friendly active packaging material for dried foods.
Antibiotic therapy for STEC O157:H7 infections induces Shiga toxin release, highlighting the need for alternative, food-compatible antimicrobial strategies. The ethanolic leaf extract of Vitex negundo L. was characterized by UPLC-qTOF-MS/MS, integrated with Feature-Based Molecular Networking (GNPS), which putatively annotated 56 metabolites. Computational screening of all annotated compounds against the Stx1 and Stx2 B-subunits identified compound 8, an iridoid glycoside, as the top-ranked candidate with predicted Stx1-binding potential (ΔG = -6.9 kcal/mol; Ki = 8.7 µM). Over 100 ns of molecular dynamics, the Stx1 B-subunit backbone remained conformationally stable (Cα RMSD < 1.5 Å) and 2–3 intermolecular hydrogen bonds with compound 8 were maintained; ADMET prediction returned a gut-restricted profile. A sweet potato starch-based film incorporating the crude extract showed antibacterial activity against E. coli O157:H7 and complete visual disintegration within 28 days of soil burial, indicating preliminary potential as an eco-friendly active packaging material for dried foods.
Although Metschnikowia pulcherrima shows promise as a biocontrol agent, its upstream development remains limited. We maximized the viable cell count of M. pulcherrima strain APC 1.2 by employing a Central Composite Design (CCD) to optimize pH and temperature. Preliminary screening identified sucrose at 20 g/L and 1600 mg YAN/L yeast extract as optimal for viable cell count (CFU/mL), with further increases in YAN providing no additional benefit. The highest viable cell count was achieved at milder temperatures (18–22 °C), where the strain grew robustly across a wide pH range (3.59–6.41), while higher temperatures increased sensitivity to acidic pH and reduced viable cell count. The strain was confirmed to be Crabtree-negative, showing no ethanol production during aerobic growth on glucose. The proposed workflow enabled reproducible optimization of cultivation parameters, providing a scalable framework for industrial applications, and laying the groundwork for future studies on non-conventional yeasts in biocontrol and industrial biotechnology.
High production costs remain a major obstacle to the commercialization of cell-based foods, primarily due to the reliance on costly pharmaceutical-grade reagents and infrastructure. This study introduces a practical solution using a dual technical/regulatory approach. We developed iCoater, a food-grade coating agent manufactured via terminal heat sterilization, a standard in food processing. This process results in no detectable bacterial growth and 12-month stability without filtration. In performance tests using primary duck liver-derived cells, iCoater showed efficacy in cell attachment and proliferation. Furthermore, we confirmed that this performance and low endotoxin levels were maintained when the tested water sources covered a wide hardness range from soft (< 1.0 mg/L) to hard (1468 mg/L) water. By aligning the food-grade manufacturing process, we proposed the regulatory pathway under Japanese legislation, identifying "gelatin-processed food" as a realistic and transparent labeling strategy. This integrated approach provides a practical reference for future developments in cellular agriculture.
