High-level production of a cold-active B-mannanase from Bacillus subtilis Bs5 and its molecular cloning and expression

Mannanases can be useful in the food, feed, pulp and paper industries. In this research a Bacillus subtilis strain (named Bs5) which produced hign-level β-mannanase was isolated. Maximum level of β-mannanase (1231.41 U/mL) was reached when B. subtilis Bs5 was grown on konjac powder as the carbon source for nine hours at 32°C. The β-mannanase was a typical cold-active enzyme and its optimal temperature of 35°C was the lowest among those of the known mannanases from bacteria. In addition, the optimal pH was 5.0 and much wide pH range from 3.0–8.0 was also observed in the β-mannanase. These properties make the β-mannanase more attractive for biotechnological applications. The DNA sequence coding the β-mannanase was cloned and the open reading frame consisted of 1089 bp encoding 362 amino acids. A phylogenetic tree of the β-mannanase based on the similarity of amino acid sequences revealed that the β-mannanase formed a cluster with the β-mannanases of B. subtilis, which was separated from the mannanases of fungi and other bacteria The β-mannanase gene could be expressed in Escherichia coli and the recombinant β-mannanase was characterized by western blot. This study provided a new source of carbohydrate hydrolysis enzyme with novel characteristics from B. subtilis.