Exo-polygalacturonase enzyme produced by Aspergillus sojae ATCC 20235 was purified using three-phase partitioning (TPP), an emerging bio-separation technique where a single step as compared to the classical multi-step purification was used. Using this technique, crude enzyme solution (pH 6.6) saturated to 30% (w/v) with ammonium sulphate and with a crude extract to tert-butanol ratio of 1:1 (v/v) at 25• C resulted in 25.5% recovery of exo-polygalacturonase with a 6.7-fold purification. The purified enzyme was characterized with respect to its activity and stability at various pH and temperature ranges. Optimum pH and temperature for maximum activity were determined as pH 4 and 55• C. The enzyme was stable at both acidic and alkaline pH for 2 h at 30• C. The thermal stability study showed that the purified enzyme had an inactivation energy of 68.41 kcal/mol and a half-life (t 1/2 ) value of 3.6 h at 75• C presenting a large thermal stability. The kinetic constants K m and V max using polygalacturonic acid as substrate were 0.75 g l −1 and 1.14 mol min −1 , respectively. SDS-PAGE profiling revealed that the purified exopolygalacturonase had two bands with the molecular weights of 36 and 53 kDa. The enzyme was completely inhibited in the presence of Mn 2+ and SDS and induced significantly by EDTA, glycerol and -mercaptoethanol.
a b s t r a c tCrude exo-polygalacturonase enzyme (produced by Aspergillus sojae), significant for industrial processes, was characterized with respect to its biochemical and thermal properties. The optimum pH and temperature for maximum crude exo-polygalacturonase activity were pH 5 and 55°C, respectively. It retained 60-70% of its activity over a broad pH range and 80% of its initial activity at 65°C for 1 h. The thermal stability study indicated an inactivation energy of E d = 152 kJ mol
À1. The half lives at 75 and 85°C were estimated as 3.6 and 1.02 h, respectively. Thermodynamic parameters, DH * , DS * and DG * , were determined as a function of temperature. The kinetic constants K m and V max , using polygalacturonic acid as substrate, were determined as 0.424 g l À1 and 80 lmol min
À1, respectively. SDS-PAGE profiling revealed three major bands with molecular weights of 36, 53 and 68 kDa. This enzyme can be considered as a potential candidate in various applications of waste treatment, in food, paper and textile industries.
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