Characterization, Gene Cloning, and Heterologous Expression of β-Mannanase from a Thermophilic Bacillus subtilis

Bacillus subtilis BCC41051 producing a thermostable beta-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular beta-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pI value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60 degrees C, respectively. The enzyme was stable over a pH range of 5.0-11.5, and at temperatures of up to 60 degrees C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg2+ (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).