Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

Article abstract of DOI:10.1002/cbic.201900721

Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non-aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4-oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael-type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its “Michaelase” activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The “hotspot” position Ala33 was then randomized in a highly enantioselective, but ethanol-sensitive 4-OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ-nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability-landscape-guided enzyme engineering for efficient biocatalysis in non-aqueous solvents.

Full text of DOI:10.1002/cbic.201900721

Accepted Article
Title: Tuning Enzyme Activity for Nonaqueous Solvents: Engineering
of an Enantioselective ‘Michaelase’ for Catalysis in High
Concentrations of Ethanol
Authors: Chao Guo, Lieuwe Biewenga, Max Lubberink, Ronald van
Merkerk, and Gerrit J. Poelarends
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To be cited as: ChemBioChem 10.1002/cbic.201900721
Link to VoR: http://dx.doi.org/10.1002/cbic.201900721
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10.1002/cbic.201900721
ChemBioChem
Tuning Enzyme Activity for Nonaqueous Solvents: Engineering
of an Enantioselective ‘Michaelase’ for Catalysis in High
Concentrations of Ethanol
Chao Guo,[a] Lieuwe Biewenga,[a] Max Lubberink,[a] Ronald van Merkerk,[a] and
Gerrit J. Poelarends*[a]
[a]
C. Guo,+ L. Biewenga,+ M. Lubberink,b R. van Merkerk, Prof. Dr. G.J. Poelarends
Department of Chemical and Pharmaceutical Biology,
Groningen Research Institute of Pharmacy, University of Groningen,
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
E-mail: g.j.poelarends@rug.nl
[+]
[b]
These authors contributed equally to this work.
Present address: School of Chemistry, Manchester Institute of Biotechnology, The
University of Manchester, 131 Princess Street, M1 7DN, Manchester (UK).
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ChemBioChem
ABSTRACT
Enzymes have evolved to function under aqueous conditions and may not exhibit features
essential for biocatalytic application, such as the ability to function in high concentrations of
an organic solvent. Consequently, protein engineering is often required to tune an enzyme for
catalysis in non-aqueous solvents. In this study, we have used a collection of nearly all single
mutants of 4-oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful
Michael-type additions of acetaldehyde to various nitroolefins, to investigate the effect of
each mutation on the ability of this enzyme to retain its ‘Michaelase’ activity in elevated
concentrations of ethanol. Examination of this mutability landscape allowed the identification
of two hotspot positions, Ser-30 and Ala-33, at which mutations are beneficial for catalysis in
high ethanol concentrations. The ‘hotspot’ position Ala-33 was then randomized in a highly
enantioselective, but ethanol-sensitive 4-OT variant (L8F/M45Y/F50A) to generate an
improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and
efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40%
ethanol (permitting high substrate loading) to give the desired γ-nitroaldehyde product in
excellent isolated yield (89%) and enantiopurity (ee = 98%). The presented work
demonstrates the power of mutability-landscape-guided enzyme engineering for efficient
biocatalysis in non-aqueous solvents.
Keywords
Enzyme engineering; biocatalysis; solvents; mutability landscape; Michael addition
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Introduction
The enzyme 4-oxalocrotonate tautomerase (4-OT) from Pseudomonas putida mt-2 catalyzes
the tautomerization of 2-hydroxy-2,4-hexadienedioate (1) to 2-oxo-3-hexenedioate (2) as part
of a metabolic pathway for the degradation of aromatic hydrocarbons (Scheme 1A).[1,2] In
addition, 4-OT can promiscuously catalyze several C-C bond-forming reactions, including
Michael-type additions and aldol condensations, yielding precursors for important classes of
pharmaceuticals.[3–7] For instance, the 4-OT catalyzed Michael-type addition of acetaldehyde
(3) to nitroalkenes 4a and 4b yields γ-nitroaldehydes 5a and 5b, important precursors for the
γ-aminobutyric acid analogues phenibut (R-6a) and pregabalin (S-6b), respectively (Scheme
1B).[5] Hence, several enzyme engineering studies have been performed to improve the
activity and enantioselectivity of 4-OT for this reaction.[8,9]
Scheme 1. A) Tautomerization reaction naturally catalyzed by 4-OT. B) Michael-type
addition of acetaldehyde (3) to nitroalkenes 4a and 4b, promiscuously catalyzed by 4-OT.
Products 5a and 5b are precursors for phenibut (R-6a) and pregabalin (S-6b), respectively.
Solubilization of substrates 4a and 4b requires the use of cosolvents. Since enzymes have
evolved to function under aqueous conditions, high concentrations of cosolvents can
significantly affect their catalytic performance and eventually result in enzyme
1
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precipitation.[10] In this study, we used a collection of nearly all single-mutant variants of 4-
OT to investigate the effect of each mutation on the ability of the enzyme to retain its
‘Michaelase’ activity in elevated concentrations of ethanol. Ethanol was selected as cosolvent
because it is readily accessible from biorenewable sources and can also function as a
precursor for 3.[11] Randomization of the identified ‘hotspot’ position Ala-33 in the context of
a previously engineered highly enantioselective, but ethanol-sensitive, 4-OT variant
(L8F/M45Y/F50A) afforded an improved enzyme variant (4-OT L8F/A33I/M45Y/F50A)
with high ethanol stability, allowing efficient and enantioselective Michael-type addition
reactions in 40% (v/v) ethanol. As such, our work provides an interesting example of how
targeted mutagenesis of a single amino acid can radically modify the cosolvent stability of an
enzyme, allowing efficient catalysis in high concentrations of ethanol.
Results
In order to identify ‘hotspot’ positions of 4-OT at which mutations are beneficial for catalysis
in high concentrations of ethanol, a defined collection of 1040 single-mutant variants of 4-
OT[8] was screened using cell-free extracts (CFEs) prepared from cultures each expressing a
different 4-OT mutant. The Michael-type addition of 3 to 4a was used as a model reaction in
screening owing to the marked absorbance of 4a at 320 nm. Control experiments
demonstrated no significant difference between the effect of ethanol on the reaction catalyzed
by purified 4-OT or 4-OT present in CFE (Figure S1). The ‘Michaelase’ activity of each
single-mutant variant of 4-OT was measured using either 5% or 25% ethanol as cosolvent and
the remaining activity at 25% ethanol, compared to that at 5% ethanol, was graphically
represented in a mutability landscape for solvent tolerance (Figure 1). Increasing the
concentration of ethanol from 5% to 25% reduced the ‘Michaelase’ activity of wild-type 4-OT
by approximately 50% (Figures 1 and 2A). Interestingly, analysis of the ethanol-tolerance
2
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mutability landscape revealed two ‘hotspot’ positions, Ser-30 and Ala-33, at which single
mutations resulted in enzyme variants that showed more than 70% residual ‘Michaelase’
activity at 25% ethanol (Figure 1). Notably, the crystal structure of wild-type 4-OT does not
provide an immediate explanation for the improved ethanol tolerance caused by mutations at
these two positions (Figure S2), illustrating the importance of mutability-landscape navigation
to identify functional ‘hotspot’ positions. Three single mutants, 4-OT S30C, S30Y and A33D,
which showed high ethanol tolerance, were purified and the effect of ethanol and other
cosolvents on the ‘Michaelase’ activity was tested (Figure 2). Interestingly, 4-OT S30C, S30Y
and A33D also showed tolerance towards other cosolvents such as DMSO and isopropanol,
suggesting that these mutations convey general cosolvent resistance. Notably, while the 4-OT
variants perform well up to 40% DMSO, visible protein precipitation with concomitant loss of
activity was observed at DMSO concentrations ≥50% (v/v).
Figure 1. Ethanol-tolerance mutability landscape of 4-OT. The horizontal axis of the data
matrix represents the residue positions of 4-OT. The vertical axis represents all 20 canonical
amino acids. The wild-type amino acid at each position is indicated with a bold square. White
squares indicate that this mutant is not present in the collection. The color of the square
indicates the residual ‘Michaelase’ activity of a specific single-mutant variant of 4-OT for the
addition of 3 to 4a in 25% ethanol, compared to that in 5% ethanol. Grey boxes indicate that
the ‘Michaelase’ activity was too low to determine the remaining activity.
3
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Figure 2. Michael-type addition of 3 to 4a catalyzed by purified wild-type 4-OT or 4-OT
mutants in the presence of different cosolvent concentrations. A) ethanol, B) methanol, C)
1,3-propanediol, D) DMSO, E) isopropanol, F) tert-butanol. The activity of each mutant is
normalized to the activity in the presence of 5% cosolvent.
We next investigated if we could use the information from the solvent-tolerance mutability
landscape to engineer a previously constructed highly enantioselective 4-OT variant,
L8F/M45Y/F50A (4-OT FYA),[9] to function in high concentrations of ethanol. As single
mutants at ‘hotspot’ position Ala-33 generally exhibited higher ‘Michaelase’ activity than
those at ‘hotspot’ position Ser-30, we focused our mutagenesis strategy on position Ala-33.[8]
In the context of 4-OT FYA, residue Ala-33 was mutated to all possible amino acids and the
nineteen enzyme variants were expressed and purified to homogeneity. Initially, we tested all
variants for visible precipitation upon incubation (1 h) of the enzyme with increasing
concentrations of ethanol (up to 50%). The parental enzyme 4-OT FYA rapidly precipitated
when incubated with ethanol concentrations equal to or greater than 10% (Figure 3D).
Interestingly, enzyme variants with isoleucine (A33I/FYA), leucine (A33L/FYA) or valine
(A33V/FYA) at position 33 could tolerate up to 50% ethanol without any visible protein
4
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precipitation after 1 hour of incubation. Notably, substitution of Ala-33 to aspartate, glutamate
or cysteine in the context of 4-OT FYA also strongly improved the stability of the enzyme in
high concentrations of ethanol, tolerating up to 40% ethanol without visible protein
precipitation.
Next, we tested the activity and enantioselectivity of these six quadruple mutants in the
presence of 5%, 30% or 50% ethanol. The Michael-type addition of 3 to 4b was used as
model reaction because the optical purity of product 5b can easily be analyzed by gas
chromatography. All six quadruple mutants proved to be highly enantioselective giving nearly
enantiopure product 5b, and, importantly, increasing ethanol concentrations do not negatively
affect enzyme enantioselectivity (Table 1). In the presence of 30% ethanol, the reactions with
the six quadruple mutants were completed within 35-70 min, whereas the reaction with the
parental enzyme (4-OT FYA) showed no conversion due to rapid protein precipitated (Table
1).
Table 1. Biocatalytic addition of 3 to 4b catalyzed by 4-OT mutants in different ethanol
concentrations.[a]
5% v/v ethanol
30% v/v ethanol
50% v/v ethanol
ee[b]
Reaction time ee[b] Reaction time ee[b] Reaction time
Enzymes
(min)[c]
(min)[c]
(min)[c]
[d]
[d]
[d]
[d]
FYA
98 (S)
98 (S)
96 (S)
98 (S)
98 (S)
98 (S)
98 (S)
40
50
30
55
30
35
35
-
-
-
-
A33D/FYA
A33E/FYA
A33C/FYA
A33I/FYA
A33L/FYA
A33V/FYA
98 (S)
96 (S)
98 (S)
98 (S)
98 (S)
98 (S)
70
60
70
35
50
45
92 (S)
98 (S)
> 360[e]
> 360[e]
[d]
[d]
-
-
98 (S)
96 (S)
98 (S)
90
180
120
5
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[a]
Assay conditions: 3 mM 4b, 100 mM 3, 73 µM 4-OT, 20 mM NaH2PO4 (pH 7.3), 0.3 mL
[b]
reaction volume
Determined by GC using a chiral stationary phase; the absolute
[c]
configuration was determined by literature comparison[8,9]
monitored by following the depletion in absorbance at 249 nm.
precipitation. [e] Reaction was not finished after 360 min.
Reaction progress was
No data due to protein
[d]
The best mutant, 4-OT A33I/FYA, catalyzed the Michael-type addition reaction practically as
efficient in 40% ethanol as in 10% ethanol (Figure 3B). Moreover, pre-incubation of 4-OT
A33I/FYA in 50% ethanol for 10 h resulted in only 25% loss of activity (Figure 3A). On the
contrary, the parental enzyme (4-OT FYA) rapidly lost its activity upon incubation in 10%
60
ethanol (Figure 3A, B). Interestingly, an increase in the T50 of approximately 6 °C was
observed for 4-OT A33I/FYA compared to 4-OT FYA, indicating that 4-OT A33I/FYA is
also somewhat more thermostable than 4-OT FYA (Figure 3C). Finally, to further
demonstrate the synthetic usefulness of 4-OT A33I/FYA, a semi-preparative scale reaction
was performed, using 40% ethanol as cosolvent, which allowed for the solubilization of 15
mM 4a. Using a 6.7-fold excess of 3 over 4a, the reaction was finished within 200 min.
Product R-5a was obtained in excellent isolated yield (89%) and enantiopurity (ee = 98%)
(Figures S3 and S4). Taken together, these results demonstrate that 4-OT can be engineered to
efficiently catalyze enantioselective Michael-type reactions in ethanol concentrations up to
40%.
6
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Figure 3. Characterization of 4-OT A33I/FYA (FIYA). A) Enzyme activity for the Michael-
type addition of 3 to 4a after pre-incubation (1, 2, 4, 6, 8 or 10 h) in the presence of 10, 30 or
50% ethanol. The data is normalized to the activity of the enzyme without pre-incubation. B)
Progress curves of 4-OT A33I/FYA- and 4-OT FYA-catalyzed Michael-type additions in the
presence of different ethanol concentrations. C) Temperature-induced inactivation profiles of
4-OT A33I/FYA and 4-OT FYA. The enzyme activity after incubation for 60 min at 30 °C
was set as 100%. D) Photograph of cuvettes in which 4-OT FYA (190 µg/mL, top five
cuvettes) and 4-OT A33I/FYA (190 µg/mL, bottom five cuvettes) were incubated for 1 h in
20 mM NaH2PO4 buffer (pH 7.3) containing 10%, 20%, 30%, 40% or 50% (v/v) ethanol
(cuvette 1 to 5, respectively, from left to right). 4-OT FYA shows rapid precipitation in buffer
containing >10% ethanol.
7
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Discussion
Enzymes are highly attractive catalysts for organic synthesis because of their unparalleled
enantio-, regio- and chemoselectivity. Given that enzymes have evolved to operate in the mild
aqueous environment of the cell, they are usually not fit for preparative biocatalysis in the
presence of high concentrations of organic cosolvents required for substrate solubilization.[10]
A solution to this problem is the engineering of enzymes to improve their cosolvent tolerance.
Rational enzyme engineering towards increased cosolvent tolerance is still very challenging
due to our relatively poor understanding of the interactions between enzymes and solvent
molecules.[12–14] Currently employed rational engineering strategies include stabilization of
flexible regions, introduction of new cysteine bridges and modification of access tunnels.[14–17]
An important strategy to guide enzyme-engineering efforts is to make use of mutability
landscapes.[18–21] By screening a large collection of nearly all single mutants of an enzyme,
important information is obtained on single mutations or residue positions that influence a
desired characteristic of the enzyme. Here we have applied mutability-landscape guided
enzyme engineering to improve the ethanol tolerance of 4-OT. Screening of a collection of
nearly all single-mutant variants of 4-OT revealed that mutations at particularly positions Ser-
30 and Ala-33 resulted in improved ethanol tolerance. Interestingly, a previously reported 4-
OT variant with 3.5-fold increased ‘Michaelase’ activity, 4-OT A33D, also showed improved
ethanol tolerance.[8] We used the information from the ethanol-tolerance mutability landscape
to further engineer a previously constructed highly enantioselective 4-OT variant (FYA) that
exhibits poor ethanol stability. All 19 possible variants at position Ala-33 in the context of 4-
OT FYA were constructed, expressed and purified. Remarkably, from this small, focused set
of quadruple mutants, six mutants showed strongly improved ethanol tolerance. It is
interesting to note that all six variants, including 4-OT A33D/FYA, are highly
enantioselective towards the synthesis of S-5b, similar to the parental mutant 4-OT FYA.[9]
8
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Conversely, the single mutant 4-OT A33D markedly improved the enantioselectivity towards
the opposite enantiomer R-5b.[8]
Incubation of the best mutant, 4-OT A33I/FYA, for 10 h in the presence of 50% ethanol
resulted in only 25% loss of activity, whereas the parental enzyme 4-OT FYA lost all its
activity upon incubation for 1 h in 10% ethanol. 4-OT A33I/FYA also showed an increase in
thermostability compared to 4-OT FYA, an effect that has also been observed for other
enzymes that have been engineered towards increased solvent tolerance.[22,23] We further
show that 4-OT A33I/FYA can be used to efficiently catalyze the Michael-type addition of 3
to 4a in the presence of 40% ethanol, which permitted the use of a higher substrate loading
(up to 15 mM 4a). Product R-5a could be obtained in good isolated yield (89%) and with
excellent enantiopurity (ee = 98%).
In conclusion, our results demonstrate the power of mutability landscapes to guide
engineering efforts to improve the cosolvent tolerance of enzymes. By specifically targeting
the identified ‘hotspot’ position Ala-33, we could engineer an ethanol-sensitive mutant, 4-OT
FYA, into a highly ethanol-resistant mutant 4-OT A33I/FYA. Further tuning of 4-OT
A33I/FYA might lead to new synthetic opportunities in almost neat organic solvents.
9
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Experimental section
Production of cell-free extract. Cell-free extracts (CFE) of 4-OT single mutants were
prepared according to a reported procedure.[8]
Construction of the ethanol-tolerance mutability landscape. The CFEs prepared from cells
producing 4-OT single-mutant variants were used in two reactions, containing either 5% or
25% v/v ethanol. The following reaction conditions were used: CFE (20% v/v), 3 (50 mM),
4a (0.5 mM) in 20 mM NaH2PO4 buffer (pH 7.3), 100 µL final volume. The reactions were
performed in 96-well microtiter plates (MTP) (UV-star µclear, Greiner Bio-one), covered
with UV-transparent plate seals (VIEWsealTM, Greiner Bio-one). To ensure proper mixing of
the reagents, the plate was shaken (60 s at 500 rpm) immediately after all reaction
components were added. The reaction progress was monitored in a plate reader by measuring
the depletion in absorbance at 320 nm, corresponding to the concentration of 4a, for 60 min
with a 60 s data interval. The slope of the linear part of the curve was determined for both the
reactions. The remaining enzymatic activity was determined by dividing the slope of the
reaction in 25% v/v ethanol by the slope of the reaction in 5% v/v ethanol.
4-OT purification. The purification of 4-OT single mutants[24] and 4-OT quadruple mutants[9]
are based on previously reported procedures. All purified proteins were >90% pure as
assessed by SDS-PAGE. All purified mutants were analyzed by electron spray ionization
(ESI) mass spectrometry to confirm the correct mass of the enzyme. The purified protein was
flash-frozen in liquid nitrogen and stored at -80 °C until further use.
UV-spectroscopic assay for the enzymatic activity of 4-OT single mutants in different
organic solvents. The enzymatic activities of the 4-OT mutants and wild-type 4-OT was
monitored by following the decrease in absorbance at 320 nm, which corresponds to the
10
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depletion of 4a. Purified enzyme (150 µg, 73 µM) was incubated in a 1 mm cuvette with 3
(50 mM) and 4a (2 mM) in 20 mM NaH2PO4 (pH 7.3; 0.3 mL final volume).
Construction of nineteen Ala-33 mutants of 4-OT L8F/M45Y/F50A. Ala-33 was
randomized by Quikchange technology using the gene encoding 4-OT L8F/M45Y/F50A
cloned in the pET20b vector as the template. The following two primers were used: 5’-
GCTCCCTGGATNNKCCGCTGACCAG-3’
and
5’-
CTGGTCAGCGGMNNATCCAGGGAGC-3’. After transformation of the DNA into E. coli
cells, random colonies were picked from an agar plate, and the mutant 4-OT genes were
sequenced by Macrogen Europe (Meibergdreef 31, 1105AZ, Amsterdam, the Netherlands)
until all of the 19 quadruple mutants were obtained.
Activity assays of the six best quadruple mutants. The enzymatic activities of the 4-OT
quadruple mutants and 4-OT L8F/M45Y/F50A were monitored by following the decrease in
absorbance at 249 nm, which corresponds to the depletion of 4b. Purified enzyme (150 µg, 73
µM) was incubated in a 1 mm cuvette with 3 (100 mM) and 4b (3 mM) in 20 mM NaH2PO4
buffer (pH 7.3; 0.3 mL final volume). After the reactions were completed, product 5b was
extracted with ethyl acetate (400 µL) and analyzed by gas chromatography using an Astec
CHIRALDEX G-TA column, isocratic 125 °C. Retention time S-5b: 25.6 min, retention time
R-5b: 26.9. The assignment of the absolute configuration was based on earlier reported data.[9]
Determination of T5060. 4-OT L8F/M45Y/F50A and 4-OT L8F/A33I/M45Y/F50A (50 µL of
2 mg/mL in 20 mM NaH2PO4, pH 7.3) were incubated in 0.2-mL PCR tubes at temperatures
ranging from 30 °C to 90 °C for 60 min in a thermal cycler. After incubation, the enzymes
were cooled on ice for 10 min followed by equilibration at 25 °C for 10 min. Samples were
centrifuged to remove any precipitated protein. The residual ‘Michaelase’ activity for the
addition of 3 to 4a was tested in a plate reader. Following conditions were used: 25 µL of
enzyme supernatant, 50 mM 3, 0.5 mM 4a, 5% v/v ethanol in 20 mM NaH2PO4 buffer (pH
11
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7.3), 100 µl final volume. The ‘Michaelase’ activities were normalized to that obtained after
60 min incubation at 30 °C.
Stability of 4-OT L8F/M45Y/F50A and 4-OT L8F/A33I/M45Y/F50A upon incubation
with increasing ethanol concentrations. 4-OT L8F/M45Y/F50A and 4-OT
L8F/A33I/M45Y/F50A (1 mL of 1.5 mg/mL in 20 mM NaH2PO4, pH 7.3) were incubated in
20 mM NaH2PO4 buffer (pH 7.3) containing 10%, 30% or 50% v/v ethanol in a water bath of
25 °C. Aliquots of enzyme (80 µL) were taken at different time intervals and centrifuged to
remove any aggregated protein. 50 µL of the supernatant was used to test the residual
enzymatic activity. The reaction mixture consisted of the following: 3 (50 mM), 4a (2 mM,
from a 40 mM stock solution in 100% (v/v) ethanol) in 20 mM NaH2PO4 buffer (pH 7.3), 0.3
mL final volume. Depletion of 4a was monitored by following the decrease in absorbance at
320 nm in time. The activities were normalized to the activity measured without incubation of
the enzyme.
Semi-preparative scale synthesis. To a 50 mL round bottom flask was added: 6 mL ethanol,
112 µL 3, 12 mL buffer (20 mM NaH2PO4, pH 6.5) containing 4-OT L8F/A33I/M45Y/F50A.
The reaction was initiated by the addition of 2 mL ethanol containing 150 mM 4a. The final
concentrations were: 3 (100 mM), 4a (15 mM), 4-OT L8F/A33I/M45Y/F50A (75 µM, based
on monomer concentration), and 40% (v/v) ethanol in 20 mM NaH2PO4 buffer (pH 6.5). The
reaction progress was monitored using UV-spectrophotometric analysis. At timely intervals, a
sample of 30 µL was collected from the reaction mixture and diluted to 300 µL with 20 mM
NaH2PO4 buffer and a full spectrum from 200 nm to 500 nm was recorded. After 200 min, the
reaction was finished. The reaction mixture was extracted 3x with ethyl acetate. The
combined organic layers were washed with brine and dried over anhydrous Na2SO4. The
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organic layer was concentrated in vacuo, yielding 5a without any further purification (51.5
mg, 89% yield). The aldehyde functionality of 5a was derivatized to a cyclic acetal according
to a reported procedure[5]. The enantiopurity of derivatized 5a was determined by reverse
phase HPLC using a chiralpak AD-RH column (150 mm x 4.6 mm, Daicel) MeCN/water
70:30. Retention time R-5a: 7.8 min, S-5a: 10.8 min.
Acknowledgement
We acknowledge financial support from the Netherlands Organization of Scientific Research
(VICI grant 724.016.002) and the European Union’s Horizon 2020 Research and Innovation
Programme under grant agreement No 635595 (CarbaZymes).
13
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ChemBioChem
TOC graphic and text
Loving alcohol! In this study, we generated a mutability landscape of 4-oxalocrotonate
tautomerase (4-OT) to identify ‘hotspot’ positions at which mutations are beneficial for
catalysis in high concentrations of ethanol. Randomization of one of these ‘hotspot’ residues
in a highly enantioselective, but ethanol-sensitive, enzyme variant (4-OT FYA) resulted in an
improved enzyme variant (4-OT A33I/FYA) with high ethanol stability, allowing efficient
and enantioselective catalysis of Michael reactions in 40% ethanol.
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