Comparative Abilities and Optimal Conditions for .BETA.-Glycosidase Enzymes to Hydrolyse the Glucuronide, Glucoside, and N-Acetylglucosaminide Conjugates of Bile Acids.

Abstract

Enzymatic hydrolyses were described for three variants of glycosidic conjugated bile acids with one beta-glucuronidase (Helix pomatia), three beta-glucosidase (almonds, sweet almonds, and Escherichia coli), and four beta-N-acetylglucosaminidase (jack beans, bovine kidney, human placenta, and Diplococcus pneumoniae) preparations. The substrates include the beta-glucuronide, beta-glucoside, and beta-N-acetylglucosaminide conjugates of bile acids related to hyodeoxycholic, murideoxycholic, chenodeoxycholic, and ursodeoxycholic acids possessing a sugar moiety at position C-3, C-6 or C-7. The comparative abilities and optimal conditions for the beta-glycosidases to catalyze the hydrolyses of the substrates were clarified by changing pHs and incubation times. Hydrolysis rates of the bile acid glycosides with beta-glycosidase treatments were influenced by both the source of the enzyme preparations and the conjugated position of a sugar moiety in the substrates, and the 3-glucoside and 3-N-acetylglucosaminide conjugates were usually hydrolyzed more efficiently than their corresponding 6- and 7-analogs. Escherichia coli and jack bean enzymes were chosen to hydrolyse the glucosidic and N-acetylglucosaminidic conjugated bile acids, respectively.

Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high‐resolution mass spectrometer for the purpose of doping control

“…After optimization of the hydrolysis condition in terms of the volume of β ‐glucuronidase added and the incubation time required, it was found that the hydrolysis efficiency for the deconjugation of glucuronide and glucoside forms of IOX4 to the free form were approximately 98% and 90% for 1 h incubation, respectively. Such observation that glucoside conjugates could be hydrolyzed by β ‐glucuronidase was consistent with that reported by our research group earlier 39 . In addition, our study showed that the stabilities of IOX4 in urine and plasma were about 70–90% after 1‐h incubation with 50 μl of β ‐glucuronidase.…”

Analysis of human C24 bile acids metabolome in serum and urine based on enzyme digestion of conjugated bile acids and LC-MS determination of unconjugated bile acids

“…The inclusion of enzymes in sample preparation did not introduce interferences. Because a longer incubation time was necessary for GLU1 to hydrolyse glucuronides linked at C-3 or C-7 [33], we used excess amounts of enzymes, SUL1 50 U, GLU1 500 U and/or CH 100 U/50 μL sample, and a longer incubation time, 37 °C for 6 h, in the final protocols. As illustrated in ESM Fig.…”

General Methods for the Extraction, Purification, and Measurement of Steroids by Chromatography and Mass Spectrometry

“…Bile acids are excreted in more complex forms than most other steroids, being not only conjugated to glucuronic and sulphuric acids but also joined, via a peptide bond at C24 to either glycine or taurine. Hydrolysis of bile acid glycosides is influenced by the source of the enzyme, position of sugar moiety, enzyme activity and incubation conditions (Momose et al, 1997a), but is usually attempted using cholylglycine hydrolase (e.g. Paauw et al, 1996;Gatti et al, 1997).…”