Purification and some Properties of Keratinolytic Protease Produced by Pseudomonas sp. KP-364.

A keratinolytic protease was purified from the culture medium of Pseudomonas sp. KP-364 by use of an assay of the hydrolysis of feather keratin. Membrane ultrafiltration and DEAE-cellulose ion-exchange resin and Sephadex G-150 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinolytic protease relative to that in the original medium was approximately 72-fold high. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephadex G-150 chromatography indicated that the purified keratinolytic protease is monomeric and has a molecular weight of 36 kDa. The optimal pH and temperature of the keratinolytic protease activity were 6.6 and 37 C, respectively, and the keratinolytic protease was relatively stable at pH value from 3.0 to 10.0 at 37 C for 1hour. The keratinolytic protease was inhibited by EDTA and EGTA, indicating that the keratinolytic protease was a kind of metalloprotease that require Li+ for cofactor.

Pseudomonas sp. KP-364가 생산하는 Keratinolytic Pretense의 정제 및 성질

경기도 일대의 가금류 공장부근 토양로부터 keratinolytic protease 생산성이 우수한 균주 KP-364를 선별하여 본 효소를 정제하고 일반적인 특성을 조사하였다. 본 효소는 ulkafiltration, ammonium sulfate fiactionation, DEAE-cellulose ion-exchange chromatography, Sephadex G-150 gel filtration 등을 통하여 정제되었으며 회수율은 25.2%이었다 SDS-PAGE와 gel filtration으로. 단일성과 분자량을 추정한 결과 전기영동 상에 단일 band를 나타내었으며 분자량은 약 36,000 dalton이였으며 1개의 subunit로_구성되어 있었다. 효소반응의 최적조건을 검토한 결과 최적 pH는 6.5, pH 3.0에서 10.0까지 90%이상의 활성을 나타냈으며 반응 최적 온도는 $37^{\circ}C$이였고 $60^{\circ}C$에서 1시간동안 80%이상의 활성을 유지하였다. 정제된 효소의 활성은$ FeSo_4$, KCI, $Li_2$$SO_4$를 첨가하였을 때 증가하였으며 $Ag_2$$SO_4$, $CuCl_2$, $HgCl_2$에 의해 저해되었다. 또한 EDTA, EGTA에 의해 저해되는 것으로 보아 metalloprotease의 일종이라고 판단되며 $Li^{ +}$를 cofactor로 함유하고 있는 것으로 조사되었다.