Discovery of a thermostable eugenol oxidase for vanillin biosynthesis from vanillyl alcohol via resting whole-cell catalysis.

Bacterial eugenol oxidases (EUGOs) belong to the 4-phenol oxidase subfamily of the VAO/PCMH flavoprotein family and are versatile biocatalysts offering potential for use in biotechnology and organic synthesis. In this work, a thermostable EUGO from Capillimicrobium sp. (CsEUGO) was identified and overexpressed in E. coli cells as a whole-cell biocatalyst for the biosynthesis of vanillin from vanillyl alcohol. The E. coli-CsEUGO cells oxidized vanillyl alcohol aerobically, producing only vanillin, indicating excellent reaction selectivity. Furthermore, the cells retained 63% and 57% of their activity after 6 and 12 h at 40 °C, and 40% of their activity at 50 °C. Under optimal reaction conditions, the cells converted a 200 mM concentration of substrate to vanillin, with a yield of 93%. With a fed-batch feeding method, a total of 208 mM vanillin was produced, affording a productivity of 1.0 g L-1 h-1 and a yield of 83%. Furthermore, it was found that the E. coli-CsEUGO whole-cell biocatalyst is active on several phenolic substrates, indicating a broad catalytic substrate scope. The research highlights a new biocatalyst that can selectively oxidize vanillyl alcohol, leading to the production of value-added vanillin. Additionally, it is applicable for catalytic hydroxylation, oxidative deamination, and dehydrogenation in organic synthesis.