Expression of rice (Oryza sativa L. var. Nipponbare) alpha-galactosidase genes in Escherichia coli and characterization.
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.
Li
Suhong
S
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Kim
Wook-Dong
WD
Kaneko
Satoshi
S
Prema
Prukutty A
PA
Nakajima
Mitsutoshi
M
Kobayashi
Hideyuki
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D030541:Databases, Genetic; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D000519:alpha-Galactosidase