Though radioactivity is widely used in bioassays due to its high sensitivity and convenience, safety and environmental concerns have accelerated efforts to eliminate its use. Firefly luciferase accords the most efficient production of chemical luminescence, which is an effective alternative to radioactivity. Recent improvements in the enzymatic chemistry which yield greater luminescent intensity with simper reaction kinetics further improve its commercial utility. Examining the structure and function of several divergent isozymes of beetles luciferases is proposed to gain a broader perspective of their properties, and to develop one or more isozymes which are better suited to commercial applications. Phase I of this research demonstrated feasibility by successfully developing efficient methods for a) luciferase expression in E. coli, b) mutagenesis and selection for novel phenotypes, and c) structural and enzymological analysis of modified enzymes. Phase II will focus on characterizing the recombinant luciferases, examining their relative structural relationships, and employing mutagenesis to enhance their properties and provide a method for future rational enzyme development in the absence of 3 dimensional structural information. In Phase III, specific bioassay and other commercial applications will be developed. %%% Radioactivity is commonly used in biochemical analysis, such as medical and industrial diagnostics, because it is convenient and provides high sensitivity. However, alternatives to radioactivity y are being sough due to concerns for over safety and environmental impact. the light emitted from fireflies is an extremely efficient signal, and the chemistry of this system has been recently advanced to accord a viable. In addition to efficient light production, however, commercial practicality requires that the components of the system be physically robust. The enzyme isolated from fireflies, luciferase, is the least stable of these components. Phase II research is proposed to improve this situation by two approaches: 1) examine luciferases from other luminous beetles for enzymes with greater structure. The feasibility of these approaches has been previously established in Phase I research. It is further hoped that the methods developed in the Phase II research will be applicable to the commercial development of other enzymes. In Phase III, the luciferases developed in Phase II will be adapted to novel biochemical procedures.
