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Characterization of inulolytic enzymes from the Jerusalem artichoke–derived Glutamicibacter mishrai NJAU-1

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Abstract

The rhizosphere context of inulin-accumulating plants, such as Jerusalem artichoke (Helianthus tuberosus), is an ideal starting basis for the discovery of inulolytic enzymes with potential for bio fructose production. We isolated a Glutamicibacter mishrai NJAU-1 strain from this context, showing exo-inulinase activity, releasing fructose from fructans. The growth conditions (pH 9.0; 15 °C) were adjusted, and the production of inulinase by Glutamicibacter mishrai NJAU-1 increased by 90% (0.32 U/mL). Intriguingly, both levan and inulin, but not fructose and sucrose, induced the production of exo-inulinase activity. Two exo-inulinase genes (inu1 and inu2) were cloned and heterologously expressed in Pichia pastoris. While INU2 preferentially hydrolyzed longer inulins, the smallest fructan 1-kestose appeared as the preferred substrate for INU1, also efficiently degrading nystose and sucrose. Active site docking studies with GFn- and Fn-type small inulins (G is glucose, F is fructose, and n is the number of β (2–1) bound fructose moieties) revealed subtle substrate differences between INU1 and INU2. A possible explanation about substrate specificity and INU’s protein structure is then suggested.

Key points

• A Glutamicibacter mishrai strain harbored exo-inulinase activity.

• Fructans induced the inulolytic activity in G. mishrai while the inulolytic activity was optimized at pH 9.0 and 15 °C.

• Two exo-inulinases with differential substrate specificity were characterized.

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All relevant data are within the manuscript.

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Acknowledgements

We thank Plant editors for valuable comments regarding the manuscript text. This research was supported by grants from the National Key Research and Development Program of China (2020YFD0900704), the Guidance Foundation from the Sanya Institute of Nanjing Agricultural University (NAUSY-MS16), Foreign Experts Project (X202006), Six Talent Peaks Project in Jiangsu Province (SWYY-058), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD program, 809001). The authors acknowledge Arnout Voet (Laboratory of Biomolecular Modelling and Design, KU Leuven, Belgium) for providing access to GOLD and MOE software. The authors acknowledge FWO Vlaanderen for the financial support.

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Authors and Affiliations

  1. Jiangsu Key Lab of Marine Biology, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China

    Dan Lian, Shuo Zhuang, Chen Shui, Shicheng Zheng & Mingxiang Liang

  2. Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, Jiangsu, China

    Yanhong Ma

  3. College of Grassland and Environmental Sciences, Xinjiang Agricultural University, Ürümqi, 830052, Xinjiang, China

    Zongjiu Sun

  4. Laboratory of Molecular Plant Biology and KU Leuven Plant Institute, KU Leuven, Kasteelpark Arenberg 31, 3001, Louvain, Belgium

    Jaime R. Porras-Domínguez & Wim Van den Ende

  5. Department of Bioengineering, Faculty of Engineering, Marmara University, Istanbul, 34722, Turkey

    Ebru Toksoy Öner

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  1. Dan Lian
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  2. Shuo Zhuang
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  3. Chen Shui
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  4. Shicheng Zheng
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  5. Yanhong Ma
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  6. Zongjiu Sun
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  7. Jaime R. Porras-Domínguez
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  8. Ebru Toksoy Öner
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  9. Mingxiang Liang
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  10. Wim Van den Ende
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Contributions

Liang MX: conceptualization, supervision and project administration. Lian D, Zhuang S, Shui C, Zheng SC: investigation. Lian D: methodology and writing—original draft preparation. Ma YH, Sun ZJ, Toksoy Öner E, Van den Ende W: resources, writing—reviewing and editing. Jaime R. Porras-Domínguez: modeling and docking.

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Correspondence to Mingxiang Liang.

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Lian, D., Zhuang, S., Shui, C. et al. Characterization of inulolytic enzymes from the Jerusalem artichoke–derived Glutamicibacter mishrai NJAU-1. Appl Microbiol Biotechnol 106, 5525–5538 (2022). https://doi.org/10.1007/s00253-022-12088-6

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