Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

被引:138
作者
Wang, Cheng Tao
Ji, Bao Ping
Li, Bo
Nout, Rob
Li, Ping Lan
Ji, Hong
Chen, Long Fei
机构
[1] China Agr Univ, Coll Food Sci & Nutrit Engn, Beijing 100083, Peoples R China
[2] Univ Wageningen & Res Ctr, Dept Agrotechnol & Food Sci, Lab Food Microbiol, NL-6703 HD Wageningen, Netherlands
关键词
amidolytic activity; fibrin-specificity; fibrinolytic subtilisin; serine protease; thrombolytic agent;
D O I
10.1007/s10295-006-0111-6
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 degrees 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), beta-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-PhepNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the B beta-chains and A alpha-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.
引用
收藏
页码:750 / 758
页数:9
相关论文
共 36 条
  • [1] Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles, Catharsius molossus
    Ahn, MY
    Hahn, BS
    Ryu, KS
    Kim, JW
    Kim, I
    Kim, YS
    [J]. THROMBOSIS RESEARCH, 2003, 112 (5-6) : 339 - 347
  • [2] THE FIBRIN PLATE METHOD FOR ESTIMATING FIBRINOLYTIC ACTIVITY
    ASTRUP, T
    MULLERTZ, S
    [J]. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1952, 40 (02) : 346 - 351
  • [3] The future of thrombolysis in the treatment of acute myocardial infarction
    Bode, C
    Runge, MS
    Smalling, RW
    [J]. EUROPEAN HEART JOURNAL, 1996, 17 : 55 - 60
  • [4] BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
  • [5] Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1
    Chang, CT
    Fan, MH
    Kuo, FC
    Sung, HY
    [J]. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 2000, 48 (08) : 3210 - 3216
  • [6] Fibrino(geno)lytic properties of purified hementerin, a metalloproteinase from the leech Haementeria depressa
    Chudzinski-Tavassi, AM
    Kelen, EMA
    de Paula Rosa, AP
    Loyau, S
    Sampaio, CAM
    Bon, C
    Anglés-Cano, E
    [J]. THROMBOSIS AND HAEMOSTASIS, 1998, 80 (01) : 155 - 160
  • [7] Biochemical and molecular modeling analysis of the ability of two p-aminobenzamidine-based sorbents to selectively purify serine proteases (fibrinogenases) from snake venoms
    De-Simone, SG
    Correa-Netto, C
    Antunes, OAC
    De-Alencastro, RB
    Silva, FP
    [J]. JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 2005, 822 (1-2): : 1 - 9
  • [8] FUJITA M, 1995, BIOL PHARM BULL, V18, P1387
  • [9] FUJITA M, 1995, BIOL PHARM BULL, V18, P1194
  • [10] PURIFICATION AND CHARACTERIZATION OF A STRONG FIBRINOLYTIC ENZYME (NATTOKINASE) IN THE VEGETABLE CHEESE NATTO, A POPULAR SOYBEAN FERMENTED FOOD IN JAPAN
    FUJITA, M
    NOMURA, K
    HONG, K
    ITO, Y
    ASADA, A
    NISHIMURO, S
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1993, 197 (03) : 1340 - 1347