High-level production, purification and characterization of a thermostable β-mannanase from the newly isolated Bacillus subtilis WY34

A newly isolated Bacillus subtilis WY34 produced high level of mannanase (1105 U/ml) when grown on konjac powder as the carbon source at 50 degrees C. The beta-mannanase was purified 5.4-fold to homogeneity with a final recovery of 20.3% and a specificity of 8302.4 U/mg protein. The purified mannanase appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 39.6 kDa. It was identified as a glycoprotein by periodic acid-Schiff staining with a carbohydrate content of 13.1%. The first 10 N-terminal amino acid sequence (HTVSPVNPNA) of the purified enzyme showed high homology (90% identity) with the N-terminal region of P-mannanase from B. subtilis NM-39. The optimal temperature and pH for marmanase activity was 65 degrees C and pH 6.0, respectively. The marmanase activity was stable within pH 5.5-10.1. It was stable up to 60 degrees C at pH 6.0. The mannanase was highly specific towards locust bean gum, but exhibited very low activity towards starch, CMC and birchwood xylan. Apparent K values of the marmanase for locust bean gum, guar gum and konjac powder were 7.6, 10.5 and 27.4 mg ml(-1), respectively. Copra mannan was degraded mainly to mannotetraose, mannobiose and mannotriose when incubated with the marmanase, suggesting that the marmanase is an endomannanase and is suitable for mannooligosaccharide production. These unique properties of the purified P-mannanase from B. subtilis WY34 make this enzyme attractive for biotechnological applications. (c) 2006 Elsevier Ltd. All rights reserved.