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

摘要

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 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 32.2IU/ml in culture supernatant was achieved at 120h 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.