Journal of Applied Phycology

, Volume 25, Issue 4, pp 1111–1122 | Cite as

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

  • Sonali Banerjee
  • Radha Prasanna
  • Suvendra Nath Bagchi


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.


Anabaena fertilissima Caseinolytic protease Cyanobacteria D-Dimer γ-Dimer Fibrin(ogen) hydrolysis Plasmin 



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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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Sonali Banerjee
    • 1
  • Radha Prasanna
    • 2
  • Suvendra Nath Bagchi
    • 1
  1. 1.Department of Biological SciencesRani Durgavati UniversityJabalpurIndia
  2. 2.Division of MicrobiologyIndian Agricultural Research InstituteNew DelhiIndia

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