Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) for purification of xylanase.
It is shown that eudragit S-100, a copolymer of methylacrylic acid and methylmethacrylate, undergoes three-phase partitioning. It was found that 95% eudragit S-100 could be recovered as the interfacial precipitate by using 30% (w/v) ammonium sulfate, 1:1 ratio of t-butanol to polymer solution at 40 degrees C. Three-phase partitioning of proteins uses simultaneous addition of ammonium sulfate and t-butanol to precipitate proteins in an interfacial layer separating the aqueous phase and organic solvent. Exploiting the affinity of xylanases towards eudragit S-100, it was possible to purify xylanase from Aspergillus niger; 60% recovery of activity with 95-fold purification could be obtained by this process. The purified enzyme showed A single band on SDS-PAGE. The technique shows promise to develop into a general method that could be termed "macroaffinity ligand-facilitated three-phase partitioning (MLFTPP).
Sharma
Aparna
A
Chemistry Department, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India.
Gupta
M N
MN
D001234:Aspergillus niger / Q000201:enzymology; D002458:Cell Fractionation / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D004789:Enzyme Activation; D008024:Ligands; D011109:Polymethacrylic Acids; D012680:Sensitivity and Specificity; D013696:Temperature; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis / Q000302:isolation & purification*
D008024:Ligands; D011109:Polymethacrylic Acids; C038300:methylmethacrylate-methacrylic acid copolymer; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase