title: A novel platelet-activating factor acetylhydrolase discovered in a metagenome from the earthworm-associated microbial community.
abstract: Metagenomics is an emerging field for mining the bioresources for new biomolecules for potential application in biotechnology and biomedicine. In the present study, a novel acetylhydrolase (Est13) was detected during the function-based screening of a metagenomic library established from the DNA extracted from the cellulose-depleting microbial community set up with an earthworm cast. Analysis showed that Est13 exhibited some similarities with a human and parasite platelet-activating factor acetylhydrolase (PAF-AH) belonging to the SGNH hydrolase superfamily. Biochemical characterization of the purified recombinant enzyme using substrates common for hydrolases of this superfamily demonstrated that Est13 hydrolysed p-nitrophenyl acetate quite efficiently, with a k(cat) /K(M) value of 3209 mM(-1) s(-1). The Est13 showed highest activity at pH 8.0 and 40°C, conditions in which it is relatively stable compared with known PAF-AHs. In vitro functional analysis of the platelet-activating factor hydrolysis showed a dose- and time-dependent inhibition of platelet aggregation in the range of 2-4 µM, making this enzyme a potential candidate for biomedical applications.
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E-30100 Murcia, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Navarro-Fernández
authors.forename: Taras Y
authors.initials: TY
authors.lastname: Nechitaylo
authors.forename: José A
authors.initials: JA
authors.lastname: Guerrero
authors.forename: Olga V
authors.initials: OV
authors.lastname: Golyshina
authors.forename: Francisco
authors.initials: F
authors.lastname: García-Carmona
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sánchez-Ferrer
authors.forename: Peter N
authors.initials: PN
authors.lastname: Golyshin
mesh_terms: D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015698:Genomic Library; D006801:Humans; D006868:Hydrolysis; D054892:Metagenome; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000382:microbiology*; D010974:Platelet Aggregation; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase