Bacterial luciferase of Vibrio harveyi MAV: Purification, characterization and crystallization

Abstract A new procedure for the large-scale purification of luciferase from Vibrio harveyi MAV was developed with a view to the subsequent crystallization of the enzyme. Homogeneous luciferase was obtained in four steps. The purification procedure included the following steps: (a) ion exchange chromatography (IEX) with two coupled columns, S-Sepharose Fast Flow (cation exchange) followed by Q-Sepharose Fast Flow (anion exchange); (b) hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose CL-4B; (c) adsorption chromatography (AC) on hydroxyapatite; and (d) affinity chromatography (AFC)) on Ω-Aminohexylagarose. The enzyme was purified 34-fold with a yield of 29% and a specific activity of 1.8 × 10 11 LU mg −1 . Optimal activity of the enzyme was found to be at pH 6.8 and 30°C. Luciferase was inhibited by diethylcarbonate, phenylmethylsulfonylfluoride, and diethyl-p-nitrophenyl-phosphate. A cysteine residue is involved in binding FMNH 2 , whereas aldehyde binding occurs in the presence of serine and histidine residues. The enzyme was crystallized in various forms depending upon the different precipitation agents used. Two types of crystals were found: needles in ammonium sulfite and rhombics in ammonium sulfate.