High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris
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A novel, high-level expression, thermostable mannan endo-1,4-beta-mannosidase is urgently needed for industrial applications.
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.
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.
KeywordsHigh-density fermentation High-level expression Industry Mannanase Oligosaccharides Pichia pastoris Thermophilic
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).
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.
- Fu L, Zhai C, Kang LX et al (2012) Cloning of the gene encoding endo-1,4-β-mannosidase from Bacillus subtilis HB002 and expression in Pichia pastoris. Sciencepaper Online.http://www.paper.edu.cn/releasepaper/content/201211-323. Accessed 19 Nov 2012