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.

Characterization of a new oxidant-stable serine protease isolated by functional metagenomics

“…Both plasmid libraries, differing only in their average insert size (7 and 12 kb), had been successfully screened for lipolytic (Biver and Vandenbol 2013) and antimicrobial activities (Biver et al 2013). Further analysis of the small-insert library allowed us to identify one positive clone on agar plates supplemented with 1 g/l AZCL-casein.…”

“…Several non-secreted proteins have been identified by functional metagenomics, probably as a result of membrane leakage after prolonged growth (Biver and Vandenbol 2013; Phrommao et al 2011). Its absence in the culture supernatant after one night does not mean, however, that SBcas3.3 is a membrane-anchored or intracellular protease when produced in its original host.…”

Discovery and expression of a Pseudomonas sp. esterase as a novel biocatalyst for the efficient biosynthesis of a chiral intermediate of pregabalin

“…For the latter, which was also a highly enantioselective enzyme, its maximal activity was obtained at 35°C, pH 10.0, and it exerted high enzymatic activity at temperatures from 30 to 45°C. Many other microbial esterases belonging to Family VIII also adapted to moderate temperatures and alkaline pHs including two cold-active esterases, EstM-N1 and EstM-N2, which showed optimal activities at 20 and 30°C, respectively, at pH 9.0 [44], as well as esterases SBLip1 and SBLip2 which showed highest activities at 35 and 45°C, at pH 10.0, respectively [45].…”

“…By locking the stereocenter earlier and enabling the racemization of (R)-CNDE, a significant reduction of the E-factor from 86 to 8 was achieved [19]. Zheng et al attempted the stereospecific hydrolysis of racemic CNDE using a newly isolated Morgarella morganii strain ZJB-09203, to provide (S)-CCMA in 45.3% yield and 95% ee (1.5 M substrate loading) with wild-strain resting cells [21]. Li et al described the kinetic resolution of CNDE catalyzed by Escherichia coli overexpressing an S88T/A99N/V116D mutant, to afford (S)-CCMA in 42.4% yield and 98% ee with a substrate loading of 1 M [22].…”

Bioremediation potential of microorganisms derived from petroleum reservoirs

“…Metagenomic libraries are a valuable tool often used in studies for the characterization of poorly understood environments, such as deep oceans (Quaiser et al, 2011;Dick et al, 2013), deserts (Neveu et al, 2011, forests (Faoro et al, 2011Biver and Vandenbol, 2013), and petroleum reservoirs . The application of genetically modified organisms (GMOs) in contaminated environments has been described previously (Sayler et al, 1999;Ripp et al, 2000).…”