Aryl‐Alcohol‐Oxidase‐Mediated Synthesis of Piperonal and Other Valuable Aldehydes

Abstract

The use of fungal aryl‐alcohol oxidases in biocatalysis is still modest, despite their advantageous capability to produce valuable aldehydes via oxidation of the respective alcohols without the need for costly external cofactors. For biocatalytic application, enzyme stability in the presence of organic solvents used for substrate solubilization has to be investigated, and process limitations due to low oxygen supply and the accumulation of the by‐product H2O2 must be addressed. In this study, we showed that the aryl‐alcohol oxidase PeAAO2 from Pleurotus eryngii P34 remained active and stable in the presence of up to 30% (v/v) various organic solvents and up to 500 mM H2O2. The potential of this biocatalyst was explored based on conversion of piperonyl alcohol to the fragrance compound piperonal. After reaction optimization, product titers of up to 245 mM were achieved within 3 h. Addition of catalase was imperative to re‐introduce O2 as co‐substrate into the reaction, thereby diminishing oxygen limitation. On a preparative scale, space‐time‐yield of 9.5 g/l/h was achieved and 244.6 mg piperonal (85% yield) with >99% purity were isolated via simple crystallization from n‐hexane extract. Under optimized reaction conditions four other substrates, cumic alcohol, 2‐thiophenemethanol, trans,trans‐2,4‐heptadienol and trans‐2‐cis‐6‐nonadienol, at concentrations of up to 300 mM were converted to the corresponding aldehydes within 20 h. Our results demonstrate that PeAAO2 is a versatile and promising biocatalyst for the production of valuable aldehydes.

Enzymatic reactions towards aldehydes: An overview

“…62 In a follow-up study, conditions for a preparative scale reaction were optimized to reach a space-time yield of 9.5 g L −1 h −1 for piperonal (244.6 mg; 85% yield) and further applied for the oxidation of cumic alcohol, thiophene-2-yl-methanol, (E,E)-2,4-heptadienol and (E)-2-(Z)-6-nonadienol (Figure 1). Up to 300 mM of these substrates were converted to the corresponding aldehydes within 20 h. 63 A new thermotolerant aryl alcohol oxidase from Moesziomyces antarcticus (MaAAOx) was recently described as accepting a broad range of primary benzylic alcohols, aliphatic allylic alcohols and furan derivatives like HMF. Among oxidation products were odorous compounds such as benzaldehyde, cuminaldehyde, piperonal and perillaldehyde.…”

“…This aldehyde was enzymatically prepared from piperonylic acid with CAR as the key enzyme in an E. coli ‐based living cell catalyst with as space–time yield of 1.5 g L −1 h −1 222 . Starting from the respective alcohol, space–time yield of 9.5 g L −1 h −1 was achieved using AAOx 63 . In both reports, piperonal was isolated in high yield and >99% purity via simple crystallization from n ‐hexane extracts.…”

Bienzymatic Cascade Combining a Peroxygenase with an Oxidase for the Synthesis of Aromatic Aldehydes from Benzyl Alcohols

“…Alcohol oxidases catalyse the aerobic oxidation of benzyl alcohols (Scheme 1B) [9]. Particularly, aryl alcohol oxidases (AAOx), have gained considerable interest in recent years [10][11][12][13][14][15][16][17]. An apparent drawback of using AAOx is the stoichiometric formation of H 2 O 2 impairing the biocatalyts' robustness.…”

Structure-Based Redesign of a Methanol Oxidase into an “Aryl Alcohol Oxidase” for Enzymatic Synthesis of Aromatic Flavor Compounds

“…In the past few years, aryl alcohol oxidases (AAOx, EC 1.1.3.7) have gained considerable interest as catalysts for the selective and mild oxidation of benzyl alcohols to the corresponding aldehydes. , The recombinant expression of functional fungal enzymes in procariotic hosts such as Escherichia coli, however, still is challenging due to the lack of post-translational modifications such as glycosylation, etc. − Therefore, it is critical to find alternative catalysts to aromatic alcohol oxidase. Alcohol oxidases (AOx, E.C.…”