Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae.
A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.
Berka
R M
RM
Novo Nordisk Biotech, Inc., Davis, California, USA. ramb@novo.dk
Schneider
P
P
Golightly
E J
EJ
Brown
S H
SH
Madden
M
M
Brown
K M
KM
Halkier
T
T
Mondorf
K
K
Xu
F
F
D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018388:Codon, Terminator; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D006835:Hydrazones / Q000378:metabolism; D042845:Laccase; D003904:Mitosporic Fungi / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000235:genetics
D018388:Codon, Terminator; D004271:DNA, Fungal; D006835:Hydrazones; C009370:syringaldazine; D010088:Oxidoreductases; D042845:Laccase; D000516:alpha-Amylases