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Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids

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Abstract

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 ~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 K M and k cat for ABTS were of 6.5 ± 0.2 μM and 83 s−1, for SGZ of 4.3 ± 0.2 μM and 100 s−1, and for 2,6-DMP of 56.7 ± 1.0 μM and 28 s−1. Highest oxidizing activity towards ABTS was obtained at 85°C. However, after 1 h incubation of CotA at 70°C and 80°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.

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Acknowledgments

The thank Prof. Dr. U. Beifuss (University of Hohenheim, Germany) for providing us with dehydrodisinapic acid dilactone. This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 706), the Ministerium für Wissenschaft, Forschung und Kunst des Landes Baden-Württemberg and the Fonds der Chemie (Germany).

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Authors and Affiliations

  1. Institute of Technical Biochemistry, University of Stuttgart, 70569, Stuttgart, Germany

    Katja Koschorreck, Sven M. Richter, Rolf D. Schmid & Vlada B. Urlacher

  2. Institute of Physical Chemistry, University of Stuttgart, 70569, Stuttgart, Germany

    Augusta B. Ene & Emil Roduner

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  1. Katja Koschorreck
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  2. Sven M. Richter
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  3. Augusta B. Ene
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  4. Emil Roduner
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  5. Rolf D. Schmid
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  6. Vlada B. Urlacher
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Correspondence to Vlada B. Urlacher.

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Koschorreck, K., Richter, S.M., Ene, A.B. et al. Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids. Appl Microbiol Biotechnol 79, 217–224 (2008). https://doi.org/10.1007/s00253-008-1417-2

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  • DOI: https://doi.org/10.1007/s00253-008-1417-2

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