Purification and cloning of a rhamnogalacturonase tolerant to an acetylated rhamnogalacturonan

J. Normand; P. Delavault; J.-F. Thibault; E. Bonnin

Purification and cloning of a rhamnogalacturonase tolerant to an acetylated rhamnogalacturonan

In: Pectins and pectinases

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J. Normand

J. Normand ¹Unité de Recherche Biopolymères, Interactions, Assemblages, INRA Nantes, BP 71627, 44316 Nantes Cedex 03, France

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P. Delavault

P. Delavault ²Laboratoire de Biologie et Pathologie Végétale, Université de Nantes, 44322 Nantes, France

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J.-F. Thibault

J.-F. Thibault ¹Unité de Recherche Biopolymères, Interactions, Assemblages, INRA Nantes, BP 71627, 44316 Nantes Cedex 03, France

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, and

E. Bonnin

E. Bonnin ²Laboratoire de Biologie et Pathologie Végétale, Université de Nantes, 44322 Nantes, France

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Type:: Chapter

Pages:: 115–125

DOI:: https://doi.org/10.3920/9789086866779_009

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A rhamnogalacturonase acting on acetylated substrate was detected in the commercial preparation Driselase^®, 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 fulllength 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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Pectins and pectinases

E-Book ISBN:: 9789086866779

Publisher:: Wageningen Academic

Print Publication Date:: 30 Oct 2009

Subjects
- Life Sciences
  - General
  - Food & Health

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Purification and cloning of a rhamnogalacturonase tolerant to an acetylated rhamnogalacturonan

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Pectins and pectinases

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