Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: De Santi, Concetta; Altermark, Bjørn; Pierechod, Marcin; Ambrosino, Luca; de Pascale, Donatella; Willassen, Nils-Peder
• Is Part Of: BMC biochemistry. Volume 17:Issue 1(2016)
• Page Start: 1
• Page End: 13
• Publication Date: 2016-12
• Subjects: Metagenomics libraries -- Cold-active esterase -- Salt -- Homology modeling -- Biotechnological applications; Biochemistry -- Periodicals; 572.05
• DOI: 10.1186/s12858-016-0057-x
• Languages: English
• ISSNs: 1471-2091

Abstract

Abstract Background The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields. Results Here we present a novel esterase gene (lip3 ) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis 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 conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified fromEscherichia coli, preferentially hydrolyzed short and medium lengthp -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-mercaptoethanol and 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 the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.