Biochemical and molecular genetic characterisation of a novel laccase produced by the aquatic ascomycete Phoma sp. UHH 5-1-03.
A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer ( approximately 150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography-tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.
Junghanns
C
C
University of Louvain, Louvain-la-Neuve, Belgium.
Pecyna
M J
MJ
Böhm
D
D
Jehmlich
N
N
Martin
C
C
von Bergen
M
M
Schauer
F
F
Hofrichter
M
M
Schlosser
D
D
D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007526:Isoelectric Point; D042845:Laccase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055503:Protein Multimerization; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry; D014871:Water Microbiology
D004269:DNA, Bacterial; D042845:Laccase