Biochemical characterization of an extracellular polygalacturonase from Trichoderma harzianum.
An extracellular polygalacturonase (PGII) from Trichoderma harzianum was purified to homogeneity by two chromatography steps using DEAE-Sepharose and Sephacryl S-200. The molecular weight of T. harzianum PGII was 31,000 Da by gel filtration and SDS-PAGE. PGII had isoelectric point of 4.5 and optimum pH of 5.0. PGII was very stable at the pH 5.0. The extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (DE). PGII had very low activity toward non-pectic polysaccharides. The apparent K(m) value and K(cat) value for hydrolyzing polygalacturonic acid (PGA) were 3.4 mg/ml and 592 s(-1), respectively. PGII was found to have temperature optimum at 40 degrees C and was approximately stable up to 30 degrees C for 60 min of incubation. All the examined metal cations showed inhibitory effects on the enzyme activity. A 1,10-phenanthroline, Tween 20, Tween 80, Triton X-100 and SDS had no effect on the enzyme activity. The rate of enzyme catalyzed reduction of viscosity of solutions of PGA or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. The storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to 1 year. These properties of T. harzianum PGII with appreciable activity would be potentially novel source of enzyme for food processing.
Mohamed
Saleh A
SA
Molecular Biology Department, National Research Centre, Cairo, Egypt. saleh38@hotmail.com
Farid
Nevin M
NM
Hossiny
Ebtsam N
EN
Bassuiny
Roqaya I
RI
D004795:Enzyme Stability; D007700:Kinetics; D010368:Pectins / Q000378:metabolism*; D011096:Polygalacturonase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014242:Trichoderma / Q000201:enzymology* / Q000378:metabolism
D010368:Pectins; D011096:Polygalacturonase