Identification and characterization of some Aspergillus pectinolytic glycoside hydrolases

Keywords: Aspergillusniger , Arabidopsis thaliana , homogalacturonan, rhamnogalacturonan, xylogalacturonan, xylogalacturonan hydrolase, exo-polygalacturonasePectinases are used for many food applications, in particular for the manufacture of fruit juices. However, the array of pectin modifying enzymes as available today is insufficient to completely degrade pectic polysaccharides from plants, which consequently can cause problems in food processing. As the genome sequence of Aspergillusniger indicated the presence of more pectin modifying enzymes than previously known, research was carried out to identify, produce, and characterize novel pectinases from this species.From the complete inventory of the pectinolytic glycoside hydrolase family 28 of A.niger a new gene group of seven exo-acting enzymes was found. Three of these enzymes (PGXA, PGXB, PGXC) were biochemically identified from which it was demonstrated that PGXB and PGXC act as an exo-polygalacturonase while PGXA rather acts like an exo-xylogalacturonan hydrolase.The xylogalacturonan hydrolase (XGH) was thoroughly investigated for its action towards a xylogalacturonan (XGA) derived from gum tragacanth by isolation and characterization of the produced oligosaccharides. Also XGH activity towards XGA in the saponified modified 'hairy' regions (MHR-s) of pectin from apples and potatoes was investigated. The enzyme predominantly released the di-saccharide GalAXyl from these substrates which illustrates the preference of XGH to act between two xylosylated GalA residues. However this enzyme was also able to release low substituted XGA oligosaccharides as well as linear GalA oligosaccharides, which shows its tolerance for unsubstituted GalA residues in its active site.By using XGH as analytical tool, the presence of XGA could also be demonstrated in the stem and the leaves of Arabidopsis thaliana , which shows that the presence of this polymer is not strictly confined to storage tissues or reproductive organs of plants as was previously thought to be the case

Xylogalacturonan exists in cell walls from various tissues of Arabidopsis thaliana

Evidence is presented for the presence of xylogalacturonan (XGA) in Arabidopsis thaliana. This evidence was obtained by extraction of pectin from the seeds, root, stem, young leaves and mature leaves of A. thaliana, followed by treatment of these pectin extracts with xylogalacturonan hydrolase (XGH). Upon enzymatic treatment, XGA oligosaccharides were primarily produced from pectin extracts obtained from the young and mature leaves and to a lesser extent from those originating from the stem of A. thaliana. The oligosaccharide GalA3Xyl was predominantly formed from these pectin extracts. No XGA oligosaccharides were detected in digests of pectin extracts from the seeds and roots. A low number of XGA oligosaccharides was obtained from pectins of A. thaliana. This indicates a uniform distribution of xylose in XGA from A. thaliana. The predominant production of GalA3Xyl, as well as the release of linear GalA oligosaccharides pointed to a lower degree of xylose substitution in XGA from A. thaliana than in XGA from apple and potato. The estimated amount of XGA accounted for approximately 2.5%, 7% and 6% (w/w) of the total carbohydrate in the pectin fraction of the stem, young leaves and mature leaves, respectively