Application of β-glucuronidase-immobilised silica gel formulation to microfluidic platform for biotransformation of β-glucuronides (original) (raw)
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Journal of chromatography. B, Biomedical sciences and applications, 1998
An immobilized enzyme reactor based upon beta-glucuronidase (BG-IMER) has been developed for the on-line deconjugation of substrates. The activity of the BG-IMER and its applicability to on-line deconjugation was investigated. The BG-IMER was coupled to a reversed-phase column (C8 or C18) and the latter column was used to separate substrates and products eluted from the beta-glucuronidase reactor. The activity of the BG-IMER was followed by measurement of percent deconjugation and the parameters investigated were: substrate concentration, pH (4 to 6), temperature (r.t., 37 degrees C), enzyme-substrate contact time using flow-rates of 0.1 to 1.0 min/min (15-1.5 min). The glucuronides used in the evaluation of the BG-IMER were: 4-methylumbelliferyl-beta-D-glucuronide, p-acetaminophen-beta-D-glucuronide, 3'-azido-3'-deoxythymidine-beta-D-glucuronide, phenyl-beta-D-glucuronide, chloramphenicol-beta-D-glucuronide, estradiol-17-beta-D-glucuronide and morphine-beta-D-glucuronide. T...
2019
International audiencea β-glucuronidases (GUS) are exoglycosidases of considerable therapeutic interest cleaving β-D-glucuronides and fund in the lysosomes, in the tumoral microenvironnement and expressed by the gut microbiota. We hypothesis that the GUS tetravalent nature may confer a specific susceptibility to clusterized substrates and developed mono-, di, and octavalent glucuronide substrates releasing fluorescent amino-coumarines after GUS hydrolysis. GUS showed a much lower catalytic efficiency in hydrolyzing the clustered glucuronides due to a significantly lower enzymatic velocity and affinity for the substrates. GUS was 52-fold less efficient in hydrolyzing GlcA substrates presented on an octameric silsequioxane (COSS) compared to a monovalent GlcA of similar chemical structure. Thus, kinetic and thermodynamic data of GUS hydrolysis towards multivalent glucuronides were easily obtained with these new types of enzymatically-triggered probes with self-immolative linkers. More...
2018
β-glucuronidase enzyme belongs to the hydrolase family which perform breakdown of complex carbohydrates. β- glucuronidases are large biomolecule consists of polypeptide chains and are widely included eukaryotes and prokaryotes, however their parasitic sources are extremely constrained. β-glucuronidase from fungal source could be profoundly valuable as a result of its high selectivity and stability over extensive variety of pH and temperature which makes it exceptionally reasonable for mechanical applications. Number of studies have been conducted to test the enzyme stability by immobilization which can easily be detached from biomaterials and nano particles for repeated use in the chemical reaction. β-glucuronidase is also comprehensively use in industry for protein catalyzed reactions. For this purpose enzyme was isolated and cloned from many sources such as Penicillium purpurogenum Li-3 ZnO-NP, sodium alginate, sepabeads etc. These studies revealed that β-glucuronidase shows maxim...
Pharmaceutics
Glucuronides hydrolysis by intestinal microbial β-Glucuronidases (GUS) is an important procedure for many endogenous and exogenous compounds. The purpose of this study is to determine the impact of experimental conditions on glucuronide hydrolysis by intestinal microbial GUS. Standard probe 4-Nitrophenyl β-D-glucopyranoside (pNPG) and a natural glucuronide wogonoside were used as the model compounds. Feces collection time, buffer conditions, interindividual, and species variations were evaluated by incubating the substrates with enzymes. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. Fresh feces showed the highest hydrolysis activities. Sonication increased total protein yield during enzyme preparation. The pH of the reaction system increased the activity in 0.69–1.32-fold, 2.9–12.9-fold, and 0.28–1.56-fold for mouse, rat, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hy...
Stabilization of b-Glucuronidase by Immobilization in Magnetic-Silica Hybrid Supports
Catalysts
β-Glucuronidases are a class of enzymes that catalyze the breakdown of complex carbohydrates. They have well documented biocatalytic applications in synthesis, therapeutics, and analytics that could benefit from enzyme immobilization and stabilization. In this work, we have explored a number of immobilization strategies for Patella vulgata β-Glucuronidase that comprised a tailored combination of biomimetic silica (Si) and magnetic nanoparticles (MNPs). The individual effect of each material on the enzyme upon immobilization was first tested. Three different immobilization strategies for covalent attachment on MNPs and different three catalysts for the deposition of Si particles were tested. We produced nine different immobilized preparations and only two of them presented negligible activity. All the preparations were in the micro-sized range (from 1299 ± 52 nm to 2101 ± 67 nm of hydrodynamic diameter). Their values for polydispersity index varied around 0.3, indicating homogeneous ...
Catalysts
The enzymatic conversion of biomass-derived compounds represents a key step in the biorefinery flowsheet, allowing low-temperature high-efficiency reactions. β-Glucosidases are able to hydrolyze cellobiose into glucose. Wrinkled silica nanoparticles (WSNs) were demonstrated to be a good support for the immobilization of β-glucosidases, showing better performance than free enzymes in batch reaction; on the other hand, immobilized enzyme microreactors (IEMs) are receiving significant attention, because small quantities of reagents can be used, and favorable heat and mass transfer can be achieved with respect to conventional batch systems. In this work, we prepared, characterized, and tested structured enzymatic reactor compounds by a honeycomb monolith, a WSN washcoat, and β-glucosidases as the active phase. Powder and structured materials were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), N2 physisorption, thermogravimetric analysis (TGA...