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Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases by Travis R. Besanger; Richard J. Hodgson; James R.A. Green; John D. Brennan is a Chemistry article available to read on EtoBox.

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Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low muL/min range. Using the enzyme gamma-glutamyl transpeptidase (gamma-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns w

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Author

Travis R. Besanger; Richard J. Hodgson; James R.A. Green; John D. Brennan

Publisher

Elsevier Science; Elsevier ; Elsevier BV (ISSN 0003-2670)

Published

2006

Language

EN

Field

Chemistry (Physical Sciences)