식품으로서 안전하게 섭취하여 혈전증을 사전에 예방하거나 개선할 수 있도록 하기 위하여, 전통적 방법으로 제조한 청국장으로부터 미생물을 분리하여 혈전분해 효소활성이 우수한 미생물을 선발·동정한 결과, Bacillus amyloliquefaciens HC188이라 명명하였다. 선발미생물이 생산하는 혈전분해 효소를 분리 및 정제한 결과, 50.7배의 정제도와 5.5%의 수율을 나타내었고, 혈전분해 효소단백질의 분자량은 22.3 kDa 이었으며, N-terminal 아미노산 서열은 Ala-Gln-Ser-Val-Pro-Tyr-Gly-Val-Ser-Gln-Ile-Lys-Ala-Pro-Ala로 분석되었다. 효소의 최적반응 pH와 온도는 pH 8.0과 40oC로 나타났으며, pH 6.0-8.0과 20-40oC 사이에서 효소가 비교적 안정하였다. 금속이온에 대한 영향은 2 mM과 5 mM 농도의 CoCl2와 CaCl2의 금속이온이 존재할 때 효소활성이 증가하였으며, inhibitor로서 EDTA와 PMSF에 의해 효소활성이 저해되므로 청국장에서 분리한 B. amyloliquefaciens HC188가 생성한 혈전분해 효소는 metallo-serine protease로 사료되었다.

A bacterium producing a fibrinolytic enzyme was isolated from Cheonggukjang. The bacterium was identified as a strain of Bacillus amyloliquefaciens by 16S rDNA analysis and designated as B. amyloliquefaciens HC188. The optimum culture medium appeared to be one containing 0.5% (w/v) maltose and 0.5% (w/v) soytone. Bacterial growth in the optimal medium at 37oC reached the stationary phase after 27 hours of incubation and the fibrinolytic enzyme showed optimum activity at 24 hours. The enzyme was purified by 20-80% ammonium sulfate precipitation, CM Sepharose fast flow ion exchange chromatography, and Sephacryl S-200HR column chromatography. Its specific activity was 38359.3 units/mg protein and the yield was 5.5% of the total activity of the crude extracts. The molecular weight was 24.7 kDa and the amino acids of the N terminal sequence were AQSVPYGVSQIKAPA. The fibrinolytic enzyme activity had an optimum temperature of 40oC and an optimum pH of 8.0, and the enzyme was stable in the ranges 20-40oC and pH 6.0-8.0. Enzyme activity was increased by Ca2+ and Co2+ but inhibited by Cu2+, EDTA, and PMSF. It is suggested that the purified enzyme is a metallo-serine protease.