Purification, characterization, and cDNA cloning of a novel alpha-galactosidase from Mortierella vinacea.
A novel alpha-galactosidase, designated alpha-galactosidase II, was isolated from the culture filtrate of Mortierella vinacea. The molecular size of the purified enzyme estimated by gel filtration was 60 kDa, which agreed with that, 51-62 kDa, estimated by SDS-PAGE. The enzyme was thermolabile at neutral pH, but the addition of BSA to the enzyme solution at the concentration of 0.01% increased its stability considerably. The enzyme appears to be novel because it showed a distinct substrate specificity from other microbial alpha-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan, that is, the enzyme liberated not only side-chain alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotetraose but also terminal alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotriose. In addition, the enzyme acted on galactomannans effectively. alpha-Galactosidase II cDNA was cloned and its nucleotides sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 376 amino acid residues with a molecular mass of 41,334 Da. The derived amino acid sequence of the enzyme showed 31-49% sequence similarity with those of alpha-galactosidases from other origins.
Shibuya
H
H
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Kobayashi
H
H
Sato
T
T
Kim
W S
WS
Yoshida
S
S
Kaneko
S
S
Kasamo
K
K
Kusakabe
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009090:Mucorales / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D000519:alpha-Galactosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018076:DNA, Complementary; D000519:alpha-Galactosidase