Clean Enzymatic Oxidation of 12α-Hydroxysteroids to 12-Oxo-Derivatives Catalyzed by Hydroxysteroid Dehydrogenase

Article abstract of DOI:10.1002/adsc.201900144

The C12 specific oxidation of hydroxysteroids is an essential reaction required for the preparation of pharmaceutical ingredients like ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA), which can be synthesized by Wolff-Kishner reduction of the obtained 12-oxo-hydroxysteroids. 12α-hydroxysteroid dehydrogenases (12α-HSDHs) have been shown to perform this reaction with high yields, under mild conditions and without the need of protection and deprotection steps, required in chemical synthesis. Here, the recombinant expression and biochemical characterization of the nicotinamide adenine dinucleotide (NAD⁺)-dependent HSDH from Eggerthella lenta (El12α-HSDH) are reported. This enzyme shows comparable properties with the well-known nicotinamide adenine dinucleotide phosphate (NADP⁺)-dependent enzyme from Clostridium sp. 48–50. In order to perform a viable and atom efficient enzymatic hydroxysteroid oxidation, NAD(P)H oxidase (NOX) was employed as cofactor regeneration system: NOX uses oxygen (O₂) as sacrificial substrate and produces only water as side product. 10 mM of cholic acid was fully and selectively converted to 12-oxo-CDCA in 24 h. The possibility to employ this system on UCA and 7-oxo-deoxycholic acid (7-oxo-DCA) as substrates was additionally investigated. The performance of the El12α-HSDH was evaluated also in combination with a “classical” regeneration system (oxaloacetate/malate dehydrogenase) showing full conversion in 4 h. Finally, the feasibility of a catalytic aerobic-NAD⁺-dependent enzymatic oxidation was shown on a preparative scale (oxidation of CA to 12-oxo-CDCA) employing the El12α-HSDH-NOX system in a segmented-flow-reactor. (Figure presented.).

Full text of DOI:10.1002/adsc.201900144

Title: Clean Enzymatic Oxidation of 12α-Hydroxysteroids to 12-Oxo-
Derivatives Catalyzed by Hydroxysteroid Dehydrogenase
Authors: Fabio Tonin, Natalia Alvarenga, JiaZheng Ye, Isabel Arends,
and Ulf Hanefeld
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
COMMUNICATION
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Clean Enzymatic Oxidation of 12α-Hydroxysteroids to 12-Oxo-
Derivatives Catalyzed by Hydroxysteroid Dehydrogenase
a
a
a
a,b
Fabio Tonin , Natália Alvarenga , Jia Zheng Ye , Isabel W.C.E. Arends and Ulf
a
Hanefeld *
a
Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
Present address: Faculty of Science, Utrecht University, Budapestlaan 6, 3584 CD Utrecht, The Netherlands. E-mail:
b
U.Hanefeld@tudelft.nl
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. The C12 specific oxidation of hydroxysteroids is digestion of fatty acids, to reduce cholesterol
an essential reaction required for the preparation of absorption and, in cases of cholestatic diseases, to
[
3]
pharmaceutical ingredients like ursodeoxycholic acid
stimulate the liver functions. CDCA has been used
for the same treatments, but its side effects made the
use in clinical treatment less desiderable than
(
UDCA) and chenodeoxycholic acid (CDCA), which can
be synthesized by Wolff-Kishner reduction of the obtained
2-oxo-hydroxysteroids. 12α-hydroxysteroid
1
[4]
UDCA.
However, in the last years several
dehydrogenases (12α-HSDHs) have been shown to perform
this reaction with high yields, under mild conditions and
without the need of protection and deprotection steps,
pharmaceutical properties of this compound have
been discovered and explored (e.g. CDCA is used for
the treatment of Cerebrotendinous Xanthomatosis
required in chemical synthesis. Here, the recombinant
[5]
expression and biochemical characterization of the (CTX), a rare genetic metabolic disorder).
nicotinamide adenine dinucleotide (NAD )-dependent Nowadays, these two compounds are produced by
+
HSDH from Eggerthella lenta (El12α-HSDH) are reported. chemical C12 dehydroxylation of cholic acid (CA) in
This enzyme shows comparable properties with the well-
a 5-step synthesis: after the protection of the
carboxylic group, a selective protection of the 3- and
known nicotinamide adenine dinucleotide phosphate
+
(
NADP )-dependent enzyme from Clostridium sp. 48-50.
7-OH groups with acetic anhydride and pyridine is
In order to perform a viable and atom efficient enzymatic
hydroxysteroid oxidation, NAD(P)H oxidase (NOX) was
employed as cofactor regeneration system: NOX uses
performed (92% yield). The removal of the 12-OH
group is achieved by a redox route. Oxidation with
CrO
formed keto group by Wolff-Kishner reduction (82%
selectively converted to 12-oxo-CDCA in 24 h. The yield) finally give CDCA with an overall yield of
3
(98% yield) and subsequent reduction of the
oxygen (O
2
) as sacrificial substrate and produces only
water as side product. 10 mM of cholic acid was fully and
[6]
possibility to employ this system on UCA and 7-oxo- 65%.
deoxycholic acid (7-oxo-DCA) as substrates was The selective oxidation of the 12-OH group can be
additionally investigated. The performance of the El12α-
achieved
by
using
a
12α-hydroxysteroid
HSDH was evaluated also in combination with a “classical”
regeneration system (oxaloacetate/malate dehydrogenase)
showing full conversion in 4 h. Finally, the feasibility of a
[7]
dehydrogenase (12α-HSDH). Using this enzyme,
the 12-hydroxy group of CA can be specifically
oxidized to the corresponding ketone (forming 12-
oxo-CDCA) without the need of protection steps or
the toxic Cr (VI). The product can afterwards
undergo Wolff-Kishner reduction to form CDCA
+
catalytic aerobic-NAD -dependent enzymatic oxidation
was shown on a preparative scale (oxidation of CA to 12-
oxo-CDCA) employing the El12α-HSDH-NOX system in a
segmented-flow-reactor.
(
Scheme 1). The advantages of enzymatic oxidation
+
Keywords: Bile acids; NAD -dependent; 12α-
include the reduction of waste and mild reaction
conditions with high conversion and yield utilizing
hydroxysteroid dehydrogenases; NAD(P)H oxidase; Flow-
reactor
[8]
catalysts easily produced by microorganisms. The
1
2α-HSDHs belong to the family of oxidoreductases
+
+
with NAD or NADP as electron acceptor. The
NADP -dependent activity is distributed among the
strains of the genus Clostridium, while the NAD -
+
Ursodeoxycholic
acid
(UDCA)
and
+
chenodeoxycholic acid (CDCA) are widely used as
pharmaceutical ingredients.[1] UDCA can be
employed to treat gallstones,[2] to improve the
dependent activity has been observed and reported in
Eubacterium sp.
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
Scheme 1. Oxidation of 12α-OH group of hydroxysteroids catalysed by 12α-HSDH.
+
In this work, the gene corresponding to the NAD+-
Several reports describe the NADP -dependent 12α-
HSDH from Clostridium sp. 48-50 (C12α-HSDH) as
biocatalyst for the production of 12-oxo
dependent 12α-HSDH from Eggerthella lenta
(El12α-HSDH) was characterized. Although this
[
9]
+
[12]
hydroxysteroids. However, the use of a NAD -
dependent enzyme for this reaction would be more
enzymatic activity was identified earlier,
its
biocatalytic potential was not investigated to date.
The identified gene coding for the El12α-HSDH
+
+
desirable: in comparison to NADP , NAD is more
stable, naturally more abundant, cheaper and easier to
regenerate by employing additional enzymes and
sacrificial substrates.[ In addition, several studies
have recently reported the development of a fully
(
GenBank: WP_114518444.1) showed 57% of
+
sequence identity with the NADP -dependent C12α-
10]
HSDH (GenBank: WP_044992937), 53% with the
+
NADP -dependent enzyme from Eggerthella
+
NAD -dependent process for the production of 12-
CAG:298 (GenBank: CDD59475.1) and differs from
the reported enzyme Elen-2515 (GenBank:
ACV56470.1) by a single amino acid (P41S).
Multiple sequence analyses (BLASTp), 3D structure
oxo-UDCA,[ opening up the possibility that the
11]
same cofactor could be used for both epimerization
+
(
employing NAD -dependent 7α- and 7βHSDH) and
the 12-OH oxidation step.
modelling (using SWISS-MODEL) and docking
+
[13]
A NAD -dependent 12α-HSDH from unknown
analyses (AutoDock VINA)
were carried out in
source has been commercialized by Genzyme
Biochemicals and employed in several biocatalytic
studies,[ however no DNA or protein sequences
were annotated for this enzyme.
order to investigate the protein sequence/structure
relationship of this enzyme. Particularly, the NADP+
binding motif (G39 and R40) in C12α-HSDH, is
8c]
+
exchanged to a NAD binding motif (D39 and L40)
Figure 1. (A) Hypothetical
binding mode of the NADH
inside the 3D model of El12α-
HSDH active site: the putative
formation of a hydrogen bond
between the aspartate side-
chain (D39) and the 2’-OH
group of ribose (distance 1.9 -
2
.3 Å). (B) Docking analysis of
CA in the catalytic pocket of
El12α-HSDH. The 3D structure
model of the El12α-HSDH was
obtained
MODEL
using
SWISS-
(
https://swissmodel.expasy.org/
interactive), employing the
crystal structure of the putative
SDR
from
Burkholderia
xenovorans (PDB ID: 5JY1.1)
as template. Docking analyses
were performed employing
[
13]
AutoDock Vina
Supplementary Material).
(see
2
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
(
Supplementary Figure 1). This change in the amino
K
m
value on the performances of the enzymes is of
acid sequence is frequently involved in the
lower relevance as concentrations are typically higher
(< 5 mM). Indeed, to achieve similar conversions,
larger amounts of El12α-HSDS where therefore
necessary (Table 2).
determination of the cosubstrate specificity of these
enzymes.[
11]
The 3D model of the El12α-HSDH
structure showed a homotetrameric oligomeric state,
conserved among the group of SDR oxidoreductases,
The enzymatic activities in the presence of the not-
preferred cofactor were also assayed and, as predicted
by the in silico analysis of the sequence/structural
+
confirming the predicted NAD -binding (Figure 1A).
Docking analysis of CA in the active site of the
El12α-HSDH showed correct positioning of the 12α-
OH group with respect to the catalytic residues (S145
and Y158) and the nicotinamide cofactor (Figure 1B).
Therefore, the bioinformatic data suggested that the
+
relationship, El12α-HSDH shows a strict NAD -
dependent activity (Figure 2A). Notably, since the K
m
+
value for NAD is 0.91 mM, a decrease of the activity
was observed when lower amounts of cofactor were
employed.
+
sequence was coding for a NAD -dependent 12α-
HSDH.
Table 1. Kinetic parameters of the recombinant C12α-
HSDH and El12α-HSDH
In order to evaluate the catalytic performances of the
El12α-HSDH and directly compare them with the
one of the NADP -dependent system reported in
literature, C12α-HSDH and El12α-HSDH were
recombinantly expressed in E. coli BL21 (DE3) cells
as N-His-tagged proteins and purified by a single
HiTrap chelating chromatography step ( 95%
purity, as determined by SDS-PAGE analysis,
Supplementary Figure 2).
+
kcat
Km
kcat/K
m
-1
(
s )
(mM)
CAa)
97.7 ±2.4
0.121 ±0.014 807.3 ±113.2
0.252 ±0.022 248.9 ±22.4
7
7
-oxo-DCAa) 62.7 ±0.2
UCAa)
DCAa)
113.8 ±2.8 0.239 ±0.025 476.0 ±61.4
198.9 ±11.5 0.548 ±0.117 362.9 ±98.4
El12α-HSDH was expressed in higher volumetric
yield than the C12α-HSDH (215 and 26 mg/Lculture
NADP+b)
78.6 ±2.4
0.073 ±0.009 1077 ±165
,
respectively), making it more suitable for its
industrial employment (Purification tables are
provided in Supplementary Table 1). El12α-HSDH
and C12α-HSDH showed enzymatic activity (under
standard conditions) of 59.2 and 30.1 U mg ,
respectively.
Notably, the position of the His-tag plays a crucial
role in the activity of these enzymes: when a C-His-
C12α-HSDH was expressed and purified, it showed a
low enzymatic activity (0.1 U mg ). This loss of
activity was probably due to the C-His-Tag
CAa)
146.1 ±4.0 0.636 ±0.062 229.8 ±28.6
-oxo-DCAa) 90.0 ±2.3
UCAa)
DCAa)
0.461 ±0.047 195.3 ±24.8
0.399 ±0.060 218.1 ±41.3
0.673 ±0.130 40.9 ±10.1
87.0 ±3.4
27.5 ±1.5
-1
NAD+b)
122.4 ±10.5 0.919 ±0.164 133.2 ±35.2
a)
Kinetic parameters determined in presence of 1 mM of
+
b)
NAD(P) ; Kinetic parameters determined in presence of 1
mM of CA. Activities were spectrophotometrically
measured by following the production of NAD(P)H at 340
nm. All the activities were performed in presence of 50
mM KPi buffer, pH 8.0 and 10% MeOH at 25 °C. kcat
values were calculated considering a MW of 111 and 113
kDa for C12α-HSDH and El12α-HSDH, respectively.
-1
“
masking” of the C-terminal proline residue, which
has an essential role in the binding of bivalent cations
necessary for the enzyme oligomerization. Similar
behaviour was observed in other C-terminal proline
proteins.[ Additionally, the C-His-Tag was located
close to the active site of the enzyme, restricting the
accessibility of the substrate in the catalytic pocket
14]
The activity and the stability of the obtained enzymes
were evaluated at different pH values, different
MeOH concentrations and temperatures. Unlike the
C12α-HSDH, which showed a pH optimum at pH
8.0, the optimal pH for El12α-HSDH activity was 10
(Figure 2B). However, stability studies showed that
this enzyme was less stable at this pH value, retaining
43% of its initial activity when incubated for 24 hour
at pH 10 (Supplementary Figure 4A). Thus, we
conclude that the optimal working pH for both
enzymes is 8.0.
(
3
further details are provided in Supplementary Figure
and Supplementary Table 2).
Kinetic parameters of the two enzymes were
evaluated with different substrates (CA, 7-oxo-
deoxycholic acid (7-oxo-DCA), UCA and
deoxycholic acid (DCA)) in the presence of β-
nicotinamide cofactors (NAD and NADP , for
El12α-HSDH and C12α-HSDH, respectively) (Table
+
+
1
).
Interestingly, El12α-HSDH was less hampered by the
cosolvent, retaining 100% of its activity in presence
of 10% of MeOH (Figure 2C). Additionally, no
significant decrease of activity was observed after 24
The C12α-HSDH showed higher affinity to both
substrate and cosubstrate. On the other hand, El12α-
HSDH displayed higher Vmax for CA and 7-oxo-
DCA: in biocatalytic reactions, the influence of the
3
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
h of incubation of the enzyme under the same
conditions (Supplementary Figure 4B).
and NADPH, (198 and 223 mU/mgfreeze-dried powder,
respectively). In order to demonstrate the
applicability of this enzymatic cascade
Supplementary Scheme 1A), bioconversion reactions
(
-
1
were set up with 0.1 U mL of 12α-HSDHs, NOX
(
mM) and NAD(P) (0.2, 0.5 and 1 mM).
-1
0.5 mg mL ), different amounts of CA (5, 10 and 20
+
The C12α-HSDH-NOX system was able to fully
convert 5 and 10 mM of CA into 12-oxo-CDCA in 8
and 48 h, respectively (Figure 3A). However, when
higher concentration (20 mM) of CA where
employed only 85% of conversion to the desired
product was achieved in 48 h. Notably, the reaction
rates observed were not influenced by the different
+
concentrations of NADP employed in the reactions,
which can be explained by the low K of the enzyme
m
for the cofactor (73 µM), making the enzymatic
reactions proceed at rates close to Vmax even at low
cosubstrate concentrations (Table 2; entries 1-3).
Figure 2. Activity of C12α-HSDH (black) and El12α-
HSDH (grey) under different conditions. (A) activity in
presence of different concentrations of nicotinamide
cofactors, 1 mM CA, 10% MeOH and 50 mM KPi pH 8.0.
(
B) Effect of pH on the enzymatic activity at 25 °C. The
value at pH 8.0 is taken as 100%. (C) Effect of MeOH on
the enzymatic activity at pH 8.0, 25 °C. The value in
absence of MeOH is taken as 100%. (D) Effect of
temperature on the enzymatic activity at pH 8.0. The value
at 25 °C is taken as 100%. In all the cases, activities were
+
determined in presence of 1 mM CA and 1 mM NAD(P)
by measuring the NAD(P) reduction at 340 nm.
+
Finally, the influence of the temperature on the
enzymatic activity of these two enzymes was
evaluated. As previously reported, C12α-HSDH is a
thermostable protein, showing
a
temperature
optimum of 75 °C and retaining 97% of its initial
activity after 24 h of incubation at 37 °C. On the
other hand, El12α-HSDH showed a temperature
optimum of 55 °C (Figure 2D), but its activity was
halved after incubation at 37 °C for 24 h. However,
both enzymes were stable when incubated at 25 °C
for 24 h (Supplementary Figure 4C).
The biocatalytic properties of the two 12α-HSDHs
were evaluated in batch mode reactions. In order to
regenerate the oxidised nicotinamide cofactors,
NAD(P)H oxidase (NOX), a commercial enzyme that
Figure 3: Bioconversion time-courses employing (A)
C12α-HSDH and (B) El12α-HSDH of: (green lines) 20
mM, (red lines) 10 mM and (blue lines) 5 mM of CA in
presence of (from the lightest to the darkest tone) 0.2, 0.5
+
uses O
In comparison with other classical regeneration
systems, the NOX system has the advantage that O
2
for the oxidation of NAD(P)H was applied.
and 1 mM NAD(P) . All reactions were carried out
-
1
employing 0.1 U/mL of enzyme (2.6 µg mL and 3.5 µg
-
1
mL of C12α-HSDS and El12α-HSDS, respectively) and
2
-
1
0
.5 mg mL of NOX in 50 mM KPi, pH 8.0 and 10%
as sacrificial substrate is gaseous and generates only
water as side product, leading to cleaner reactions and
MeOH at 25 °C.
[15]
simplifying the downstream processes.
As
On the other hand, in the presence of 1 mM NAD+,
El12α-HSDH converted 5 and 10 mM of CA in 12-
observed from the biochemical characterization,
NOX shows comparable activity with both NADH
4
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
oxo-CDCA in 6 and 24 h, respectively (Figure 3B).
Under the same conditions, 20 mM of substrate were
converted to the desired product (94% conversion).
these recombinant enzyme.
Having established the time course of the reaction,
experiments focused on the scope of the enzymatic
system (Table 2).
+
Different to its NADP -dependent homologue, the
reaction rate decreased when lower amounts of
cosubstrate were applied. However, except when 20
mM of CA were incubated in the presence of 0.2 mM
The systems C12α-HSDH-NOX and El12α-HSDH-
NOX were applied for the preparation of 7,12-dioxo-
LCA, 12-oxo-UDCA and 12-oxo-LCA (using 7-oxo-
DCA, UCA and DCA as substrate, respectively;
entries 7-12). In comparison to the reaction with CA
as substrate, lower conversions and rates were
observed with both enzymatic systems. However,
+
NAD(P) , the catalytic performances of the El12α-
HSDH were better than those of the C12α-HSDH,
leading to higher conversions and lower reaction
times (Table 2, entries 4-6). Again, the disadvantage
in the use of the El12α-HSDH is partially
compensated by the high volumetric production of
+
when a “classical” NAD -regeneration system
(
oxaloacetate and malate dehydrogenase (MDH –
Table 2. Bioconversion table
[
Substrate] Regeneration Scale
Conversion(time)
(%)(h)
Entry
Enzyme
Reaction
12-oxo-CDCA
(
mM)
10
20
20a)
10
20
20a)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
20
system
NOX
(mg)
10
20
20
10
20
20
10
10
10
10
10
10
10
10
10
10
1
2
3
4
5
6
7
8
9
C12α-HSDH
C12α-HSDH
C12α-HSDH
El12α-HSDH
El12α-HSDH
El12α-HSDH
CA
CA
CA
CA
CA
CA
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
54(6)
36(6)
32(6)
80(6)
26(6)
12(6)
10(6)
28(6)
43(6)
12(6)
96(24)
72(24)
65(24)
>99(24)
78(24)
48(24)
34(24)
76(24)
56(24)
94(24)
20(24)
12-oxo-CDCA
12-oxo-CDCA
12-oxo-CDCA
12-oxo-CDCA
12-oxo-CDCA
7,12-dioxo-LCA
12-oxo-UDCA
12-oxo-LCA
NOX
NOX
NOX
NOX
NOX
C12α-HSDH 7-oxo-DCA
NOX
C12α-HSDH
C12α-HSDH
UCA
DCA
NOX
NOX
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
El12α-HSDH 7-oxo-DCA
7,12-dioxo-LCA
12-oxo-UDCA
12-oxo-LCA
NOX
El12α-HSDH
El12α-HSDH
El12α-HSDH
UCA
DCA
CA
NOX
3
2
(6)
(6)
NOX
3
(24)
12-oxo-CDCA
7,12-dioxo-LCA
12-oxo-UDCA
12-oxo-LCA
OxAc + MDH
OxAc + MDH
OxAc + MDH
OxAc + MDH
>99(2)
El12α-HSDH 7-oxo-DCA
85(2)
56(2)
53(2)
>99(4)
>99(4)
>99(4)
El12α-HSDH
El12α-HSDH
El12α-HSDH
UCA
DCA
CA
12-oxo-CDCA
7,12-dioxo-LCA
12-oxo-UDCA
12-oxo-LCA
OxAc + MDH 200
OxAc + MDH 1000
OxAc + MDH 200
OxAc + MDH 200
>99(4)
El12α-HSDH 7-oxo-DCA
>99(4)
>99(4)
>99(4)
34(6)b)
23(6)c)
El12α-HSDH
El12α-HSDH
C12α-HSDH
C12α-HSDH
El12α-HSDH
El12α-HSDH
El12α-HSDH
El12α-HSDH
UCA
DCA
CA
12-oxo-CDCA
12-oxo-CDCA
12-oxo-CDCA
12-oxo-CDCA
12-oxo-CDCA
12-oxo-CDCA
NOX
NOX
NOX
NOX
NOX
NOX
25
25
CA
CA
25
18(6)b)
c)
CA
25
9
(6)
CA
300
600
78(6)d)
28(6)d)
>99(24)d)
68(24)d)
CA
a)[NAD(P)+]=0.2 mM; b)Reaction in autoclave under oxygen pressure (3 bar); c)Reaction in autoclave under ambient
d)
-1
-1
atmosphere; Reaction in flow-reactor. All the reactions were carried out employing 0.1 U mL of enzyme (2.6 µg mL
-
1
-1
+
and 3.5 µg mL of C12α-HSDS and El12α-HSDS, respectively), 0.5 mg mL of NOX and 0.5 mM NAD(P) in 50 mM
KPi, pH 8.0 and 10% MeOH at 25 °C. In all the cases we observe a reaction selectivity of 100%. Reaction times are shown
in the brackets.
5
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
[
6]
Supplementary Scheme 1B) was used, all three
substrates were fully converted in 4 h by El12α-
HSDH (entries 13-16). The same performance was
observed on preparative scale (entries 17-20). The
comparison between the regeneration systems
suggests that the rate limiting enzyme of 12α-HSDH-
NOX cascade is the cofactor regeneration by NOX.
For the pure chemical route we calculated an atom
[17]
[18]
efficiency of 49% and an E-factor of 55.4.
In addition to these undesired values, the route
requires the use of the highly toxic and carcinogenic
CrO . The combination of 12α-HSDH and NOX
3
showed high atom efficiency (96%) and a low E-
factor (2.5). In comparison, the “classical” MDH
regeneration system showed a lower (but still
acceptable) atom efficiency (75%) and an E-factor
value of 4.4. These calculations do not take in
account the waste produced for the production and
purification of the enzymes and chemicals or for the
Since NOX uses O as electron acceptor, the poor
2
solubility of this gas in aqueous environment can
+
limit the NAD(P) -regeneration rate. In order to
investigate the O
out in an autoclave (with 3 bar of pure O
2
2
limitation, reactions were carried
gas; entries
1 and 23). Increased conversions were obtained
2
downstream
process.
The
alternative
when comparing the reaction under O
2
pressure with
chemo/enzymatic route for the production of 12-oxo-
UDCA (chemical oxidation of CA to dehydrocholic
acid (DHCA) followed by reduction with 3α- and 7β-
HSDHs in presence of formate dehydrogenase ) is
less atom economic (57%). Although the E-factor
value of this system (5.2) is comparable with the 12α-
HSDH-MDH system proposed here, the chemical
oxidation is still performed employing toxic and
environmentally hazardous reagents, making it less
desirable for an industrial scale.
control reactions (in autoclave under atmospheric
pressure; entries 22 and 24). However, with both
enzymes, conversion values lower than those
obtained in the rotatory shaker were observed,
suggesting that the stirring method employed in the
autoclave reactor is not optimal (possibly because of
the mechanical stress that leads to the inactivation of
the biocatalysts).
[19]
A promising technology that enables high O
2
transfer
rates without strain on the enzymatic structure is the
In conclusion, the clean oxidation of several 12α-
hydroxysteroids to 12-oxo derivatives by employing
flow-reactor.[
16]
Preparative reactions employing
El12α-HSDH-NOX system was recirculated for 24 h
in a flow-system (Supplementary Figure 5). This
system (entry 25-26) performed equally well as the
batch reactions on the rotatory shaker (analytical
scale; entry 4-5, respectively). 10 mM of CA were
fully converted into 12-oxo-CDCA in 24 h. However,
when a high substrate loading was applied (20 mM
CA) only 13.6 mM of product were obtained.
1
2α-HSDH-NOX system has been shown. This one-
step enzymatic transformation avoids the use of
protection groups and toxic oxidants (CrO ) required
3
by the chemical synthesis and, in comparison with
other enzymatic route, does not need sacrificial
substrates that complicate downstream processing. In
+
+
addition, the substitution of a NADP - for a NAD
_
dependent enzymatic system was achieved.
Combined, these factors improve the sustainability of
CDCA and UDCA production.
+
These data show the feasibility of a NAD -dependent
process for the specific oxidation of 12-OH
hydroxysteroids to 12-oxo-hydroxysteroids catalysed
by recombinantly expressed El12α-HSDH. The
biosynthetic potential of this enzyme is similar to the
one of the widely used C12α-HSDH, with the
advantage of using a cheaper and more stable
Experimental section
Cloning, recombinant expression and purification of
1
2α hydroxysteroid dehydrogenases (12α-HSDHs)
+
cosubstrate, NAD .
The synthetic cDNAs encoding for the El12α-HSDH and
for the C12α-HSDH were designed by in silico back
translation of the amino acid sequence reported in the
GenBank database (Accession no. WP_114518444.1 and
AET80684.1, respectively). The genes were subcloned into
the pET28a(+) plasmid, using NcoI (CCATGG) and XhoI
The combination of 12α-HSDH and NOX represents
a promising system for this biocatalytic conversion:
C12α-HSDH and El12α-HSDH, coupled with NOX
+
for NAD(P) regeneration, showed similar TTN and
-1
-1
ToF (330000 and 2.6 s vs. 250000 and 2.9 s for the
C12α-HSDH and El12α-HSDH, respectively).
However, when El12α-HSDH was coupled with a
(
(
CTCGAG) restriction sites resulting in 6.1-kb constructs
pET28-El12α-HSDH and pET28-C12α-HSDH). Six
codons (encoding for six additional histidines) were added
to the 5′-end of the 12α-HSDH gene during the subcloning
process. The codon usage of the synthetic gene was
optimized for expression in Escherichia coli and produced
by BaseClear.
HSDH enzymes were expressed in BL21(DE3) E. coli
cells and purified by HiTrap chelating affinity
chromatograpy employing the same procedure described in
a preceding paper[11a]
+
classical regeneration system for NAD , a ToF of
1
1
7.4 s- was calculated, confirming that the low
activity of NOX is limiting the reaction rate of these
systems. Future research will be conducted in order to
+
find a more robust O
regeneration system.
2
dependent NAD(P)
Green metrics values were calculated in order to
compare the different synthetic routes.
.
6
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
Activity and kinetic measurements
Acknowledgments:
1
2α-HSDHs enzymatic activities in the crude extract and
of the purified enzyme were determined at 25 °C using 1.0
mM CA, 1.0 mM NAD(P) , in 50 mM KPi buffer, pH 8.0
F.T. and N.A. gratefully acknowledge the ONE-FLOW project
(https://one-flow.org) that has received funding from the
European Union's FET-Open research and innovation actions
+
and 10% methanol (v/v). The production of NAD(P)H was
followed at 340 nm (extinction coefficient of NAD(P)H is
(proposal 737266 - ONE-FLOW). We thank Morten van der
Schie and all the BOC group of TU Delft for the scientific
discussions.
−
1
−1
6
,220 M ·cm ). One unit (U) was defined as the amount
of enzyme producing 1 µmol of product per minute at
5 °C and at pH 8.0. Blank measurements were performed
2
+
in absence of CA, NAD(P) and enzyme.
References:
Biocatalytic reactions
All bioconversions were carried out employing 0.1 U mL
-
1
[1]
[2]
T. Ikegami, Y. Matsuzaki, Hepatol. Res. 2008, 38,
23-131.
-
1
1
of purified C12α-HSDS (2.6 µg mL ) or El12α-HSDS (3.5
-1
-1
a) A. Crosignani, P. M. Battezzati, K. D. Setchell,
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NOX(001) (Prozomix Limited, Haltwhistle, UK - NOX),
1
0% MeOH and 50 mM KPi buffer at pH 8.0. Analytical
scale conversions were carried out in a 15 mL tube filled
with 2 mL of reaction mixture. Reactions were incubated
at 25 °C on rotator shaker (45 rpm) (IntelliMixer, Neolab
Migge GmbH, Heidelberg, DE). Reaction in autoclave
were performed employing 6 mL of reaction mixture in 10
mL metal vessels (HP Chemscan 8, HEL Ltd,
Borehamwood, UK), magnetically stirred (200 rpm), at
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2
5 °C under 3 bar of pure oxygen pressure. Control
reactions were performed in autoclave vessels under
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+
employing the “classical” NAD -regeneration system were
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-
1
-1
dehydrogenase from pig heart (6 U mL , 5 µg mL , Sigma
-
1
Aldrich, Saint Louis, USA) and 2.6 µg mL of C12α-
HSDS or 3.5 µg mL of El12α-HSDS. Samples (50 μL)
-
1
[
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water/CH
3
CN/TFA (70/30/0.1) solution and centrifuged at
1
4000 xg for 2 min. 10 μL of the obtained samples were
8
10.
analysed by HPLC (Supplementary Material).
Flow reactor experiments
Silicon reactor coils (2 mm ID) with a volume of 60 mL
were constructed. The reaction mixture (75 mL) containing
M. del Mar Amador, M. Masingue, R. Debs, F.
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+
-1
[6]
[7]
1
0 mM CA, 0.5 mM of NAD , 0.1 U mL of El12α-
-
1
HSDH and 0.5 mg mL of NOX was introduced (3.5 mL
min ) via a peristaltic pump (120U, Watson Marlow,
-
1
F. Tonin, I. W. C. E. Arends, Beilstein J. Org.
Chem. 2018, 14, 470.
-
1
Falmouth, UK), while the pure oxygen flow (5 mL min )
was controlled by a mass flow controller (EL-FLOW,
Bronkhorst, Ruurlo, NL), resulting in a segmented flow.
The reaction mixture was recirculated for 24 h (at 21 °C)
and, at fixed times analysed by HPLC.
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Abbreviations:
[
[
9]
CA, Cholic acid; CDCA, Chenodeoxycholic acid; DCA,
Deoxycholic acid; DHCA, dehydrocholic acid; LCA,
Lithocholic acid; UCA, Ursocholic acid; UDCA,
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HSDH,
Hydroxysteroid
1
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dehydrogenase; MDH, Malate Dehydrogenase; NOX,
NAD(P)H oxidase; OxAc, Oxaloacetate; SDR, Short-chain
Dehydrogenase/Reductase; ToF, Turnover frequency;
TTN, Total turnover number.
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8
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Advanced Synthesis & Catalysis
10.1002/adsc.201900144
COMMUNICATION
Clean Enzymatic Oxidation of 12α-
Hydroxysteroids to 12-Oxo-Derivatives
Catalyzed by Hydroxysteroid Dehydrogenase
Adv. Synth. Catal. Year, Volume, Page – Page
a
a
a
Fabio Tonin , Natália Alvarenga , Jia Zheng Ye ,
a,b
a
Isabel W.C.E. Arends and Ulf Hanefeld *
9
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