Bioprospecting microbial genomes to expand the biocatalytic toolbox of rubber oxygenases.

Surveying a Pseudomonas aeruginosa-derived oxidoreductase activity

Background and Objectives: Flavoprotein monooxygenases (FPMOs) participate in various biological processes, including lignin degradation, natural product biosynthesis, and xenobiotic detoxification. This study aimed to investigate the heterologous expression of FPMO and assess its functional activity by examining its capacity to inactivate ampicillin. Materials and Methods: In silico analyses were performed to predict the enzyme’s secondary and tertiary structures. The target gene was isolated from Pseudomonas aeruginosa, cloned into the pET-22b vector, and expressed in Escherichia coli BL21 (DE3). Heterologous protein expression was examined using SDS–PAGE, and the protein was purified through nickel-affinity chromatography. Enzymatic activity was measured by spectrophotometric monitoring of NADPH oxidation at 340 nm. Antibacterial activity was tested using an agar well-diffusion assay, measuring the inhibition zone diameter in E. coli treated with ampicillin. Results: Homology modeling indicated that the 3D structure of FPMO is very similar to cyclohexanone monooxygenase, supporting its classification as a Baeyer–Villiger monooxygenase (BVMO). Molecular docking proposed that ampicillin might be a substrate for FPMO, with predicted interactions at Ile141 and Val159. Enzymatic assays confirmed that FPMO catalyzes the oxidation of ampicillin, using FAD and NADPH as cofactors. Additionally, well-diffusion tests showed decreased ampicillin antibacterial activity after treatment. The enzyme’s activity was further confirmed using an ampicillin inactivation assay. Conclusion: The heterologously expressed protein was functionally active. These findings suggest that the studied FPMO may play a role in antibiotic resistance in P. aeruginosa by oxidatively inactivating ampicillin.

Expression and biochemical characterization of a patatin-like medium-chain esterase Rv1063c from Mycobacterium tuberculosis

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. 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. 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. 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.

Genome mining of Trp-anthranilic acid cyclic dipeptides in Aspergillus driven by analysis of NRPS adenylation domains

A novel family of fungal protein biosurfactants: Discovery and sustainable production.

Protein-based biosurfactants remain underexplored compared to glycolipids and lipopeptides, despite their unique interfacial properties and self-assembly behavior. PAC3, a surface-active protein produced by the marine fungus Acremonium sclerotigenum, exhibits dual behavior as both a biosurfactant and bioemulsifier. For this reason, it can be seen as a high molecular weight proteinaceous compound, able to efficiently reduce surface tension. Here, we identify PAC3 as the first member of a previously unrecognized family of fungal protein biosurfactants. The complete amino acid sequence of PAC3 was determined through a combined de novo transcriptomic and mass spectrometry approach, revealing an 83-residue protein that lacks the canonical eight-cysteine motif typical of hydrophobins, the most surface-active proteins known. Sequence, phylogenetic, and structural analyses revealed a distinct fold and amphipathic architecture, with a negatively charged surface and a hydrophobic planar region, providing a molecular basis for its strong interfacial activity. The identification of homologous sequences across fungi supports the existence of a novel protein family. Notably, we show through spectroscopy and confocal microscopy that PAC3 fibrils exhibit deep-blue intrinsic fluorescence, a property recently associated with amyloid architecture. To support industrial application, we developed a simplified downstream process based on methanol/chloroform extraction, reducing costs while preserving functionality. In parallel, the use of waste frying oil enhanced fungal biomass production and supported efficient PAC3 synthesis, demonstrating a sustainable production strategy. Overall, this study introduces a new class of fungal biosurfactant proteins and provides a foundation for their biotechnological exploitation.

Screening of Actinomycetes from Rhizosphere Soils for L-glutaminase Production

Actinomycetes are filamentous, Gram-positive bacteria of major ecological and biomedical importance due to their exceptional ability to produce diverse bioactive secondary metabolites encoded by numerous biosynthetic gene clusters. In the present study, actinomycetes were isolated from rhizospheric and non-rhizospheric soil samples and systematically characterized using morphological, biochemical, and molecular approaches. The isolates were screened for antimicrobial, antioxidant, and L-glutaminase-producing potential, followed by 16S rRNA gene sequencing for taxonomic identification. Several isolates exhibited strong antibacterial activity against clinically significant pathogens, including Staphylococcus aureus (NCIM 2073) and Escherichia coli (NCTC 9001). Antioxidant potential evaluated using the DPPH radical scavenging assay confirmed the presence of redox-active metabolites. Efficient production of L-glutaminase, an amidohydrolase enzyme was observed. To complement the experimental findings, in silico molecular docking studies were performed to investigate the interaction of selected bioactive compounds with relevant target proteins, providing mechanistic insights into binding affinity and molecular stability. The docking results revealed favourable binding energies and key hydrogen-bond and hydrophobic interactions, supporting the observed biological activities. Overall, the integrated experimental and in silico approach highlights soil-derived actinomycetes as versatile and sustainable bioresources with significant pharmaceutical and biotechnological potential, emphasizing their role in combating antimicrobial resistance and enabling the rational development of novel therapeutic and industrial products.

Mining, Cloning, and Bioinformatic Analysis of Amidase Genes from Three Brewing Microorganisms

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.