Purification of a bacterial pullulanase on a fluidized bed of calcium alginate beads.
Pullulanase from Bacillus acidopullulyticus was purified on a packed bed and a fluidized bed of calcium alginate beads. The binding of enzyme activity to the medium was found to follow Langmuir isotherm pattern. The maximum binding capacity was 1476 U ml(-1) matrix and the dissociation constant was 142 U ml(-1). The dynamic binding capacities at 5% breakthrough in the packed and fluidized beds were 472 U ml(-1) and 644 U ml(-1), respectively. In the packed bed as well as the fluidized bed, an activity recovery of more than 95% with fold purification in the range of 46-59 was observed. The elution with a competitive inhibitor, viz. maltose, and high-fold purification indicate an affinity-based process. The purification process worked equally well with columns of bed volumes of 3.8 and 10 ml.
Roy
Ipsita
I
Chemistry Department, Indian Institute of Technology, Hauz Khas, New Delhi.
Gupta
Munishwar N
MN
D000327:Adsorption; D000464:Alginates / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D020723:Glucuronic Acid; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D006603:Hexuronic Acids
D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D006026:Glycoside Hydrolases; C100181:pullulanase