title: Characterization of a novel thermostable patatin-like protein from a Guaymas basin metagenomic library.
abstract: Deep-sea hydrothermal vents are a natural habitat for thermophiles, in which contain plenty of enzymes that can function at high temperatures. In this work, we constructed a fosmid library in Escherichia coli using metagenomic DNA isolated from a chimney sample collected in the hydrothermal vents in Guaymas Basin. The library was screened for lipolytic activity and positive clones were subjected to subcloning. A novel patatin-like protein (PLP) that exhibited less than 45 % identity in amino acid sequence to known enzymes was obtained. Common features of the patatin-like proteins, such as four conserved blocks, were detected. Interestingly, there was an Ala at site 42 in PLP instead of the first Gly-residue in the consensus sequence Gly-X-Ser-X-Gly found in other PLP homologs. The active sites of PLP were Ser44 and Asp160. Spectrophotometric assays with different p-nitrophenyl esters demonstrated a preference for p-nitrophenyl butyrate (C4) and p-nitrophenyl decanoate (C10). Moreover, PLP demonstrated optimal activity at 70 °C and at pH 9.0 (Tris-HCl). The activation energy from the linear Arrhenius plot was found to be 38.3 ± 0.9 kJ/mol. The K m and V max of PLP for C4 were 304 ± 38 μM and 14 ± 0.38 μmol min(-1) mg(-1), respectively. Gene-mining of the metagenome dataset that was generated by pyrosequencing the same chimney sample resulted in identification of 20 PLP homolog gene fragments, which could represent promising examples of this category of thermostable proteins.
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Dongchuan Road 800, 200240, Shanghai, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Fu
authors.forename: Ying
authors.initials: Y
authors.lastname: He
authors.forename: Fangdi
authors.initials: F
authors.lastname: Xu
authors.forename: Qun
authors.initials: Q
authors.lastname: Ma
authors.forename: Fengping
authors.initials: F
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
mesh_terms: D020816:Amino Acid Motifs; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D030562:Databases, Protein; D015723:Gene Library; D060086:Hydrothermal Vents / Q000382:microbiology*; D054892:Metagenome
chemical_list: D002265:Carboxylic Ester Hydrolases