Abstract
Crude pectinase extract (FI), obtained from Aspergillus niger, was partially purified by ammonium sulphate precipitation at saturation of 0-20% (FIIa), 20-80% (FIIb) and 80-100% (FIIc). While all precipitated fractions exhibited pectin methyl esterase (PME), beta -1,3-glucanase, polyphenol esterase (PPE), polygalacturonase (PG) and beta -galactosidase activities, fraction FIIa contained the majority of PME and beta -1,3-glucanase activities. However, fraction FIIc contained the highest PPE, PG and beta -galactosidase activities, whereas fraction FIIb contained the least of all these activities. Electrophoretic analyses of the partially purified FIIc fraction demonstrated the presence of one major band with a molecular weight of 17.5 kDa and five minor bands with molecular weights in the range from 7.2 to 20.1 kDa. The purification procedure resulted in a 2.1-fold increase in PPE activity compared to that of the crude extract. The optimum pH and temperature for PPE hydrolytic activity were 5.25 and 40 degree C, respectively. PPE reduced the oxidation, by mushroom tyrosinase, of catechin and chlorogenic acid used as substrates to brownish coloured products, however, the rate of oxygen uptake during the oxidation reaction was constant. The PPE inhibitory effect on tyrosinase activity was 12 times higher with catechin than with chlorogenic acid. In addition, PPE exhibited a competitive and a mixed type of inhibition for tyrosinase with chlorogenic acid and catechin, respectively. High-performance liquid chromatography analyses of enzymic end-products suggested that, in the case of chlorogenic acid, the inhibitory mechanism of PPE was probably due to the formation of substances that acted as inhibitors to tyrosinase activity. Moreover, the results indicated that the inhibitory mechanism of PPE on tyrosinase, using catechin as substrate, was not the same as that for chlorogenic acid.