DOI QR코드

DOI QR Code

Optimization, Purification, and Characterization of Haloalkaline Serine Protease from a Haloalkaliphilic Archaeon Natrialba hulunbeirensis Strain WNHS14

  • Ahmed, Rania S (Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technological Applications) ;
  • Embaby, Amira M (Institute of Graduate Studies and Research, Biotechnology Department, Alexandria University) ;
  • Hassan, Mostafa (Institute of Graduate Studies and Research, Environmental Studies Department, Alexandria University) ;
  • Soliman, Nadia A (Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technological Applications) ;
  • Abdel-Fattah, Yasser R (Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technological Applications)
  • Received : 2019.04.12
  • Accepted : 2019.07.19
  • Published : 2021.06.28

Abstract

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The BoxBehnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.

Keywords

Acknowledgement

The authors are extremely grateful to the City for Scientific Research and Technological Applications, Alexandria, Egypt for providing financial help and facilities to complete this work.

References

  1. Abdel-Fattah YR, El-Enshasy HA, Soliman NA, 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. https://doi.org/10.4014/jmb.0806.380
  2. Adams MWW, Kelly RM. 1995. Enzymes from microorganisms in extreme environments. Chem. Eng. News. 17: 32-42. https://doi.org/10.1021/cen-v073n051.p032
  3. Akolkar A, Bharambe N, Trivedi S, Desai A. 2009. Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon, Halobacterium sp. SP1(1). Lett. Appl. Microbiol. 48: 77-83. https://doi.org/10.1111/j.1472-765X.2008.02492.x
  4. Castillo AM, Gutierrez MC, Kamekura M, Ma Y, Cowan DA, Jones BE, et al. 2006. Halovivax asiaticus gen. nov., sp. nov., a novel extremely halophilic archaeon isolated from Inner Mongolia, China. Int. Syst. Evol. Microbiol. 56: 765-770. https://doi.org/10.1099/ijs.0.63954-0
  5. Chauhan B, Gupta R. 2004. Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14. Process Biochem. 39: 2115-2122. https://doi.org/10.1016/j.procbio.2003.11.002
  6. D'Alessandro CP, De Castro RE, Gimenez MI, Paggi RA. 2007. Effect of nutritional conditions on extracellular protease production by the haloalkaliphilic archaeon Natrialba magadii. Lett. Appl. Microbiol. 44: 637-642. https://doi.org/10.1111/j.1472-765X.2007.02122.x
  7. DasSarma S, DasSarma P. 2001. Halophiles, eLS. John Wiley and Sons Ltd, Chichester.
  8. Desmarais D, Jablonski PE, Fedarko NS, Roberts MF. 1997. 2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea. J. Bacteriol. 179: 3146-3153. https://doi.org/10.1128/jb.179.10.3146-3153.1997
  9. Fan H, Xue Y, Ma Y, Ventosa A, Grant WD. 2004. Halorubrum tibetense sp. nov., a novel haloalkaliphilic archaeon from Lake Zabuye in Tibet, China. Int. Syst. Evol. Microbiol. 54: 1213-1216. https://doi.org/10.1099/ijs.0.03032-0
  10. Ferrero MA, Castro GR, Abate CM, Baigori MD, Sineriz F. 1996. Thermostable alkaline proteases of Bacillus licheniformis MIR 29: isolation, production and characterization. Appl. Microbiol. Biotechnol. 45: 327-332.
  11. Ghorbel B, Sellami-Kamoun A, Nasri M. 2003. Stability studies of protease from Bacillus cereus BG1. Enzyme Microb. Technol. 32: 513-518. https://doi.org/10.1016/S0141-0229(03)00004-8
  12. Gimenez MI, Studdert CA, Sanchez JJ, De Castro RE. 2000. Extracellular protease of Natrialba magadii: purification and biochemical characterization. Extremophiles 4: 181-188. https://doi.org/10.1007/s007920070033
  13. Gomes J, Steiner W. 2004. The biocatalytic potential of extremo philes and extremozymes. Food Technol. Biotechnol. 42: 223-235.
  14. Gupta M, Aggarwal S, Navani NK, Choudhury B. 2015. Isolation and characterization of a protease-producing novel haloalkaliphilic bacterium Halobiforma sp. strain BNMIITR from Sambhar lake in Rajasthan, India. Ann. Microbiol. 65: 677-686. https://doi.org/10.1007/s13213-014-0906-z
  15. Gupta M, Sharma P, Dev K, Sourirajan A. 2016. Halophilic bacteria of Lunsu produce an array of industrially important enzymes with salt tolerant activity. Biochem. Res. Int. 2016: 1-10.
  16. Hacene H, Rafa F, Chebhouni N, Boutaiba S, Bhatnagar T, Baratti JC, et al. 2004. Biodiversity of prokaryotic microflora in El Golea Salt lake, Algerian Sahara. J. Arid Environ. 58: 273-284. https://doi.org/10.1016/j.jaridenv.2003.08.006
  17. Hu L, Pan H, Xue Y, Ventosa A, Cowan DA, Jones BE, et al. 2008. Halorubrum luteum sp. nov., isolated from Lake Chagannor, Inner Mongolia, China. Int. Syst. Evol. Microbiol. 58: 1705-1708. https://doi.org/10.1099/ijs.0.65700-0
  18. Joshi RH, Dodia MS, Singh SP. 2008. Production and optimization of a commercially viable alkaline protease from a haloalkaliphilic bacterium. Biotechnol. Bioprocess Eng. 13: 552-559. https://doi.org/10.1007/s12257-007-0211-9
  19. Kamekura M, Seno Y. 1990. A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1. Biochem. Cell Biol. 68: 352-359. https://doi.org/10.1139/o90-048
  20. Kembhavi AA, Kulkarni A, Pant A. 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
  21. Konig H, Stetter KO. 1989. Archaeobacteria, in Bergey's Manual of Systematic Bacteriology, pp. 2171-2253. Vol. 3, J.T. Staley MP, Bryant N Pfennig, and J.G. Holt, Editors. 1989, Williams and Wilkens, Baltimore.
  22. Kumar CG. 2002. Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus. Lett. Appl. Microbiol. 34: 13-17. https://doi.org/10.1046/j.1472-765x.2002.01044.x
  23. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  24. Lanyi JK. 1974. Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol. Rev. 38: 272-290. https://doi.org/10.1128/br.38.3.272-290.1974
  25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. https://doi.org/10.1016/S0021-9258(19)52451-6
  26. Madern D, Ebel C, Zaccai G. 2000. Halophilic adaptation of enzymes. Extremophiles 4: 91-98. https://doi.org/10.1007/s007920050142
  27. Manikandan M, Kannan V, Velikonja BH, Pasic L. 2011. Optimization of growth medium for protease production by Haloferax Lucentensis VKMM 007 by response surface methodology. Braz. J. Microbiol. 42: 818-824. https://doi.org/10.1590/s1517-838220110002000050
  28. Mortez E, Krogh TN, Vorum H, Gorg A. 2001. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 1: 1359-1363. https://doi.org/10.1002/1615-9861(200111)1:11<1359::AID-PROT1359>3.0.CO;2-Q
  29. Oren A. 2007. Biodiversity in highly saline environments, in: Gerday, C., Glansdorff, N. (eds.), pp 223-231. Physiology and Biochemistry of Extremophiles. ASM Press, Washington.
  30. Oren A. 2002. Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. J. Ind. Microbiol. Biotechnol. 28: 56-63. https://doi.org/10.1038/sj/jim/7000176
  31. Paggi RA, Martone CB, Fuqua C, De Castro RE. 2003. Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus. FEMS Microbiol. Lett. 221: 49-52. https://doi.org/10.1016/S0378-1097(03)00174-5
  32. Palsaniya P, Mishra R, Beejawat N, Sethi S, Gupta BL. 2012. Optimization of alkaline protease production from bacteria isolated from soil. J. Microbiol. Biotechnol. Res. 2: 858-865.
  33. Patel R, Dodia M, Singh SP. 2005. Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp., Production and optimization. Process Biochem . 40: 3569-3575. https://doi.org/10.1016/j.procbio.2005.03.049
  34. Patel S, Saraf M. 2015. Perspectives and application of halophilic enzymes. pp 403-419. In Maheshwari, D.K., Saraf, M. (Eds.), Halophiles: Biodiversity and Sustainable Exploitation. Springer International Publishing, Cham.
  35. Rahman RNZA, Geok LP, Basri M, Salleh AB. 2005. Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K. Bioresour. Technol. 96: 429-436. https://doi.org/10.1016/j.biortech.2004.06.012
  36. Sanchez-Porro C, Mellado E, Bertoldo C, Antranikian G, Ventosa A. 2003. Screening and characterization of the protease CP1 produced by the moderately halophilic bacterium Pseudoalteromonas sp. strain CP76. Extremophiles 7: 221-228. https://doi.org/10.1007/s00792-003-0316-9
  37. Schiralid C, Giuliano M, De Rosa M. 2002. Perspectives on biotechnological applications of archaea. Archaea 1: 75-86. https://doi.org/10.1155/2002/436561
  38. Selim S, Hagagy N, Aziz MA, El-Meleigy ES, Pessione E. 2014. Thermostable alkaline halophilic-protease production by Natronolimnobius innermongolicus WN18. Nat. Prod. Res. 28: 1476-1479. https://doi.org/10.1080/14786419.2014.907288
  39. Shi W, Tang X-F, Huang Y, Gan F, Tang B, Shen P. 2006. An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7: Gene cloning, expression and characterization. Extremophiles 10: 599-606. https://doi.org/10.1007/s00792-006-0003-8
  40. Sinha R, Khare SK. 2013. Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: Differential role of metal ions in stability and activity. Bioresour. Technol. 145: 357-361. https://doi.org/10.1016/j.biortech.2012.11.024
  41. Studdert CA, Herrera Seitz MK, Plasencia Gil MI, Sanchez JJ, de Castro RE. 2001. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease. J. Basic Microbiol. 41: 375-383. https://doi.org/10.1002/1521-4028(200112)41:6<375::AID-JOBM375>3.0.CO;2-0
  42. Vidyasagar M, Prakash SB, Sreeramulu K. 2006. Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101. Lett. Appl. Microbiol. 43: 385-391. https://doi.org/10.1111/j.1472-765X.2006.01980.x
  43. VijayAnand S, Hemapriya J, Selvin J, Kiran S. 2010. Production and optimization of haloalkaliphilic protease by an extremophile-Halobacterium sp. Js1, isolated from thalassohaline environment. Glob. J. Biotechnol. Biochem. 5: 44-49.
  44. Woese CR, Fox GE. 1977. Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Natl. Acad. Sci. 74: 5088- 5090. https://doi.org/10.1073/pnas.74.11.5088
  45. Xu Y, Zhou P, Tian X. 1999. Characterization of two novel haloalkaliphilic archaea Natronorubrurn bangense gen. nov., sp. nov. and Natronorubrurn tibetense gen. nov., sp. nov. Int. Syst. Evol. Bacteriol. 49: 261-266. https://doi.org/10.1099/00207713-49-1-261