[en] in order to isolate novel organic solvent-tolerant (oSt) lipases, a metagenomic library was built using dna derived from a temperate forest soil sample. a two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. the deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described oSt lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/b hydrolase subgroup (abh04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (c4) compounds, behaving as an esterase rather than a lipase. the RBest1 esterase was purified and biochemically characterized. the optimal esterase activity was observed at ph 6.5 and at temperatures ranging from 38 to 45 °c. enzymatic activity, determined by hydrolysis of p‐nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. noteworthy, RBest1 remains significantly active at high ionic strength. these findings suggest that RBest1 possesses the ability of oSt enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.
Disciplines :
Microbiology
Author, co-author :
Berlemont, Renaud
Spee, Olivier
Delsaute, Maud
Lara, Yannick ; Université de Liège > Centre d'ingénierie des protéines
Schuldes, Jörg
Simon, Carola
Power, Pablo
Daniel, Rolf
Galleni, Moreno ; Université de Liège > Département des sciences de la vie > Macromolécules biologiques
Language :
English
Title :
Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification
Bell G, Janssen AEM, Halling PJ. Water activity fails to predict critical hydration level for enzyme activity in polar organic solvents: Interconversion of water concentrations and activities. Enz Microb Technol 1997; 20: 471-7.
Bergman NH, Anderson EC, Swenson EE, Niemeyer MM, Miyoshi AD, Hanna PC. Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation. J Bacteriol 2006; 188: 6092-100.
Berlemont R, Delsaute M, Pipers D, D'Amico S, Feller G, Galleni M, Power P. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples. ISME J 2009; 3: 1070-81.
Bhaya D, Grossman AR, Steunou AS, Khuri N, Cohan FM, Hamamura N, Melendrez MC, Bateson MM, Ward DM, Heidelberg JF. Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses. ISME J 2007; 1: 703-13.
Byun JS, Rhee JK, Kim DU, Oh JW, Cho HS. Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library. Acta Crystallogr Sect F Struct Biol Cryst Commun 2006; 62: 145-7.
Cieslinski H, Bialkowskaa A, Tkaczuk K, Dlugolecka A, Kur J, Turkiewicz M. Identification and molecular modeling of a novel lipase from an Antarctic soil metagenomic library. Pol J Microbiol 2009; 58: 199-204.
Couto GH, Glogauer A, Faoro H, Chubatsu LS, Souza EM, Pedrosa FO. Isolation of a novel lipase from a metagenomic library derived from mangrove sediment from the south Brazilian coast. Genet Mol Res 9: 514-23.
Chu X, He H, Guo C, Sun B. Identification of two novel esterases from a marine metagenomic library derived from South China Sea. Appl Microbiol Biotechnol 2008; 80: 615-25.
Daniel R. The soil metagenome--a rich resource for the discovery of novel natural products. Curr Opin Biotechnol 2004; 15: 199-204.
Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32: 1792-7.
Felsenstein J. An alternating least squares approach to inferring phylogenies from pairwise distances. Syst Biol 1997; 46: 101-11.
Ferencko L, Cote MA, Rotman B. Esterase activity as a novel parameter of spore germination in Bacillus anthracis. Biochem Biophys Res Commun 2004; 319: 854-8.
Frostegard A, Courtois S, Ramisse V, Clerc S, Bernillon D, Le Gall F, Jeannin P, Nesme X, Simonet P. Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol 1999; 65: 5409-20.
Gupta A, Khare SK. Enzymes from solvent-tolerant microbes: useful biocatalysts for non-aqueous enzymology. Crit Rev Biotechnol 2009; 29: 44-54.
Handelsman J, Liles M, Mann D, Riesenfeld C, Goodman RM. Cloning the metagenome: culture-independent access to the diversity and functions of the uncultivatedmicrobial world. In: Wren B, Dorrell N, editors. Methods in Microbiology - Functional Microbial Genomics, 1st edition. New York, Academic Press, 2002, p. 241-55.
Henne A, Daniel R, Schmitz RA, Gottschalk G. Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl Environ Microbiol 1999; 65: 3901-7.
Henne A, Schmitz RA, Bomeke M, Gottschalk G, Daniel R. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl Environ Microbiol 2000; 66: 3113-6.
Hong KS, Lim HK, Chung EJ, Park EJ, Lee MH, Kim JC, Choi GJ, Cho KY, Lee SW. Selection and characterization of forest soil metagenome genes encoding lipolytic enzymes. J Microbiol Biotechnol 2007; 17: 1655-60.
Hugenholtz P, Goebel BM, Pace NR. Impact of cultureindependent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 1998; 180: 4765-74.
Hun CJ, Rahman RN, Salleh AB, Basri M. A newly isolated organic solvent tolerant Bacillus sphaeriucs 205y producing organic solvent-stable lipase. Biochem Eng J 2003; 15: 147-51.
Jeon JH, Kim JT, Kim YJ, Kim H., Lee HS, Kang SG, Kim SJ, Lee JH. Cloning and characterization of a new coldactive lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol 2009; 81: 865-74.
Kim YH, Kwon EJ, Kim SK, Jeong YS, Kim J, Yun HD, Kim H. Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library. Biochem Biophys Res Commun 393: 45-9.
Kim YJ, Choi GS, Kim SB, Yoon GS, Kim YS, Ryu YW. Screening and characterization of a novel esterase from a metagenomic library. Protein Expr Purif 2006; 45: 315-323.
Klibanov AM. Improving enzymes by using them in organic solvents. Nature 2001; 409: 241-6.
Koeller KM, Wong CH. Enzymes for chemical synthesis. Nature 2001; 409: 232-40.
Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 1987; 30: 81-7.
Lee MH, Lee CH, Oh TK, Song JK, Yoon JH. 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 2006; 72: 7406-9.
Lee SW, Won K, Lim HK, Kim JC, Choi GJ, Cho KY. Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl Microbiol Biotechnol 2004; 65: 720-6.
Masayama A, Kuwana R, Takamatsu H, Hemmi H, Yoshimura T, Watabe K, Moriyama R. A novel lipolytic enzyme, YcsK (LipC), located in the spore coat of Bacillus subtilis, is involved in spore germination. J Bacteriol 2007; 189: 2369-75.
Meilleur C, Hupe JF, Juteau P, Shareck F. Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library. J Ind Microbiol Biotechnol 2009; 36: 853-61.
Moreira D, Rodriguez-Valera F, Lopez-Garcia P. Analysis of a genome fragment of a deep-sea uncultivated Group II euryarchaeote containing 16S rDNA, a spectinomycinlike operon and several energy metabolism genes. Environ Microbiol 2004; 6: 959-69.
Otero C, Fernandez-Perez M, Hermoso JA, Ripoll MM. Activation in the family of Candida rugosa isolipases by polyethylene glycol. J Mol Cat B: Enzymatic 2005; 32: 225-9.
Rahman RN, Chin JH, Salleh AB, Basri M. Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y. Mol Genet Genomics 2003; 269: 252-260.
Sellek GA, Chaudhuri JB. Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb Technol 1999; 25: 471-82.
Serdakowski AL, Dordick JS. Enzyme activation for organic solvents made easy. Trends Biotechnol 2008; 26: 48-54.
Setlow P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 2006; 101: 514-25.
Sulong MR, Abdul Rahman RN, Salleh AB, Basri M. A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene. Protein Expr Purif 2006; 49: 190-5.
Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007; 24: 1596-9.
Zhou J, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Appl Environ Microbiol 1996; 62: 316-22.
