Abstract
High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.
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Bornscheuer UT, Kazlauskas RJ, 1999. Hydrolases in organic synthesis: regio- and stereoselective biotransformations. Wiley-VCH Weinheim, New York
Verger R, 1997. ‘Interfacial activation’ of lipases: facts and artifacts. Trends Biotechnol 15, 32–38
Bornscheuer UT, 2002. Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26, 73–81
Schmid RD, Verger R, 1998. Lipases: interfacial enzymes with attractive applications. Angew Chem Int Ed 37, 1608–1633
Beisson F, Tiss A, Rivière C, et al. 2000. Methods for lipase detection and assay: a critical review. Eur J Lipid Sci Technol 102, 133–153
Mateos JC, Ruiz K, Rodriguez JA, et al. 2007. Mapping substrate selectivity of lipases from thermophilic fungi. J Mol Catal B 49, 104–112
Ramírez L, Arrizon J, Cardador A, et al. 2008. A new microplate screening method for the simultaneous activity quantification of feruloyl esterases, tannases, and chlorogenate esterases. Appl Biochem Biotechnol 151, 711–723
Janes LE, Lowendahl AC, Kazlauskas RJ, 1998. Quantitative screening of hydrolase libraries using pH indicators: Identifying active and enantioselective hydrolases. Chem Eur J 4, 2324–2331
Brockman HL, 1981. Triglyceride lipase from porcine pancreas. Methods Enzymol 71, 619–627
Desnuelle P, Constantin MJ, Baldy J, 1955. Technique potentiométrique pour la mesure de l’activité de la lipase pancréatique. Bull Sté Chim Biol 37, 285–290
Entressangles B, Desnuelle P, 1968. Action of pancreatic lipase on aggregated glyceride molecules in an isotopic system. Biochim Biophys Acta 159, 285–295
Ferrato F, Carrière F, Sarda L et al (1997) A critical re-evaluation of the phenomenon of “interfacial activation”. In: Dennis E, Rubin B (eds) Methods enzymol. Academic Press, New York, 286:327–347
Sarda L, Desnuelle P, 1958. Action de la lipase pancréatique sur les esters en émulsion. Biochim Biophys Acta 30, 513–521
Alahverdjieva V, Ivanova M, Verger R, et al. 2005. A kinetic study of the formation of b-cyclodextrin complexes with monomolecular films of fatty acids and glycerides spread at the air/water interface. Colloid Surf B 42, 9–20
Acknowledgments
Eduardo Mateos-Díaz and Ángeles Camacho Ruiz acknowledges their Masters and PhD scholarships received from CONACYT. Lipid-free porcine pancreatic powder was a generous gift from Dr. Frédéric Carrière from Laboratory of Enzymology at Interfaces and Physiology of Lipolysis UPR-CNRS 9025 (Marseille, France).
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Mateos-Díaz, E., Rodríguez, J.A., de los Ángeles Camacho-Ruiz, M., Mateos-Díaz, J.C. (2012). High-Throughput Screening Method for Lipases/Esterases. 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_5
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DOI: https://doi.org/10.1007/978-1-61779-600-5_5
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