Large scale production of vanillin using an eugenol oxidase from Nocardioides sp. YR527

“Bacterial eugenol oxidases (EUGOs) entered recently the scientific spotlight as these versatile biocatalysts were reported to transform lignin degradation products into valuable platform chemicals. Here we describe the second member of the enzyme family, EUGO from Nocardioides sp. YR527 (NspEUGO) and investigated its biocatalytic potential in direct comparison with EUGO from Rhodococcus jostii RHA1. NspEUGO was found to be most active on vanillyl alcohol derivatives (up to 5.6 ± 0.3 s-1) while for eugenol the highest affinity was observed (K_M: 33.1 ± 4.1 μM). For the catalyzed reactions, a high ambiguity was observed in dependency of the pH: The highest k_cat of 16.0 ± 0.3 s-1 was found at pH 9.5 while the best long-term performance was detected at pH 6.0 (~300,000 TTN). To stabilize the enzyme, immobilization on a total of 14 carrier materials was conducted and the performance was investigated in two reactor types for large scale application. This resulted in the successful production of vanillin at 1 g.L-1.h-1 in a packed bed reactor.”

Highlights:

• The authors opted for covalent immobilization of NspEUGO on ECR8415F beads and deployed the heterogeneous biocatalyst by use of a SpinChem rotating bed reactor (RBR) into a solution of 105 mM vanillyl alcohol in a glycine-NaOH solution at pH 9.5 that was aerated with 0.2 L/min.
• After 50 h reaction time, a total of 2.1 g substrate/product mixture was isolated (55% extraction efficiency) which contained 43% of the intended product vanillin.
• For the use of immobilized NspEUGO in the RBR, a total turnover number of 182,800 ± 6,500 per active site was estimated. This represents a 1.5-fold increase compared to the free enzyme in the same buffer conditions.
• Although good overall biocatalyst stability, reusability, and ease of deployment in the RBR, the authors identified product inhibition as severe limitation to the biocatalytic system and thus had to switch to a continuous processing strategy in a packed bed flow reactor.