Purification and characterisation of polygalacturonases from a commercial Aspergillus niger preparation.
The polygalacturonase (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) activity of Pectinol is resolved into two fractions (E1 and E2) of about equal total activity on DEAE-cellulose. These fractions are purified from other pectinolytic enzyme activity by Sephadex G-75 chromatography. Both E1 and E2 reduce the viscosity of polygalacturonate by 50% after 7% of the glycosidic bonds are hydrolysed. Their activities are not affected by iodoacetate (1 mM) or EDTA (10 mM). E1 and E2 have different molecular weights (35 000 and 85 000, respectively) and different electrophoretic mobilities on sodium dodecyl sulphate polyacrylamide gels. Their pH (4.1 and 3.8 respectively) and ionic strength optima and specific activities also differ. Both enzymes are inhibited at similar rates by diethyl pyrocarbonate at pH 6 but only E2 is protected from this inhibition by 2% (w/v) polygalacturonate. The rate of change of protein absorbance at 250 nm accompanying this inhibition, and the residues are essential for the activities of both E1 and E2. About 2 molecules of carbethoxyhistidine per subunit of E2 and 0.6 molecules per subunit of E1 are present in the completely inhibited enzymes.20
D001230:Aspergillus / Q000201:enzymology*; D001234:Aspergillus niger / Q000201:enzymology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000302:isolation & purification; D006639:Histidine / Q000032:analysis; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D011096:Polygalacturonase / Q000302:isolation & purification / Q000378:metabolism
D006898:Hydroxylamines; D046911:Macromolecular Substances; D006639:Histidine; D006026:Glycoside Hydrolases; D011096:Polygalacturonase; D004047:Diethyl Pyrocarbonate