title: Improving the activity of immobilized subtilisin by site-specific attachment to surfaces.
abstract: Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors. The protease subtilisin BPN' was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties. To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis. This cysteine residue was designed to be away from the active site of the enzyme. The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports. This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase. Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
authors.forename: W
authors.initials: W
authors.lastname: Huang
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: D
authors.initials: D
authors.lastname: Bhattacharyya
authors.forename: L G
authors.initials: LG
authors.lastname: Bachas
mesh_terms: D001407:Bacillus; D001665:Binding Sites; D015374:Biosensing Techniques; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D013381:Subtilisins / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D013381:Subtilisins