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Expression and Characterization of Hyperthermotolerant Xylanases, SyXyn11P and SyXyn11E, in Pichia pastoris and Escherichia coli

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Abstract

Both Syxyn11P and Syxyn11E, two codon-optimized genes encoding glycoside hydrolase (GH) family 11 hyperthermotolerant xylanases (designated SyXyn11P and SyXyn11E), were synthesized and inserted into pPIC9KM and pET-28a(+) vectors, respectively. The resulting recombinant expression vectors, pPIC9KM-Syxyn11P and pET-28a(+)-Syxyn11E, were transformed into Pichia pastoris GS115 and Escherichia coli BL21, respectively. The maximum activities of two recombinant xylanases (reSyXyn11P and reSyXyn11E) expressed in P. pastoris and E. coli reached 30.9 and 17.8 U/ml, respectively. The purified reSyXyn11P and reSyXyn11E displayed the same pH optimum at 6.5 and pH stability at a broad range of 4.5–9.0. The temperature optimum and stability of reSyXyn11P were 85 and 80 °C, higher than those of reSyXyn11E, respectively. Their activities were not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn2+ and Ag+. The K m and V max of reSyXyn11P toward birchwood xylan were 4.3 mg/ml and 694.6 U/mg, whose K m was close to that (4.8 mg/ml), but whose V max was much higher than that (205.6 U/mg) of reSyXyn11E. High-performance liquid chromatography analysis indicated that xylobiose and xylotriose as the major products were excised from insoluble corncob xylan by reSyXyn11P.

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Acknowledgments

This work was supported by the National Nature Science Foundation of China (No. 31101229). The authors are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China

    Jianfang Li, Huimin Zhang, Chunjuan Wang & Yunhai Dong

  2. Wuxi Medical School, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China

    Minchen Wu

  3. School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China

    Lijuan Zhu & Peng Zhang

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  1. Jianfang Li
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  2. Huimin Zhang
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  4. Chunjuan Wang
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  5. Yunhai Dong
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  6. Lijuan Zhu
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  7. Peng Zhang
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Corresponding author

Correspondence to Minchen Wu.

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Jianfang Li and Huimin Zhang contributed equally to this work

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Li, J., Zhang, H., Wu, M. et al. Expression and Characterization of Hyperthermotolerant Xylanases, SyXyn11P and SyXyn11E, in Pichia pastoris and Escherichia coli . Appl Biochem Biotechnol 172, 3476–3487 (2014). https://doi.org/10.1007/s12010-014-0786-5

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  • DOI: https://doi.org/10.1007/s12010-014-0786-5

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