Abstract
Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.
Similar content being viewed by others
References
Mine Y, Wong AHK, Jiang B (2005) Fibrinolytic enzymes in Asian traditional fermented foods. Food Rev In 38:243–250
Moukhametova LI, Aisina RB, Lomakina GY, Varfolomeev SD (2002) Properties of the urokinase-type plasminogen activator modified with phenylglyoxal. Russ J Bioorg Chem 28(4):278–283
Bode C, Runge M, Smalling RW (1996) The future of thrombolysis in the treatment of acute myocardial infarction. Eur Heart J 17:55–60
Fujita M, Hong K, Ito Y, Fujii R, Kariya K, Nishimuro S (1995) Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat. Biol Pharm Bull 18:1387–1391
Kim W, Choi K, Kim Y (1996) Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK11-4 screened from Chungkook-Jang. Appl Environ Microbiol 62:2482–2488
Zheng ZL, Zuo ZY, Liu ZG (2005) Construction of a 3D model of nattokinase, a novel fibrinolytic enzyme from Bacillus natto: a novel nucleophilic catalytic mechanism for nattokinase. J Mol Graph 23:373–380
Wang F, Wang C, Li M (2005) Crystal structure of earthworm fibrinolytic enzyme component B: a novel, glycosylated two-chained trypsin. J Mol Biol 348:671–685
Jia YH, Jin Y, Lu QM, Li DS (2003) Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom. Acta Biochem S 35(8):689–694
De-Simone SG, Correa-Netto C, Antunes OAC (2005) Biochemical and molecular modeling analysis of the ability of two p-aminobenzamidine-based sorbents to selectively purify serine proteases (fibrinogenases) from snake venoms. J Chromatogr B Analyt Technol Biomed Life Sci 822:1–9
You WK, Sohn YD, Kim KY, Park DH (2004) Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans. Insect Biochem Mol Biol 34:239–250
Ahn MY, Hahn BS, Ryu KS (2003) Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles, Catharsius molossus. Thromb Res 112:339–347
Chudzinski-Tavassi AM, Kelen EM, De Paula Rosa AP (1998) Fibrino(geno)lytic properties of purified hementerin, a metalloprotease from the leech Haementeria depressa. Thromb Haemost 80:155–160
Sumi H, Hamada H, Tsushima H (1987) A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a typical and popular soybean food in the Japanese diet. Experientia 43:1110–1111
Sumi H, Hamada H, Nakanishi K, Hiratani H (1990) Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol 84:139–143
Sumi H, Yanagisawa Y, Yatagai C (2004) Natto bacillus as oral fobrinolytic agent: Nattokinase activity and the ingestion effect of Bacillus natto. Food Sci Tec 10(1):17–20
Fujita M, Hong K, Ito Y (1995) Transport of NK across of the rat intestinal tract. Biol Pharm Bull 18(9):1194–1196
Wang CT, Ji BP, Li B, Ji H (2003) Isolation of Douchi fibrinolytic enzyme producing strain DC33 from Chinese Douchi and primary analysis of the enzyme property. Food Sci (China) 6:34–38
Krieg Nr, Holt JG (1984) Bergey’s manual of systematic bacteriology, Williams and Wilkins, Baltimore 1:1104–1139
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Astrup T, MuEllertz S (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346–351
Price Nc, Stevens L (1989) Fundamentals of enzimology 2nd edn. Oxford university press, Oxford
Peng Y, Huang Q, Zhang RH, Zhang YZ (2003) Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. Comp Biochem Physiol B Biochem Mol Biol 134:45–52
Glazer AN (1967) Esteratic reactions catalyzed by subtilisins. J Biol Chem 242(3):433–436
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685
Kim HK, Kim GT, Kim DK, Choi WA (1997) Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermented fish. J Ferment Bioen 84:307–312
Wong SL, Price CW, Goldfarb DS, Doi RH (1984) The subtilisin E gene of Bacillus subtilis is transcribed from a sigma-37 promoter in vivo. Proc Natl Acad Sci USA 81:1184–1188
Peng Y, Yang XJ, Zhang YZ (2005) Microbial fibrinolytic enzymes: an overview of source, production, properties and thrombolytic activity in vivo. Appl Microbiol Biotechnol 0175–7598, 1432–0614 (Online). DOI: 10.1007/s00253-005-0159-7
Matsuo O, Rijken DC, Collen D (1981) Thrombolysis by human tissue plasminogen activator and urokinase in rabbits with experimental pulmonary embolus. Nature 291(5816):590–591
Wiman B, Wallen P (1975) On the primary structure of human plasminogen and plasmin. Purification and characterization of cyanogen-bromide fragments. Eur J Biochem 57:387–394
Lijnen HR, Cock FD, Hoef BV, Schlott B, Collen D (1994) Characterization of the interaction between plasminogen and staphylokinase. Eur J Biochem 224:143–149
Fujita M, Nomura K, Hong K, Ito Y, Asada A (1993) Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem Biophys Res Commun 197:1340–1347
Takahashi M, Sekine T, Kuba N, Nakamori S, Yasuda M (2004) The production of recombinant APRP, an alkaline protease derived from Bacillus pumilus TYO-67, by in vitro refolding of pro-enzyme fixed on a solid surface. J Biochem 136(4):549–556
Chang CT, Fan MH, Kuo FC, Sung HY (2000) Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J Agric Food Chem 48:3210–3216
Seo JH, Lee SP (2004) Production of fibrinolytic enzyme(KK) from soybean grits fermented by Bacillus firmus NA-1. J Med Food 7(4):442–449
Kim JH, Kim YS (1999) A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea. Biosci Biotechnol Biochem 63:2130–2136
Smith EL, Delange RJ, Evans WH, Landon M (1968) Subtilisin Carlsberg V. The complete sequence, comparison with subtilisin BPN’, evolutionary relationships. J Biol Chem 243:2184–2191
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wang, C.T., Ji, B.P., Li, B. et al. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi . J IND MICROBIOL BIOTECHNOL 33, 750–758 (2006). https://doi.org/10.1007/s10295-006-0111-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10295-006-0111-6