Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Production and Characterization of an Alkaline Protease from Bacillus licheniformis MH31

  • Yu, Jeong-Hyeon (Department of Agricultural Chemistry, College of Agriculture and Lifesciences, Chungnam National University) ;
  • Jin, Hyun-Seok (Namyang Dairy Products Co., Ltd. R&D Center) ;
  • Choi, Woo-Young (Department of Agricultural Chemistry, College of Agriculture and Lifesciences, Chungnam National University) ;
  • Yoon, Min-Ho (Department of Agricultural Chemistry, College of Agriculture and Lifesciences, Chungnam National University)
  • Published : 2006.12.31
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Abstract

A alkalophilic strain, Bacillus licheniformis MH31 producing an alkaline protease was isolated from mine soil of Boryeong in Korea. Production of a high level of alkaline protease was achieved 42 h after incubation when the bacterium was grown at pH 9.0 and $35^{\circ}C$ in Horikoshi medium supplemented with 0.5%(w/v) starch and 1%(w/v) skim milk as carbon and nitrogen source, respectively. The molecular weight of partially purified enzyme was estimated to be 30 kDa by SDS-PAGE and its optimum pH was pH 10. The enzyme showed optimum temperature at $50^{\circ}C$, and was stable up to $60^{\circ}C$ after 1 h incubation. The protease was strongly inhibited by 1 mM of PMSF which was known well as strong inhibitor of serine proteases, but almost not inhibited by 5 mM of EDTA and 1,10-phenanthroline. When the protein hydrolysis products of 1% skim milk by partially purified protease was compared with available commercial proteases using HPLC analysis, most of hydrolysis products were detected below molecular weight of 10,000 and the hydrolysis ratio of purified enzyme was 24.8% lower than those(above 32%) of commercial proteases.

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References

  1. Atalo, K. and Gashe, B. A. (1993) Protease production by a thermophilic Bacillus species (P-001A) which degrades various kinds fibrous protein. Biotechnol. Lett. 15, 1151-1156 https://doi.org/10.1007/BF00131207
  2. Lee, C. H., Kwon, T. J., Kang, S. M., Suh, H. H., Kwon, G. S., Oh, H. M. and Yoon, B. D. (1994) Production and characterization of an alkaline protease from an isolate, Xanthomonas sp. YL-37. Kor. J. Appl. Microbiol. Biotechnol. 22, 515-521
  3. Malathi, S. and Chakraborty, R. (1991) Production of alkaline protease by a new Aspergillus flavus isolated under solid-substrate fermentation conditions for use as a depilation agent. Appl. Environ. Microbiol. 27, 712-716
  4. Chen, S. T., Kao, C. L. and Whang, K. T. (1995) Alkaline protease catalysis of a secodary amine to form a petide bond. Int. J. Peptide Prot. Res. 46, 314-319
  5. Anwar, A. and Saleemuddin, M. (1998) Alkaline protease: A review. Bioresource Technology 64, 175-183 https://doi.org/10.1016/S0960-8524(97)00182-X
  6. Rahman, R. N. Z. A., Kamaruzaman, C. N. R., Basri, A. M., Yunus, W. M. Z.W. and Salleh, A. B. (1994) Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1. Appl. Microbiol. Biotechnol. 40, 822-827 https://doi.org/10.1007/BF00173982
  7. Jacobs, M., Eliasson, M., Uhelen, M. and Flock, J. I. (1985) Cloning, sequencing and expression of Subtilisin Carlsberg from Bacillus lichenifomis. Nucleic Acids Review. 13, 8913-8926 https://doi.org/10.1093/nar/13.24.8913
  8. Johnvesly, B. and Naik, G. R. (2001) Production of Bleachstable and halo-tolerant alkalie protease by an alkalophilic Bacillus pumilus JB05 isolated from cement industry effluents. J. Microbiol. Biotechnol. 11(4), 558-563
  9. Hameed, A., Natt, M. A. and Evans, C. S. (1996) Production of alkaline protease by a Bacillus subtilis isolate for use as being enzyme in leather treatment. World Journal of Microbiol. Biotechnol. 12, 289-291 https://doi.org/10.1007/BF00360930
  10. Horikoshi, K. (1971a) Production of alkaline protease by alkalophilic Bacillus No. 221. Agri. Biol. Chem. 35, 1407- 1414 https://doi.org/10.1271/bbb1961.35.1407
  11. Fujiwara, N., Masui, A. and Imanaka, T. (1993) Purification and properties of highly thermostable alkaline protease from an alkalophilic and thermophilic Bacillus sp. J. Biotechnol. 30, 245-256 https://doi.org/10.1016/0168-1656(93)90117-6
  12. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein determination. J. Biol. Chem. 193, 265-275
  13. Saitou, N. and Nei, M. (1987). The neighbor-joining method: a now method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425
  14. Kimura, M. (1983) The Neutral Theory of Molecular Evolution, pp. 41-46. Cambridge: Cambridge University Press
  15. Calik, P., Tomln, G. C., Oliver, S. G. and Ozdamar, T. H. (2003) Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network. Enzyme and Microbial Technology. 32, 706-720 https://doi.org/10.1016/S0141-0229(03)00030-9
  16. Ferrero, M. A., Cstro, G. R., Abate, C. M., Baigori, M. D. and Sineriz, F. (1996) Themostable alkaline protease of Bacillus licheniformis MIR-29: Isolation, production and characterization. Appl. Microbiol. Biotechnol. 45, 327-332 https://doi.org/10.1007/s002530050691
  17. Kumar C. G., Tiwari M. P. and Jany K. D. (1999) Novel alkaline serine proteases from aklophlic Bacillus spp.: purification and some properties. Process Biochem. 34, 441-449 https://doi.org/10.1016/S0032-9592(98)00110-1
  18. Margesin. R., Palma, N., Knauseder, F. and Schinner, F. (1992) Purification of an alkaline protease produced by a psychrotrophic Bacillus sp. J. Biotechnol. 24, 203-206 https://doi.org/10.1016/0168-1656(92)90124-R