Browse > Article

Purification and Characterization of a Subtilisin D5, a Fibrinolytic Enzyme of Bacillus amyloliquefaciens DJ-5 Isolated from Doenjang  

Choi, Nack-Shick (Enzyme Based Fusion Technology Research Team, Jeonbuk Branch Institute, Korea Research Institute Bioscience & Biotechnology (KRIBB))
Chung, Dong-Min (Systemic Proteomics Research Center, Korea Research Iinstitute Bioscience & Biotechnology (KRIBB))
Han, Yun-Jon (Enzyme Based Fusion Technology Research Team, Jeonbuk Branch Institute, Korea Research Institute Bioscience & Biotechnology (KRIBB))
Kim, Seung-Ho (Systemic Proteomics Research Center, Korea Research Iinstitute Bioscience & Biotechnology (KRIBB))
Song, Jae-Jun (Enzyme Based Fusion Technology Research Team, Jeonbuk Branch Institute, Korea Research Institute Bioscience & Biotechnology (KRIBB))
Publication Information
Food Science and Biotechnology / v.18, no.2, 2009 , pp. 500-505 More about this Journal
Abstract
The fibrinolytic enzyme, subtilisin D5, was purified from the culture supernatant of the isolated Bacillus amyloliquefaciens DJ-5. The molecular weight of subtilisin D5 was estimated to be 30 kDa. Subtilisin D5 was optimally active at pH 10.0 and $45^{\circ}C$. Subtilisin D5 had high degrading activity for the A$\alpha$-chain of human fibrinogen and hydrolyzed the $B{\beta}$-chain slowly, but did not affect the $\gamma$-chain, indicating that it is an $\alpha$-fibrinogenase. Subtilisin D5 was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it belongs to the serine protease. The specific activity (F/C, fibrinolytic/caseinolytic activity) of subtilisin D5 was 2.37 and 3.52 times higher than those of subtilisin BPN' and Carlsberg, respectively. Subtilisin D5 exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. The first 15 amino acid residues of the N-terminal sequence of subtilisin D5 are AQSVPYGISQIKAPA; this sequence is identical to that of subtilisin NAT and subtilisin E.
Keywords
Bacillus amyloliquefaciens; doenjang; fibrinolytic enzyme; subtilisin D5;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
Times Cited By Web Of Science : 5  (Related Records In Web of Science)
Times Cited By SCOPUS : 6
연도 인용수 순위
1 Sumi H, Hamada H, Nakanishi K, Hiratani H. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol.-Sasel. 84: 139-143 (1990)   DOI   ScienceOn
2 Chang CT, Fan MH, Kuo FC, Sung HY. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J. Agr. Food Chem. 48: 3210-3216 (2000)   DOI   ScienceOn
3 Kim SH, Choi NS, Lee WY, Lee JW, Kim DH. Isolation of Bacillus strains secreting fibrinolytic enzymes from doenjang. Korean J. Microbiol. 34: 87-90 (1998)
4 Chun J, Bae KS. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. Anton. Leeuw. Int. J. G. 78: 123-127 (2000)   DOI   ScienceOn
5 Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425 (1987)   PUBMED
6 Hung CC, Huang KF, Chiou SH. Characterization of one novel venom protease with $\beta$-fibrinogenase activity from the Taiwan habu (Trimeresurus mucrosquamatus): Purification and cDNA sequence analysis. Biochem. Bioph. Res. Co. 205: 1707-1715 (1994)   DOI   ScienceOn
7 Laemmli UK. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685 (1970)   DOI   PUBMED   ScienceOn
8 Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, Chen LF. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional douchi. J. Ind. Microbiol. Biot. 33: 750-758 (2006)   DOI   ScienceOn
9 Siigur E, Aaspollu A, Tu AT, Siigur J. cDNA cloning and deduced amino acid sequence of fibrinolytic enzyme (lebetase) from Vipera lebetina snake venom. Biochem. Bioph. Res. Co. 224: 229-236 (1996)   DOI   ScienceOn
10 Lee JW, Seu JH, Rhee IK, Jin I. Purification and characterization of brevinase, a heterogeneous two-chain fibrinolytic enzyme from the venom of Korean snake, Agkistrodon blomhoffii brevicaudus. Biochem. Bioph. Res. Co. 260: 665-670 (1999)   DOI   ScienceOn
11 Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. Strain CK 11-4 screened from cheonggukjang. Appl. Environ. Microb. 62: 2482-2488 (1996)   PUBMED
12 Simth EL, Delange RJ, Evans WH, Landon M. Subtilisin Carlsberg V. The complete sequence, comparison with subtilisin BPN', evolutionary relationships. J. Biol. Chem. 243: 2184-2191 (1968)   PUBMED
13 Peng Y, Huang Q, Zhang R, Zhang Y. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douche, a traditional Chinese soybean food. Comp. Biochem. Phys. B 134: 45-52 (2003)   DOI   ScienceOn
14 Kim SB, Lee DW, Cheigh CI, Choe EA, Lee SJ, Hong YH, Choi HJ, Pyun YR. Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean. Tempeh. J. Ind. Microbiol. Biot. 33: 436-444 (2006)   DOI   ScienceOn
15 Takahashi M, Sekine T, Kuba N, Nakamori S, Yasuda M. 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: 549-556 (2004)   DOI   ScienceOn
16 Matsubara K, Sumi H, Hori K, Miyazawa K. Purification and characterization of two fibrinolytic enzymes from a marine green alga, Codium intricatum. Comp. Biochem. Phys. B 119: 177-181 (1998)   DOI   ScienceOn
17 Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto; a typical and popular soybean food in the Japanese diet. Experientia 43: 1110-1111 (1987)   DOI   PUBMED   ScienceOn
18 Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membrane. J. Biol. Chem. 262: 10035-10038 (1987)   PUBMED
19 Lu F, Sun L, Lu Z, Bie X, Fang Y, Liu S. Isolation and identification of an endophytic strain EJS-3 producing novel fibrinolytic enzyme. Curr. Microbiol. 54: 435-439 (2007)   DOI   ScienceOn
20 Matsubara K, Hori K, Matsuura Y, Miyazawa K. Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum. Comp. Biochem. Phys. B 125: 137-143 (2000)   DOI   ScienceOn
21 Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791 (1985)   DOI   ScienceOn
22 Felsenstein J. PHYLIP (phylogenetic inference package) version 3.5c. Department of Genetics, University of Washington, Seattle, WA, USA (1993)
23 Kim SH, Choi NS. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from doenjang. Biosci. Biotech. Bioch. 64: 1722-1725 (2000)   DOI   ScienceOn
24 Sumi H, Nakajima N, Yatagai C. A unique strong fibrinolytic enzyme (Katsuwokinase) in Skipjack 'shiokara', a Japanese traditionall fermented food. Comp. Biochem. Phys. B 112: 543-547 (1995)   DOI   ScienceOn
25 Roberts MS, Nakamura LK, Cohan FM. Bacillus mojavensis sp. nov., distinguishable from Bacillus subtilis by sexual isolation, divergence in DNA sequence, and differences in fatty acid composition. Int. J. Syst. Bacteriol. 44: 256-264 (1994)   DOI   ScienceOn
26 Asrup T, Müllertz S. The fibrin plate method for estimating fibrinolytic activity. Arch. Bioch. Biophys. 40: 346-351 (1952)   DOI   ScienceOn
27 Kim HK, Kim GT, Kim DK, Choi WA, Park SH, Jeong YK, Kong IS. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermented fish. J. Ferment. Bioeng. 84: 307-312 (1997)   DOI   ScienceOn
28 Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem. Bioph. Res. Co. 197: 1340-1347 (1993)   DOI   ScienceOn
29 Lane DJ. 16S/23S rRNA sequencing. pp. 115-175. In: Nucleic Acid Techniques in Bacterial Systematics. Stackebrandt E, Goodfellow M (eds). John Wiley and Sons, New York, NY, USA (1991)
30 Wong SL, Price CW, Goldfarb DS, Doi RH. The subtilisin E gene of Bacillus subtilis is transcribed from a sigma-37 promoter in vivo. P. Natl. Acad. Sci. USA 81: 1184-1188 (1984)   DOI   ScienceOn
31 Matsubara K, Hori K, Matsuura Y, Miyazawa K. A fibrinolytic enzyme from a marine green alga, Codium latum. Phytochemistry 52: 993-999 (1999)   DOI   ScienceOn
32 Chun J. Computer-assisted classification and identification of actinomycetes. PhD thesis, University of Newcastle, Newcastle upon Tyne, UK (1995)
33 Rochelle PA, Fry JC, Parkes RJ, Weightman AJ. DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities. FEMS Microbiol. Lett. 100: 59-66 (1992)   DOI
34 Jukes TH, Cantor CR. Evolution of protein molecules. pp. 21-132. In: Mammalian Protein Metabolism. Munro HN (ed). Academic Press, Inc., New York, NY, USA (1969)
35 Seo JH, Lee SP. Production of fibrinolytic enzyme (KK) from soybean grits fermented by Bacillus firmus NA-1. J. Med. Food 7:442-449 (2004)   DOI   ScienceOn