Study On Extraction And Purification Of Characteristic Enzyme β-Glucuronidase From E.Coli

This paper studied the activities of β -glucuronidase( β -G) in different culture condition, p-Nitrophenyl- β -glucuronide(PNPG) was used as substrate. Best condition for E.coli to produce β -G was detected and β -G was extracted and purified.It was found that β - G activity of not-freeze-thawing was better than freeze-thawing method, submerged culture was better than slant method, shaking in liquid culture 6 hours was better than 8 days, culture two was better than culture one and culture three. Best condition for E.coli to produce β -G was: β -G in culture two after shaking for 6 hours. Culture two was composed by Peptone 3%, Sodium Glycerophosphate 5%, pH 6.8.By a combination of ammonium sulphate fraction, gel filtration chromatography and Ca3(PO4)2 adsorption chromatography, β - G was purified about 15.3 times and achieved with an recovery rate of 5.1%.The purity and molecular weight of β - G was determined by SDS-PAGE combined with Coomassie brilliant blue and Fuchsin basic staining. Density of acrylamide gel was 12%. SDS-PAGE analysis yield two bands of 72KD and 19KD in extraction and one band of 72KD in purification, band of 72KD was subunit of β -G.