| The use of binding peptides and binding proteins in the construction of ligand-enzyme conjugates for binding assays and for the affinity-based immobilization of enzymes and bacterial cells has been investigated. To this end, a genetically engineered peptide-enzyme conjugate was developed, consisting of the streptavidin binding peptide Strep-tag II and bacterial alkaline phosphatase as the marker enzyme. The peptide-enzyme conjugate was used to detect biotin, biocytin, and desthiobiotin. Though the detection limits were high relative to other methods, due to the low binding affinity of the peptide relative to biotin and its analogues, the dose-response curves obtained clearly show that peptide mimics can be used to detect non-peptide analytes.; The reversible calcium-dependent binding of calmodulin was used to develop a reversible binding scheme for the enzyme organophophorous hydrolase (OPH). In order to utilize this reversible binding to immobilize OPH, the gene coding for vu-1 calmodulin was fused to the N-terminus of the opd gene, which codes for OPH. The resulting calodulin-OPH fusion protein was immobilized on a cellulose membrane functionalized with the phenothiazine derivative TAPP. The enzyme fusion, once immobilized, retained a significant amount of its specific activity, and could be repeatedly loaded, unloaded, and reloaded onto the membrane.; Calmodulin was also used as an affinity tail for the immobilization of the thermostable phenol hydroxylase of Geobacillus stearothermophilus BR 219. It was proposed that the thermostable properties of calmodulin might allow it to be used in an affinity-based immobilization scheme for thermostable enzymes. Calmodulin was therefore genetically fused to the N-terminus of the phenol hydroxylase, and the enzyme fusion was successfully immobilized on TAPP-cellulose. Calmodulin was able to remain bound to the membrane at the optimum temperature of the phenol hydroxylase (55°C).; Finally, a method for immobilizing bacterial cells on surfaces through the binding interaction of the antimicrobial peptide cecropin-P1 was developed. The antimicrobial peptide cecropin-P1 was covalently attached to microplate well surfaces, and used to immobilized E. coli 0157:H7 and K-12 cells, which were subsequently detected by an anti- E. coli-HRP conjugate. The data clearly demonstrated that the measured signals were dependent upon both the concentration of viable cells and the amount of cecropin-P1 coupled to the surface. |
