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Purification and characterization of a fibrino(geno)lytic protease from cultured natural isolate of a cyanobacterium, Anabaena fertilissima

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Abstract

An isolate of the cyanobacterium Anabaena from paddy fields was cultured and identified as Anabaena fertilissima based on morphometric features and 16S rRNA gene sequence matching. Cell extracts prepared using bead beater hydrolyzed casein. The caseinolytic protease with native molecular mass of 49 kDa was purified using ammonium sulfate fractionation, hydrophobic, affinity and ion-exchange chromatography, and gel filtration. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified protease was resolved in 17-kDa homologue of microcompartment protein and 27-kDa fragment of unknown protein. The enzyme in native state was digested with gelatin and fibrin in substrate gels producing bands corresponding to ca. 49 kDa. Moreover, a plasmin-specific substrate d-Val-Leu-Lys p-nitroanilide was also hydrolyzed with apparent K m = 0.18 mM and V max = 4.9 × 10−7 M s−1; while Ca2+ stimulated, phenylmethanesulfonyl fluoride, leupeptin, and chelators completely abolished the amidolytic activity. The enzyme exhibited pH and temperature stability over a wide range. Upon incubation with fibrinogen, the Aα- and Bβ-chains preferentially cleaved, though the products thus resolved on SDS-PAGE moved at masses different from those of thrombin- and plasmin hydrolysates, and unlike thrombin, cross-linking of fibrinopeptides was not observed. In the plate assays, fibrinolysis was revealed at comparable strengths to that of plasmin, and the dissolute so obtained upon SDS-PAGE lacked bands corresponding to γ-dimer. Consequently, the degraded D-Dimer peptides appeared. The cyanobacterial protease displayed several unique properties not found in microbial and snake venom fibrinolytic enzymes.

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References

  • Agrawal MK, Zitt A, Bagchi D, Weckesser J, Bagchi SN, von Elert E (2005) Characterization of proteases in gut of Daphnia magna and their inhibition by Microcystis aeruginosa PCC7806. Environ Toxicol 20:314–322

    Article  PubMed  CAS  Google Scholar 

  • Andrews P (1965) The gel filtration behavior of proteins related to their molecular weights over a wide range. Biochem J 96:595–606

    PubMed  CAS  Google Scholar 

  • Astrup T, Mullertz S (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346–351

    Article  PubMed  CAS  Google Scholar 

  • Cannon GC, Bradburne CE, Aldrich CE, Baker SH, Heinhorst S, Shively JM (2001) Microcompartments in prokaryotes: carboxysomes and polyhedra. Appl Environ Microbiol 67:5351–5361

    Article  PubMed  CAS  Google Scholar 

  • Chang AK, Kim HY, Park JE, Acharya P, Park IS, Yoon SM, You HJ, Hahm KS, Park JK, Lee JS (2005) Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis. J Bact 187:6906–6916

    Google Scholar 

  • Chiu HF, Yang SP, Kuo YL, Lai YS, Chou TC (2006) Mechanisms involved in the antiplatelet effect of C-phycocyanin. Br J Nutr 95:435–440

    Article  PubMed  CAS  Google Scholar 

  • Datta G, Dong A, Witt J, Tu AT (1995) Biochemical-characterization of basilase, a fibrinolytic protease from Cratolus basiliscus. Arch Biochem Biophys 317:365–373

    Article  PubMed  CAS  Google Scholar 

  • de Souza DLN, Gomes MSR, Ferreira FB, Rodrigues RS, Ache DC, Richardson M, Borges MH, Rodrigues VM (2012) Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom. Comp Biochem Physiol B 161:102–109

    Article  Google Scholar 

  • Desikachary TV (1959) Cyanophyta. ICAR Monographs, New Delhi

    Google Scholar 

  • Dong Y, Huang X, Wu XY, Zhao J (2000) Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120. J Bact 182:1575–1579

    Article  PubMed  CAS  Google Scholar 

  • Francis S, Markland J (1998) Snake venom fibrinogenolytic and fibrinolytic enzymes: an updated inventory. Thromb Haemost 79:668–674

    Google Scholar 

  • Funk C, Hauβühl K, Adamska I (2001) Family of Deg/Htr proteases in the cyanobacterium Synechocystis sp. PCC6803: investigations toward their expression and function. In: Larkum T, Critchley C (eds) PS 2001 Proceedings: 12th International Congress on Photosynthesis. CSIRO, Melbourne, Australia, pp S8–S042

    Google Scholar 

  • Ghosh SK, Bagchi D, Bagchi SN (2008) Proteolytic activity in Microcystis aeruginosa PCC7806 is inhibited by a trypsin-inhibitory cyanobacterial peptide with a partial structure of microviridin. J Appl Phycol 20:1045–1052

    Article  CAS  Google Scholar 

  • Gupta V, Natarajan C, Chaudhary V, Kumar A, Sharma E, Sharma J, Bhatnagar AK, Prasanna R (2012) Analyses of diversity among fungicidal Anabaena strains. J Appl Phycol. doi:10.1007/s10811-012-9793-5

  • Kelly SL, Adams SA, Robson SC, Kirsch RE, Shephard EG (1994) Fibrinogenolysis by a neutrophil membrane protease generates an Aα1-21 fragment. Biochem J 298:689–695

    PubMed  CAS  Google Scholar 

  • Kim SH, Choi NS, Lee WY (1998) Fibrin zymography: a direct analysis of fibrinolytic enzyme on gels. Anal Biochem 263:115–116

    Article  PubMed  CAS  Google Scholar 

  • Kim HC, Choi BS, Sapkota K, Kim S, Lee HJ, Yoo JC, Kim SJ (2011) Purification and characterization of a novel, highly potent fibrinolytic enzyme from Paecilomyces tenuipes. Process Biochem 46:1545–1553

    Article  CAS  Google Scholar 

  • Ko HJ, Yan JP, Zhu L, Qi YP (2004) Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comp Biochem Physiol C 137:65–74

    Article  Google Scholar 

  • Kyung-Ju H, Choi KH, Kim MJ, Park CC (2007) Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal. J Microbiol Biotechnol 17:1469–1476

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lockau A, Massalsky B, Dirmeir A (1988) Purification and partial characterization of a calcium-stimulated protease from the cyanobacterium, Anabaena variabilis. Eur J Biochem 172:433–438

    Article  PubMed  CAS  Google Scholar 

  • Lowry OH, Rosenbrough NJ, Forr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Madhyastha HK, Radha KS, Sugiki M, Omura S, Maruyama M (2006) Purification of c-phycocyanin from Spirulina fusiformis and its effect on the induction of urokinase-type plasminogen activator from calf pulmonary endothelial cells. Phytomedicine 13:564–569

    Article  PubMed  CAS  Google Scholar 

  • Martinez J, Azam F (1993) Aminopeptidase activity in marine chroococcoid cyanobacteria. App Environ Microbiol 59:3701–3707

    CAS  Google Scholar 

  • Matsubara K, Sumi H, Hori K, Miyazawa K (1998) Purification and characterization of two fibrinolytic enzymes from a marine green alga, Codium intricatum. Comp Biochem Physiol B 119:177–181

    Article  Google Scholar 

  • Matsubara K, Matsuura Y, Sumi H, Hori K, Miyazawa K (1999) A fibrinolytic enzyme from the green alga Codium latum activates plasminogen. Fish Sci 68:455–457

    Article  Google Scholar 

  • Nanni B, Balestreri E, Dainese E, Cozzeni I, Felicioli R (2001) Characterization of a specific phycocyanin-hydrolyzing protease purified from Spirulina platensis. Res Microbiol 156:259–266

    Article  CAS  Google Scholar 

  • Park SE, Li MH, Kim JS, Sapkota K, Kim JE, Choi BS, Yoon YH, Lee JC, Lee HH, Kim CS, Kim SJ (2007) Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia. Biosci Biotech Biochem 71:2214–2222

    Article  CAS  Google Scholar 

  • Patel GK, Kawale AA, Sharma AK (2012) Purification and physicochemical characterization of a serine protease with fibrinolytic activity from latex of a medicinal herb Euphorbia hirta. Plant Physiol Biochem 52:104–111

    Article  PubMed  CAS  Google Scholar 

  • Perez-Llorens JL, Benitez E, Vergara JJ, Berges JA (2003) Characterization of proteolytic enzyme activities in macroalgae. Eur J Phycol 38:55–64

    Article  Google Scholar 

  • Pizzo SV, Schwartz ML, Hill RL, McKee PA (1973) The effect of plasmin on the subunit structure of human fibrin. J Biol Chem 248:4574–4583

    PubMed  CAS  Google Scholar 

  • Ramos MV, Viana CA, Silva AFB, Freitas CDT, Figueiredo IST, Oliveira RSB, Alencar NMN, Lima-Filho JVM, Kumar VL (2012) Proteins derived from latex of C. procera maintain coagulation homeostasis in septic mice and exhibit thrombin- and plasmin-like activities. Naunyn-Schmeideberg’s Arch Pharmacol. doi:10.1007/s00210-012-0733-3

  • Richter R, Hejazi M, Kraft R, Ziegler K, Lockau W (1999) Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartic acid (cyanophycin): molecular cloning of the gene of Synechocystis sp. 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme. Eur J Biochem 263:163–169

    Article  PubMed  CAS  Google Scholar 

  • Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure culture of cyanobacteria. J Gen Microbiol 111:1–61

    Article  Google Scholar 

  • Schwert WH, Takenaka Y (1955) A spectrophotometric determination of trypsin and chymotrypsin. Biochim Biophys Acta 16:570–575

    Article  PubMed  CAS  Google Scholar 

  • Stanne TM, Pojidaeva E, Andersson FI, Clarke AK (2007) Distinctive types of ATP-dependent Clp proteases in cyanobacteria. J Biol Chem 282:14394–14402

    Article  PubMed  CAS  Google Scholar 

  • Sugimoto S, Fujii T, Morimiya T, Johdo O, Nakamura T (2007) The fibrinolytic activity of the novel protease derived from a Tempeh producing fungus Fusarium sp. BLB. Biosci Biotech Biochem 71:2184–2189

    Google Scholar 

  • Tao CW, Ji BP, Li B, Nout R, Li PL, Ji H, Chen LF (2006) Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol 33:750–758

    Article  Google Scholar 

  • Uesegi Y, Usuki H, Iwabuchi M, Hatanaka T (2011) Highly potent fibrinolytic serine protease from Streptomyces. Enzyme Microb Technol 48:7–12

    Article  Google Scholar 

  • Vieira DF, Watanabe L, Santana CD, Marcussi S, Sampaio SV, Soares AM, Arni RK (2004) Purification and characterization of jarassin-I, a thrombin-like enzyme from Bothrops jararaca snake venom. Acta Biochem Biophys Sin (Shanghai) 36:798–802

    Article  CAS  Google Scholar 

  • Wiman B, Collen D (1979) On the mechanism of the reaction between human α2-antiplasmin and plasmin. J Biol Chem 254:9291–9297

    PubMed  CAS  Google Scholar 

  • Yada E, Nagata H, Noguchi Y, Kodera Y, Nishima H, Inada Y, Matsushima A (2005) An arginine specific protease from Spirulina platensis. Mar Biotechnol 7:474–480

    Article  PubMed  CAS  Google Scholar 

  • Yang G, Hu B, Zhao J (2011) Specific degradation of photosystem II D1 protein by a protease (Alr3815) in heterocysts of the cyanobacterium Anabaena sp. PCC7120. Chin Sci Bull 56:1068–1070

    Article  CAS  Google Scholar 

  • Zhang P, Sicora CI, Vorontsove N, Allahverdiyeva Y, Battchikova N, Nixon PJ, Aro EM (2007) FtsH protease is required for induction of inorganic carbon acquisition complexes in Synechocystis sp. PCC6803. Mol Microbiol 65:728–740

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank the Head of the Department of Biological Sciences, R. D. University, Jabalpur, for providing lab facilities; S. Rajagopal, School of Life Sciences, Hyderabad University, Hyderabad, for MALDI-TOF MS analysis; and Vishal Gupta and Santosh Babu, Division of Microbiology, IARI, New Delhi, for helping with 16S rDNA-based identification and related bioinformatic analyses. The financial support by the Department of Biotechnology, Government of India, New Delhi (project no. BT/PR11135/PBD/17/582/2008) is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Biological Sciences, Rani Durgavati University, Jabalpur, 482001, India

    Sonali Banerjee & Suvendra Nath Bagchi

  2. Division of Microbiology, Indian Agricultural Research Institute, New Delhi, 110012, India

    Radha Prasanna

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  1. Sonali Banerjee
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Correspondence to Suvendra Nath Bagchi.

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Banerjee, S., Prasanna, R. & Bagchi, S.N. Purification and characterization of a fibrino(geno)lytic protease from cultured natural isolate of a cyanobacterium, Anabaena fertilissima . J Appl Phycol 25, 1111–1122 (2013). https://doi.org/10.1007/s10811-012-9946-6

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  • DOI: https://doi.org/10.1007/s10811-012-9946-6

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