Advertisement

Applied Biochemistry and Biotechnology

, Volume 162, Issue 3, pp 830–842 | Cite as

Production and Stability of Protease from Candida buinensis

  • Daniela de Araújo Viana
  • Carolina de Albuquerque Lima
  • Rejane Pereira Neves
  • Cristina Souza Mota
  • Keila Aparecida Moreira
  • José Luiz de Lima-Filho
  • Maria Taciana Holanda Cavalcanti
  • Attilio Converti
  • Ana Lúcia Figueiredo PortoEmail author
Article

Abstract

Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations carried out at variable temperature, pH, and soybean flour concentration, according to a 23 full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL) when cultivated at 35 °C, pH 6.5, and using soybean flour concentrations in the range 0.1–0.5% (w/v). The cell-free supernatants showed the highest activity at 25 °C and pH 7.0, and satisfactory stability in the ranges 25–30 °C and pH 7–9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; ΔH* = 37.3 kJ/mol; ΔS* = −197.5 J/mol K; ΔG* = 101 kJ/mol).

Keywords

Candida buinensis Protease production Soybean flour medium Raw milk 

Notes

Acknowledgments

The present work was financed by the Fundação de Amparo à Ciência do Estado de Pernambuco (FACEPE) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors are also grateful to LIKA (Biotechnology Laboratory)–Federal University of Pernambuco (UFPE) and Federal Rural University of Pernambuco (UFRPE).

References

  1. 1.
    De Azeredo, L. A. I., Freire, D. M. G., Soares, R. M. A., Leite, S. G. F., & Coelho, R. R. (2004). Production and partial characterization of thermophilic proteases from Streptomyces sp. isolated from Brazilian cerrado soil. Enzyme Microbial Technology, 34, 354–358.CrossRefGoogle Scholar
  2. 2.
    Chi, Z., Ma, C., Wang, P., & Li, H. F. (2007). Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresource Technology, 98, 534–538.CrossRefGoogle Scholar
  3. 3.
    Poza, M., De Miguel, T., Sieiro, C., & Villa, T. G. (2001). Characterization of a broad pH range protease of Candida caseinolytica. Journal of Applied Microbiology, 91, 916–921.CrossRefGoogle Scholar
  4. 4.
    Gupta, R., Beg, Q. K., & Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59, 15–32.CrossRefGoogle Scholar
  5. 5.
    Celik, E., & Calik, P. (2004). Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species. Biotechnology Progress, 20, 491–499.CrossRefGoogle Scholar
  6. 6.
    Rao, R. S., Jyothi, C. P., Prakasham, R. S., Rao, C. S., Sarma, P. N., & Rao, L. V. (2006). Strain improvement of Candida tropicalis for the production of xylitol: biochemical and physiological characteristics of wild-type and mutant strain CT-OMV5. Journal of Microbiology, 44, 113–120.Google Scholar
  7. 7.
    Porto, A. L. F., Campos-Takaki, G. M., & Lima-Filho, J. L. (1996). Effects of culture conditions on protease production by Streptomyces clavuligerus growing on soybean flour medium. Applied Biochemistry and Biotechnology, 60, 115–122.CrossRefGoogle Scholar
  8. 8.
    Moreira, K. A., Cavalcanti, M. T. H., Duarte, H. S., Tambourgi, E. B., Melo, E. H. M., Silva, V. L., et al. (2001). Partial characterization of proteases from Streptomyces clavuligerus using an inexpensive medium. Brazilian Journal of Microbiology, 32, 215–220.Google Scholar
  9. 9.
    Moreira, K. A., Albuquerque, B. F., Teixeira, M. F. S., Porto, A. L. F., & Lima-Filho, J. L. (2002). Application of protease from Nocardiopsis sp. as a laundry detergent additive. World Journal of Microbiology and Biotechnology, 18, 307–312.CrossRefGoogle Scholar
  10. 10.
    Moreira, K. A., Porto, T. S., Teixeira, M. F. S., Porto, A. L. F., & Lima-Filho, J. L. (2003). New alkaline protease from Nocardiopsis sp.: Partial purification and characterization. Process Biochemistry, 39, 67–72.CrossRefGoogle Scholar
  11. 11.
    Cavalcanti, M. T. H., Teixeira, M. F. S., Lima-Filho, J. L., & Porto, A. L. F. (2004). Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp. Bioresource Technology, 93, 29–35.CrossRefGoogle Scholar
  12. 12.
    Cavalcanti, M. T. H., Martinez, C. R., Furtado, V. C., Neto, B. B., Teixeira, M. F. S., Lima-Filho, J. L., et al. (2005). Milk-clotting protease production by Nocardiopsis sp. in an inexpensive medium. World Journal of Microbiology and Biotechnology, 21, 151–154.CrossRefGoogle Scholar
  13. 13.
    Buzini, P., & Martini, A. (2002). Extracellular enzymatic activity profiles in yeast and yeast-like strains isolated from tropical environments. Journal of Applied Microbiology, 93, 1020–1025.CrossRefGoogle Scholar
  14. 14.
    Ramírez-Zavala, B., Mercado-Flores, Y., Hernández-Rodríguez, C., & Villa-Tanaca, L. (2004). Purification and characterization of a lysine aminopeptidase from Kluveromyces marxianus. FEMS Microbiology Letters, 235, 369–375.Google Scholar
  15. 15.
    Pereira-Dias, S., Potes, M. E., Marinho, A., Malfeito-Ferreira, M., & Loureiro, V. (2000). Characterization of yeast flora isolated from an artisanal Portuguese ewes' cheese. International Journal of Food Microbiology, 60, 55–63.CrossRefGoogle Scholar
  16. 16.
    Corbo, M. R., Lanciotti, R., Albenzio, M., & Sinigaglia, M. (2001). Occurrence and characterization of yeast isolated from milk and dairy products of Apulia region. International Journal of Food Microbiology, 69, 147–152.CrossRefGoogle Scholar
  17. 17.
    Wojtatowicz, M., Chrzanowska, J., Juszczyk, P., Skiba, A., & Gdula, A. (2001). Identification and biochemical characteristics of yeast microflora of Rokpol cheese. International Journal of Food Microbiology, 69, 135–140.CrossRefGoogle Scholar
  18. 18.
    Barnett, J. A., Payne, R. W., & Yarrow, D. (1990). Yeasts: characteristics and identification (2nd ed.). Cambrige: University Press.Google Scholar
  19. 19.
    Ray, M. K., Uma devi, K., Seshu Kumar, G., & Shivaji, S. (1992). Extracellular protease from the antarctic yeast Candida humicola. Applied and Environmental Microbiology, 58, 1918–1923.Google Scholar
  20. 20.
    Lima, C. A., Rodrigues, P. M. B., Porto, T. S., Viana, D. A., Lima Filho, J. L., Porto, A. L. F., et al. (2009). Production of a collagenase from Candida albicans URM3622. Biochemical Engineering Journal, 43, 315–320.CrossRefGoogle Scholar
  21. 21.
    Viljoen, B. C. (2001). The interaction between yeasts and bacteria in dairy environments. International Journal of Food Microbiology, 69, 37–44.CrossRefGoogle Scholar
  22. 22.
    Lacaz, C. S., Porto, E., Martins, J. E. C., Heins-Vaccari, E. M., & Melo, N. T. (2002). Tratado de Micologia Médica (9th ed.). São Paulo: Sarvier.Google Scholar
  23. 23.
    Christensen, W. B. (1946). Urea decomposition as a means of differentiating Proteus and Paracolor cultures from each other and from Salmonella and Shigella types. Journal of Bacteriology, 52, 461–466.Google Scholar
  24. 24.
    Leighton, T. J., Doi, R. H., Warren, R. A. J., & Lelen, R. A. (1973). The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis. Journal of Molecular Biology, 76, 103–122.CrossRefGoogle Scholar
  25. 25.
    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
  26. 26.
    Ariahu, C. C., & Ogunsua, A. O. (2000). Thermal degradation kinetics of thiamine in prewinkle based formulated low acidity foods. International Journal of Food Science & Technology, 35, 315–321.CrossRefGoogle Scholar
  27. 27.
    Eyring, H. J. (1935). The activated complex in chemical reactions. Journal of Chemical Physics, 3, 107–115.CrossRefGoogle Scholar
  28. 28.
    Abdelgadir, W. S., Hamad, S. H., Moller, P. L., & Jakobsen, M. (2001). Characterisation of the dominant microbiota of Sudanese fermented milk Rob. International Dairy Journal, 11, 63–70.CrossRefGoogle Scholar
  29. 29.
    Narvhus, J. A., & Gadaga, T. H. (2003). The role of interaction between yeasts and lactic acid bacteria in African fermented milks: A review. International Journal of Food Microbiology, 86, 51–60.CrossRefGoogle Scholar
  30. 30.
    Szekeres, A., Kredics, L., Antal, Z., Kevei, F., & Manczinger, L. (2004). Isolation and characterization of protease overproducing mutants of Trichoderma harzianum. FEMS Microbiology Letters, 233, 215–222.CrossRefGoogle Scholar
  31. 31.
    Chellappan, S., Jasmin, C., Basheer, S. M., Elyas, K. K., Baht, S. G., & Chandrasekaran, M. (2006). Production, purification and partial characterization of a novel protease from marine Engyodontium album BTMFS10 under solid state fermentation. Process Biochemistry, 41, 956–961.CrossRefGoogle Scholar
  32. 32.
    Joo, H., & Chang, C. (2005). Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: Pptimization and some properties. Process Biochemistry, 40, 1263–1270.CrossRefGoogle Scholar
  33. 33.
    Bolumar, T., Sanz, Y., Aristoy, M. C., & Toldra, F. (2003). Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii. Applied and Environmental Microbiology, 69, 227–232.CrossRefGoogle Scholar
  34. 34.
    Okamoto, M., Yonejima, Y., Tsujimoto, Y., Suzuki, Y., & Watanabe, K. (2001). A Thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1. Applied Microbiology and Biotechnology, 57, 103–108.CrossRefGoogle Scholar
  35. 35.
    Nakayama, T., Tsuruoka, N., Akai, M., & Nishino, T. (2000). Thermostable collagenolytic activity of a novel thermophilic isolate, Bacillus sp. strain NTAP-1. Journal of Bioscience and Bioengineering, 89, 612–614.CrossRefGoogle Scholar
  36. 36.
    Harrington, D. J. (1996). Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infection Immunology, 64, 1885–1891.Google Scholar
  37. 37.
    Ma, C., Ni, X., Chi, Z., Ma, L., & Gao, L. (2007). Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources. Marine Biotechnology, 9, 343–351.CrossRefGoogle Scholar
  38. 38.
    Viana Marques, D. A., Oliveira, R. P. S., Perego, P., Porto, A. L. F., Pessoa, A., Jr., & Converti, A. (2009). Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060. Enzyme Microbial Technology, 45, 169–173.CrossRefGoogle Scholar

Copyright information

© Humana Press 2010

Authors and Affiliations

  • Daniela de Araújo Viana
    • 1
  • Carolina de Albuquerque Lima
    • 1
  • Rejane Pereira Neves
    • 2
  • Cristina Souza Mota
    • 2
  • Keila Aparecida Moreira
    • 1
  • José Luiz de Lima-Filho
    • 1
  • Maria Taciana Holanda Cavalcanti
    • 1
  • Attilio Converti
    • 3
  • Ana Lúcia Figueiredo Porto
    • 4
    Email author
  1. 1.Federal University of Pernambuco, Biotecnology LaboratoryCampus UniversitárioRecifeBrazil
  2. 2.Micology DepartmentFederal University of PernambucoRecifeBrazil
  3. 3.Department of Chemical and Process EngineeringUniversity of GenoaGenoaItaly
  4. 4.Federal Rural University of PernambucoDepartment of Animal Physiology and MorphologyDois IrmãosBrazil

Personalised recommendations