CLONING AND HIGH-LEVEL EXPRESSION OF ALPHA-GALACTOSIDASE CDNA FROM PENICILLIUM-PURPUROGENUM

The cDNA coding for Penicillium purpurogenum alpha-galactosidase (alph a Gal) was cloned and sequenced. The deduced amino acid sequence of th e alpha-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino ac id sequence of the enzyme showed similarity to eukaryotic alpha Gals f rom plants, animals, yeasts, and filamentous fungi. The highest simila rity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yea st GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximatel y 0.2 g/liter. The recombinant enzyme purified to homogeneity was high ly glycosylated, showed slightly higher specific activity, and exhibit ed properties almost identical to those of the native enzyme from P. p urpurogenum in terms of the N-terminal amino acid sequence, thermoacti vity, pH profile, and mode of action on galacto-oligosaccharides.