Biotechnology Letters

, Volume 40, Issue 9–10, pp 1395–1406 | Cite as

Structural and functional characterization of a novel lipolytic enzyme from a Brazilian Cerrado soil metagenomic library

  • Paula Istvan
  • Amanda Araújo Souza
  • Aisel Valle Garay
  • Debora Farage Knupp dos Santos
  • Gideane Mendes de Oliveira
  • Renata Henrique Santana
  • Fabyano Alvares Cardoso Lopes
  • Sonia Maria de Freitas
  • João Alexandre Ribeiro Gonçalves Barbosa
  • Ricardo Henrique KrügerEmail author
Original Research Paper



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.


Cerrado 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).

Supporting information

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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (XLSX 11 kb)
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Supplementary material 2 (TIFF 132 kb)
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Supplementary material 3 (XLSX 9 kb)
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Supplementary material 4 (TIFF 6147 kb)
10529_2018_2598_MOESM5_ESM.tif (267 kb)
Supplementary material 5 (TIFF 267 kb)


  1. Adler AJ, Greenfield NJ, Fasman GD (1973) Circular dichroismus and optical rotatory dispersion of proteins and polypeptides. Methods Enzymol 27:675–735CrossRefPubMedGoogle Scholar
  2. Altschul S, Madden T, Schäffer A et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedPubMedCentralGoogle Scholar
  3. Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343(Pt 1):177–183CrossRefPubMedPubMedCentralGoogle Scholar
  4. Arpigny JL, Jendrossek D, Jaeger KE (1998) A novel heat-stable lipolytic enzyme from Sulfolobus acidocaldarius DSM 639 displaying similarity to polyhydroxyalkanoate depolymerases. FEMS Microbiol Lett 167:69–73. CrossRefPubMedGoogle Scholar
  5. Bairoch A, Boeckmann B (1994) The SWISS-PROT protein sequence data bank: current status. Nucleic Acids Res 22:3578–3580CrossRefPubMedPubMedCentralGoogle Scholar
  6. Berman HM (2000) The Protein Data Bank. Nucleic Acids Res 28:235–242. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bhattacharya D, Cheng J (2013) 3Drefine: consistent protein structure refinement by optimizing hydrogen bonding network and atomic-level energy minimization. Proteins 81:119–131. CrossRefPubMedGoogle Scholar
  8. Böhm G, Muhr R, Jaeinicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 5:191–195CrossRefPubMedGoogle Scholar
  9. Bonelli FS, Jonas A (1993) Reaction of lecithin: cholesterol acyltransferase with a water soluble substrate: effects of surfactants. Biochim Biophys Acta 1166:92–98CrossRefPubMedGoogle Scholar
  10. Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26:73–81. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Borrelli GM, Trono D (2015) Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications. Int J Mol Sci 16:20774–20840. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Buchfink B, Xie C, Huson DH (2014) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60. CrossRefPubMedGoogle Scholar
  13. de Castro AP, Quirino BF, Allen H et al (2011) Construction and validation of two metagenomic DNA libraries from Cerrado soil with high clay content. Biotechnol Lett 33:2169–2175. CrossRefPubMedGoogle Scholar
  14. DeLano WL (2002) The PyMOL molecular graphics system. Schrödinger LLC wwwpymolorg, version 1.
  15. Delmont TO, Robe P, Cecillon S et al (2011) Accessing the soil metagenome for studies of microbial diversity. Appl Environ Microbiol 77:1315–1324. CrossRefPubMedGoogle Scholar
  16. Dewan SS (2017) Global markets for enzymes in industrial applications.
  17. dos Santos DFK, Istvan P, Noronha EF et al (2015) New dioxygenase from metagenomic library from Brazilian soil: insights into antibiotic resistance and bioremediation. Biotechnol Lett 37:1809–1817. CrossRefPubMedGoogle Scholar
  18. Dukunde A, Schneider D, Lu M et al (2017) A novel, versatile family IV carboxylesterase exhibits high stability and activity in a broad pH spectrum. Biotechnol Lett 39:577–587. CrossRefPubMedGoogle Scholar
  19. Feller G, Thiry M, Gerday C (1990) Sequence of a lipase gene from the antarctic psychrotroph Moraxella TA144. Nucleic Acids Res 18:6431. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Ferrer M, Bargiela R, Martínez-Martínez M et al (2016) Biodiversity for biocatalysis: a review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes. Biocatal Biotransform 2422:1–15. CrossRefGoogle Scholar
  21. Gu X, Wang S, Wang S et al (2015) Identification and characterization of two novel Esterases from a metagenomic library. Food Sci Technol Res 21:649–657. CrossRefGoogle Scholar
  22. Hide W, Chan A, Li WH (1992) Structure and evolution of the lipase superfamily. J Lipid Res 33:167–178PubMedGoogle Scholar
  23. Hriscu M, Chiş L, Toşa M, Irimie FD (2013) pH-Profiling of thermoactive lipases and esterases: caveats and further notes. Eur J Lipid Sci Technol 115:571–575. CrossRefGoogle Scholar
  24. Hyatt D, Chen G-L, LoCascio PF et al (2010) Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform 11:119. CrossRefGoogle Scholar
  25. Jeon JH, Kim JT, Kang SG et al (2009) Characterization and its potential application of two esterases derived from the arctic sediment metagenome. Mar Biotechnol 11:307–316. CrossRefPubMedGoogle Scholar
  26. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kumar S, Tsai C-J, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng Des Sel 13:179–191. CrossRefGoogle Scholar
  28. Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. CrossRefPubMedGoogle Scholar
  29. Lee MH, Lee S-W (2013) Bioprospecting potential of the soil metagenome: novel enzymes and bioactivities. Genom Inform 11:114. CrossRefGoogle Scholar
  30. Lee MH, Khan R, Tao W et al (2018) Soil metagenome-derived 3-hydroxypalmitic acid methyl ester hydrolases suppress extracellular polysaccharide production in Ralstonia solanacearum. J Biotechnol 270:30–38. CrossRefPubMedGoogle Scholar
  31. Lenfant N, Hotelier T, Velluet E et al (2013) ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: tools to explore diversity of functions. Nucleic Acids Res 41:423–429. CrossRefGoogle Scholar
  32. Lovell SC, Davis IW, Arendall WB et al (2003) Structure validation by Calpha geometry: phi, psi and Cbeta deviation. Proteins 50:437–450. CrossRefPubMedGoogle Scholar
  33. Newman JR, Fuqua C (1999) Broad-host-range expression vectors that carry the l-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227:197–203. CrossRefPubMedGoogle Scholar
  34. Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217. CrossRefPubMedGoogle Scholar
  35. Ovchinnikov S, Park H, Varghese N et al (2017) Protein structure determination using metagenome sequence data. Science 355:294–298. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Pereira MR, Mercaldi GF, Maester TC et al (2015) Est16, a new esterase isolated from a metagenomic library of a microbial consortium specializing in diesel oil degradation. PLoS ONE 10:1–16. CrossRefGoogle Scholar
  37. Price MN, Dehal PS, Arkin AP (2010) FastTree 2 - Approximately maximum-likelihood trees for large alignments. PLoS ONE. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42:W320–W324. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78:1606–1619. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Schuck P (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 320:104–124. CrossRefPubMedGoogle Scholar
  41. Torsvik V, Goksoyr J, Daae FL (1990) High diversity in DNA of soil bacteria. Appl Environ Microbiol 56:782–787. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Tyzack JD, Furnham N, Sillitoe I et al (2017) Understanding enzyme function evolution from a computational perspective. Curr Opin Struct Biol 47:131–139. CrossRefPubMedGoogle Scholar
  43. Valle A, Pérez-Socas LB, Canet L et al (2018) Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation. Sci Rep 8:1–18. CrossRefGoogle Scholar
  44. Webb B, Sali A (2014) Protein structure modeling with MODELLER. Methods Mol Biol 1137:1–15. CrossRefPubMedGoogle Scholar
  45. Xing MN, Zhang XZ, Huang H (2012) Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis. Biotechnol Adv 30:920–929. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Yang X, Wu L, Xu Y et al (2018) Identification and characterization of a novel alkalistable and salt-tolerant esterase from the deep-sea hydrothermal vent of the East Pacific Rise. Microbiologyopen. CrossRefPubMedGoogle Scholar
  47. Yin DL, Bernhardt P, Morley KL et al (2010) Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase. Biochemistry 49:1931–1942. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Paula Istvan
    • 1
  • Amanda Araújo Souza
    • 2
  • Aisel Valle Garay
    • 2
  • Debora Farage Knupp dos Santos
    • 1
  • Gideane Mendes de Oliveira
    • 2
  • Renata Henrique Santana
    • 3
  • Fabyano Alvares Cardoso Lopes
    • 1
  • Sonia Maria de Freitas
    • 2
  • João Alexandre Ribeiro Gonçalves Barbosa
    • 2
  • Ricardo Henrique Krüger
    • 1
    Email author
  1. 1.Laboratório de Enzimologia, Departamento de Biologia Celular, Instituto Central de Ciências SulUniversidade de Brasília – UnBBrasíliaBrazil
  2. 2.Laboratório de Biofísica, Departamento de Biologia CelularUniversidade de BrasíliaBrasíliaBrazil
  3. 3.Instituto Federal de BrasíliaBrasíliaBrazil

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