An Improved Spectrophotometric Method for Toluene‐4‐Monooxygenase Activity
Name
Chemistry A European J - 2023 - Baskaran - An Improved Spectrophotometric Method for Toluene‐4‐Monooxygenase Activity.pdf
Description
Published version
Size
955.33 KB
Format
Adobe PDF
Checksum (MD5)
41495a33b16db664deda9c0ebb0be73d
Author(s)
Baskaran, Barathkumar
Gill, Thomas M
Furst, Ariel L
Date Issued
April 3, 2023
Journal
Chemistry – A European Journal
Publisher
Wiley
Citation
Baskaran, Barathkumar, Gill, Thomas M and Furst, Ariel L. 2023. "An Improved Spectrophotometric Method for Toluene‐4‐Monooxygenase Activity." Chemistry – A European Journal, 29 (19).
Version
Final published version
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-naphthol. Previously, such analysis was only possible with chromatographic separation, increasing the cost and complexity of analysis. The efficacy of our improved workflow was evaluated by monitoring the activity of a toluene-4-monooxygenase enzyme from Pseudomonas mendocina KR-1. Our colorimetric regioisomer quantification was found to be consistent with chromatographic analysis by HPLC. The development and validation of a quantitative colorimetric assay for monooxygenase activity that enables regioisomeric distinction and quantification represents a significant advance in analytical methods to monitor enzyme activity. By maintaining facile, low-cost, high-throughput readout while incorporating quantification, this assay represents an important alternative to more expensive chromatographic quantification techniques.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. Center for Environmental Health Sciences
Terms of Use
Creative Commons Attribution-Noncommercial
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1002/chem.202203322
