Production and Characterization of Thermostable Protease from Bacillus licheniformis Isolated from Korean Traditional Soybean Paste

재래식 된장에서 분리된 Bacillus licheniformis의 내열성 Protease 특성과 생산성

  • Bae, Young Eun (Department of Food Science & Biotechnology, Woosong University) ;
  • Yoon, Ki-Hong (Department of Food Science & Biotechnology, Woosong University)
  • 배영은 (우송대학교 식품생물과학과) ;
  • 윤기홍 (우송대학교 식품생물과학과)
  • Received : 2012.10.16
  • Accepted : 2012.11.16
  • Published : 2012.12.31


Among 63 Bacillus strains grown at $60^{\circ}C$ from sixteen samples of homemade Korean soybean paste, one strain was selected for producing the thermostable protease. The isolate has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. Culture filtrate of the isolate showed maximal protease activity at the reaction condition of $60-65^{\circ}C$ and pH 11. The culture filtrate retained more than 87% of initial protease activity after incubation for 30 min at $60^{\circ}C$ without substrate. In order to develop the medium composition, effects of ingredients including nitrogen sources, carbon sources, metal ions and phosphate were examined for protease production of the isolate. Lactose and soytone peptone were the most effective carbon and nitrogen source for the enzyme production. After the late logarithmic growth phase the isolate began to produce the protease, and the maximum protease productivity was reached to 550 unit/ml in the optimized medium consisting of lactose (3%), soytone peptone (1.5%), $MgSO_4$ (0.1%), $K_2HPO_4$ (0.03%), and $KH_2PO_4$ (0.03%) at 28 h of incubation.


Bacillus licheniformis;properties;protease productivity;soybean paste


  1. Abdel-Fattah, Y.R., El-Enshasy, H.A., Soliman, N.A., and El-Gendi, H. 2009. Bioprocess development for production of alkaline protease by Bacillus pseudofirmus Mn6 through statistical experimental designs. J. Microbiol. Biotechnol. 19, 378-386.
  2. Abusham, R.A., Rahman, R.N., Salleh, A.B., and Basri, M. 2009. Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand. Microb. Cell Fact. 8, 20.
  3. Bang, S.H. and Jeong, I.-S. 2011. Characterization of an alkaline protease from an alkalophilic Bacillus pseudofirmus HS-54. Kor. J. Microbiol. 47, 194-199.
  4. Brar, S.K., Verma, M., Tyagi, R.D., Surampalli, R.Y., Barnabe, S., and Valero, J.R. 2007. Bacillus thuringiensis proteases: Production and role in growth, sporulation and synergism. Process Biochem. 42, 773-790.
  5. Deng, A., Wu, J., Zhang, Y., Zhang, G., and Wen, T. 2010. Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour. Technol. 101, 7100-7106.
  6. Gupta, R., Beg, Q.K., Khan, S., and Chauhan, B. 2002. An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl. Microbiol. Biotechnol. 60, 381-395.
  7. Haddar, A., Fakhfakh-Zouari, N., Hmidet, N., Frikha, F., Nasri, M., and Kamoun, A.S. 2010. Low-cost fermentation medium for alkaline protease production by Bacillus mojavensis A21 using hulled grain of wheat and sardinella peptone. J. Biosci. Bioengneer. 110, 288-294.
  8. Hadj-Ali, N.E., Agrebi, R., Ghorbel-Frikha, B., Sellami-Kamoun, A., Kanoun, S., and Nasri, M. 2007. Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolated Bacillus licheniformis NH1. Enzyme Microb. Technol. 40, 515-523.
  9. Hmidet, N., Ali, N.E.-H., Hadder, A., Kanoun, S., Alya, E.-K., and Nasri, M. 2009. Alkaline proteases and thermostable alpha-amylase co-produced by Bacillus licheniformis NH1: characterization and potential application as detergent additive. Biochem. Engineer. J. 47, 71-79.
  10. Hmidet, N., Ali, N.H., Zouari-Fakhfakh, N., Haddar, A., Nasri, M., and Sellemi-Kamoun, A. 2010. Chicken feathers: a complex substrate for the co-production of alpha-amylase and proteases by B. licheniformis NH1. J. Ind. Microbiol. Biotechnol. 37, 983-990.
  11. Horikoshi, K. 1990. Enzymes of alkalophiles. pp. 275-294. In Fogarty, W.M. and Kelly, C.T. (eds.), Microbial enzyme and biotechnology, 2nd ed. Elsevier Applied Science Publishers, Barking, UK.
  12. Johnvesly, B. and Naik, G.R. 2001. Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem. 37, 139-144.
  13. Juyigbe, F.M. and Ajele, J.O. 2008. Some properties of extracellular protease from Bacillus licheniformis LBBL-11 isolated from iru, a traditionally fermented African locust bean condiment. African J. Biochem. Res. 2, 206-210.
  14. Kanekar, P.P., Nilegaonkar, S.S., Sarnaik, S.S., and Kelkar, A.S. 2002. Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India. Bioresour. Technol. 85, 87-93.
  15. Kim, S.S., Lee, J.-H., Ahn, Y.-S., Kim, J.-H., and Kang, D.-K. 2003. A fibrinolytic enzyme from Bacillus amyloliquefaciens D4-7 isolated from Chungkook-Jang; it's characterization and influence of additives on thermostability. Kor. J. Microbiol. Biotechnol. 31, 271-276.
  16. Koma, D., Yamanaka, H., Moriyoshi, K., Ohmoto, T., and Sakai, K. 2007. Overexpression and characterization of thermostable serine protease in Escherichia coli encoded by the ORF TTE0824 from Thermoanaerobacter tengcongensis. Extremophiles 11, 769-779.
  17. Kumar, C.G. 2002. Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus. Lett. Appl. Microbiol. 34, 13-17.
  18. Kumar, D. and Bhalla, T.C. 2004. Purification and characterization of a small size protease from Bacillus sp. APR-4. Indian J. Exp. Biol. 42, 515-521.
  19. Lee, S.-K., Bae, D.-H., Kwon, T.-J., Lee, S.-B., Lee, H.-H., Park, J.-H., Heo, S., and Johnson, M.G. 2001. Purification and characterization of a fibrinolytic enzyme from Bacillus sp. KDO-13 isolated from soybean paste. J. Microbiol. Biotechnol. 11, 845-852.
  20. Li, S., He, B., Bai, Z., and Ouyang, P. 2009. A novel organic solvent-stable alkaline protease from organic solvent-tolerant Bacillus licheniformis YP1A. J. Mol. Catal. B Enzym. 56, 85-88.
  21. Liu, C.H., Chiu, C.S., Ho, P.L., and Wang, S.W. 2009. Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto. J. Appl. Microbiol. 107, 1031-1041.
  22. Mabrouk, S.S., Hashem, A.M., El-Shayeb, N.M.A., Ismail, A.-M.S., and Abdel-Fattah, A.F. 1999. Optimization of alkaline protease productivity by Bacillus licheniformis ATCC 21415. Bioresour. Technol. 69, 155-159.
  23. Nilegaonkar, S.S., Zambare, V.P., Kanekar, P.P., Dhakephalkar, P.K., and Sarnaik, S.S. 2007. Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour. Technol. 98, 1238-1245.
  24. Prakash, M., Banik, R.M., and Koch-Brandt, C. 2005. Purification and characterization of Bacillus cereus protease suitable for detergent industry. Appl. Biochem. Biotechnol. 127, 143-155.
  25. Sellami-Kamoun, A., Haddar, A., Ali, N.E.-H., Ghorbel-Frikha, B., Kanoun, S., and Nasri, M. 2008. Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laudry detergent formulations. Microbiol. Res. 163, 299-306.
  26. Shivanand, P. and Jayaraman, G. 2009. Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast. Process Biochem. 44, 1088-1094.
  27. Son, H.-J. 2005. Cultural conditions for protease production by a feather-degrading bacterium, Bacillus megaterium F7-1. Kor. J. Microbiol. Biotechnol. 33, 315-318.
  28. Sookkheo, B., Sinchaikul, S., Phutrakul, S., and Chen, S.T. 2000. Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33. Protein Expr. Purif. 20, 142-151.
  29. Sun, C., Jin, C., Yang, S., and Zhang, S. 1999. Purification and properties of genetic expressing product of thermostable protease from Bacillus stearothermophilus HY-69. Chin. J. Biotechnol. 15, 15-21.
  30. Yoon, K.-H., Lee, M.S., Park, B.W., Park, Y.-H., Kim, H., Kim, J.H., and Kim, M.S. 2006. Enzyme production of a protease-producing strain, Bacillus sp SH-8 isolated from insect-eating plant. Kor. J. Microbiol. Biotechnol. 34, 323-328.
  31. Yoon, K.-H. and Shin, H.Y. 2010. Medium optimization for the protease production by Bacillus licheniformis isolated from Cheongkookjang. Kor. J. Microbiol. Biotechnol. 38, 385-390.

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