Aug 2026· EAS Journal of Biotechnology and Genetics· Vol 8, pp. 50-58· 0 citations
TL;DR
This study enriches the genetic reservoir of brewing-origin EC hydrolases and provides essential molecular and structural foundations for future solubility engineering and enzyme preparation development.
Abstract
Ethyl carbamate (EC), a Group 2A carcinogen, is unavoidably formed during fermentation and poses a persistent safety concern in alcoholic beverages and fermented foods. Although enzymatic degradation offers a mild and substrate-specific strategy, EC hydrolases suitable for brewing environments remain scarce, and heterologous expression of known candidates often results in inactive inclusion bodies. In this study, we mined amidase genes from three brewing-associated microorganisms—Bacillus velezensis, Clavispora lusitaniae, and Saccharomyces cerevisiae—based on the conserved GGSSGG motif of the AS-family amidases. Three full-length genes, amiE (1458 bp), amdA (1632 bp), and amd08 (1665 bp), were successfully cloned into pET-28a vectors. Prokaryotic expression revealed that AmiE (52.63 kDa) and AmdA (60.76 kDa) were predominantly deposited as insoluble inclusion bodies, while Amd08 (61.39 kDa) was not detected due to apparent gene silencing. To circumvent these expression barriers, we performed comprehensive bioinformatic analyses. All three proteins were predicted as stable, hydrophilic molecules with theoretical pI values of 5.12–5.47 and negative GRAVY indices. Secondary structures were dominated by α-helices and random coils, and homology modeling confirmed the presence of intact GGSSGG motifs and Lys-Ser-Ser catalytic triads in each protein. Multidimensional model validation further supported their stereochemical reliability. Collectively, this study enriches the genetic reservoir of brewing-origin EC hydrolases and provides essential molecular and structural foundations for future solubility engineering and enzyme preparation development.
The accumulation of acetaminophen (APAP) in agricultural soils and water systems poses risks to ecosystems and public health. This study characterizes Acinetobacter sp. DL27, an APAP-degrading strain with broad temperature and pH adaptability, demonstrating its bioremediation potential in soil and wastewater. Through high-resolution mass spectrometry, we identified three novel metabolic intermediates, thereby refining the bacterial APAP degradation pathway. Multiomics analysis elucidated metabolic coordination and stress-tolerance mechanisms, leading to the identification of a novel amidase (AdA). Recombinant AdA exhibited activity over 10–60 °C and pH 4.0–10.0, with a Km of 8.96 ± 1.03 μM and a kcat/Km of 9.04 μM–1s–1, indicating higher catalytic efficiency than previously reported APAP amidases. Molecular dynamics simulations and site-directed mutagenesis confirmed that a Ser161–Ser185–Lys82 triad constitutes the catalytic center driving amide cleavage. These findings provide mechanistic insights into bacterial APAP biodegradation and highlight the practical application potential of strain DL27 and AdA.
Boxiang Kou, Yufei Yue, Lei Wang et al.· Journal of Agricultural and...· 0 citations
Pyrethroid residues in tea have emerged as a significant safety concern due to their high lipophilicity and persistence, while efficient and mild enzymatic removal resources remain scarce. In this study, two novel esterase genes, gene3341 and gene2566, were identified from Lysinibacillus pakistanensis VF-2, both belonging to the α/β-hydrolase superfamily with a typical catalytic triad and conserved Gly-X-Ser-X-Gly motif. The recombinant enzymes Est3341 and Est2566 were successfully expressed in Escherichia coli, and exhibited β-cypermethrin (β-CY) degradation efficiencies of 77.97% and 71.92% within 7 days, respectively. Biochemical characterization demonstrated that Est3341 was a cold-adapted esterase with optimal activity at 25 °C and pH 7.0, enabling efficient degradation under mild conditions suitable for heat-sensitive tea matrices. Notably, Est3341 degraded 98.68% of high-concentration β-CY within 36 h, and its activity was significantly enhanced by Ca2+ and Mn2+. In tea leaf application, Est3341 removed 55.01% of β-CY, 59.40% of cypermethrin, and 46.23% of deltamethrin residues within 20 min at 25 °C. GC-MS analysis revealed that Est3341 hydrolyzed β-CY's ester bonds to yield 3-phenoxybenzaldehyde and other characteristic intermediates. This study provides a high-efficiency, mild-condition, tea-adapted esterase resource for green removal of pyrethroid residues in agricultural products, offering a promising strategy for food safety and environmental bioremediation.
Wanting Wang, Hu Zhou, Jie Tang et al.· Pesticide Biochemistry and P...· 0 citations
Aflatoxin B1 is a potent Group I carcinogen that contaminates food and feed, necessitating efficient detoxification strategies. This study isolated a novel bacterial strain, Enterobacter sp. HNGD-822 from soil, which efficiently degrades AFB1. Genome analysis identified two novel multicopper oxidase (laccase) genes, EbMCO1 and EbMCO2. The recombinant enzymes were heterologously expressed, exhibiting optimal activity at 50 °C and pH 7.0, with strict Cu2+ dependence. Within 12 h, EbMCO1 degraded over 99% of AFB1, while EbMCO2 also showed high activity under the same conditions. Both enzymes primarily converted AFB1 into AFQ1 and epi-AFQ1, products that exhibited significantly reduced hepatotoxicity, developmental toxicity, and oxidative stress in transgenic zebrafish. Applied to artificially contaminated peanut meal matrices, EbMCO1 and EbMCO2 achieved degradation efficiencies of 89.35% and 82.66%, respectively, without altering nutritional quality. This work presents novel laccases with promising potential for the biocontrol of AFB1 in the feed and food industries.
Yan Zhang, Yiqian Zhang, Nazish Muzaffar et al.· Journal of Agricultural and...· 0 citations
Biosurfactants are amphiphilic biomolecules that are produced by microorganisms and have important industrial, environmental, and biomedical applications because of their biodegradability, low toxicity, and high surface activity. In the present study, the potential of biosurfactant production by a
Lactobacillus helveticus
indigenous strain (MTCC 5463) was investigated using integrated
in silico
, molecular, biochemical, and analytical approaches focused on the conserved non-ribosomal peptide synthetase (NRPS) adenylation domain. Comparative sequence and domain analysis of the
Bacillus velezensis
FZB42 reference identified domains for adenylation, condensation, and peptidyl carrier proteins (PCP) that are shared by biosurfactant biosynthetic genes. The AMP-binding domain was amplified, and then the resulting fragment was directionally cloned into the pET-28a expression vector and expressed in
Escherichia coli
BL21(DE3). A recombinant 14-kDa AMP-binding protein, which was associated with the production of a biosurfactant-like compound, was produced. The recombinant biosurfactant was extracted using chloroform:methanol solvent systems and found to have significant emulsification activity against petrol, diesel, mustard oil, and refined oil, and also its E24 value was found to be stable for 1 week. Thin-layer chromatography confirmed the presence of lipidic and glycolipid components, while FTIR analysis indicated the presence of hydroxyl, amide, aliphatic, and carbohydrate-associated functional groups, which are characteristics of a glycolipopeptide structure. The hydrophobic lipid architecture of the biosurfactant was supported by GC–MS profiling, which identified the presence of significant amounts of C16/C18 long-chain fatty acid derivatives and amides. Moreover, ¹H NMR and ¹³C NMR analyses indicated the presence of peptide bonds, glycerol/carbohydrate groups, and long hydrocarbon chains, giving further confirmation of the amphiphilic glycolipopeptide nature of the recombinant biosurfactant. In conclusion, the study shows that conserved NRPS adenylation domains can be utilized to produce biosurfactants in probiotic lactic acid bacteria and suggests that the recombinant glycolipopeptide biosurfactants produced by
L. helveticus
MTCC 5463 have potential for use in the industry.
Unknown authors· Frontiers in Bacteriology· 0 citations
ABSTRACT Polyethylene terephthalate (PET) waste represents a major environmental challenge due to limited recycling solutions. Thermophilic bacteria from geothermal environments harbor diverse enzymatic machinery adapted to extreme conditions, offering promising biocatalysts for plastic degradation; however, biological resources from Peru and other South American countries remain scarce. We characterized four bacterial strains isolated from two geothermal sites in Cajamarca, Peru, screened for PET hydrolysis at 50°C. Whole‐genome sequencing using hybrid assembly achieved near‐complete circular genomes. GTDB‐Tk classification identified three species: Neobacillus thermocopriae (strain 19A), Bacillus licheniformis (strains 16P and BI2), and Brevibacillus agri (strain BI8). Quantitative assays revealed that strain 16P achieved the highest mass loss (0.598%), followed by strain BI8 (0.449%). ATR‐FTIR analysis of the incubated sheets showed a significant reduction of the ester carbonyl index in strains 16P, 19A, and BI8 relative to both non‐incubated PET and an abiotic control, whereas strain BI2 did not differ from the controls, indicating preferential modification of ester bonds at the sheet surface. Genome mining and structure‐based homology searches identified multiple candidate enzymes similar to validated PETases and carboxylesterases, including PETase46‐like homologs in strains BI8 and 16P and a terephthalate‐active carboxylesterase homolog in strain 16P. Molecular docking supported the conservation of catalytic geometry and substrate‐binding sites in these candidates. This work represents one of the first systematic genomic and structural characterizations of putative PET‐hydrolases in Peruvian geothermal bacteria, expanding knowledge of extremophile diversity and advancing thermostable enzymes for sustainable plastic waste management.
Marco A Rivera-Jacinto, Claudia Rodríguez-Ulloa, Sara R Briones-Ramírez et al.· MicrobiologyOpen· 0 citations
Insights are provided into putative glycosyl hydrolase candidates for efficient lignocellulosic waste pre-treatment and significant predicted halophilic and thermostable properties are revealed, suggesting these putative enzymes may endure industrial conditions.
Zhen Wei Tan, Muhamad Naim Abd Malek, Kah Yaw Ee et al.· Journal of General and Appli...· 0 citations
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