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

BMC Biotechnology

Yangchun Xu¹  Qirong Shen¹  Waseem Raza¹  Dongyang Liu¹  Zhong Wei¹  Haiping Zheng¹  Shuixian Li¹  Hanpeng Liao¹ 

[1]Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, China

关键词: Penicillium oxalicum;    Pichia pastoris;    Expression system;    Gene cloning;    Endo-1,4-β-mannanase;   

Others  :  1084479
DOI  :  10.1186/s12896-014-0090-z

 received in 2014-07-12, accepted in 2014-10-09,  发布年份 2014

【 摘 要 】

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 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-β-mannanase in Pichia pastoris is suitable for various biotechnology applications.

【 授权许可】

   
2014 Liao et al.; licensee BioMed Central Ltd.

【 图 表 】

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