Identification of fibrinogen-induced nattokinase WRL101 from Bacillus subtilis WRL101 isolated from Doenjang

Abstract

To increase the fibrinolytic enzyme (nattokinase WRL101) in Bacillus subtilis WRL101, bovine fibrin or fibrinogen (1.0%, w/v) were added in the tryptic soy broth as a substrate. The fibrinolytic activity cultured in the fibrinogen-contained medium was increased. On the other hand, fibrin decreased the activity. Using the medium condition, nattokinase WRL101 (fibrinogen-induced nattokinase WRL101, FIN-WRL101) was isolated by commercial chromatographic techniques and its biochemical characteristics were investigated. The molecular weight of FIN-WRL101 was estimated to be 29 kDa. FIN-WRL101 was optimally active at pH 11.0 and 47°C. It had high degrading activity for the Aα-chain and B-chain of human fibrinogen, but did not affect the -chain, indicating that it is an -fibrinogenase. FIN-WRL101 was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it belongs to the serine protease. FIN-WRL101 exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. Its nucleotide and amino acid sequences were determined.

Partial Purification and Biochemical Evaluation of Protease Fraction (MA-1) from Mycoleptodonoides aitchisonii and Its Fibrinolytic Effect

“…Fibrinogen subchain specificity tended to differ for each enzyme. For example, WRL101 derived from Bacillus strain has substrate specificity for Bβ-chain [ 39 ]. The AprE127 enzyme derived from the Bacillus strain had substrate specificity similar to that of MA-1.…”

Biotechnology, Bioengineering and Applications of Bacillus Nattokinase

“…Although nattokinase was first isolated from the Japanese food natto, similar fibrinolytic enzyme-producing strains are also available from other traditionally fermented foods: Bacillus subtilis Natto B-12 [ 17 ] and Bacillus subtilis JNFE0126 [ 18 ] were isolated from natto; Bacillus amyloliquefaciens DC-4 [ 19 ], Bacillus subtilis LD-8547 [ 20 ] and Bacillus sublitis DC33 [ 21 ] were obtained from Chinese traditional fermented food tempeh; Bacillus subtilis LSSE-62 [ 22 ] was obtained from Chinese soybean paste; and Bacillus sp. strain CK 11-4 [ 23 ] and Bacillus subtilis WRL101 [ 24 ] were isolated from Chungkook-Jang, a traditional Korean fermented food. In addition to fermented food sources, Bacillus cereus VITSDVM3 [ 25 ], isolated from rust, was also confirmed as a potent nattokinase producer ( Table 1 ).…”

Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy

“…Fermented soybeans have been consumed as traditional foods in many Asian countries, and indeed, they are sources of nattokinase and other fibrinolytic enzymes. These includes natto, a Japanese traditional fermented soybean obtained from fermentation with Bacillus subtilis G8 and Bacillus subtilis (Chang et al, 2012;Lucy et al, 2019), moromi, oncom and gambus (Indonesian traditional fermented soybean obtained from fermentation with Bacillus cereus, Bacillus subtilus, Bacillus pumilus, and Stenotrophomonas sp, respectively) (Afifah et al, 2014;Nailufar et al, 2016;Stephani et al, 2017;Syahbanu et al, 2020) as well as douchi and doufuru (Chinese traditional fermented soybean obtained from fermentation with Bacillus (Ok and Choi, 2005;Wang et al, 2006;Jo et al, 2011;Wei et al, 2011;Yuan et al, 2012;Chen et al, 2013;Heo et al, 2013;Park et al, 2013;Gad et al, 2014;Thokchom and Joshi, 2014;Bi et al, 2015;Huy et al, 2016;Liu et al, 2016;Nailufar et al, 2016;Hu et al, 2019;Ito, 2020;Syahbanu et al, 2020 (Shirasaka et al, 2012;Biji et al, 2016;Vijayaraghavan et al, 2016a;Vijayaraghavan et al, 2016b;Vijayaraghavan et al, 2019), seafood fermented with Bacillus sp, Bacillus. velezensis BS2, Bacillus pumilus BS15 and Bacillus coagulans (Anh et al, 2015;Prihanto and Firdaus, 2019;Yao et al, 2019;Kim et al, 2020) and liquors obtained from fermentation with Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus-Genus (Lapsongphon et al, 2013;Johnson e...…”