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Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein

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Abstract

A xyloglucanase of the GH74 family was identified in the thermophilic fungus strain Myceliophthora thermophila VKPM F-244, and its gene sequence was optimized for cloning and expression in Pichia pastoris. The recombinant xyloglucanase MtXgh74 exhibited the highest activity toward tamarind seed xyloglucan with a K M value of 0.51 ± 0.06 mg/mL. The activities on barley β-glucan and carboxymethylcellulose were about 4 and 2%, respectively, compared to xyloglucan. Maximum xyloglucanase activity was observed at 70–75 °C and pH 6.5. After pre-incubation at 50 °C, pH 6.0 for 3 h, the enzyme retained 100% of its activity. The half-life of MtXgh74 at 60 °C, pH 6.0 was 40 min. In P. pastoris, MtXgh74 was produced in glycosylated form. The enzyme production in a 1 L bioreactor resulted in a yield of 118 U/mL or 5.3 g/L after 51 h fermentation. Kinetic studies of the hydrolysis product formation suggest that MtXgh74 has an endo-processive mode of action. The final products were the standard xyloglucan building blocks XXXG, XXLG, XLXG, and XLLG. Additionally, MtXgh74 hydrolyzed various linkages within the xyloglucan building blocks XXXG, XXLG, and XLXG (except XLLG) producing diverse low molecular weight oligosaccharides which may be identified by MALDI-TOF as XG, XX, XXG/GXX/XGX, XXX, LG, LX/XL, XLX/XXL, LLG, GXXXG, GXLLG, XLLGX. The unique combination of different activities within one enzyme along with its high thermostability and specificity toward xyloglucan makes MtXgh74 a promising candidate enzyme for industrial applications.

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Author notes

  1. Oksana V. Berezina, Jonathan Herlet, and Sergey V. Rykov contributed equally to this work.

Authors and Affiliations

  1. State Research Institute of Genetics and Selection of Industrial Microorganisms, 1-st Dorojniy pr. 1, Moscow, Russian Federation, 117545

    Oksana V. Berezina, Sergey V. Rykov, Artem Zavyalov, Dmitriy Kozlov, Liliya Sakhibgaraeva, Irina Krestyanova & Sergey V. Yarotsky

  2. Department of Microbiology, Technical University Munich, Emil-Ramann-Str. 4, 85354, Freising, Germany

    Jonathan Herlet, Petra Kornberger, Wolfgang H. Schwarz, Vladimir V. Zverlov & Wolfgang Liebl

  3. Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq. 2, Moscow, Russian Federation, 123182

    Vladimir V. Zverlov

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  1. Oksana V. Berezina
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  2. Jonathan Herlet
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Correspondence to Oksana V. Berezina.

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The article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This study was funded by the Ministry of Education and Science of the Russian Federation (grant number 14.628.21.0001, identification number RFMEFI62814X0001) and the German Federal Ministry of Education and Research (grant number FKZ: 031A556).

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Berezina, O.V., Herlet, J., Rykov, S.V. et al. Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein. Appl Microbiol Biotechnol 101, 5653–5666 (2017). https://doi.org/10.1007/s00253-017-8297-2

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