Advertisement

Production, Partial Purification, and Biochemical Characterization of a Thermotolerant Alkaline Metallo-protease from Staphylococcus sciuri

  • Rasha Abu-KhudirEmail author
  • Maha M. Salem
  • Nanis Gamal Allam
  • Ehab M. M. Ali
Article
  • 101 Downloads

Abstract

Protease-producing Staphylococcus sciuri was isolated from poultry soil samples and culture conditions for protease production were optimized. The isolated protease showed a maximum activity of 235.1 U/ml. Enzyme purification procedure involved ammonium sulphate precipitation and Sephacryl S-200 HR gel filtration chromatography (GFC). The purification process resulted in the production of three protease fractions namely protease І (metallo-alkaline protease), II, and IІІ. The metallo-alkaline protease was purified to 25.49-fold with specific activity of 982.22 U/mg and 3.76% recovery. The partially purified metallo-protease was optimally active at pH 10.0 and 70 °C and exhibited thermal stability up to 50 °C. The protease activity was enhanced by Ca2+ and Mg2+, completely inhibited by Hg2+ and Cu2+, and significantly reduced by EDTA. The protease showed significant stability towards various surfactants, including SDS. The Km and Vmax values were 0.68 mg/ml and 166.66 nmol of azocasein/ml/h, respectively, while the activation energy (Ea) was 3.07 Kcal/mol. Hence, it is evident that the produced protease possesses unique characteristics and could be a plausible candidate for various industrial and biotechnological applications.

Keywords

Protease Bacterial isolates Poultry waste Purification Characterization 

Notes

Acknowledgements

No funding sources had been involved in the conduction and/or preparation of this article.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Rao, M. B., Tanksale, A. M., Ghatge, M. S., & Deshpande, V. V. (1998). Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev, 62(3), 597–635.Google Scholar
  2. 2.
    Lopez-Otin, C., & Bond, J. S. (2008). Proteases: Multifunctional enzymes in life and disease. J Biol Chem, 283(45), 30433–30437.Google Scholar
  3. 3.
    da Silva, R. R. (2017). Bacterial and fungal proteolytic enzymes: Production, catalysis and potential applications. Appl Biochem Biotechnol, 183(1), 1–19.Google Scholar
  4. 4.
    Craik, C. S., Page, M. J., & Madison, E. L. (2011). Proteases as therapeutics. Biochem J, 435(1), 1–16.Google Scholar
  5. 5.
    Eatemadi, A., Aiyelabegan, H. T., Negahdari, B., Mazlomi, M. A., Daraee, H., Daraee, N., Eatemadi, R., & Sadroddiny, E. (2017). Role of protease and protease inhibitors in cancer pathogenesis and treatment. Biomed Pharmacother, 86, 221–231.Google Scholar
  6. 6.
    Godfrey, T., & West, S. (1996). Industrial enzymology (2nd ed.). New York, N.Y.: Macmillan Publishers Inc.Google Scholar
  7. 7.
    Gupta, R., Beg, Q. K., & Lorenz, P. (2002). Bacterial alkaline proteases: Molecular approaches and industrial applications. Appl Microbiol Biotechnol, 59(1), 15–32.Google Scholar
  8. 8.
    Nigam, P. S. (2013). Microbial enzymes with special characteristics for biotechnological applications. Biomolecules, 3(3), 597–611.Google Scholar
  9. 9.
    Pratush, A., Gupta, A., & Bhalla, T. C. (2013). Microbial proteases: Prospects and challenges. Dehradun: Bhalla publishers.Google Scholar
  10. 10.
    Niyonzima, F. N., & More, S. (2015). Detergent-compatible proteases: Microbial production, properties, and stain removal analysis. Prep Biochem Biotechnol, 45(3), 233–258.Google Scholar
  11. 11.
    Uyar, F., Porsuk, I., Kizil, G., & Ince Yilma, E. (2011). Optimal conditions for production of extracellular protease from newly isolated Bacillus cereus strain CA15. EurAsia J BioSci, 5, 1–9.Google Scholar
  12. 12.
    Leighton, T. J., Dor, R. H., Warren, R. A., & Kelln, R. A. (1973). The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis. J Mol Biol, 76(1), 103–122.Google Scholar
  13. 13.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem, 193(1), 265–275.Google Scholar
  14. 14.
    Weissenberg, R. (1938). Studies on virus diseases of fish: I. Lymphocystis disease of the orange filefish (Aleutera schoepfii). Am J Epidemiol, 28(3), 455–462.Google Scholar
  15. 15.
    Lineweaver, H., & Burk, D. (1934). The determination of enzyme dissociation constants. J Am Chem Soc, 56, 658–666.Google Scholar
  16. 16.
    Segel, I. H. (1976). Biochemical calculations (2nd ed.). New York: John Wiley and sons.Google Scholar
  17. 17.
    Yoo, A. Y., & Park, J. K. (2016). Isolation and characterization of a serine protease-producing marine bacterium Marinomonas arctica PT-1. Bioprocess Biosyst Eng, 39(2), 307–314.Google Scholar
  18. 18.
    Abdel-Rhman, S. H. M., El-Mahdy, A. M., & Abdelmegeed, E. S. A. (2014). Optimization of protease production by Ps. aeruginosa PAO1 and physico-chemical characterization of the enzyme. J Am Sci, 10(7), 62–72.Google Scholar
  19. 19.
    Akram, M., Shafaat, S., Bukhari, D. A., & Rehman, A. (2014). Characterization of a thermostable alkaline protease from Staphylococcus aureus S2 isolated from chicken waste. Pak J Zool, 46(4), 1125–1132.Google Scholar
  20. 20.
    Revathi, D., & Palanisany, A. (2015). Production, purification and characterization of protease from Yersinia sp. and Staphylococcus sp. Int J Adv Res, 3(7), 424–434.Google Scholar
  21. 21.
    Bholay, A. D., More, S. Y., Patil, V. B., & Patil, N. (2012). Bacterial extracellular alkaline proteases and its industrial applications. I Res J Biological Sci, 1(7), 1–5.Google Scholar
  22. 22.
    Oh, Y., Shih, I. I., Tzeng, Y., & Wang, S. (2000). Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes. Enzym Microb Technol, 27(1–2), 3–10.Google Scholar
  23. 23.
    Al-Abdalall, A. H., & Al-Khaldi, E. M. (2016). Production of alkaline proteases by alkalophilic Bacillus subtilis during recycling animal and plant wastes. Afr J Biotechnol, 15(47), 2698–2702.Google Scholar
  24. 24.
    Saggu, S. K., & Mishra, P. C. (2017). Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil. PLoS One, 12(11), e0188724.Google Scholar
  25. 25.
    Ahmetoglu, N., Matpan Bekler, F., Acer, O., Guven, R. G., & Guven, K. (2015). Production, purification and characterization of thermostable metallo-protease from newly isolated Bacillus sp. KG5. EurAsia J BioSci, 9, 1–11.Google Scholar
  26. 26.
    Thakur, S., Sharma, N. K., Thakur, N., Savitri, & Bhalla, T. C. (2016). Organic solvent tolerant metallo protease of novel isolate Serratia marcescens PPB-26: Production and characterization. 3 Biotech, 6(2), 180.Google Scholar
  27. 27.
    Nisha, N. S., & Divakaran, J. (2014). Optimization of alkaline protease production from Bacillus subtilis NS isolated from sea water. Afr J Biotechnol, 13(16), 1707–1713.Google Scholar
  28. 28.
    Gaur, S., Agrahari, S., & Wadhwa, N. (2010). Purification of protease from Pseudomonas thermaerum GW1 isolated from poultry waste site. Open Microbiol J, 4, 67–74.Google Scholar
  29. 29.
    Olajuyigbe, F. M., & Kolawole, A. O. (2011). Purification and partial characterization of a thermostable alkaline protease from Bacillus licheniformis LHSB-05 isolated from hot spring. Afr J Biotechnol, 10(55), 11703–11713.Google Scholar
  30. 30.
    Thaz, C. J., & Jayaraman, G. (2014). Stability and detergent compatibility of a predominantly β-sheet serine protease from halotolerant B. aquimaris VITP4 strain. Appl Biochem Biotechnol, 172(2), 687–700.Google Scholar
  31. 31.
    Rawway, M., Taha, T. M., Eltokhey, A., & Abdul-Raouf, U. M. (2015). Optimization, partial purification and characterization of halo-thermophilic alkaline protease from moderately halophilic bacterium AH10 isolated from Alexandria (Egypt). Int J Curr Microbiol App Sci, 4(11), 304–317.Google Scholar
  32. 32.
    Dos Santos Aguilar, J. G., & Sato, H. H. (2018). Microbial proteases: Production and application in obtaining protein hydrolysates. Food Res Int, 103, 253–262.Google Scholar
  33. 33.
    Hammami, A., Fakhfakh, N., Abdelhedi, O., Nasri, M., & Bayoudh, A. (2018). Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing. Int J Biol Macromol, 108, 56–68.Google Scholar
  34. 34.
    Xin, Y., Sun, Z., Chen, Q., Wang, J., Wang, Y., Luogong, L., Li, S., Dong, W., Cui, Z., & Huang, Y. (2015). Purification and characterization of a novel extracellular thermostable alkaline protease from Streptomyces sp. M30. J Microbiol Biotechnol, 25(11), 1944–1953.Google Scholar
  35. 35.
    Sharma, K. M., Kumar, R., Panwar, S., & Kumar, A. (2017). Microbial alkaline proteases: Optimization of production parameters and their properties. J Genet Eng Biotechnol, 15(1), 115–126.Google Scholar
  36. 36.
    Proceedings of the Burapha University International Conference (2015). Bangsaen, Chonburi, Thailand: Burapha University, 2015, 759–767Google Scholar
  37. 37.
    Yildirim, V., Baltaci, M. O., Ozgencli, I., Sisecioglu, M., Adiguzel, A., & Adiguzel, G. (2017). Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: A potential additive for detergents. J Enzyme Inhib Med Chem, 32(1), 468–477.Google Scholar
  38. 38.
    Mothe, T., & Sultanpuram, V. R. (2016). Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus. 3 Biotech, 6(1), 53.Google Scholar
  39. 39.
    Patil, U., Mokashe, N., Chaudhari, A. (2016). Detergent-compatible, organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: Purification and characterization. Prep Biochem Biotechnol, 46(1), 56–64.Google Scholar
  40. 40.
    Yilmaz, B., Baltaci, M. O., Sisecioglu, M., & Adiguzel, A. (2016). Thermotolerant alkaline protease enzyme from Bacillus licheniformis A10: Purification, characterization, effects of surfactants and organic solvents. J Enzyme Inhib Med Chem, 31(6), 1241–1247.Google Scholar
  41. 41.
    David, A., Singh Chauhan, P., Kumar, A., Angural, S., Kumar, D., Puri, N., & Gupta, N. (2018). Coproduction of protease and mannanase from Bacillus nealsonii PN-11 in solid state fermentation and their combined application as detergent additives. Int J Biol Macromol, 108, 1176–1184.Google Scholar
  42. 42.
    Mhamdi, S., Ktari, N., Hajji, S., Nasri, M., & Sellami Kamoun, A. (2017). Alkaline proteases from a newly isolated Micromonospora chaiyaphumensis S103: Characterization and application as a detergent additive and for chitin extraction from shrimp shell waste. Int J Biol Macromol, 94(Pt A), 415–422.Google Scholar
  43. 43.
    Nilegaonkar, S. S., Zambare, V. P., Kanekar, P. P., Dhakephalkar, P. K., Sarnaik, S. S. (2007). Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour Technol, 98(6), 1238–1245.Google Scholar
  44. 44.
    Waghmare, S. R., Gurav, A. A., Mali, S. A., Nadaf, N. H., Jadhav, D. B., Sonawane, K. D. (2015). Purification and characterization of novel organic solvent tolerant 98 kDa alkaline protease from isolated Stenotrophomonas maltophilia strain SK. Prot Exp Purif, 107, 1–6.Google Scholar
  45. 45.
    Annamalai, N., Rajeswari, M. V., Balasubramanian, T. (2014). Extraction, purification and application of thermostable and halostable alkaline protease from Bacillus alveayuensis CAS 5 using marine wastes. Food Bioprod Process, 92(4), 335–342.Google Scholar
  46. 46.
    Chittoor, J. T., Balaji, L., Jayaraman, G. (2016). Optimization of Parameters that Affect the Activity of the Alkaline Protease from Halotolerant Bacterium, Bacillus acquimaris VITP4, by the Application of Response Surface Methodology and Evaluation of the Storage Stability of the Enzyme. Iran J Biotechnol, 14(1), 23–32.Google Scholar
  47. 47.
    Guleria, S., Walia, A., Chauhan, A., Shirkot, C. K. (2016). Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere. J Basic Microbiol, 56(2), 138–152.Google Scholar
  48. 48.
    Chatterjee, J., Giri, S., Maity, S., Sinha, A., Ranjan, A., Rajshekhar, Gupta, S. (2015). Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation. Biotechnol Appl Biochem, 62(5), 709–718.Google Scholar
  49. 49.
    Elbanna, K., Ibrahim, I. M., Revol-Junelles, A. M. (2015). Purification and characterization of halo-alkali-thermophilic protease from Halobacterium sp. strain HP25 isolated from raw salt, Lake Qarun, Fayoum, Egypt. Extremophiles, 19(4):763–774.Google Scholar
  50. 50.
    Zhang, Z., Hao, H., Tang, Z., Zou, Z., Zhang, K., Xie, Z., Babe, L., Goedegebuur, F., Gu, X. (2015). Identification and Characterization of a New Alkaline Thermolysin-Like Protease, BtsTLP1, from Bacillus thuringiensis Serovar Sichuansis Strain MC28. J Microbiol Biotechnol, 25(8), 1281–1290.Google Scholar
  51. 51.
    Kotlar, C., Ponce, A., Roura, S. (2015). Characterization of a novel protease from Bacillus cereus and evaluation of an eco-friendly hydrolysis of a brewery by product. J Inst Brew, 121(4), 558–565.Google Scholar
  52. 52.
    Iqbal, A., Hakim, A., Hossain, M. S., Rahman, R. M., Islam, K., Azim, M. F., Ahmed, J., Assaduzzaman, M., Hoq, M. M., Azad, A. K. (2018). Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2. J Genet Eng Biotechnol, 6, 29–37.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Chemistry DepartmentBiochemistry Branch, Faculty of Science, Tanta UniversityTantaEgypt
  2. 2.Chemistry DepartmentCollege of Science, King Faisal UniversityAl-HofufSaudi Arabia
  3. 3.Botany DepartmentMicrobiology Unit, Faculty of Science, Tanta UniversityTantaEgypt
  4. 4.Department of BiochemistryFaculty of Science, King Abdulaziz UniversityJeddahSaudi Arabia

Personalised recommendations