Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene.
Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO(4) and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k(cat)/K(m)) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s(-1) mM(-1), respectively, with K(m) values of 4.2, 67.8, and 104.9 microM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15 degrees C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.
Martin
C
C
UFZ, Department of Environmental Microbiology, Helmholtz Centre for Environmental Research - UFZ, Permoserstr. 15, 04318, Leipzig, Germany. claudia.martin@arcormail.de
Pecyna
M
M
Kellner
H
H
Jehmlich
N
N
Junghanns
C
C
Benndorf
D
D
von Bergen
M
M
Schlosser
D
D
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D005858:Germany; D007526:Isoelectric Point; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny
D042845:Laccase