Bacterial Luciferase of Vibrio harveyi MAV: Purification, Characterization, and Application

Abstract

In the medical and biotechnological industry is an increasing interest for bioluminescent systems for example as analytical tools and genetic markers. We introduced a new procedurefora large scale purification of luciferase from Vibrio harveyi MAV in view of subsequentcrystallization of the enzyme. In four steps the luciferase was obtained in greater than 99 % purity using the chromatography equipment (FPLC-System)from Pharmacia/LKB, Freiburg, Germany. Homogeneity of the luciferase was proved by SDS-PAGE andsilver staining. The enzyme was purified 34fold to a yield of 29 % and a specific activity of 1.8*10'' [LU/mg]. The optimum activity of luciferase was detected at pH 6.8 and 30°C.Theisoelectric point proved to be at pH 4.7. Treatment with the inhibitors diethylcarbonate, phenylmethylsulfonyifluoride, and diethyl-p-nitrophenylphosphate until unknownasluciferase inhibitors, affected the activity with the same extent and nearly the samevelocity as other inhibitors, reacting with histidine and cysteine residues at the active center of luciferase. The enzymecrystallized depending upondifferent precipitation agentsin various forms. We obtained twotypesof crystals, needles in ammonium sulfite and rhombic-like in ammonium sulfate.