Bioprocess and Biosystems Engineering

, Volume 30, Issue 5, pp 297–304 | Cite as

Production of inulinase by solid-state fermentation: effect of process parameters on production and preliminary characterization of enzyme preparations

  • Marcio Mazutti
  • Gustavo Ceni
  • Marco Di Luccio
  • Helen Treichel
Original Paper


This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 × 1010 cells mL−1 and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds−1 h−1. The maximum activity was obtained at pH 5.0 and 55.0°C. Within the investigated range, the enzyme extract was more thermostable at 50.0°C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol−1. The extract showed highest stability from pH 4.5 to 4.8. Apparent K m and V max are 7.1 mM and 17.79 M min−1, respectively.


Solid-state fermentation Inulinase Thermostability Kluyveromyces 



The authors thank FAPERGS for the financial support for this work and scholarships and Laboratório de Engenharia de Bioprocessos (FEA/UNICAMP) for providing the strain used in this work.


  1. 1.
    Nigam P, Singh D (1994) Solid state (substrate) fermentation systems and their application in biotechnology. J Basic Microbiol 34:404–423CrossRefGoogle Scholar
  2. 2.
    Pandey A, Soccol CR, Mitchell DA (2000) New development in solid state fermentation: I—bioprocesses and bioproducts. Process Biochem 35:1153–1169CrossRefGoogle Scholar
  3. 3.
    Pandey A, Soccol CR, Nigam P, Soccol VT (2000) Biotechnological potential of agro-industrial residues: I sugarcane bagasse. Bioresour Technol 74:81–87CrossRefGoogle Scholar
  4. 4.
    Pandey A (1992) Recent developments in solid state fermentation. Process Biochem 27:109–117CrossRefGoogle Scholar
  5. 5.
    Robinson T, Nigam P (2003) Bioreactor design for protein enrichment of agricultural residues by solid state fermentation. Biochem Eng J 13:197–203CrossRefGoogle Scholar
  6. 6.
    Pandey A (2003) Solid state fermentation. Biochem Eng J 13:81–84CrossRefGoogle Scholar
  7. 7.
    Barrios-González J, González H, Mejía A (1993) Effect of size particle, packing density and agitation on penicillin production in solid state fermentation. Biotechnol Adv 11:539–547CrossRefGoogle Scholar
  8. 8.
    Dyaz-Godynez G, Soriano-Santos J, Augur C, Viniegra-Gonzalez G (2001) Exopectinases produced by Aspergillus niger in solid state and submerged fermentation: a comparative study. J Ind Microbiol Biotechnol 26:271–275CrossRefGoogle Scholar
  9. 9.
    Selvakumar P, Pandey A (1999) Solid state fermentation for the synthesis of inulinase from Staphylococcus sp. and Kluyveromyces marxianus. Process Biochem 34:851–858CrossRefGoogle Scholar
  10. 10.
    Zhang L, Zhao C, Zhu D, Ohta Y, Wanga Y (2004) Purification and characterization of inulinase from Aspergillus niger AF10 expressed in Pichia pastoris. Protein Expr Purif 35:272–275CrossRefGoogle Scholar
  11. 11.
    Otha K, Suetsugu N, Nakamura T (2002) Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96. J Biosci Bioeng 94:78–80CrossRefGoogle Scholar
  12. 12.
    Vandamme EJ, Derycke DG (1983) Microbial inulinases: fermentation process, properties and application. Adv Appl Microbiol 29:139–176CrossRefGoogle Scholar
  13. 13.
    Fachin D, Loey AMV, Nguyen BL, Verlent I, Indrawati I, Hedricks ME (2002) Comparative study of the inactivation kinetics of pectinmethylesterase in tomato juice and purified form. Biotechnol Prog 18:739–744CrossRefGoogle Scholar
  14. 14.
    Rodrigues MI, Iemma AF (2005) Planejamento de Experimentos e Otimização de Processos: Uma estratégia seqüencial de planejamentos. Casa do Pão, Campinas, BrazilGoogle Scholar
  15. 15.
    Miller GL (1959) Use of dinitrosalisylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  16. 16.
    Santisteban-Silva BOY, Maugeri F (2005) Agitation, aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus. Enzyme Microb Tech 36:717–724CrossRefGoogle Scholar
  17. 17.
    Mazutti M, Bender JP, Treichel H, Di Luccio M (2006) Optimization on inulinase production by solid state fermentation using sugar cane as substrate. Enzyme Microb Technol 39:56–59CrossRefGoogle Scholar
  18. 18.
    Ikasari L, Mitchell DA (1994) Protease production by Rhizopus oligosporus in solid-state fermentation. World J Microbiol Biotechnol 10:320–324CrossRefGoogle Scholar
  19. 19.
    Saxena S, Saxena RK (2004) Statistical optimization of tannase production from Penicillium variable using fruits (chebulic myrobalan) of Terminalia chebula. Biotechnol Appl Biochem 39:99–106CrossRefGoogle Scholar
  20. 20.
    Bender JP, Mazutti MA, Oliveira D, Treichel H, Di Luccio M (2006) Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation. Appl Biochem Biotech 32:951–958CrossRefGoogle Scholar
  21. 21.
    Brand D, Pandey A, Rodriguez-Leon JA, Roussos S, Brand I, Soccol CR (2001) Packed bed column fermenter and kinetic modelling for upgrading the nutritional quality of coffee husk in solid state fermentation. Biotechnol Prog 17:1065–1070CrossRefGoogle Scholar
  22. 22.
    Pessoa A, Vitolo M (1999) Inulinase from Kluyveromyces marxianus: culture medium composition and enzyme extraction. Braz J Chem Eng 16:237–245CrossRefGoogle Scholar
  23. 23.
    Cazetta ML, Martins PMM, Monti R, Contiero J (2005) Yacon (Polymnia sanchifolia) extract as a substrate to produce inulinase by Kluyveromyces marxianus var. bulgaricus. J Food Eng 66:301–305CrossRefGoogle Scholar
  24. 24.
    Cruz-Guerrero A, Garcia-Peña I, Barzana E, Racia-Garibay M, Gómez-Ruiz L (1995) Kluyveromyces marxianus CDBB-L-278: a wild inulinase hyperproducing strain. J Ferment Bioeng 80:159–163CrossRefGoogle Scholar
  25. 25.
    Catana R, Ferreira BS, Cabral JM S, Fernandes P (2005) Immobilization of inulinase for sucrose hydrolysis. Food Chem 91:517–520CrossRefGoogle Scholar
  26. 26.
    Gill PK, Manhas RK, Singh P (2006) Comparative analysis of thermostability of extracellular inulinase activity from Aspergillus fumigatus with commercially available (Novozyme) inulinase. Bioresour Technol 97:355–358CrossRefGoogle Scholar
  27. 27.
    Fullbrook PD (1996) Practical applied kinetics. In: Godfrey T, West S (eds) Industrial enzymology, 2nd edn. Stockholm Press, New York, pp 483–540Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Marcio Mazutti
    • 1
  • Gustavo Ceni
    • 1
  • Marco Di Luccio
    • 1
  • Helen Treichel
    • 1
  1. 1.Department of Food EngineeringUniversidade Regional Integrada do Alto Uruguai e das Missões Campus de ErechimErechimBrazil

Personalised recommendations