High-level production of a cold-active B-mannanase from Bacillus subtilis BS5 and its molecular cloning and...

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