Purification and Characterization of a Thermostable α-Galactosidase from Thielavia Terrestris NRRL 8126 in Solid State Fermentation


Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for a-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified a-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the a-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the a-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.


  1. 1.

    Ademark, P., Larsson, M., Tjerneld, F., Stalbrand, H. (2001) Multiple α-galactosidases from Aspergillus niger: purification, characterization and substrate specificities. Enzyme Microb. Technol. 29, 441–448.

    CAS  Article  Google Scholar 

  2. 2.

    Akaogi, M., Ohmura, N., Suzuki, T., Kotwal, S. M., Gote, M. M., Sainkar, S. R., Khan, M. I., Khire, J. M. (1988) Production of α-galactosidase by thermophilic fungus Humicola sp. in solid-state fermentation and its application in soyamilk hydrolysis. Process Biochem. 33, 337–343.

    Google Scholar 

  3. 3.

    Aleksieva, P., Tchorbanov, B., Nacheva, L. (2010) High yield production of a-galactosidase extracted from Penicillium chrysogenum and Aspergillus niger. Biotechnol. & Biotechnol. Eq. 24, 1620–1623.

    CAS  Article  Google Scholar 

  4. 4.

    Anisha, G. S., John, R. P., Prema, P., Pandey, A. (2010) Investigation on a-galactosidase production by Streptomyces griseoloalbus in a forcefully aerated packed-bed bioreactor operating in solid-state fermentation condition. Appl. Biochem. Biotechnol. 160, 421–427.

    CAS  Article  Google Scholar 

  5. 5.

    Asano, N., Ishii, S., Kizu, H., Ikeda, K., Yasuda, K., Kato, A., Martin, O. R., Fan, J. Q. (2000) In vitro inhibition and intracellular enhancement of lysosomal a-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. Eur. J. Biochem. 267, 4179–4186.

    CAS  Article  Google Scholar 

  6. 6.

    Awan, M. S., Jalal, F., Ayub, N., Akhtar, M., W., Rajoka, M. I. (2009) Production and characterization of a-galactosidase by a multiple mutant of Aspergillus niger in solid-state fermentation. Food Technol. Biotechnol. 47, 370–380.

    CAS  Google Scholar 

  7. 7.

    Bradford, M. A. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248–254.

    CAS  Article  Google Scholar 

  8. 8.

    Bulpin, P. V., Gidley, M. J., Jeffcoat, R., Underwood, D. R. (1990) Development of a biotechnological process for the modification of galactomannan polymers with plant a-galactosidase. Carbohydr. Polym. 12, 155–168.

    CAS  Article  Google Scholar 

  9. 9.

    Dey, P. M., Patel, S., Brownleader, M. D. (1993) Induction of a-galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonobola) gum. Biotechnol. Appl. Biochem. 17, 361–371.

    CAS  PubMed  Google Scholar 

  10. 10.

    El-Gindy, A. A. (2002) Production, purification and some properties of a-galactosidase from Aspergillus niger. Aft. J. Mycol. Biotechnol. 10, 1–9.

    CAS  Google Scholar 

  11. 11.

    El-Gindy, A. A., Ali, U. F., Ibrahim, Z. M., Isaac, G. S. (2008) A cost-effective medium for enhanced production of extracellular a-galactosidase in solid substrate cultures of Aspergillus awamori and A. carbonarius. Aust. J. Basic Appl. Sci. 2, 880–899.

    CAS  Google Scholar 

  12. 12.

    Gaillard, B. D. E. (1965) Comparison of the hemicelluloses from plants belonging to two different plant families. Phytochem. 4, 631–634.

    CAS  Article  Google Scholar 

  13. 13.

    Glantz, A. S. (1992) Primer of biostatistics. McGraw Hill, Inc., USA.

    Google Scholar 

  14. 14.

    Holker, F., Hofer, M., Lenz, J. (2004) Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Appl. Microbiol. Biotechnol. 64, 175–186.

    CAS  Article  Google Scholar 

  15. 15.

    Jermyn, M. A. (1955) Cellulose and hemicellulose. In: Peach, K., Tracey, M. V. (eds), Modern Methods of Plant Analysis. 2, 197–224.

    Google Scholar 

  16. 16.

    Kotwal, S. M., Khan, M. I., Khire, J. M. (1995) Production of thermostable a-galactosidase from thermophilic fungus Humicola sp. J. ind. Microbiol. 15, 116–120.

    CAS  Article  Google Scholar 

  17. 17.

    Kotwal, S. M., Gote, M. M., Sainker, S. R., Khan, M. I., Khire, J. M. (1998) Production of a-galactosidase from the thermophilic fungus Humicola sp. in solid state fermentation and its application in soymilk hydrolysis. Process Biochem. 33, 337–343.

    CAS  Article  Google Scholar 

  18. 18.

    Kotwal, S. M., Gote, M. M., Khan, M. I., Khire, J. M. (1999) Production, purification and characterization of a constitutive intracellular a-galactosidase from the thermophilic fungus Humicola sp. J. Ind. Microbial. Biotechnol. 23, 661–667.

    CAS  Article  Google Scholar 

  19. 19.

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

    CAS  Article  Google Scholar 

  20. 20.

    Lineweaver, H., Burk, D. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658–666.

    CAS  Article  Google Scholar 

  21. 21.

    Liu, C. Q., Chen, Q. H., Tang, B., Ruan, H., He, G. Q. (2007) Response surface methodology for optimizing the fermentation medium of a-galactosidase in solid-state fermentation. Lett. Appl. Microbiol. 45, 206–212.

    CAS  Article  Google Scholar 

  22. 22.

    Malanchuk, V. M., Buglova, T. T., Varbanets, L. D. (2001) Study of functional groups in the active center of a-galactosidase of Penicillium sp. 23. Mikrobiolohichnyi zhurnal (Kiev, Ukraine. (1993) 63, 9–18.

    CAS  PubMed  Google Scholar 

  23. 23.

    Manzanares, P., de Graaff H. L., Visser, J. (1998) Characterization of galactosidases from Aspergillus niger: purification of a novel a-galactosidase activity. Enzyme Microb. Technol. 22, 383–390.

    CAS  Article  Google Scholar 

  24. 24.

    Mulimani, V., Ramalingam, H. (1995) Enzymatic hydrolysis of raffinose, stachyose in soybean milk by a-galactosidase from Gibberella fujikuroi. Biochem. Molecular Biol. International 36, 897–905.

    CAS  Google Scholar 

  25. 25.

    Ozsoy, N., Berkkan, H. (2003) Production and characterization of a-galactosidase from Aspergillus flavipes. Cell Biochem. Fund. 21, 387–389.

    CAS  Article  Google Scholar 

  26. 26.

    Palmer, T. (1991) Extraction and Purification of Enzymes. In: Palmer, T. (ed.) Understanding Enzymes, Ellis Horwood. Ltd., London, pp. 301–317.

    Google Scholar 

  27. 27.

    Pessela, B. C. C., Fernandez-Lafuente, R., Torres, R., Mateo, C., Fuentes, M., Filho, M., Vian, A., Garcia, J. L., Guisan, J. M., Carrascosa, A. V. (2007) Production of a thermoresistant α-galactosidase from Thermus sp. strain T2 for food processing. Food Biotechnol. 21, 91–103.

    CAS  Article  Google Scholar 

  28. 28.

    Peterson, E. A., Sober, H. A. (1962) Column chromatography of protein: Substituted cellulases. In: S. Colowich, N. Kapllan (eds) Methods in Enzymology. Vol. 5, Academic Press, New York, pp. 3–27.

    CAS  Article  Google Scholar 

  29. 29.

    Plummer, D. T. (1978) The practice of column chromatography In: Palmer, T. (ed.) An Introduction to Practical Biochemistry. McGraw-Hill Book Company, New York, pp. 61–66.

    Google Scholar 

  30. 30.

    Puchart, V., Vrsanskâ, M., Bhat, M. K., Biely, P. (2000) Purification and characterization of a-galactosidase from a thermophilic fungus Thermomyces lanuginosus. Biochim. Biophys. Acta 1524, 27–37.

    CAS  Article  Google Scholar 

  31. 31.

    Rezessy-Szabó, J. M., Bujna, E., Hoschke, A. (2002) Effect of different carbon and nitrogen sources on a-galactosidase activity originated from Thermomyces lanuginosus CBS 395. 62/B. Acta Alimentaria 31, 73–82.

    Article  Google Scholar 

  32. 32.

    Rezessy-Szabó, J. M., Nguyen, Q. D., Hoschke, Á., Braet, C., Hajós, G., Claeyssens, M. (2007) A novel thermostable a-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395. 62/b: Purification and characterization. Biochim. Biophys. Acta (BBA)-General Subjects. (1770) 1, 55–62.

    Article  Google Scholar 

  33. 33.

    Shankar, S. K., Mulimani, V. H. (2007) α-Galactosidase production by Aspegillus oryzae in solid state fermentation. Biores. Technol. 98, 958–961.

    CAS  Article  Google Scholar 

  34. 34.

    Sinitsyna, O. A., Fedorova, E. A., Vakar, I. M., Kondratieva, E. G., Rozhkova, A. M., Sokolova, L. M., Bubnova, T. M., Okunev, O. N., Chulkin, A. M., Vinetsky, Y. P., Sinitsyn, A. P. (2008) Isolation and characterization of extracellular α-galactosidases from Penicillium canescens. Biochemistry 73, 97–106. (In Russian).

    CAS  PubMed  Google Scholar 

  35. 35.

    Svastits-Dücső, L., Nguyen, Q. D., Lefler, D. D., Rezessy-Szabó, J. M. (2009) Effects of galactomannan as carbon source on production of a-galactosidase by Thermomyces lanuginosus: Fermentation, purification and partial characterization. Enzyme Microb. Technol. 5, 367–371.

    Article  Google Scholar 

  36. 36.

    Wang, C. L., Li, D. F., Lu, W. Q., Wang, Y. H., Lai, C. H. (2004) Influence of cultivating conditions on the ?-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation. Lett. Appl. Microbiol. 39, 369–375.

    CAS  Article  Google Scholar 

  37. 37.

    Zeilinger, S., Kristufek, D., Arisan-Atac, I., Hodits, R., Kubicek, C. P. (1993) Conditions of formation, purification, and characterization of an a-galactosidase of Trichoderma reesei RUT C-30. Appl. Environ. Microbiol. 59, 1347–1353.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Eman M. Fawzi.

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Cite this article

Saad, R.R., Fawzi, E.M. Purification and Characterization of a Thermostable α-Galactosidase from Thielavia Terrestris NRRL 8126 in Solid State Fermentation. BIOLOGIA FUTURA 63, 138–150 (2012). https://doi.org/10.1556/ABiol.63.2012.1.11

Download citation


  • Hemicelluloses
  • α-galactosidase
  • thermostability
  • Thielavia terrestris