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Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas
Berlemont, Renaud; Spee, Olivier; Delsaute, Maud; Lara, Yannick; Schuldes, Jörg; Simon, Carola; Power, Pablo; Daniel, Rolf; Galleni, Moreno.
  • Berlemont, Renaud; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Spee, Olivier; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Delsaute, Maud; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Lara, Yannick; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Schuldes, Jörg; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Simon, Carola; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Power, Pablo; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Daniel, Rolf; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
  • Galleni, Moreno; University of Liège. Centre for Protein Engineering. Laboratory of Biological Macromolecules.
Rev. argent. microbiol ; 45(1): 3-12, mar. 2013. graf, tab
Article in English | LILACS | ID: lil-672048
ABSTRACT
In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.
RESUMEN
Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.
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Full text: Available Index: LILACS (Americas) Main subject: Esterases / Metagenomics / Lipase Country/Region as subject: Europa Language: English Journal: Rev. argent. microbiol Journal subject: Microbiology Year: 2013 Type: Article Affiliation country: Belgium

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Full text: Available Index: LILACS (Americas) Main subject: Esterases / Metagenomics / Lipase Country/Region as subject: Europa Language: English Journal: Rev. argent. microbiol Journal subject: Microbiology Year: 2013 Type: Article Affiliation country: Belgium