Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions

Division of Biochemical Engineering, Institute of Food Technology 1 and Centre of Applied Genetics 2 , University of Agricultural Sciences Vienna (Universität für Bodenkultur Wien, BOKU), Muthgasse 18, A-1190 Wien, Austria Institute of Biochemical Technology and Microbiology, University of Technology (TU Wien), Getreidemarkt 9, A-1060 Wien, Austria 3 Author for correspondence: Dietmar Haltrich. Tel: +43 1 36006 6275. Fax: +43 1 36006 6251. e-mail: haltrich{at}edv2.boku.ac.at The major laccase isoenzyme LAP2 secreted by the white-rot basidiomycete Trametes pubescens in response to high copper concentrations was purified to apparent electrophoretic homogeneity using anion-exchange chromatography and gel filtration. The monomeric protein has a molecular mass of 65 kDa, of which 18% is glycosylation, and a pI value of 2·6. The pH optima of the laccase depend on the substrates oxidized and show bell-shaped pH activity profiles with an optimum of 3–4·5 for phenolic substrates such as 2,6-dimethoxyphenol or syringaldazine, while the non-phenolic substrates ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and ferrocyanide show a monotonic pH profile with a rate increasing with decreasing pH. The catalytic efficiencies k cat / K m determined for some of its substrates were 48 x 10 6 , 47 x 10 6 , 20 x 10 6 and 7 x 10 6 M -1 s -1 for ABTS, syringaldazine, ferrocyanide and oxygen, respectively. Furthermore, the gene lap2 encoding the purified laccase was cloned and its nucleotide sequence determined. The gene consists of 1997 bp, with the coding sequence interrupted by eight introns and flanked by an upstream region in which putative CAAT, TATA, MRE and CreA consensus sequences were identified. Based on Northern analysis containing total RNA from both induced and uninduced cultures, expression of lap2 is highly induced by copper, which is also corroborated by an increase in laccase activity in response to copper. A stimulating effect of various other heavy metal ions on laccase synthesis was also observed. In addition to induction, a second regulatory mechanism seems to be repression of lap2 transcription by glucose. Keywords: white-rot fungus, ligninolytic enzymes, polyphenol oxidase, induction, copper Abbreviations: ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate); 2,6-DMP, 2,6-dimethoxyphenol; HSE, heat-shock element; MRE, metal-responsive element; STRE, stress-responsive element a The GenBank accession numbers for the lap2 and lap1a genes rep