Three-phase partitioning of alpha-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques.
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.
Dhananjay
S K
SK
Department of Biochemistry, Gulbarga University, Gulbarga, Karnataka, 585106, India. dhananjay_sk@rediffmail.com
Mulimani
V H
VH
D000645:Ammonium Sulfate / Q000737:chemistry; D001236:Aspergillus oryzae / Q000737:chemistry / Q000201:enzymology*; D005591:Chemical Fractionation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D005233:Fatty Alcohols / Q000737:chemistry; D005285:Fermentation; D005590:Fractional Precipitation; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification*
D003470:Culture Media; D005233:Fatty Alcohols; D005656:Fungal Proteins; D000519:alpha-Galactosidase; D000645:Ammonium Sulfate