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High-Throughput Screen for Aromatic Hydroxylation

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The oxidation of aromatic compounds is important in producing chemical intermediates for the chemical and pharmaceutical industries ( 1 , 2 ). Conventional aromatic oxidation reactions are prone to byproduct formation and often require heavy-metal catalysts, extremes of temperature and pressure, and explosive reagents ( 3 ). In contrast, biocatalysts such as mono- and dioxygenases perform the same chemistry in water at ambient conditions, usually with higher regioselectivity than the analogous chemical process. Because of these inherent advantages, there has been increasing interest in applying aromatic oxygenases to chemical processes and bioremediation ( 46 ). Discovery and engineering of these enzymes is critical since the available natural enzymes are often not immediately suitable for these applications. Here, three colorimetric assays for hydroxylated aromatic compounds are discussed that can be implemented in high-throughput and thus are useful for biocatalyst discovery and engineering by directed evolution. These include assays employing Gibbs’ reagent, 4-aminoantipyrine (4-AAP) and Fast Violet B (FVB) (Fig. 1 ). These methods should enable the optimization of oxygenases to industrially relevant substrates and realistic process conditions.
 
Fig. 1.  Chemistry of assay methods. ( A ) Coupling of Gibbs’ reagent to a phenolic compound ( 17 ). ( B ) Coupling of 4-aminoantipyrine to a phenolic compound ( 16 ). ( C ) Coupling of Fast Violet B to a phenolic compound ( 18 ). ( D ) Chemistry performed by dioxygenases to yield a cis-dihydrodiol. ( E ) Dehydrogenation of a cis -dihydrodiol to form a catechol. ( F ) Acidification of a cis -dihydrodiol to form phenols.

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