Abstract
Seven lipolytic genes were isolated and sequenced from a metagenomic library that was constructed following biomass enrichment in a fed-batch bioreactor submitted to high temperature (50–70°C) and alkaline pH (7–8.5). Among those sequences, lipIAF1-6 was chosen for further study and cloned in Streptomyces lividans 10–164. The G+C content within the sequence was 64.3%. The encoded protein, LipIAF1-6, was related to various putative lipases previously identified in different genome sequences. Homology of LipIAF-6 with the different lipases did not exceed 31%. The optimum pH (8.5) and temperature (60°C) of the purified enzyme were in agreement with the enrichment conditions. Furthermore, the enzyme was thermostable for as long as 30 min at 70°C. The maximum activity of the purified lipase was 4,287 IU/mg towards p-nitrophenyl (p-NP) butyrate (60°C; pH 8.5). LipIAF1-6 does not seem to need the presence of metal ions for its activity. The enzyme was slightly inhibited by 10 mM CoCl2 (14%), HgCl2 (12%), and dithiothreitol (DTT) (15%). The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) reduced activity by 39% and 71% when incubated at concentrations of 1 and 10 mM, respectively. Finally, LipIAF1-6 was stable in different organic solvents, and against several surfactants and oxidative agents commonly found in detergent formulations. These results are quite encouraging for further use of this enzyme in different industrial processes.
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Abdel-Fattah YR, Gaballa AA (2008) Identification and over-expression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli. Microbiol Res 163(1): 13–20. doi:10.1016/j.micres.2006.02.004
Alloue WA, Aguedo M, Destain J, Ghalfi H, Blecker C et al (2008) Les lipases immobilisées et leurs applications. Biotechnol Agron Soc Environ 12(1):57–68
Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183
Bora L, Kalita MC (2007). Production and optimization of thermostable lipase from a thermophilic Bacillus sp. LBN 4. Int J Microbiol 4(1)
Boston M, Requadt C, Danko S, Jarnagin A, Ashizawa E et al (1997) Structure and function engineered Pseudomonas mendocina lipase. Methods Enzymol 284:298–317
Choo DW, Kurihara T, Suzuki T, Soda K, Esaki N (1998) A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11–1: gene cloning and enzyme purification and characterization. Appl Environ Microbiol 64(2):486–491
Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R, Wittwer P (2005) Metagenomic gene discovery: past, present and future. Trends Biotechnol 23(6):321–329. doi:10.1016/j.tibtech.2005.04.001
Demirjian DC, Moris-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5(2):144–151
Diaz M, Ferreras E, Moreno R, Yepes A, Berenguer J, Santamaria R (2008) High-level overproduction of Thermus enzymes in Streptomyces lividans. Appl Microbiol Biotechnol 79(6):1001–1008. doi:10.1007/s00253-008-1495-1
Elend C, Schmeisser C, Leggewie C, Babiak P, Carballeira JD et al (2006) Isolation and biochemical characterization of two novel metagenome-derived esterases. Appl Environ Microbiol 72(5):3637–3645. doi:10.1128/Aem.72.5.3637-3645.2006
Entcheva P, Liebl W, Johann A, Hartsch T, Streit WR (2001) Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia. Appl Environ Microbiol 67 (1):89–99. doi:10.1128/AEM.67.1.89-99.2001
Fan Z, Yue C, Tang Y, Zhang Y (2009) Cloning, sequence analysis and expression of bacterial lipase-coding DNA fragments from environment in Escherichia coli. Mol Biol Rep 36(6):1515–1519. doi:10.1007/s11033-008-9344-y
Fickers P, Destain J, Thonart P (2008) Les lipases sont des hydrolases atypiques: principales caractéristiques et applications. Biotechnol Agron Soc Environ 12(2):119–130
Fojan P, Jonson PH, Petersen MTN, Petersen SB (2000) What distinguishes an esterase from a lipase: a novel structural approach. Biochimie 82(11):1033–1041
Handelsman J (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68(4):669. doi:10.1128/Mbr.68.4.669-685.2004
Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzyme Microbiol Technol 39(2):235–251. doi:10.1016/j.enzmictec.2005.10.016
Healy FG, Ray RM, Aldrich HC, Wilkie AC, Ingram LO, Shanmugam KT (1995) Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose. Appl Microbiol Biotechnol 43(4):667–674
Henne A, Schmitz RA, Bomeke M, Gottschalk G, Daniel R (2000) Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl Environ Microbiol 66(7):3113–3116
Hurtubise Y, Shareck F, Kluepfel D, Morosoli R (1995) A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins. Mol Microbiol 17(2):367–377
Ishida M, Yoshida M, Oshima T (1997) Highly efficient production of enzymes of an extreme thermophile, Thermus thermophilus: a practical method to overexpress GC-rich genes in Escherichia coli. Extremophiles 1(3):157–162
Jaeger KE, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Ann Rev Microbiol 53:315–351
Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13(4):390–397
Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15(1):29–63
Kademi A, Ait-Abdelkader N, Fakhreddine L, Baratti J (2000) Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans. Appl Microbiol Biotechnol 54(2):173–179
Kim YJ, Choi GS, Kim SB, Yoon GS, Kim YS, Ryu YW (2006) Screening and characterization of a novel esterase from a metagenomic library. Protein Expr Purif 45(2):315–323. doi:10.1016/j.pep.2005.06.008
Lee MH, Lee CH, Oh TK, Song JK, Yoon JH (2006) Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases. Appl Environ Microbiol 72(11):7406–7409. doi:10.1128/Aem.01157-06
Li H, Zhang X (2005) Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1. Protein Expr Purif 42(1):153–159. doi:10.1016/j.pep.2005.03.011
Li X, Qin L (2005) Metagenomics-based drug discovery and marine microbial diversity. Trends Biotechnol 23(11):539–543. doi:10.1016/j.tibtech.2005.08.006
Lorenz P, Eck J (2005) Metagenomics and industrial applications. Nat Rev Microbiol 3(6):510–516. doi:10.1038/nrmicro1161
Lykidis A, Mavromatis K, Ivanova N, Anderson I, Land M et al (2007) Genome sequence and analysis of the soil cellulolytic actinomycete Thermobifida fusca YX. J Bacteriol 189(6):2477–2486. doi:10.1128/JB.01899-06
Mead DA, Szczesna-Skorupa E, Kemper B (1986) Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng 1(1):67–74
Meilleur C, Hupe JF, Juteau P, Shareck F (2009) Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library. J Ind Microbiol Biotechnol 36(6):853–861
Niehaus F, Bertoldo C, Kahler M, Antranikian G (1999) Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 51(6):711–729
Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol VT (1999) The realm of microbial lipases in biotechnology. Biotechnol Appl Biochem 29:119–131
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
Rashid N, Shimada Y, Ezaki S, Atomi H, Imanaka T (2001) Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A. Appl Environ Microbiol 67(9):4064–4069
Rosenau F, Jaeger KE (2000) Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion. Biochimie 82(11):1023–1032
Rossi M, Ciaramella M, Cannio R, Pisani FM, Moracci M, Bartolucci S (2003) Extremophiles 2002. J Bacteriol 185(13):3683–3689. doi:10.1128/Jb.185.13.3683-3689.2003
Ruiz C, Javier Pastor FI, Diaz P (2003) Isolation and characterization of Bacillus sp. BP-6 LipA, a ubiquitous lipase among mesophilic Bacillus species. Lett Appl Microbiol 37(4):354–359
Salameh MA, Wiegel J (2007) Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl Environ Microbiol 73(23):7725–7731. doi:10.1128/AEM.01509-07
Schmeisser C, Steele H, Streit WR (2007) Metagenomics, biotechnology with non-culturable microbes. Appl Microbiol Biotechnol 75(5):955–962. doi:10.1007/s00253-007-0945-5
Sharma AK, Tiwari RP, Hoondal GS (2001) Properties of a thermostable and solvent stable extracellular lipase from a Pseudomonas sp. AG-8. J Basic Microbiol 41 (6):363–366. doi:10.1002/1521-4028(200112)41:6<363::AID-JOBM363>3.0.CO;2-C
Sharma R, Chisti Y, Banerjee UC (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19 (8):627–662
Sibille N, Favier A, Azuaga AI, Ganshaw G, Bott R et al (2006) Comparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase. Protein Sci 15(8):1915–1927. doi:10.1110/Ps.062213706
Snellman EA, Sullivan ER, Colwell RR (2002) Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1. Eur J Biochem 269(23):5771–5779
Soliman NA, Knoll M, Abdel-Fattah YR, Schmid RD, Lange S (2007) Molecular cloning and characterization of thermostable esterase and lipase from Geobacillus thermoleovorans YN isolated from desert soil in Egypt. Process Biochem 42(7):1090–1100. doi:10.1016/j.procbio.2007.05.005
Sugihara A, Ueshima M, Shimada Y, Tsunasawa S, Tominaga Y (1992) Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia. J Biochem 112(5):598–603
Tesch C, Nikoleit K, Gnau V, Gotz F, Bormann C (1996) Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes. J Bacteriol 178(7):1858–1865
van den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6(3):213–218. doi:10.1016/S1369-5274(03)00060-2
Wang GYS, Graziani E, Waters B, Pan WB, Li X et al (2000) Novel natural products from soil DNA libraries in a streptomycete host. Organic Lett 2(16):2401–2404. doi:10.1021/Ol005860z
Yan YL, Yang J, Dou YT, Chen M, Ping SZ et al (2008) Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proc Natl Acad Sci USA 105(21):7564–7569. doi:10.1073/pnas.0801093105
Acknowledgments
This work was funded by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada. A. Côté is a recipient of a scholarship from Fondation Armand-Frappier. We are grateful to Raymonde Jetté, Guillaume Brault and François-Xavier Lussier for helpful discussions.
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Côté, A., Shareck, F. Expression and characterization of a novel heterologous moderately thermostable lipase derived from metagenomics in Streptomyces lividans . J Ind Microbiol Biotechnol 37, 883–891 (2010). https://doi.org/10.1007/s10295-010-0735-4
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DOI: https://doi.org/10.1007/s10295-010-0735-4