Li S, Kim WD, Kaneko S, Prema PA, Nakajima M, et al.
Bioscience, biotechnology, and biochemistry •
Two putative alpha-galactosidase genes from rice (Oryza sativa L. var. Nipponbare) belonging to glycoside hydrolase family 27 were cloned and expressed in Escherichia coli. These enzymes showed alpha-galactosidase activity and were purified by Ni Sepharose column chromatography. Two purified recombinant alpha-galactosidases (alpha-galactosidase II and III; alpha-Gal II and III) showed a single protein band on SDS-PAGE with molecular mass of 42 kDa. These two enzymes cleaved not only alpha-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose, and stachyose, but also liberated the galactosyl residues attached to the O-6 position of the mannosyl residue at the reducing-ends of mannobiose and mannotriose. In addition, these enzymes clipped the galactosyl residues attached to the inner-mannosyl residues of mannopentaose. Thus, alpha-Gal II catalyzes efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.
Shibuya H, Nagasaki H, Kaneko S, Yoshida S, Park GG, et al.
Applied and environmental microbiology •
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.
