Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate.
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 degrees C and 50 degrees 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.
Normand
Jessica
J
INRA, Unité de Recherche Biopolymères, Interactions, Assemblages, BP 71627, 44316, Nantes, Cedex 03, France.
Ralet
Marie-Christine
MC
Thibault
Jean-François
JF
Rogniaux
Hélène
H
Delavault
Philippe
P
Bonnin
Estelle
E
D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D013058:Mass Spectrometry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D010368:Pectins / Q000378:metabolism; D020072:Polyporales / Q000201:enzymology*; D013696:Temperature
D005656:Fungal Proteins; D009844:Oligosaccharides; C473998:rhamnogalacturonan J1; D010368:Pectins; D006026:Glycoside Hydrolases; C090358:rhamnogalacturonan rhamnohydrolase