3-Glucuronidase purification

Since then it has been established that emulsin is a mixture of enzymes comprising in addition to /3-glucosidase28 the enzymes a-galactosidase, a-mannosidase, /3-glucuronidase and /3-(N-acetyl)-glucosaminidase. Concerning the process of purification of the Rohferment and the mode of differentiation of the constituent enzymes of emulsin, the most informa-... 

J. W. Owens, P. Stahl, Purification and Characterization of Rat Liver Microsomal /3-Glucuronidase , Biochim. Biophys. Acta 1976, 438, 474 - 486. 

Evangelista, R.L., Kusnadi, A.R., Howard, J.A., andNikolov, Z.L. (1998). Process and economic evaluation of the extraction and purification of recombinant beta-glucuronidase from transgeiuc com. Biotechnol. Progress 14 807-614. 

Using immobilized -glucuronidase reactors, estriol and estradiol glucuronides have been determined in urine by a column-switching technique (270, 271). Both glucuronides were hydrolyzed by the immobilized enzyme at pH 7. The steroid mixture was subsequently separated by gradient elution on a reversed-phase column, to be finally detected by UV absorbance at 280 nm. In this procedure, the activity of enzyme did not alter even after 150 h continuous run and exposure to a mobile phase containing 10% methanol. When a separate reversed-phase precolumn was inserted in the LC system, additional sample purification and shorter analysis time could be attained (272). 

The only non-mammalian /8-glucuronidase that has been subjected to systematic purification is the enzyme from sheep-rumen microorganisms. After repeated ammonium sulfate fractionation, Marsh101 obtained a colorless preparation with a specific activity of 1,900 (400-fold purification and 2% recovery). The final product gave a linear, specific-property, solubility test from which a figure of 2,200 was derived for the ultimate specific activity of the enzyme, but it was considered that the enzyme may have formed a solid solution with inactive protein. 

Khandke, K. M., Vithayathil, P. J., andMurthy, S. K., Purification and characterization of an alpha-D-glucuronidase from a thermophilic fungus, Thermoascus aurantiacus. Arch Biochem Biophys 1989, 274 (2), 511-7. 

Another product of esterase cleavage is p-nitrophenol (PNP), which is generated in the human body by the degradation of parathion, parathion-methyl and parathion-ethyl. Some methods for the determination of PNP in urine are summarized in Table 9.5. PNP can, for example, be determined in the urine of occupationally exposed subjects by means of GC-ECD. Sample preparation involves acid hydrolysis, extraction with diethyl ether, derivatization with diazoethane and purification on silica gel columns. The LOD was 20 pg L with a recovery of 85-98%. This method has also been used in a study on selected pesticide residues and metabolites in urine from a survey of the US general population.In the context of another survey study, a more complicated technique to detect PNP in urine has recently been used by Hill et involving GC-MS-MS with positive chemical ionization after derivatization with l-chloro-3-iodopropane. The authors also used this method for the determination of the metabolite TCP and 10 other analytes in urine.Sample preparation involved hydrolysis with p-glucuronidase, several extraction steps using different solvents and purification by SPE (silica gel column). The LOD was 1 pg L with an inter-assay RSD... 

Purification of rat-liver p-D-glucuronidase Sulphide-disulphide interchange chromatography in the separation of plasma proteins... 

The levels of /3-D-glucuronidase activity in skin fibroblasts from cases of cystic fibrosis and controls have been compared. The purification (470 fold, 46 % yield, 6 stages) of the saline extractable jS-D-glucuronidase from human leucocytes and some of its catalytic and molecular properties have been reported. Comparisons of these properties were made with those of other human and non-human jS-o-glucuronidases. The subcellular distribution of glycoside hydrolases including jS-o-glucuronidase in human polymorphonuclear leucocytes has been determined. ... 

Matrix for the immobilization of laccase Purification of jS-D-galactosidase and jS-D-glucuronidase isoenzymes from normal human liver by affinity chromatography... 

DEAE-cellulose column chromatography has contributed much to the advancement of purification and separation of multiple forms of glucuronidase (Moore and Lee, 1960). 

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