alpha-Galactosidase from cultured rice (Oryza sativa L. var. Nipponbare) cells.
The alpha-galactosidase from rice cell suspension cultures was purified to homogeneity by different techniques including affinity chromatography using N-epsilon-aminocaproyl-alpha-D-galactopyranosylamine as the ligand. From 11 l of culture filtrate, 28.7 mg of purified enzyme was obtained with an overall yield of 51.9%. The cDNA coding for the alpha-galactosidase was cloned and sequenced. The enzyme was found to contain 417 amino acid residues composed of a 55 amino acid signal sequence and 362 amino acid mature alpha-galactosidase; the molecular weight of the mature enzyme was thus calculated to be 39,950. Seven cysteine residues were also found but no putative N-glycosylation sites were present. The observed homology between the deduced amino acid sequences of the mature enzyme and alpha-galactosidases from coffee (Coffea arabica), guar (Cyamopsis tetragonolooba), and Mortierella vinacea alpha-galactosidase II were over 73, 72, and 45%, respectively. The enzyme displayed maximum activity at 45 degrees C when p-nitrophenyl-alpha-D-galactopyranoside was used as substrate. The rice alpha-galactosidase and Mortierella vinacea alpha-galactosidase II acted on both the terminal alpha-galactosyl residue and the side-chain alpha-galactosyl residue of the galactomanno-oligosaccharides.
Kim
Wook-Dong
WD
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305-8572, Japan.
Kobayashi
Osamu
O
Kaneko
Satoshi
S
Sakakibara
Yoshikiyo
Y
Park
Gwi Gun
GG
Kusakabe
Isao
I
Tanaka
Hideo
H
Kobayashi
Hideyuki
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008969:Molecular Sequence Data; D012275:Oryza / Q000166:cytology / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D000519:alpha-Galactosidase