Skip to main content
SpringerLink
Log in

The Preparation and Properties of Recombinant Forms of GH74 Family Xyloglucanase from the Trichoderma reesei Fungus

  • PRODUCERS, BIOLOGY, SELECTION, AND GENE ENGINEERING
  • Published:
Applied Biochemistry and Microbiology Aims and scope Submit manuscript

Abstract

Cloning and expression of the full-length endo-processive-type xyloglucanase from the Trichoderma reesei (TrXeg74A) fungus, as well as its catalytic domain TrXeg74A-CD, in the Penicillium verruculosum B1-537 recipient strain have been carried out. P. verruculosum is a highly effective producer of cellulases. The levels of protein secretion after culturing the obtained recombinant strains in a laboratory fermenter were 35.4 and 31.4 g/L, respectively. TrXeg74A accounted for at least 30% of the total protein, while TrXeg74A-CD was expressed to a much lesser extent. Both forms of the recombinant enzyme were isolated in purified state and their properties were studied. TrXeg74A and TrXeg74A-CD were characterized by a similar degree of processivity when exposed to tamarind xyloglucan and the same Michaelis constant (0.35-0.38 g/L), close to that for the native enzyme (0.30 g/L), while the catalytic constant for TrXeg74A-CD was 1.5 times higher than the corresponding parameter for full-length xyloglucanase. The obtained new recombinant P. verruculosum strains can be useful in the development of composite enzyme preparations for efficient hydrolysis of renewable lignocellulosic raw materials.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.

Similar content being viewed by others

REFERENCES

  1. Rashmi, R. and Siddalingamurthy, K.R., Microbial xyloglucanases: a comprehensive review, Biocatal. Biotransf., 2018, vol. 36, no. 4, pp. 280–295. https://doi.org/10.1080/10242422.2017.1417394

    Article  CAS  Google Scholar 

  2. Zavyalov, A.V., Rykov, S.V., Lunina, N.A., Sushkova, V.I., Yarotsky, S.V., and Berezina, O.V., Plant polysaccharide xyloglucan and enzymes that hydrolyze it, Russ. J. Bioorg. Chem., 2019, vol. 45, no. 7, pp. 845–859. https://doi.org/10.1134/S1068162019070148

    Article  CAS  Google Scholar 

  3. Attia, M.A. and Brumer, H., Recent structural insights into the enzymology of the ubiquitous plant cell wall glycan xyloglucan, Curr. Opin. Struct. Biol., 2016, vol. 40, no. 5, pp. 43–53. https://doi.org/10.1016/j.sbi.2016.07.005

    Article  CAS  PubMed  Google Scholar 

  4. Lopes, D.C.B., Carraro, C.B., Silva, R.N., and de Paula, R.G., Molecular characterization of xyloglucanase cel74a from Trichoderma reesei, Int. J. Mol. Sci., 2021, vol. 22, p. 4545. https://doi.org/10.3390/ijms22094545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Grishutin, S.G., Gusakov, A.V., Markov, A.V., Ustinov, B.B., Semenova, M.V., and Sinitsyn, A.P., Specific xyloglucanases as a new class of polysaccharide-degrading enzymes, Biochim. Biophys. Acta, Gen. Subj., 2004, vol. 1674, no. 3, pp. 268–281. https://doi.org/10.1016/j.bbagen.2004.07.001

    Article  CAS  Google Scholar 

  6. Matsuzawa, T. and Yaoi, K., GH74 xyloglucanases: structures and modes of activity, Trends Glycosci. Glycotechnol., 2016, vol. 28, pp. E63–E70. https://doi.org/10.4052/tigg.1510.1E

    Article  Google Scholar 

  7. Arnal, G., Stogios, P.J., Asohan, J., Skarina, T., Savchenko, A., and Brumer, H., Structural enzymology reveals the molecular basis of substrate regioselectivity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase, Biochem. J., 2018, vol. 475, no. 24, pp. 3963–3978. https://doi.org/10.1042/BCJ20180763

    Article  CAS  PubMed  Google Scholar 

  8. Arnal, G., Stogios, P.J., Asohan, J., Attia, M.A., Skarina, T., Viborg, A.H., Henrissat, B., Savchenko, A., and Brumer, H., Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74, J. Biol. Chem., 2019, vol. 294, no. 36, pp. 13233–13247. https://doi.org/10.1074/jbc.RA119.009861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gusakov, A.V., Additional sequence and structural characterization of an endo-processive GH74 xyloglucanase from Myceliophthora thermophila and the revision of the EC 3.2.1.155 entry, Biochim. Biophys. Acta, Gen. Subj., 2020, vol. 1864, no. 3, p. 129511. https://doi.org/10.1016/j.bbagen.2020.129511

    Article  CAS  PubMed  Google Scholar 

  10. Teeri, T.T., Crystalline cellulose degradation: new insight into the function of cellobiohydrolases, Trends Biotechnol., 1997, no. 5, pp. 160–167. https://doi.org/10.1016/S0167-7799(97)01032-9

  11. Sinitsyn, A.P., Sinitsyna, O.A., Zorov, I.N., and Rozhkova, A.M., Exploring the capabilities of the Penicillium verruculosum expression system for the development of producers of enzymes for the effective degradation of renewable plant biomass: a review, Appl. Biochem. Microbiol., 2020, no. 6, pp. 638–646. https://doi.org/10.1134/S0003683820060162

  12. Volkov, P.V., Gusakov, A.V., Rubtsova, E.A., Rozhkova, A.M., Matys, V.Y., Nemashkalov, V.A., and Sinitsyn, A.P., Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species, Biochimie, 2019, vol. 157, pp. 123–130. https://doi.org/10.1016/j.biochi.2018.11.010

    Article  CAS  PubMed  Google Scholar 

  13. Aslanidis, C. and de Jong, P.J., Ligation-independent cloning of PCR products (LIC-PCR), Nucleic Acids Res., 1990,vol. 18, no. 20, pp. 6069–6074. https://doi.org/10.1093/nar/18.20.6069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Aleksenko, A.Y., Makarova, N.A., Nikolaev, I.V., and Clutterbuck, A.J., Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene, Curr. Genet., 1995, vol. 28, no. 5, pp. 474–477. https://doi.org/10.1007/BF00310818

    Article  CAS  PubMed  Google Scholar 

  15. Sinitsyn, A.P., Osipov, D.O., Rozhkova, A.M., Bushina, E.V., Dotsenko, G.S., Sinitsyna, O.A., Kondrat’eva, E.G., Zorov, I.N., Okunev, O.N., Nemashkalov, V.A., Matys, V.Y., and Koshelev, A.V., The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials, Appl. Biochem. Microbiol., 2014, vol. 50, no. 8, pp. 761–772. https://doi.org/10.1134/S0003683814080055

    Article  CAS  Google Scholar 

  16. Peterson, G.L., Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall, Anal. Biochem., 1979, vol. 100, no. 2, pp. 201–220. https://doi.org/10.1016/0003-2697(79)90222-7

    Article  CAS  PubMed  Google Scholar 

  17. Gusakov, A.V., Semenova, M.V., and Sinitsyn, A.P., Mass spectrometry in the study of extracellular enzymes produced by filamentous fungi, J. Anal. Chem., 2010, vol. 65, no. 14, pp. 1446–1461. https://doi.org/10.1134/S1061934810140030

    Article  CAS  Google Scholar 

  18. Nelson, N., A photometric adaptation of the Somogyi method for the determination of sugars, J. Biol. Chem., 1944, vol. 153, pp. 375–379.

    Article  CAS  Google Scholar 

  19. Sinitsyna, O.A., Bukhtoyarov, F.E., Gusakov, A.V., Okunev, O.N., Bekkarevitch, A.O., Vinetsky, Y.P., and Sinitsyn, A.P., Isolation and properties of major components of Penicillium canescens extracellular enzyme complex, Biochemistry (Moscow), 2003, vol. 68, no. 11, pp. 1200–1209.

    CAS  PubMed  Google Scholar 

  20. Gusakov, A.V., Markov, A.V., Grishutin, S.G., Semenova, M.V., Kondratyeva, E.G., and Sinitsyn, A.P., Viscometric method for assaying of total endodepolymerase activity of pectinases, Biochemistry (Moscow), 2002, vol. 67, no. 6, pp. 676–682.

    CAS  PubMed  Google Scholar 

  21. Stals, I., Sandra, K., Devreese, B., van Beeumen, J., and Claeyssens, M., Factors influencing glycosylation of Trichoderma reesei cellulases. II. N-glycosylation of Cel7A core protein isolated from different strains, Glycobiology, 2004, vol. 14, no. 8, pp. 725–737. https://doi.org/10.1093/glycob/cwh081

    Article  CAS  PubMed  Google Scholar 

  22. Dotsenko, A.S., Gusakov, A.V., Volkov, P.V., Rozhkova, A.M., and Sinitsyn, A.P., N-Linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity, Biotechnol. Bioeng., 2016, vol. 113, no. 2, pp. 283–291. https://doi.org/10.1002/bit.25812

    Article  CAS  PubMed  Google Scholar 

  23. Dotsenko, A.S., Gusakov, A.V., Rozhkova, A.M., Sinitsyna, O.A., Nemashkalov, V.A., and Sinitsyn, A.P., Effect of N-linked glycosylation on the activity and other properties of recombinant endoglucanase IIa (Cel5A) from Penicillium verruculosum. Protein Eng. Des. Sel., 2016, vol. 29, no. 11, pp. 495–501. https://doi.org/10.1093/protein/gzw030

    Article  CAS  PubMed  Google Scholar 

  24. Gusakov, A.V., Dotsenko, A.S., Rozhkova, A.M., and Sinitsyn, A.P., N-Linked glycans are an important component of the processive machinery of cellobiohydrolases, Biochimie, 2016, vol. 132, no. 1, pp. 102–108. https://doi.org/10.1016/j.biochi.2016.11.004

    Article  CAS  PubMed  Google Scholar 

  25. Sinitsyna, O.A., Fedorova, E.A., Pravilnikov, A.G., Rozhkova, A.M., Skomarovsky, A.A., Matys, V.Yu., Bubnova, T.M., Okunev, O.N., Vinetsky, Yu.P., and Sinitsyn, A.P., Isolation and properties of xyloglucanases of Penicillium sp., Biochemistry (Moscow), 2010, vol. 75, no. 1, pp. 41–49. https://doi.org/10.1134/S0006297910010062

    Article  CAS  PubMed  Google Scholar 

  26. Morozova, V.V., Gusakov, A.V., Andrianov, R.M., Pravilnikov, A.G., Osipov, D.O., and Sinitsyn, A.P., Cellulases of Penicillium verruculosum, Biotechnol. J., 2010, vol. 5, no. 8, pp. 871–880. https://doi.org/10.1002/biot.201000050

    Article  CAS  PubMed  Google Scholar 

  27. Berezina, O.V., Rykov, S.V., Polyakova, A.K., Bozdaganyan, M.E., Sidochenko, A.V., Baudrexl, M., Schwarz, W.H., Zverlov, V.Z., and Yarotsky, S.V., Strategic aromatic residues in the catalytic cleft of the xyloglucanase MtXgh74 modifying thermostability, mode of enzyme action, and viscosity reduction ability, Appl. Microbiol. Biotechnol., 2021, vol. 105, no. 4, pp. 1461–1476. https://doi.org/10.1007/s00253-021-11106-3

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

The work was carried out within the framework of the Molecular Design, Structural and Functional Analysis and Regulation of Enzyme Systems, Cellular Structures, and Bionanomaterials: Fundamentals and Applications in Technology, Medicine, Environmental Protection research project, state registration number 121041500039-8.

Author information

Authors and Affiliations

  1. Department of Chemistry, Moscow State University, 119991, Moscow, Russia

    A. V. Gusakov, D. A. Klimov, O. A. Sinitsyna & A. P. Sinitsyn

  2. Fundamentals of Biotechnology Federal Research Center, Russian Academy of Sciences, 119071, Moscow, Russia

    E. G. Kondratyeva, A. M. Rozhkova, P. V. Volkov, I. A. Shashkov & A. P. Sinitsyn

Authors
  1. A. V. Gusakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. A. Klimov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E. G. Kondratyeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. O. A. Sinitsyna
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. M. Rozhkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. P. V. Volkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. I. A. Shashkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. P. Sinitsyn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. V. Gusakov.

Ethics declarations

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This work does not contain any studies involving human and animal subjects.

CONFLICT OF INTEREST

The authors of this work declare that they have no conflicts of interest.

Additional information

Translated by I. Gordon

Publisher’s Note.

Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

The electronic version of the article contains additional material available free of charge on the journal's website http://www.biotech-jr.ru.

Abbreviations: CBH, cellobiohydrolase; CBM, cellulose-binding module; CMC, carboxymethylcellulose; EG, endoglucanase; GlcNAc, N-acetylglucosamine; MCC, microcrystalline cellulose; PEG, polyethylene glycol; RS, reducing sugars; TrXeg74A, full-length (recombinant) Trichoderma reesei xyloglucanase; TrXeg74A-CD, TrXeg74A catalytic domain.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gusakov, A.V., Klimov, D.A., Kondratyeva, E.G. et al. The Preparation and Properties of Recombinant Forms of GH74 Family Xyloglucanase from the Trichoderma reesei Fungus. Appl Biochem Microbiol 59, 999–1007 (2023). https://doi.org/10.1134/S0003683823070025

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0003683823070025

Keywords:

Search

Navigation