|An automated modular microsystem for enzymatic digestion with gut-on-a-chip applications
Haan, P. de; Ianovska, M.A. ; Mathwig, K. ; Bouwmeester, H. ; Verpoorte, E. - \ 2020
In: 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017. - Chemical and Biological Microsystems Society (21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 ) - ISBN 9780692941836 - p. 1593 - 1594.
Digestion - Enzyme kinetics - Gut-on-a-chip - Organ-on-a-chip
Gut-on-a-chip models have gained attention as replacements for other cell-based assays or animal studies in drug development or toxicological studies. These models aim to provide a more accurate representation of the in vivo situation in form and function; however, no digestive processes have been included in these systems so far. This work describes a miniaturized digestive system based on artificial digestive juices that digest liquid samples in a series of three microreactors. After optimization of the pH value of juices and mixtures, samples leading to fluorescent products were digested to demonstrate enzyme functionality and to determine kinetic parameters.
A xylenol orange-based screening assay for the substrate specificity of flavin-dependent para-phenol oxidases
Ewing, Tom A. ; Noord, Aster Van; Paul, Caroline E. ; Berkel, Willem J.H. Van - \ 2018
Molecules 23 (2018)1. - ISSN 1420-3049
Enzyme kinetics - Flavoprotein - Oxidase - Screening assay - Substrate specificity
Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.