Efficient chemoselective biohydrogenation of 1,3-diaryl-2-propen-1-ones catalyzed by Saccharomyces cerevisiae yeasts in biphasic system

Article abstract of DOI:10.1016/j.molcatb.2010.01.010

A series of chalcones (1-9) was synthesized by base catalyzed aldol condensation with 50-94% yields. These α,β-unsaturated carbonyl compounds were used as substrates in biotransformation reactions mediated by three industrial Saccharomyces cerevisiae strains in biphasic systems. Several reaction parameters were evaluated, such as yeast concentration, temperature, pH, substrate concentration, organic solvent, volume of aqueous and organic phases and the influence of substituent groups on chalcones 1-9. The biotransformation was chemoselective and formed only the corresponding saturated ketones. The highest conversion (>99%) to the dihydrochalcone was obtained at 30-45°C and pH above 5.5, while the cellular and substrate concentrations also showed a strong influence on the biohydrogenation reaction. Organic solvents with log. P >3.2 (hexane or heptane) were the most appropriate, and 40-80% of aqueous phase allowed the highest conversions probably by maintaining the yeast enzymes catalytically active. The influence of substituents on rings A and B of chalcones 1-9 was low and no correlation between the donor or withdrawing electron groups was observed.

Full text of DOI:10.1016/j.molcatb.2010.01.010

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 157–163
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Efficient chemoselective biohydrogenation of 1,3-diaryl-2-propen-1-ones
catalyzed by Saccharomyces cerevisiae yeasts in biphasic system
Vanessa Dutra Silvaa, Boris Ugarte Stambukb, Maria da Grac¸ a Nascimentoa,∗
a Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis 88040-900, Brazil
b Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis 88040-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2009
Received in revised form 7 January 2010
Accepted 8 January 2010
Available online 18 January 2010
A series of chalcones (1–9) was synthesized by base catalyzed aldol condensation with 50–94% yields.
These ␣,␤-unsaturated carbonyl compounds were used as substrates in biotransformation reactions
mediated by three industrial Saccharomyces cerevisiae strains in biphasic systems. Several reaction param-
eters were evaluated, such as yeast concentration, temperature, pH, substrate concentration, organic
solvent, volume of aqueous and organic phases and the influence of substituent groups on chalcones 1–9.
The biotransformation was chemoselective and formed only the corresponding saturated ketones. The
highest conversion (>99%) to the dihydrochalcone was obtained at 30–45 ◦C and pH above 5.5, while the
cellular and substrate concentrations also showed a strong influence on the biohydrogenation reaction.
Organic solvents with log P >3.2 (hexane or heptane) were the most appropriate, and 40–80% of aqueous
phase allowed the highest conversions probably by maintaining the yeast enzymes catalytically active.
The influence of substituents on rings A and B of chalcones 1–9 was low and no correlation between the
donor or withdrawing electron groups was observed.
Keywords:
␣,␤-Unsaturated carbonyl compounds
Saccharomyces cerevisiae
Biphasic system
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
cate that the microbiological reduction of these compounds in
many cases gives a mixture of saturated ketone or aldehyde, sat-
Whole cells of various microorganisms have been employed
to catalyze the asymmetric reduction of ketones, aldehydes, ␤-
ketoesters, imines and ␣,␤-unsaturated systems with C C bonds
activated by strongly polarizing groups, such as nitro, carbonyl,
or hydroxyl groups [1–8]. Whole cells are useful tools in the
production of chiral synthons as they are able to catalyze reac-
tions exhibiting high chemo-, regio- and enantioselectivity [9,10].
The application of whole cells (growing or resting cells) in the
reduction reactions has clear advantages, as the separation of
enzyme and the regeneration of NADH are no longer required [11].
These biocatalysts are also able to work under mild conditions,
such as room temperature and atmospheric pressure, minimizing
problems of isomerization, racemization, epimerization and rear-
rangement that may occur during traditional chemical catalysis.
Therefore, biocatalytic asymmetric synthesis attracts considerable
attention due to its simple, cheap and benign methodologies that
combine green chemistry with high efficiency [12–14].
urated alcohol or allylic alcohol, indicating that several enzymes
may catalyze the reduction of C C and C O double bonds competi-
tively. For example, the reduction of 2-ethylhexen-2-enal mediated
by baker’s yeast in aqueous media gave a mixture of the corre-
sponding saturated alcohol and allylic alcohol in a ratio of 46:54
[19]. In the case of the reduction of an ␣,␤-disubstituted enone
(3-methyl-4-phenyl-3-buten-2-one) mediated by whole cells of
Rhodotorula rubra M18D3 yeast in a 2% glucose solution the cor-
responding (S)-saturated chiral ketones were the major products
with 32–49% conversion yields; while the corresponding saturated
and unsaturated alcohols were produced in small amounts [20].
Rodrigues et al. used Phicia stipitis CCT 2617 yeast adsorbed on
Amberlite XAD-7 for the reduction in aqueous media of an acyclic
␣-methyleneketone (2-ethyl-1-phenylprop-2-en-1-one), obtain-
ing the corresponding (S)-unsaturated alcohol as the single product
(eep >99%) with a conversion yield of 65% [21].
As shown above, water is the natural solvent of choice for
whole-cell biocatalysis. However, performing the reduction of ␣,␤-
unsaturated carbonyl compounds with whole cells in water has
several drawbacks, including low solubility of organic substrates
and products, undesired side reactions such as hydrolysis, and dif-
ficult separation of products [22]. One of the technical solutions to
this problem is the application of two liquid-phase reaction systems
in which the substrate solubilizes in the organic solvent [23,24], and
Quite a large number of papers and reviews have been pub-
lished describing the microbiological reduction of ␣,␤-unsaturated
carbonyl compounds [15–18]. The available literature data indi-
∗
Corresponding author. Tel.: +55 48 37216844; fax: +55 48 37216850.
E-mail address: graca@qmc.ufsc.br (M.G. Nascimento).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.01.010

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V.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 157–163
Scheme 1. The biohydrogenation of chalcones 1–9 catalyzed by S. cerevisiae.
thus this organic phase reservoir permits the storage of a high con-
centration of substrates or products. Furthermore, this approach
allows in situ removal of the products, thus improving the over-
all process productivity [25]. The disadvantage of organic–aqueous
biphasic systems is the effect of solvent toxicity on the activity and
stability of the biocatalyst [26]. In whole-cell systems, the solvent
might be incorporated within the membrane lipids, which results in
the disruption of membrane functions, inactivation or denaturation
of membrane-bound enzymes, collapse of transport mechanisms,
or even complete cell lysis [25,27]. Therefore, one of the key factors
in the implementation of an effective aqueous–organic bioconver-
sion is the selection of an appropriate solvent, which has to be
biocompatible, to provide an adequate substrate pool and prod-
uct sink, exhibiting the required mass transfer characteristics. An
empirical rule, correlating the toxicity of the organic solvent with
its log P values, is in current use, and the log P values are considered
a measure of the solvent hydrophobicity. However, this correlation
is still not completely understood and does not hold true for all
organic solvents [28–31].
In this report whole cells of Saccharomyces cerevisiae were
used for the biohydrogenation of the activated double bonds
of chalcones in a biphasic system to obtain the correspond-
ing dihydrochalcones. These compounds belong to the flavonoid
family and display an impressive array of biological activities
such as anti-malarial, anti-cancer, anti-tuberculosis, cardiovascu-
lar, anti-leishmanial, anti-hyperglycemic, anti-inflammatory and
anti-fungal [32–35]. The operational conditions for the biohydro-
genation of chalcone 1, such as amount of biocatalyst, reaction
temperature, pH values, substrate concentration, organic solvents,
and effect of the volume ratio of the aqueous phase to the organic
phase (Vaq/Vorg), were analyzed to improve the conversion into
the corresponding dihydrochalcones. The influence of substituent
groups on both aromatic rings of chalcones 2–9 was also examined
(Scheme 1).
2.2. Chemicals
The following chemicals were used as received. Ben-
zaldehyde (>99%), and potassium phosphate buffer were
purchased from Vetec; 3ꢀ,4ꢀ-(methylenedioxy)acetophenone
(>98%),
3,4-(methylenedioxy)benzaldehyde
(>99%),
3,4-
dimethoxybenzaldehyde (>99%), 3,4-dimethoxyacetophenone
(>98%), and 4-methoxybenzaldehyde (>98%) from Aldrich-Sigma;
acetophenone (>98%) was purchased from Riedel-di-Haen; 4ꢀ-
methoxyacetophenone (>98%) and sodium borohydride from
Merck; 4-nitroacetophenone (97%), 4-nitrobenzaldehyde (>98%)
and citric acid from Acros; sodium hydroxide from Grupo Química
and deuterium chloroform and acetone-d6 from Cambridge Isotope
Laboratories. All organic solvents were obtained from commercial
sources and were analytical grade.
2.3. Preparation of 1,3-diaryl-2-propen-1-ones 1–9
The 1,3-diaryl-2-propen-1-ones 1–9 (Scheme 1) were prepared
by aldol condensation in a medium with 50% (m/v) KOH using
equimolar amounts of substituted benzaldehyde with substituted
acetophenones according to the typical procedures described in the
literature [34]. The yields varied from 60 to 94%.
(2E)-1,3-diphenyl-2-propen-1-one (1): 3.5 g, 80% yield, m.p.
53–54 ◦C (55–56 ◦C) [39];
(2E)-1-(4-methoxyphenyl)-3-phenyl-2-propen-1-one (2): 3.5 g,
70% yield, m.p. 100 ◦C (104–105 ◦C) [33];
(2E)-1-(3,4-dimethoxyphenyl)-3-phenyl-2-propen-1-one
4.1 g, 72% yield, m.p. 82–83 ◦C (85–87 ◦C) [40];
(2E)-1-(1,3-benzodioxol-5-yl)-3-phenyl-2-propen-1-one
4.3 g, 78% yield, m.p. 97 ◦C (97–98 ◦C) [41];
(3):
(4):
(2E)-1-(4-nitrophenyl)-3-phenyl-2-propen-1-one (5): 3.2 g, 60%
yield, m.p. 146–147 ◦C (145–147 ◦C) [40];
(2E)-3-(4-methoxyphenyl)-1-phenyl-2-propen-1-one (6): 3.1 g,
62% yield, m.p. 70 ◦C (75–77 ◦C) [39];
2. Experimental
(2E)-3-(3,4-dimethoxyphenyl)-1-phenyl-2-propen-1-one
4.7 g, 84% yield, m.p. 83–84 ◦C (87–89 ◦C) [40];
(2E)-3-(1,3-benzodioxol-5-yl)-1-phenyl-2-propen-1-one
4.7 g, 94% yield, m.p. 118 ◦C (117 ◦C) [39];
(7):
2.1. Microorganisms
(8):
Three commercial S. cerevisiae yeast strains were used in their
“active-dry-yeast” form: a baker’s yeast (BY) from Fleischmann,
and two industrial yeast strains (CAT-1 and PE-2) which are cur-
rently being used for efficient ethanol production in Brazil [36].
These two industrial fuel ethanol yeasts have been commercially
available since the late 1990s, distributed initially by Lallemand Inc.
from Canada [37], and more recently by LNF Latino Americana Ltda.
from Brazil [38]. The pellets of dehydrated yeast cells were directly
used in the biohydrogenation reactions as described below.
(2E)-3-(4-nitrophenyl)-1-phenyl-2-propen-1-one (9): 3.1 g, 84%
yield, m.p. 160 ◦C (160–163 ◦C) [39].
2.4. Preparation of racemic alcohols
The racemic alcohols were prepared from the chalcones 1–9 by
reduction with NaBH4 as described on the literature and were used
as standards on the 1H NMR and chiral gas chromatography analysis
[42,43]. For example, the racemic alcohol (R,S)-1,3-diphenyl-2-

V.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 157–163
159
propen-1-ol was obtained in 84% yield, after purification. m.p.
49 ◦C (56–57 ◦C) [43]. 1H NMR (CDCl3, 400 MHz) ␦ (ppm) 7.26–7.40
(10H, m), 6.66 (1H, dd), 6.40 (1H,m), 5.14 (1H,m), 1.61 (1H, s).
IR (KBr) cm−1 3345, 3027, 1599, 1493, 1450, 1010, 966, 746, 696.
Chiral-GC tR 18.1 and 18.9 min.
2.5. Biohydrogenation reactions
The biohydrogenation of 1,3-diaryl-2-propen-1-ones 1–9 in a
biphasic system was performed in a 125-mL shake flask. In general
2–6 g of active dry yeast were suspended in 0–60 mL of potas-
sium phosphate/citric acid buffer (adjusted to the desired pH), and
0–60 mL of organic solvent containing 0.5–5.0 mmol of substrate
was added, being the total volume of 60 mL (and Vaq/Vorg expressed
in %). The reaction mixture was incubated at 25–50 ◦C with con-
stant magnetic stirring. Aliquots were withdrawn at specified time
intervals from the reaction mixture, extracted with diethyl ether
(2× 10 mL), and analyzed by gas chromatography to evaluate the
percentage of conversion. Substrate and product peak areas were
compared, and the sum of both areas was considered as 100%.
The dihydrochalcone 10, was prepared using 6 g of active dry
yeast, 1.0 mmol of chalcone 1 in 30 mL of n-hexane and 30 mL of
potassium phosphate/citric acid buffer (Vaq/Vorg 1:1%), at pH 5.5
and 35 ◦C. After 3 h reaction the mixture was extracted with diethyl
ether (4× 20 mL), and after the solvent evaporation the crude prod-
uct was crystallized in absolute ethanol forming 10 in 85% yield. 1
H
NMR (CDCl3, 400 MHz) ␦ (ppm) 7.24–8.00 (10H, m), 3.33 (2H, t),
3.16 (2H,t). IR (KBr) cm−1 2922, 2853, 1450, 1171, 741, 694. Chiral-
GC tR 14.4 min.
2.6. Analytical methods
The reactions were monitored by thin-layer chromatogra-
phy (TLC) using n-hexane:ethyl acetate (9:1, v/v) as the eluent,
and a gas chromatograph (GC-14B Shimadzu) equipped with
a
chiral column (CP-chirasil-Dex CB, packed ␤-cyclodextrin,
25 m × 0.25 mm × 0.25 mm, CHROMOPACK) and H2 was used as
the carrier gas with a pressure of 75 kPa. The temperatures of
the injector and detector were 250 ◦C and 275 ◦C, respectively. A
column was set to temperature ramps of 100–150 ◦C (10 ◦C/min)
and 150–200 ◦C (5 ◦C/min). The retention times of dihydrochalcone
10, chalcone 1 and the corresponding racemic alcohol (R,S)-
1,3-diphenyl-2-propen-1-ol were 14.4, 17.3 and 18.1–18.9 min,
respectively. The substrate and products were fully characterized
by infrared spectra (PerkinElmer FT-IR 1600) and 1H NMR (Varian
AC 400F-400 MHz). Melting points were determined on a Micro-
química melting point apparatus and were not corrected.
Fig. 1. Time course of (2E)-1,3-diphenyl-2-propen-1-one biohydrogenation in a
water/n-hexane biphasic system using 33.3 g/L (ꢀ), 66.7 g/L (᭹), and 100 g/L (ꢁ) of
whole yeast cells of BY (A), CAT-1 (B), and PE-2 (C). Reaction conditions: 208.0 mg
(1 mmol) substrate, 30 mL potassium phosphate/citric acid buffer (0.2 M/0.1 M, pH
4.5), 30 mL n-hexane (Vaq/Vorg 1:1%), 25 ◦C.
3. Results and discussion
We initially synthesized a series of 1,3-diaryl-2-propen-1-ones
by base catalyzed aldol condensation, and applied them in bio-
hydrogenation reactions using S. cerevisiae cells in water/organic
solvent biphasic systems. Biphasic systems, consisting of an aque-
ous phase and water immiscible organic phase, are often chosen to
avoid the inhibition of the reaction by substrate and products that
may occur in aqueous systems [24,44]. Three commercial active
dry yeasts were used, including a baker’s yeast (BY) and two fuel
ethanol strains (CAT-1 and PE-2) selected for their excellent yields
and high tolerance towards industrial stresses [36].
The biohydrogenation of (2E)-1,3-diphenyl-2-propen-1-one (1)
in a water/n-hexane biphasic system by all three yeasts was
completely chemoselective, producing only the corresponding
saturated ketone 1,3-diphenyl-1-propanone (10). The choice of n-
hexane to be used as organic solvent was based on our previous
results on the reduction of ethyl acetoacetate by yeasts, and some
otherliteraturedata[4,11,27]. No alcoholwasdetected by chiral-GC
analysis, even after 168 h of reaction, and the dihydrochalcone pro-
duced was also confirmed by IR and 1H NMR analysis. In fact, the
only known report describing the hydrogenation of chalcones by
microorganisms also obtained the corresponding saturated ketone
when the reaction was catalyzed by Corynebacterium equi IFO3730
in aqueous medium [45,46].
Fig. 1 shows the conversion rates of 1 into 10 as a function of time
and yeast cell concentration. The initial rates increased with the cell
concentration, and the highest values were obtained using 100 g/L
of whole yeast cells. All three biocatalysts were capable of similar
and highly efficient reduction of this chalcone, with initial reac-
tion rates of 6.6, 7.0 and 7.6 × 10−5 mmol L−1 min−1 for strains BY,
CAT-1 and PE-2, respectively. Considering these results, the reac-

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V.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 157–163
tion time of 3 h and a cell concentration of 100 g/L of BY, CAT-1 and
PE-2 were used for the subsequent experiments aiming at the opti-
mization of other operational conditions for the biohydrogenation
of (2E)-1,3-diphenyl-2-propen-1-one mediated by whole cells of
S. cerevisiae. After defining the best reaction conditions, a study of
substituent effects on both rings A and B of the chalcones 1–9 was
also performed.
and the highest conversions into product (>99%) were achieved at
temperatures between 35 and 45 ◦C (Fig. 2B). The different conver-
sions obtained, especially at 30 ◦C, may reflect a better performance
of the industrial yeast strains PE-2 and CAT-1 at this temperature
[36]. Nevertheless, a further increase in temperature led to a drop
in the conversion into 10, especially for PE-2, indicating some loss
of the yeast cell activity at higher temperatures.
Similar results were obtained by Matsuda et al. in the asymmet-
ric reduction of acetophenone in aqueous solution using free or
immobilized cells of Geotrichum candidum NBRC 5767 with yields
of 85–89% (eep >99%) at temperatures of 30–40 ◦C [50]. From these
results a temperature of 35 ◦C was considered as appropriate for
the biohydrogenation of 1 into its product 10 by whole yeast cells
in water/organic solvent biphasic systems.
3.1. Effect of substrate concentration
Some previous reports have demonstrated that a change in the
substrate concentration in the medium may affect the reaction rate,
and sometimes the stereoselectivity of a particular process, as sub-
strate inhibition might occur at different substrate concentrations
for different enzymes present in the yeasts [47,48]. Thus, different
substrate concentrations ranging from 8.3 to 83.3 mM were used in
the biohydrogenation of 1 in the n-hexane/water biphasic system
mediated by S. cerevisiae yeasts.
3.3. Effect of aqueous medium pH
Another important parameter which must be evaluated in bio-
catalyzed reactions is the pH of the reaction medium, as all enzymes
have an optimal pH at which the reaction rate is maximized. Devi-
ations in pH from the optimal value can lead to decreased activity
due to changes in the ionization of amino acid residues at the active
site of the enzyme, while larger deviations in pH leads to denatura-
tion of the enzyme protein itself. Therefore, pH is among the most
significant factors affecting enzyme-catalyzed reactions [47,49,51].
The effect of the pH values of the aqueous medium for the reduc-
tion of (2E)-1,3-diphenyl-2-propen-1-one (1) in a water/n-hexane
biphasic system was studied in a pH range from 3.5 to 13.0 (Fig. 3A).
The conversion into 1,3-diphenyl-1-propanone (10) mediated by
yeasts was strongly inhibited at acidic pH (pH 3.5), but increased
significantly at pH 4.5, and reached maximum values (>99% con-
version) at pH 5.5 or higher (Fig. 3A), using any of the three yeast
strains. As described previously, deviations in pH from the opti-
mal values can decrease the activity and the conversion degrees
into products, but this effect was not observed in this reaction
mediated by yeast cells. These results indicating a wide range of
stability in relation to pH (especially at basic pH) are of great inter-
est, and might reflect the high stress tolerance that these industrial
yeasts have [36]. A buffer with pH 5.5 was considered to be the
optimum condition for the biohydrogenation of chalcone 1 into its
corresponding saturated ketone 10 mediated by yeast cells.
As observed in Fig. 2A, the conversions into 10 remained
constant as the substrate concentration increased from 8.3 to
25 mM, forming the product at >99% conversion. At higher sub-
strate concentrations, however, the conversion into 10 decreased,
especially in the case of BY yeast cells, but still reached conver-
sions of 75 and 77% at the highest substrate concentration tested
using CAT-1 or PE-2 yeasts, respectively. Wang et al. described a
similar effect of substrate concentration on the asymmetric reduc-
tion of 4ꢀ-methoxyacetophenone with immobilized Rhodotorula sp.
AS22.2241 cells in aqueous buffer, where an increase in substrate
concentration above 40 mM led to a marked drop in the maximum
substrate conversions [44].
3.2. Effect of temperature
The temperature in an enzyme-catalyzed process may increase
the reaction rate because a higher temperature accelerates molec-
ular collisions between the enzyme and the substrate, but
inactivation of the enzyme at higher temperatures can also
occur [49]. To evaluate the influence of temperature on the bio-
hydrogenation of (2E)-1,3-diphenyl-2-propen-1-one (1) in the
water/n-hexane biphasic system with yeast cells, the temperature
of the reaction was varied from 20 to 50 ◦C.
When the temperature increased from 20 to 35 ◦C, the forma-
tion of product 10 mediated by the yeast cells increased slightly,
Lou et al. described a similar pH effect in the biosynthesis of
optically active organosilyl alcohol via asymmetric reduction of acyl
Fig. 2. Influence of substrate concentration (A) and temperature (B) on the biohydrogenation of (2E)-1,3-diphenyl-2-propen-1-one in water/n-hexane biphasic system
mediated by whole yeast cells of BY (ꢀ), CAT-1 (ꢁ) and PE-2 ( ). Reaction conditions as given in Fig. 1, except for cell concentration (100 g/L), time (3 h), and the indicated
substrate concentrations or temperatures. In the case of (A) a pH of 5.5 and temperature of 35 ◦C were used.

V.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 157–163
161
Fig. 3. Influence of buffer pH (A) and of the percentage of volumetric buffer aqueous phase (B) on the reduction of (2E)-1,3-diphenyl-2-propen-1-one in water/n-hexane
biphasic system mediated by whole yeast cells of BY (ꢀ), CAT-1 (ꢁ) and PE-2 ( ). Reaction conditions as in Fig. 2A but with the indicated buffer pH (A), or volumetric buffer
percentage (B), and 1 mmol substrate.
silane catalyzed by immobilized S. cerevisiae mutant cells, where
high conversions (83–95%) and eep (93–96%) were obtained also in
a broad pH range (pH 5.5–9.0) [47].
known, log P denotes the hydrophobicity of the organic solvents
[28,29].
Fig. 4 shows that the conversion in 1,3-diphenyl-1-propanone
(10) mediated by yeast cells increased with an increase in the log P
value of the organic solvent. The highest conversion rates (99%)
were achieved using n-heptane (log P 4.0), but n-hexane (log P 3.5)
or cyclohexane (log P 3.2) also proved to be suitable solvents for
this reaction, forming 10 with 98% conversion. Using toluene (log P
2.5), only moderate conversions were obtained, while using sol-
vents with log P values lower than 2, such as chloroform (2.0) or
dichloromethane (1.5), the product was not formed. Probably cell
membranes are easily destroyed by organic solvents with low log P
values, and this autolysis of the yeast cells will certainly limit the
biocatalytic process [11,24,48,53].
The order of the solvents in which the highest
conversions were obtained was as follows: n-heptane > n-
hexane > cyclohexane > toluene. This data is also in agreement
with those reported for the baker’s yeast reduction of ethyl
acetoacetate in organic–aqueous biphasic systems, with complete
conversions in 24 h with eep of 92.1% in a water/n-hexane system
[4,48].
3.4. Effect of volumetric buffer phase ratio (Vaq/Vorg
)
In an aqueous–organic solvent biphasic system, the volumetric
phase ratio influences the interfacial area, which in turn affects bio-
transformation rates [52]. Generally speaking, enzymes and active
cells may be inactivated once in direct contact with organic solvents
due to their toxicity [47]. Thus, the effect of the volume ratio of the
aqueous phase to the organic phase (Vaq/Vorg) on the biohydro-
genation of 1 catalyzed by BY, CAT-1 and PE-2 strains was studied
in water/n-hexane biphasic system with varying the Vaq/Vorg values
maintaining a total volume of 60 mL (Fig. 3B).
The volumetric percentage of the aqueous buffer phase to the
organic solvent phase (Vaq/Vorg, %) substantially affected the con-
version of 1 into 10. The conversions increased significantly with
an increase in the Vaq/Vorg, reaching higher levels in the range
of 50–80% Vaq/Vorg (Fig. 3B). A further rise in the Vaq/Vorg ratio
led to a small decrease in the reduction of 1, possibly owing to
the lower substrate solubilization in the aqueous phase. Similar
results were obtained by Cruz et al. for the whole-cell bioconver-
sion of ␤-sitosterol in aqueous–organic two-phase systems [23],
and by Bie et al. in the bioconversion of methyltestosterone by
Arthobacter simplex AS.1.94 [53]. A volumetric phase ratio of 50%
(corresponding to 30 mL of each solvent) was considered to be the
optimum condition under which a >99% conversion of 1 to 10 was
obtained using yeast cells in the water/organic solvent biphasic
system.
3.5. Influence of organic solvents
The feasibility of biphasic bioconversion systems is dependent
both on the organic phase toxicity and on its ability to act as a
reservoir for the substrate and product [28]. The organic solvent
mediates substrate and product partitioning between the organic
phase and aqueous phase, which is beneficial to maintain a high
catalytic activity of the cells present in the aqueous phase, par-
ticularly when the solvent has a low toxicity toward the microbe
[11,24]. In order to study this effect, a series of organic solvents
was chosen covering a wide range of log P values, with special
emphasis on those which are hydrophobic and are often assumed
to be more biocompatible in the biocatalytic process. As is well
Fig. 4. Reduction of (2E)-1,3-diphenyl-2-propen-1-one catalyzed by whole yeast
cells of BY (ꢀ), CAT-1 (ꢁ), or PE-2 ( ) in water/organic solvent biphasic sys-
tems containing either dichloromethane (log P = 1.5), chloroform (2.0), toluene (2.5),
cyclohexane (3.2), n-hexane (3.5), or n-heptane (4.0) as organic solvents. Reaction
conditions as in Fig. 2A except for a substrate concentration of 16.7 mM.

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V.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 157–163
Table 1
Chemoselective
4. Conclusions
biohydrogenation
of
chalcones
with
yeasts.a
This is the first report related to the use of whole cells of three S.
cerevisiae industrial yeast strains for the biohydrogenation of 1,3-
diaryl-2-propen-1-ones (1–9) in water/organic solvent biphasic
systems, producing the corresponding saturated ketones (10–19).
After optimizing the reaction conditions, ketone 10 was obtained
with high conversions (>99%). The influence of substituent groups
on rings A and B of chalcones 1–9 was, in general, small and no
correlation between the donor or withdrawing electron groups
was observed. The use of microorganisms as biocatalysts in the
biotransformation of chalcones is an important strategy to obtain
dihydrochalcones with high chemoselectivity, under mild reaction
conditions, using low cost reagents and with low environmental
pollution.
.
Chalcones
R1
R2
Conversion (%)b
BY
CAT-1
PE-2
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
79
49
57
74
62
97
16
64
94
92
56
30
91
45
97
2
96
50
40
91
47
94
8
4-OCH3
3,4-di-OCH3
3,4-(OCH2O)
4-NO2
H
H
H
H
4-OCH3
3,4-di-OCH3
3,4-(OCH2O)
4-NO2
49
74
47
63
Acknowledgements
a
Reaction conditions: 16.7 mM substrate, 100 g/L yeasts, 30 mL potassium phos-
phate/citric acid buffer (0.2 M, 0.1 M pH 5.5), 30 mL n-hexane (Vaq/Vorg 1:1%), 35 ◦C,
This study was supported in part by Universidade Federal
de Santa Catarina, and the Brazilian funding agencies CNPq
(#479812/2006-3), CAPES, and FAPESP (#04/10067-6). We are also
grateful to Fermentec Ltda (Brasil) for the donation of yeast strains
CAT-1 and PE-2.
1 h.
b
Determined by chiral-GC.
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