Abstract

Research Article

Production and characterization of staphylokinase enzyme from Staphylococcus aureus ASIA4

Nourah Hassan Alzahrani and Fareed Shawky El-Shenawy*

Published: 17 September, 2020 | Volume 4 - Issue 1 | Pages: 027-035

Six clinical Staphylococcus aureus strains isolated from different clinical samples. Isolates ASIA1 and ASIA2 isolated from urine samples of urinary tract infected patients; ASIA3 isolated from swab samples of burn abscess patients at Assiut University hospital as well as ASIA4, ASIA5 and ASIA6 obtained from blood samples of different cancer patients at South Egypt Cancer Institute. All isolates showed varied abilities to produce halo zones of hydrolysis with different diameters on blood agar, heated plasma agar, casein agar and skim milk agar plates along with different clot lyses percent. Staphylococcus aureus ASIA3, ASIA4 and ASIA6 produced 4.83, 5.98 and 2.08 U/mL of staphylokinase on tryptone soy broth reduced to 1.95, 2.08 and 1.70 U/mL on casein hydrolysate yeast extract broth, respectively. On the other hand, Staphylococcus aureus ASIA1, ASIA2 and ASIA5 gave 2.20, 2.93 and 3.65 U/mL on CYEB compared to 2.10, 1.88 and 3.41 U/mL on TSB as production medium. The staphylokinase yielded from the hyperactive producer Staphylococcus aureus ASIA4 was increased for 7.64-fold (from 2.08 U/mL to 15.88 U/mL) on the optimized fermentation medium composed of 5.0 g sucrose as carbon source, 10.0 g soy bean as nitrogen source, 5.0 g NaCl, K2HPO4 5.0 g and pH 7.0 that inoculated with isolate ASIA4 and incubated for 24 h at 35 °C. Moreover, Staphylokinase activity reached its peak at the optimal enzymatic reaction conditions which were reaction time 25 min, casein as substrate, reaction pH 8.0, reaction temperature 40 °C. In addition it retained 100% of its activity at temperature ranged between 15 and 45 °C and pH ranged from pH 6.0 to 9.0. EDTA inhibited the enzyme activity by 3.0% to 32.2% with increasing its values from 30.0 to 90.0 mM. MgCl2 at a concentration of 30 mM increased the enzyme activity by 4% and then slightly decreased at higher concentrations but NaCl was potent staphylokinase activator at concentrations lower than 90 mM.

Read Full Article HTML DOI: 10.29328/journal.apps.1001024 Cite this Article Read Full Article PDF

Keywords:

Staphylokinase; Staphylococcus aureus; Thrombolytic agents; Optimization; Characterization

References

  1. Min W, Yao C, Wenliang F, Minji Z, Yuanyuan W,et al. Construction of a novel staphylokinase (SAK) mutant with low immunogenicity and its evaluation in rhesus monkey. Int J Biol Macromol. 2020; 146: 781-789. https://pubmed.ncbi.nlm.nih.gov/31730959/
  2. Nedaeinia R, Faraji H, Javanmard SH, et al. Bacterial staphylokinase as a promising third-generation drug in the treatment for vascular occlusion. Mol Biol Rep. 2020; 47: 819-841. https://pubmed.ncbi.nlm.nih.gov/31677034/
  3. Shah NR, Panchal HK. Screening of staphylokinase producing Staphylococcus spp. from different sources. GJBB. 2019; 8: 194-197.
  4. Deepa K, Faujdar SS, Azmi W, Mehrishi P, Solanki S. Screening and optimization of staphylokinase from Staphylococcus aureus isolated from nasal swab of healthy students in Himachal Pradesh University, India. Biomed Biotechnol Res J. 2019; 3: 228-232.
  5. Jasim HM, Dellol RA, Hamzah AS. Optimum conditions of staphylokinase production cloned in E. coli Jm109 (De3). Int J Curr Microbiol Appl Sci. 2015; 4: 10-19.
  6. Faraji H, Soltani F, Ramezani M, et al. Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro. Biotechnol Lett. 2020; 42: 103-114. https://pubmed.ncbi.nlm.nih.gov/31686286
  7. Chandrappa CP, Singh M, Chandrasekar N, Govindappa M. Production and partial purification of staphylokinase from Staphylococcus hominis. Research Journal of Pharmaceutical, Biological and Chemical Sciences (RJPBCS). 2017; 8: 77-86.
  8. Lemaire S. Intracellular Staphylococcus aureus, an emerging links to persistent and relapsing infections: factors influencing the activity of antimicrobials against intracellular S. aureus. Doctoral Thesis, Université Catholique de Louvain. 2008; 14: 766-777.
  9. Atlas M, Parks C, Brown A. Laboratory manual of experimental microbiology. Mosby–Year–Book, Inc., 1995; USA.
  10. Collee J, Miles R, Watt B. Tests for the identification of bacteria. In: Practical medical microbiology. Collee J, Fraser A, Marmion B, Simmons A. (eds.), 4th edition, 1996; Churchill Livingstone, U.K.
  11. Harly J, Prescott L. Laboratory exercises microbiology. 5th Ed, WCB, 2002; The Mcgraw-Hill Companies. New York.
  12. Kateete DP, Kimani CN, Katabazi FA, Okeng A, Okee MS, et al. Identification of Staphylococcus aureus: DNase and mannitol salt agar improve the efficiency of the tube coagulase test. Ann Clin Microbiol Antimicrob. 2010; 9: 23.
  13. Devriese LA, Kerckhovea AV. Comparison of methods used for testing staphylokinase (fibrinolysin) production in Staphylococcus strains. Antonie van Leeuwenhoek. 1980; 46: 457-465. https://pubmed.ncbi.nlm.nih.gov/6453557/
  14. Kondo I, Fujise K. Serotype B staphylococcal bacteriophage singly converting staphylokinase. Infect Immun. 1977; 18: 266–272. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163009/
  15. Pulicherla KK, Gadupudi GS, Rekha VP, Seetharam B, Rao K. International Journal of Advanced Science and Technology. 2011; 30.
  16. Shagufta NB, Ravi M, Subhashchandra MG, Jayaraj YM. Screening of staphylokinase producing Staphylococcus aureus from clinical samples. Int J Res Biological Sci. 2014; 4: 46-48.
  17. Devi S, Mohanasrinivasan V, Vaishnavi B, Selvarajan E, Naine SJ. Optimization studies for enhanced production of streptokinase by Streptococcus equisimilis UVM6 C. J Pure Appl Microbiol. 2013; 7: 1-5.
  18. Medveďová A, Valík Ľ, Sirotná Z, Czech DL. Growth characterization of Staphylococcus aureus in milk: a quantitative approach. Food Sci. 2009; 27: 443–453.
  19. Sutherland JP, Bayliss AJ, Roberts TA. Predictive modelling of growth of Staphylococcus aureus: the effects of temperature, pH and sodium chloride. Int J Food Microbiol. 1994; 21: 217-236. https://pubmed.ncbi.nlm.nih.gov/8024974/
  20. Singh D, Chand AE, Goel S. Prevalence of MRSA among Staphylococcus aureus isolated from patients of urinary tract infection along with its antibiogram. Int J Med Sci Current Res. 2019; 2: 364-370.
  21. Abdulzahra AA, Al-Jameel R, Akool MA-Z, Al-Jameel DS. Prevalence of toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus isolated from patient combined psoriasis with urinary tract infections (UTIs) and gastroenteritis in age groups between 8-18 years. Sys Rev Pharm. 2020; 11: 80-85.
  22. El-Gendy MMA, Abdel-Wahhab KG, Mannaa FA, Farghaly AA, El-Bondkly AMA. Carcinogenic activities and sperm abnormalities of methicillin resistance Staphylococcus aureus and inhibition of their virulence potentials by ayamycin. Applied biochemistry and biotechnology. 2017; 183: 833-852. https://pubmed.ncbi.nlm.nih.gov/28389766/
  23. El-Gendy MMAA, El-Bondkly AMA, Keera AA, Ali AM. Incidence of methicillin-resistant Staphylococcus aureus (MRSA) in microbial community of cancer patients and evaluation of their resistant pattern. Arabian J Sci Engineering. 2018a; 43: 83-92.
  24. El-Gendy MMA, Mohamed ZK, Hekal NZ, Ali FM, Yousef AEM. Production of bioactive metabolites from different marine endophytic Streptomyces species and testing them against methicillin-resistant Staphylococcus aureus (MRSA) and cancer cell lines. BioTechnologia. 2018b; 99: 13–35.
  25. Morello JK, Mizer HE, Granato PA. Laboratory manual and work book in microbiology applications to patient care. 2006; 8thed. Mc Graw Hill.
  26. Kotra SR, Kumar A, Rao S, Pulicherla KK. Statistical optimization of media components for enhanced production of the recombinant staphylokinase variant from salt inducible E.Coli GJ1158. Int J Bio-Sci Bio-Technol. 2012; 4: 27-40.
  27. El-Bondkly AAM, El-Gendy MMAA, El-Bondkly EAM, El-Bondkly AAM. Biodiversity and biological activity of the fungal microbiota derived from the medicinal plants Salvia aegyptiaca L. and Balanties aegyptiaca L. Biocatalysis and Agricultural Biotechnology. 2020; 28: 101720.
  28. Nguyen THT, Quyen DT. High-level expression, purification and properties of a fully active even glycosylated staphylokinase variant SakfC from Staphylococcus aureus QT08 in Pichia. African J Microbiol Res. 2012; 6: 2129-2136.
  29. Schlott B, Hartmann M, Gührs K, et al. High yield production and purification of recombinant staphylokinase for thrombolytic therapy. Nat Biotechnol. 1994; 12: 185-189. https://pubmed.ncbi.nlm.nih.gov/7764434/
  30. Yarzábal LA. Modulation of staphylokinase-dependent plasminogen activation by mono- and divalent ions. Brazilian Journal of Medical and Biological Research. 1999; 32: 39-43.
  31. El-Bondkly AAM, El-Gendy MMAA, El-Bondkly AMA. Construction of efficient recombinant strain through genome shuffling in marine endophytic Fusarium sp. ALAA-20 for improvement lovastatin production using agro-industrial wastes. Arab J Sci Eng. 2020.
  32. El-Bondkly AAM, El-Gendy MMAA, El-Bondkly EAM, Ahmed AM. Biodiversity and biological activity of the fungal microbiota derived from the medicinal plants Salvia aegyptiaca L. and Balanties aegyptiaca L. Biocatalysis and Agricultural Biotechnology. 2020; 28: 101720.

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