Communications
doi.org/10.1002/cbic.202100183
ChemBioChem
indicated that piperine was not a direct substrate, but most
likely cleaved by co-oxidation during linoleic acid oxidation
(Scheme 1). Recent work suggested that various members of
the catalytic cycle of lipoxygenases might interact with
unsaturated substrates in co-oxidation reactions.[8] In the
presented case, the initial linoleic acid hydroperoxide radicals
may abstract hydrogens from the unsaturated bridge of
piperine paving the way for an autoxidative insertion of an
3,4-methylenedioxycinnamaldehyde concentrations, respec-
tively.
During the first three hours, over 60% of the maximal
product concentration was obtained (Figure 2e). The following
decrease of the biotransformation rate was most likely the
result of a linoleic acid limitation, as it was completely degraded
after 16 h (Figure S5). Higher linoleic acid concentrations of a
fed-batch regime may be applied. In addition, higher enzyme
concentrations may be used, as they led to increased product
formation (Figure S7).
LOXPsa1 was further examined for bioconversion of other
alkenes. The aryl alkenes trans-anethole, (E)-methyl isoeugenol,
and α-methylstyrene were converted to the expected olfactants
p-anisaldehyde, veratraldehyde, and acetophenone, respectively
(Figure 2f and Scheme S1). The highest product concentration
was identified for α-methylstyrene followed by trans-anethole
(about two-fold lower) and (E)-methyl isoeugenol (about six-
fold lower).
oxygen molecule. As
a stable dioxene- or hydroperoxo-
intermediate was not found, the exact mechanistic route
remains obscure. In the mycelium and crude extract, fungal
PUFAs most likely initialized the co-oxidation process as
substrates, which would well explain the activity loss during the
initial purification attempts and the LMMF-dependency (Fig-
ure 1a). In contrast to the results for the crude extract, addition
of Mn2+ had no influence on the biotransformation yield of
LOXPsa1 (Figures 1b and 2a). A second, Mn2+-dependent enzyme
may participate in the piperine conversion. This remains to be
elucidated in a follow-up study.
To increase the biotransformation yield, different PUFAs and
concentrations were examined as well as the influence of pH,
temperature, piperine concentration, and incubation time
(Figures 2b–e and S6). Biotransformation experiments with
linoleic and α-linolenic acid showed that the product concen-
tration increased significantly with rising PUFA concentrations
(up to 17.5-fold; exemption: 2.5 mM α-linolenic acid) (Fig-
ure 2b). The PUFA concentration was the parameter with the
highest effect on the biotransformation yield. These findings
support the co-oxidative character of the piperine cleavage
reaction. Linoleic acid at the highest concentration (2.5 mM)
In summary, the biocatalytic generation of piperonal using
piperine as substrate was achieved by a co-oxidation reaction
catalyzed by LOXPsa1 in the presence of linoleic acid. In addition,
a
second
aroma
compound,
3,4-methylenedioxycinnamaldehyde, was generated, which also
offered a vanilla-like odor. Separation of both aldehydes may be
achieved by adsorption to zeolithes as shown, for example, for
limonene and carvone.[21] Alternatively, a combined application
could be envisaged due to the similar odor attributes. Although
the improved reaction conditions increased the product
concentrations, further optimization is needed. Besides higher
linoleic acid concentrations, monokaryotic daughter-strains of
P. sapidus are an option, as they showed higher LOX activities[22]
and higher product concentrations after piperine transforma-
tion (Figure S8). In addition, some of the daughter strains
achieved
the
highest
piperonal
(25 μM)
and
3,4-methylenedixycinnamaldehyde concentrations (53 μM) and
was thus used for all subsequent assays.
Analysis of the piperine biotransformation revealed a pH
favored
the
formation
of
piperonal
over
°
optimum of 7 (Figure S6) and a temperature optimum of 30 C
3,4-methylenedioxycinnamaldehyde. As LOXPsa1 converted fur-
ther aryl alkenes to their respective odor-active aldehydes, it
showed potential as biocatalyst for aroma production. However,
further optimization is needed to improve product concen-
trations for a potential industrial application.
(overall product concentration, Figure 2c). These results agreed
with the optima reported for the linoleic acid oxidation by
LOXPsa1.[14] However, the product ratio of piperonal to 3,4-
methylenedioxycinnamaldehyde increased from 0.5 to 0.65 at
°
37 C (Figure 2c). This most likely resulted from thermodynamic
effects,[20] which disfavor the cleavage of the second double
bond and hence 3,4-methylenedioxycinnamaldehyde formation Experimental Section
at higher temperatures. As piperonal is the more valuable
Experimental details are given in the Supporting Information.
°
cleavage product, 37 C was considered as optimal for piperonal
synthesis and used for the following experiments. Temperature
was the only parameter that effected the product-ratio
(Figures 2b–e and S6).
Acknowledgements
Additional experiments showed a linear increase in product
concentration with rising piperine concentrations (Figure 2d,
coefficient of determination R2 �0.90). Concentrations higher
than 1.6 mM piperine were not investigated due to the lack of
solubility. An increase of the incubation time to 48 h resulted in
This research was funded by the BMBF cluster Bioeconomy
International 2015, grant number 031B0307A. We thank B. Fuchs
for her preparatory work for this project and C. Theobald and N.
Püth
for
the
olfactory
analysis
of
the
highest
overall
piperonal
(41 μM)
and
3,4-methylenedioxycinnamaldehyde. Open access funding enabled
and organized by Projekt DEAL.
3,4-methylenedioxycinnamaldehyde concentrations (56 μM)
(Figure 2e). Thus, improving the reaction conditions (linoleic
acid and piperine concentration, reaction temperature and
time) achieved a 24- and 15-fold increase of the piperonal and
ChemBioChem 2021, 22, 1–6
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