Abstract
The strain HK-12 was enriched and isolated from naturally fermented soybean for the production of fibrinolytic enzyme and the proteome of this enzyme induced during the incubation period was analyzed. The activity of fibrinolytic enzyme derived from supernatants of the HK-12 culture was performed by fibrin plate method for solid fibrinolytic activity. As the result, the fibrinolytic activity of HK-12 grown on the nutrient agar media was about 2.3 times greater than that of plasmin used as standard. The purified enzyme was prepared by a series of purification process including ammonium sulfate precipitation, DEAE-cellulose, Sephadex chromatography. The molecular weight of the enzyme was determined to approximately 23kDa with SDS-PAGE. In order to examine which strain HK-12 proteins increased or decreased during the incubation period, 2-DE analysis was performed. Protein spot #1 significantly expressed on the 2-DE gel of bacteria cultivated for 36-hrs was analysed. As the result of protein sequence analysis using MALDI-TOF MS, one protein was identified as serine protein kinase (PrkA).
자연발효된 청국장으로부터 혈전용해활성을 가지는 세균 Bacillus licheniformis HK-12를 농화분리하여, 배양기간 동안 생산된 혈전용해활성을 가지는 단백질에 대해 프로테옴 분석을 실시하였다. B. licheniformis HK-12를 액체 영양배지에 접종하여 얻어진 배양상등액을 피브린 평판법(fibrin plate method)을 사용하여 효소활성을 측정하였다. 그 결과, HK-12의 혈전용해 활성은 대조구인 plasmin보다 약 2.3배정도 높은 활성을 나타내었다. 효소는 배양상등액을 ammonium sulfate 침전, DEAE-cellulose chromatography, Sephadex chromatography 등을 수행하여 분리정제하였으며, 정제된 혈전용해효소의 분자량은 SDS-PAGE를 통해 약 23 kDa로 측정되었다. 배양시간에 따른 HK-6의 세포외 단백질의 변화를 2-D PAGE 분석을 통하여 분석하였다. 그 결과 36시간배양 후에 가장 현저하게 유도된 spot #1을 분리하였으며, MALDI-TOF MS를 이용하여 단백질 동정을 실시한 결과, 유도된 단백질의 아미노산 서열은 $^1EKKIEKYREEEORLK^{15}$으로서, serine protein kinase (PrkA) (AAU22526)로 확인되었다.
