Characterization of a Novel Fibrinolytic Enzyme, BsfA, from Bacillus subtilis ZA400 in Kimchi Reveals Its Pertinence to Thrombosis Treatment

Abstract

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40°C), metal ions (Mn(2+), Ca(2+), K(2+), and Mg(2+)), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

Purification and characterization of a novel, highly potent fibrinolytic enzyme from Bacillus subtilis DC27 screened from Douchi, a traditional Chinese fermented soybean food

“…However, DFE27 showed significant differences from them in terms of strain source, optimum substrate specificity, fibrinolytic action mode or N-terminal sequence. For example, the N-terminal 24-amino-acids sequence of DFE27 was different from that of other fibrinolytic enzymes originated from other Asian fermented foods, such as natto 31,35 , fermented soybean 34 , doen-jang 28 , Chungkook-jang 41 and kimchi 33 . In addition, even though showing identical action mode, the DFE27 exhibited different N-terminal sequence or substrate hydrolysis characteristic with other Bacillus fibrinolytic enzyme originated from Douchi , such as fibrinolytic enzyme FS33 and another enzyme from Bacillus sp.…”

“…subtillis isolated from Douchi . Most fibrinolytic enzymes from food have a molecular mass between 28 and 30 kDa, and the molecular mass of DFE27 (29 kDa) was similar to that of Subtilisin DFE (28 kDa) 18 , BsfA (28.4 kDa) 33 , Subtilisin QK-2 (28 kDa) 34 and NAT(30 kDa) 35 . Furthermore, the activity of DFE27 was obviously inhibited by PMSF, proving that it is a serine protease.…”

Understanding the ontogeny and succession of Bacillus velezensis and B. subtilis subsp. subtilis by focusing on kimchi fermentation

“…In particular, B. subtilis subsp. subtilis and B. velezensis are the most frequently isolated bacilli from fermented foods or soil 5 , 28 , 29 .…”

Mapping of residues of fibrinogen cleaved by protease II of Bacillus thuringiensis var. israelensis IMV B-7465

“…These properties allowed authors to conclude the potential anti-thrombotic use of the enzymes in peptide-based cardiovascular drug development [7][8][9]12]. Some of them were already tested in animal models [8] or were cloned for these purposes [13].…”