Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids.
A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The enzyme has a molecular weight of approximately 65 kDa and demonstrates activity towards canonical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants KM and kcat for ABTS were of 6.5+/-0.2 microM and 83 s(-1), for SGZ of 4.3+/-0.2 microM and 100 s(-1), and for 2,6-DMP of 56.7+/-1.0 microM and 28 s(-1). Highest oxidizing activity towards ABTS was obtained at 85 degrees C. However, after 1 h incubation of CotA at 70 degrees C and 80 degrees C, a residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid by CotA was observed, and highest activity of CotA was found towards sinapic acid.
Koschorreck
Katja
K
Institute of Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany.
Richter
Sven M
SM
Ene
Augusta B
AB
Roduner
Emil
E
Schmid
Rolf D
RD
Urlacher
Vlada B
VB
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052160:Benzothiazoles / Q000378:metabolism*; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006835:Hydrazones / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism; D042845:Laccase / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism*
D001426:Bacterial Proteins; D052160:Benzothiazoles; D006835:Hydrazones; D062385:Hydroxybenzoates; D013451:Sulfonic Acids; C009370:syringaldazine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C533003:CotA protein, Bacillus licheniformis; D042845:Laccase; C017616:phenolic acid