Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of Two Thermostable Proteases from the Thermophilic Fungus Chaetomium thermophilum

  • Li, An-Na (Department of Environmental Biology, Shandong Agricultural University) ;
  • Ding, AI-Yun (Department of Environmental Biology, Shandong Agricultural University) ;
  • Chen, Jing (Department of Environmental Biology, Shandong Agricultural University) ;
  • Liu, Shou-An (Department of Environmental Biology, Shandong Agricultural University) ;
  • Zhang, Ming (Department of Environmental Biology, Shandong Agricultural University) ;
  • Li, Duo-Chuan (Department of Environmental Biology, Shandong Agricultural University)
  • Published : 2007.04.30
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Abstract

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by tractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63kDa protease (PRO63) at pH5.0. The optimum temperature for the two proteases was $65^{\circ}C$. The PRO33 had a $K_m$ value of 6.6mM and a $V_{max}$ value of $10.31{\mu}mol/l/min$, and PRO63 l7.6mM and $9.08{\mu}mol/l/min$, with casein as substrate. They were thermostable at $60^{\circ}C$. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at $70^{\circ}C$ for 1h. The thermal stability of the two enzymes was significantly enhanced by $Ca^{2+}$. The residual activity of PRO33 and PRO63 at $70^{\circ}C$ after 60min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing $Ca^{2+}$. These properties make them applicable for many biotechnological purposes.

Keywords

References

  1. Aunstrup, K. 1980. Proteniases. In A. H. Rose (ed.), Microbial Enzymes and Bioconversions. Academic Press: London, New York, Toronto, Sydney and San Francisco
  2. Bazarzhapov, B. B., E. V. Lavrent'eva, Y. E. Dunaevskii, E. N. Bilanenko, and B. B. Namsaraev. 2006. Extracellular proteolytic enzymes of microscopic fungi from thermal springs of the Barguzin Valley (Northern Baikal region). Appl. Biochem. Microbiol. 42: 186-189 https://doi.org/10.1134/S000368380602013X
  3. Burlini, N., P. Magnani, A. Villa, F. Macchi, P. Tortora, and A. Guerritore. 1992. A heat-stable serine protease from the extreme thermophilic archaebacterium Sulfolobus solfataricus. Biochim. Biophys. Acta 1122: 238-292
  4. Chakrabarti, S. K., N. Matsumura, and R. S. Ranu. 2000. Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJRA 6.2). Curr. Microbiol. 40: 239-244 https://doi.org/10.1007/s002849910048
  5. Chen, X. G., O. Stabnikova, J. H. Tay, J. Y. Wang, and S. T. L. Tay. 2004. Thermoactive extracellular proteases of Geobacillus caldoproteolyticus, sp. nov., from sewage sludge. Extremophiles 8: 489-498 https://doi.org/10.1007/s00792-004-0412-5
  6. Connaris, H., D. A. Cowan, and R. J. Sharp. 1991. Heterogeneity of proteases from the hypermophilic archaeobacterium Pyroccus furiosus. J. Gen. Microbiol. 137: 1193-1199 https://doi.org/10.1099/00221287-137-5-1193
  7. Cooney, D. G. and R. Emerson. 1964. Thermophilic Fungi. An Account of Their Biology, Activities and CLASSIFICATION. W. H. Freeman and Co, San Francisco
  8. Cowan, D. A. and R. M. Daniel. 1982. Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile. Biochim. Biophys. Acta 705: 239-305
  9. Cowan, D. A., K. A. Smolenski, R. M. Daniel, and H. W. Morgan. 1987. An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88$^{\circ}C$. Biochemi. J. 247: 121-133 https://doi.org/10.1042/bj2470121
  10. Cronlund, A. and J. H. Woychik. 1986. Effect of microbial rennets on meat protein fraction. J. Agric. Food Chem. 34: 502-505 https://doi.org/10.1021/jf00069a033
  11. Domsch, K. H., W. Gams, and T. H. Anderson. 1980. Compendium of Soil Fungi. Acdemic Press, London
  12. Escobar, J. and S. Barnett. 1995. Synthesis of acid protease from Mucor miehei: Integration of production and recovery. Process Biochem. 30: 695-700 https://doi.org/10.1016/0032-9592(95)00005-4
  13. Gary, S. K. and B. N. Johri. 1994. Rennet: Current trends and future research. Food Rev. Int. 10: 313-335 https://doi.org/10.1080/87559129409541005
  14. Gary, S. K. and B. N. Johri. 1999. Proteolytic enzymes. In Johri, B. N., T. Satyanarayana, and T. Olsen, (eds.), Thermophilic Moulds in Biotechnology. Dordrecht, Boston and London: Kluwer Academic Publishers
  15. Hasbay, I. and Z. B. Ogel. 2002. Production of neutral and alkaline extracellular proteases by the thermophilic fungus, Scytalidium thermophilum, grown on microcrystalline cellulose. Biotechnol. Lett. 24: 1107-1110 https://doi.org/10.1023/A:1016026904687
  16. Hashimoto, H., T. Iwaasa, and T. Yokotusha. 1973. Some properties of acid protease from the thermophilic fungus, Penicillium dupnti K1014. Appl. Microbiol. 25: 578-583
  17. Hasnain, S., K. Adeli, and A. C. Storer. 1992. Purification and characterization of an extracellular thio-containing serine proteinase from Thermomyces lanuginosus. Biochem. Cell Biol. 70: 117-122 https://doi.org/10.1139/o92-017
  18. Hyeung, J., K. Byoung, P. Yu, and K. Yu. 2002. A novel subtilisin-like serine protease from Thermoanaerobacter yonseiensis KB-1: Its cloning, expression, and biochemical properties. Extremophiles 6: 233-243 https://doi.org/10.1007/s00792-001-0248-1
  19. Imanaka, T., K. Sasaki, and M. Takagi. 1986. A new way of enhancing the thermal stability of proteases. Nature 324: 695-697 https://doi.org/10.1038/324695a0
  20. Jensen, B., P. Nebelong, J. Olsen, and M. Reeslev. 2002. Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus. Biotechnol. Lett. 24: 41-45 https://doi.org/10.1023/A:1013805232462
  21. Karavaeva, N. N., M. N. Zakirov, and N. G. Mukhiddinova. 1975. Partial purification and some properties of protease from Torula thermophila. Biochimiya 40: 909-914
  22. Kim, J. M., M. C. Yang, and J. S. Hyung. 2005. Preparation of feather digest as fertilizer with Bacillus pumillus KHS-1. J. Microbiol. Biotechnol. 15: 427-476
  23. Kristjansson, J. K. 1989. Thermophilic organisms as sources of themostable enzymes. Trends Biotechnol. 7: 349-353 https://doi.org/10.1016/0167-7799(89)90035-8
  24. Laemmli, U. K. 1970. Clearage of structural proteins during the assembly of the head of becteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  25. Lee, J. S., S. B. Hyung, and S. P. Sang. 2006. Purification and characterization of two novel fibrinolytic proteases from mushroom, Fomitella fraxinea. J. Microbiol. Biotechnol. 16: 264-271
  26. Leon, D. K., G. V. Wilfried, J. S. Roland, M. S. Ruth, A. Garabed, O. John, and M. V. Willem. 2002. Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans. Extremophiles 6: 185-194 https://doi.org/10.1007/s007920100239
  27. Li, D. C., Y. J. Yang, C. Y. Shen. 1997. Protease production by the thermophilic fungus Thermomyces lanuginosus. Mycol. Res. 101: 18-22 https://doi.org/10.1017/S0953756296002109
  28. Li, D. C., M. Lu, Y. L. Li, and J. Lu. 2003. Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT2. Enzyme Microb. Technol. 33: 932-938 https://doi.org/10.1016/S0141-0229(03)00245-X
  29. Lin, X. L. and J. Tang. 1990. Purification, characterization and gene cloning of Theropsin, a thermostable acid protease from Sulfolobus acidocaldarius. J. Biol. Chem. 265: 1490-1495
  30. Maheshwari, R., G. Bharadwaj, and M. K. Bhat. 2000. Thermophilic fungi: Their physiology and enzymes. Microbiol. Mol. Biol. Rev. 64: 461-488 https://doi.org/10.1128/MMBR.64.3.461-488.2000
  31. Mishra, R. and R. Maheshwari. 1996. Amylases of the thermophilic fungus Thermomyces lanuginosus: Their purification, properties, action on starch and response to heat. J. Biosci. 2: 653-672
  32. Nam, G. W., D. W. Lee, H. S. Lee, N. J. Lee, B. C. Kim, E. A. Choe, J. K. Hwang, M. T. Suhartono, and Y. R. Pyun. 2002. Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch. Microbiol. 178: 538-547 https://doi.org/10.1007/s00203-002-0489-0
  33. North, W. J. 1982. Comparative biochemistry of the proteinases of eukaryotic microorganisms. Microbiol. Rev. 46: 308-340
  34. 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 https://doi.org/10.1007/s002530100731
  35. Ong, P. S. and G. M. Gaucher. 1973. Protease production by thermophilic fungi. Can. J. Microbiol. 19: 129-133 https://doi.org/10.1139/m73-019
  36. Ong, P. S. and G. M. Gaucher. 1976. Production, purification and characterization of thermomycolase, the extracellular serine protease of thermophilic fungus Malbranchea pulchella var. sulfurea. Can. J. Microbiol. 22: 165-176 https://doi.org/10.1139/m76-023
  37. Outtrup, H. and C. O. L. Boye. 1990. Microbial proteinases and biotechnology. In Fogarty, W. M. (ed.), Microbial Enzymes and Biotechnology, 2nd Ed. London and New York, Elsevier Science Publishers
  38. Rick, W. 1974. Chymotrypsin. In Bergmeyer, H. U. (ed.), Methods of Enzymatic Analysis. Berlin, New York, and London: Academic Press
  39. Sabine, R. and A. Garabed. 2001. Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophiles 5: 399-408 https://doi.org/10.1007/s007920100209
  40. Shenolikar, S. and K. J. Stevenson. 1982. Purification and partial characterization of a thiol proteinase from the thermophilic fungus Humicola lanuginosus. Biochem. J. 205: 147-152 https://doi.org/10.1042/bj2050147
  41. Thakur, M. S., N. G. Karanth, and K. Nand. 1990. Production of fungal rennet by Mucor miehei using solid state fermentation. Appl. Microbiol. Biotechnol. 32: 409-413 https://doi.org/10.1007/BF00903774
  42. Ward, O. P. 1983. Protease. In Fogarty, W. M. (ed.), Microbial Enzymes in Biotechnology. London: Applied Science Publisher