title: Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes.
abstract: A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40 °C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.
authors.affiliation: Departamento de Bioquímica y Genómica Microbianas, Instituto de Investigaciones Biológicas "Clemente Estable", Montevideo, Uruguay.
authors.forename: María Cecilia
authors.initials: MC
authors.lastname: Rodríguez
authors.affiliation: Departamento de Bioquímica y Genómica Microbianas, Instituto de Investigaciones Biológicas "Clemente Estable", Montevideo, Uruguay.
authors.forename: Inés
authors.initials: I
authors.lastname: Loaces
authors.affiliation: Departamento de Bioquímica y Genómica Microbianas, Instituto de Investigaciones Biológicas "Clemente Estable", Montevideo, Uruguay.
authors.forename: Vanesa
authors.initials: V
authors.lastname: Amarelle
authors.affiliation: Laboratorio de Ecología Microbiana, Instituto de Investigaciones Biológicas "Clemente Estable", Montevideo, Uruguay.
authors.forename: Daniella
authors.initials: D
authors.lastname: Senatore
authors.affiliation: Departamento de Genómica, Instituto de Investigaciones Biológicas "Clemente Estable", Montevideo, Uruguay.
authors.forename: Andrés
authors.initials: A
authors.lastname: Iriarte
authors.affiliation: Departamento de Bioquímica y Genómica Microbianas, Instituto de Investigaciones Biológicas "Clemente Estable", Montevideo, Uruguay.
authors.forename: Elena
authors.initials: E
authors.lastname: Fabiano
authors.affiliation: Departamento de Bioquímica y Genómica Microbianas, Instituto de Investigaciones Biológicas "Clemente Estable", Montevideo, Uruguay.
authors.forename: Francisco
authors.initials: F
authors.lastname: Noya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D004950:Esterases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D056186:Metagenomics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012417:Rumen / Q000382:microbiology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004950:Esterases