A surfactant tolerant laccase of Meripilus giganteus.
A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K ( m ) values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k (cat)/k (m) (s(-1) mM(-1)). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.
Institut für Lebensmittelchemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167, Hannover, Germany. gunnar.schmidt@lci.uni-hannover.de
Gunnar
G
Schmidt
Ulrich
U
Krings
Manfred
M
Nimtz
Ralf G
RG
Berger
D052160:Benzothiazoles; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D055453:Coriolaceae / Q000201:enzymology*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013451:Sulfonic Acids; D013501:Surface-Active Agents / Q000378:metabolism*; D013696:Temperature
D052160:Benzothiazoles; D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D013451:Sulfonic Acids; D013501:Surface-Active Agents; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D042845:Laccase