title: Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.
abstract: 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 α/β 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.
authors.affiliation: Centre for Protein Engineering, University of Liège, Sart-Tilman, Belgium.
authors.forename: Renaud
authors.initials: R
authors.lastname: Berlemont
authors.forename: Olivier
authors.initials: O
authors.lastname: Spee
authors.forename: Maud
authors.initials: M
authors.lastname: Delsaute
authors.forename: Yannick
authors.initials: Y
authors.lastname: Lara
authors.forename: Jörg
authors.initials: J
authors.lastname: Schuldes
authors.forename: Carola
authors.initials: C
authors.lastname: Simon
authors.forename: Pablo
authors.initials: P
authors.lastname: Power
authors.forename: Rolf
authors.initials: R
authors.lastname: Daniel
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
mesh_terms: D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry; D002087:Butyrates / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004950:Esterases / Q000145:classification / Q000302:isolation & purification*; D005858:Germany; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008049:Lipase / Q000145:classification / Q000302:isolation & purification*; D008066:Lipolysis; D056186:Metagenomics; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014197:Trees; D014280:Triglycerides / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002087:Butyrates; D011994:Recombinant Proteins; D012492:Salts; D012997:Solvents; D014280:Triglycerides; D004247:DNA; D004950:Esterases; D008049:Lipase