Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in Bacillus subtilis and characterization of the recombinant enzyme.
A novel alkaline mannanase Man26A has been found in the culture of an alkaliphilic Bacillus sp. strain JAMB-750 and the optimal pH for the mannanase activity of the enzyme was around pH 10 (J Biol Macromol 4: 67-74, 2004). This optimal pH is the highest among those of the mannanases reported to date. The gene man26A coding the enzyme was cloned from the genomic DNA of strain JAMB-750 and sequenced. It encodes a protein of 997 amino acids including a signal peptide. The N-terminal half (Glu27-Val486) of the enzyme exhibited moderate similarities to other mannanases belonging to glycoside hydrolase family 26, such as the enzymes from Cellvibrio japonicus (37% identity), Cellulomonas fimi (33% identity), and Bacillus sp. strain AM-001 (28% identity). The C-terminal half was found to contain four domains. The first, second, third, and fourth domains exhibited similarities to the carbohydrate-binding module, the mannan-binding module, the Homo sapiens collagen type IX alpha I chain, and the membrane anchor region of Gram-positive surface proteins, respectively. Its recombinant mannanase was produced extracellularly using Bacillus subtilis as the host. The optimal pH for the mannanase activity of the recombinant enzyme was around pH 10. The enzyme was very resistant to surfactants, for example, SDS up to 2.0% (w/v).
Hatada
Yuji
Y
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka 237-0061, Japan. hataday@jamstec.go.jp
Takeda
Nobuhiro
N
Hirasawa
Kazumichi
K
Ohta
Yukari
Y
Usami
Ron
R
Yoshida
Yasuhiko
Y
Grant
William D
WD
Ito
Susumu
S
Horikoshi
Koki
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001407:Bacillus / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D044902:beta-Mannosidase / Q000737:chemistry*
D000596:Amino Acids; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D044902:beta-Mannosidase