Rapid neutral protease purification by dye-affinity membrane chromatography

Dye-affinity hollow fibres were prepared by radiation-induced grafting of polymers with different hydrophilicity degree on to the membranes and dye attachment after reaction of the grafted epoxy groups with ammonia to produce amino groups. Membranes grafted with glycidyl methacrylate/dimethyl acrylamide (GMA/DMAA) 1/3 showed better chromatographic properties than those grafted with GMA. The dye Yellow HE-4R was selected for purification of a neutral protease from Flavourzyme™ on the basis of the maximum capacity of the membranes obtained. No leakage of the dye from the membranes was evidenced after incubation in 8 M urea in 0.5 M NaOH for 2 months. From isotherms developed with membranes grafted with GMA/DMAA 1/3, a maximum capacity of 24220 U/ml membrane and a dissociation constant of 91 U/ml was calculated. Elution with 2 M NaCl at pH 4 allowed quantitative recovery of the adsorbed protease. A yield of 72% with a purification factor of 3.7 was achieved by working at 5 ml/min for the load, wash and regeneration steps and 1 ml/min for the elution step. Under these conditions, productivity was 1828 U/h ml, far higher than that attained with the same dye attached to a soft gel: 304 U/h ml [Iannucci NB, Navarro del Cañizo AA, Cascone O. Purification of neutral protease by dye-affinity chromatography. Appl Biochem Biotechnol 2003;104:173–84].