Improved Biocatalytic Activity of the Debaryomyces Species in Seawater

Article abstract of DOI:10.1002/cctc.201900558

Biocatalysis is an environmentally friendly strategy widely used in the chemical and pharmaceutical industries. One of its approaches consists in utilizing whole cells, which usually involves the consumption of large quantities of water. Oceans cover a large part of the Earth's surface, and constitute an important reservoir that can be used as an alternative to freshwater in chemical reactors. This work analyzed the behavior of several yeast halotolerant species belonging to genera Debaryomyces and Schwanniomyces in both freshwater and seawater. It concluded that these microorganisms were more resistant to organic solvents/compounds in the second medium. Their metabolic activity was also greater, which made the reduction reaction of several prochiral ketones more effective. Besides, yeasts cells displayed better performance in subsequent recycling steps, which could help reduce the costs of the process.

Full text of DOI:10.1002/cctc.201900558

Accepted Article
Title: Improved Biocatalytic Activity of the Debaryomyces Species in
Seawater
Authors: Cecilia Andreu and Marcel·lí del Olmo
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Improved Biocatalytic Activity of the Debaryomyces Species in
Seawater
[a]
[b]
Cecilia Andreu* and Marcel·lí del Olmo*
Abstract: Biocatalysis is an environmentally friendly strategy widely used in the chemical and pharmaceutical industries. One of its approaches
consists in utilizing whole cells, which usually involves the consumption of large quantities of water. Oceans cover a large part of the Earth's
surface, and constitute an important reservoir that can be used as an alternative to freshwater in chemical reactors. This work analyzed the
behavior of several yeast halotolerant species belonging to genera Debaryomyces and Schwanniomyces in both freshwater and seawater. It
concluded that these microorganisms were more resistant to organic solvents/compounds in the second medium. Their metabolic activity was
also greater, which made the reduction reaction of several prochiral ketones more effective. Besides, yeasts cells displayed better performance
in subsequent recycling steps, which could help reduce the costs of the process.
Introduction
The chemical and pharmaceutical industries are becoming
increasingly more concerned about environmental problems,
and new strategies are being introduced to reduce the negative
impact of usual production processes. In fact, a change has been
made in the objectives required for a chemical reaction, from the
traditional concepts of efficiency and selectivity that focused
mainly on chemical yield, to others where priorities are the
maximal utilization of raw materials, low pollutant production, and
eliminating the use of toxic and/or hazardous substances.[1]
Accordingly, biocatalysis, defined as the use of enzymes or whole
the present-day, biocatalysis is considered an expertise area in
most process chemistry departments in large pharma and fine
chemical companies,[
technology.[
10]
and is often known as green
11]
Choosing an appropriate solvent for a chemical process is one of
the most important factors in sustainable chemistry. Water is one
of the solvents most preferred by the pharmaceutical industry
because it is safe, non-toxic and incurs none of the typical risks
[12]
related to organic solvents. However, the low solubility of the
hydrophobic substrates in water entails large quantities of water
being used, which is another problem in industrial processes that
does not benefit the environment and an important one to
[
2]
cells as catalysts for industrial synthetic chemistry , offers
multiple advantages over conventional chemical catalysis.[3]
Whole cells and enzymes are nature's sustainable, renewable and
biodegradable catalysts, which are used under mild temperature,
pressure and pH conditions. Water is employed as an
investigate.[
13]
Thus seawater has been proposed as a bio-
sustainable alternative to using freshwater.[ Oceans cover 71%
of the Earth's surface, and seawater is a widely available
inexpensive resource. Oceans also constitute the most important
reservoir of biodiversity in nature, with microorganisms adapted
to high salinity. They host about 2 million species and densities of
10 eukaryotic cells and 10 prokaryotic cells per milliliter
of seawater, which represents an important resource of
biocatalysts to produce bioactive compounds.[ Therefore, the
characterization of new marine microorganisms with potential
biotechnological applications to perform bioprocesses under
14]
[
4]
environmentally friendly solvent, and no heavy metals are
used.[5] The good control over chemo-, regio- and stereo-
selectivity renders unnecessary the protection and deprotection
steps that are often required in conventional organic routes, which
results in shorter syntheses.[6] For all these reasons, nowadays
biocatalysis is considered a powerful tool that meets the Green
Chemistry principles introduced by Anastas.[7]
6
12
15]
Biocatalysis has evolved from academic curiosity to an industrially
attractivetechnologyfortheenantioselectivesynthesisofAPIs.[8] Indeed
it has become a key technology to manufacture chiral drugs.[9] In
[16]
salinity conditions has recently become an important challenge.
Extensive research has also been conducted to analyze the
physico-chemical features of their enzymes. Among them, it is
worth mentioning the excellent stability to high ionic strength, a
property that has been correlated with increased tolerance for
organic solvents.[
[
[
a]
b]
Dr. C. Andreu
17]
Departament de Química Orgànica
Universitat de València (UVEG)
Vicent Andrés Estellés s.n., E-46100, Burjassot, València, Spain
E-mail: cecilia.andreu@uv.es
Recently, the ability of whole cells of marine and other
halotolerant yeasts to perform biocatalytic processes in seawater
has been described. Rocha et al. have reported how whole cells
of seven marine fungi strains are able to carry out the asymmetric
reduction of 2-chloro-1-phenylethanone in an artificial seawater
Dr. M. del Olmo
Departament de Bioquímica i Biologia Molecular
Universitat de València (UVEG)
Dr. Moliner 50, E-46100, Burjassot, València, Spain
E-mail: m.del.olmo@uv.es
[
18]
medium.
Serra et al. have demonstrated that several
Meyerozyma guilliermondii and Rhodotorula mucilaginosa strains,
isolated at different deep-sub-seaflor sediment depths in New
Zealand,[19] are able to reduce aromatic ketones with high molar
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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ChemCatChem
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conversions and moderate-to-high enantioselectivity in both
freshwater and seawater. [14d] More recently, this research group
has also reported the use of one of these strains, Meyerozyma
guilliermondii LM2 (UBOCC-A-214008), for the efficient
hydrolysis of benzonitrile and the dynamic kinetic resolution of
mandelonitrile in seawater.[20] We have also demonstrated the
ability of halotolerant yeast Debaryomyces etchellsii to produce
almost the same as in the control experiment, probably due to low
solubility and, therefore, to the content of the solvents in these
media. With the other non miscible solvent, chloroform,
differences between both media were bigger than 4-fold in
seawater for D. fabryi, around 1.6-fold for S. etchellsii and about
1.34-fold for S. polymorphus. For D. hansenii, no statistically
significant differences were observed. The cell viability for
miscible solvents was, in most cases, higher in seawater than in
freshwater, and this difference was bigger for Debaryomyces
strains (D. fabryi and D. hansenii). Particularly large differences
were detected for DMF in both strains and for acetonitrile in D.
fabryi. Likewise, the cells of all the considered strains were more
thermostable in seawater. The biggest difference between both
media was found in such situation for D. fabryi, followed by S.
polymorphus, while the smallest difference was detected in D.
hansenii.
(
R)-(-)-phenylacetylcarbinol, a chiral precursor of the drug
ephedrine, in seawater. Under these conditions, the conversion
rate of the starting material in (R)-PAC was higher, with minimum
by-products production compared to freshwater. It was also
possible to increase the benzaldehyde load in the reaction
medium by at least twofold, and cells maintained their activity after
several recycling rounds.[14e]
The present work analyzes the potentiality of several
Debaryomyces halotolerant yeast strains as cell factories for
biocatalytic processes using seawater as
a
solvent.
Debaryomyces hansenii isolates have been tradicionally obtained
from several foods such as cheese or sausages, cold sea
water,[21] and in salterns on the Namibian Skeleton coast and the
Great Salt Lake exposed to seasonal low temperatures,[22] and
present a high biotechnological potential.[23] Other less known
strains related to D. hansenii considered herein include D.
D. fabryi
1
00
80
60
hansenii var. fabryi,
Schwanniomyces
etchellsii
and S.
40
polymorphus var. polymorphus. These four strains have been
shown to be able to grow in the presence of NaCl concentrations
up to at least 7.5% (w/v).[14e]
2
0
0
D. hansenii
100
8
0
0
Results and Discussion
6
4
0
The halotolerant strains of genera Debaryomyces and
Schwanniomyces are more resistant to different organic
solvents in seawater than in freshwater
20
0
S.etchellsii
The low solubility of the organic substrates in water is a problem
in biocatalytic processes, and it is sometimes beneficial to use
organic solvents to improve it. The correlation found between high
stability to elevated ionic strength and tolerance to organic
solvents in enzymes produced by halophilic microorganisms,[17]
encouraged us to screen the viability of Debaryomyces and
Schwanniomyces species with halotolerant behavior in the
presence of several organic solvents.
1
00
8
0
60
4
0
20
0
Five water miscible (acetonitrile -ACN-, N,N-dimethylformamide -
DMF-, dimethyl sulfoxide -DMSO-, ethanol -EtOH- and
tetrahydrofuran -THF-) and three non-miscible (chloroform –Chl-,
heptane –Hept- and toluene –Tol-) solvents were chosen and
added in different amounts to the freshwater or seawater
containing yeast cells, as described in the Experimental Section.
After 24 h at 30 °C, the viability of these cells was determined by
the trypan blue exclusion test.[24] The same procedure was
applied to determine the thermostability of these yeasts in
freshwater/seawater by maintaining cells at 40 °C for 1 day. A
parallel sample of yeasts in freshwater/seawater at 30 °C was
used as the control in all the experiments (see the Experimental
Section).
S.polymorphus
1
00
8
0
60
4
0
20
0
Condition
The results in Figure 1 show different behaviors when using non
miscible or miscible solvents. Thus for all the strains, the viability
in toluene or heptane saturated in freshwater or seawater was
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Figure 1. Viability of Debaryomyces and Schwanniomyces species in
freshwater (orange) and seawater (blue) at 40 °C or in the presence of different
organic solvents. Experiments were carried out in triplicate. Average and
standard deviation data are shown.
Ability for reducing phenylacetone in seawater compared to
freshwater in Debaryomyces and Schwanniomyces species
in the presence of organic solvents
To explore the correlation between cell viability and metabolic
activity, we decided to study the capability of these cells to carry
out the reduction of pro-chiral ketones into chiral alcohols, a well-
known reaction carried out frequently by yeast whole cells. To
check the effect of the above-mentioned water miscible organic
solvents in freshwater or seawater containing 2% glucose as the
reaction media, the reduction of 1-phenylpropan-2-one
Figure 2. Conversion of 1a into 1b in freshwater (orange) and seawater
(blue) under different conditions: I: 30 °C, II: 40 °C, III: ACN (4%) 30 °C,
(
phenylacetone) 1a was chosen as the benchmark (Scheme 1)
IV: DMF (6%) 30 °C.
EtOH and THF were not included, due to the low viability
observed in the presence of these solvents. Nor was DMSO
considered, because not much difference in viability was
observed between freshwater and seawater in most cases. Table
Another important factor in biocatalyzed reactions is the substrate
charge due to its toxicity for cells. As with organic solvents, it may
differ in both freshwater and seawater. In fact our previous work
1
and Figure 2 show the conversion of substrate 1a into alcohol
1
b in presence of ACN and DMF, and also when the reaction was
found than benzaldehyde was more toxic for the S. etchellsii cells
carried out at 40 °C. All these data together indicate that being S.
etchellsii and S. polymorphus better biocatalysts than D. fabryi
and D. hansenii for this reaction, the latter are more sensitive for
the nature of the water present in the medium.
grown in a freshwater medium than in a seawater one.[14e] Hence
the toxicity of the substrate of this reaction was analyzed in the
four strains considered herein determining the lethal dosis LD50
.
The obtained results are shown in Table 2. In all the considered
strains, except for S. polymorphus, lower toxicity was observed in
seawater than in freshwater, and the differences were statistically
significant for the Debaryomyces strains when a p-value lower
than 0.01 was considered for this purpose.
Table 2. LD50 of strains considered in this work for substrate 1a.
Scheme 1. Model reaction for screening the biocatalytic ability of the yeasts
D. fabryi
D. hansenii
S. etchellsii
S. polymorphus
used in this work.
Freshwater
Seawater
t-test[
3.51 ± 0.22
4.25 ± 0.05
0,00465485
2.71 ± 0.03
3.94 ± 0.06
5,21919E-06
3.59 ± 0.31
4.36 ± 0.07
0,04341733
3.16 ± 0.03
3.15 ± 0.16
0,97127844
a]
The data shown correspond to the mean and standard deviation of at least three
independent experiments.
Table 1. Effect of some water miscible solvents or temperature on the
reduction of 1a biocatalyzed by several Debaryomyces and
Schwanniomyces strains.
[a] p-value was determined by the t-test of the Microsoft Excel package by
considering two cues and samples of a different variance.
Conditions[a]
D.
fabryi
D.
hansenii
0.44
0.70
0.18
1.16
0
S.
etchellsii
1.48
1.61
0.12
S.
polymorphus
3.14
[
b]
[b]
[b]
[b]
FWD
SWD
0.12
0.32
0
0.30
0
A close inspection made of the above-described data indicated
that, in general, for the four strains explored herein the use of high
temperature or the addition of organic solvents was detrimental
for biocatalytic conversions, although cells displayed more
resistance to these conditions in seawater than in freshwater.
Moreover, the transformation of substrate 1a was also more
effective, but behaviour was not the same for them all. In the
considered Debaryomyces strains the differences in all these
aspects appeared to be bigger and/or more statistically significant.
Accordingly, further studies were carried out with strains D.
hansenii and D. fabryi without any addition of cosolvents.
3.12
1.97
3.55
1.12
FWD-40 °C
SWD-40 °C
FWD-4% ACN
SWD-4% ACN
FWD-6% DMF
SWD-6% DMF
1.37
1.68
0.12
0.08
0.12
0.23
0
0.11
1.59
0.46
0.39
1.83
0.97
1.96
[
a] Reactions were carried out using 50 OD600 units of whole cells in 10 mL
of reaction medium in freshwater or seawater, containing glucose (2%)
FWD/SWD) and ACN (4%) or DMF (6%) when it is indicated. 1a (37.2
(
mol, 5mg) was employed as the substrate. These mixtures were stirred at
150 rpm in an orbital shaker for 24 h at 30 °C or 40 °C. Experiments were
carried out in triplicate, where the standard deviation was lower than 5% of
the mean value shown in this table. [b] Conversion expressed as mol (1b)
-
1
mg (cells dry weight). Cells dry weight was determined after the
lyophilization of the aliquots containing 50 OD600 of whole cells.
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ChemCatChem
10.1002/cctc.201900558
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mixtures were stirred at 150 rpm in an orbital shaker for 24 h (see the
Experimental Section). Experiments were carried out in triplicate and the
standard deviation was below 5% of the mean value shown in this table. [b]
Substrate scope
The reactions with both Debaryomyces yeast strains under the
previously described standard conditions (see Experimental
Section) were carried out using several ketones as substrates
-1
Conversion expressed asmol (alcohol) mg (dry weight cells). Cell dry weight
was determined after the lyophilization of the aliquots containing 50 OD600 units
of whole cells. [c] Conversion was not expressed by numerical numbers
because the reduction of the substrate was complete and no comparison was
possible.
(
Scheme 2 and Table 3).
The LD50 value was also determined for strains D. hansenii and
D. fabryi for substrate 4a, and the results were consistent with the
above-described data about the transformation of this substrate
in the two considered media. As shown in Table 4, the value was
higher in seawater, with statistically significant differences
compared to the data found in freshwater. For 3a, the LD50 value
could not be determined because of its low solubility in water.
However, it is worth mentioning that viability in the presence of
the concentration used for the reduction processes was around
Scheme 2. The additional substrates used herein. The derived alcohols are
respectively named 2b, 3b, 4b and 5b in the text.
9
9% in both freshwater and seawater in the two considered
The conversion of ketones depended on their reactivity. For both
strains, the least electrophilic ketone, acetophenone (2a), was
almost recovered after a 24-hour reaction, and the most activated,
trifluoroacetophenone (5a), was totally reduced, even when 10
OD600 units of whole cells were used, except for when biocatalysis
was carried out in the presence of DMF (6%). In this case,
conversion was 6.7 and 11.9 mols (5b) mg-1 of cell dry weight in
freshwater and seawater respectively for D. fabryi, and 5.4 and
Debaryomyces strains.
Table 4. LD50 of the Debaryomyces strains considered herein for substrate
4a.
3.7 mols (5b) mg-1 of cells dry weight for D. hansenii. The
D. fabryi
2.21 ± 0.06
2.95 ± 0.04
D. hansenii
2.73 ± 0.13
3.91 ± 0.12
0,0001035
1
activated ketone 4-nitroacetophenone 3a was converted in more
extent than 1a and the results were always better when seawater
was used as the reaction medium instead of freshwater. Finally,
phenoxyacetone 4a was totally reduced when D. hansenii was the
catalyst, except in the presence of DMF in freshwater (1.55 mols
Freshwater
Seawater
t-test[a]
0,00614412
-1
(
4b) mg of cells dry weight). The conversions of this substrate
The data shown correspond to the mean and standard deviation of at least three
independent experiments.
with D. fabryi were incomplete, and values were always higher in
seawater (Table 3).
[a] p-value was determined by the t-test of the Microsoft Excel package by
considering two cues and samples of a different variance.
Enantioselectivity determination of the obtained alcohols
using freshwater/seawater.
Table 3. Effect of some water miscible solvents or temperature on the
reduction of 3a and 4a biocatalyzed by the Debaryomyces strain used
herein.
The results described in the previous sections confirmed the
interest of using 2% glucose in seawater as a reaction medium
for the biocatalytic reduction of several substrates employing
whole cells of strains D. hansenii and D. fabryi. The next study
aim was to set up semi-preparative reactions in 100 mL of a
freshwater or seawater medium using 500 OD600 units of cells
at 30 °C. As no reaction was detected with substrate 2a, and no
differences between both media were observed in the most
reactive ketone 5a, a comparison was made with substrates 1a,
3a and 4a. The substrate concentration was selected from the
above-mentioned LD50 data to keep it below the value found for
Strain
Condition[a]
3b[b]
SWD
4b[b]
SWD
FWD
1.51
0.49
0.38
FWD
2.28
0.22
0,47
D. fabryi
30 °C
1.67
1.74
0.48
3.87
3.87
2.15
4
4
6
3
4
4
6
0 °C
% ACN
% DMF
0 °C
0.43
0.54
0.54
3.36
D. hansenii
nd[c]
nd[c]
nd[c]
nd[c]
0 °C
1.81
1.04
0.49
2.5
nd[c]
nd[c]
1.55
nd[c]
nd[c]
nd[c]
both strains. Accordingly, for substrates 1a and 4a, 1.5 mg mL
-
1
were added. For 3a, the concentration corresponding to the
% ACN
% DMF
1.21
0.9
-1
maximal solubility of this substrate (0.61 mg mL , according to
our analyses) was employed. From these experiments,
conversion, yield and enantiomeric excess were determined
under both conditions. As expected from the LD50 data, the
results for both strains were equal or better when
transformations were carried out in seawater (Table 5). For
[
a] Reactions were carried out using 50 OD600 units of whole cells in 10 mL of
reaction medium in freshwater or seawater containing glucose (2%)
FWD/SWD) at 30/40 °C or at 30 °C in the presence of ACN (4%) or DMF (6%).
7.2 mol of substrates 3a/4a were used as the starting material. These
(
3
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10.1002/cctc.201900558
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enantiomeric excess, values were similar and good for each
strain in both media for substrates 1a and 4a. With substrate 3a,
enantiomeric excess was always bad, and was worse in
freshwater when D. fabryi was used, and in seawater when D.
hansenii was the employed biocatalyst. Interestingly,
enantiopreference also differed for each yeast.
3) continued to be apparent in further recycling steps. With
substrate 4a, conversion progressively decreased in freshwater
from R1, but it was completed in seawater for the first three
recycling rounds and decreased to 57% in the fourth one. Finally
for 3a, the pattern was similar to that described for 4a.
D. fabryi
.
Table 5. Comparison of the results in freshwater and seawater in
semipreparative reactions
Strain
Product/time
h)
FWD
SWD
(
Conversion[a]/yield[b]
Conversion[a]/yield[b]
/ee (%)-absolute
/
ee (%)-absolute
configuration[
12/10/85-(S)
39/35/35-(R)
100/83/65-(S)
46/38/90-(S)
100/86/35-(S)
100/82/92-(S)
c]
[c]
configuration
D. fabryi
1b/48
19/14/85-(S)
78/65/57-(R)
100/85/63-(S)
76/66/89-(S)
100/93/20- (S)
100/82/92-(S)
3
4
b/48
b/24
D. hansenii
D. hansenii
1b/48
3
4
b/48
b/24
Reactions were carried out using 500 OD600 units of whole cells in 100 mL of
the reaction medium in freshwater or seawater containing glucose (2%)
(FWD/SWD). These mixtures were stirred at 150 rpm in an orbital shaker for 24-
48 h at 30 °C. Experiments were carried out in triplicate and the standard
deviation was below 5% of the mean value shown in this table.
1
[
a] Conversion determined by HNMR from the reaction crude. [b] Isolated yield
R0
R1
R2
R3
R4
after chromatographic purification. [c] Enantiomeric excess was determined by
chiral HPLC, and absolute configuration by the comparison of rotatory power
with the values described in the literature (see the Experimental Section).
Recycling round
Figure 3. Conversions through several rounds of recycling cells of D. fabryi
and D. etchellsii during biocatalytic activity over substrates 1a, 3a and 4a.
The above figure shows the average value in three replicates. The standard
deviation was lower than 5% in all cases.
Effect of using freshwater/seawater in the reaction medium
on recycling yeast cells
A previous work employed whole cells of S. etchellsii to produce
(
R)-PAC and we found that using seawater to prepare the
During these experiments, the viability of yeast cells was
determined at the end of some recycling rounds. The found data
(Supporting Information, Table 3) are consistent with the
transformation results shown in Figure 3.
medium reaction could help to reduce the costs of the process
because it allowed yeast cells to be reutilized several times.[14e]
To determine if this was not particular case, this sort of
experiments were also carried out herein. For this purpose, after
a 24- or a 48-hour reaction cells were centrifuged and washed,
and new reaction mixtures were sequentially set up. These
studies were run in parallel by incubating cells in FWD and SWD.
The results in Figure 3 and Table 2 of the Supporting Information
show that the experiments which used seawater in the growth
and reaction media allowed cells to be more efficiently recycled.
In both media, D. fabryi surprisingly displayed a better ability to
convert substrate 1a in rounds R1, R2 and R3 than when fresh
cells were used (Table 5 and R0 in Figure 3); conversion was
always better in each cycle in the presence of seawater. To
reduce substrate 3a in freshwater, the activity of the cell
population dramatically diminished in the first round, and
transformation was not achieved at all in the second round.
Conversely when using seawater, cells could be reused for two
more rounds, although conversion progressively reduced. For
substrate 4a, the first recycling done in freshwater significantly
decreased but not in seawater.
Discussion
Environmental problems due to pollution and to diminishing
primary resources have favored the emergence and
development of Green Chemistry, understood as the design of
chemical products and processes to reduce or eliminate the use
and generation of hazardous substances.[7] In this context,
biocatalysis is a good alternative to many catalytic processes that
employ conventional chemicals.
A few recent reports have demonstrated the possibility of using
seawater as an alternative to freshwater in biocatalytic
processes.[14d-e,18,20] The present work aimed to explore its scope
and potential use in industry in-depth. To this end, the effect of
the medium in the metabolic activity of several halotolerant yeast
strains belonging to the genera Debaryomyces and
Schwanniomyces was checked. Their resistance to organic
solvents in freshwater and seawater was compared, and their
When considering D. hansenii, the initially found differences
with substrate 1a between both media (Table 5 and R0 in Figure
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ChemCatChem
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FULL PAPER
catalytic activity in both media was followed via the reduction of Conclusions
prochiral ketones.
It was observed that when the four studied yeasts were incubated
in a seawater medium, they displayed increased thermotolerance
and wider viability in the presence of organic solvents than when
a freshwater medium was used (Figure 1). Consistently with this
finding, the results described in Table 1 and Figure 2 demonstrate
that they were able to carry out the reduction of model substrate
The use of halotolerant Debaryomyces strains as biocatalysts in
organic synthesis processes that imply using lots of water looks
promising thanks to their robustness in saline media. The use of
seawater did not lead to any adverse effect and could, in fact,
even improve some of the parameters considered when analyzing
chemical conversion performance. Its abundance and availability
makes less necessary the use of cosolvents that negatively affect
the yield and could be non-environmentally friendly. Moreover,
the higher resistance of yeast cells in seawater to organic
substrates and the possibility of further cell recycling steps
provide an interesting strategy to reduce costs by making the
process competitive.
1a in seawater. Indeed the percentage of conversion was, under
most of tested conditions, better than when freshwater was
employed. The same effect was also observed when the yeast
cell toxicity of 1a was studied by measuring parameter LD50
(
Table 2).
Some particularly relevant differences were found in the
Debaryomyces species. Thus more attention was paid to them by
extending the scope of the process to a small family of ketones
with different electronic properties. As expected, no differences
were observed when the very electrophilic substrate 5a was used
due to the almost total conversion of the starting material in both
media. Bigger differences were observed with the less reactive
substrate 1a, and with others that display intermediate reactivity
Experimental Section
General
The seawater used in the experiments came from the
Mediterranean Sea (El Perelló beach, València, Spain). It was
sterilized by autoclaving under standard conditions and was
maintained at 18 ºC. The water salinity for this area, determined
by weighing the solid residue after lyophilization, was around 4%
(
3a or 4a, Table 3). Using these ketones as the starting material,
and by increasing the substrate charge, greater activity was once
again proven when the reaction medium was prepared in
seawater (Table 5). In agreement with these results, the LD50
measurements of the two strains for these substrates were
enhanced in seawater (Tables 2 and 4). It is worth mentioning that,
despite the greater thermotolerance and catalytic activity
observed in this medium when the reactions were carried out at
(
w/v). The sodium content, analyzed by Flame Emission
Spectroscopy (589.0 nm, Thermo Scientific model iCE 3000
-1
Series), was 12.46±0.17 g L , and pH ranged from 7.5 to 8.5,
depending on the batch.
40 °C with low charge of substrates (Figures 1 and 2, Tables 1
All the commercially available reagents were purchased from
Sigma-Aldrich. Product purification by flash chromatography was
performed on Merck silica gel 60 (particle size: 0.040-0.063 mm).
Thin-layer chromatography (TLC) was carried out on Merck silica
and 3), the increase in their concentration was deleterious and
transformation in this case was very poor (data not shown).
Moreover, the reuse capability notably improved in the seawater
medium (Figure 3 and Table 2 Supporting Information). With D.
fabryi some particularly relevant results were obtained with
substrate 4a. It was possible to reuse the same cells in at least
four cycles in seawater and very good activity was maintained
plates 60 F254
.
Products were characterized by NMR spectra recorded in a
Bruker DRX 300 spectrometer using deuterated chloroform as a
solvent. Chemical shifts are reported in ppm in relation to the
residual solvent peak. Absolute configurations were determined
by making comparisons with the optical rotations reported in the
(
conversion 68%), while reuse was only possible for one cycle in
freshwater. Despite not being so noteworthy, the same effect was
observed with substrates 1a and 3a. Enantioselectivity was
maintained in each cycle with alcohol 1b (around 88% in both
freshwater and seawater), but a slight decrease was observed for
alcohols 3b (from 57 to 45) and 4b (from 63 to 50) (Supporting
Information Table 2).
D. hansenii displayed similar behavior and the differences found
in the conversion of substrates 3a and 4a were more relevant (3a
was converted into 2.5% and 4a into 10% in freshwater in the third
round, while the conversion in seawater was 100% in both cases).
Enantioselectivity was almost the same in alcohols 1b and 4b
literature, which were performed in
a Perkin Elmer 241
Polarimeter at λ=589 nm. Enantiomeric excess was established
by High-Performance Liquid Chromatography (HPLC) with a
Merck Hitachi Lachrom system using either a Chiralcel ODH or a
Chiralpak IC column. Racemic alcohols 1b-5b, synthesized by
[25]
conventional methods, were used as standards.
Strains and culture conditions
The yeast strains used for this work are described in Supporting
Information Table 1.
Cells were grown overnight to an OD600 ranging from 15 to 50
(
around 93% and 90%, respectively), and this value increased for
alcohol 3b in seawater in each cycle (from 20% in round R0 to
1% in round R3). As we cannot presently offer any explanation
5
(
(
depending on the strain) by incubation in an orbital shaker
shaking diameter 25 mm, 150 rpm) at 30 ºC in a 50 mL
for this, further studies are necessary to understand this
interesting fact.
The catalytic activity in the subsequent recycling rounds
correlated with viability data determined by the trypan blue
exclusion assay at the end of each round. Supporting
Information Table 3 shows such results obtained for substrate
Erlenmeyer flask containing 10-20 mL of YPD medium (1% w/v
yeast extract, 2% w/v peptone, 2% w/v glucose) or SW-YPD
medium (with the same YPD composition but prepared with
seawater). The latter medium was filtered, sterilized and
supplemented with chloramphenicol to a final concentration of
4
a.
-1
1
00 g mL . To avoid any differential effect of this antibiotic on
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ChemCatChem
10.1002/cctc.201900558
FULL PAPER
yeast growth, it was also added to the YPD medium prior to the
inoculation of cells.
Cells’ ability to be recycled was also analyzed. Reactions,
performed under the conditions described above, were carried out
in parallel using FWD or SWD. After 24 h or 48 h, each one was
stopped by centrifuging. The supernatant was removed, the pellet
was washed with the same medium (10 mL) and the reactions
were set up again under the same conditions using fresh solutions.
The process was repeated for several rounds. In each one, the
crude material was analyzed to determine the conversion like that
explained above.
Viability determination by the blue trypan assay
Experiments were run to determine the viability of yeast cells. The
volume that corresponded to 5 OD600 units was incubated in 1 mL
of water or seawater in an Eppendorf tube at 30 ºC for 24 h in the
presence of 10% (v/v) ethanol, 6% (v/v) DMF, 4% (v/v)
acetonitrile, 12% (v/v) DMSO, 8% (v/v) THF and saturated
(+)-(S)-1-Phenyl-2-propanol (1b)
-1
-1
solutions of chloroform (7.95 mg mL ), heptane (3.4 g mL ) and
Colorless oil
H-RMN (300 MHz, CDCl
-1
[26]
1
3
toluene (526 g mL ).
To determine the effect of media on
3
): δ= 1.2, (d, J(H,H)= 6 Hz, 3H; CH
3
),
3
cells’ heat resistance, experiments were also set up using
freshwater or seawater at 40 °C. The results were always
compared with a sample control of these cells incubated at 30 °C
in freshwater or seawater. The same procedure was followed to
determine viability in the presence of the substrates used herein;
in this case, several concentrations of them, ranging between 0.3
mg mL-1 and 7 mg mL , were employed, and LD50 was calculated
from the obtained data. Viability was determined by the trypan
blue exclusion assay, which allows the direct identification and
enumeration of live (unstained) and dead (blue) cells in a given
population.[24] Experiments were carried out in triplicate.
1.6 (broad s, 1H; OH), 2.6 (dd, J(H,H)= 13Hz, 8Hz, 1H; CH
2
), 2.7
3
(dd, J(H,H)= 13Hz, 5Hz, 1H; CH
2
), 3.92 (m, 1H; CH), 7.11-7.26
): δ= 22.7, 45.7,
68.8, 126.4, 128.5, 129.4, 138.5 ppm; ODH Chiralcel column,
(m, 5H; CH Ar) ppm; 13C-RMN (75 MHz, CDCl
3
-1
(Hex/iprOH 95:5, flow= 0.5 mL min , wavelength= 214 nm), (S)=
20= + 34.7 (c= 1 in CHCl
14 min (major), (R)= 15 min (minor); [] ,
D 3
91% ee), (lit +42.2, c= 1 in CHCl ).
3
-1
[27]
(+)-(R)-1-(4-Nitrophenyl)ethanol ((+)-3b); (-)-(S)-1-(4-
Nitrophenyl)ethanol ((-)-3b)
Yellow oil
1
3
H-RMN (300 MHz, CDCl
3
): δ= 1.5 (d, J(H,H)= 6.4 Hz, 3H; CH
3
),
3
Reduction of ketones 1a-5a biocatalyzed by whole cells of
halotolerant strains of Debaryomyces and Schwanniomyces
genera. General procedure
2.3 (broad s, 1H; OH), 5.0 (q, J(H,H)= 6.4 Hz, 1H; CH), 7.5 (d,
3
3
J(H,H)= 8.7 Hz, 2H; CH Ar), 8.16 (d, J(H,H)= 8.7 Hz, 2H; CH Ar)
ppm; 13C-RMN (75 MHz, CDCl
): δ= 25.4, 69.4, 123.7, 126.1,
47.1, 153.1 ppm; IC Chiralpak column (Hex/iprOH 95:5, flow=
3
1
-1
The volume that corresponded to 50 OD600 units (or 10 when 5a
was the reaction substrate) from the above-described overnight
cultures was centrifuged, and cells were washed and
resuspended in the reaction medium. For this purpose, FWD (2%
0.3 mL min , wavelength= 214 nm), (R)= 40 min, (S)= 42 min.
[] , 57% ee, R), (lit +35.1, c= 1 in CHCl
20= + 21 (c= 1 in CHCl
).
[]
CHCl
D
3
3
[28]
20= -11 (c= 1 in CHCl
).[29]
D
3
, 35% ee, S), (lit -20.5, c= 1.46 in
(
w/v) glucose in freshwater) or SWD (2% (w/v) glucose in
3
seawater) was employed. The pellet was suspended in a final 10
mL volume (including the substrate and any additive considered
besides the reaction medium) in 50-mL Erlenmeyer flasks. In all
cases, the mixture was incubated at 30 °C (or alternatively at
(+)-(S)-1-Phenoxy-2-propanol (4b)
Colorless oil
1
3
H-RMN (300 MHz, CDCl
3
) δ=1.3 (d, J(H,H)= 6 Hz, 3H; CH
3
),
), 3.9
3
40 °C) for 30 min, with orbital shaking before adding the substrate
2.15 (broad s, 1H; OH), 3.8 (dd, J(H,H)= 9Hz, 8Hz, 1H; CH
2
3
up to a final concentration of 3.72 mM. The mixture was
maintained with orbital shaking for 24 h at the same temperature.
Then it was centrifuged (3 min at 3000 rpm) and the aqueous
supernatant was extracted with methylene chloride (2x8 mL). The
organic phases were combined and dried over sodium sulfate.
After solvent evaporation, the crude material was analyzed by
1HNMR. The percentage of both the obtained alcohol and the
remaining ketone was determined by integration. Conversions
were expressed as mol (alcohol) mg-1 dry weight (cells). Cell dry
weight was determined after the lyophilization of the aliquots
containing 50 OD600 of whole cells.
(dd, J(H,H)= 9Hz, 3Hz, 1H; CH
2
), 4,20 (m, 1H; CH), 6.9 (m, 3H;
): δ=
18.7, 66.3, 73.2, 114.6, 121.1, 129.5, 158.5 ppm; ODH Chiralcel
CH Ar), 7.15 (m, 2H; CH Ar) ppm; 13C-RMN (75 MHz, CDCl
3
-
1
column (Hex/iprOH 95:5, flow= 1 mL min , wavelength= 214 nm),
3
(R) 13 min (minor), (S) 31 min (major); [] ,
20= + 28 (c 0.8 in CHCl
92% ee), (lit +30.7, c= 1.32 in CHCl ).
3
D
[
18]
(±)-2,2,2-trifluoro-1-phenylethan-1-ol (5b)
Colorless oil
1
H-RMN (300 MHz, CDCl
J(H,F)= 6Hz, 1H; CH), 7.4-7.5(m, 5H; CH Ar) ppm; C-RMN (75
3
) δ= 2.8 (broad s, 1H, OH), 4.9 (q,
3
13
2
1
In order to determine enantiomeric excess and to fully
characterize the obtained alcohols, analogous experiments were
carried out in 500 mL Erlenmeyer flasks in fresh water or seawater
3
MHz, CDCl ): δ= 72.8 (q, J(C,F)= 31,5 Hz), 124.2 (q, J(C,F)=
275 Hz), 127.4, 128.6, 129.5, 133.9 ppm.
(
100 mL). The amount of cells was incremented to 500 OD600 and
the final substrate concentration was 1.5 mg mL-1 (substrates 1a
and 4a) and 0.61 mg mL-1 (substrate 3a). The crude material
obtained after extracting the aqueous supernatant was purified by
column chromatography and fully characterized by NMR, chiral
HPLC and optical rotation measures. All the experiments were
carried out in triplicate.
Acknowledgements
This work was supported by the Universitat de València (UV-INV-
AE15-323062). We gratefully acknowledge Dr J. Ramos for
providing us with the Debaryomyces hansenii strain CBS767 and
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ChemCatChem
10.1002/cctc.201900558
FULL PAPER
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FULL PAPER
Cecilia Andreu* and Marcel·lí del Olmo*
Page No. – Page No.
Improved Biocatalytic Activity of the
Debaryomyces Species in Seawater
Seawater in biocatalytic reactions: The use of seawater increases the cells’
resistance of Debaryomyces species to organic solvents/compounds. It also
improves their metabolic activity allowing bigger yields in reactions catalyzed by
them. The possibility of further cell recycling steps provides an interesting strategy to
reduce costs and makes the process more competitive.
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