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 (temied FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, kcat/KM, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.
Wang
J
J
Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington 40506-0055, USA.
Bhattacharyya
D
D
Bachas
L G
LG
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli; D005818:Genetic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism*
D017931:DNA Primers; D004800:Enzymes, Immobilized; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; D013381:Subtilisins