Three-phase partitioning of α-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques

Abstract

Simple, attractive and versatile technique, three-phase partitioning (TPP) was used to purify alpha-galactosidase from fermented media of Aspergillus oryzae. The various conditions required for attaining efficient purification of the alpha-galactosidase fractions were optimized. The addition of n-butanol, t-butanol, and isopropanol in the presence of ammonium sulfate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The single step of three-phase partitioning, by saturating final concentration of ammonium sulfate (60%) with 1:1 t-butanol, gave activity recovery of 92% with 12-fold purification at second phase of TPP. The final purified enzyme after TPP showed considerable purification on SDS-PAGE with a molecular weight of 64 kDa. The enzyme after TPP showed improved activity in organic solvents. Results are compared with conventional established processes for the purification of alpha-galactosidase produced by Aspergillus oryzae and overall the proposed TPP technique resulted in 70% reduction of purification cost compared to conventional chromatographic protocols.

Purification and Recovery of Invertase from Potato Tubers (Solanum tuberosum) by Three Phase Partitioning and Determination of Kinetic Properties of Purified Enzyme

“…They were also unscalable, time consuming, expensive multistep protocols [2][3][4][5][9][10][11][12]. Three phase partitioning has relatively recent use for bioseperation of enzymes and inhibitors [13][14][15][16][17]. It uses ammonium sulfate with certain saturation to precipitate the protein and tbutanol is added to make three phase layers and to remove lipids, phenolic compounds and some detergents [13,18].…”

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

“…preferencyjne gromadzenie się laktoferyny w fazach dolnych, podczas gdy pozostałe białka, po wysoleniu w tych warunkach, były obecne w fazach środkowych. Podobną zależność zaobserwowali Dhananjay i Mulimani [9] podczas ekstrakcji α-galaktozydazy w układach trójfazowych. Wpływ pH serwatki na stopień oczyszczenia i wydajność odzysku laktoferyny przedstawiono na rys.…”

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

“…From the previous works showed that ammonium sulfate were superior to other salts [4], [9], [11], [14] and [15]. This might be due to the Hofmeister series, which is a classification of ions based on their salting-out ability [31] and [32].…”

“…Dhananjay et al [4] found that the optimum TPP condition for α-galactosidase was the use of 30% ammonium sulfate along with 1: 1 ratio of t-butanol to aqueous crude extract provided the recovery around 99%. From the previous works showed that ammonium sulfate were superior to other salts [4], [9], [11], [14] and [15].…”

“…TPP is a concentrating or dewatering step and some enzymes have enhanced catalytic activities in these conditions within short periods of time (about 1 h) [4]. TPP has been widely used to separate and purify various enzymes such as Aspergillus oryzae [4], protease/amylase inhibitor from wheat germ [5] and ragi (Eleusine coracana) [6], phospholipase D from Dacus carota [7], α-galactosidase form tomato [8], and pepino (Solanum muricatum) [9], invertase from baker's yeast [10], and tomato [11], proteases from papaya peels [12], Calotropis procera latex [13] and giant catfish viscera [14], inulinase from Aspergillus niger [15]. Rawdkuen et al [14] showed the best condition for separating of alkaline proteases from farmed giant catfish viscera that was consisted of the crude enzyme extract to t-butanol ratio of 1.0: 0.5 in the presence of 50% (NH 4 ) 2 SO 4 .…”