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 f rom the thermophilic fungus Myceliophthora thermophila, and the nucleo tide sequence of this gene was determined. The deduced amino acid sequ ence of M. thermophila laccase (MtL) shows homology to laccases from d iverse fungal genera. A vector containing the M. thermophila laccase c oding region, under transcriptional control of an Aspergillus oryzae a -amylase gene promoter and terminator, was constructed for heterologou s expression in A. oryzae. The recombinant laccase expressed in A. ory zae was purified to electrophoretic homogeneity by anion-exchange chro matography. Amino-terminal sequence data suggests that MtL is synthesi zed as a preproenzyme. The molecular mass was estimated to be approxim ately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Car bohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copp er oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper at oms per subunit. With syringaldazine as a substrate, MtL has optimal a ctivity at pH 6.5 and retains nearly 100% of its activity when incubat ed at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.