alpha-Glucosidase from a strain of deep-sea Geobacillus: a potential enzyme for the biosynthesis of complex carbohydrates.
An alpha-glucosidase from Geobacillus sp. strain HTA-462, one of the deepest sea bacteria isolated from the sediment of the Mariana Trench, was purified to homogeneity and estimated to be a 65-kDa protein by SDS-PAGE. At low ion strength, the enzyme exists in the homodimeric form (130 kDa). It is a thermo- and alkaline-stable enzyme with a half-life of 13.4 h and a maximum hydrolytic activity at 60 degrees C and pH 9.0 in 15 mM glycine-NaOH buffer. The enzyme exclusively hydrolyzed alpha-1,4-glycosidic linkages of oligosaccharides in an exo-type manner. The enzyme had an overwhelming transglycosylation activity and glycosylated various non-sugar molecules when maltose was used as a sugar donor. It converted maltose to isomaltose. The gene encoding the enzyme was cloned and sequenced. The recombinant enzyme could be extracellularly overproduced by Bacillus subtilis harboring its gene and preserved the primary properties of the native enzyme. Site-directed mutagenesis experiments showed that Asp98 is essential for the enzyme activity in addition to Asp199, Asp326, and Glu256.
Hung
Vo Si
VS
Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, Kanagawa, 237-0061, Japan. vohungsi@jamstec.go.jp
Hatada
Yuji
Y
Goda
Saori
S
Lu
Jie
J
Hidaka
Yuko
Y
Li
Zhijun
Z
Akita
Masatake
M
Ohta
Yukari
Y
Watanabe
Kenji
K
Matsui
Hirokazu
H
Ito
Susumu
S
Horikoshi
Koki
K
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D004795:Enzyme Stability; D019015:Geologic Sediments / Q000382:microbiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D000520:alpha-Glucosidases