Characterization of immobilized β-Glucuronidase in aqueous and mixed solvent systems

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Abstract

β-Glucuronidase (β-d-glucuronide glucuronosohydrolase, EC 3.2.1.31) was covalently attached to alkylamine-controlled pore glass via a glutaraldehyde immobilization scheme. The activity of this immobilized β-glucuronidase was studied with respect to several kinetic parameters in comparison with the behavior of the soluble enzyme. Km values for p-nitrophenyl glucuronide, estriol-3-glucuronide, and estriol-16β-glucuronide were determined. For each substrate the Km was essentially the same, 0.2 mM, and this value did not change when the enzyme was immobilized. The soluble and immobilized enzyme both displayed a relatively broad pH maximum centered at pH 6.8 for all substrates. Several organic-aqueous mixtures including methanol, ethanol, acetonitrile and ethylene glycol were tested, and their effects on the activity of immobilized β-glucuronidase were similar to those found for the soluble enzyme. Long-term (1 year) storage stability tests of the immobilized enzyme were carried out. The immobilized enzyme retained 40% of its initial activity after 1 year and was very robust towards most of the organic solvents tested.

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