title: Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest soil metagenomic library.
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.
authors.affiliation: Unité de Microbiologie et Génomique, Gembloux Agro-Bio Tech, Université de Liège, Avenue Maréchal Juin 6, 5030 Gembloux, Belgium. Sophie.Biver@ulg.ac.be
authors.forename: Sophie
authors.initials: S
authors.lastname: Biver
authors.forename: Micheline
authors.initials: M
authors.lastname: Vandenbol
mesh_terms: D000595:Amino Acid Sequence; D005075:Biological Evolution; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008066:Lipolysis; D056186:Metagenomics; D008969:Molecular Sequence Data; D012492:Salts / Q000494:pharmacology; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature; D014197:Trees; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D012492:Salts; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; D008049:Lipase; D001618:beta-Lactamases