Vanillin is an important compound in the food and cosmetic industries and as an intermediate in pharmaceutical and functionalized polymer synthesis. 12000 tons of vanillin are produced each year for these uses. There are no high-throughput methods for measuring vanillin that are moisture-tolerant even with the large amounts of vanillin used industrially, which makes vanillin quantification slower than it could be. Additionally, the two current industrial methods for producing vanillin use toxic reagents or generate hazardous byproducts. Biocatalytic methods for producing vanillin are attractive as more sustainable alternatives to reduce toxic materials in vanillin synthesis. Herbaspirillum seropedicae carotenoid cleavage oxygenase (HsCCO) is a mononuclear non-heme iron enzyme that converts isoeugenol and 4-vinylguaiacol with O2 into vanillin. These two gaps in vanillin production guide the objectives of this project: 1) developing a moisture-tolerant UV-Vis based derivatization assay for vanillin using acetophenone to allow for high-throughput vanillin concentration determination and 2) characterizing the kinetics of vanillin production by HsCCO to work towards greener vanillin synthesis. The moisture-tolerant vanillin assay uses acetophenone to perform a crossed aldol reaction on vanillin, which creates a derivatization product that has an absorbance peak at 465 nm that can be quantified. Vanillin assay product structure is verified by NMR and HPLC-MS. HsCCO steady-state reaction kinetics with isoeugenol and 4-vinylguaiacol are probed using the acetophenone derivatization assay for vanillin. HsCCO transient-state reaction kinetics are probed using stopped-flow absorbance spectroscopy. Development of a high-throughput method for measuring vanillin and studying a biocatalytic method for vanillin synthesis will hopefully allow vanillin production to be more efficient and sustainable.

High-throughput analysis of vanillin concentration through derivatization with acetophenone followed by ultraviolet-visible light spectroscopy and its use in studying biocatalytic methods for vanillin synthesis

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ACS Fall 2022

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