Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food
Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food
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DOI：
10.1016/s1096-4959(02)00183-5
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发表时间：
2003-01-01
影响因子：
2.2
通讯作者：
Zhang, YZ
中科院分区：
文献类型：
--
作者：
Peng, Y;Huang, Q;Zhang, YZ
从中国传统的大豆发酵食品豆豉中分离到一株产纤溶酶的解淀粉芽孢杆菌DC-4。从B的上清液中纯化纤溶酶（枯草杆菌蛋白酶DFE）。DC-4菌株培养液中的纤维蛋白溶解酶具有嗜热性、亲水性和较强的纤维蛋白溶解活性。经SDS-PAGE和等电聚焦电泳证实，DFE为均一的枯草杆菌蛋白酶，分子量为28000 Da，pI为8.0。最适反应pH值为9.0，最适反应温度为48 ℃。枯草杆菌蛋白酶DFE不仅能水解纤维蛋白，而且对多种合成底物，特别是Suc-Ala-Ala-Pro-Phe-pNA和苯甲磺酰氟都能完全抑制其纤溶活性。这些结果表明，枯草杆菌蛋白酶DFE是一种枯草杆菌蛋白酶家族的丝氨酸蛋白酶，类似于来自纳豆芽孢杆菌的纳豆激酶。其N端前24个氨基酸残基为AQSVPYGVSQIKAPALHSQGFTGS，与枯草杆菌蛋白酶K-54相同，与NK和CK不同。枯草杆菌蛋白酶DFE基因序列分析结果表明，枯草杆菌蛋白酶DFE是一种新的纤溶酶。(C)2002年爱思唯尔科技有限公司All rights reserved.
Bacillus amyloliquefaciens DC-4, which produces a strongly fibrinolytic enzyme, was isolated from douchi, a traditional Chinese soybean-fermented food. A fibrinolytic enzyme (subtilisin DFE) was purified from the supernatant of B. amyloliquefaciens DC-4 culture broth and displayed thermophilic, hydrophilic and strong fibrinolytic activity. Subtilisin DFE was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of 28000 Da and a pl of 8.0. The optimal reaction pH value and temperature were 9.0 and 48 degreesC, respectively. Subtilisin DFE not only hydrolyzed fibrin but also several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA, and phenylmethylsulfony fluoride can completely inhibit its fibrinolytic activity. These results indicated that subtilisin DFE is a subtilisin-family serine protease, similar to nattokinase from Bacillus natto. The first 24 amino acid residues of the N-terminal sequence of subtilisin DFE were AQSVPYGVSQIKAPALHSQGFTGS, which is identical to that of subtilisin K-54, and different from that of NK and CK. Results from subtilisin DFE gene sequence analysis showed that subtilisin DFE is a novel fibrinolytic enzyme. (C) 2002 Elsevier Science Inc. All rights reserved.