Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate.
Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.
Mondal
Kalyani
K
Chemistry Department, Indian Institute of Technology, Delhi Hauz Khas, New Delhi 110016, India.
Sharma
Aparna
A
Gupta
Munishwar Nath
MN
D000464:Alginates / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism; D020723:Glucuronic Acid / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism*; D008024:Ligands; D012680:Sensitivity and Specificity
D000464:Alginates; D006603:Hexuronic Acids; D008024:Ligands; D020723:Glucuronic Acid; D006026:Glycoside Hydrolases; D005087:Glucan 1,4-alpha-Glucosidase; C100181:pullulanase