Cloning and high-level expression of alpha-galactosidase cDNA from Penicillium purpurogenum.
The cDNA coding for Penicillium purpurogenum alpha-galactosidase (alphaGal) was cloned and sequenced. The deduced amino acid sequence of the 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 acid sequence of the enzyme showed similarity to eukaryotic alphaGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.
Shibuya
H
H
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
Nagasaki
H
H
Kaneko
S
S
Yoshida
S
S
Park
G G
GG
Kusakabe
I
I
Kobayashi
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000519:alpha-Galactosidase