Characterization of an Alkaline Protease from an Alkalophilic Bacillus pseudofirmus HS-54

호알칼리성 Bacillus pseudofirmus HS-54가 생산하는 알칼리성 Protease의 특성

  • Bang, Seong-Ho (Department of Biological Sciences, Hanseo University) ;
  • Jeong, In-Sil (Department of Biological Sciences, Hanseo University)
  • 방성호 (한서대학교 생명과학과) ;
  • 정인실 (한서대학교 생명과학과)
  • Received : 2011.08.19
  • Accepted : 2011.09.05
  • Published : 2011.09.30

Abstract

An alkalophilic bacterium producing alkaline protease was isolated from waste water and solar saltern sample and identified as Bacillus pseudofirmus HS-54 based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The HS-54 protease was purified to homogeneity using ammonium sulfate precipitation, DEAE cellulose column chromatography, and sephadex G-100 gel filtration with a 4.0 purification fold. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 27 kDa. The optimal pH and temperature for the purified protease activity were 10.0 and $50^{\circ}C$, respectively. The purified enzyme was relatively stable at the pH range of 6.0-11.0 and at the temperature below $50^{\circ}C$. This enzyme was activated by $Ca^{2+}$ and $Mg^{2+}$ and inhibited by $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Al^{3+}$, $Ag^{2+}$. And this enzyme was strongly inhibited by PMSF, suggesting that it belongs to the serine protease superfamily.

Keywords

Bacillus pseudofirmus HS-54;alkaline protease;purification;serine protease

Acknowledgement

Supported by : 한서대학교

References

  1. Adinarayana, K., P. Ellaiah, and S.D. Prasad. 2003. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS Pharm. Sci. Tech. 4, 1-9.
  2. Arulmani, M., K. Aparanjini, K. Vasanthi, P. Arumugam, M. Arivuchelvi, and P.T. Kalaichelvan. 2007. Purification and partial characterization of serine protease from thermostable alkaliphilic Bacillus laterosporus AK-1. World J. Microb. Technol. 23, 475-481. https://doi.org/10.1007/s11274-006-9249-7
  3. Banerjee, U.C., R.K. Sani, W. Azmi, and R. Soni. 1999. Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Bichem. 35, 213-219. https://doi.org/10.1016/S0032-9592(99)00053-9
  4. Banika, R.M. and M. Prakash. 2004. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res. 159, 135-140. https://doi.org/10.1016/j.micres.2004.01.002
  5. Bradford, M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  6. Chung, S.S., Y.U. Shin, H.J. Kim, G.H. Jin, and H.H. Lee. 2001. Transfomation of an alkaline protease over-producer, Vibrio mettschnikovii strain RH530, and improvement of plasmid stability by the par locus. J. Microbiol. Biotechnol. 11, 222-228.
  7. Dhandapani, R. and R. Vijayaragvan. 1994. Production of thermophilic, extracellular alkaline protease by Bacillus steareothermophilus AP-4. J. Microbiol. Biotechnol. 10, 33-35. https://doi.org/10.1007/BF00357559
  8. Dodia, M.S., C.M. Rawal, H.G. Bhimani, R.H. Joshi, S.K. Khare, and S.P. Singh. 2008. Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6. J. Ind. Microbiol. Biotechnol. 35, 121-131. https://doi.org/10.1007/s10295-007-0273-x
  9. Donaghy, J.A. and A.M. Mckay. 1993. Production and properties of alkaline protease by Aurebasdum pullulans. J. Appl. Bacteriol. 60, 662-666.
  10. Giri, S.S., V. Sukumaran, S.S. Sen, M. Oviya, B.N. Banu, and P.K. Jena. 2011. Purification and partial characterization of a detergent and oxidizing agent stable alkaline protease from a newly isolated Bacillus subtilis VSG-4 of tropical soil. J. Microbiol. 49, 455-461. https://doi.org/10.1007/s12275-011-0427-4
  11. Gupta, R., Q.K. Beg, and P. Rorenze. 2002. Bacterial alkaline proteases : molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59, 15-32. https://doi.org/10.1007/s00253-002-0975-y
  12. Gupta, R., K. Gupta, R. Saxena, and S. Khan. 1999. Bleach-stable alkaline protease from Bacillus sp. Biotechnol. Lett. 21, 135-138. https://doi.org/10.1023/A:1005478117918
  13. Haddar, A., R. Agreibi, A. Bougatef, N. Hmide, A. Sellami- Kamoun, and M. Nasri. 2009. Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent. Bioresour. Technol. 100, 3366-3373. https://doi.org/10.1016/j.biortech.2009.01.061
  14. Hadi-Ali, N.E., R. Agrebi, B. Ghobel-Frikha, A. Sellami- Kamoun, S. Kanoun, and M. Nasri. 2007. Biochemical and molecular characterization of a detergent stable alkaline protease from a newly isolated B. licheniformis NH1. Enzyme Microb. Technol. 40, 515-523. https://doi.org/10.1016/j.enzmictec.2006.05.007
  15. Horikoshi, K. 1971. Production of alkaline enzymes by alkalophilic microorganisms, Part I, alkaline protease produced by Bacillus No. 221. Agric. Biol. Chem. 36, 1407-1414.
  16. Horikoshi, K. 1990. Enzymes of alkalophiles, pp. 275-294. Microbial Enzyme and Biotechnology, 2nd ed. MO : Elsevier Applied Science, Amsterdam, Netherlands.
  17. Horikoshi, K. 1996. Alkalophiles : from an industrial point of view. FEMS Microbiol. 18, 259-270. https://doi.org/10.1111/j.1574-6976.1996.tb00242.x
  18. Jaouadi, B., S. Ellouz-Chaabouni, M. Rhimi, and S. Bejar. 2008. Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency. Biochimie. 90, 1291-1305. https://doi.org/10.1016/j.biochi.2008.03.004
  19. Joo, H.S., C.G. Kumar, C.G. Park, K.T. Kim, S.R. Paik, and C.S. Chang. 2002. Optimization of the production of an extracellular alkaline protease from Bacillus horokoshi. Process Biochem. 38, 155-159. https://doi.org/10.1016/S0032-9592(02)00061-4
  20. Kazan, D., A.A. Denizci, M.N.K. Oner, and A. Erarslan. 2005. Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42. J. Ind. Microbiol. Biotechnol. 32, 335-344. https://doi.org/10.1007/s10295-005-0260-z
  21. Kembhavi, A.A., A. Kulkarni, and A. Pant. 1993. Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM No. 64. Appl. Biochem. Biotechnol. 38, 83-92. https://doi.org/10.1007/BF02916414
  22. Kumar, C.G. and H. Takagi. 1999. Microbial alkaline protease: from a bioindustrial viewpoint. Biotechnol. Adv. 17, 561-594. https://doi.org/10.1016/S0734-9750(99)00027-0
  23. Laemmli, U.K. 1971. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
  24. Lee, S. and D.J. Jang. 2001. Progressive rearrangement of subtilisin Carlsberg into orderly and inflexible conformation with $Ca^{2+}$ binding. Biophy. J. 81, 2972-2978. https://doi.org/10.1016/S0006-3495(01)75937-1
  25. Oberoi, R., Q.K. Beg, S. Puri, R.K. Saxeana, and R. Gupta. 2001. Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp. World J. Microbiol. Biotechnol. 17, 493-497. https://doi.org/10.1023/A:1011918806201
  26. Panouille, M., J.F. Thibault, and E. Bonnin. 2006. Cellulase and protease preparations can extract pectins from various plant by product. J. Argric. Food Chem. 54, 8926-8935. https://doi.org/10.1021/jf0617824
  27. Patel, R.K., M.S. Dodia, R.H. Joshi, and S.P. Singh. 2006. Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. Process Biochem. 41, 2002-2009. https://doi.org/10.1016/j.procbio.2006.04.016
  28. Rao, M.B., A.M. Tanksale, M.S. Ghatge, and V.V. Deshpande. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597-635.
  29. Shah, K., K. Mody, J. Keshri, and B. Jha. 2010. Purification and characterization of a solvent, detergent and oxidizing agent tolerant protease from Bacillus cereus isolated from the Gulf of Khambhat. J. Mol. Catal. B: Enzym. DOI: 10.1016/j.molcatb. 2010.07.010.
  30. Shin, Y.U., G.S. Lee, J.H. Jo, and H.Y. Lee. 2010. Strain development for the overproduction of alkaline protease from Vibrio metschnikovii by molecular evolution. Kor. J. Microbiol. 46, 383-388.
  31. Uchida, H., D. Kondo, S. Yamashita, T. Tanaka, L.H. Tran, H. Nagano, and T. Uwajima. 2004. Purification and properties of a protease produced by Bacillus subtilis CN2 isolated from a Vietnamese fish sauce. World J. Microbiol. Biotechnol. 20, 579-582. https://doi.org/10.1023/B:WIBI.0000043168.15067.44
  32. Yanagida, N., T. Uozumi, and T. Beppu. 1986. Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli. J. Bacteriol. 166, 937-944. https://doi.org/10.1128/jb.166.3.937-944.1986