Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis M
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Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6 Li-Li Man 1 & Dian-Jun Xiang 2 & Chun-Lan Zhang 1
# Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi. The presence of aprN, a gene-encoding nattokinase, was verified with PCR method. The predicted amino acid sequence was aligned with homologous sequences, and a phylogenetic tree was constructed. Nattokinase was sublimated with ammonium sulfate, using a DEAE-Sepharose Fast Flow column, a CM-Sepharose Fast Flow column and a Sephadex G-75 gel filtration column. SDS-PAGE analysis indicated that the molecular weight of the purified nattokinase from Bacillus subtilis MX-6 was about 28 kDa. Fermentation of Bacillus subtilis MX-6 nattokinase showed that nattokinase production was maximized after 72 h; the diameter of clear zone reached 21.60 mm on the plasminogen-free fibrin plate. Nattokinase production by Bacillus subtilis MX-6 increased significantly after supplementation with supernatant I, supernatant II and soy peptone but decreased substantially after the addition of amino acids. This result indicated that the nattokinase production by B. subtilis MX-6 might be induced by soybean polypeptides. The addition of MgSO4 and CaCl2 increased B. subtilis MX-6 nattokinase production. Keywords Nattokinase . Screening . Bacillus subtilis . Induction
Introduction Cardiovascular diseases (CVDs) that result from the coagulation of fibrin have a severe impact on human health [1–3]. Thrombolytic enzymes are therefore often used to treat CVDs [4–6]. Thrombolytic agents may be divided into two categories based on their effects: one type contains urokinase and a tissuetype plasminogen activator (t-PA), which decomposes fibrin by Li-Li Man, Dian-Jun Xiang and Chun-Lan Zhang contributed equally to this work. * Dian-Jun Xiang [email protected] Li-Li Man [email protected] Chun-Lan Zhang [email protected] 1
College of Life Science, Inner Mongolia University for Nationalities, Tongliao 028042, People’s Republic of China
2
College of Agriculture, Inner Mongolia University for Nationalities, Tongliao 028042, People’s Republic of China
transforming the passivating proenzyme plasminogen into plasmin [7, 8]. The other thrombolytic type is a plasmin-like protein that degrades the fibrin directly and dissolves thrombin entirely. Both types of fibrinolytic agents have been used extensively in the treatment of CVDs but have nonetheless several drawbacks including high cost, possible bleeding complications and risk of acute coronary reocclusion [9, 10]. In light of these limitations, the fibrinolytic enzymes produced by various microbes deserve further attention. It is possible to acquire these microbial fibrinolytic enzymes rapidly, reducing the energy input, processing time and overall cost of CVD treatment [11]. The fibrinolyt
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