Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan.

Two exo-polygalacturonases (EC 3.2.1.67) were purified from a commercial Aspergillus niger enzyme preparation by ammonium sulfate precipitation, preparative electrofocusing, anion-exchange and size-exclusion chromatographies. The enzymes had molar masses of 82 kDa (exo-PG1) and 56 kDa (exo-PG2). Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Addition of 0.01 mM HgCl(2) increased the exo-PG2 activity 3.4 times but did not affect exo-PG1. Analysis of the reaction products of (reduced) pentagalacturonate by high-performance anion-exchange chromatography revealed that both enzymes split the substrate from the non-reducing end in a multi-chain attack mode. Exo-PG1 had a broad specificity towards oligogalacturonates with different degrees of polymerisation, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid and Xyl-GalA disaccharide, as identified by electrospray ionization-ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing galacturonic acid and acetylated galacturonic acid, as shown by ESI-ITMS.

Assessment of methanolysis for the determination of composite sugars of gelling carrageenans and agarose by HPLC.

The characterization of the main composite sugars of commercial gelling red algae galactans (agarose, iota and kappa carrageenans) by methanolysis and separation of the methyl glycosides produced by high performance liquid chromatography is described. The methanolysis (methanolic hydrochloric acid strength, temperature, and reaction time) was optimized in order to release monosaccharides in near quantitative yield. The results were compared to those obtained by (1) gas chromatography of the alditol acetates of the neutral sugars released by acid hydrolysis and (2) specific colorimetric determination of the acid-labile 3,6-anhydrogalactosyl residue. Conditions such as methanolic 0.125 M HCl for 1 h at 85 degrees C were sufficient to release all of the galactosidic and 3,6-anhydrogalactosidic bonds for iota carrageenan without apparent degradation of the anhydrogalactosyl unit. However, with the same conditions, the yields of 3,6-anhydrogalactosyl residues were 80 and 70% for kappa carrageenan and agarose, respectively. These yields were not improved by stronger conditions. At the opposite extreme, under very mild methanolysis conditions such as methanolic 0.01 M HCl at 100 degrees C for 1 h, agars and gelling carrageenans were well differentiated by the respective determination of agarobiose- and carrabiose-dimethyl acetal which are well-separated on octadecyl reversed phase HPLC columns with water as eluent.

Integration of inulin determination in the AOAC method for measurement of total dietary fibre.

A suitable modification of the standard AOAC method for the measurement of dietary fibre is proposed to quantitatively include beta-fructans in the determination of the soluble dietary fibre fraction and as a consequence in the related total dietary fibre fraction. The standard AOAC method is modified by including a preheated commercial inulinase, Novozym SP 230, to the amyloglucosidase incubation step. It was previously outlined that this commercial inulinase contains some pectolytic activity. It is now demonstrated that a heat pretreatment of this enzyme preparation at 60 degrees C for 2 h eliminates this pectolytic activity while keeping sufficient activity to hydrolyse all the inulin from the soluble fibre fraction.

Variability of cell wall polysaccharides composition and hemicellulose enzymatic profile in an apple progeny.

The genetic variability of apple cell walls polysaccharides chemical composition and structure was assessed in a progeny of 141 individuals harvested over 2 years. The variability of the hemicelluloses oligosaccharides released by glucanase was analyzed by MALDI-TOF MS. The genetic contribution was distinguished from harvest year as well as from parental crossing patterns and scab resistance selection. Results showed that harvest year had a major impact on cell wall polysaccharide composition and structure. Within each harvest, genetic effect impact more significantly cell wall polysaccharide chemistry than does reciprocal crossing or early scab selection. Uronic acids, glucose, galactose and xylose contents as well as some glucomannan and xyloglucan structures have a high heritability. This first cell wall chemotyping of an apple progeny opens the way for future searches of genetic markers for the chemical variability of cell wall polysaccharides.