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Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from Caldicellulosiruptor bescii maximizes enzyme activity and irreversibly binds to amorphous cellulose

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Abstract

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo-1,4-β-glucanase CelD (85019 Da). The N-terminal region of the protein includes a signal peptide and a catalytic module of glycoside hydrolase family 5 (GH5), followed by a carbohydrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo-1,4-β-glucanase with maximum activity on barley β-glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4–11, temperatures of 0–75°C, and NaCl concentrations of 0–5 M. Only 100% formamide or 1% SDS have been able to remove the protein.

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Abbreviations

GH:

glycoside hydrolase

SP:

signal peptide

Cat5:

catalytic module of glycoside hydrolase family 5

CBM28:

carbohydrate-binding module of family 28

SLH:

surface-layer homology module

BCC:

bacterial crystalline cellulose

PAG:

polyacrylamide gel

CMC:

carboxymethyl cellulose

SDS:

sodium dodecyl sulfate

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Authors and Affiliations

  1. Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia

    G. A. Velikodvorskaya, L. A. Chekanovskaya, N. A. Lunina, I. A. Dvortsov & V. V. Zverlov

  2. All-Russia Research Institute for Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow, 127550, Russia

    O. V. Sergienko & V. G. Lunin

  3. Gamalei Research Institute for Epidemiology and Microbiology, Ministry of Healthcare of the Russian Federation, Moscow, 123098, Russia

    V. G. Lunin

  4. Department of Microbiology, Technische Universität München, Freising, 85350, Germany

    V. V. Zverlov

Authors
  1. G. A. Velikodvorskaya
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  2. L. A. Chekanovskaya
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  3. N. A. Lunina
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  4. O. V. Sergienko
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  5. V. G. Lunin
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  6. I. A. Dvortsov
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  7. V. V. Zverlov
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Correspondence to N. A. Lunina.

Additional information

Original Russian Text © G.A. Velikodvorskaya, L.A. Chekanovskaya, N.A. Lunina, O.V. Sergienko, V.G. Lunin, I.A. Dvortsov, V.V. Zverlov, 2013, published in Molekulyarnaya Biologiya, 2013, Vol. 47, No. 4, pp. 667–673.

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Velikodvorskaya, G.A., Chekanovskaya, L.A., Lunina, N.A. et al. Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from Caldicellulosiruptor bescii maximizes enzyme activity and irreversibly binds to amorphous cellulose. Mol Biol 47, 581–586 (2013). https://doi.org/10.1134/S0026893313040158

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  • DOI: https://doi.org/10.1134/S0026893313040158

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