Bacillus amyloliquefaciens HC188이 생산하는 혈전분해 효소의 정제 및 특성

Purification and Characterization of a Fibrinolytic Enzyme Produced by Bacillus amyloliquefaciens HC188

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 혈전분해효소를 분리 및 정제한 결과, 50.7배의 정제도와 5.5%의 수율을 나타내었고, 혈전분해 효소단백질의 분자량은 22.3 kDa이었으며, 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 $40^{\circ}C$로 나타났으며, pH 6.0-8.0과 $20-40^{\circ}C$ 사이에서 효소가 비교적 안정하였다. 금속이온에 대한 영향은 2 mM과 5 mM 농도의 $CoCl_2$와 $CaCl_2$의 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 $37^{\circ}C$ reached the stationary phase after 27 h of incubation and the fibrinolytic enzyme showed optimum activity at 24 h. 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 $40^{\circ}C$ and an optimum pH of 8.0, and the enzyme was stable in the ranges $20-40^{\circ}C$ and pH 6.0-8.0. Enzyme activity was increased by $Ca^{2+}$ and $Co^{2+}$ but inhibited by $Cu^{2+}$, EDTA, and PMSF. It is suggested that the purified enzyme is a metallo-serine protease.