T.S. Zhuk et al.
Bioorganic Chemistry 108 (2021) 104651
Fig. 1. Model compounds benzaldehyde (1), p-nitrobenzaldehyde (2), cyclohexanone (3), adamantanone (4) utilized for screening procedure (A) and the workflow
of the screening (B, MC = main culture).
efficient “green” catalysts [31]. On the other hand, fungal secretomes
consist of a complex mixture of enzymes of different nature that are able
to catalyse a broad range of chemical transformations, and are thus not
expected to be selective if whole-cell cultures are utilized. Nevertheless,
examination of whole-cell transformations is typically the first step in
revealing the catalytic properties of enzymes.
purchased from Merck KGaA (Darmstadt, Germany).
2.2. Organisms
The stock culture of the fungi Dichomitus albidofuscus (CBS, 321.75),
Marasmius cohortalis (DSMZ, 8257), Pleurotus sapidus (DSMZ, 8266),
Flammulina velutipes (DSMZ, 1658), Daedalea quercina (DSMZ, 4953),
Ischnoderma benzoinum (CBS, 231.97), Pleurotus cornucopiae (DSMZ,
5342), Pleurotus ostreatus (DSMZ, 1833), and Dichomitus squalens (DSMZ,
9615) were maintained on a solid medium containing 15 g⋅Lꢀ 1 malt
extract (Fluka, Neu-Ulm, Germany) and 15 g⋅Lꢀ 1Agar-Agar (Roth,
Karlsruhe, Germany).
In this study, we reveal the reductive potential of whole-cell cultures
of several white-rot fungi. In particular, Dichomitus squalens was chosen
as this fungus has been shown to degrade chlorophenoxyacetic acids
[32], and its carboxylate reductase was recently characterized [33].
Additionally, Dichomitus albidofuscus, which is able to oxidase aliphatic
compounds [34], Ischnoderma benzoinum that transforms L-phenylala-
nine to benzaldehyde [35], and Daedalea quercina whose laccases are
able to decolorize synthetic dyes efficiently [36] were selected for the
present study. Whereas the chemistry of Daedalea quercina and Mar-
asmius cohortalis remains largely undisclosed, fungi of the Pleurotus
family, namely P. sapidus, P. ostreatus, and P. cornucopiae, display an
enormously rich chemistry and various applications in biotechnology
[20,37–39] and are also subject of our scrutiny.
2.3. Submerged cultures
The culture medium was prepared by dissolving malt extract (30 g)
in 1 L of deionised water. For preparation of the precultures, a 1 cm2
agar plug from the leading mycelial edge was transferred into 100 mL
medium (250 mL Erlenmeyer flask) and then homogenized with a T 25
digital Ultra-Turrax homogenizer (IKA, Staufen, Germany; 30 s, 10.000
r⋅minꢀ 1). The precultures were grown on an incubation shaker (Orbi-
tron, Infors HAT, Bottmingen, Switzerland; 150 r⋅minꢀ 1, deflection 25
mm) under exclusion of light at 24 ◦C for 7 days. Afterwards, the pre-
cultures were homogenized, and 10% (v/v) of the homogenate was
inoculated for submerged cultivation into 400 mL (1000 mL Erlenmeyer
flask), 200 mL (500 mL Erlenmeyer flask) or 40 mL (100 mL Erlenmeyer
flask) medium.
2. Materials and methods
2.1. Chemicals
Benzaldehyde, benzoic acid, cyclohexanone, adamantanone, 3-
chloro-4-methoxybenzaldehyde,
m-methylbenzaldehyde,
m-nitrobenzaldehyde,
vera-
traldehyde,
m-anisaldehyde,
p-nitro-
benzaldehyde,
p-bromobenzaldehyde,
2-heptenal,
dodecanal,
undecanal, m-nitrobenzoic acid, p-nitrobenzoic acid, vanillic acid, cin-
namonic acid, ferulic acid, p-coumaric acid (for synthesis), benzyl
alcohol, cyclohexanol, 2-adamantanol, 3-chloro-4-methoxybenzyl
alcohol, m-methylbenzyl alcohol, veratryl alcohol, m-anisyl alcohol, m-
nitrobenzyl alcohol, p-nitrobenzyl alcohol, p-bromobenzyl alcohol, 1-
heptanol, 1-dodecanol, 1-undecanol, vanillin, dihydroconiferyl
alcohol, 1-hydroxy-3-phenylpropanol-1-ol, 1-hydroxy-3-(4-hydrox-
yphenyl)propan (as standards for compound identification) were
2.4. Screening procedure
The substrate (0.5 mmol) was added to submerged cultures of the
respective fungus (40 mL) on the 3rd culture day. The reaction mixture
was incubated on an incubation shaker at 150 r⋅minꢀ 1 (deflection 25
mm) under exclusion of light at 24 ◦C for 4 days. Afterwards, 5 g of NaCl
was added to the medium, and the mixture was stirred for 10 min at 800
rpm (magnetic stirrer). For extraction 25 mL of Et2O was added, and the
2