An Improved Spectrophotometric Method for Toluene-4-Monooxygenase Activity.

ABSTRACT: Monooxygenases, an important class of enzymes, have been the subject of enzyme engineering due to their high activity and versatile substrate scope. Reactions performed by these biocatalysts have long been monitored by a colorimetric method involving the coupling of a dye precursor to naphthalene hydroxylation products generated by the enzyme. Despite the popularity of this method, we found the dye product to be unstable, preventing quantitative readout. By incorporating an extraction step to solubilize the dye produced, we have improved this assay to the point where quantitation of enzyme activity is possible. Further, by incorporating spectral deconvolution, we have, for the first time, enabled independent quantification of the two possible regioisomeric products: 1-naphthol and 2-naphth

SUBMITTER: Baskaran B 

PROVIDER: S-EPMC10423644 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature