DOI QR코드

DOI QR Code

Purification and Characterization of Manganese-Dependent Alkaline Serine Protease from Bacillus pumilus TMS55

  • Received : 2010.09.01
  • Accepted : 2010.10.23
  • Published : 2011.01.28

Abstract

The purification and characterization of a $Mn^{2+}$-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be $60^{\circ}C$. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. $Mn^{2+}$ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants ($H_2O_2$, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.

Keywords

References

  1. Akel, H., F. Al-Quadan, and T. K. Yousef. 2009. Characterization of a purified thermostable protease from hyperthermophilic Bacillus strain HUTBS71. Eur. J. Sci. Res. 31: 280-288.
  2. Alexander, K. T. W. 1988. Enzymes in the tannery - catalysts for progress? J. Am. Leather Chem. Assoc. 83: 287-316.
  3. Annapurna, R. A., N. K. Chandrababu, N. Samivelu, C. Rose, and N. M. Rao. 1996. Eco-friendly enzymatic dehairing using extracellular protease from Bacillus species isolate. J. Am. Leather Chem. Assoc. 91: 115-119.
  4. Anwar, A. and M. Saleemuddin. 2000. Alkaline protease from Spilosoma obliqua: Potential applications in bio-formulations. Biotechnol. Appl. Biochem. 31: 85-89. https://doi.org/10.1042/BA19990078
  5. Chandrasekaran, M. 1997. Industrial enzymes from marine microorganisms: The Indian scenario. J. Mar. Biotechnol. 5: 86-89.
  6. Dayanandan, A., J. Kanagaraj, L. Sounderraj, R. Govindaraju, and G. S. Rajkumar. 2003. Application of an alkaline protease in leather processing: An ecofriendly approach. J. Cleaner Prod. 11: 533-536. https://doi.org/10.1016/S0959-6526(02)00056-2
  7. Geok, L. P., C. N. A. Razak, R. N. Z. Abd Rahman, M. Basri, and A. B. Salleh. 2003. Isolation and screening of an extracellular organic solvent-tolerant protease producer. Biochem. Eng. J. 13: 73-77. https://doi.org/10.1016/S1369-703X(02)00137-7
  8. Ghorbel-Frikha, B., A. Sellami-Kamoun, N. Fakhfakh, A. Haddar, L. Manni, and M. Nasri. 2005. Production and purification of a calcium-dependent protease from Bacillus cereus BG1. J. Ind. Microbiol. Biotechnol. 32: 186-194. https://doi.org/10.1007/s10295-005-0228-z
  9. Green, G. H. 1952. Unhairing by means of enzymes. J. Soc. Leather Technol. Chem. 36: 127-134.
  10. Gupta, R., K. Gupta, R. K. Saxena, and S. Khan. 1999. Bleachstable, alkaline protease from Bacillus sp. Biotechnol. Lett. 21: 135-138. https://doi.org/10.1023/A:1005478117918
  11. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams. 1994. Bergey's Manual of Determinative Bacteriology 9 Ed. Williams and Wilkins, Waverly, Baltimore, MD.
  12. Horikoshi, K. 1999. Alkaliphiles: Some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63: 735-750.
  13. Huang, Q., Y. Peng, X. Li, H. Wang, and Y. Zhang. 2003. Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr. Microbiol. 46: 169-173. https://doi.org/10.1007/s00284-002-3850-2
  14. Jaouadi, B., S. Ellouz-Chaabouni, M. Rhimi, and S. Bejar. 2008. Biochemical and molecular characterization of a detergentstable 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
  15. Johnvesly, B. and G. R. Naik. 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. https://doi.org/10.1016/S0032-9592(01)00191-1
  16. Kim, C. J., J. S. Lee, S. H. Choi, and M. J. Oh. 1997. Enzyme detergent using alkaline protease produced by Halomonas sp. ES-10. Kor. J. Appl. Microbiol. Biotechnol. 25: 51-55.
  17. Kobayashi, T., Y. Hakamada, S. Adachi, J. Hitomi, T. Yoshimatsu, K. Koike, S. Kawai, and S. Ito. 1995. Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl. Microbiol. Biotechnol. 43: 473-481. https://doi.org/10.1007/BF00218452
  18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  19. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.
  20. Marsal, A., J. Cot, E. G. Boza, P. J. Celma, and A. M. Manich. 1999. Oxidizing unhairing process with hair recovery. Part I. Experiments on the prior hair immunization. J. Soc. Leather Technol. Chem. 83: 310-315.
  21. Nilegaonkar, S. S., V. P. Zambare, P. P. Kanekar, P. K. Dhakephalkar, and S. S. Sarnaik. 2007. Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour. Technol. 98: 1238-1245. https://doi.org/10.1016/j.biortech.2006.05.003
  22. North, M. J. 1982. Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiol. Rev. 46: 308-340.
  23. Ogino, H. and H. Ishikawa. 2001. Enzymes which are stable in the presence of organic solvents. J. Biosci. Bioeng. 91: 109-116. https://doi.org/10.1016/S1389-1723(01)80051-7
  24. Ogino, H., M. Yamada, F. Watanabe, H. Ichinose, M. Yasuda, and H. Ishikawa. 1999. Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems. J. Biosci. Bioeng. 88: 513-518. https://doi.org/10.1016/S1389-1723(00)87668-9
  25. Olivera, N., C. Sequeiros, F. Sineriz, and J. Breccia. 2006. Characterization of alkaline proteases from a novel alkali-tolerant bacterium Bacillus patagoniensis. World J. Microbiol. Biotechnol. 22: 737-743. https://doi.org/10.1007/s11274-005-9099-8
  26. Rahman, R. N. Z. R. A., L. P. Geok, M. Basri, and A. B. Salleh. 2006. An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: Enzyme purification and characterization. Enzyme Microb. Technol. 39: 1484-1491. https://doi.org/10.1016/j.enzmictec.2006.03.038
  27. Ramasami, T., J. R. Rao, N. K. Chandrababu, K. Parthasarathi, P. G. Rao, P. Saravanan, R. Gayatri, and K. J. Sreeram. 1999. Beamhouse and tanning operations: Process chemistry revisited. J. Soc. Leather Technol. Chem. 83: 39-45.
  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. Reddy, L. V. A., Y. J. Wee, and H. W. Ryu. 2008. Purification and characterization of an organic solvent and detergent-tolerant novel protease produced by Bacillus sp. RKY3. J. Chem. Technol. Biotechnol. 83: 1526-1533. https://doi.org/10.1002/jctb.1946
  30. Reddy, L. V. A., Y. J. Wee, J. S. Yun, and H. W. Ryu. 2008. Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches. Bioresour. Technol. 99: 2242-2249. https://doi.org/10.1016/j.biortech.2007.05.006
  31. Rheinheimer, G. 1992. Aquatic Microbiology, 4th Ed. John Wiley & Sons, New York.
  32. Saeki, K., J. Hitomi, M. Okuda, Y. Hatada, Y. Kageyama, M. Takaiwa, et al. 2002. A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease. Extremophiles 6: 65-72. https://doi.org/10.1007/s007920100224
  33. Sarath, G., R. S. Dela Motte, and F. W. Wagner. 1989. Enzyme assay methods, pp. 25-55. In R. J. Beynon and J. S. Bond (eds.). Proteolytic Enzymes. A Practical Approach. IRL Press, Oxford, England Inc., New York, Tokyo.
  34. Steele, D. B., M. J. Fiske, B. P. Steele, and V. C. Kelley. 1992. Production of a low-molecular-weight, alkaline-active, thermostable protease by a novel, spiral-shaped bacterium, Kurthia spiroforme, sp. nov. Enzyme Microb. Technol. 14: 358-360. https://doi.org/10.1016/0141-0229(92)90003-7
  35. Syed Ibrahim, K., D. Bakkiyaraj, R. James, T. Ganesh Babu, and S. T. Karutha Pandian. 2009. Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2. Plasmid 62: 183-190. https://doi.org/10.1016/j.plasmid.2009.08.001
  36. Yossan, S., A. Reungsang, and M. Yasuda. 2006. Purification and characterization of alkaline protease from Bacillus megaterium isolated from Thai fish sauce fermentation process. Science Asia 32: 377-383. https://doi.org/10.2306/scienceasia1513-1874.2006.32.377

Cited by

  1. A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization vol.168, pp.4, 2012, https://doi.org/10.1007/s12010-012-9828-z
  2. Extracellular Proteome of a Highly Invasive Multidrug-resistant Clinical Strain of Acinetobacter baumannii vol.11, pp.12, 2011, https://doi.org/10.1021/pr300496c
  3. <i>Bacillus pumilus</i>: Possible Model for the Bioweapon <i>Bacillus anthracis</i> vol.2, pp.3, 2011, https://doi.org/10.4236/aim.2012.23048
  4. Subtilisin-like proteinase secreted by the Bacillus pumilus KMM 62 strain at different growth stages vol.38, pp.2, 2012, https://doi.org/10.1134/s1068162012020082
  5. Covalent Immobilization of Trypsin on a Novel Aldehyde-Terminated PAMAM Dendrimer vol.33, pp.7, 2011, https://doi.org/10.5012/bkcs.2012.33.7.2181
  6. Bacterial Community of Koumiss from Mongolia Investigated by Culture and Culture-Independent Methods vol.28, pp.4, 2011, https://doi.org/10.1080/08905436.2014.964253
  7. PURIFICATION AND PROPERTIES OF DETERGENT-COMPATIBLE EXTRACELLULAR ALKALINE PROTEASE FROMScopulariopsisspp. vol.44, pp.7, 2014, https://doi.org/10.1080/10826068.2013.854254
  8. Production, purification and characterization of a thermostable alkaline serine protease from Bacillus lichniformis NMS-1 vol.6, pp.3, 2015, https://doi.org/10.5897/ijbmbr2014.0199
  9. Characterization of a metalloprotease from an isolate Bacillus thuringiensis 29-126 in animal feces collected from a zoological garden in Japan vol.59, pp.4, 2016, https://doi.org/10.3839/jabc.2016.063
  10. Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus vol.6, pp.1, 2011, https://doi.org/10.1007/s13205-016-0377-y
  11. Thermotolerant alkaline protease enzyme from Bacillus licheniformis A10: purification, characterization, effects of surfactants and organic solvents vol.31, pp.6, 2011, https://doi.org/10.3109/14756366.2015.1118687
  12. Purification and Characterization of Halophilic Organic Solvent Tolerant Protease from Marine Bacillus sp. APCMST-RS7 and Its Antioxidant Potentials vol.87, pp.1, 2011, https://doi.org/10.1007/s40011-015-0603-0
  13. A novel bacteriocin from Enterococcus faecalis 478 exhibits a potent activity against vancomycin-resistant enterococci vol.12, pp.10, 2011, https://doi.org/10.1371/journal.pone.0186415
  14. Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2 vol.16, pp.1, 2011, https://doi.org/10.1016/j.jgeb.2017.10.011
  15. Purification and characterization of the thermostable protease produced by Serratia grimesii isolated from channel catfish vol.99, pp.5, 2019, https://doi.org/10.1002/jsfa.9451
  16. Applications of enzymes in leather processing vol.18, pp.3, 2011, https://doi.org/10.1007/s10311-020-00971-5
  17. NanoSIMS Imaging of Bioaccumulation and Subcellular Distribution of Manganese During Oyster Gametogenesis vol.55, pp.12, 2011, https://doi.org/10.1021/acs.est.1c02393
  18. Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil vol.11, pp.1, 2011, https://doi.org/10.1038/s41598-021-92197-w