Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Chemistry. 2023 Apr 3;29(19):e202203322. doi: 10.1002/chem.202203322. Epub 2023 Feb 27.
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.
单加氧酶是一类重要的酶，由于其高活性和广泛的底物范围，一直是酶工程的研究对象。这些生物催化剂所进行的反应长期以来一直通过比色法进行监测，该方法涉及将染料前体与酶产生的萘羟化产物偶联。尽管这种方法很流行，但我们发现染料产物不稳定，无法进行定量读数。通过加入萃取步骤以溶解生成的染料，我们已经改进了该测定法，使得酶活性的定量成为可能。此外，通过光谱解卷积，我们首次能够独立定量两种可能的区域异构体产物：1-萘酚和 2-萘酚。以前，这种分析只能通过色谱分离进行，增加了分析的成本和复杂性。通过监测来自门多萨假单胞菌 KR-1 的甲苯-4-单加氧酶的活性来评估我们改进的工作流程的效果。我们的比色区域异构体定量与 HPLC 的色谱分析一致。开发和验证用于监测酶活性的单加氧酶活性的定量比色测定法，能够区分和定量区域异构体，这代表了分析方法在监测酶活性方面的重大进展。通过保持简便、低成本、高通量的读数，同时进行定量，该测定法代表了对更昂贵的色谱定量技术的重要替代方法。
