Biotechnology and Bioprocess Engineering

, Volume 16, Issue 3, pp 549–560 | Cite as

Production and characterization of a collagenolytic serine proteinase by Penicillium aurantiogriseum URM 4622: A factorial study

  • Carolina A. Lima
  • José L. Lima Filho
  • Benício B. Neto
  • Attilio Converti
  • Maria G. Carneiro da Cunha
  • Ana L. F. Porto
Research Paper
First Online:
Received:
Revised:
Accepted:

Abstract

A 24 full factorial design was used to identify the main effects and interactions of the initial medium pH, soybean flour concentration, temperature and orbital agitation speed on extracellular collagenase production by Penicillium aurantiogriseum URM4622. The most significant variables for collagenase production were soybean flour concentration and initial medium pH that had positive main effects, and temperature that had a negative one. Protein concentration in soybean flour revealed to be a significant factor for the production of a collagenase serine proteinase. The most favorable production conditions were found to be 0.75% soybean flour, pH 8.0, 200 rpm, and 28°C, which led to a collagenase activity of 164 U. The enzyme showed an optimum activity at 37°C and pH 9.0, was stable over wide ranges of pH and temperature (6.0 ∼ 10.0 and 25 ∼ 45°C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonylfluoride. The firstorder rate constants for collagenase inactivation in the crude extract, calculated from semi-log plots of the residual activity versus time, were used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* d = 107.4 kJ/mol and ΔH* d = 104.7 kJ/mol). The enzyme is probably an extracellular neutral serine collagenase effective on azocoll, gelatin and collagen decomposition.

Keywords

collagenase enzyme production Penicillium aurantiogriseum submerged culture factorial design 
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References

  1. 1.
    Sumantha, A., C. Larroche, and A. Pandey (2006) Microbiology and industrial biotechnology of food grade proteases-a perspective. Food Technol. Biotech. 44: 211–220.Google Scholar
  2. 2.
    Sandhya, C., A. Sumantha, G. Szakacs, and A. Pandey (2005) Comparative evaluation of neutral protease production by Aspergillus oryzae in submerged and solid-state fermentation. Proc. Biochem. 40: 2689–2694.CrossRefGoogle Scholar
  3. 3.
    Ravanti, L. and V. M. Kähäri (2000) Matrix metalloproteases in wound repair. Int. J. Mol. Med. 6: 391–407.Google Scholar
  4. 4.
    Kang, S., Y. B. Jang, Y. J. Choi, and J. Kong (2005) Purification and properties of a collagenolytic protease produced by marine bacterium Vibrio vulnificus CYK279H. Biotechnol. Bioproc. Eng. 10: 593–598.CrossRefGoogle Scholar
  5. 5.
    Kim, M., S. E. Hamilton, L. W. Guddat, and C. M. Overall (2007) Plant collagenase: Unique collagenolytic activity of cysteine proteases from ginger. Biochim. Biophys. Acta 1770: 1627–1635.CrossRefGoogle Scholar
  6. 6.
    Erdeve, O., B. Atasay, and S. Arsan (2007) Collagenase application for amputation in a preterm. Pediatr. Dermatol. 24: 195–196.CrossRefGoogle Scholar
  7. 7.
    Jin, B., H. J. Alter, Z. C. Zhang, J. W. Shih, J. M. Esteban, T. Sun, Y. S. Yang, Q. Qiu, X. L. Liu, L. Yao, H. D. Wang, and L. F. Cheng (2005) Reversibility of experimental rabbit liver cirrhosis by portal collagenase administration. Lab. Invest. 85: 992–1002.CrossRefGoogle Scholar
  8. 8.
    Matsushita, O., C. M. Jung, J. Minami, S. Katayama, N. Nishi, and A. Okabe (1998) A study of the Collagen-binding Domain of a 116-kDa Clostridium histolyticum Collagenase. J. Biol. Chem. 273: 3643–3648.CrossRefGoogle Scholar
  9. 9.
    Tran, L. H. and H. Nagano (2002) Isolation and characteristics of Bacillus subtilis CN2 and its collagenase production. J. Food Sci. 67: 1184–1187.CrossRefGoogle Scholar
  10. 10.
    Nakayama, T., N. Tsuruoka, M. Akai, and T. Nishino (2000) Thermostable collagenolytic activity of a novel Thermophilic isolate, Bacillus sp. Strain NTAp-1. J. Biosci. Bioeng. 89: 612–614.CrossRefGoogle Scholar
  11. 11.
    Lauer-Fields, J. L., D. Juska, and G. B. Fields (2002) Matrix metalloproteinases and collagen catabolism. Biopolymers 66: 19–32.CrossRefGoogle Scholar
  12. 12.
    Tamai, E., S. Miyata, H. Tanaka, H. Nariya, M. Suzuki, O. Matsushita, N. Hatano, and A. Okabe (2008) High-level expression of his-tagged clostridial collagenase in Clostridium perfringens. Appl. Microbiol. Biotechnol. 80: 627–635.CrossRefGoogle Scholar
  13. 13.
    Yakovleva, M. B., T. L. Khoang, and Z. K. Nikitina (2006) Collagenolytic activity in several species of Deuteromycetes under various storage conditions. Appl. Biochem. Micro. 42: 431–434.CrossRefGoogle Scholar
  14. 14.
    Lima, C. A., P. M. B. Rodrigues, T. S. Porto, D. A. Viana, J. L. Lima Filho, A. L. F. Porto, and M. G. C. Cunha (2009) Production of a collagenase from Candida albicans URM3622. Biochem. Eng. J. 43: 315–320.CrossRefGoogle Scholar
  15. 15.
    Rao, Y. K., S. Lu, B. Liu, and Y. Tzeng (2006) Enhanced production of an extracellular protease from Beauveria bassiana by optimization of cultivation processes. Biochem. Eng. J. 28: 57–66.CrossRefGoogle Scholar
  16. 16.
    Joo, H. S., G. C. Park, K. M. Kim, S. R. Paik, and C. S. Chang (2001) Novel alkaline protease from the Polychaeta, Periserrula leucophryna: Purification and characterization. Proc. Biochem. 36: 893–900.CrossRefGoogle Scholar
  17. 17.
    Joo, H. and C. Chang (2005) Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: Optimization and some properties. Proc. Biochem. 40: 1263–1270.CrossRefGoogle Scholar
  18. 18.
    Bruns, R. E., I. S. Scarminio, and B. de Barros Neto (2006) Statistical Design-Chemometrics. pp. 1–412. Elsevier, Amsterdam, The Netherlands.Google Scholar
  19. 19.
    Wang, Q., Y. Hou, Z. Xu, J. Miao, and G. Li (2008) Optimization of cold-active protease production by the psychrophilic bacterium Colwellia sp. NJ341 with response surface methodology. Bioresource Technol. 99: 1926–1931.CrossRefGoogle Scholar
  20. 20.
    Porto, A. L. F., G. M. Campos-Takaki, and J. L. Lima-Filho (1996) Effects of culture conditions on protease production by Streptomyces clavuligerus growing soy bean flour medium. Appl. Biochem. Biotechnol. 60: 115–122.CrossRefGoogle Scholar
  21. 21.
    Chavira, R. J., T. J. Burnett, and J. H. Hageman (1984) Assaying proteinases with azocoll. Anal. Biochem. 136: 4446–4450.Google Scholar
  22. 22.
    Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gardener, M. D. Prevenano, C. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76–85.CrossRefGoogle Scholar
  23. 23.
    Endo, A., S. Murakawa, and H. Shimizu (1987) Purification and properties of collagenase from Streptomyces species. J. Biochem. 102: 163–177.Google Scholar
  24. 24.
    Rosen, H. (1975) A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67: 10–15.CrossRefGoogle Scholar
  25. 25.
    Moore, S. and W. H. Stein (1948) Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176: 367–388.Google Scholar
  26. 26.
    Leighton, T. J., R. H. Doi, R. A. J. Warren, and R. A. Lelen (1973) The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis. J. Mol. Biol. 76: 103–122.CrossRefGoogle Scholar
  27. 27.
    James, G. T. (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal. Biochem. 86: 574–579.CrossRefGoogle Scholar
  28. 28.
    Roels, J. A. (1983) Energetics and kinetics in biotechnology. pp. 163–203. Biomedical Press, Elsevier, Amsterdam, Netherlands.Google Scholar
  29. 29.
    Ariahu, C. C. and A. O. Ogunsua (2000) Thermal degradation kinetics of thiamine in prewinkle based formulated low acidity foods. Int. J. Food Sci. Technol. 35: 315–321.CrossRefGoogle Scholar
  30. 30.
    Eyring, H. J. (1935) The activated complex in chemical reactions. J. Chem. Phys. 3: 107–115.CrossRefGoogle Scholar
  31. 31.
    Elibol, M. and A. R. Moreira (2005) Optimizing some factors affecting alkaline protease production by a marine bacterium Teredinobacter turnirae under solid substrate fermentation. Proc. Biochem. 40: 1951–1956.CrossRefGoogle Scholar
  32. 32.
    Chellappan, S., C. Jasmin, S. M. Basheer, and K. K. Elyas (2006) Production, purification and partial characterization of a novel protease from marine Engyodontium album BTMFS10 under solid state fermentation. Proc. Biochem. 41: 956–961.CrossRefGoogle Scholar
  33. 33.
    Laxman, R. S., A. P. Sonawane, S. V. More, B. S. Rao, M. V. Rele, V. V. Jogdand, V. V. Deshpande, and M. B. Rao (2005) Optimization and scale up of production of alkaline protease from Conidiobolus coronatus. Proc. Biochem. 40: 3152–3158.CrossRefGoogle Scholar
  34. 34.
    Anandan, D., W. N. Marmer, and R. L. Dudley (2007) Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii. J. Ind. Microbiol. Biotechnol. 34: 339–347.CrossRefGoogle Scholar
  35. 35.
    Chi, Z., C. Ma, P. Wang, and H. F. Li (2007) Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresource Technol. 98: 534–538.CrossRefGoogle Scholar
  36. 36.
    Chi, Z. and S. Zhao (2003) Optimization of medium and cultivation conditions for pullulan production by a new pullulan-producing yeast. Enz. Microb. Technol. 33: 206–221.CrossRefGoogle Scholar
  37. 37.
    Groudieva, T., M. Kambourova, H. Yusef, M. Royter, R. Grote, H. Trinks, and G. Antranikian (2004) Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen. Extremophiles 8: 475–488.CrossRefGoogle Scholar
  38. 38.
    Zardetto, S. (2005) Effect of modified atmosphere packaging at abuse temperature on the growth of Penicillium aurantiogriseum isolated from fresh filled pasta. Food Microbiol. 22: 367–371.CrossRefGoogle Scholar
  39. 39.
    Sakurai, Y., H. Inoue, W. Nishii, T. Takahashi, Y. Lino, M. Yamamoto, and K. Takahashi (2009) Purification and characterization of a major collagenase from Streptomyces parvulus. Biosci. Biotechnol. Biochem. 73: 21–28.CrossRefGoogle Scholar
  40. 40.
    Tsuruoka, N., T. Nakayama, M. Ashida, H. Hemmi, M. Nakao, H. Minakata, H. Oyama, K. Oda, and T. Nishino (2003) Collagenolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain NTAP-1: Purification, characterization, gene cloning, and heterologous expression. Appl. Environ. Microb. 69: 162–169.CrossRefGoogle Scholar
  41. 41.
    Petrova, D., A. Derekova, and S. Vlahov (2006) Purification and properties of individual collagenases from Streptomyces sp. strain 3B. Folia Microbiol. 51: 93–98.CrossRefGoogle Scholar
  42. 42.
    Viana, D. A., C. A. Lima, R. P. Neves, C. S. Mota, K. A. Moreira, J. L. Lima-Filho, M. T. H. Cavalcanti, A. Converti, and A. L. F. Porto (2010) Production and stability of protease from Candida buinensis. Appl. Biochem. Biotechnol. 162: 830–842.CrossRefGoogle Scholar
  43. 43.
    Moreno, J. M., M. Arroyo, M. J. Hernáiz, and J. V. Sinisterra (1997) Covalent immobilization of pure isoenzymes from lipase of Candida rugosa. Enz. Microb. Technol. 21: 552–558.CrossRefGoogle Scholar
  44. 44.
    Porto, T. S., C. S. Porto, M. T. H. Cavalcanti, J. L. Lima Filho, P. Perego, A. L. F. Porto, A. Converti, and A. Pessoa Jr (2006) Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract. Biotechnol. Progr. 22: 1637–1642.Google Scholar
  45. 45.
    Ramírez-Zavala, B., Y. Mercado-Flores, C. Hernández-Rodríguez, and L. Villa-Tanaca (2004) Purification and characterization of a lysine aminopeptidase from Kluveromyces marxianus. FEMS Microbiol. Lett. 235: 369–375.Google Scholar
  46. 46.
    Ma, C., X. Ni, Z. Chi, L. Ma, and L. Gao (2007) Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources. Mar. Biotechnol. 9: 343–351.CrossRefGoogle Scholar
  47. 47.
    De Vicente, J. I., D. De Arringa, P. Del Valle, J. Soler, and A. P. Eslava (1996) Purification and characterization of an extracellu lar aspartate protease from Phycomyces blakesleeanus. Fungal Genet. Biol. 20: 115–124.CrossRefGoogle Scholar
  48. 48.
    Okamoto, D. N., M. Y. Kondo, J. A. N. Santos, S. Nakajima, K. Hiraga, K. Oda, M. A. Juliano, L. Juliano, and I. E. Gouvea (2009) Kinetic analysis of salting activation of a subtilisin-like halophilic protease. Biochim. Biophys. Acta 1794: 367–373.Google Scholar
  49. 49.
    Fusek, M., X. L. Lin, and J. Tang (1990) Enzymic properties of thermopsin. J. Biol. Chem. 265: 1496–1501.Google Scholar
  50. 50.
    Rossi, F. G., M. Z. Ribeiro, A. Converti, M. Vitolo, and A. Pessoa Jr. (2003) Kinetic and thermodynamic aspects of glucose-6-phosphate dehydrogenase activity and synthesis. Enz. Microb. Technol. 32: 107–113.CrossRefGoogle Scholar
  51. 51.
    Ikegaya, K., S. Sugio, K. Murakami, and K. Yamanouchi (2003) Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium-dependent stability. Biotechnol. Bioeng. 81: 187–192.CrossRefGoogle Scholar
  52. 52.
    Villa, A., L. Zecca, P. Fusi, S. Colombo, G. Tedeschi, and P. Tortora (1993) Structural features responsible for kinetic thermal stability of a carboxipeptidase from the archaebacterium Sulfolobus solfataricus. Biochem. J. 295: 827–831.Google Scholar
  53. 53.
    Kristjansson, M. M. and J. E. Kinsella (1990) Alkaline serine proteinase from Thermomonospora fusca YX. Biochem. J. 270: 51–55.Google Scholar
  54. 54.
    Nagano, H. and K. A. To (1999) Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2. Biosci. Biotechnol. Biochem. 63: 181–183.CrossRefGoogle Scholar
  55. 55.
    Okamoto, M., Y. Yonejima, Y. Tsujimoto, Y. Suzuki, and K. Watanabe (2001) A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1. Appl. Microbiol. Biotechnol. 57: 103–108.CrossRefGoogle Scholar
  56. 56.
    Uesugi, Y., J. Arima, H. Usuki, M. Iwabuchi, and T. Hatanaka (2008) Two bacterial collagenolytic serine proteases have different topological specificities. Biochim. Biophys. Acta 1784: 716–726.Google Scholar
  57. 57.
    Sukhosyrova, E. A., Z. K. Nikitina, M. B. Yakovleva, E. V. Veshchikova, and V. A. Bykov (2003) Characteristics of collagenolytic enzymes secreted by deuteromycete fungi Aspergillus flavus. B. Exp. Biol. Med. 135: 447–451.CrossRefGoogle Scholar
  58. 58.
    Wu, Q., C. Li, C. Li, H. Chen, and L. Shuliang (2010) Purification and characterization of a novel collagenase from Bacillus pumilus Col-J. Appl. Biochem. Biotechnol. 160: 129–139.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • Carolina A. Lima
    • 1
  • José L. Lima Filho
    • 1
    • 2
  • Benício B. Neto
    • 3
  • Attilio Converti
    • 4
  • Maria G. Carneiro da Cunha
    • 1
    • 2
  • Ana L. F. Porto
    • 1
    • 5
  1. 1.Laboratory of Immunopathology Keizo Asami (LIKA)Federal University of Pernambuco-UFPE, PEPernambucoBrazil
  2. 2.Department of BiochemistryFederal University of Pernambuco-UFPE, PEPernambucoBrazil
  3. 3.Department of Fundamental ChemistryFederal University of Pernambuco-UFPE (UFPE)PernambucoBrazil
  4. 4.Department of Chemical and Process EngineeringUniversity of GenoaGenoaItaly
  5. 5.Department of Morphology and Animal PhysiologyFederal Rural University of Pernambuco-UFRPE, PEPernambucoBrazil

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