Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Abstract

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45°C, pH of 4.8, reaction time of 15min. The enzyme was stable within the pH range of 4.0–5.5 for 1h. At 4°C it retained 50% activity after 108h but at room temperature it lost its 50% activity after 3h. The addition of Mn2+, K+, Zn2+, Ca2+ and Al3+ inhibited the enzyme activity; it increased in the presence of Mg2+ and Cu2+ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The Km and Vmax values of the enzyme were found to be 0.083mg/ml and 18.21μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T650 from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.