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Background  Endo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries.  Penicillium oxalicum  is a powerful hemicellulase-producing fungus (Bioresour Technol 123:117-124, 2012); however, few previous studies have focused on the cloning and expression of the endo-1,4-β-mannanase gene from  Penicillium oxalicum .    Results  A gene encoding an acidophilic thermostable endo-1,4-β-mannanase (E.C. 3.2.1.78) from  Penicillium oxalicum  GZ-2, which belongs to glycoside hydrolase family 5, was cloned and successfully expressed in  Pichia pastoris  GS115. A high enzyme activity (84.4 U mL −1 ) was detected in the culture supernatant. The recombinant endo-1,4-β-mannanase (rPoMan5A) was tagged with 6 × His at its C-terminus and purified using a Ni-NTA Sepharose column to apparent homogeneity. The purified rPoMan5A showed a single band on SDS-PAGE with a molecular mass of approximately 61.6 kDa. The specific activity of the purified rPoMan5A was 420.9 U mg −1  using locust bean gum as substrate. The optimal catalytic temperature (10 min assay) and pH value for rPoMan5A are 80°C and pH 4.0, respectively. The rPoMan5A is highly thermostable with a half-life of approximately 58 h at 60°C at pH 4.0. The  K   m   and  V   max   values for locust bean gum, konjac mannan, and guar gum are 7.6 mg mL −1  and 1425.5 μmol min −1  mg −1 , 2.1 mg mL −1  and 154.8 μmol min −1  mg −1 , and 2.3 mg mL −1  and 18.9 μmol min −1  mg −1 , respectively. The enzymatic activity of rPoMan5A was not significantly affected by an array of metal ions, but was inhibited by Fe 3+  and Hg 2+ . Analytical results of hydrolytic products showed that rPoMan5A could hydrolyze various types of mannan polymers and released various mannose and manno-oligosaccharides, with the main products being mannobiose, mannotriose, and mannopentaose.    Conclusion  Our study demonstrated that the high-efficient expression and secretion of acid stable and thermostable recombinant endo-1, 4-β-mannanase in  Pichia pastoris  is suitable for various biotechnology applications.

bmc biotechnology

A new acidophilic thermostable endo-1,4-β-mannanase from Penicillium oxalicum GZ-2: cloning, characterization and functional expression in Pichia pastoris