Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries

摘要

Background: The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomesof unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range ofbiotechnological and pharmaceutical fields.Results: Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomiclibraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on thebasis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme.The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conservedpentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of thesubstrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the externalsurface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared withhomologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified fromEscherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate beingp-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0.Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanoland high concentrations of salt. The enzyme was also highly activated by NaCl.Conclusions: The biochemical characterization and homology model reveals a novel esterase originating from themarine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerantenzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate forbiotechnological applications such as organic synthesis reactions and cheese ripening processes