LccA, an archaeal laccase secreted as a highly stable glycoprotein into the extracellular medium by Haloferax volcanii.
Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic 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(-)(1) 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-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO(4). Optimal oxidation of ABTS and SGZ was at 45 degrees C and pH 6 and pH 8.4, respectively. The apparent K(m) values for SGZ, bilirubin, and ABTS were 35, 236, and 670 muM, with corresponding k(cat) 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 degrees C of 31.5 h.
Uthandi
Sivakumar
S
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA.
Saad
Boutaiba
B
Humbard
Matthew A
MA
Maupin-Furlow
Julie A
JA
D000595:Amino Acid Sequence; D052160:Benzothiazoles / Q000378:metabolism; D002241:Carbohydrates / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D019622:Haloferax volcanii / Q000201:enzymology* / Q000235:genetics; D006835:Hydrazones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics; D013451:Sulfonic Acids / Q000378:metabolism
D052160:Benzothiazoles; D002241:Carbohydrates; D006023:Glycoproteins; D006835:Hydrazones; D013451:Sulfonic Acids; C009370:syringaldazine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D042845:Laccase