Applied Biochemistry and Biotechnology

, Volume 177, Issue 6, pp 1252–1271 | Cite as

Comparative Secretome Analysis of Aspergillus niger, Trichoderma reesei, and Penicillium oxalicum During Solid-State Fermentation

  • Weili Gong
  • Huaiqiang Zhang
  • Shijia Liu
  • Lili Zhang
  • Peiji Gao
  • Guanjun Chen
  • Lushan Wang


Filamentous fungi such as Aspergillus spp., Trichoderma spp., and Penicillium spp. are frequently used to produce high concentrations of lignocellulosic enzymes. This study examined the discrepancies in the compositions and dynamic changes in the extracellular enzyme systems secreted by Aspergillus niger ATCC1015, Trichoderma reesei QM9414, and Penicillium oxalicum 114-2 cultured on corn stover and wheat bran. The results revealed different types and an abundance of monosaccharides and oligosaccharides were released during incubation, which induced the secretion of diverse glycoside hydrolases. Both the enzyme activities and isozyme numbers of the three fungal strains increased with time. A total of 279, 161, and 183 secretory proteins were detected in A. niger, T. reesei, and P. oxalicum secretomes, respectively. In the A. niger secretomes, more enzymes involved in the degradation of (galacto)mannan, xyloglucan, and the backbone of pectin distributed mostly in dicots were detected. In comparison, although P. oxalicum 114-2 hardly secreted any xyloglucanases, the diversities of enzymes involved in the degradation of xylan and β-(1,3;1,4)-d-glucan commonly found in monocots were higher. The cellulase system of P. oxalicum 114-2 was more balanced. The degradation preference provided a new perspective regarding the recomposition of lignocellulosic enzymes based on substrate types.


Aspergillus niger Trichoderma reesei Penicillium oxalicum Glycoside hydrolase Solid-state fermentation Secretome 


Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12010_2015_1811_MOESM1_ESM.tif (1.9 mb)
Figure S1 Dynamic zymography of the extracellular proteases of the three filamentous fungal strains during the 5-day solid-state fermentation. (TIFF 1985 kb)
12010_2015_1811_MOESM2_ESM.tif (2 mb)
Figure S2 Enzymes related to the degradation of cellulose, hemicellulose (xyloglucan, galactomannan, xylan, and (1,3;1,4)-β-d-glucan), and pectin (homogalacturon, xylogalacturonan, and rhamnogalacturon І) detected in the secretomes. The number of plus signs (+) with different colors represents the number of specific enzymes in different strains. (TIFF 2086 kb)


  1. 1.
    Adav, S. S., Chao, L. T. and Sze, S. K. (2012) Quantitative secretomic analysis of Trichoderma reesei strains reveals enzymatic composition for lignocellulosic biomass degradation. Molecular & Cellular Proteomics, 11, M111. 012419.Google Scholar
  2. 2.
    Adav, S. S., Li, A. A., Manavalan, A., Punt, P., & Sze, S. K. (2010). Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes. Journal of Proteome Research, 9, 3932–3940.CrossRefGoogle Scholar
  3. 3.
    Alfaro, M., Oguiza, J. A., Ramírez, L., & Pisabarro, A. G. (2014). Comparative analysis of secretomes in basidiomycete fungi. Journal of Proteomics, 102, 28–43.CrossRefGoogle Scholar
  4. 4.
    Andersen, M. R., Giese, M., Ronald, P., & Nielsen, J. (2012). Mapping the polysaccharide degradation potential of Aspergillus niger. BMC Genomics, 13, 313.CrossRefGoogle Scholar
  5. 5.
    Braaksma, M., Martens-Uzunova, E. S., Punt, P. J., & Schaap, P. J. (2010). An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data. BMC Genomics, 11, 584.CrossRefGoogle Scholar
  6. 6.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  7. 7.
    Burton, R. A., Gidley, M. J., & Fincher, G. B. (2010). Heterogeneity in the chemistry, structure and function of plant cell walls. Nature Chemical Biology, 6, 724–732.CrossRefGoogle Scholar
  8. 8.
    Chundawat, S. P. S., Beckham, G. T., Himmel, M. E., & Dale, B. E. (2011). Deconstruction of lignocellulosic biomass to fuels and chemicals. Annual Review of Chemical and Biomolecular Engineering, 2, 121–145.CrossRefGoogle Scholar
  9. 9.
    Cosgrove, D. J. (2005). Growth of the plant cell wall. Nature Reviews Molecular Cell Biology, 6, 850–861.CrossRefGoogle Scholar
  10. 10.
    da Costa Sousa, L., Chundawat, S. P., Balan, V., & Dale, B. E. (2009). ‘Cradle-to-grave’ assessment of existing lignocellulose pretreatment technologies. Current Opinion in Biotechnology, 20, 339–347.CrossRefGoogle Scholar
  11. 11.
    Delmas, S., Pullan, S. T., Gaddipati, S., Kokolski, M., Malla, S., Blythe, M. J., Ibbett, R., Campbell, M., Liddell, S., & Aboobaker, A. (2012). Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing. PLoS Genetics, 8, e1002875.CrossRefGoogle Scholar
  12. 12.
    DeMartini, J. D., Pattathil, S., Miller, J. S., Li, H., Hahn, M. G., & Wyman, C. E. (2013). Investigating plant cell wall components that affect biomass recalcitrance in poplar and switchgrass. Energy & Environmental Science, 6, 898–909.CrossRefGoogle Scholar
  13. 13.
    Dodd, D., & Cann, I. K. (2009). Enzymatic deconstruction of xylan for biofuel production. GCB Bioenergy, 1, 2–17.CrossRefGoogle Scholar
  14. 14.
    Doyle, S. (2011). Fungal proteomics: from identification to function. FEMS Microbiology Letters, 321, 1–9.CrossRefGoogle Scholar
  15. 15.
    Foreman, P. K., Brown, D., Dankmeyer, L., Dean, R., Diener, S., Dunn-Coleman, N. S., Goedegebuur, F., Houfek, T. D., England, G. J., & Kelley, A. S. (2003). Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. Journal of Biological Chemistry, 278, 31988–31997.CrossRefGoogle Scholar
  16. 16.
    Gao, D., Uppugundla, N., Chundawat, S. P., Yu, X., Hermanson, S., Gowda, K., Brumm, P., Mead, D., Balan, V., & Dale, B. E. (2011). Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides. Biotechnology for Biofuels, 4, 5.CrossRefGoogle Scholar
  17. 17.
    Gao, P., Qu, Y., Zhao, X., Zhu, M., & Duan, Y. (1997). Screening microbial strain for improving the nutritional value of wheat and corn straws as animal feed. Enzyme & Microbial Technology, 20, 581–584.CrossRefGoogle Scholar
  18. 18.
    Girard, V., Dieryckx, C., Job, C., & Job, D. (2013). Secretomes: the fungal strike force. Proteomics, 13, 597–608.CrossRefGoogle Scholar
  19. 19.
    Glass, N. L., Schmoll, M., Cate, J. H., & Coradetti, S. (2013). Plant cell wall deconstruction by ascomycete fungi. Annual Review of Microbiology, 67, 477–498.CrossRefGoogle Scholar
  20. 20.
    Griffin, T. J., Gygi, S. P., Ideker, T., Rist, B., Eng, J., Hood, L., & Aebersold, R. (2002). Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae. Molecular & Cellular Proteomics, 1, 323–333.CrossRefGoogle Scholar
  21. 21.
    Häkkinen, M., Arvas, M., Oja, M., Aro, N., Penttilä, M., Saloheimo, M., & Pakula, T. M. (2012). Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates. Microbial Cell Factories, 11, 134.CrossRefGoogle Scholar
  22. 22.
    Henrissat, B., Coutinho, P. M. and Davies, G. J. (2001), in Plant cell walls, Springer, pp. 55–72.Google Scholar
  23. 23.
    Herold, S., Bischof, R., Metz, B., Seiboth, B., & Kubicek, C. P. (2013). Xylanase gene transcription in Trichoderma reesei is triggered by different inducers representing different hemicellulosic pentose polymers. Eukaryotic Cell, 12, 390–398.CrossRefGoogle Scholar
  24. 24.
    Herpoel-Gimbert, I., Margeot, A., Dolla, A., Jan, G., Molle, D., Lignon, S., Mathis, H., Sigoillot, J. C., Monot, F., & Asther, M. (2008). Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnology for Biofuels, 1, 18.CrossRefGoogle Scholar
  25. 25.
    Himmel, M. E., Ding, S.-Y., Johnson, D. K., Adney, W. S., Nimlos, M. R., Brady, J. W., & Foust, T. D. (2007). Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science, 315, 804–807.CrossRefGoogle Scholar
  26. 26.
    Jiang, L., He, L., & Fountoulakis, M. (2004). Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Journal of Chromatography A, 1023, 317–320.CrossRefGoogle Scholar
  27. 27.
    Juhasz, T., Szengyel, Z., Reczey, K., Siika-Aho, M., & Viikari, L. (2005). Characterization of cellulases and hemicellulases produced by Trichoderma reesei on various carbon sources. Process Biochemistry, 40, 3519–3525.CrossRefGoogle Scholar
  28. 28.
    Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature, 227, 680–685.CrossRefGoogle Scholar
  29. 29.
    Lahjouji, K., Storms, R., Xiao, Z., Joung, K.-B., Zheng, Y., Powlowski, J., Tsang, A., & Varin, L. (2007). Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor. Applied Microbiology and Biotechnology, 75, 337–346.CrossRefGoogle Scholar
  30. 30.
    Liao, H., Li, S., Wei, Z., Shen, Q., & Xu, Y. (2014). Insights into high-efficiency lignocellulolytic enzyme production by Penicillium oxalicum GZ-2 induced by a complex substrate. Biotechnology for Biofuels, 7, 162.CrossRefGoogle Scholar
  31. 31.
    Liepman, A. H., Wightman, R., Geshi, N., Turner, S. R., & Scheller, H. V. (2010). Arabidopsis—a powerful model system for plant cell wall research. The Plant Journal, 61, 1107–1121.CrossRefGoogle Scholar
  32. 32.
    Liu, D., Li, J., Zhao, S., Zhang, R., Wang, M., Miao, Y., Shen, Y., & Shen, Q. (2013). Secretome diversity and quantitative analysis of cellulolytic Aspergillus fumigatus Z5 in the presence of different carbon sources. Biotechnology for Biofuels, 6, 1–16.CrossRefGoogle Scholar
  33. 33.
    Liu, G., Zhang, L., Wei, X., Zou, G., Qin, Y., Ma, L., Li, J., Zheng, H., Wang, S. and Wang, C. (2013) Genomic and secretomic analyses reveal unique features of the lignocellulolytic enzyme system of Penicillium decumbens. PloS One, 8, e55185.Google Scholar
  34. 34.
    Martens-Uzunova, E. S., & Schaap, P. J. (2009). Assessment of the pectin degrading enzyme network of Aspergillus niger by functional genomics. Fungal Genetics and Biology, 46, S170–S179.CrossRefGoogle Scholar
  35. 35.
    Martinez, D., Berka, R. M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S. E., Chapman, J., Chertkov, O., Coutinho, P. M., & Cullen, D. (2008). Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nature Biotechnology, 26, 553–560.CrossRefGoogle Scholar
  36. 36.
    Marx, I. J., van Wyk, N., Smit, S., Jacobson, D., Viljoen-Bloom, M., & Volschenk, H. (2013). Comparative secretome analysis of Trichoderma asperellum S4F8 and Trichoderma reesei Rut C30 during solid-state fermentation on sugarcane bagasse. Biotechnology for Biofuels, 6, 1–13.CrossRefGoogle Scholar
  37. 37.
    Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.CrossRefGoogle Scholar
  38. 38.
    Parry, N., Beever, D., OWEN, E., VANDENBERGHE, I., Van Beeumen, J., & Bhat, M. (2001). Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. The Biochemical Journal, 353, 117–127.CrossRefGoogle Scholar
  39. 39.
    Pel, H. J., de Winde, J. H., Archer, D. B., Dyer, P. S., Hofmann, G., Schaap, P. J., Turner, G., de Vries, R. P., Albang, R., Albermann, K., Andersen, M. R., Bendtsen, J. D., Benen, J. A., van den Berg, M., Breestraat, S., Caddick, M. X., Contreras, R., Cornell, M., Coutinho, P. M., Danchin, E. G., Debets, A. J., Dekker, P., van Dijck, P. W., van Dijk, A., Dijkhuizen, L., Driessen, A. J., d'Enfert, C., Geysens, S., Goosen, C., Groot, G. S., de Groot, P. W., Guillemette, T., Henrissat, B., Herweijer, M., van den Hombergh, J. P., van den Hondel, C. A., van der Heijden, R. T., van der Kaaij, R. M., Klis, F. M., Kools, H. J., Kubicek, C. P., van Kuyk, P. A., Lauber, J., Lu, X., van der Maarel, M. J., Meulenberg, R., Menke, H., Mortimer, M. A., Nielsen, J., Oliver, S. G., Olsthoorn, M., Pal, K., van Peij, N. N., Ram, A. F., Rinas, U., Roubos, J. A., Sagt, C. M., Schmoll, M., Sun, J., Ussery, D., Varga, J., Vervecken, W., van de Vondervoort, P. J., Wedler, H., Wosten, H. A., Zeng, A. P., van Ooyen, A. J., Visser, J., & Stam, H. (2007). Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nature Biotechnology, 25, 221–231.CrossRefGoogle Scholar
  40. 40.
    Qu, Y., Gao, P., & Wang, Z. (1984). Screening of catabolite repression-resistant mutants of cellulase producing Penicillium spp. Acta Mycol Sinica, 3, 238–243.Google Scholar
  41. 41.
    Ribeiro, D. A., Cota, J., Alvarez, T. M., Bruchli, F., Bragato, J., Pereira, B. M., Pauletti, B. A., Jackson, G., Pimenta, M. T., Murakami, M. T., Camassola, M., Ruller, R., Dillon, A. J., Pradella, J. G., Paes Leme, A. F. and Squina, F. M. (2012) The Penicillium echinulatum secretome on sugar cane bagasse. PloS One, 7, e50571.Google Scholar
  42. 42.
    Ribeiro, D. A., Cota, J., Alvarez, T. M., Bruechli, F., Bragato, J., Pereira, B. M., Pauletti, B. A., Jackson, G., Pimenta, M. T., & Murakami, M. T. (2012). The Penicillium echinulatum secretome on sugar cane bagasse. PloS One, 7, e50571.CrossRefGoogle Scholar
  43. 43.
    Saykhedkar, S., Ray, A., Ayoubi-Canaan, P., Hartson, S. D., Prade, R., & Mort, A. J. (2012). A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover. Biotechnology for Biofuels, 5, 52.CrossRefGoogle Scholar
  44. 44.
    Scheller, H. V., & Ulvskov, P. (2010). Hemicelluloses. Annual Review of Plant Biology, 61, 263–289.CrossRefGoogle Scholar
  45. 45.
    Schuster, A., & Schmoll, M. (2010). Biology and biotechnology of Trichoderma. Applied Microbiology and Biotechnology, 87, 787–799.CrossRefGoogle Scholar
  46. 46.
    Sriranganadane, D., Waridel, P., Salamin, K., Reichard, U., Grouzmann, E., Neuhaus, J. M., Quadroni, M., & Monod, M. (2010). Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH. Journal of Proteome Research, 9, 3511–3519.CrossRefGoogle Scholar
  47. 47.
    Stricker, A. R., Mach, R. L., & De Graaff, L. H. (2008). Regulation of transcription of cellulases-and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei). Applied Microbiology and Biotechnology, 78, 211–220.CrossRefGoogle Scholar
  48. 48.
    Sweeney, M. D., & Xu, F. (2012). Biomass converting enzymes as industrial biocatalysts for fuels and chemicals: recent developments. Catalysts, 2, 244–263.CrossRefGoogle Scholar
  49. 49.
    van den Brink, J., & de Vries, R. P. (2011). Fungal enzyme sets for plant polysaccharide degradation. Applied Microbiology and Biotechnology, 91, 1477–1492.CrossRefGoogle Scholar
  50. 50.
    Wilson, D. B. (2011). Microbial diversity of cellulose hydrolysis. Current Opinion in Microbiology, 14, 259–263.CrossRefGoogle Scholar
  51. 51.
    Xing, S., Li, G., Sun, X., Ma, S., Chen, G., Wang, L., & Gao, P. (2013). Dynamic changes in xylanases and β-1, 4-endoglucanases secreted by Aspergillus niger An-76 in response to hydrolysates of lignocellulose polysaccharide. Applied Biochemistry and Biotechnology, 171, 832–846.CrossRefGoogle Scholar
  52. 52.
    Zhang, Q., Zhang, X., Wang, P., Li, D., Chen, G., Gao, P. and Wang, L. (2014) Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence-assisted carbohydrate electrophoresis. Electrophoresis.Google Scholar
  53. 53.
    Zhang, X., Liu, N., Yang, F., Li, J., Wang, L., Chen, G., & Gao, P. (2012). In situ demonstration and quantitative analysis of the intrinsic properties of glycoside hydrolases. Electrophoresis, 33, 280–287.CrossRefGoogle Scholar
  54. 54.
    Zhang, Y.-H. P., & Lynd, L. R. (2003). Cellodextrin preparation by mixed-acid hydrolysis and chromatographic separation. Analytical Biochemistry, 322, 225–232.CrossRefGoogle Scholar
  55. 55.
    Zhao, X., Zhang, L., & Liu, D. (2012). Biomass recalcitrance. Part I: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose. Biofuels, Bioproducts and Biorefining, 6, 465–482.CrossRefGoogle Scholar
  56. 56.
    Zhao, Z., Liu, H., Wang, C., & Xu, J.-R. (2013). Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi. BMC Genomics, 14, 274.CrossRefGoogle Scholar
  57. 57.
    Zhou, J.-Y., Schepmoes, A. A., Zhang, X., Moore, R. J., Monroe, M. E., Lee, J. H., Camp, D. G., Smith, R. D., & Qian, W.-J. (2010). Improved LC−MS/MS spectral counting statistics by recovering low-scoring spectra matched to confidently identified peptide sequences. Journal of Proteome Research, 9, 5698–5704.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Weili Gong
    • 1
  • Huaiqiang Zhang
    • 1
  • Shijia Liu
    • 1
  • Lili Zhang
    • 1
  • Peiji Gao
    • 1
  • Guanjun Chen
    • 1
  • Lushan Wang
    • 1
  1. 1.The State Key Laboratory of Microbial TechnologyShandong UniversityJinanChina

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