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

Concetta De Santi,Bjørn Altermark,Luca Ambrosino,Nils-Peder Willassen,Marcin Miroslaw Pierechod,Donatella De Pascale

doi:10.1186/s12858-016-0057-x

Abstract

BackgroundThe 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.ResultsHere 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 from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-016-0057-x) contains supplementary material, which is available to authorized users.

Highlights

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
These enzyme families are all characterized by a catalytic triad consisting of a nucleophilic serine, a catalytic acid and a histidine residue which is located in a conserved GlyXaa-Ser-Xaa-Gly pentapeptide that forms a sharp elbow in the center of the α/β-fold [14]
Construction of metagenomic libraries and screening for lipolytic enzymes Three small fosmid libraries were created from marine sediment samples

Summary

Introduction

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. Lipolytic enzymes can be grouped into 8 different families based on their sequence, structure and biological functions [13]. These enzyme families are all characterized by a catalytic triad consisting of a nucleophilic serine, a catalytic acid (aspartate or glutamate) and a histidine residue which is located in a conserved GlyXaa-Ser-Xaa-Gly pentapeptide that forms a sharp elbow in the center of the α/β-fold [14]. Lipases can be distinguished from esterases by exhibiting the interfacial activation [15] Both enzymes have a secondary structural elements, called lids, that change conformation to accommodate the substrates [16, 17]. There are exceptions such as the previously characterized Candida antarctica Lip B [18]

Methods

Results

Discussion

Conclusion