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首页> 外文期刊>Journal of industrial microbiology & biotechnology >Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi
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Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

机译：分离自中国传统豆chi的枯草芽孢杆菌DC33纤溶酶的纯化和鉴定

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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-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bbeta-chains and Aalpha-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.
机译：从中国传统的大豆发酵食品八宝豆池中分离出产生新型纤溶酶的枯草芽孢杆菌DC33。使用各种色谱步骤的组合，将强纤维蛋白特异性酶枯草杆菌蛋白酶FS33纯化至电泳均一。枯草杆菌蛋白酶FS33的最佳温度，pH值和pI分别为55℃，8.0和8.7。在还原和非还原条件下通过SDS-PAGE测得的分子量为30kDa。该酶显示出的纤溶活性水平比枯草杆菌蛋白酶嘉士伯高约六倍。该酶N末端序列的前15个氨基酸残基是A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A，与其他已知的纤溶酶不同。 5 mM苯基甲烷磺酰氟（PMSF）和1 mM大豆胰蛋白酶抑制剂（SBTI）完全抑制了枯草杆菌蛋白酶FS33的酰胺水解活性，但1,4-二硫苏糖醇（DTT），β-巯基乙醇和对羟基巯基苯甲酸（PHMB）并未完全抑制枯草杆菌蛋白酶FS33的酰胺分解活性。影响酶的活性;因此，丝氨酸和色氨酸在酶的活性位点是必需的。枯草杆菌蛋白酶FS33的最高亲和力是对N-Succ-Ala-Ala-Pro-Phe-pNA。因此，该酶被认为是枯草杆菌蛋白酶样丝氨酸蛋白酶。纤溶酶对纤维蛋白原的Bbeta链和Aalpha链具有很高的降解活性，并且还作用于血液的血栓和纤溶因子，例如纤溶酶原，尿激酶，凝血酶和激肽释放酶。因此枯草杆菌蛋白酶FS33能够以两种方式降解纤维蛋白凝块，即（a）通过纤溶酶原形成活性纤溶酶和（b）通过直接纤溶作用。

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