Simultaneous refolding/purification of xylanase with a microwave treated smart polymer.
Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble-insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.
Roy
Ipsita
I
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Mondal
Kalyani
K
Sharma
Aparna
A
Gupta
Munishwar N
MN
D000345:Affinity Labels; D001234:Aspergillus niger / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008872:Microwaves; D011108:Polymers / Q000378:metabolism / Q000528:radiation effects; D011489:Protein Denaturation; D017510:Protein Folding; D014995:Xylosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D011108:Polymers; D014995:Xylosidases