An enzymatic tandem reaction to produce odor-active fatty aldehydes

Abstract

Aldehydes represent a versatile and favored class of flavoring substances. A biocatalytic access to odor-active aldehydes was developed by conversion of fatty acids with two enzymes of the α-dioxygenase pathway. The recombinant enzymes α-dioxygenase (α-DOX) originating from Crocosphaera subtropica and fatty aldehyde dehydrogenase (FALDH) from Vibrio harveyi were heterologously expressed in E. coli, purified, and applied in a coupled (tandem) repetitive reaction. The concept was optimized in terms of number of reaction cycles and production yields. Up to five cycles and aldehyde yields of up to 26% were achieved. Afterward, the approach was applied to sea buckthorn pulp oil as raw material for the enzyme catalyzed production of flavoring/fragrance ingredients based on complex aldehyde mixtures. The most abundant fatty acids in sea buckthorn pulp oil, namely palmitic, palmitoleic, oleic, and linoleic acid, were used as substrates for further biotransformation experiments. Various aldehydes were identified, semi-quantified, and sensorially characterized by means of headspace-solid phase microextraction-gas chromatography-mass spectrometry-olfactometry (HS-SPME-GC-MS-O). Structural validation of unsaturated aldehydes in terms of double-bond positions was performed by multidimensional high-resolution mass spectrometry experiments of their Paternò-Büchi (PB) photoproducts. Retention indices and odor impressions of inter alia (Z,Z)-5,8-tetradecadienal (Z,Z)-6,9-pentadecadienal, (Z)-8-pentadecenal, (Z)-4-tridecenal, (Z)-6-pentadecenal, and (Z)-8-heptadecenal were determined for the first time. KEY POINTS: • Coupled reaction of Csα-DOX and VhFALDH yields chain-shortened fatty aldehydes. • Odors of several Z-unsaturated fatty aldehydes are described for the first time. • Potential for industrial production of aldehyde-based odorants from natural sources.

Keywords: Biotransformation; Fatty aldehyde dehydrogenase (FALDH); Fatty aldehydes; Flavoring production; α-Dioxygenase (α-DOX).

Fig. 1

Catalytic cycle yielding chain-shortened fatty aldehydes and acids by oxidative decarboxylation via α-dioxygenase (α-DOX) and successive oxidation via fatty aldehyde dehydrogenase (FALDH)

Fig. 2

Optimization of biotransformation: a Comparison of incubation times, b enzyme unit ratios Csα-DOX:VhFALDH [U/L] (4:1 = 50:12.5, 8:1 = 100:12.5, 12:1 = 150:12.5, 16:1 = 200:12.5), c total enzyme activity concentrations. Error bars indicate standard deviations

Fig. 3

Yield of aldehydes per gram substrate supplemented for biotransformation experiments with Csα-DOX and VhFALDH. Error bars indicate standard deviations

Fig. 4

Exemplary PB reaction, of the analyte (Z)-8-heptadecenal with 3-acetylpyridine. HCD experiments initiated fragmentation of PB-products yielding diagnostic α- and ω-ions confirming the double-bond position of the aldehyde between C₈ and C₉