title: Characterization of a new and thermostable esterase from a metagenomic library.
abstract: A new gene encoding an esterase (designated as EstEP16) was identified from a metagenomic library prepared from a sediment sample collected from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 249 amino acid residues. It was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified to homogeneity. The monomeric EstEP16 presented a molecular mass of 51.7 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. When p-nitrophenyl butyrate was used as a substrate, recombinant EstEP16 exhibited highest activity at pH 8.0 and 60°C. The recombinant enzyme retained about 80% residual activity after incubation at 90°C for 6 h, which indicated that EstEP16 was thermostable. Homology modeling of EstEP16 was developed with the monoacylglycerol lipase from Bacillus sp. H-257 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of a typical catalytic triad. The activity of EstEP16 was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism.
authors.affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, People's Republic of China.
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Zhu
authors.forename: Jianbo
authors.initials: J
authors.lastname: Li
authors.forename: Huinong
authors.initials: H
authors.lastname: Cai
authors.forename: Hui
authors.initials: H
authors.lastname: Ni
authors.forename: Anfeng
authors.initials: A
authors.lastname: Xiao
authors.forename: Luhong
authors.initials: L
authors.lastname: Hou
mesh_terms: D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D019015:Geologic Sediments; D006863:Hydrogen-Ion Concentration; D060086:Hydrothermal Vents; D054892:Metagenome; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D004950:Esterases
keywords: Characterization; Esterase; Metagenome