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Expression of Bacillus subtilis MA139 β-mannanase in Pichia pastoris and the Enzyme Characterization

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Abstract

The 1014 nucleotides long gene-encoding β-mannanase from Bacillus subtilis strain MA139 was cloned using PCR. To obtain high expression levels in Pichia pastoris, the β-mannanase gene was optimized according to the codon usage bias of P. pastoris and fused downstream of GAP promoter. The reconstituted plasmid pGAP-mann was transformed into P. pastoris X-33 strain to constitutively express β-mannanase. When cultured at 28 °C for 3 days protein yields up to 2.7 mg/mL was obtained with the enzyme activity of up to 230 U/mL. In comparison, wild-type gene product yielded 1.9 mg/mL and 170 U/mL, respectively indicating that the protein yield and enzyme activity were significantly improved by codon modification. After purification, the enzyme properties were characterized. The optimal activity was at pH 6.0 and 50 °C. In the pH range of 3.0 to 9.0, β-mannanase showed above 60% of its peak activity. Among the numerous ions tested copper significantly inhibited the enzyme activity. These results suggested that codon-optimized β-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

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Acknowledgements

This work was supported by the National High Technology Research and Development Program (2007AA100601), the Program for New Century Excellent Talents in University (NCET-07-0807), and the Project of State Key Laboratory of Animal Nutrition (2004DA125184 (team) 0806).

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Authors and Affiliations

  1. College of Animal Science and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, People’s Republic of China

    Jiayun Qiao, Bing Dong & Yunhe Cao

  2. China National Feed Quality Supervision and Testing Center, Beijing, 100081, People’s Republic of China

    Zhenghua Rao

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  1. Jiayun Qiao
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  2. Zhenghua Rao
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  3. Bing Dong
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  4. Yunhe Cao
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Correspondence to Bing Dong or Yunhe Cao.

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Qiao, J., Rao, Z., Dong, B. et al. Expression of Bacillus subtilis MA139 β-mannanase in Pichia pastoris and the Enzyme Characterization. Appl Biochem Biotechnol 160, 1362–1370 (2010). https://doi.org/10.1007/s12010-009-8688-7

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  • DOI: https://doi.org/10.1007/s12010-009-8688-7

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