Purification, Kinetic, and Thermodynamic Characteristics of an Exo-polygalacturonase from Penicillium notatum with Industrial Perspective.
An extracellular exo-polygalacturonase (exo-PG) produced by Penicillium notatum was purified (3.07-folds) by ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Two distinct isoforms of the enzyme, namely exo-PGI and exo-PGII, were identified during column purification with molecular weights of 85 and 20 kDa, respectively, on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme displayed its optimum activity at pH 6.0 and 50 °C and was found to be stable in the slightly acidic pH (ranging from 4.5 to 6.0). Michaelis-Menten parameters, i.e., Km (app)and Vmaxfor pectin hydrolysis, were calculated to be 16.6 mg/mL and 20 μmol/mL/min, respectively. The enzyme followed biphasic deactivation kinetics. Phase I of the exo-PGI showed half-lives of 6.83 and 2.39 min at 55 and 80 °C, respectively, whereas phase II of the enzyme exhibited a half-life of 63.57 and 22.72 min at 55 and 80 °C, respectively. The activation energy for denaturation was 51.66 and 44.06 kJ/mol for phase I and phase II of the exo-PGI, respectively. The enzyme activity was considerably enhanced by Mn2+, whereas exposure to a hydrophobic environment (urea and sodium azide solution) drastically suppressed the enzyme activity. Results suggest that exo-PGI might be considered as a potential candidate for various applications, particularly in the food and textile industries.
Environmental & Material Chemistry Laboratory, Department of Chemistry, University of Agriculture, Faisalabad, 38040, Pakistan.|Department of Chemistry, Government College Women University, Faisalabad, Pakistan.
Faiza
F
Amin
Environmental & Material Chemistry Laboratory, Department of Chemistry, University of Agriculture, Faisalabad, 38040, Pakistan.
Haq Nawaz
HN
Bhatti
Industrial Biotechnology Laboratory, Department of Biochemistry, University of Agriculture, Faisalabad, 38040, Pakistan. bilaluaf@hotmail.com.
Muhammad
M
Bilal
Industrial Biotechnology Laboratory, Department of Biochemistry, University of Agriculture, Faisalabad, 38040, Pakistan.
Muhammad
M
Asgher
D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D006868:Hydrolysis; D010368:Pectins / Q000737:chemistry*; D010408:Penicillium chrysogenum / Q000201:enzymology*; D011096:Polygalacturonase / Q000737:chemistry / Q000302:isolation & purification; D013816:Thermodynamics
D005656:Fungal Proteins; D010368:Pectins; D011096:Polygalacturonase
Catalytic activity; Exo-polygalacturonase; Kinetics; Purification; Thermal inactivation; Thermodynamic parameters