돼지 신장으로부터 디펩티다제의 부분정제 및 그에 대한 신규 카바페넴계 항생물질 DWP20418의 안정성 평가

The Stability Test of New Carbapenem DWP20418 and Partial Purification and Characterization of Renal Dehydropeptidase-I

Dehydropeptidase-I (DHP-I) was solubilized from porcine kidney by treatment with n-butanol and partially purified 19.25 fold by (NH4)2SO4 precipitation, DEAE-Sepharose CL-6B ion exchange chromatography and Sephacryl S-300 HR chromatography with an overall yield of 19.16. DHP-I showed its optimal activity at pH 7.5 and 25oC. Its activity was stable under neutral and alkaline conditions, but was disappeared under acidic condition. And DHP-I was heat-labile and its activity remained at 45oC for 3hrs. The enzyme was not inhibited by dicationic ions, while its activity was increased by Co2+(1mM) and Zn2+ (0.1mM). The enzyme was inhibited by EDTA and N-ethylmaleimide. The relative molecular mass of DHP-I was estimated to be approximately 100kDa by gel filtration chromatography. The KAPPAm value of DHP-I for glycyldehydrophenylalanine (GDHP) was 1.98mM. DWP20418 [(1R, 5S, 6S)-6-[1-(R)-Hydroxyethyl]-1-methyl-2-[(2S, 4S)-2-(piperazinylcarbonyl)-1-(R)-hydroxyethyl)pyrrolidine-4-thio]carbapen-2-em-3-carboxylic acid], compared with meropenem (MEPM), was rather easily hydrolized by DHP-I, while it was four times more resistant than imipenem (IPM) to DHP-I.