Purification of neutral protease by dye affinity chromatography.
Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25 degrees C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8 degrees C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25 degrees C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.
Iannucci
Nancy B
NB
Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
Navarro del Cañizo
Agustín A
AA
Cascone
Osvaldo
O
D000345:Affinity Labels; D002846:Chromatography, Affinity / Q000379:methods*; D004396:Coloring Agents; D010450:Endopeptidases / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification*; D004789:Enzyme Activation; D008024:Ligands; D011786:Quality Control; D012680:Sensitivity and Specificity; D013696:Temperature
D000345:Affinity Labels; D004396:Coloring Agents; D008024:Ligands; D010450:Endopeptidases; C412470:flavourzyme