Staphylococcus epidermidis urease의 정제 및 생화학적 특성에 관한 연구

Abstract

본 연구에서는 피부상재균이며 기회병원균이기도 한 Staphylococcus epidermidis ATCC12228로부터 urease효소를 4단계 크로마토그라피 방법을 사용하여 1,127배 정제하고 그 생화학적인 특성을 규명하였다. 정제된 urease 효소는 SDS-PACE 전기영동분석 및 gel-filtration 크로마토그라피를 이용한 천연분자량 분석결과, 67, 16.1 및 12.7 kDa의 3개 subunit가 3량체로 회합되어 존재하는 것으로 나타났으며 catalytic unit 당 2.2개의 니켈 원소를 함유하는 것으로 측정되었다. 정제된 효소의 비활성은 993.8 U/mg, <TEX>$K_m$</TEX>값은 8.5mM로 각각 산출되었다. Staphylococcus epidermidis is a coagulase-negative, gram-positive bacterium that normally inhabits the human skin. S. epidermidis is also known to be an opportunistic pathogen in infections of various indwelling medical devices. This report describes purification and characterization of the urease of S. epidermidis urease, which may act as a virulence factor. The urease from S. epidermidis was purified 1,127 fold by using DEAE-Sepharose, Phenyl-Sepharose, Mono-Q and Superdex HR200 column chromatography. The specific activity of the purified enzyme was 993.8 U/mg. Michaelis constant(<TEX>$K_m$</TEX>) of the enzyme was estimated to be 8.5 mM urea by using Lineweaver-Burke double reciprocal plot. The native molecular weight of the urease was shown to be 255 kD by using Superose 6HR gel filtration chromatography and the purified enzyme contained 2.2 nickel ions per catalytic unit. The overall stoichiometry of the enzyme subunits appears to be <TEX>$(\alpha\beta\gamma)_3$</TEX>, which is consistent with the enzymes from other bacteria sources.