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Biotechnology Letters

, Volume 37, Issue 9, pp 1853–1859 | Cite as

High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris

  • Shi Yu
  • Zhezhe Li
  • Yaping Wang
  • Wanping Chen
  • Lin Fu
  • Wei Tang
  • Chen Chen
  • Yunyun Liu
  • Xue Zhang
  • Lixin Ma
Original Research Paper

Abstract

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.

Keywords

High-density fermentation High-level expression Industry Mannanase Oligosaccharides Pichia pastoris Thermophilic 

Notes

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Nos. 31100057, 31172320 and 31340036); the National 973 Program of China (No. 2013CB910801); the Nature Science Fund for Creative Research Groups of Hubei Province of China (No. 2012FFA034); and the Foundation for High and New Technology Industrial Innovative Research Groups of the Wuhan Science and Technology Bureau’s Department (No. 2014070504020239).

Supporting information

Supplementary Fig. 1—Effect of Endo H on the activity (filled circle) at different temperatures of MAN-P. MAN-P was incubated with Endo H at 37 °C for 3 h, diluted in a buffer of 0.1 M citric acid and 0.2 M Na2HPO4 (pH 5.0), and then incubated at different temperatures for 5 min. The enzyme activity at 80 °C was taken as 100 %. The absolute enzyme activity corresponding to 100 % was 334.8 U/ml. Values are the means of three independent experiments ± standard deviations.

Supplementary Table 1-Primers used for synthesis of the MAN-P gene.

Supplementary material

10529_2015_1848_MOESM1_ESM.pdf (34 kb)
Supplementary material 1 (PDF 33 kb)
10529_2015_1848_MOESM2_ESM.pdf (36 kb)
Supplementary material 2 (PDF 35 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Shi Yu
    • 1
  • Zhezhe Li
    • 1
  • Yaping Wang
    • 1
  • Wanping Chen
    • 1
  • Lin Fu
    • 1
  • Wei Tang
    • 1
  • Chen Chen
    • 1
  • Yunyun Liu
    • 1
  • Xue Zhang
    • 1
  • Lixin Ma
    • 1
  1. 1.Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life SciencesHubei UniversityWuhanPeople’s Republic of China

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