A novel beta-mannanase with high specific activity from Bacillus circulans CGMCC1554: gene cloning, expression and enzymatic characterization.
A DNA fragment of 2,042 bp containing a novel beta-mannanase gene, man5A, was identified from the genome of the mannan-degrading bacterium Bacillus circulans CGMCC1554. The open reading frame of man5A comprised 978 bp encoding a protein of 326 amino acids with a predicted molecular weight of 32 kDa. The amino acid sequence of the encoded mannanase, MAN5A, showed the highest identity (78.5%) to beta-mannanases belonging to glycosyl hydrolases family 5. The gene man5A was efficiently expressed in Escherichia coli and Pichia pastoris with the highest activity of 541 U/ml in a 3-L fermenter. Recombinant MAN5A purified from E. coli had a high specific activity of 4,839 U/mg, which is much higher than that of enzymes that showed high sequence identity. The enzyme showed maximum activity at pH 7.6 and 60 degrees C and resistance to trypsin. After hydrolysis of LBG, oligomannosides accounted for 76% of the hydrolysis products. All these properties collectively make MAN5A a better candidate than current mannanases for use in the food and feed industry.
Yang
Peilong
P
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
Li
Yanan
Y
Wang
Yaru
Y
Meng
Kun
K
Luo
Huiying
H
Yuan
Tiezheng
T
Bai
Yingguo
Y
Zhan
Zhichun
Z
Yao
Bin
B
D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D044902:beta-Mannosidase