Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (termed FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Baci llus amyloliquefaciens. The fusion protein SET-FLAG was immobilized to nonp orous polystyrene and silica beads both in a site-directed and a random fas hion. Site-directed immobilization was achieved by employing the interactio n between protein A and a monoclonal antibody specific for the FLAG peptide , while random immobilization was obtained by using glutaraldehyde as a cro ss-linking reagent. The activity of the immobilized enzymes was compared. I t was found that the site-directed subtilisin had higher catalytic efficien cy, k(cat)/K-M, which was more than 7-fold of that of the randomly immobili zed enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.