Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang

Abstract

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70؇C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gln-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-NA). A similar result was also obtained from fibrin plate analysis.

Characteristics of fermentation and bioavailability of isoflavones in Korean soybean paste (doenjang) with application of Bacillus sp. KH‐15

“…If a fibrinolytic enzyme is produced by food-grade micro-organisms in a fermented food, the food can be consumed daily to prevent thrombosis and other related diseases. In this respect, recent studies were carried out by researchers (Sumi et al, 1987;Nakajima et al, 1993;Kim et al, 1996). We also isolated Bacillus sp.…”

“…Caseinolytic activity was determined by the modified fibrin plate method (Kim et al, 1996). Caseinolytic activity was assayed by the following procedure.…”

Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4

“…Fibrinolytic activity was determined by using both the modified plasminogen-free fibrin plate and the plasminogen-rich fibrin plate methods [2,18]. Fibrinogen [5 ml of 0.6% (wt/vol); Sigma] solution in a 50 mM sodium phosphate buffer (pH 7.4) was mixed with the same volume of 2% (wt/vol) agarose solution and 0.1 ml of thrombin solution (10 NIH unit/ml; Sigma) in a Petri dish.…”

“…Fibrinolytic enzymes dissolve the blood clots formed from fibrinogen by thrombin. When clots are not lysed, they accumulate in blood vessels, and cause thrombosis leading to myocardial infraction and other cardiovascular diseases [1,2]. The major thrombolytic agents are classified into two types.…”

“…It was purified from Bacillus natto screened from natto, a traditional Japanese soybean fermented food [5,6]. Subsequently, many fibrinolytic enzymes were identified in different traditional fermented foods, such as CK from Chungkook-Jang [2], subtilisin DJ-4 from Doen-Jang [1] in Korea, and subtilisin DFE from douchi in China [7]. All of these enzymes are subtilisin group proteins and belong to the same bacterial strain, Bacillus sp.…”