Abstract
Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.
Highlights
The global trade of industrial enzymes has been estimated to be 2.3 billion dollars [1]
All clones were screened for lipolytic activity based on the hydrolytic activity of emulsified tributyrin (1%), resulting in 30 positive lipolytic clones that showed a clear zone around the colonies, which corresponded to approximately one lipolytic gene per 4.9 Mb DNA
The residues involved in the substrate-pocket and family classification are shown in bold and the catalytic triad residues are denoted with asterisks (*)
Summary
The global trade of industrial enzymes has been estimated to be 2.3 billion dollars [1]. The enzyme market has been estimated to be € 3.4 billion with an annual growth of 6.5 to 10% [2] Within this market, lipolytic enzymes have attracted enormous attention due to their wide biotechnological applications. Lipolytic enzymes have attracted enormous attention due to their wide biotechnological applications They are members of the broad family of proteins containing an alpha/beta hydrolase fold and can be classified according to their substrate preferences as lipases (EC 3.1.1.3) that hydrolyse water-insoluble long-chain acylglycerols (C > 10) or esterases (EC 3.1.1.1) that hydrolyse water-soluble short-chain acylglycerols (C 10). Lipolytic enzymes are involved in catalyzing esters hydrolysis, ester synthesis, transesterification and other reactions. Esterases can show high regio- and stereospecificity, can hydrolyse a wide range of substrates, do not require the presence of cofactors, and can be stable and active in organic solvents [7,8]
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