LACCASE FROM THE WHITE-ROT FUNGUS TRAMETES-VERSICOLOR - CDNA CLONING OF ICC1 AND EXPRESSION IN PICHIA-PASTORIS

A cDNA coding for laccase was isolated from the ligninolytic fungus Tr ametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, w hich encodes a laccase isoenzyme of 498 amino-acid residues preceded b y a 22-residue signal peptide. The lcc1 cDNA was cloned into the vecto r pHIL-D2 for expression in Pichia pastoris under the control of the A OX1 promoter. Transformants were found to secrete active recombinant e nzyme after induction with methanol. The use of growth medium buffered to pH 6.0 and control of pH during cultivation were found to be impor tant, or even necessary, for obtaining activity in liquid cultures. Th e effect of exchanging the native secretion signal for the Saccharomyc es cerevisiae alpha-factor pre-pro secretion signal was studied by clo ning the portion encoding the mature enzyme into the vector pPIC9. The activity obtained for the construct encoding the native laccase signa l sequence was found to be seven-fold higher than for the construct en coding the alpha-factor secretion signal. Utilisation of the P. pastor is pep4 mutant strain SMD1168 was found to provide a two-fold higher l evel of activity compared with P. pastoris GS115.