Use of polyethylene glycol and high-performance liquid chromatography for preparative separation of Aspergillus ficuum acid phosphatases.
Proteins of Aspergillus ficuum culture filtrate were sequentially fractionated with 4, 9, 15, 19, 24, 30 and 36% polyethylene glycol (PEG) into seven acid phosphatases (APases) with 93% and 52% overall recoveries of activity and protein, respectively. Crude extract was also separated into seven APase peaks on a 30 cm x 2.5 cm I.D. anion-exchange column using 0.1 M Tris-HCl (pH 8.0) and a 0-0.4 M KCl gradient as the eluent, but their resolution was incomplete. However, when individual PEG precipitates were injected on to the column, each APase was eluted in a single, large peak resulting in 85% recovery and fifteen-fold purification of APase activity over the PEG precipitates. Use of PEG prior to HPLC separations also reduced the separation time to half and allowed a tenfold increase in sample load with complete resolution. The APases in PEG fractions and their corresponding HPLC peaks varied significantly in their kinetic parameters, including substrate specificity and pH optimum. The method developed is most beneficial for the isolation of these closely related APases from microbial or other sources for further molecular biology studies.
Hamada
J S
JS
US Department of Agriculture, Southern Regional Research Center, New Orleans, LA 70179.
D000135:Acid Phosphatase / Q000302:isolation & purification*; D001230:Aspergillus / Q000201:enzymology*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D011092:Polyethylene Glycols
D011092:Polyethylene Glycols; D000135:Acid Phosphatase