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Chapitre D'ouvrage Année : 2009

Purification and cloning of a rhamnogalacturonase tolerant to an acetylated rhamnogalacturonan

Résumé

A rhamnogalacturonase acting on acetylated substrate was detected in the commercial preparation Driselase (R), an enzymatic mixture derived from the basidiomycete Irpex lacteus. The enzyme was purified by hydrophobic interaction chromatography, gel filtration and preparative isoelectric focusing, resulting in the isolation of different rhamnogalacturonan hydrolases having various isoelectric points. SDS-PAGE analysis of the fractions revealed a similar molar mass around 55 kDa and mass spectrometry analysis after trypsin cleavage did not permit to find difference in the aminoacid sequence of the different isoforms. In order to produce a recombinant rhamnogalacturonase using a molecular approach to produce high amount of protein, PCR primers were designed based on a sequence alignment of six known rhamnogalacturonases from ascomycetes. Using 5' and 3' RACE experiments, a full-length cDNA was isolated and the corresponding putative aminoacid sequence shared significant identities not only with ascomycete rhamnogalacturonases but also with the purified rhamnogalacturonase from Irpex lacteus.
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Dates et versions

hal-02822966 , version 1 (06-06-2020)

Identifiants

  • HAL Id : hal-02822966 , version 1
  • PRODINRA : 31079
  • WOS : 000321697600009

Citer

Jessica Normand, Philippe Delavault, Jean-François Thibault, Estelle Bonnin. Purification and cloning of a rhamnogalacturonase tolerant to an acetylated rhamnogalacturonan. Pectins and pectinases, Wageningen Academic Publishers, 2009, 978-90-8686-108-8. ⟨hal-02822966⟩
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