CTS-9307518 Kentucky Research Foundation D. Allen Butterfield ABSTRACT Methods for the immobilization of enzymes often lead to reduced activity due to conformational changes, unfavorable orientations and other factors. A new technique for more specific and uniform attachment with spacers will be explored in this project on membrane bioreactors. Site-directed mutagenesis is to be used in modifying the genetic code for a cysteine residue and then expressing the variant enzyme (subtilsin). Functional groups at one terminus of the spacer compound will be reacted with amino or hydroxyl moieties in a polymeric membrane while the other terminus will contain a thiol to be oxidized to the disulfide in completing the immobilization. The immobilized enzyme and intermediate structures will be characterized by NMR, x-ray fluorescence, spin-label studies, and other methods. Catalytic behavior is to be examined under various reaction conditions including aqueous and organic solvents. Immobilization of enzymes facilitates a continuous mode of operation and aids the purification of bioproducts. This fundamental research could help improve the production processes in many biotechnology companies.
