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Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate

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Abstract

A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site.

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Acknowledgments

We gratefully thank Audrey Geairon and David Ropartz from the ‘‘Biopolymers-Interaction-Structural Biology” platform located at the INRA Center of Nantes (http://www.nantes.inra.fr/plateformes_et_plateaux_techniques/plateforme_bibs) for the mass spectrometry analyses. Sylviane Daniel is acknowledged for her technical assistance.

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Authors and Affiliations

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

    Jessica Normand, Marie-Christine Ralet, Jean-François Thibault, Hélène Rogniaux & Estelle Bonnin

  2. Laboratoire de Biologie et Pathologie Végétales, Université de Nantes, Nantes-Atlantique Universités, EA 1157, UFR Sciences et Techniques, 2 rue de la Houssinière, 44322, Nantes, Cedex 3, France

    Jessica Normand & Philippe Delavault

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  1. Jessica Normand
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  2. Marie-Christine Ralet
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  3. Jean-François Thibault
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  4. Hélène Rogniaux
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  5. Philippe Delavault
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  6. Estelle Bonnin
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Correspondence to Estelle Bonnin.

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Fig. S1

Full MS spectrum of the final digest of acetylated RG degraded with the RG-hydrolase (PPT 130 kb)

Table S1

Retention time and response factor of standard oligomers (DOC 33 kb)

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Normand, J., Ralet, MC., Thibault, JF. et al. Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate. Appl Microbiol Biotechnol 86, 577–588 (2010). https://doi.org/10.1007/s00253-009-2310-3

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  • DOI: https://doi.org/10.1007/s00253-009-2310-3

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