Characterization and gene cloning of a novel beta-mannanase from alkaliphilic Bacillus sp. N16-5.
An alkaline beta-mannanase was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme had optimum activity at pH 9.5 and 70 degrees C. It was composed of a single polypeptide chain with a molecular weight of 55 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 4.3. The enzyme efficiently hydrolyzed galactomannan and glucomannan, producing a series of oligosaccharides and monosaccharides. The beta-mannanase gene (manA) contained an open reading frame (ORF) of 1,479 bp, encoding a 32-amino acids signal peptide, and a mature protein of 461 amino acids, with a calculated molecular mass of 50,743 Da. Strain N16-5 ManA, deduced from the manA ORF, exhibited relatively high amino acid similarity to the members of the glycosyl hydrolase family 5. The eight conserved active-site amino acids in family 5 glycosyl hydrolase were found in the deduced amino acid sequence of strain N16-5 ManA.
Ma
Yanhe
Y
Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, China.
Xue
Yanfen
Y
Dou
Yuetan
Y
Xu
Zhenghong
Z
Tao
Wenyi
W
Zhou
Peijin
P
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004269:DNA, Bacterial; D008670:Metals; D044902:beta-Mannosidase