Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library

Abstract

A novel lipolytic gene, estq7, was identi ed from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910 S -1 , respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Homology modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking reveled the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property, and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

Molecular Cloning and Characterization of a New Family VI Esterase from an Activated Sludge Metagenome

“…Due to the development of metagenomic libraries from various complex samples such as soils [12][13][14][15][16], compost [17][18][19], activated sludge [20][21][22][23], and sediments [24][25][26], many novel lipolytic enzymes have been discovered. Tremendous genetic resources from a number of unculturable microorganisms might be utilized more effectively and successfully when the metagenomic approach is used in the discovery of novel genes.…”