TY - JOUR
T1 - Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag
AU - Wang, Jianquan
AU - Bhattacharyya, D.
AU - Bachas, L. G.
PY - 2001
Y1 - 2001
N2 - 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 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.
AB - 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 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.
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U2 - 10.1007/s002160000622
DO - 10.1007/s002160000622
M3 - Article
C2 - 11293705
AN - SCOPUS:0034935569
SN - 0937-0633
VL - 369
SP - 280
EP - 285
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 3
ER -