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A Recombinant Highly Thermostable β-Mannanase (ReTMan26) from Thermophilic Bacillus subtilis (TBS2) Expressed in Pichia pastoris and Its pH and Temperature Stability

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Abstract

A gene encoding a highly thermostable β-mannanase from a thermophilic Bacillus subtilis (TBS2) was successfully expressed in Pichia pastoris. The maximum activity of the recombinant thermostable β-mannanase (ReTMan26) was 5435 U/mL, which was obtained by high-density, fed-batch cultivation after 168-h induction with methanol in a 50-L bioreactor. The protein yield reached 3.29 mg/mL, and the protein had a molecular weight of ~42 kDa. After fermentation, ReTMan26 was purified using a 10-kDa cut-off membrane and Sephadex G-75 column. The pH and temperature optima of purified ReTMan26 were pH 6.0 and 60 °C, respectively, and the enzyme was stable at pH 2.0–8.0 and was active at 20–100 °C. HPLC analysis of the products of locust bean gum hydrolysis showed that the mannan-oligosaccharide content was 62.5%. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min, which was higher than any other β-mannanase reported up to now, suggesting its potential for industrial applications.

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Acknowledgements

This work was supported by the open project of Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University (grant no. KLIB-KF201510), and the Fundamental Research Funds for the Central Universities (grant no. JUSRP111A24), the 111 Project (No. 111-2-06).

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, No. 1800 Lihu Avenue, Wuxi, 214122, China

    Zhangcai Luo, Guoying Li, Yao Du & Xiaobin Yu

  2. School of Biological Sciences, Ludong University, Yantai, 264025, China

    Jing Miao

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Correspondence to Xiaobin Yu.

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Luo, Z., Miao, J., Li, G. et al. A Recombinant Highly Thermostable β-Mannanase (ReTMan26) from Thermophilic Bacillus subtilis (TBS2) Expressed in Pichia pastoris and Its pH and Temperature Stability. Appl Biochem Biotechnol 182, 1259–1275 (2017). https://doi.org/10.1007/s12010-017-2397-4

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