Laceases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laceases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a lacease (LccA) from the halophillc archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 ± 10 mU - ml-1) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U - mg with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3- ethylbenzothlazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO4. Optimal oxidation of ABTS and SGZ was at 45°C and pH 6 and pH 8.4, respectively. The apparent Km values for SGZ, bilirubin, and ABTS were 35, 236, and 670 μM, with corresponding kcat values of 22, 29, and 10 s-1, respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50°C of 31.5 h. © 2010, American Society for Microbiology. All Rights Reserved.
