Degradation of Differently Substituted Xylogalacturonans by Endoxylogalacturonan Hydrolse and Endopolygalacturonases

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Abstract
A method was developed to make xylogalacturonans (XGAs) with different degrees of xyloslyation from gum tragacanth (XGA-25, XGA-29, XGA-35 and XGA-47), using alkali treatment at 4°C and acid treatment at 100°C. Ester linkages as well as fucose and arabinose substituents could selectively be removed by this procedure. Galactosyl- and xylosyl-linkages appeared to be more stable, while some backbone degradation of the galacturonan took place upon prolonged acid treatment. Using XGA-35, endoxylogalacturonan hydrolase (XGH) from Aspergillus tubingensis, expressed in Kluyveromyces lactis, was characterised with respect to kinetic parameters, temperature and pH effects. XGA-25 and XGA-47 were degraded with endopolygalacturonases (PGs) from Aspergillus niger (PG1, PG2), from A. tubingensis (PF-arf), from Kluyveromyces fragilis (PG-kluyv) and XGH from A. tubingensis. The activity of the different PGs decreased with increasing degrees of xylosylation. However, for each PG a different tolerance for the presence of side chains was observed. PG-arf and PG1 were hindered most by xylosyl branching, whereas XGH appeared to have a requirement for xylosylation and was almost not active towards polygalacturonic acid. The degradability of xylogalacturonans by XGH increased with higher degrees of xylosylation. Typically, a highly substituted xylogalacturonan from pea was almost resistant to XGH treatment. XGH produces a distinctive set of oligosaccharides from XGA, which is different from the hydrolysis products of PG action. Saponified modified hairy regions from apple (MHR-S) containing xylogalacturonan, were partially degraded by XGH. A combination of XGH and rhamnogalacturonan hydrolase was able to fully degrade the high molecular weight fraction of MHR-S. The two enzymes acted additively, no synergy being observed.