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 β-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 β-mannanase. Among the parameters investigated in shaking flask cultures, the pH value of medium had significant influence on the production of β-mannanase by recombinant P. pastoris. β-Mannanase produced at pH 7.0 was 6.7 times of that at pH value of 6.0. The highest β-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 β-mannanase was purified and characterized. The purified β-mannanase produced by P. pastoris has optimum pH of 10.0 and optimum temperature of 70 °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 β-mannanase. These properties make the recombinant β-mannanase more useful in the detergent industries, the pulp and paper processing and other industrial processes.