Biocatalysed reduction of carboxylic acids to primary alcohols in aqueous medium: A novel synthetic capability of the zygomycete fungus Syncephalastrum racemosum

Article abstract of DOI:10.1016/j.molcatb.2015.03.010

Abstract The zygomycete fungus Syncephalastrum racemosum shows the remarkable capability of reducing carboxylic acids to primary alcohols in water medium, at ambient temperature and pressure. The reaction does not require molecular hydrogen, and in most cases affords quantitative transformations. The results herein reported highlight the basic molecular scaffold that can be accepted by the fungus, the effects due to substituents, and also the possibility that carboxylic acids can be generated in the reaction medium by enzymatic hydrolysis of the corresponding methyl esters. This biocatalysed reduction implements the scarcely supplied enzymatic toolbox for the conversion of carboxylic groups into primary alcohols, which can be exploited for the optimisation of sustainable synthetic procedures.

Full text of DOI:10.1016/j.molcatb.2015.03.010

Journal of Molecular Catalysis B: Enzymatic 116 (2015) 83–88
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biocatalysed reduction of carboxylic acids to primary alcohols in
aqueous medium: A novel synthetic capability of the zygomycete
fungus Syncephalastrum racemosum
Elisabetta Brennaa,b,∗, Flavia Cannavalea, Michele Crottia, Fabio Parmeggiania,
Alice Romagnoloc, Federica Spinac, Giovanna Cristina Varesec
a Politecnico di Milano, Dipartimento di Chimica, Materiali, Ingegneria Chimica, Via Mancinelli 7, I-20131 Milano, Italy
b Istituto di Chimica del Riconoscimento Molecolare – CNR, Via M. Bianco 9, I-20131 Milano, Italy
c Department of Life Sciences and Systems Biology, University of Turin, viale P. A. Mattioli 25, I-10125 Turin, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The zygomycete fungus Syncephalastrum racemosum shows the remarkable capability of reducing car-
boxylic acids to primary alcohols in water medium, at ambient temperature and pressure. The reaction
does not require molecular hydrogen, and in most cases affords quantitative transformations. The results
herein reported highlight the basic molecular scaffold that can be accepted by the fungus, the effects due
to substituents, and also the possibility that carboxylic acids can be generated in the reaction medium by
enzymatic hydrolysis of the corresponding methyl esters. This biocatalysed reduction implements the
scarcely supplied enzymatic toolbox for the conversion of carboxylic groups into primary alcohols, which
can be exploited for the optimisation of sustainable synthetic procedures.
Received 29 January 2015
Received in revised form 10 March 2015
Accepted 12 March 2015
Available online 20 March 2015
Keywords:
Biocatalysis
Carboxylic acids
Esters
© 2015 Elsevier B.V. All rights reserved.
Alcohols
Reduction
Filamentous fungi
1. Introduction
group reduction to primary alcohols [4]. Nevertheless, the devel-
opment of a cost-effective, and highly selective catalyst for this
The reduction of esters and carboxylic acids to primary alcohols
is a key reaction of organic chemistry [1], which has widespread
application in the manufacturing processes of fine and bulk chem-
icals. This transformation still relies on the stoichiometric use of
metal hydride reagents and their derivatives [2], often with the
requirement of extensive heating, and anhydrous conditions. The
highly exothermic hydrolytic work up is generally troublesome,
and yields voluminous precipitates. Recently, transition metal
catalysed hydrogenation reactions [3] employing molecular hydro-
gen as a reducing agent have been investigated in order to address
the waste challenge connected with hydride promoted reductions.
However, only a limited number of these new procedures work
at room temperature and low hydrogen pressure. Other chemo-
catalytic methods, such as the metal-catalysed hydrosilylation of
esters, have received considerable interest as a tool for carboxylic
transformation is still desirable because most of the known pro-
cedures either require expensive silanes or have limited functional
group tolerance.
As for biocatalysed methods, this is a very challenging task,
because the reduction of acids is characterised by a very low
redox potential, and it has been described up to now only for a
very limited number of microorganisms and cultured plant cells.
The two classes of enzymes which have been reported so far as
biocatalysts of this reaction are [5]: carboxylic acid reductases
(CARs, E.C.1.2.1.30) from aerobic sources, such as bacteria, fungi
and plants, and aldehyde ferredoxin oxidoreductases (AORs, E.C.
1.2.99.6) from anaerobic bacteria and archaea.
In 1969 Gross et al. isolated a CAR [6] from Neurospora crassa
and performed preliminary studies on the reaction mechanism. The
reduction was found to be adenosine triphosphate (ATP), Mg2+ and
NADPH dependent [6c]. An homologous enzyme was isolated and
purified by Rosazza and co-workers from Nocardia species NRRL
5646 in 1997 [7]. This CAR enzyme was overexpressed, employed
successfully in biocatalytic reductions [7a,c], and its reaction mech-
anism was fully elucidated [7d]. Recently [8], the conversion of
3,4-dihydroxyphenylacetic acid into 3-hydroxytyrosol has been
∗
Corresponding author at: Politecnico di Milano, Dipartimento di Chimica, Mate-
riali, Ingegneria Chimica, Via Mancinelli 7, I-20131 Milano, Italy.
Tel.: +39 02 23993077; fax: +39 02 23993180.
E-mail address: mariaelisabetta.brenna@polimi.it (E. Brenna).
http://dx.doi.org/10.1016/j.molcatb.2015.03.010
1381-1177/© 2015 Elsevier B.V. All rights reserved.

84
E. Brenna et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 83–88
performed in whole Escherichia coli BL21(DE3) cells overexpressing
CAR from Nocardia and phosphopantetheinyl transferase from E.
coli, with the concomitant action of an endogenous E. coli aldehyde
reducing activity. In 2013 [9], Turner et al. described the conversion
of fatty acids into the corresponding alcohols, by coexpression of a
CAR from Mycobacterium marinum and an aldehyde reductase from
Synechocystis species.
column (30 m × 0.25 mm × 0.25 ␮m, Agilent), employing the fol-
lowing temperature program: 60 ◦C (1 min)/6 ◦C min−1/150 ◦C
(1 min)/12 ◦C min−1/280 ◦C (5 min). The identity of the reduction
products was established by comparison with reference samples
(either commercially available or prepared by lithium aluminium
hydride reduction of the corresponding methyl esters).
Sodium carboxylates C6–C10 were transformed into the corre-
sponding alcohols by growing cultures of the fungus Colletotrichum
gloeosporioides, but the method lacks general applicability to dif-
ferent substrates [10]. Some examples of aerobic reductions of
aromatic carboxylic acids by fungi have been described, especially
in connection with the conversion of ferulic acid into vanillin [5].
Resting cells of Clostridium thermoaceticum and Clostridium
formicoaceticum were described to reduce [11] carboxylic acids and
carboxylates to alcohols at the expense of carbon monoxide or, less
efficaciously, in the presence of formate. The enzymes responsi-
ble for the carboxylic acid reduction in these two organisms were
isolated and characterised to be highly oxygen sensitive tungsten
enzymes (W-AORs). In spite of the efficiency of the reaction, the
requirement for strictly anaerobic conditions makes this alterna-
tive poorly appealing for preparative applications.
In 1999, it was first reported [12a] that the hyperthermophilic
archaea Pyrococcus furiosus reduced carboxylic acids with starch
as carbon and energy source at 90 ◦C. Recently, the use of this
microorganism has been reconsidered [12b], because it contains
suitable enzymes to couple the oxidation of molecular hydrogen
(catalysed by hydrogenases) to the reduction of carboxylic acids to
aldehydes (mediated by an AOR). The presence of endogenous alco-
hol dehydrogenases promotes the final conversion of aldehydes
into primary alcohols. A biocatalytic hydrogenation procedure has
been optimised for the reduction of carboxylic acids at 40 ◦C under
5 bar molecular hydrogen pressure. The same authors have also
described the possibility to employ syngas instead of hydrogen to
promote this P. furiosus-mediated reduction, thus hypothesising
the presence in this microorganism of enzymes capable of coupling
CO oxidation to acid reduction [12c].
2.2. Fungal strains
The strain of the zygomycete fungus S. racemosum MUT 2770
employed for this work is preserved at Mycotheca Universitatis Tau-
rinensis (MUT, Department of Life Sciences and Systems Biology,
University of Turin).
2.3. Biotransformation experiments
Fungal strains were pre-grown in Petri dishes containing malt
extract solid medium (MEA: 20 g L−1 glucose, 20 g L−1 malt extract,
20 g L−1 agar, 2 g L−1 peptone) from which the inoculum for liquid
cultures was set up. The fungus was inoculated as conidia suspen-
sion (1 × 106 conidia/mL) in 50 mL flasks containing 40 mL of malt
extract liquid medium. Flasks were incubated at 25 ◦C and were
maintained under agitation (110 rpm).
After 2 days of pre-growth, a 500 mM solution of the substrate
in DMSO was added, to a starting substrate concentration (c0) of
1–5 mM. For each substrate, three biological replicates were run.
The experiment was run for 3 days after the addition of the sub-
strates, during which time 1 mL samples were taken, at specified
intervals (usually 24, 48, and 72 h). Each sample was extracted
with EtOAc (500 ␮L), the organic phase was dried over anhydrous
Na2SO4 and analysed by means of GC/MS. In some cases (see Section
2.4) the isolation of the reduced product has been carried out.
For each set of biotransformations, one flask was used to mea-
sure the initial biomass and pH before the addition of the substrate.
These parameters were also evaluated at the end of the experiment
for all the flasks. The liquid media was separated from the biomass
by filtration and was used for pH measurement while the mycelia
were dried at 60 ◦C for 24 h to measure the biomass dry weight.
As for eukaryotic cells, nine different plant cell cultures have
also been successfully employed on several carboxylic acids, but
only in modest yields and with long pre-culture times and reaction
times [13].
2.4. Isolation procedure of biotransformation products
We now wish to report on the possibility of preparing primary
alcohols by reduction of carboxylic acids in a very mild and efficient
way, using the whole cells of the zygomycete fungus Syncepha-
lastrum racemosum in aqueous medium, at room temperature and
pressure. In some cases the carboxylic acids can be generated in
the reaction medium by enzymatic hydrolysis of the correspond-
ing methyl esters. This peculiar reducing activity was discovered
during a functional screening of the enzymatic capabilities of a
selection of filamentous fungi of the Mycotheca Universitatis Tauri-
nensis (MUT), performed on model substrates in the aim of finding
new biocatalysts for synthetic applications.
In the case of benzoic acid, methyl benzoate, phenylacetic
acid, methyl phenylacetate, 3-phenylpropanoic acid, phenoxy-
acetic acid, methyl phenoxyacetate, 2-furoic acid, 2-(thiophen-2-
yl)acetic acid, methyl 2-(thiophen-2-yl)acetate, 2,4-hexadienoic
acid, methyl 2,4-hexadienoate, and methyl octanoate the isolation
of the final products was carried out. For benzoic acid, 2-(thiophen-
2-yl)acetic acid and 2,4-hexadienoic acid c0 = 1 mM was employed,
while for all the other substrates c0 = 5 mM was used (in both cases
the reaction total volume was 40 mL). After 72 h, the reaction mix-
ture was filtered to remove the biomass. The filtrate was extracted
with EtOAc (3 × 20 mL), dried on Na2SO4, and concentrated under
reduced pressure. When the conversion was not complete (GC/MS
analysis) the residue was purified either by treatment with a sat-
urated NaHCO3 solution or by column chromatography (silica gel,
elution with hexane and increasing amount of EtOAc).
2. Experimental
2.1. Materials and equipment
Benzyl alcohol: from benzoic acid (3.8 mg, 89%) and from
methyl benzoate (19.9 mg, 92%). 1H NMR (400 MHz, CDCl3, TMS):
ı = 7.35–7.25 (m, 5H, aromatic hydrogens), 4.67 (s, 2H, CH2). 13C
NMR (100 MHz, CDCl3, TMS): ı = 141.2, 128.3, 127.3, 126.9, 65.0.
GC/MS: tR = 6.46 min, m/z 108 (M+, 95), 91 (20), 79 (100).
1-Phenylethanol: from phenylacetic acid (9.0 mg, 37%) and from
methyl phenylacetate (6.3 mg, 26%). 1H NMR (400 MHz, CDCl3,
TMS): ı = 7.35–7.15 (m, 5H, aromatic hydrogens), 3.85 (t, 2H,
J = 6.6 Hz, PhCH2CH2OH), 3.85 (t, 2H, J = 6.6 Hz, PhCH2CH2OH). 13C
The carboxylic acids employed for the work were commer-
cial products purchased from Sigma–Aldrich. The corresponding
methyl and ethyl esters were either commercial products from
Sigma–Aldrich or were prepared by Fischer esterification. Stock
solutions (500 mM) of each substrate in DMSO were employed for
the biotransformations.
GC/MS analyses were performed on an Agilent HP 6890 gascro-
matograph equipped with a 5973 mass detector and an HP-5-MS

E. Brenna et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 83–88
85
Fig. 1. Percentage yields of the primary alcohols obtained by bioreduction of substituted benzoic acids (black bars) and methyl esters (white bars), determined by GC analysis
of the crude mixture after 72 h reaction time.
NMR (100 MHz, CDCl3, TMS): ı = 138.4, 129.3, 128.6, 126.5, 63.2,
39.4. GC/MS: tR = 8.19 min, m/z 122 (M+, 27), 91 (100), 65 (15).
3-Phenylpropanol: from 3-phenylpropanoic acid (18.4 mg, 68%).
1H NMR (400 MHz, CDCl3, TMS): ı = 7.25–7.10 (m, 5H, aromatic
hydrogens), 3.60 (t, 2H, J = 6.4 Hz, PhCH2CH2CH2OH), 2.65 (t, 2H,
J = 7.2 Hz, PhCH2CH2CH2OH), 1.83 (m, 2H, PhCH2CH2CH2OH). 13C
NMR (100 MHz, CDCl3, TMS): ı = 141.5, 128.4, 128.3, 125.9, 62.5,
34.4, 32.1. GC/MS: tR = 12.38 min, m/z 136 (M+, 20), 117 (100), 91
(95).
2-Phenoxyethanol: from phenoxyacetic acid (25.9 mg, 94%) and
from methyl phenoxyacetate (26.4 mg, 96%). 1H NMR (400 MHz,
CDCl3, TMS): ı = 7.28 (m, 2H, aromatic hydrogens), 6.93 (3H, m,
aromatic hydrogens), 4.08 (m, 2H, PhOCH2CH2OH), 3.94 (m, 2H,
PhOCH2CH2OH). 13C NMR (100 MHz, CDCl3, TMS): ı = 158.6, 129.4,
121.0, 114.6, 69.2, 61.2. GC/MS: tR = 11.82 min, m/z 138 (M+, 27), 94
(100), 77 (32).
3. Results and discussion
Filamentous fungi are
a huge group of organisms with
great biodiversity. They have heterogeneous, complex and a-
specific enzymatic patterns, which make them potentially valuable
reagents for biocatalysis. They are able to grow readily using simple
carbon sources and with respect to many bacteria they can work at
room temperature and pressure. S. racemosum belongs to Zygomy-
cota phylum and particularly the strain MUT 2770 was isolated
from air. Since it is not a human pathogen, there are no drawbacks
to its use both in laboratory and in industrial plants.
We carried out a preliminary investigation of the substrate scope
of S. racemosum reduction by using unsubstituted aromatic and
aliphatic carboxylic acids, in order to define the basic structural
scaffold of the substrate that could be accepted and transformed
by the fungus. The corresponding methyl and ethyl esters were
also employed as starting materials, in order to verify the possibil-
ity to slowly generate the carboxylic acid in the reaction medium
by enzymatic hydrolysis.
The reactions were performed by addition of the starting
compound (generally 5 mM final concentration in the reaction
medium) to a pre-grown culture of S. racemosum in MEA medium,
and incubated at 25 ◦C. The conversion values determined by
GC/MS analysis and the corresponding isolation yields are reported
in Table 1. Benzoic acid, 2-(thiophen-2-yl)acetic acid, and 2,4-
hexadienoic acid were not reduced when the starting substrate
concentration (c0) was 5 mM, whereas their conversion was
nearly complete when a lower substrate loading was employed
(c0 = 1 mM). The reaction occurred on aromatic, heteroaromatic
and aliphatic derivatives, either acids or methyl esters, whereas
ethyl esters were not transformed. Remarkably, the presence of
an electron-rich heteroaromatic ring does not prevent the reduc-
tion.After the preliminary screening, the effects on the bioreduction
due to the substituents of the aromatic ring were investigated.
The data of the biotransformations of substituted benzoic and
phenylacetic acids and of the corresponding esters are reported
in Figs. 1 and 2, revealing quite a broad tolerance towards non-
natural substrates. The methoxy group seems to be the substituent
of the molecular skeleton best tolerated by the fungus. The three
regioisomeric toluic acids and the corresponding methyl esters,
and the three nitrobenzoic acids and methyl esters were recov-
ered unreacted (data not shown in Fig. 1). On the contrary,
the methyl and nitro groups did not have a negative effect on
the reduction of phenylacetic acid derivatives. For this latter
Furan-2-ylmethanol: from 2-furoic acid (35.3 mg, 90%). 1H NMR
(400 MHz, CDCl3, TMS): ı = 7.38 (m, 1H, heteromatic hydrogen),
6.32 (m, 1H, heteroaromatic hydrogen), 6.27 (m, 1H, heteroaro-
matic hydrogen), 4.59 (s, 2H, CH2OH). 13C NMR (100 MHz, CDCl3,
TMS): ı = 153.6, 142.5, 110.5, 107.8, 57.0. GC/MS: tR = 3.92 min, m/z
98 (M+, 100), 81 (60), 63 (60).
2-(Thiophen-2-yl)ethanol: from 2-(thiophen-2-yl)acetic acid
(3.7 mg, 72%) and from methyl 2-(thiophen-2-yl)acetate (24.6 mg,
96%). 1H NMR (400 MHz, CDCl3, TMS): ı = 7.20 (m, 1H, heteroaro-
matic hydrogen), 6.99 (m, 1H, heteroaromatic hydrogen), 6.90 (m,
1H, heteroaromatic hydrogen), 3.85 (t, 2H, J = 6.2 Hz, CH2OH), 3.02
(t, 2H, J = 6.2 Hz, CH2CH2OH). 13C NMR (100 MHz, CDCl3, TMS):
ı = 140.5, 127.0, 125.8, 124.0, 63.4, 33.3. GC/MS: tR = 9.47 min, m/z
128 (M+, 30), 110 (5), 97 (100).
(E,E)-2,4-Hexadien-1-ol: from (E,E)-2,4-hexadienoic acid
(2.9 mg, 74%) and from methyl (E,E)-2,4-hexadienoate (4.51 mg,
23%). 1H NMR (400 MHz, CDCl3, TMS): ı = 6.15 (dd, 1H, J = 15.0,
10.5 Hz, CH = C), 5.95 (dd, 1H, J = 15.0, 10.5 Hz, CH = C), 5.59 (m,
2H, 2CH = C), 4.00 (d, 2H, J = 6.0 Hz, CH2OH), 1.68 (d, 2H, J = 6.2 Hz,
CH3C = ). 13C NMR (100 MHz, CDCl3, TMS): ı = 130.7, 129.5, 128.7,
125.6, 66.5, 17.4. GC/MS: tR = 5.04 min, m/z 98 (M+, 62), 83 (78), 55
(100).
1-Octanol: from methyl octanoate (9.10 mg, 35%). 1H NMR
(400 MHz, CDCl3, TMS): ı = 3.63 (t, 2H, J = 6.6 Hz, CH2OH), 1.56 (t,
2H, J = 6.5 Hz, CH2), 1.40-1.20 (m, 10H, 5 CH2), 0.88 (t, 3H, J = 7.1 Hz,
CH3). 13C NMR (100 MHz, CDCl3, TMS): ı = 63.1, 32.8, 31.8, 29.4,
29.2, 25.8, 22.6, 14.0. GC/MS: tR = 8.36 min, m/z 112 (M+–18, 5), 84
(53), 70 (70), 56 (100).

86
E. Brenna et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 83–88
Table 1
Bioreduction of unsubstituted aromatic and aliphatic carboxylic acids and estersa.
Substrate
Product
c0 (mM)b
Conversion (%)c
Isolation yield (%)d
PhCOOH
PhCOOH
PhCOOMe
PhCOOEt
PhCH2OH
PhCH2OH
PhCH2OH
–
5
1
5
5
0
>99
>99
0
–
89
92
PhCH2COOH
PhCH2COOMe
PhCH2COOEt
PhCH2CH2OH
PhCH2CH2OH
–
5
5
5
44
37
26
32 [47]e
0
PhCH2CH2COOH
PhCH2CH2COOMe
PhCH2CH2COOEt
PhCH2CH2CH2OH
–
–
5
5
5
73
0
0
68
PhOCH2COOH
PhOCH2COOMe
PhOCH2CH2OH
PhOCH2CH2OH
5
5
>99
>99
94
96
2-Furoic acid
Methyl 2-furoate
2-Furfurol
2-Furfurol
5
5
>99
8
90
2-(Thiophen-2-yl)acetic acid
2-(Thiophen-2-yl)acetic acid
Methyl 2-(thiophen-2-yl)acetate
2-(Thiophen-2-yl)ethanol
2-(Thiophen-2-yl)ethanol
2-(Thiophen-2-yl)ethanol
5
1
5
0
89
>99
–
72
96
Octanoic acid
Methyl octanoate
–
5
5
0
1-Octanol
45 [26]e
35
(E,E)-2,4-Hexadienoic acid
(E,E)-2,4-Hexadienoic acid
(E,E)-Methyl 2,4-hexadienoate
(E,E)-2,4-Hexadien-1-ol
(E,E)-2,4-Hexadien-1-ol
(E,E)-2,4-Hexadien-1-ol
5
1
5
0
>99
32
–
74
23
a
All experiments were performed as triplicates, deviations were always below 5% (see Section 2 for reaction conditions).
Starting substrate concentration.
Percentage yields of the primary alcohol, determined by GC analysis of the crude mixture after 72 h reaction time (no degradation or side-product formation was observed).
When conversion was not complete, purification by treatment with sat. NaHCO3 solution or column chromatography was performed.
The percentage of the carboxylic acid obtained by ester hydrolysis is reported in square brackets.
b
c
d
e
class, the chlorine atom either in ortho, meta or para position
(data not shown in Fig. 2) prevented the reaction, whereas this
inhibitory effect was decidedly of a lesser extent in the benzoic
acid series.
The time course of the reaction (Fig. 3a and b) was investi-
gated for phenoxyacetic acid and its methyl ester, which were both
completely reduced to the corresponding primary alcohol in 72 h
reaction time.
which probably diffuses across the cell membrane, and is then
transformed by reduction.
The results collected in Table 1 and in Figs. 1 and 2 highlight that
for most acid/ester pairs only the carboxylic acid was transformed.
In a few cases, both the acid and the methyl ester were converted
into primary alcohols. Benzoic acid and some other carboxylic acids
were recovered unreacted when c0 = 5 mM was employed, whereas
the corresponding methyl esters were reduced either totally or
partially. For the latter derivatives, the slow release of the acid
by hydrolysis in the fermentation medium likely allowed it to be
These data show that the hydrolysis of the ester occurs in
the reaction medium to generate in situ the carboxylic acid,
Fig. 2. Percentage yields of the primary alcohols obtained by bioreduction of substituted phenylacetic acids (black bars) and methyl esters (white bars), determined by GC
analysis of the crude mixture after 72 h reaction time.

E. Brenna et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 83–88
87
Scheme 1. Catalytic cycle for the reduction of benzoic acid to benzaldehyde by CAR isolated from Nocardia sp. NRRL 5646. HS-CAR represents phosphopantetheinylated CAR
with the free terminal thiol group (see Ref. [14]).
reduced before its concentration could reach the critical value pos-
sibly toxic or inhibitory for the fungus. To support this hypothesis,
the bioreduction of benzoic acid was investigated by decreasing the
substrate starting concentration. With c0 = 2.5 mM, 3% of benzalde-
hyde was observed after 24 h, and 97% of unreacted acid. The use of
c0 = 1 mM gave 28% of benzaldehyde, 63% of benzyl alcohol and 9%
of benzoic acid at 2 h reaction time, and complete conversion into
benzyl alcohol after 4 h.
The detection of benzaldehyde as an intermediate of benzoic
acid reduction is in agreement with the presence a CAR, whose
accepted reduction mechanism is reported in Scheme 1 [14].
The adenylation of benzoic acid occurs at the N-terminal
domain of the enzyme by reaction with ATP, yielding the activated
intermediate benzoyl-AMP and a pyrophosphoric acid unit. The
nucleophilic attack of the phosphopantetheine thiol on benzoyl-
AMP releases AMP and gives a covalently bound thioester. The
latter is then reduced by NADPH at the C-terminal domain, releas-
ing benzaldehyde, NADP+ and the free HS-CAR ready for another
reduction cycle.
In the investigations led on the reaction mechanism of CAR from
Neurospora crassa [6c], the formation of the activated intermediate
had been described to be inhibited by the presence of hydroxy-
lamine. The inhibition of the exchange reaction of benzoic acid
with ATP by hydroxylamine was confirmed also in the case of S.
racemosum: benzoic acid was not reduced when the reaction was
performed with 1 mM substrate concentration in the presence of
1 mM hydroxylamine.
4. Conclusions
The S. racemosum-catalysed reductions of carboxylic acids,
either employed as starting compounds or produced from esters
by enzymatic hydrolysis, shows relevant advantages over known
chemical methods. For example, the reduction of phenoxyacetic
acid is described in the literature according to the following pro-
cedures: (i) with stoichiometric amounts of either titanium(IV)
chloride and sodium borohydride (14 h) [15] or diisopropoxytita-
nium(III) tetrahydroborate (4 h) [16] at ambient temperature; (ii)
with lithium aluminum hydride in refluxing THF [17]; (iii) with
copper(II) triflate (16 h) at 80 ◦C [18]; (iv) by reaction of the corre-
sponding thiol ester with tetrabutylammonium borohydride (3 h)
in refluxing chloroform [19]; (v) by reduction of the 6-nitro-1-
(2-nitrophenylsulphonyloxy)benzotriazole derivative with sodium
borohydride [20]. In the case of methyl benzoate, besides tradi-
tional hydride reductions, procedures based on the use of molecular
hydrogen and ruthenium catalysts have been reported, most com-
monly at 100 ◦C and 50 bar [21].
The biotrasformation with S. racemosum whole cells system
has all those characteristics of diminished environmental bur-
den, reduced costs, and increased safety that are necessary to
Fig. 3. Time course of S. racemosum-catalysed reduction of phenoxyacetic acid (a)
and methyl phenoxyacetate (b).

88
E. Brenna et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 83–88
increment the sustainability of manufacturing processes [22]. It
does not require a controlled atmosphere: neither oxygen has to
be excluded, nor hydrogen has to be supplied. The reaction occurs
in water medium with a minimum amount of organic solvents, with
neither by-product formation, nor energy consumption for heating
systems. Non-hazardous materials are employed under safe reac-
tion conditions with reduced risk of exposure, explosions and fires,
and without production of toxic waste. The reaction is easily per-
formed on a semi-preparative scale (ca. 30 mg substrate), and the
work-up for product recovery requires only a simple extraction
procedure: neither any particular quenching, nor the filtration of
metal salts as byproducts is needed.
Work has to be done to increase the reaction throughput, by
optimising the fermentation conditions, and eventually isolating
the enzymes responsible for the transformation. The chemose-
lectivity of the biocatalysed procedure has to be evaluated on
dicarboxylic acid derivatives and on substrates showing other func-
tional groups, in order to define the synthetic potential of the
reduction.
This kind of reaction enriches the enzymatic toolbox [23] which
is made available to organic synthetic chemists, and which is
still very scarce in the supply of biocatalysts for the reduction of
carboxylic acids into primary alcohols. It can be considered for
the development of green processes [24] for the biotechnological
conversion of fatty acids, recovered from biomasses, into fatty alco-
hols, which are chemical commodities currently employed on a
large scale for the production of fragrances, emollients, detergents,
and food additives. S. racemosum-catalysed reduction can be also
employed for the preparation of natural flavors and fragrances,
because it satisfies the requisites established by European regu-
lations for the natural labelling of the corresponding products.
[3] (a) P.A. Dub, T. Ikariya, ACS Catal. 2 (2012) 1718–1741;
(b) S. Werkmeister, K. Junge, M. Beller, Org. Process Res. Dev. 18 (2014)
289–302.
[4] S. Das, K. Möller, K. Junge, M. Beller, Chem. Eur. J. 17 (2011) 7414–7417.
[5] K. Napora-Wijata, G.A. Strohmeier, M. Winkler, Biotechnol. J. 9 (2014) 822–843.
[6] (a) G.G. Gross, M.H. Zenk, Eur. J. Biochem. 8 (1969) 413–419;
(b) G.G. Gross, M.H. Zenk, Eur. J. Biochem. 8 (1969) 420–425;
(c) G.G. Gross, Eur. J. Biochem. 31 (1972) 585–692.
[7] (a) T. Li, J.P.N. Rosazza, J. Bacteriol. 179 (1997) 3482–3487;
(b) T. Li, J.P.N. Rosazza, J. Ind. Microbiol. Biotechnol. 25 (2000) 328–332;
(c) A. He, T. Li, L. Daniels, I. Fotheringham, J.P.N. Rosazza, Appl. Environ. Micro-
biol. 70 (2004) 1874–1881;
(d) P. Venkitasubramanian, L. Daniels, J.P.N. Rosazza, in: R.N. Patel (Ed.), Bio-
catalysis in the Pharmaceutical and Biotechnology Industries, CRC Press, Taylor
& Francis Group, Boca Raton, 2006.
[8] K. Napora-Wijata, K. Robins, A. Osorio-Lozada, M. Winkler, ChemCatChem 6
(2014) 1089–1095.
[9] M.K. Akhtar, N.J. Turner, P.R. Jones, Proc Natl. Acad. Sci. U.S.A. 110 (2013) 87–92.
[10] G. Fronza, C. Fuganti, P. Grasselli, S. Servi, G. Zucchi, M. Barbeni, J. Chem. Soc.
Chem. Commun. (1995) 439–450.
[11] (a) H. Simon, H. White, H. Lebertz, I. Thanos, Angew. Chem. 99 (1987) 785–787;
(b) L. Fraisse, H. Simon, Arch. Microbiol. 150 (1988) 381–386;
(c) H. White, G. Strobl, R. Feicht, H. Simon, Eur. J. Biochem. 184 (1989) 89–96;
(d) H. White, C. Huber, R. Feicht, H. Simon, Arch. Microbiol. 159 (1993) 244–249.
[12] (a) E.C.D. van den Ban, H.M. Willemen, H. Wassink, C. Laane, H. Haaker, Enzym.
Microb. Technol. 25 (1999) 251–257;
(b) Y. Ni, P.-L. Hagedoorn, J.-H. Xu, I.W.C.E. Arends, F. Hollmann, Chem. Com-
mun. 48 (2012) 12056–12058;
(c) Y. Ni, P.-L. Hagedoorn, J.-H. Xu, I.W.C.E. Arends, F. Hollmann, J. Mol. Catal. B:
Enzym. 103 (2014) 52–55.
[13] R. Villa, F. Molinari, J. Nat. Prod. 71 (2008) 693–696.
[14] P. Venkitasubramanian, L. Daniels, J.P.N. Rosazza, J. Biol. Chem. 282 (2007)
478–485.
[15] S. Kano, Y. Tanaka, E. Sugino, S. Hibino, Synthesis (1980) 695–697.
[16] S. Chandrasekaran, R. Ravikumar, J. Org. Chem. 61 (1996) 826–828.
[17] X. Cheng, T. Morton, V. Nguyen, J. Am. Chem. Soc. 114 (1992) 7127–7132.
[18] G. Chen, Y. Zhang, W. Dayoub, M. Lemaire, Y. Zhang, Tetrahedron 68 (2012)
7400–7407.
[19] H. Liu, W. Luo, Synth. Commun. 19 (1989) 387–392.
[20] T. Okawara, N. Ikeda, T. Yamasaki, M. Furukawa, Mitsuru, Chem. Pharm. Bull.
36 (1988) 3628–3631.
[21] See for example: K. Aoki, Y. Ino, K. Ishida, T. Kobayashi, W. Kuriyama, T. Mat-
sumoto, O. Ogata, T. Saito, N. Sayo, S. Tanaka, Org. Process Res. Dev. 16 (2012)
166–171.
References
[22] C. Jiménez-González, D.J.C. Constable, C.S. Ponder, Chem. Soc. Rev. 41 (2012)
1485–1498.
[23] B.M. Nestl, S.C. Hammer, B.A. Nebel, B. Hauer, Angew. Chem. Int. Ed. 53 (2014)
3070–3095.
[1] P.G. Andersson, I.J. Munslow (Eds.), Modern Reduction Methods, Wiley-VCH,
Weinheim, 2008;
J. Magano, J.R. Dunetz, Org. Process Res. Dev 16 (2012) 1156–1184.
[2] J. Seyden-Penne, Reductions by Alumino and Borohydrides in Organic Synthe-
sis, 2nd ed., Wiley, New York, 1997.
[24] R.A. Sheldon, Green Chem. 16 (2014) 950–962.