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.
Unité de Recherche sur les Polysaccharides, leurs Organisations et Interactions, INRA, Nantes, France. sakamoto@biochem.osakafu-u.ac.jp
T
T
Sakamoto
E
E
Bonnin
B
B
Quemener
J-F
JF
Thibault
D001234:Aspergillus niger / Q000201:enzymology*; D027461:Beta vulgaris; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006603:Hexuronic Acids / Q000032:analysis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000378:metabolism; D018532:Pisum sativum; D010368:Pectins / Q000378:metabolism*; D011096:Polygalacturonase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis; D013379:Substrate Specificity; D013696:Temperature
D006603:Hexuronic Acids; D009844:Oligosaccharides; D011134:Polysaccharides; C096230:pentagalacturonic acid; C465879:xylogalacturonan; C007819:galacturonic acid; D010368:Pectins; D011096:Polygalacturonase; C003181:polygalacturonic acid