한국의 전통 대두발효식품인 청국장에서 혈전용해능이 우수한 미생물을 분리하였으며, 이를 동정한 결과 B.amyloliquefaciens D4로 명명하였다. 혈전용해효소의 대량분비를 위해 N-methyl-N-nitro-N-nitrosoguanidine을 사용한 돌연변이를 유도하여 B. amyloliquefaciens D4-7 변이주를 얻었으며, plasmin 1 U/ml 6.5배 정도의 높은 혈전분해능을 나타내었다. B. amyloliquefaciens D4-7는 분리대두단백배지에서 혈전용해효소 분비능이 가장 우수하였다. B. amyloliquelaciens D4-7가 생산하는 혈전용해효소의 최적 활성조건은 pH 10, 5$0^{circ}C$이었고, pH 7.0 에서 pH 11 사이에서 상대적으로 안정하였으며, $50^{circ}C$에서 30분간 방치하였을 경우 50%의 활성이 유지되었다. 또한, NaCl, glycerol 또는glucose 첨가에 의해 혈전용해효소의 저장안정성이 높아지는 것을 확인할 수 있었다.
Bacillus amyloliquefaciens D4, which produces a strongly fibrinolytic enzyme, was isolated from Chungkook-Jang, a traditional Korean soybean-fermented food. B. amyloliquefaciens D4 was mutated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) to yield a series of mutants with increasing levels of fibrinolytic enzyme production. After mutation, a mutant D4-7 was obtained with fibrinolytic activity about eight times stronger than the parent strain. The fibrinolytic activity of B. amyloliquefaciens D4-7, reached a maximum, when the producer was cultivated in 2% Isolated Soy Protein (ISP) broth for 48 h at $37^{circ}C$. Compared to commercial fibrinolytic enzymes, the cell-free culture supernatant of B. amyloliquefaciens D4-7 showed stronger activity than plasmin and streptokinase. The optimum temperature and pH were $50^{circ}C$ and 10.0 and thermostability was increased by the addition of glycerol, glucose, and NaCl.
