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

Background: Endo-1,4-beta-mannanase is an enzyme that can catalyze the random hydrolysis of beta-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-beta-mannanase gene from Penicillium oxalicum. Results: A gene encoding an acidophilic thermostable endo-1,4-beta-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-beta-mannanase (rPoMan5A) was tagged with 6 x 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 degrees C and pH 4.0, respectively. The rPoMan5A is highly thermostable with a half-life of approximately 58 h at 60 degrees 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 mu mol min(-1) mg(-1), 2.1 mg mL(-1) and 154.8 mu mol min(-1) mg(-1), and 2.3 mg mL(-1) and 18.9 mu mol min(-1) mg(-1), respectively. The enzymatic activity of rPoMan5A was not significantly affected by an array of metal ions, but was inhibited by Fe3+ and Hg2+. 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-beta-mannanase in Pichia pastoris is suitable for various biotechnology applications.