Purification and Characterization of Aryl Acylamidase from Pseudomonas sp.

Pseudomonas sp. Aryl Acylamidase의 정제 및 성질

  • 황인균 (한국식품의약품안전청) ;
  • 방원기 (고려대학교 자연자원대학 응용생명환경화학과)
  • Published : 1998.10.01

Abstract

Aryl acylamidase [EC 3.5.1.13] present in an acetaminophen-assimilating Pseudomonas sp. has been purified to a homogeneity using series of ammonium sulfate fractionation, DEAE-Sephacel anion exchange, Phenyl-Sepharose CL-4B hydrophobic, and Sephadex G-100 gel-permeation chromatography. The molecular weight, which was estimated by gel-permeation filtration and sodium dodecyl sulfate polyacylamide gel electrophoresis, was about 57 kDa and 56 kDa, respectively, indicating that this enzyme is a monomeric protein. The optimum pH was 10.5 and the optimum temperature was 40$^{\circ}C$. After incubation of the enzyme at 50$^{\circ}C$ for 30 min, residual activity of the enzyme was 34% compared to its original activity. The Km values for acetaminophen and 4'-nitroactanilide were 0.10 mM and 0.11 mM, respectively.

Acetaminophen 자화성 Pseudomonas sp.에 존재하는 aryl acylamidase[EC 3.5.1.13]는 ammonium sulfate fractionation, DEAE-Sephacel anion exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic interaction chroamtography 및 Sephadex G-100 gel-permeation chromatography를 통해 순수 정제되었다. 정제된 효소는 SDS-PAGE상에서 분자량을 측정한 결과 56 kDa, Sephadex G-100 gel-permeation chromatography로 측정한 결과 57 kDa이었으며, 따라서 단일한 subunit로 구성된 효소이었다. 정제된 효소의 최대 활성을 위한 pH와 온도는 각각 pH 10.5와 4$0^{\circ}C$이였다. 5$0^{\circ}C$에서 30분간 처리시 34%의 잔존활성을 나타내었다. Acetaminophen과 4'-nitroacetanilide쉐 대한 Km값은 각각 0.10 mM과 0.11 mM 이었다.

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