ABSTRACT
OBJECTIVE: To isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features. RESULTS: Of 6720 clones evaluated, Clone W (10,000 bp) presented lipolytic activity and four predicted coding sequences, one of them LipW. Characterization of a predicted esterase/lipase, LipW, showed 28% sequence identity with an arylesterase from Pseudomonas fluorescens (pdb|3HEA) from protein database (PDB). Phylogenetic analysis showed LipW clustered with family V lipases; however, LipW was clustered in different subclade belonged to family V, suggesting a different subgroup of family V. In addition, LipW presented a difference in family V GH motif, a glycine replaced by a serine in GH motif. Estimated molecular weight and stokes radius values of LipW were 29,338.67-29,411.98 Da and 2.58-2.83 nm, respectively. Optimal enzyme activity was observed at pH 9.0-9.5 and at 40 °C. Circular dichroism analysis estimated secondary structures percentages as approximately 45% α-helix and 15% ß-sheet, consistent with the 3D structure predicted by homology. CONCLUSION: Our results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.
Subject(s)
Esterases/genetics , Esterases/metabolism , Soil Microbiology , Amino Acid Motifs , Brazil , Circular Dichroism , Cloning, Molecular , Esterases/chemistry , Metagenome , Models, Molecular , Molecular Weight , Phylogeny , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, FluorescenceABSTRACT
Antibiotic resistance has become a major concern for human and animal health, as therapeutic alternatives to treat multidrug-resistant microorganisms are rapidly dwindling. The problem is compounded by low investment in antibiotic research and lack of new effective antimicrobial drugs on the market. Exploring environmental antibiotic resistance genes (ARGs) will help us to better understand bacterial resistance mechanisms, which may be the key to identifying new drug targets. Because most environment-associated microorganisms are not yet cultivable, culture-independent techniques are essential to determine which organisms are present in a given environmental sample and allow the assessment and utilization of the genetic wealth they represent. Metagenomics represents a powerful tool to achieve these goals using sequence-based and functional-based approaches. Functional metagenomic approaches are particularly well suited to the identification new ARGs from natural environments because, unlike sequence-based approaches, they do not require previous knowledge of these genes. This review discusses functional metagenomics-based ARG research and describes new possibilities for surveying the resistome in environmental samples.
Subject(s)
Drug Resistance, Microbial/genetics , Environment , Metagenome/genetics , Metagenomics/methods , Animals , Anti-Bacterial Agents/classification , Anti-Bacterial Agents/pharmacology , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Base Sequence , Culture Techniques , DNA, Bacterial , Drug Resistance, Bacterial/genetics , Drug Resistance, Bacterial/physiology , Genes, Bacterial/genetics , HumansABSTRACT
OBJECTIVES: Putative new dioxygenases were identified in a metagenomic ß-lactam-resistance screening and, given their key role on aromatic metabolism, we raise the hypothesis that these enzymes maybe concomitantly related to antibiotic resistance and aromatic degradation. RESULTS: ORFs of three putative dioxygenases were isolated from resistant metagenomic clones. One of them, CRB2(1), was subcloned into pET24a expression vector and subjected to downstream phenotypic and bioinformatics analyses that demonstrated the "dual effect" of our metagenomic dioxygenase, on antibiotic and aromatic resistance. Furthermore, initial characterization assays strongly suggests that CRB2(1) open-reading frame is an extradiol-dioxygenase, most probably a bicupin domain gentisate 1,2-dioxygenase. This observation is, to our knowledge, the first description of a metagenomic dioxygenase and its action on ß-lactam resistance. CONCLUSION: Unraveling the diversity of antibiotic resistance elements on the environment could not only identify new genes and mechanisms in which bacteria can resist to antibiotics, but also contribute to biotechnology processes, such as in bioremediation.
