Polygalacturonases exopolygalacturonases

The molecular masses of polygalacturonase and exopolygalacturonase were approximately determined by gel chromatography on Superose 12 using FPLC device (Pharmacia, Sweden) and the Calibration proteins II kit (Boehringer-Mannheim, Germany). 

The activities of pectic enzymes present in cultivation medium (98 mg of protein extracted from 2.5 1 of pectin medium) were poor, not leading to the clarification of cultivation medium indicating the cleavage of pectate chains, with values 0.024 pmol/min.mg for polygalacturonase, 0.004 pmol/min.mg for exopolygalacturonase, 0.034 pmol/min.mg for pectinesterase and 0.005 pmol/min.mg for pectin lyase. The production of individual pectic enzymes was dependent on the C-source used in the cultivation medium (Tab. 1). 

The pH optima determination showed at least three pH regions with increased polygalacturonase activity (pH optima 4.0, 4.6 and pH 5.6) the pH 4.0 being the pH optimum of exopolygalacturonase (Fig. 4). 

The approximate determination of molecular masses of polygalacturonases present in the medium (Fig. 5) showed two activity peaks corresponding the values of about 40 kDa for polygalacturonase and 50 kDa for exopolygalacturonase. 

Fig. 5. The approximate molecular mass determination of polygalacturonase [(0—0) - substrate 0.5% pectate, pH 4.6] and exopolygalacturonase [( — ) - substrate 1.0 pmol/ml of di(D-galactosiduronic) acid, pH 4.0] on Superose 12 column (FPLC device). Flow rate 0.5 ml/min. System 0.05 M phosphate buffer pH 7.0, 0.15 M NaCl. Standarts Ferritin (450 kDa), Katalase (240 kDa), Aldolase (158 kDa), Albumin (68 kDa), Albumin (45 kDa), Chymotrypsinogen A (25 kDa), Cytochrome C (12.5 kDa).

Mill (140, 141) isolated two exo-polygalacturonases from mycelial extracts of Aspergillus niger which diflFered in pH optima, stability, and activation by metal ions. Exopolygalacturonase I had a pH optimum of 4.5 and was fully active only in the presence of Hg ions. A ninefold increase in activity occurred in the presence of IfxM HgCb the Km was 6.2 X lO M. Dialysis against 2,3-dimercaptopropanol removed all activity in absence of Hg Various other divalent cations and chloride ion did... 

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