Characterization of three-phase partitioned exo-polygalacturonase from Aspergillus sojae with unique properties

Abstract

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.

Three-phase partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera

“…In TPP of phospholipase D from Dacus carota, lowering the temperature from 25 to 4ºC led to a decrease in the activity yield by 13% but no change in the specific activity of the enzyme in the precipitate [7]. Dogan et al [36] also reported improved yields and purification of exo-polygalacturonase using TPP at 25ºC rather than 37ºC. At 25ºC gave a 25.5% recovery of with a 6.7-fold purification, only 8.8%…”

“…recovery and 0.7-fold purification was obtained at 37ºC [36]. However, Rajeeva et al [34] showed that there was no significance in recovery and purification fold of laccase by TPP at 5ºC compare to 35ºC.…”

Aspergillus sojae Tarafından Üretilen Poligalakturonazın Kısmi Saflaştırılması için Kromatografik Bir Yaklaşım

“…In addition to its high specific activity, SEC purification of 3 rd IEXC pooled fraction revealed the highest partial yield (36%) and purification fold (2.00). According to a study by Dogan and Tari [38], PG from A. sojae ATCC 20235 strain was purified with a recovery of 25.5% and 6.7 fold purification by threephase partitioning in which the enzyme solution was mixed with ammonium sulphate and tert-butanol. In another study by Nagai et al [37] the two step purification including cation exchange and size exclusion chromatography for Aspergillus awamori resulted in only 3.04% recovery with a higher purification fold (345) and specific activity (487 U/mg) than the findings in this study.…”

Separation of Lactoferrin From Whey in Three-Phase Extraction Systems (Tpp Method)

“…Skrajnie małe bądź duże stężenia siarczanu amonu powodowały zmniejszenie wielkości parametrów ekstrakcji. W literaturze na temat ekstrakcji białek w układach trójfazowych odnotowuje się, że PF oraz R% mogą osiągać wielkości maksymalne przy tym samym stopnia wysycenia solą [4,10] względnie, że korzystniejsze jest większe stężenie soli do zwiększenia wydajności odzysku aniżeli do podwyższenia stopnia oczyszczenia [17].…”

“…Obserwowano to przy oczyszczaniu α-galaktozydazy z Lycopersicon esculentum [4], egzopoligalakturonazy pochodzącej z Aspergillus sojae [10] oraz ekstrakcji peroksydazy z Ipomoea palmata [17]. Z kolei w trakcie oczyszczania pektynazy z owoców pomidora przyjęto stosunek surowego ekstraktu do tert-butanolu równy 1 : 2 [25].…”