Abstract
The use of metagenomic techniques for enzyme discovery constitutes a powerful approach. Functional screens, in contrast to sequence homology search, enable us to select enzymes based on their activity. It is noteworthy that they additionally guarantee the identification of genes coding for enzymes that exhibited no sequence similarity to known counterparts from public databases and that even do not match any putative catalytic residues, involved in the selected catalytic function. Therefore, this strategy not only provides new enzymes for new biotechnological applications, but also allows functional assignment of many proteins, found in abundance in the databases, currently designated as “hypothetical” or “conserved hypothetical” proteins. In the past decade, there has been an exponential increase in the design of functional screening programmes, the majority of them established for hydrolases and oxidoreductases. Here, functional screening methods that guarantee the greatest enzyme diversity, for mining esterases and lipases, are described.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Steele HL, Jaeger KE, Daniel R et al (2009) Advances in recovery of novel biocatalysts from metagenomes. J Mol Microbial Biotechnol 16:25–37
Schnoes AM, Brown SD, Babbitt PC (2009) Annotation error in public databases: misannotation of molecular function in enzyme superfamilies. PLoS Comput Biol 5:e1000605M–E
Fernández-Arrojo L, Guazzaroni ME, López-Cortés N et al (2010) Metagenomic era for biocatalyst identification. Curr Opin Biotechnol 21:725–733
Vieites JM, Ghazi A, Beloqui A et al (2009) Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome. Microb Biotechnol 2:31–39
Sharma VK, Kumar N, Prakash T et al (2010) MetaBioME: a database to explore commercially useful enzymes in metagenomic datasets. Nucleic Acids Res 38(Database issue):D468–D472
Vieites JM, Guazzaroni ME, Beloqui A et al (2010) Molecular methods to study complex microbial communities. In: Streit W, Daniel R (eds) Metagenomics: Methods and Protocols. Springer: Hatfield, UK. Meth Mol Biol 668:1–37
Miller RB, Karn RC (1980) A rapid spectrophotometric method for the determination of esterase activity. J Biochem Biophys Meth 3:345–54
Ferrer M, Golyshina OV, Chernikova TN et al (2005) Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ Microbiol 7:1996–2010
Torres D, Fraaije M (2007) Discovery, redesign and applications of Baeyer-Villiger monooxygenases. In: Matsuda T (ed) Future directions in Biocatalysis, 1st edn. Elsevier, Amsterdam
Anderson JA (1939) The use of tributyrin agar in dairy bacteriology. Ber 3 Int Mikrobiol Kongress 3:726–728
Lawrence RC, Fryer TF, Reiter B (1967) Rapid method for the quantitative estimation of microbial lipases. Nature 213:1264–1265
Mackenzie RD et al (1967) Rapid colorimetric micromethod for free fatty acids. J Lipid Res 8:589–597
Höfelmann M, Kittsteiner-Eberle R, Schreier P (1983) Ultrathin-layer agar gels: a novel print technique for ultrathin-layer isoelectric focusing of enzymes. Anal Biochem 128:217–222
Kouker G, Jaeger KE (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53:211–213
Price MF, Wilkinson ID, Gentry LO (1982) Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia 20:7–14
Borst A, Fluit AC (2003) High levels of hydrolytic enzymes secreted by Candida albicans isolates involved in respiratory infections. J Med Microbiol 52:971–974
Acknowledgements
This research was supported by the Spanish CSD2007-00005 project and FEDER funds, ERA-Net PathoGenoMics (BFU2008-04398-E), by the European Community Project MAGICPAH (FP7-KBBE-2009-245226) and MAMBA (FP7-KBBE-2008-226977) and by CONACYT CB-2008-01 (101784) and PROMEP (47410061).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Reyes-Duarte, D., Ferrer, M., García-Arellano, H. (2012). Functional-Based Screening Methods for Lipases, Esterases, and Phospholipases in Metagenomic Libraries. In: Sandoval, G. (eds) Lipases and Phospholipases. Methods in Molecular Biology, vol 861. Humana Press. https://doi.org/10.1007/978-1-61779-600-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-61779-600-5_6
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-599-2
Online ISBN: 978-1-61779-600-5
eBook Packages: Springer Protocols