In diagnostic enzyme immunoassays (EIA), enzymes are conjugated with either antigen or antibody to amplify the presence of antigen-antibody complexes. Currently, beta-galactosidase, horseradish peroxidase, and alkaline phosphatase are the three most widely used enzymes in EIA. These enzymes each have their respective advantages and disadvantages. The ideal enzyme should have very high catalytic efficiency and be able to use low concentrations of chromogenic or fluorescent substrates which yield very low blank background. This ideal enzyme should make enzyme immunoassays more sensitive and rapid. The short-term objective of this proposal is to generate beta-galactosidase with high catalytic efficiency.
The specific aims are to characterize the possibilities and limitations of a promising technique for isolating enzymes of different properties, such as high kcat or lower Km. In preliminary studies, this technique had produced beta- galactosidase with high kcat. The technique to be employed involves selective procedures following random mutations in a chemostat environment. This approach has the advantage over site-directed mutagenesis in that detailed structural information on the enzyme of interest is not necessary. Moreover, the evolutionary approach proposed makes available to us an unlimited repertoire of mutations and their combinations, rather than being limited by preconceived notions as to the optimal structure desired.
