Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation

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.

Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications

“…Additionally, we evidenced the highly conserved HGGG motif in the EST3 sequence upstream of the serine motif; according to some authors, this motif is involved in hydrogen-bonding interactions that stabilize the oxyanion hole and plays a role in catalysis (Hårdeman and Sjöling 2007;Bunterngsook et al 2010). Interestingly, Est 16, a previously isolated esterase from the same library (Pereira et al 2015) that shares 30 % of identity with EST3, was clustered with bacterial lipolytic family V, evidencing the diversity of enzymes that can be isolated from similar sources.…”

“…Metagenomic libraries from contaminated soils have proven to be great sources of many lipolytic enzymes (Elend et al 2006;Glogauer et al 2011;Garcia et al 2015; Kim et al The enzyme assays were performed on 50-mM phosphate buffer, pH 6.0, at 41°C. The small letters on the top of the values (a, b, c, d) indicates the significant difference between each condition performed in the experiment, according to ANOVA and Tukey's test at 5 % probability Pereira et al 2015). In the present work, we characterized the esterase EST3 isolated from a metagenomic library derived from soil contaminated with petroleum hydrocarbons, which belongs to family IV of the lipolytic enzymes.…”

“…Recently, we have performed a screening for clones exhibiting lipolytic activity on agar containing tributyrin as the indicator substrate, which resulted in the identification of 30 positive clones. From one of the positive clones, a new esterase member of family V of bacterial lipolytic enzymes, designated as Est16, was cloned, overexpressed and characterized (Pereira et al 2015).…”

Descrição do resistoma de solo de Cerrado stricto sensu e o potencial de dioxigenases metagenômicas na resistência antimicrobiana e em processos de biorremediação

“…Todavia, a metagenômica funcional ainda é uma técnica robusta para identificar novos biocatalisadores de interesse provenientes de diferentes amostras ambientais. Exemplos recentes incluem enzimas com atividade amilolítica, celulolítica e lipolítica, com diversas aplicações industriais (Xu et al, 2014;Alnoch et al, 2015;Kanokratana et al, 2015;Kim et al, 2015;Pereira et al, 2015;Prive et al, 2015;Su et al, 2015;Vester et al, 2015), e também aquelas com potencial na utilização em processos de biorremediação (Lee et al, 2008;Nagayama et al, 2015). Esta abordagem é também uma ferramenta interessante na bioprospecção de antimicrobianos (O'mahony et al, 2015;Takeshige et al, 2015).…”

Expressão e caracterização estrutural e funcional de sequências genômicas codificadoras de enzimas lipolíticas.

“…O solo contaminado foi coletado de uma antiga fábrica de lubrificantes localizada na cidade de Ribeirão Preto -SP (ACCORSINI et al, 2012) e foi utilizado para o desenvolvimento de um consórcio microbiano, em meio enriquecido com óleo diesel, (PAIXÃO et al, 2010) e posterior construção da biblioteca. Em trabalho realizado anteriormente (PEREIRA et al, 2015) foram prospectadas enzimas lipolíticas nos 4224 clones da biblioteca, sendo identificados 30 clones positivos.…”