Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323.
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 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0-5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3h. The addition of Mn(2+), K(+), Zn(2+), Ca(2+) and Al(3+) inhibited the enzyme activity; it increased in the presence of Mg(2+) and Cu(2+) ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K(m) and V(max) values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/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 %T(650) 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.
Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India.
Shashi
S
Kant
Anuja
A
Vohra
Reena
R
Gupta
D001234:Aspergillus niger / Q000201:enzymology*; D002850:Chromatography, Gel; D003300:Copper / Q000737:chemistry; D004795:Enzyme Stability; D005638:Fruit / Q000737:chemistry; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D027845:Malus / Q000737:chemistry; D008970:Molecular Weight; D010368:Pectins / Q000737:chemistry; D010636:Phenols / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry; D011096:Polygalacturonase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D031647:Psidium / Q000737:chemistry; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D005656:Fungal Proteins; D010636:Phenols; D010936:Plant Extracts; D013438:Sulfhydryl Compounds; D003300:Copper; D010368:Pectins; D011096:Polygalacturonase; D008274:Magnesium; C003181:polygalacturonic acid