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Recombination of Thermo-Alkalistable, High Xylooligosaccharides Producing Endo-Xylanase from Thermobifida fusca and Expression in Pichia pastoris

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Abstract

For xylooligosaccharide (XO) production, endo-xylanase from Thermobifida fusca was modified by error-prone PCR and DNA shuffling. The G4SM1 mutant (S62T, S144C, N198D, and A217V) showed the most improved hydrolytic activity and was two copies expressed in Pichia pastoris under the control of GAP promoter. The maximum xylanase activity in culture supernatants was 165 ± 5.5 U/ml, and the secreted protein concentration reached 493 mg/l in a 2-l baffled shake flask. After 6× His-tagged protein purification, the specific activity of G4SM1 was 2036 ± 45.8 U/mg, 2.12 times greater than that of wild-type enzyme. Additionally, G4SM1 was stable over a wide pH range from 5.0 to 9.0. Meanwhile, half-life of G4SM1 thermal inactivation at 70 °C increased 8.5-fold. Three-dimensional structures suggest that two amino acid substitutions, S62T and S144C, located at catalytic domain may be responsible for the enhanced activity and thermostability of xylanase. Xylobiose was the dominant end product of xylan hydrolysis by G4SM1. Due to its attractive biochemical properties, G4SM1 has potential value in commercial XO production.

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Acknowledgments

This work was supported in part by IAEA Coordinated Research Projects (No. 16327/R0), the National Natural Science Foundation of China (30971702), the Natural Science Foundation of Zhejiang Province (3090247), and research grants from the Science and Technology Department of Zhejiang Province, China (2010C32008).

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Authors and Affiliations

  1. Institute of Dairy Science, College of Animal Science, Zhejiang University, Hangzhou, 310058, China

    Qian Wang, Wen Du, Jia-Kun Wang & Jian-Xin Liu

  2. College of Life Science, Zhejiang University, Hangzhou, 310058, China

    Xiao-Yan Weng

  3. Department of Food Science, College of Life Science, China JiLiang University, Hangzhou, 310018, China

    Ming-Qi Liu

  4. Institute of Dairy Science, Zhejiang University, Yuhangtang Road 866#, Hangzhou, Zhejiang, China

    Jia-Kun Wang

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Correspondence to Jia-Kun Wang.

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Wang, Q., Du, W., Weng, XY. et al. Recombination of Thermo-Alkalistable, High Xylooligosaccharides Producing Endo-Xylanase from Thermobifida fusca and Expression in Pichia pastoris . Appl Biochem Biotechnol 175, 1318–1329 (2015). https://doi.org/10.1007/s12010-014-1355-7

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  • DOI: https://doi.org/10.1007/s12010-014-1355-7

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