Inducible and constitutive expression of a novel thermostable alkaline β-mannanase from alkaliphilic Bacillus sp N16-5 in Pichia pastoris and characterization of the recombinant enzyme

A novel thermostable alkaline beta-mannanase from alkaliphilic Bacillus sp. N16-5 was expressed successfully in Pichia pastoris GS115. The combined usage of inducible and constitutive promoters (AOX1 and GAP) enhanced the expression of beta-mannanase. Among the parameters investigated in shaking flask cultures, the pH value of medium had significant influence on the production of beta-mannanase by recombinant P. pastoris. beta-Mannanase produced at pH 7.0 was 6.7 times of that at pH value of 6.0. The highest beta-mannanase activity of 32.2 IU/ml in culture supernatant was achieved at 120 h of cultivation in BMGY medium (pH 7.0). The recombinant beta-mannanase was purified and characterized. The purified beta-mannanase produced by P. pastoris has optimum pH of 10.0 and optimum temperature of 70 degrees C, which are very close to those of the native enzyme from alkaliphilic Bacillus sp. N16-5. However, much higher thermal stability and pH stability were observed in recombinant beta-mannanase. These properties make the recombinant beta-mannanase more useful in the detergent industries, the pulp and paper processing and other industrial processes. (C) 2008 Elsevier Inc. All rights reserved.