Skip to main content

Advertisement

SpringerLink
Log in

A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

A new cellulase producer strain of Penicillium digitatum (RV 06) was previously obtained from rotten maize grains. This work aim was to optimize the production and characterize this microorganism produced cellulase. A CMCase maximum production (1.6 U/mL) was obtained in stationary liquid culture, with an initial pH of 5.0, at 25 °C, with 1% lactose as carbon source, and cultured for 5 days. The produced enzyme was purified by ammonium sulfate precipitation and exclusion chromatography. The purified enzyme optimal temperature and pH were 60 °C and 5.2, respectively. The experimental Tm of thermal inactivation was 63.68 °C, and full activity was recovered after incubation of 7 h at 50 °C. The purified 74 kDa CMCase presented KM for CMC of 11.2 mg/mL, Vmax of 0.13 μmol/min, kcat of 52 s−1, and kcat/KM of 4.7 (mg/mL)−1 s−1. The purified enzyme had a high specificity for CMC and p-nitrophenyl cellobioside and released glucose and cellobiose as final products of the CMC hydrolysis. The enzyme trypsin digestion produced peptides whose masses were obtained by MALDI-TOF/TOF mass spectrometry, which was also used to obtain two peptide sequences. These peptide sequences and the mass peak profile retrieved a CBHI within the annotated genome of P. digitatum PD1. Sequence alignments and phylogenetic analysis confirmed this enzyme as a CBHI of the glycoside hydrolase family 7. The P. digitatum PD1 protein in silico structural model revealed a coil and β-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme.

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
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Singh, R., Kumar, M., Mittal, A., & Mehta, P. K. (2016). Microbial enzymes: industrial progress in 21st century. 3 Biotech, 6(2), 174.

    PubMed  PubMed Central  Google Scholar 

  2. Adrio, J. L., & Demain, A. L. (2014). Microbial enzymes: Tools for biotechnological processes. Biomolecules, 4(1), 117–139.

    PubMed  PubMed Central  Google Scholar 

  3. Kuhad, R. C., Gupta, R., & Singh, A. (2011). Microbial cellulases and their industrial applications. Enzyme Research, 2011, 280696.

    PubMed  PubMed Central  Google Scholar 

  4. Dashtban, M., Schraft, H., & Qin, W. (2009). Fungal bioconversion of lignocellulosic residues; opportunities & perspectives. International Journal of Biological Sciences, 5(6), 578–595.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhang, P., Himmel, M. E., & Mielenz, J. R. (2006). Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances, 24, 452–481.

    CAS  Google Scholar 

  6. Payne, C. M., Knott, B. C., Mayes, H. B., Hansson, H., Himmel, M. E., Sandgren, M., Stahlberg, J., & Beckham, G. T. (2015). Fungal cellulases. Chemical Reviews, 115, 1308–1448.

    CAS  PubMed  Google Scholar 

  7. Gusakov, A. V., & Sinitsyn, A. P. (2012). Cellulases from Penicillium species for producing fuels from biomass. Biofuels, 3(4), 463–477.

    CAS  Google Scholar 

  8. Faria, C. B., Abe, C. A. L., Silva, C. N., Tessmann, D. J., & Barbosa-Tessmann, I. P. (2012). New PCR assays for the identification of Fusarium verticillioides, Fusarium subglutinans, and other species of the Gibberella fujikuroi complex. International Journal of Molecular Sciences, 13, 115–132.

    PubMed  Google Scholar 

  9. Abe, C. A. L., Faria, C. B., Castro, F. F., Souza, S. R., Santos, F. C., Silva, C. N., Tessmann, D. J., & Barbosa-Tessmann, I. P. (2015). Fungi isolated from maize (Zea mays L.) grains and production of associated enzyme activities. International Journal of Molecular Sciences, 16, 15328–15346.

    CAS  PubMed  Google Scholar 

  10. Eckert, J. W., & Eaks, I. L. (1989). Postharvest disorders and diseases of citrus fruits. In W. Reuther, E. C. Calavan, & G. E. Carman (Eds.), The citrus industry (Vol. 5, pp. 179–250). Oakland: University of California Press.

    Google Scholar 

  11. Mandels, M., & Weber, J. (1969). The production of cellulases. Advances in Chemistry, 95, 391–414.

    CAS  Google Scholar 

  12. Canteri, M. G., Althaus, R. A., Virgens Filho, J. S., & Giglioti, E. A. (2001). SASM -Agri: sistema para análise e separação de médias em experimentos agrícolas pelos métodos Scoft-Knott, Tukey e Duncan. Brazilian Journal of Agrocomputation, 1, 18–24.

  13. Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry, 59, 257–268.

    CAS  Google Scholar 

  14. Miller, G. L. (1959). Use of Dinitrosalicylic acid reagent for determination of deducing sugar. Analytical Chemistry, 31(3), 426–428.

    CAS  Google Scholar 

  15. Zhang, Y.-H. P., Cui, J., Lynd, L. R., & Kuang, L. R. (2006). A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules, 7(2), 644–648.

    CAS  PubMed  Google Scholar 

  16. Deshpande, M. V., Eriksson, K.-E., & Pettersson, L. G. (1984). An assay for selective determination of exo-1,4,-β-glucanases in a mixture of cellulolytic enzymes. Analytical Biochemistry, 138, 48l–487l.

  17. Bradford, M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye-binding. Analytical Biochemistry, 72, 248–254.

    CAS  PubMed  Google Scholar 

  18. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.

    CAS  PubMed  Google Scholar 

  19. Chaabouni, S. E., Mechichi, T., Limam, F., & Marzouki, N. (2005). Purification and characterization of two low molecular weight endoglucanases produced by Penicillium occitanis mutant Pol 6. Applied Biochemistry and Biotechnology, 125(2), 99–112.

    CAS  PubMed  Google Scholar 

  20. Han, S. O., Yukawa, H., Inui, M., & Doi, R. H. (2005). Molecular cloning and transcriptional and expression analysis of engO, encoding a new noncellulosomal family 9 enzyme, from Clostridium cellulovorans. Journal of Bacteriology, 187(14), 4884–4889.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Trevelyan, W. E., Procter, D. P., & Harrison, J. S. (1950). Detection of sugars on paper chromatograms. Nature, 166(4219), 444–445.

    CAS  PubMed  Google Scholar 

  22. Perkins, D. N., Pappin, D. J., Creasy, D. M., & Cottrell, J. S. (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis, 20(18), 3551–3567.

    CAS  PubMed  Google Scholar 

  23. Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7), 1870–1874.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Webb, B., & Sali, A. (2016). Comparative protein structure modeling using Modeller. Current Protocols in Bioinformatics, 54, 5.6.1–5.6.37.

    Google Scholar 

  25. Moroz, O. V., Maranta, M., Shaghasi, T., Harris, P. V., Wilson, K. S., & Davies, G. J. (2015). The Three-dimensional structure of the cellobiohydrolase Cel7A from Aspergillus fumigatus at 1.5 A resolution. Acta Crystallographica. Section F, Structural Biological Communications, 71(Pt 1), 114–20. L.

    CAS  Google Scholar 

  26. Collaborative Computational project, number 4. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallographica. Section D, Biological Crystallography, 50(Pt 5), 760–763.

    Google Scholar 

  27. Cheng, J., Saigo, H., & Baldi, P. (2006). Large-scale prediction of disulphide bridges using kernel methods, two-dimensional recursive neural networks, and weighted graph matching. Proteins, 62(3), 617–629.

    CAS  PubMed  Google Scholar 

  28. McNicholas, S., Potterton, E., Wilson, K. S., & Noble, M. E. M. (2011). Presenting your structures: The CCP4MG molecular-graphics software. Acta Cryst, D67, 386–394.

    Google Scholar 

  29. Böhm, G., Muhr, R., & Jaenicke, R. (1992). Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Engineering, 5(3), 191–195.

    PubMed  Google Scholar 

  30. Cantor, C. R., & Schimmel, P. R. (1980). Biophysical chemistry. San Francisco: W.H. Freeman Co..

    Google Scholar 

  31. Prasanna, H. N., Ramanjaneyulu, G., & Rajasekhar Reddy, B. R. (2016). Optimization of cellulase production by Penicillium sp. 3 Biotech, 6, 162.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Jeya, M., Joo, A.-R., Lee, K.-M., Sim, W.-I., Oh, D.-K., Kim, Y.-S., Kim, I.-W., & Lee, J.-K. (2010). Characterization of endo-β-1,4-glucanase from a novel strain of Penicillium pinophilum KMJ601. Applied Microbiology and Biotechnology, 85(4), 1005–1014.

    CAS  PubMed  Google Scholar 

  33. Das, A., & Ghosh, U. (2009). Solid-state fermentation of waste cabbage of Penicillium notatum NCIM NO-923 for production and characterization of cellulases. Journal of Scientific and Industrial Research, 68, 714–718.

    CAS  Google Scholar 

  34. Sindhu, R., Suprabha, N. G., & Shashidhar, S. (2011). Media engineering for the production of cellulase from Penicillium species (SBSS 30) under solid state fermentation. Biotechnology, Bioinformatics and Bioengineering, 1(3), 343–349.

    Google Scholar 

  35. Jørgensen, H., Mørkeberg, A., Krogh, K. B. R., & Olsson, L. (2005). Production of cellulases and hemicellulases by three Penicillium species: effect of substrate and evaluation of cellulose adsorption by capillary electrophoresis. Enzyme Microbial Technology, 36, 42–48.

    Google Scholar 

  36. Pol, D., Laxman, R. S., & Rao, M. (2012). Purification and biochemical characterization of endoglucanase from Penicillium pinophilum MS 20. Indian Journal of Biochemistry & Biophysics, 49(3), 189–194.

    CAS  Google Scholar 

  37. Camassola, M., & Dillon, A. J. P. (2007). Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation. Journal of Applied Microbiology, 3, 2196–2204.

    Google Scholar 

  38. Gao, L., Wang, F., Gao, F., Wanga, L., Zhao, J., & Qu, Y. (2011). Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production. Bioresource Technology, 102(17), 8339–8342.

    CAS  PubMed  Google Scholar 

  39. Seiboth, B., Hartl, L., Pail, M., Fekete, E., Karaffa, L., & Kubicek, C. P. (2004). The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on D-galactose. Molecular Microbiology, 51(4), 1015–1025.

    CAS  PubMed  Google Scholar 

  40. Santa-Rosa, P. S., Souza, A. L., Roque, R. A., Andradea, E. V., Astolfi-Filho, S., Mota, A. J., & Nunes-Silva, C. G. (2018). Production of thermostable β-glucosidase and CMCase by Penicillium sp. LMI01 isolated from the Amazon region. Electronic Journal of Biotechnology, 31, 84–92.

    CAS  Google Scholar 

  41. Bai, H., Wang, H., Sun, J., Irfan, M., Han, M., Huang, Y., Han, X., & Yang, Q. (2013). Purification and characterization of beta 1,4-glucanases from Penicillium simplicissimum H-11. BioResources, 8(3), 3657–3671.

    Google Scholar 

  42. Das, A., Ghosh, U., Mohapatra, P. K. D., Pati, B. R., & Mondal, K. C. (2012). Study on thermodynamics and adsorption kinetics of purified endoglucanase (CMCase) from Penicillium notatum Ncim No-923 produced under mixed solid-state fermentation of waste cabbage and bagasse. Brazilian Journal of Microbiology, 43(3), 1103–1111.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Volkov, P. V., Rozhkova, A. M., Gusakov, A. V., & Sinitsyn, A. P. (2014). Homologous cloning, purification and characterization of highly active cellobiohydrolase I (Cel7A) from Penicillium canescens. Protein Expression and Purification, 103, 1–7.

    CAS  PubMed  Google Scholar 

  44. Dotsenko, A. S., Gusakov, A. V., Volkov, P. V., Rozhkova, A. M., & Sinitsyn, A. P. (2016). N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity. Biotechnology and Bioengineering, 113(2), 283–291.

    CAS  PubMed  Google Scholar 

  45. Gao, L., Gao, F., Wang, L., Geng, C., Chi, L., Zhao, J., & Qu, Y. (2012). N-Glycoform diversity of cellobiohydrolase I from Penicillium decumbens and synergism of nonhydrolytic glycoform in cellulose degradation. The Journal of Biological Chemistry, 287(19), 15906–15915.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Divne, C., Stahlberg, J., Reinikainen, T., Ruohonen, L., Pettersson, G., Knowles, J. K., Teeri, T. T., & Jones, T. A. (1994). The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science, 265(5171), 524–528.

    CAS  PubMed  Google Scholar 

  47. Jalak, J., Kurašin, M., Teugjas, H., & Väljamäe, P. (2012). Endo-exo synergism in cellulose hydrolysis. The Journal of Biological Chemistry, 287(34), 28802–28815.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Grassick, A., Murray, P. G., Thompson, R., Collins, C. M., Byrnes, L., Birrane, G., Higgins, T. M., & Tuohy, M. G. (2004). Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii. European Journal of Biochemistry, 271(22), 4495–4506.

    CAS  PubMed  Google Scholar 

  49. Shoemaker, S., Schweickart, V., Ladner, M., Gelfand, D., Kwok, S., Myambo, K., & Innis, M. (1983). Molecular cloning of exo-cellobiohydrolase I derived from Trichoderma reesei strain L27. Biotechnology, 1, 691–696.

    CAS  Google Scholar 

  50. van Arsdell, J. N., Kwok, S., Schweickart, V. L., Ladner, M. B., Gelfand, D. H., & Innis, M. A. (1987). Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei. BioTechnology, 5(1), 60–64.

    Google Scholar 

  51. Bischof, R. H., Ramoni, J., & Seiboth, B. (2016). Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei. Microbial Cell Factories, 15, 106.

    PubMed  PubMed Central  Google Scholar 

  52. Beckham, G. T., Dai, Z., Matthews, J. F., Momany, M., Payne, C. M., Adney, W. S., Baker, S. E., & Himmel, M. E. (2012). Harnessing glycosylation to improve cellulase activity. Current Opinion in Biotechnology, 23(3), 338–345.

    CAS  PubMed  Google Scholar 

  53. Davies, G. J., Wilson, K. S., & Henrissat, B. (1997). Nomenclature for sugar-binding subsites in glycosyl hydrolases. The Biochemical Journal, 321(Pt 2), 557–559.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Henrissat, B. (1998). Glycoside families. Biochemical Society Transactions, 26(2), 153–156.

    CAS  PubMed  Google Scholar 

  55. Nerinckx, W., Desmet, T., & Claeyssens, M. (2003). A hydrophobic platform as a mechanistically relevant transition state stabilizing factor appears to be present in the active centre of all glycoside hydrolases. FEBS Letters, 538(1-3), 1–7.

    CAS  PubMed  Google Scholar 

  56. Colussi, F., Serpa, V., Delabona, P. S., Manzine, L. R., Voltatodio, M. L., Alves, R., Mello, B. L., Pereira Jr., N., Farinas, C. S., Golubev, A. M., Santos, M. A., & Polikarpov, I. (2011). Purification, and biochemical and biophysical characterization of cellobiohydrolase I from Trichoderma harzianum IOC 3844 J. Journal of Microbiology Biotechnology, 21(8), 808–817.

    CAS  PubMed  Google Scholar 

  57. Muñoz, I. G., Ubhayasekera, W., Henriksson, H., Szabó, I., Pettersson, G., Johansson, G., Mowbray, S. L., & Ståhlberg, J. (2001). Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 Å resolution and homology models of the isozymes. Journal of Molecular Biology, 314, 1097–1111.

    PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Grant 001 – Ministry of Education, Brazil) for the students’ scholarships. The authors are also thankful to the Department of Biochemistry from the Federal University of Paraná, Brazil, and the Analytical Core at the State University of Maringá, for the use of its MS facility and CD equipment, respectively.

Author information

Authors and Affiliations

  1. Department of Biochemistry, State University of Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil

    Fabiane Cristina dos Santos, Marco Aurelio Schuler de Oliveira & Ione Parra Barbosa-Tessmann

  2. Department of Technology, State University of Maringá, Av. Ângelo Moreira da Fonseca, 1800, Umuarama, PR, 87506-370, Brazil

    Flavio Augusto Vicente Seixas

Authors
  1. Fabiane Cristina dos Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marco Aurelio Schuler de Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Flavio Augusto Vicente Seixas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ione Parra Barbosa-Tessmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ione Parra Barbosa-Tessmann.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PPTX 148 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

dos Santos, F.C., de Oliveira, M.A.S., Seixas, F.A.V. et al. A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization. Appl Biochem Biotechnol 192, 257–282 (2020). https://doi.org/10.1007/s12010-020-03307-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-020-03307-9

Keywords

Search

Navigation