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Heterologous expression and biochemical characterization of a thermostable endo-β-1,4-glucanase from Colletotrichum orchidophilum

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Abstract

To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55–75 °C and high thermostability (about 85% residual activity) at the temperature of 55 °C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0–5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0–6.0) at 4 °C for 24 h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and β-d-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters Vmax, Km, and Kcat/Km values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against β-d-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.

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Acknowledgements

This work was supported by the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang, P. R. China (2018R01014).

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No. 18 Chaowang Road, Hangzhou, 310014, Zhejiang, People’s Republic of China

    Hai-Yan Zhou, Jian-Bao Zhou, Xiao-Nan Yi, Yan-Mei Wang, Ya-Ping Xue, Zhi-Qiang Liu & Yu-Guo Zheng

  2. The National and Local, Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Hai-Yan Zhou, Jian-Bao Zhou, Xiao-Nan Yi, Yan-Mei Wang, Ya-Ping Xue, Zhi-Qiang Liu & Yu-Guo Zheng

  3. Zhejiang Huakang Pharmaceutical Co., LTD, 18 Huagong Road, Huabu Town, Kaihua, 324302, People’s Republic of China

    De-Shui Chen, Xin-Ping Cheng, Mian Li, Hong-Yan Wang & Kai-Qian Chen

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  1. Hai-Yan Zhou
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Zhou, HY., Zhou, JB., Yi, XN. et al. Heterologous expression and biochemical characterization of a thermostable endo-β-1,4-glucanase from Colletotrichum orchidophilum. Bioprocess Biosyst Eng 44, 67–79 (2021). https://doi.org/10.1007/s00449-020-02420-7

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