ObjectiveTo isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features.ResultsOf 6720 clones evaluated, Clone W (10,000bp) 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 29,338.67-29,411.98Da and 2.58-2.83nm, respectively. Optimal enzyme activity was observed at pH 9.0-9.5 and at 40 degrees C. Circular dichroism analysis estimated secondary structures percentages as approximately 45% -helix and 15% -sheet, consistent with the 3D structure predicted by homology.ConclusionOur results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.