Vanillin, the compound responsible for the well-known vanilla aroma, is almost exclusively produced via a chemical process, with only a small fraction extracted from natural sources, namely, the bean of the orchid Vanilla planifolia. Research is being done towards a green chemistry process to obtain natural vanillin. A model biotechnological process is described that exposes students to the essentials of a greener, chemoenzymatic synthesis of vanillin in a multiday laboratory experiment. Bacterial expression was utilized to produce the enzyme eugenol oxidase (EUGO), which was purified from cell extracts using inexpensive gravity-flow ion-exchange chromatography, and was highly visual due to the bright yellow color of the enzyme caused by its covalently-bound flavin cofactor. Students evaluated its purity with SDS?PAGE and a purification table and measured its kinetic parameters with UV?vis spectrophotometry. Vanillin was produced by EUGO through the enzymatic oxidation of vanillyl alcohol. Because of its highly visual and olfactory nature, this is an ideal experiment to expose undergraduate students to the basic principles of a biotechnological process, enzyme production and purification and elementary enzyme kinetics.
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