RESUMO
The aminopeptidase gene from Mesorhizobium SEMIA3007 was cloned and overexpressed in Escherichia coli. The enzyme called MesoAmp exhibited optimum activity at pH 8.5 and 45 °C and was strongly activated by Co2+ and Mn2+. Under these reaction conditions, the enzyme displayed Km and kcat values of 0.2364 ± 0.018 mM and 712.1 ± 88.12 s-1, respectively. Additionally, the enzyme showed remarkable stability in organic solvents and was active at high concentrations of NaCl, suggesting that the enzyme might be suitable for use in biotechnology. MesoAmp is responsible for 40% of the organism's aminopeptidase activity. However, the enzyme's absence does not affect bacterial growth in synthetic broth, although it interfered with biofilm synthesis and osmoregulation. To the best of our knowledge, this report describes the first detailed characterization of aminopeptidase from Mesorhizobium and suggests its importance in biofilm formation and osmotic stress tolerance. In summary, this work lays the foundation for potential biotechnological applications and/or the development of environmentally friendly technologies and describes the first solvent- and halo-tolerant aminopeptidases identified from the Mesorhizobium genus and its importance in bacterial metabolism.
Assuntos
Aminopeptidases/química , Aminopeptidases/farmacologia , Biofilmes/efeitos dos fármacos , Mesorhizobium/metabolismo , Sequência de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Ativação Enzimática , Evolução Molecular , Genoma Bacteriano , Genômica/métodos , Multimerização Proteica , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência de DNA , SoluçõesRESUMO
Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of α/ß hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-Ǻ resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met213 and Phe217) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues.
Assuntos
Esterases/química , Esterases/metabolismo , Metagenômica , Consórcios Microbianos/genética , Butiratos/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Estabilidade Enzimática , Esterases/genética , Esterases/isolamento & purificação , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/genética , Lipase/isolamento & purificação , Lipase/metabolismo , Lipólise , Nitrofenóis/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity against p-nitrophenyl esters of different chain lengths and highest catalytic efficiency against p-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn(+2), Li(+), EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Esterases/química , Esterases/genética , Sequência de Aminoácidos , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Esterases/metabolismo , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Metagenoma , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
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.