Aug 2026· Frontiers in Microbiology· Vol 17· 0 citations· 36 references
Medicine
TL;DR
Biochemically characterizes Rv1063c as a medium-chain-specific esterase encoded by a protein homologous to the patatin superfamily as a medium-chain-specific esterase encoded by a protein homologous to the patatin superfamily.
Abstract
Introduction Mycobacterium tuberculosis is extremely dependent upon lipid-hydrolyzing enzymes to utilize the resources of host lipids and survive within the cell. Rv1063c is a conserved hypothetical protein predicted to share sequence homology with the patatin-like family. Methods In the present study, the rv1063c gene was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Functional enzyme was obtained by solubilizing inclusion bodies in 8 mol·L−1 urea, refolding via gradient dialysis, and purifying through Ni-affinity chromatography. Results Bioinformatic homology analysis indicates Rv1063c is homologous to members of the patatin-like family containing a putative Ser52-Asp166 catalytic dyad, though this residue pair has not been experimentally validated in the present study. The recombinant Rv1063c was mainly expressed as inclusion bodies without significant soluble cytoplasmic expression. Biochemical assays demonstrated the recombinant protein exclusively hydrolyzes medium-chain pnitrophenyl esters (C8–12), with maximum hydrolytic activity toward p-NP-C8 (caprylate), followed by C10 and C12, and exhibited the highest activity at 40 °C and pH 7.0. The enzyme exhibited moderate catalytic efficiency but poor thermal stability. All catalytic data in this paper were obtained from artificial p-nitrophenyl ester substrates, which cannot fully reflect natural lipid substrate preference of mycobacteria in vivo. Discussion This work biochemically characterizes Rv1063c as a medium-chain-specific esterase encoded by a protein homologous to the patatin superfamily. Its physiological and pathogenic roles remain undetermined, and further multi-level experiments are required to clarify its biological functions.
Laccase is a class of known copper-dependent oxidase which can antagonize molds and degrade mycotoxins efficiently, but few reports exist on the structure and function of laccase from Bacillus velezensis. In this study, we identified a CotA-type laccase gene (locus tag: ctg_03438) in B. velezensis T-3, and its DON removal capacity was investigated via bioinformatics analysis, 4D-DIA quantitative proteomics, and in vitro fermentation assays. Results indicated that the CotA gene encodes an extracellular spore coat protein (CotA) that lacks typical signal peptides and transmembrane domains. Crucially, 4D-DIA quantitative proteomic analysis confirmed that CotA in B. velezensis T-3 is expressed at a high level, with 23 unique peptides covering 47.30% of the protein sequence, including a strictly proteotypic peptide (PLKEADTSRK). Molecular docking revealed a stable DON-CotA interaction (ΔG = -6.299 kcal/mol) with a 6.4 Å distance to the T1 copper center, suggesting a potential binding mode for DON recognition. Functionally, B. velezensis T-3 demonstrated potent antifungal activity (inhibition zone: 3.51 cm against Fusarium graminearum (F. graminearum)) and achieved a 30.06% reduction of DON in contaminated feed within 10 days. This study provides a safe candidate strain and molecular-level evidence for the development of biodetoxification agents.
Jiao-Lian Su, Yan-Hong Cao, Tan Dinh Ngo et al.· Biofeed Science and Technolo...· 0 citations
PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1 is characterized and a platform for characterizing cryptic gene clusters within secondary metabolic pathways is established for characterizing cryptic gene clusters within secondary metabolic pathways.
Amaan Fruitwala, Jade X Tiszler, Daniel Watson et al.· Protein Science· 0 citations
Findings suggest that the studied FPMO may play a role in antibiotic resistance in P. aeruginosa by oxidatively inactivating ampicillin by oxidatively inactivating ampicillin.
Maliheh Mohammadkhani, Shamsozoha Abolmaali, S. D. Astaneh· Iranian Journal of Microbiol...· 0 citations
Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.
Janish Kumar, Jyotisha, Rahila Qureshi et al.· International Journal of Bio...· 0 citations
Macrolides are widely used in both human and veterinary medicine, and enzymatic inactivation represents an important yet underexplored resistance mechanism beyond the well-studied Ere family. Here, we functionally characterized ECO-1, an Est-type macrolide esterase identified from
Escherichia coli
. Phylogenetic analysis placed ECO-1 as a distinct branch within the α/β-hydrolase superfamily, showing low sequence identity (37.8–46.7%) to previously characterized macrolide esterases. Heterologous expression of
eco-1
in
E. coli
selectively increased resistance to 16-membered macrolides, with marked MIC elevation for tylosin (32-fold), tildipirosin (8-fold) and tilmicosin (4-fold), while showing minimal effects on 14- and 15-membered macrolides. Consistently, IC₅₀ values for five 16-membered macrolides (tylosin, tilmicosin, tildipirosin, spiramycin and kitasamycin) increased in ECO-1-expressing strains. Using purified recombinant ECO-1, agar diffusion assays demonstrated substantial loss of antibacterial activity after enzyme treatment. ESI-MS further confirmed hydrolysis products with a characteristic + 18 Da mass shift for all five substrates, supporting ester bond cleavage as the inactivation mechanism. Genomic context analysis revealed
eco-1
embedded in a conserved multidrug resistance region associated with Tn3-family transposition and co-localized with clinically relevant resistance determinants including
bla
TEM-1
and tetracycline resistance genes (
tet(B)/tetR(B)/tet(C)
). A large-scale database search identified 136 ECO-1—positive genomes across 14 countries, predominantly in
E. coli
and
Salmonella enterica
, spanning animal, food, human, and environmental sources. Collectively, ECO-1 expands the repertoire of Est-type macrolide esterases with selective activity toward 16-membered macrolides and highlights the potential dissemination risk associated with mobile resistance gene clusters.
Tao Song, Xinyu Ye, Yani He et al.· Scientific Reports· 0 citations
In this study, the glucansucrase-encoding gene (dsr) from Weissella confusa P2 was cloned and heterologously expressed in Escherichia coli BL21(DE3). A 4542 bp fragment encoding 1510 amino acids was obtained, and the recombinant enzyme exhibited a molecular weight of approximately 161 kDa with a theoretical pI of 4.74. After Ni-IDA affinity chromatography, the specific activity reached 300.19 U/mg with a purification fold of 18.43. Enzymatic characterization revealed that the recombinant glucansucrase displayed optimal activity at pH 5.5 and 30 °C, and retained over 70% relative activity after 48 h incubation at 10-30 °C. The enzyme was significantly activated by Ca2+, while strongly inhibited by Ni2+, Cd2+, SDS, EDTA, DTT, and high concentrations of organic solvents. Kinetic analysis using sucrose as substrate yielded Km of 0.56 mM and Vmax of 4.57 U/mg, indicating exceptionally high substrate affinity. Using this recombinant enzyme, exopolysaccharide (EPS) was synthesized in vitro, achieving a yield of 59.23 ± 0.21 g/L, representing an 18.79% improvement over EPS synthesis by the wild-type strain. These findings enrich the genetic resources of glucansucrase, provide a promising biocatalyst for efficient EPS production, and establish a foundation for the application of glucansucrase and its polysaccharides products in the food and biotechnology industries.
