Purification and characterization of a fibrino(geno)lytic protease from cultured natural isolate of a cyanobacterium, Anabaena fertilissima
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.
KeywordsAnabaena 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.
- 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 187:6906–6916Google 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 161:102–109CrossRefGoogle Scholar
- Desikachary TV (1959) Cyanophyta. ICAR Monographs, New DelhiGoogle Scholar
- Francis S, Markland J (1998) Snake venom fibrinogenolytic and fibrinolytic enzymes: an updated inventory. Thromb Haemost 79:668–674Google 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–S042Google 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
- 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 17:1469–1476Google Scholar
- Martinez J, Azam F (1993) Aminopeptidase activity in marine chroococcoid cyanobacteria. App Environ Microbiol 59:3701–3707Google 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 263:163–169PubMedCrossRefGoogle 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 71:2184–2189Google Scholar