High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris
2015
Yu, Shi | Li, Zhezhe | Wang, Yaping | Chen, Wanping | Fu, Lin | Tang, Wei | Chen, Chen | Liu, Yunyun | Zhang, Xue | Ma, Lixin
OBJECTIVES: A novel, high-level expression, thermostable mannan endo-1,4-beta-mannosidase is urgently needed for industrial applications. RESULTS: The mannan endo-1,4-β-mannosidase gene (MAN) from Aspergillus niger CBS 513.88 was optimized based on the codon usage bias in Pichia pastoris and synthesized by overlapping PCR to produce MAN-P. It was expressed in P. pastoris GS115 from a constitutive expression vector pHBM-905 M. MAN-P reached 594 mg/l in shake-flasks after 192 h induction. On production in a 5 l fermenter, the yield of MAN-P reached ~3.5 mg/ml and the enzyme activity was 1612 U/ml. The enzyme exhibited a maximum activity of 3049 U/ml at 80 °C and retained 60 % enzyme activity at 80 °C for 2 h. The pH optimum was 4.5 and the enzyme was stable over the pH range 1.5–11. CONCLUSION: The thermostability of MAN-P is higher than other known fungal mannanases and the expression and thermophilic properties make MAN-P useful for industrial applications.
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