Abstract
Three new lipolytic genes were isolated from a forest soil metagenomic library by functional screening on tributyrin agar plates. The genes SBLip1, SBLip2 and SBLip5.1 respectively encode polypeptides of 445, 346 and 316 amino acids. Phylogenetic analyses revealed that SBLip2 and SBLip5.1 belong to bacterial esterase/lipase family IV, whereas SBLip1 shows similarity to class C β-lactamases and is thus related to esterase family VIII. The corresponding genes were overexpressed and their products purified by affinity chromatography for characterization. Analyses of substrate specificity with different p-nitrophenyl esters showed that all three enzymes have a preference for short-acyl-chain p-nitrophenyl esters, a feature of carboxylesterases as opposed to lipases. The β-lactamase activity of SBLip1, measured with the chromogenic substrate nitrocefin, was very low. The three esterases have the same optimal pH (pH 10) and remain active across a relatively broad pH range, displaying more than 60 % activity between pH 6 and 10. The temperature optima determined were 35 °C for SBLip1, 45 °C for SBLip2 and 50 °C for SBLip5.1. The three esterases displayed different levels of tolerance to salts, solvents and detergents, SBLip2 being overall more tolerant to high concentrations of solvent and SBLip5.1 less affected by detergents.
Similar content being viewed by others
References
Arbeli Z, Fuentes CL (2007) Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol Lett 272(2):269–275. doi:10.1111/j.1574-6968.2007.00764.x
Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343(Pt 1):177–183
Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340(4):783–795. doi:10.1016/j.jmb.2004.05.028
Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26(1):73–81
Duan CJ, Feng JX (2010) Mining metagenomes for novel cellulase genes. Biotechnol Lett 32(12):1765–1775. doi:10.1007/s10529-010-0356-z
Fazary AE, Ju Y-H (2008) The large-scale use of feruloyl esterases in industry. Biotechnol Mol Biol Rev 3(5):095–110
Hardeman F, Sjoling S (2007) Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol 59(2):524–534. doi:10.1111/j.1574-6941.2006.00206.x
Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzyme Microb Technol 39:235–251
Hasan F, Shah AA, Javed S, Hameed A (2010) Enzymes used in detergents: lipases. Afr J Biotechnol 9(31):4836–4844
Hemila H, Koivula TT, Palva I (1994) Hormone-sensitive lipase is closely related to several bacterial proteins, and distantly related to acetylcholinesterase and lipoprotein lipase: identification of a superfamily of esterases and lipases. Biochim Biophys Acta 1210(2):249–253
Hu Y, Fu C, Huang Y, Yin Y, Cheng G, Lei F, Lu N, Li J, Ashforth EJ, Zhang L, Zhu B (2010) Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment. FEMS Microbiol Ecol 72(2):228–237. doi:10.1111/j.1574-6941.2010.00851.x
Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13(4):390–397
Jeon JH, Kim SJ, Lee HS, Cha SS, Lee JH, Yoon SH, Koo BS, Lee CM, Choi SH, Lee SH, Kang SG (2011) Novel metagenome-derived carboxylesterase that hydrolyzes beta-lactam antibiotics. Appl Environ Microbiol 77(21):7830–7836. doi:10.1128/AEM.05363-11
Kelly JA, Kuzin AP (1995) The refined crystallographic structure of a DD-peptidase penicillin-target enzyme at 1.6 A resolution. J Mol Biol 254(2):223–236. doi:10.1006/jmbi.1995.0613
Ko KC, Rim SO, Han Y, Shin BS, Kim GJ, Choi JH, Song JJ (2012) Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil. J Ind Microbiol Biotechnol. doi:10.1007/s10295-011-1080-y
Matteotti C, Haubruge E, Thonart P, Francis F, De Pauw E, Portetelle D, Vandenbol M (2011) Characterization of a new beta-glucosidase/beta-xylosidase from the gut microbiota of the termite (Reticulitermes santonensis). FEMS Microbiol Lett 314(2):147–157. doi:10.1111/j.1574-6968.2010.02161.x
Nacke H, Will C, Herzog S, Nowka B, Engelhaupt M, Daniel R (2011) Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories. FEMS Microbiol Ecol 78(1):188–201. doi:10.1111/j.1574-6941.2011.01088.x
Panda T, Gowrishankar BS (2005) Production and applications of esterases. Appl Microbiol Biotechnol 67(2):160–169. doi:10.1007/s00253-004-1840-y
Pang M-D, Abdulla N, Lee C-W, Ng C-C (2008) Isolation of high molecular weight dna from forest topsoil for metagenomic analysis. Asia Pac J Mol Biol Biotechnol 16(2):35–41
Peng Q, Zhang X, Shang M, Wang X, Wang G, Li B, Guan G, Li Y, Wang Y (2011) A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea. Microb Cell Fact 10:95. doi:10.1186/1475-2859-10-95
Rajendhran J, Gunasekaran P (2008) Strategies for accessing soil metagenome for desired applications. Biotechnol Adv 26(6):576–590. doi:10.1016/j.biotechadv.2008.08.002
Ranjan R, Grover A, Kapardar RK, Sharma R (2005) Isolation of novel lipolytic genes from uncultured bacteria of pond water. Biochem Biophys Res Commun 335(1):57–65. doi:10.1016/j.bbrc.2005.07.046
Rashamuse K, Magomani V, Ronneburg T, Brady D (2009) A novel family VIII carboxylesterase derived from a leachate metagenome library exhibits promiscuous beta-lactamase activity on nitrocefin. Appl Microbiol Biotechnol 83(3):491–500. doi:10.1007/s00253-009-1895-x
Sakai Y, Ishikawa J, Fukasaka S, Yurimoto H, Mitsui R, Yanase H, Kato N (1999) A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli. Biosci Biotechnol Biochem 63(4):688–697
Sangeetha R, Arulpandi I, Geetha A (2011) Bacterial lipases as potential industrial biocatalysts: an overview. Res J Microbiol 6(1):1–24
Sezonov G, Joseleau-Petit D, D’Ari R (2007) Escherichia coli physiology in Luria–Bertani broth. J Bacteriol 189(23):8746–8749. doi:10.1128/JB.01368-07
Sheridan PP, Panasik N, Coombs JM, Brenchley JE (2000) Approaches for deciphering the structural basis of low temperature enzyme activity. Biochim Biophys Acta 1543(2):417–433
Shu Z-Y, Jiang H, Lin R-F, Jiang Y-M, Lin L, Huang J-Z (2010) Technical methods to improve yield, activity and stability in the development of microbial lipases. J Mol Catal B Enzym 62:1–8
Singh R, Gupta N, Goswami VK, Gupta R (2006) A simple activity staining protocol for lipases and esterases. Appl Microbiol Biotechnol 70(6):679–682. doi:10.1007/s00253-005-0138-z
Sorokin DY, Jones BE (2009) Improved method for direct screening of true lipase-producing microorganisms with particular emphasis on alkaline conditions. Mikrobiologiia 78(1):144–149
Steele HL, Jaeger KE, Daniel R, Streit WR (2009) Advances in recovery of novel biocatalysts from metagenomes. J Mol Microbiol Biotechnol 16(1–2):25–37. doi:10.1159/000142892
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. doi:10.1093/molbev/msr121
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680
Uchiyama T, Miyazaki K (2009) Functional metagenomics for enzyme discovery: challenges to efficient screening. Curr Opin Biotechnol 20(6):616–622. doi:10.1016/j.copbio.2009.09.010
Vulic M, Kolter R (2002) Alcohol-induced delay of viability loss in stationary-phase cultures of Escherichia coli. J Bacteriol 184(11):2898–2905
Wagner UG, Petersen EI, Schwab H, Kratky C (2002) EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity. Protein Sci 11(3):467–478. doi:10.1110/ps.33002
Wei Y, Contreras JA, Sheffield P, Osterlund T, Derewenda U, Kneusel RE, Matern U, Holm C, Derewenda ZS (1999) Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase. Nat Struct Biol 6(4):340–345. doi:10.1038/7576
Yu EY, Kwon MA, Lee M, Oh JY, Choi JE, Lee JY, Song BK, Hahm DH, Song JK (2011) Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome. Appl Microbiol Biotechnol 90(2):573–581. doi:10.1007/s00253-011-3132-7
Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62(2):316–322
Acknowledgments
We thank Christel Mattéotti for helpful discussions. Sophie Biver is a Postdoctoral Researcher of the Fonds National de la Recherche Scientifique (F.R.S-FNRS).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Biver, S., Vandenbol, M. Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest soil metagenomic library. J Ind Microbiol Biotechnol 40, 191–200 (2013). https://doi.org/10.1007/s10295-012-1217-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10295-012-1217-7