Abstract
A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42kDa. 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 0.17mM and 1910s-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Structural 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 revealed 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.
Highlights
Soil is “natural medium” for microorganisms, and 1 g of soil usually contains 106-109 microorganisms, which is the most abundant resource of microbial and provides a valuable reservoir for mining novel genes and biocatalysts (Bachar et al 2010)
Both carboxylesterases and lipases can catalyze the hydrolysis of p-nitrophenyl esters, while the former tend to hydrolyze short to medium chain and water-soluble fatty-acid esters, the latter show specificity toward long-chain and low water solubility ones
A fosmid metagenomic library was constructed with environmental DNA extracted from an agricultural soil sample
Summary
Soil is “natural medium” for microorganisms, and 1 g of soil usually contains 106-109 microorganisms, which is the most abundant resource of microbial and provides a valuable reservoir for mining novel genes and biocatalysts (Bachar et al 2010). Lipolytic enzymes (EC 3.1.1.x) belong to the α/β hydrolase family, which are ubiquitous in animals, plants and microorganisms (Jaeger and Eggert 2002) Based on their substrate preferences and structural characteristics, lipolytic enzymes were classified into carboxylesterases (EC 3.1.1.1), lipases (EC 3.1.1.3) and various Phospholipases (Arpigny and Jaeger 1999). Both carboxylesterases and lipases can catalyze the hydrolysis of p-nitrophenyl esters (pNPEs), while the former tend to hydrolyze short to medium chain and water-soluble fatty-acid esters, the latter show specificity toward long-chain and low water solubility ones. Merely a minority of isolated enzymes were successfully applied in industry (Ferrer et al 2016), researchers are still keen to screen novel lipolytic enzymes with improved properties from various environmental samples to meet the up-growing demands
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