Abstract
Functional screens have been extensively used for searching native enzymes or mutant variants in clone libraries. Esterases and lipases are the most retrieved enzymes, because they are within the more demanded industrial enzymes and because a number of simple and generic screening methods can be applied for their screen. Here, we describe the use of a generic pH indicator assay protocol which unambiguously allows detecting in high-throughput manner esterase and lipase activity and quantifying specific activities using an ester concentration above 0.5 mM. The described method is simple and generic to allow the selection of esterases and lipases targeting desired esters.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kennedy J, Flemer B, Jackson SA, Lejon DPH, Morrissey JP, O’Gara F, Dobson ADW (2010) Marine metagenomics: new tools for the study and exploitation of marine microbial metabolism. Mar Drugs 8(3):608–628. https://doi.org/10.3390/md8030608
Dalmaso GZL, Ferreira D, Vermelho AB (2015) Marine extremophiles: a source of hydrolases for biotechnological applications. Mar Drugs 13(4):1925–1965
Alcaide M, Tchigvintsev A, Martínez-Martínez M, Popovic A, Reva ON, Lafraya Á, Bargiela R, Nechitaylo TY, Matesanz R, Cambon-Bonavita M-A (2015) Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp Rimicaris exoculata by using functional metagenomics. Appl Environ Microbiol 81(6):2125–2136
Ferrer M, Martínez-Martínez M, Bargiela R, Streit WR, Golyshina OV, Golyshin PN (2016) Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends. J Microbial Biotechnol 9(1):22–34
Reyes-Duarte D, Ferrer M, García-Arellano H (2012) Functional-based screening methods for lipases, esterases, and phospholipases in metagenomic libraries. In: Sandoval G (ed) Lipases and phospholipasese. Springer, New York, NY, pp 101–113
Ferrer M, Bargiela R, Martínez-Martínez M, Mir J, Koch R, Golyshina OV, Golyshin PN (2015) Biodiversity for biocatalysis: a review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes. Biocatal Biotransformation 33(5-6):235–249
Martínez-Martínez M, Lores I, Peña-García C, Bargiela R, Reyes-Duarte D, Guazzaroni M-E, Peláez AI, Sánchez J, Ferrer M (2014) Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters. J Microbial Biotechnol 7(2):184–191. https://doi.org/10.1111/1751-7915.12107
Peña-García C, Martínez-Martínez M, Reyes-Duarte D, Ferrer M (2016) High throughput screening of esterases, lipases and phospholipases in mutant and metagenomic libraries: a review. Comb Chem High Throughput Screen 19(8):605–615
Martinez-Martinez M, Alcaide M, Tchigvintsev A, Reva O, Polaina J, Bargiela R, Guazzaroni ME, Chicote A, Canet A, Valero F, Eguizabal ER, Guerrero MD, Yakunin AF, Ferrer M (2013) Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach. Appl Environ Microbiol 79(12):3553–3562. https://doi.org/10.1128/aem.00240-13
Janes L, Löwendahl C, Kazlauskas R (1998) Rapid quantitative screening of hydrolases using pH indicators. Finding enantioselective hydrolases. Chem A Eur J 4:2317–2324
Martínez-Martínez M, Coscolín C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernández J, Bollinger A, Thies S, Méndez-García C, Popovic A, Brown G, Chernikova TN, García-Moyano A, Bjerga GEK, Pérez-García P, Hai T, Del Pozo MV, Stokke R, Steen IH, Cui H, Xu X, Nocek BP, Alcaide M, Distaso M, Mesa V, Peláez AI, Sánchez J, Buchholz PCF, Pleiss J, Fernández- Guerra A, Glöckner FO, Golyshina OV, Yakimov MM, Savchenko A, Jaeger KE, Yakunin AF, Streit WR, Golyshin PN, Guallar V, Ferrer M. (2018) Determinants and Prediction of esterase substrate promiscuity patterns. ACS Chem Biol 13:225–234.
Santiago G, Martínez-Martínez M, Alonso S, Bargiela R, Coscolín C, Golyshin PN, Guallar V, Ferrer M. (2018) Rational engineering of multiple active sites in an ester hydrolase. Biochemistry 57:2245–2255.
Acknowledgments
This project received funding from the European Union’s Horizon 2020 research and innovation program Blue Growth: Unlocking the potential of Seas and Oceans under grant agreement no. 634486 (project acronym INMARE). This research was also supported by the grants PCIN-2014-107 (within ERA NET IB2 grant nr. ERA-IB-14-030-MetaCat), PCIN-2017-078 (within the ERA-MarineBiotech grant ProBone), BIO2014-54494-R, and BIO2017-85522-R from the Spanish Ministry of Economy, Industry and Competitiveness. This research was also supported by the project CONACYT CB-2008-01 (101784). The authors gratefully acknowledge financial support provided by the European Regional Development Fund (ERDF). C. Coscolín thanks the Spanish Ministry of Economy, Industry and Competitiveness for a PhD fellowship (Grant BES-2015-073829).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Reyes-Duarte, D., Coscolín, C., Martínez-Martínez, M., Ferrer, M., García-Arellano, H. (2018). Functional-Based Screening Methods for Detecting Esterase and Lipase Activity Against Multiple Substrates. In: Sandoval, G. (eds) Lipases and Phospholipases. Methods in Molecular Biology, vol 1835. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8672-9_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8672-9_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8671-2
Online ISBN: 978-1-4939-8672-9
eBook Packages: Springer Protocols