Structural and functional characterization of a novel lipolytic enzyme from a Brazilian Cerrado soil metagenomic library
To isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features.
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% β-sheet, consistent with the 3D structure predicted by homology.
Our results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.
KeywordsCerrado Lipolytic enzymes Metagenome Soil
The authors thank National Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Apoio à Pesquisa do Distrito Federal (FAP-DF) for financial support. Istvan and Lopes acknowledge a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Online Resource 1—Annotation and taxonomic classification of the predicted coding regions of Clone W. The translated sequences of predicted CDSs (> 150 aa) were aligned with protein sequences in the SwissProt, TrEMBL, and UniRef100 databases. Only the top hits of each alignment are shown.
Online Resource 2—Sedimentation velocity. a Raw data of absorbance at 280 nm versus cell radius for 2.91 μM LipW. b Residuals plot supplied by SEDFIT showing goodness of fit. (c) Continuous sedimentation coefficient distribution, c(S) curve, obtained with a regularization procedure from data shown in panel a with a confidence level of 0.95 using SEDFIT and frictional ratios of 1.20–1.31. The partial specific volume (υ) 0.735614 ml/g for LipW was determined using SEDNTERP. Solvent (water) density (ρ = 0.99823 g/ml) and viscosity (η = 0.01002 poise) were also determined by SEDNTERP. Circles represent experimental data, and the solid line represents the best fit to the Lamm equation supplied by SEDFIT. Similar results were obtained for 2.14 and 1.56 μM LipW.
Online Resource 3—Molecular and biophysical parameters of LipW.
Online Resource 4—Amino acid sequence alignment of LipW with family V lipases. The G-X-S-M-G–G consensus motif of family V is shown, and the catalytic triad residues are denoted with asterisks.
Online Resource 5—Ramachandran plot of the LipW model.
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Conflict of interest
The authors declare that they have no conflict of interest.