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.