Oxidation of aromatic and aliphatic aldehydes to carboxylic acids by Geotrichum candidum aldehyde dehydrogenase

Article abstract of DOI:10.1016/j.tet.2020.131387

Oxidation reaction is one of the most important and indispensable organic reactions, so that green and sustainable catalysts for oxidation are necessary to be developed. Herein, biocatalytic oxidation of aldehydes was investigated, resulted in the synthesis of both aromatic and aliphatic carboxylic acids using a Geotrichum candidum aldehyde dehydrogenase (GcALDH). Moreover, selective oxidation of dialdehydes to aldehydic acids by GcALDH was also successful.

Full text of DOI:10.1016/j.tet.2020.131387

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Oxidation of aromatic and aliphatic aldehydes to carboxylic acids by Geotrichum
candidum aldehyde dehydrogenase
Tomoyasu Hoshino, Emi Yamabe, Muhammad Arisyi Hawari, Mayumi Tamura, Shuji
Kanamaru, Keisuke Yoshida, Afifa Ayu Koesoema, Tomoko Matsuda
PII:
S0040-4020(20)30549-4
https://doi.org/10.1016/j.tet.2020.131387
TET 131387
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 April 2020
Revised Date: 3 July 2020
Accepted Date: 6 July 2020
Please cite this article as: Hoshino T, Yamabe E, Hawari MA, Tamura M, Kanamaru S, Yoshida K,
Koesoema AA, Matsuda T, Oxidation of aromatic and aliphatic aldehydes to carboxylic acids by
Geotrichum candidum aldehyde dehydrogenase, Tetrahedron (2020), doi: https://doi.org/10.1016/
j.tet.2020.131387.
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Oxidation of aromatic and aliphatic
aldehydes to carboxylic acids by Geotrichum
candidum aldehyde dehydrogenase
Leave this area blank for abstract info.
Tomoyasu Hoshino, Emi Yamabe, Muhammad Arisyi Hawari, Mayumi Tamura, Shuji Kanamaru, Keisuke
Yoshida, Afifa Ayu Koesoema, Tomoko Matsuda*
Department of Life Science and Technology, Tokyo Institute of Technology

Oxidation of aromatic and aliphatic aldehydes to carboxylic acids
by Geotrichum candidum aldehyde dehydrogenase
Tomoyasu Hoshino, Emi Yamabe, Muhammad Arisyi Hawari, Mayumi Tamura, Shuji Kanamaru,
*
Keisuke Yoshida, Afifa Ayu Koesoema, Tomoko Matsuda
Department of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho,
Midori-ku, Yokohama, Kanagawa, 226-8501 JAPAN.
Corresponding author: Tel/fax: +81-45-924-5757
e-mail: tmatsuda@bio.titech.ac.jp (T. Matsuda)
Declarations of interest: none
Abstract
Oxidation reaction is one of the most important and indispensable organic reactions, so that green
and sustainable catalysts for oxidation are necessary to be developed. Herein, biocatalytic oxidation
of aldehydes was investigated, resulted in the synthesis of both aromatic and aliphatic carboxylic
acids using a Geotrichum candidum aldehyde dehydrogenase (GcALDH). Moreover, selective
oxidation of dialdehydes to aldehydic acids by GcALDH was also successful.
Keywords: Enzyme, Aldehyde dehydrogenase, Oxidation, Aldehyde, Carboxylic acid, Selectivity,
Green chemistry
1. Introduction
Oxidation reaction is an indispensable reaction for the chemical and pharmaceutical
industry, so that strong and useful oxidative reagents have been developed. However, these reactions
may have some drawbacks; these reactive reagents are necessary in a stoichiometric amount, they
can be explosive and/or contains hazardous heavy metals, and solvent waste and byproduct are
produced. To overcome these issues, the development of environmentally friendly reactions have
attracted attentions [1–4]. As a green catalyst, biocatalyst has been well known; the reaction can be
+
conducted in mild conditions, and non-explosive material such as NAD and oxygen in the air can be
used as oxidants [5–7]. Therefore, oxidation of alcohols, Baeyer-Villiger oxidation, oxidation of
1

sulfides, and hydroxylation have been widely studied [8–12], while the oxidation of aldehydes to
carboxylic acids have been studied to some extent [13–23]. For example, oxidation of heterocyclic
and aromatic aldehydes including 2-furancarboxaldehyde (furfural) and 5-hydroxymethylfurfural to
the corresponding carboxylic acids has been studied using Acetobacter rancens IFO3297, A.
pasteurianus IFO13753 and Serratia liquefaciens LF14 [13]. Especially, oxidation of
biomass-derived 5-hydroxymethylfurfural and related compounds has been extensively examined
using Comamonas testosteroni SC1588, a carbonyl reductase from Streptomyces coelicolor (ScCR),
an alcohol dehydrogenase (ADH) from Synechocystis sp. (SADH), and horse liver ADH (HLADH),
etc [14,18–22]. From 5-hydroxymethylfurfural, both 5-formyl-2-furancarboxylic acid and
2,5-furandicarboxylic acid were obtained with a yield of more than 95% using SADH and HLADH,
respectively [20]. The produced 5-formyl-2-furancarboxylic was not over oxidized to the
corresponding dicarboxylic acid by SADH. Oxidation of aliphatic and aromatic aldehydes by three
recombinant aldehyde dehydrogenases (ALDHs) originated from bovine lens (ALDH-Bov),
Escherichia coli (ALDH-Ec), and Pseudomonas putida (PP-ALDH) has been investigated, and
preparative scale oxidation of 5-hydroxymethylfurfural to give the corresponding carboxylic acid in
61% isolated yield (1.37 g) was reported [17]. Recently, α-ketoglutaric semialdehyde dehydrogenase
(KGSADH) from Azospirillum brasilense has been engineered to produce 3-hydroxypropionic acid
by the oxidation of 3-hydroxypropanal, derived from glycerol enzymatically [16]. However,
biocatalysts that have been tested for a wide variety of substrates including both aliphatic and
aromatic aldehydes with preparative synthetic study [17] is still limited, and the discovery of an
aldehyde dehydrogenase is necessary.
As a source of an enzyme for the oxidation of aldehydes, an organism that produces other
valuable enzymes has a high possibility to produce a versatile ALDH, also. Previously, our group has
discovered dehydrogenases widely used in organic synthesis from a dimorphic fungus, Geotrichum
candidum [12,24–32]. One of the enzymes from G. candidum NBRC 4597, acetophenone reductase
(GcAPRD), has been overexpressed in E. coli [27], and widely used for enantioselective organic
2

synthesis [28–32]. G. candidum glycerol dehydrogenase [33] and lipases [34,35] have been also used
widely. Focusing on the non-aqueous solvent tolerant enzymes from G. candidum, and wide
applications of its enzymes [12,24–30], an aldehyde dehydrogenase from this organism (GcALDH)
was targeted for the oxidation of aldehydes to carboxylic acids in this research. It was found that the
enzyme overexpressed in E. coli can catalyze the oxidation of various aromatic and aliphatic
aldehydes (Fig. 1). Moreover, the selective oxidation of dialdehydes to aldehydic acids by GcALDH
was also achieved. Preparative scale reactions were performed successfully.
2
2
. Results and discussion
.1. Preparation and characterization of the enzyme
The gene of GcALDH was amplified from the genomic DNA of G. candidum NBRC 4597
and cloned in an expression vector, pET-21b (+), leading to pET-21b (+)-GcALDH (the DNA
sequence and plasmid map of pET-21b (+)-GcALDH are shown in Fig. S1 and Fig. S2, respectively).
TM
The vector was transformed into E. coli Rosetta (DE3)pLysS. His-tagged GcALDH was induced
by IPTG and purified by Ni affinity chromatography by 6.5-fold in 33% yield (Table S1).
Construction of the overexpression system and purification were successful, as shown in the
SDS-PAGE of the purified enzyme (Fig. S3). The size of a single band around 60 kDa in the purified
enzyme lane is in accordance with the expected size of 66.2 kDa.
Coenzyme dependency of GcALDH was investigated. As shown in Table 1 Entry 1, the
+
oxidation activity toward benzaldehyde was observed using NAD , but no significant activity was
+
+
observed using NADP . Therefore, NAD was used for further studies. Then, the GcALDH
properties, such as the effect of pH, temperature, additives, and substrate concentration, were
investigated using the purified enzyme to optimize reaction conditions. First, the effect of pH on
GcALDH activity was examined (Fig. S4). Optimum oxidizing activity was observed in a pH range
of around 7.0 -7.5. The enzyme stability was also examined by comparing the activity before the
treatment with that after the treatment under pH between 5.0 and 10.5 at 8 °C for 1 week (Fig. S5).
3

The enzyme was stable under a wide range of pH between 5.0 and 8.5. Then, the effect of
temperature on GcALDH activity was examined (Fig. S6). The temperature with the highest activity
was 50 °C, which was 1.4 times that at 37 °C. Thermostability was also examined (Fig. S7). After 24
o
h, the activity was kept at 4 °C fully, but decreased to 62% at 30 °C and 26% at 40 C. However, it
was deactivated completely at 45 °C in 6 h and at 50 °C in 15 min. Therefore, the further synthetic
o
study was conducted at 40 C. The temperature dependency on the activity and stability of GcALDH
was compared with that of other enzymes reported so far and found to be comparable. The oxidation
o
o
of aromatic aldehydes by A. rancens or S. liquefaciens was conducted at 20 C or 30 C [13]. Knaus et
al examined temperature profile for the conversion of 2-methylpentanal and benzaldehyde by
o
o
o
ALDH-Bov, ALDH-Ec and PP-ALDH at 30 C, 40 C, and 50 C, and the reaction temperature was
o
set to 40 C to retain the enzyme stability and to avoid the evaporation of volatile aldehydes [17].
The effect of additives and metal ions on GcALDH activity was examined (Fig. S8). The
enzyme showed slightly higher activity with the addition of dithiothreitol (DTT), but lower activity
2
+
2+
2+
2+
with metals, especially with Zn and Cu ions. This is probably due to the binding of Zn and Cu
ion to the sulfur atom in the active site Cys352. The activities of other aldehyde dehydrogenases were
2
+
2+
also decreased with the addition of Zn and Cu ions [36–38]. The addition of EDTA did not
change the activity which indicates the absence of metal ion in the active site.
The effect of substrate concentration on GcALDH activity was examined with
benzaldehyde (0.25-50 mM) or pentanal (0.25-80 mM) (Fig. S9). The maximum reaction rate Vmax
,
Michaelis-Menten constant K , and inhibition constant K were determined to be 1.51 µmol/mg
m
i
protein/min, 3.63 mM, and 22.6 mM for benzaldehyde, and 2.31 µmol/mg protein/min, 6.42 mM,
and 92.9 mM for pentanal, respectively. The catalytic parameters of GcALDH were compared with
other enzymes reported so far and found to be comparable in terms of the Vmax but not for K since
m
1
unnatural substrates are used in this study. For example, for the case of Δ -pyrroline-5-carboxylate
dehydrogenase (ALDH4A1) from Saccharomyces cerevisiae, catalytic parameters, k and K ,
cat
m
−
1
1
were reported to be 1.5 s and 104 μM, respectively, using Δ - pyrroline-5-carboxylate as the
4

−
1
substrate [39]. For the wild type and mutants of KGSADH, k and K were reported to be 5-15 s
cat
m
and 0.17-1.6 mM, respectively, using 3-hydroxypropanal as the substrate [16]. As a result of the
o
characterization of GcAPRD, optimum reaction conditions were determined to be pH 7.2, 40 C, and
10 mM of substrate concentration.
2.2. Substrate specificity
Substrate specificity was investigated using the purified enzyme. It was revealed that
GcALDH has broad substrate specificity for the oxidation of a wide range of aromatic and aliphatic
aldehydes (Table 1), but it does not catalyze the oxidation of primary alcohols (Entries 33 and 34) or
reduction of carboxylic acids (Entries 35 and 36). Thus, the oxidation of aldehydes was successfully
conducted under mild conditions without any potentially explosive chemicals. Moreover, the large
difference in the relative activities between aldehydes and alcohols indicates the possibility of
chemoselective reactions. The excellent chemoselectivity was also demonstrated by the
transformation of 5-hydroxymethylfurfural to 5-hydroxymethl-2-furancarboxylic acid by
Comamonas testosteroni SC1588 [14].
Among substituted benzaldehyde analogs, substitutions at the ortho position decreased
the activities (Entries 2-6) while those at the para position with electron-withdrawing groups
increased the activities (Entries 7, 8, 10 and 11). The nucleophilic attack to the carbonyl carbon by
Cysteine 352 (Fig. S10 and S11) can be improved by the electron-withdrawing effects at the para
position, while it can be hindered sterically by the ortho substitutions. Similar results were also
observed for the reaction by ALDH-Bov and ALDH-Ec; the yields of para-substituted
benzaldehydes were much higher than those of ortho-substituted analogs, especially when the
substituents are large [17].
The activities toward benzyl aldehydes and propanals with an aromatic ring (Entries
1
5-19) largely depend on the structures. The activities toward two benzyl aldehydes (Entries 15 and
6) were lower than that toward benzaldehyde (Entry 1), while activities toward two out of three
1
5

3-arylpropanals (Entries 18 and 19) were higher than that toward benzaldehyde (Entry 1). Similarly,
the yields of oxidation of 2-phenylpropanal and (2E)-3-phenylprop-2-enal (cinnamaldehyde) by
ALDH-Bov, ALDH-Ec and PP-ALDH were low (less than 30%) while those of 3-phenylpropanal
are around 79-85% [17].
Among linear aliphatic aldehydes tested, high activities were exhibited especially with
substrates with a chain length from 5 to 8 (Entry 22-25, and 28). This is in good agreement with the
fact that the predicted natural substrate found by the blast search of the protein sequence of this
enzyme, δ-1-pyrroline-5-carboxylate, has also the chain length of 5 [39,40]. However, the activities
were lower toward branched aliphatic aldehydes with the same chain length (Entries 20-23 vs. 29-32).
These might be explained by the steric hindrance by the two phenyl-groups of phenylalanine 214 and
phenylalanine 458 at the entrance of the substrate in the enzyme structure constructed by modeling
(Fig. S10). For the case of the oxidation of aliphatic aldehydes by ALDH-Bov, ALDH-Ec, and
PP-ALDH, the yield of the oxidation of pentanal, 4-pentenal and 2-methylpentanal were >99%,
while the oxidation of longer aliphatic aldehydes resulted in moderate yields [17]. Therefore, the
substrate specificity of GcALDH is unique, so that GcALDH is useful for the reaction of aliphatic
substrates, similar to GcAPRD which is an excellent catalyst for the asymmetric reduction of
challenging aliphatic ketones [28,29].
To sum up the substrate specificity of GcALDH, wider varieties of substrates, including
both aromatic and aliphatic aldehydes, were tested and found to be accepted than those tested in most
of the previous publications which concentrated on the furfural and furan related compounds [13,14]
or 3-hydroxypropanal, a glycerol derived compound [16], except for the study by Knaus et al [17]. It
was also found that the activities largely depend on the detail of the substrate structure, so that further
studies such as determination of enzyme structure as well as investigation of docking poses of the
substrate in the enzyme active site are necessary to predict the substrate specificity and its substrate
recognition mechanism.
6

Encouraged by the high activity and wide substrate specificity of GcALDH, preparative
scale reactions were conducted for the representative substrates. The reaction proceeded smoothly,
1
13
and the products were successfully isolated, purified, and identified by the H NMR and C NMR
(Appendix section in the Supplementary Material).
2.3. Selective oxidation of dialdehydes to aldehydic acids
Selective oxidation of dialdehydes to aldehydic acids is challenging since the high
reactivity of the aldehyde group generally makes it difficult to oxidize only one of the aldehyde
groups selectively using strong chemical reagents [13]. To investigate the selectivity of GcALDH,
relative activities toward dialdehydes 1a-5a and the corresponding aldehydic acids 1b-5b were
determined (Table 2). High activities were observed for 2a-5a while there was no activity toward 1a.
Activity toward an aldehydic acid 1b was not observed, either. The reactivities of 1a and 1b are
possibly due to the steric hindrance by the ortho substitutions. The yield of the oxidation of 1a by S.
liquefaciens is also much lower than those of 2a and 3a [13].
Interestingly, the activities toward dialdehydes were much higher than those toward the
corresponding aldehydic acids for the case of terephthalaldehyde 3a and 3b (Entry 3),
4-bromoisophthalaldehyde 4a and 4b (Entry 4), and 2-bromoterephthalaldehyde 5a and 5b (Entry 5),
but not for the case of isophthalaldehyde 2a and 2b (Entry 2). To investigate the selectivity difference
between the 2a/2b and 3a/3b (Entry 2 vs. Entry 3), their kinetic parameters were examined (Table
S2). For the case of 3a and 3b, there was a large favorable difference in Vmax and small unfavorable
difference in K . For the case of 2a and 2b, there was a large unfavorable difference in K and small
m
m
favorable differences in Vmax. Therefore, the selective reaction is possible for the oxidation of 3a as
the reaction rate of 3a is much faster than that of 3b (Fig 2), while for the case of 2a, 2b is oxidized
further to the corresponding m-dicarboxylic acid as soon as formed in a small concentration due to
the smaller K for 2b than 2a. The selectivity difference between 2a/2b and 3a/3b was compared
m
with other enzymes (Table S3), while the biocatalytic selective oxidation of 4a and 5a was reported
7

for the first time to the best of our knowledge. Similar results were reported for ScCR and SADH
20]; the selectivity between 3a and 3b was higher than that between 2a and 2b. However, the
[
opposite trend was also reported for S. liquefaciens [13]; the selectivity between 2a and 2b is high
while that between 3a and 3b is not. The preferential formation of aldehydic acid 2b might be
ascribed to the difference of membrane permeability or the affinities of aldehydic acid and
dialdehyde for the enzyme. On the other hand, for the case of the whole-cell biocatalytic oxidation of
3a using Deinococcus wulumuqiensis R12, 3c was obtained in >99% yield [22].
Encouraged by the relative activity differences between the dialdehydes (3a, 4a, and 5a)
and aldehydic acids (3b, 4b, and 5b), preparative scale synthesis of 3b, 4b and 5b was conducted
Table 3). Interestingly, the reaction proceeded selectively, and the dicarboxylic acids were not
(
detected. The selectivities in a preparative scale were higher than the relative activity differences
because in the presence of dialdehydes, the reaction of aldehydic acids, competing to get into the
substrate-binding site with dialdehydes, is inhibited. Furthermore, regioselectivities for oxidation of
4a and 5a giving only one isomer of aldehydic acids, 4b and 5b, were also excellent.
3. Conclusions
It was found that GcALDH has a broad substrate specificity and can selectively oxidize
only one aldehyde group of dialdehydes. The oxidation of aldehydes under mild conditions using a
green catalyst, GcALDH, was successfully demonstrated. In the future, further studies such as
mutagenesis are required to develop this enzyme for industrial use.
8

4
4
. Experimental section
.1. General materials and methods
All chemicals, except 4a and 5a which were prepared according to the literature methods
[
41], were purchased from commercial suppliers and used without further purification. Enzyme
activity assays were performed using UV-1600-UV-Visible spectrophotometer Shimadzu (Kyoto,
1
13
Japan). The assay was performed in duplicate, and the average value was taken. H NMR and C
NMR analyses were performed on a Bruker Biospin AVANCE III 400 spectrometer at 400 MHz.
4.2. Substrate specificity
0
.10 M HEPES-NaOH buffer pH 7.2 (470 µl) and 10 mM benzaldehyde solution in 0.10 M
o
HEPES-NaOH buffer pH 7.2 (500 µl) were added to a UV cell, and incubated at 37 C for 20 min.
+
Then, an enzyme solution (20 µl) and 10 mM NAD solution (10 µl) were added to start the reaction.
+
Thus, the final concentration is 5.0 mM substrate and 0.10 mM NAD . For the assay of the reduction
+
activity toward carboxylic acids, NADH was used in substitution for NAD . The absorbance at 340
nm was monitored after 10 sec to 132 sec. One unit of enzyme is defined as µmol of NADH produced
in 1 min under the above conditions using benzaldehyde as a substrate. Relative activity was based on
benzaldehyde activity as 100% (12 mU).
4.3. Preparative scale reactions
Reactions were performed in 0.10 M HEPES-NaOH buffer (20 ml) pH 7.2 containing 10
+
mM substrate, 10 mM NAD , and 0.125 U/ml GcALDH cell-free extract for 3 h at 40 °C with a
rotational speed of 160 rpm. The reaction was quenched by thermal treatment at 95 °C for 10 min.
The reaction mixture was centrifuged at 12000 G for 5 min, and the supernatant was recovered.
NaOH (solid) was added to adjust pH >14, and extracted with diethyl ether (30 ml x 2), and the
aqueous layer was recovered. 12 N HCl (aq) was added to adjust pH < 1. The product was extracted
with diethyl ether (30 ml), dried over MgSO , evaporated under reduced pressure, purified by silica
4
9

1
13
gel column chromatography (hexane:ethyl acetate, 1:1) and characterized by H-NMR and C-NMR
analysis using CDCl or DMSO-d as a solvent. The yields are shown in Table 1 and Table 3. The
3
6
NMR spectrum is in the Appendix section of the supplementary material.
Acknowledgment
This work was partially supported by the Japan Society for the Promotion of Science, Japan
(grant number JP16K05864 and JP19K05560) to Tomoko Matsuda.
Appendix A. Supplementary Data
Supplementary data related to this article can be found.
Fig. 1 Oxidation of aldehydes by Geotrichum candidum aldehyde dehydrogenase (GcALDH)
Fig. 2 Selective oxidation of terephthalaldehyde 3a by GcALDH
10

Table 1. Substrate specificity of GcALDH
a
Entry
1
Substrate
Relative activity (%)
100
X= H
b
2
2
3
4
5
6
7
8
9
o-F
75
19
o-Cl
o-Br
8
o-CF3
o-CH3
p-F
<1
10
178
122
98
p-Cl
p-Br
10
11
12
13
14
15
p-COOCH3
p-CF3
p-CH3
p-C(CH3)3
p-OCH3
123
151
42
8
17
1
0
16
9
17
18
19
16
3
1
10
68
11

Table 1. continued
Entry
a
Substrate
Relative activity (%)
20
21
22
23
24
25
26
27
n=
1
2
3
4
5
6
7
8
123
208
302
436
434
313
197
50
28
413
29
78
30
31
32
85
101
4
3
3
4
n=
6
7
<1
<1
c
3
<
1
3
5
6
d
<1
c
3
<1
o
The activity was measured at 37 C in 0.10 M HEPES-NaOH buffer pH 7.2, containing 5.0 mM substrate and 0.1 mM
+
NAD by monitoring the absorbance at 340 nm. Relative activity was based on benzaldehyde (entry 1) activity as 100%
(
12 mU). One unit of enzyme activity was defined as the micromoles of NADH released by the oxidation of
o
a
benzaldehyde by enzyme per minute at 37 C. For preparative scale oxidation of aldehydes to carboxylic acids, the
o
reaction was conducted at 40 C for 3 h in HEPES-NaOH buffer (20 mL 0.10 M pH 7.2), containing an aldehyde (0.20
+
mmol), NAD (0.20 mmol) and GcALDH cell-free extract (2.5 U). Benzoic acid derivatives, X=H (21 mg, 85%), o-F (24
12

mg, 85%), o-Cl (10 mg, 32%), p-F (24 mg, 86%), p-Cl (25 mg, 80%), p-Br (31 mg, 77%), p-CF (27 mg, 72%), p-CH (20
3
3
mg, 74%), p-OCH (20 mg, 64%), cinnamic acid (16 mg, 54%), 3-phenylpropanoic acid (22 mg, 74%), heptanoic acid
3
b
+
+ c
(
14 mg, 54%), nonanoic acid (14 mg, 43%). NADP was used in substitution for NAD . NADH was used in substitution
+
d
+
for NAD . NADPH was used in substitution for NAD .
13

Table 2. Differences in GcALDH activities toward dialdehydes and aldehydic acids
Entry
Substrate
Relative
activity
Substrate
Relative
activity
(%)
dialdehyde
aldehydic acids
(%)
＜
1
＜1
1
2
1
a
1b
313
357
113
115
78
2
a
2b
3
4
5
7
9
9
3
a
3b
4b
4
a
5a
5b
o
The activity was measured at 37 C, in 0.10 M HEPES-NaOH buffer pH 7.2, containing 5.0 mM substrate and 0.1 mM
+
NAD . Relative activity was based on benzaldehyde activity as 100% as in Table 1.
14

Table 3. Preparative scale selective oxidation of dialdehydes to aldehydic acids
a
Substrate
Product
Isolated yield (%)
Regioselectivity (%)
b
9
7
6
0
-
3
a
3b
4b
c
1
>99 %
>99 %
4
a
d
2
5
a
5b
o
The reaction was conducted at 40 C and 160 rpm for 4 h in HEPES-NaOH buffer (20 mL, 0.10 M, pH 7.2), containing
+
a
aldehydes (0.20 mmol), NAD (0.20 mmol) and purified GcALDH (1.0 U). By-products including dicarboxylic acids
b
c
d
were not detected based on TLC analysis using UV (254 nm) lamp. 3b: 27 mg, 4b: 33 mg, 5b: 29 mg.
15

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19

Highlight
•
•
•
•
•
An enzyme, Geotrichum candidum aldehyde dehydrogenase (GcALDH), was
overexpressed.
GcALDH was used for oxidation of aldehydes to carboxylic acids for the first
time.
Dialdehydes were oxidized to aldehydic acids without further oxidation to
diacids.
Regioselectivities for oxidation of dialdehydes to aldehydic acids were
excellent.
Preparative scale reactions were performed successfully.

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
Declaration of interest: none