Potent Fibrinolytic Enzyme from a Mutant of Bacillus subtilis IMR-NK1

Abstract

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

“…1, the molecular weight of subtilisin FS33 was estimated to be 30 kDa by SDS-PAGE. This is similar as reported by Takahashi [32] for SMCE (30.0 kDa) of B. pumilus TYO-67, but lower than published for subtilisin E (55.8 kDa), subtilisin IMR-NK1 (31.5 kDa) [33], KK (38 kDa) [34], and KA38 (41 kDa) [25] and higher than for subtilisin NAT (27.7 kDa, formerly designated nattokinase, NK), CK (28.2 kDa), and subtilisin DFE (28.0 kDa). In spite of the strong homology of N-terminal sequences of subtilisin FS33, NK, and subtilisin E, they exhibit different molecular weights and amidolytic activities.…”

“…As shown in Table 2, subtilisin FS33 had the highest affinity for N-Succ-Ala-Ala-Pro-Phe-pNA; the studies achieved almost the same level of NK and subtilisin IMR-NK1 [33], their amidolytic activities for the substrate were 14.2 and 16.8 lmol min À1 l À1 , respectively. However, subtilisin FS33, NK, and CK react very differently with D-Val-Leu-Lys-pNA, with amidolytic activities of 0.84, 285.6, and 424 lmol min À1 l À1 , respectively.…”

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

“…Many bacterial Wbrinolytic enzymes were discovered from fermented foods, such as natto [3,[5][6][7], shiokara [8] in Japanese food, Chungkook-Jang [1], Doen-Jang [2], and Jeot-Gal [9] in Korean food, douchi [10] in Chinese food, and Tempeh [11] in Indonesian food. Of them Shiokara (Japan) and Jeot-Gal (Korea) are Wsh-fermented foods, which are used as important additive for improving the taste of other foods as well as being foods themselves [12].…”

Enhanced production of nattokinase from UV mutated Bacillus sp.

“…So far, many researchers have focused their efforts on the isolating and screening of microorganisms for enzyme production with high fibrinolytic activity (Chang et al, 2000), as well as on the purifying and characterizing of newly found enzyme (Kim et al, 1996). Evidences suggest that B. subtilis secrete nattokinase.…”

“…NK is primarily found from a traditional fermented soybean food named as "Natto" in Japan (Sumi, et al, 1987). It is a subtilisin-like serine protease with molecular weight of 27,000-42,000 Da (Chang, et al, 2000;Fujita, et al, 1993;Kim et al, 1996;Peng, et al, 2003). Besides in vitro tests of fibrinolytic activity, many in vivo studies had been reported.…”