Strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1

Abstract

BACKGROUND: Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for enhanced production of streptokinase fromStreptococcus uberisTNA-M1 isolated from bovine milk.

METHODS: The study was carried out on samples collected from milk sample. Primary screening and characterization is used as an excellent source for the isolation of β-hemolytic organisms. Strain improvement was done by both physical & chemical mutagenesis. The enzyme activity was checked by clot lysis assay and confirmed by fibrin plate method. The partially purified and crude enzyme were analysed by high-performance liquid chromatography. Molecular weight & enzyme purity was checked by SDS –PAGE, further confirmed by fibrin zymography.

RESULTS: Out of the 3 isolated strains, only one isolate expressed β-haemolysis with streptokinase (SK) activity. Based on the results of radial caseinolytic assay and blood clot dissolving assay, isolate TNA-M1 demonstrated the highest streptokinase activity. Based on morphological , biochemical and molecular characterization, it was identified asStreptococcus uberis and the strain was named as Streptococcus uberis TNA-M1. The results indicated that ultra-violet (UV) and ethyl methane sulfonate (EMS) were effective mutagenic agents for strain improvement ofStreptococcus uberis TNA-M1 and enhanced SK productivity. HPLC analysis was performed in order to confirm the presence of streptokinase with the similar retention time (0.875 min) with its standard (0.854) min. SDS-PAGE of the enzyme showed protein band of approximately 47 kDa and confirmed by fibrin zymography. It exhibited fibrinolytic activity, which was more potent than other fibrinolytic enzymes. Glucose and peptone were recorded to be the optimum carbon and nitrogen sources respectively.

CONCLUSION: Thus this study presents its novelty by highlighting the potential ofStreptococcus uberisTNA-M1 as a significant source for the production of fibrinolytic enzymes.

Keywords

Streptokinase / Streptococcus uberis / clot busters / mutagenesis / optimization

References

[1] Aradhye P K, Chavan  M D (2015). Production, charecterization and in-vitroStudy of fibrinolytic enzyme from locally isolated Micrococcus luteusB-07. World J Pharm Pharmaceut Sci, 4(8):775–783

[2] Baewald G, Mayer  G, Heikel R ,  Volzke K D ,  Roehlig R Decker , Koehler  W, Gerllach D (1975) Fermentative Production of streptococcus metabolites, especially streptokinase. German patent DD, 111096

[3] Banerjee A, Chisti  Y, Banerjee U C  (2004). Streptokinase--a clinically useful thrombolytic agent. Biotechnol Adv, 22(4): 287–307

[4] Blix S (1962). the effectiveness of the activators in clot lysis with special reference to fibrinolytic therapy: a new method for the determination of performed clot lysis. Acta Med Scand, 172: 386

[5] Boersma E, Mercado  N, Poldermans D ,  Gardien M ,  Vos J, Simoons  M L (2003). Acute myocardial infarction. Lancet, 361(9360): 847–858

[6] Boland A, Dundar  Y, Bagust A ,  Haycox A ,  Hill R, Mujica Mota  R, Walley T ,  Dickson R  (2003). Early thrombolysis for the treatment of acute myocardial infarction: a systematic review and economic evaluation. Health Technol Assess, 7(15): 1–136

[7] Christensen L R ,  Macleod C M  (1945). A proteolytic enzyme of se-rum: characterization, activation, and reaction with inhibitors. J Gen Physiol, 28(6): 559–583

[8] Chung D M, Kim  K E, Ahn  K H, Park  C S, Kim  D H, Koh  H B, Chun  H K, Yoon  B D, Kim  H J, Kim  M S, Choi  N S (2011). Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel. Biotechnol Lett, 33(8): 1663–1666

[9] Feldman L J (1974) Streptokinase manufacture. In German.German patent DE 2354019.

[10] Grimont P A D ,  Grimont F  (1984). Bergey’s Manual of systematic bacteriology. Baltimore: Williams and Wilkins.1.477 –484

[11] Holmström B, Seppälä  P, Vilhunen R ,  Enari T M  (1965). Streptokinase assay on large agar diffusion plates. Acta Chem Scand, 19(7): 1549–1554

[12] Huangkai Z, Shenghan  G, Wei-Hua C  (2012). EvolView, An online tool for visualizing, annotating and managing phylogenetic trees. Nucleic Acids Res, 40(W1): 569–572

[13] Hyun H H, Lee  Y B, Song  K H, Jeon  J Y, Lee  H H (1997). Strain improvement for enhanced production of streptokinase and streptodornase in Streptococcus sp. J Microbiol Biotechnol, 7: 101–106

[14] Ibrahim S A, O’Sullivan  D J (2000). Use of chemical mutagenesis for the isolation of food grade b-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus. J Dairy Sci, 83(5): 923–930

[15] KumarA, (2011). Evolutionary trend of thrombolytictheir significance, Int J Biosci Biotech, 3(1): 1–18

[15] Laemmli V K (1970). Determination of protein molecular weight in polyacrylamide gels. Nature, 227: 680–685

[16] 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

[17] Madhuri D H, Madhuri  M, Neha A S ,  Mohanasrinivasan V ,  Subathra D C  (2011) studies on screening, strain development, production and optimization of streptokinase from beta hemolytic streptococci. World J SciTechnol, 1(3):7–11

[18] Magarvey N A, Keller  J M, Bernan  V, Dworkin M ,  Sherman D H  (2004). Isolation and characterization of novel marine-derived actinomycete taxa rich in bioactive metabolites. Appl Environ Microbiol, 70(12): 7520–7529

[19] ParekhS,, Vinci V A,, StrobelR J , (2004). Improvement of microbial strains and fermentation processes. App Microbiol Biotechnol,54287–301

[19] Rodríguez P ,  Fuentes P ,  Barro M ,  Alvarez J G ,  Muñoz E ,  Collen D ,  Lijnen H R  (1995). Structural domains of streptokinase involved in the interaction with plasminogen. Eur J Biochem, 229(1): 83–90

[20] Schick L A, Castellino  F J (1974). Direct evidence for the generation of an active site in the plasminogen moiety of the streptokinase-human plasminogen activator complex. Biochem Biophys Res Commun, 57(1): 47–54

[21] Taleb T, Kunameeni  A, Ellaiah P  (2005). Isolation and mutagenesis of streptokinase producing bacteria. Am J Immunol, 1(4): 125–129

[22] Tillett W S, Garner  R L (1933). Thefibrinolytic activity of hemolytic streptococci. J Exp Med, 58(4): 485–502

[23] Tough J (2005). Thrombolytic therapy in acute myocardial infarction. Nurs Stand, 19(37): 55–64, quiz 66

[24] Uversky V N, Fink  A L (2004). Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim Biophys Acta, 1698(2): 131–153

[25] WHO (2010) Burden: mortality, morbidity and risk factors. In: Global status report on non- communicable diseases, WHO Press, Geneva.pp, 9–10

Acknowledgements

We are greatly indebted to Vellore Institute of Technology for the constant encouragement, help and support for extending necessary facilities.

Compliance with ethics guidelines

The authors declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by the any of the authors.

RIGHTS & PERMISSIONS

2017 Higher Education Press and Springer-Verlag Berlin Heidelberg