ISOLATION AND STRUCTURAL CHARACTERIZATION OF ENDO-RHAMNOGALACTURONASE-GENERATED FRAGMENTS OF THE BACKBONE OF RHAMNOGALACTURONAN-I

A combination of commercially available preparations of Aspergillus niger beta-D-galactosidase, endo-alpha-L-arabinanase, alpha-L-arabinosidase, and endo-beta-D-galactanase has been used to generate oligoglycosyl fragments of the backbone of rhamnogalacturonan I (RG-I) that had been isolated from the walls of suspension-cultured sycamore cells. The backbone-cleaving enzyme, which is present in the beta-D-galactosidase preparation, only fragments the RG-I backbone when many of the neutral oligoglycosyl side chains have been removed by the other exo- and endo-glycanases. The oligosaccharides released from the backbone were separated from the partially fragmented RG-I and then purified, as their oligoglycosyl aldonic acids, by HPAEC-PAD. Those backbone fragments with degrees of polymerization (dp's) between 2 and 11 were characterized using one- and two-dimensional H-1 NMR spectroscopy, electrospray mass spectrometry, and glycosyl-residue and glycosyl-linkage composition analyses. Two series of oligoglycosyl fragments were identified. The quantitatively predominant series has the structure alpha-D-GalpA-(1-->2)-alpha-L-Rhap-[-->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->2](n)-4-D-GalpA, and the quantitatively minor series has the structure alpha-L-Rhap-[ -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->](n)-4-D-GalpA ( n = 1-5). Thus, the enzyme preparations contain an alpha-L-rhamnosidase in addition to the endo-rhamnogalacturonase. The products of the endo-rhamnogalacturonase provide additional evidence that the backbone of RG-I is composed of the diglycosyl repeating unit: --> 4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->. The endo-rhamnogalac-turonase from the A. niger beta-D-galactosidase preparation and the endo-rhamnogalacturonase secreted by Aspergillus aculeatus [H.A. Schols et al. Carbohydr. Res., 206 (1990) 117-129] have the same substrate specificities and generate similar oligoglycosyl fragments.