Gene cloning, expression, and characterization of a novel beta-mannanase from Bacillus circulans CGMCC 1416.
A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoded 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with endo-beta-1,4-D-mannanase from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by Ni2+ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa beta-mannanase having a specific activity of 481.55 U/mg. The optimal activity of the purified protein, MANB48, was at 58 degrees C and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.
Li
Yanan
Y
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Yang
Peilong
P
Meng
Kun
K
Wang
Yaru
Y
Luo
Huiying
H
Wu
Ningfeng
N
Fan
Yunliu
Y
Yao
Bin
B
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001709:Biotechnology; D003001:Cloning, Molecular; D003470:Culture Media; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005685:Galactans / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D053149:Plant Gums / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003470:Culture Media; D005685:Galactans; D008351:Mannans; D053149:Plant Gums; D011993:Recombinant Fusion Proteins; D044902:beta-Mannosidase; C017471:locust bean gum