저온저장 원유 시료로부터 분리한 저온성 Pseudomonas sp. K101 로 부터 protease의 생산조건을 검토하고 단백질 분해효소를 정제하였다. Nutrient broth를 기초배지로 하여 효소 생산조건을 검토한 결과, glucose의 함량이 증가할수록 효소 생산도 증가하였으나. 0.5%이상의 농도에서는 오히려 효소 생산이 저해되었다. 유기 질소원으로는 peptone을 0.5%첨가한 경우 가장 우수한 효소생산을 보였으며, 배양최적 온도와 pH는 각각 30℃ 및 7.5였다. 배양액 중에 함유된 Protease는 Sephadex G-100, DEAE-cellulose 및 DEAE-SephadexA-50 column chromatography를 이용하여 단계별로 정제하였다. 정제된 단백질을 SDS-PAGE로 확인한 결과, 단일 band로 나타나 순수한 효소 단백질로 생각되었다. 이 효소는 monomer로 분자량이 약 42,000이었으며, casein에 대한 활성화 에너지는 8.91 Kcal/mole로 계산되었다.

Cultural conditions for protease production and purification of protease of Pseudomonas sp. K101, isolated from raw milk samples collected at Jeonbuk area, were studied. The higher concentration of glucose in the media showed the more enzyme activity, while its activity was reduced over 0.5% glucose concentration in Nutrient broth as a basal medium. Peptone among some organic nitrogen sources tested was most effective for protease production and its optimal concentration was 0.5%, The optimal temperature and pH for cultivation were measured to be 30℃and 7.5, respectively. The protease were purified with ammonium sulfate and chromatography on DEAE-cellulose, Sephadex G -100 and DE-AE-Sephadex A-50 in that order. The purified enzyme was homogenous in SDS-polyacrylamide gel electrophoresis analysis and consisted of a monomer protein with a molecular weight of abour 42,000. Activation energy (Ea) for casein was calcul-ated as 8.91Kcal/mole.