Efficient synthesis of Ibrutinib chiral intermediate in high space-time yield by recombinant E. coli co-expressing alcohol dehydrogenase and glucose dehydrogenase

Article abstract of DOI:10.1039/C8RA08100J

The production of (S)-N-boc-3-hydroxy piperidine (NBHP) via asymmetric bioreduction of 1-boc-3-piperidinone with reductase is impeded by the need for expensive coenzymes NAD(P)H. In order to regenerate the coenzyme in situ, the gene of alcohol dehydrogenase from Thermoanaerobacter brockii and glucose dehydrogenase from Bacillus subtilis were ligated into the multiple cloning sites of pRSFDuet-1 plasmid to construct the recombinant Escherichia BL21 (DE3) that co-expressing alcohol dehydrogenase and glucose dehydrogenase. Different culture conditions including the medium composition, inducer and pH etc were systematically investigated to improve the enzyme production. The enzyme activity was increased more than 11-fold under optimal culture condition, from 12.7 to 139.8 U L^?1. In the further work, the asymmetric reduction of 1-boc-3-piperidinone by whole cells of recombinant E. coli was systematic optimized to increase the substrate concentration and reaction efficiency. At last, S-NBHP (>99% ee) was prepared at 500 mM substrate concentration without external addition of cofactors. The conversion of S-NBHP reached 96.2% within merely 3 h, corresponding a high space-time yield around 774 g L^?1 d^?1. All these results demonstrated the potential of recombinant E. coli BL21 (DE3) coupled expressing alcohol dehydrogenase and glucose dehydrogenase for efficient synthesis of S-NBHP.

Full text of DOI:10.1039/C8RA08100J

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Efficient synthesis of Ibrutinib chiral intermediate in
high space-time yield by recombinant E. coli co-
expressing alcohol dehydrogenase and glucose
dehydrogenase†
Cite this: RSC Adv., 2019, 9, 2325
*
*
Songshuang Cao, Xiaomei Wu and Yi Xu
Yitong Chen, Baodi Ma,
The production of (S)-N-boc-3-hydroxy piperidine (NBHP) via asymmetric bioreduction of 1-boc-3-
piperidinone with reductase is impeded by the need for expensive coenzymes NAD(P)H. In order to
regenerate the coenzyme in situ, the gene of alcohol dehydrogenase from Thermoanaerobacter brockii
and glucose dehydrogenase from Bacillus subtilis were ligated into the multiple cloning sites of
pRSFDuet-1 plasmid to construct the recombinant Escherichia BL21 (DE3) that co-expressing alcohol
dehydrogenase and glucose dehydrogenase. Different culture conditions including the medium
composition, inducer and pH etc were systematically investigated to improve the enzyme production.
The enzyme activity was increased more than 11-fold under optimal culture condition, from 12.7 to 139.8
U Lꢀ1. In the further work, the asymmetric reduction of 1-boc-3-piperidinone by whole cells of
recombinant E. coli was systematic optimized to increase the substrate concentration and reaction
efficiency. At last, S-NBHP (>99% ee) was prepared at 500 mM substrate concentration without external
addition of cofactors. The conversion of S-NBHP reached 96.2% within merely 3 h, corresponding a high
Received 30th September 2018
Accepted 10th January 2019
space-time yield around 774 g Lꢀ1 ꢀ1. All these results demonstrated the potential of recombinant E.
d
DOI: 10.1039/c8ra08100j
coli BL21 (DE3) coupled expressing alcohol dehydrogenase and glucose dehydrogenase for efficient
synthesis of S-NBHP.
rsc.li/rsc-advances
ethanediol8 and ethyl (R)-4-chloro-3-hydroxybutanoate9 etc.
However, there were few reports about the synthesis of (S)-N-
1. Introduction
boc-3-hydroxy piperidine (NBHP) via asymmetric bioreduction.
Romain Lacheretz et al.10 used the tissue of Daucus carota as
biocatalyst to reduce 1-boc-3-piperidinone, and moderate chiral
purity (ee 95%) and low yield of product (73%) was obtained.
Other reductases reported to reduce 1-boc-3-piperidinone more
efficiently were the alcohol dehydrogenase from Thermoanaer-
obacter brockii,11 and KR-110 screened form a commercial
ketoreductase library.12 However, expensive exogenous coen-
zyme has to be added to the reaction system to allow the reac-
tion to proceed.
The expensive cofactor NAD(P)H was required in the bio-
reduction process, thus its regeneration attracted extensive
attention in the last few decades. As early as the 1980s,
researchers began to develop different cofactor regeneration
systems, including biological (intracellular coenzyme regener-
ation, substrate coupling method and enzyme coupling
method), electrochemical, photochemical and pure chemistry
and so on. Currently, enzyme coupling and substrate coupling
have been favoured.13,14 The reduction of the substrate and the
regeneration of the coenzyme are performed by two different
enzymes in the double enzyme-coupled system. At present,
formate dehydrogenase (FDH) and glucose dehydrogenase
Chiral hydroxyl piperidines and their derivatives are of great
importance in the pharmaceutical industry and many of them
have pharmaceutical activity, such as anti-tumor agents, anti-
biotics and anti-senile dementia agents.1 Many bioactive
molecules and active pharmaceutical ingredients (API) contain
one or more piperidine rings.2,3 Ibrutinib is a targeted anti-
cancer drug that inhibits Bruton tyrosine kinase for the treat-
ment of mantle cell lymphoma. (S)-N-boc-3-hydroxy piperidine
(NBHP) is a key intermediate in the synthesis of Ibrutinib.
Currently, several approaches have been developed for the
synthesis of chiral alcohol, including classic diastereomeric
resolution,4 asymmetric synthesis,5 and asymmetric reduction.6
Of these methods, asymmetric bioreduction has attracted
extensive attention due to the high stereoselectivity, mild
condition and 100% theoretical yield. Asymmetric bioreduction
has been successfully applied in the preparation of (S)-(4-
chlorophenyl)-(pyridin-2-yl)methanol,7
(R)-1-phenyl-1,2-
School of Chemical and Environmental Engineering, Shanghai Institute of Technology,
100 Haiquan Road, Shanghai 201418, China. E-mail: xuyi@sit.edu.cn;
mabaodi2005@163.com
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra08100j
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(GDH) mediated coenzyme regeneration system proved to be 37 ꢁC for 12 h on a rotary shaker (200 rpm). Then the seed
feasible in the industrial application.15 There are many culture was inoculated into 50 mL fermentation medium con-
successful examples using GDH to construct a coenzyme taining 50 mg Lꢀ1 kanamycin and grown at 37 ꢁC. Aer 3 h,
¨
regeneration system. For instance, Groger et al. had constructed IPTG was added to the culture with nal concentration of
a ‘Designer cell’ containing GDH from Bacillus subtilis and ADH 0.025 mM, and then the recombinant E. coli were continue
ꢁ
from Rhodococcus erythropolis, and it had been used for the cultivated at 40 C for another 10 h for protein expression.
asymmetric reduction of a variety of aryl ketones in the absence
of exogenous coenzyme.16,17 However, no such report on the
production of S-NBHP was found.
The alcohol dehydrogenase from Thermoanaerobacter brockii
(TbADH) was an attractive ADH in the biotechnology due to its
Biotech (Shanghai) Co., Ltd. The exogenous gene was ligated
high thermostability, excellent activity and enantioselectivity
2.3 Construction of co-expression system of TbADH and
BsGDH
The TbADH and BsGDH gene were synthesized by the Generay
for the reduction of 1-boc-3-piperidinone.11 In current research,
a novel two-enzyme (ADH and GDH) co-overexpressing system
was developed for efficient asymmetric reduction of 1-boc-3-
piperidinone without needing exogenous coenzyme. The
vector pRSFDuet-1 contains two multiple cloning sites and each
of them is preceded by a T7 promoter and ribosome binding
site. In present work, the gene TbADH18 and glucose dehydro-
genase from Bacillus subtilis (BsGDH)19 were designed to ligate
into the multiple cloning sites (MCS) of vector pRSFDuet-1 and
construct a recombinant E. coli BL21 (DE3) with both TbADH
and BsGDH activity. S-NBHP can be prepared via the asym-
metric bioreduction of 1-boc-3-piperidine catalyzed by the
TbADH and the coenzyme NADPH can be recycled by the
BsGDH and cosubstrate glucose in situ (Scheme 1). The enzyme
production and reaction efficiency were further improved via
the optimization of microorganism cultivation and reaction
condition, respectively.
into the rst multi clone site (MCS) via BamH/HindIII restriction
sites, and the second MCS via the NdeI/XhoI restriction sites.
The construction of recombinant pRSFDuet-1 plasmid and its
transformation into E. coli were performed according to stan-
dard protocols.
2.4 Enzyme assay
To assay the activity of recombinant E. coli for reducing 1-boc-3-
piperidinone, 10 mg of dry cell pellet was suspended in 950 mL
phosphate buffer (pH 7.0, 200 mM) which contained 0.5%
glucose. The mixture was incubated at 30 ꢁC and 12 000 rpm on
thermo-mixer compact (Zuofei Co., China) for 15 min. Then the
substrate was added into the mixture with a nal concentration
of 50 mM and the reaction was carried out for 15 minutes under
the same conditions. The sample was extracted twice with equal
volume of ethyl acetate. The combined extracts were dried over
anhydrous sodium sulfate and analyzed by gas chromatography
(GC).
2. Experimental
2.1 Chemicals
2.5 Optimization of culture conditions for recombinant E.
coli BL21 (DE3)
Starch and yeast power were purchased from Aladdin Industrial
Corporation (Shanghai, China). Tryptone was supplied by China
National Medicines Co. Ltd. (Beijing, China). Unless otherwise
stated, all other chemicals were obtained from commercial
sources and were of analytical grade.
The media composition and culture condition for recombinant
E. coli were optimized through the ‘one-factor-at-a-time’.20,21 The
factors of carbon source, nitrogen source, inducer, pH and
temperature were varied in each experiment, and the corre-
sponding cell mass and enzyme activity aer cultivation were
determined as described above.
2.2 Strains and culture conditions
The LB medium was used as seed medium, and the composi-
tion was (g Lꢀ1): yeast power 5; tryptone 10; NaCl 10; pH 7.0. The
fermentation medium was optimized based on LB medium.
Recombinant E. coli BL21 (DE3) cells was inoculated into LB
medium containing 50 mg Lꢀ1 kanamycin and cultivated at
2.6 Asymmetric reduction of N-boc-3-piperidone catalyzed
by recombinant E. coli co-expressing alcohol dehydrogenase
and glucose dehydrogenase
Whole cell of recombinant E. coli was used as biocatalyst in this
study. The collected cells were resuspended into the phosphate
buffer (pH 7.0, 200 mM) and were incubated at 30 ꢁC for 15 min.
Then, adequate amount of substrate, glucose, and NADP+ were
added into the suspension. The reaction mixture was incubated
at 30 ꢁC and 12 000 rpm on thermo-mixer compact (Zuofei Co.,
China). The reaction conditions including the initial pH,
temperature, co-solvent, the amount of glucose and NADP+ were
optimized in this research. When the substrate concentration
was increased to 500 mM, Na2CO3 solution (1 M) or ammonia
solution (5 M) was added to neutralize the gluconic acid formed
during the reaction.
Scheme 1 Asymmetric reduction of 1-boc-3-piperidinone to (S)-N-
boc-3-hydroxy piperidine by recombinant E. coli with both TbADH
and BsGDH activity.
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recombinant E. coli harboring pRSFduet-1-bsgdh-tbadh was
2.7 Analytical methods
used in the subsequent study.
The conversion of the sample was analyzed by GC (GC-2010,
Shimadzu Co., Japan) equipped with a b-DEX™ 120 Capillary
Column (30 m ꢂ 0.25 mm ꢂ 0.25 mm, Supelco, Sigma-Aldrich
Co., USA). The carrier gas is nitrogen, and total ow is 20.0
mL minꢀ1. The temperature of detector is set as 280 ꢁC and
injector's temperature is 230 ꢁC. The oven program of the
column temperature is as follows: 1 min at 100 ꢁC, rst ramp at
3.2 Optimization of culture conditions
3.2.1 Effect of carbon source on cell growth and enzyme
production. Different carbon sources were selected to substitute
the yeast in LB medium. Cell biomass and enzyme activity aer
cultivation on various medium were determined to evaluate the
effect of carbon sources. As shown in Fig. 1A, when the starch
was used as carbon source, enzyme activity was superior to that
of other carbon source. In addition, the price of starch is cheap.
The concentration of starch was optimized in further work
and the results revealed that the most appropriate concentra-
tion is 30 g Lꢀ1, and the activity is 98.4 U Lꢀ1 (Fig. 1B). With the
increase of starch concentration, the cell mass is increasing,
however a decrease of activity was observed when the starch
concentration exceed 30 g Lꢀ1. This may be due to the fact that
something from sugar metabolism affects the enzyme produc-
tion at high sugar concentration.
5
2
ꢁC minꢀꢀ11 to 150 ꢁC (hold for 2 min), and second ramp at
ꢁC min to 160 ꢁC (hold for 2 min). The whole analysis
process is 35 minutes. The retention time for 1-boc-3-
piperidinone and NBHP were 28.9 min and 30.9 min,
respectively.
The ee value of the product was analyzed by HPLC (LC 20AT,
Shimadzu Co., Japan) using a chiral OD-H column (4.6 mm ꢂ
250 mm, Daicel Co., Japan). The mobile phase was hexane/
isopropanol (97 : 3, by vol.). The extracted and dried samples
were analyzed at 27 ꢁC with the ow rate of 0.8 mL minꢀ1, and
the detection wavelength is 210 nm. The retention time for S
and R-NBHP were 12.8 min and 13.9 min, respectively.
3.2.2 Effect of nitrogen source on cell growth and enzyme
production. Nitrogen source is another important factor
affecting cell growth and enzyme production. The commonly
used nitrogen sources including inorganic and organic nitrogen
were tested and the results are shown in Fig. 2A. Generally, the
organic nitrogen is better than inorganic nitrogen. Among the
2.8 Preparative synthesis of S-NBHP
The preparative reduction of 1-boc-3-piperidinone was con-
ducted at the substrate concentration of 0.5 M in a 20 ml
aqueous buffer system containing 1 g recombinant E. coli.
Glucose of 2.7 g was added for the NADPH regeneration. The
ꢁ
reaction was carried out at 50 C and ammonia solution (5 M)
was periodical added to the reaction system for neutralizing the
gluconic acid formed during the reaction. Aer the substrate
was completely converted into product, the reaction mixture
was extracted twice with equal volumes of ethyl acetate. Then
the organic phase was combined, dried over anhydrous sodium
sulphate and then evaporated in vacuum. The obtained yellow
liquid was validated based on the chiral HPLC and 1H NMR
spectra.
3. Results and discussion
3.1 Construction of the recombinant plasmids co-expressing
alcohol dehydrogenase and glucose dehydrogenase
Two kinds of recombinant pRSFduet-1 with different position of
TbADH and BsGDH gene, pRSFduet-1-tbadh-bsgdh and
pRSFduet-1-bsgdh-tbadh were constructed (Fig. S1†) and trans-
formed into E. coli BL21(DE3). The expression of target protein
was induced by addition of 0.1 mM IPTG. The enzyme activity
for asymmetric reduction of 1-boc-3-piperidinone was deter-
mined as described in 2.4 to compare the efficiencies of these
two kinds recombinant E. coli and the results were shown in the
Table S1†. Both of the specic activity and activity of recombi-
Fig. 1 Effect of carbon source (A) and its concentration (B) on cell
nant E. coli harboring pRSFduet-1-tbadh-bsgdh was inferior to mass (-) and enzyme activity (,). (A). Different carbon source were
added into the medium. The recombinant E. coli was cultivated at the
the recombinant E. coli harboring pRSFduet-1-bsgdh-tbadh. The
medium with different carbon source. After inoculation at 37 ꢁC for 2 h,
enzymes expression of the recombinant E. coli harboring
IPTG (0.1 mM) was added, and then cultivated at 30 ꢁC for 12 h. (B). The
pRSFduet-1-bsgdh-tbadh was further veried by SDS-PAGE (as
recombinant E. coli was cultivated at the medium with different starch
concentration. After inoculation at 37 ꢁC for 2 h, IPTG (0.1 mM) was
shown in Fig. S2†), and the results showed that both TbADH
and BsGDH expressed as soluble proteins. Therefore, the added, and then cultivated at 30 ꢁC for 12 h.
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Fig. 3 Effect of induction time (A) and induction concentration (B) on
cell mass (-) and enzyme activity (,). (A). Medium cultivated at 37 ꢁC,
200 rpm for 0–5 h, then IPTG (0.1 mM) was added, cultivated at 30 ꢁC,
200 rpm for 10 h. (B). Medium cultivated at 37 ꢁC, 200 rpm for 3 h, then
different concentrations of induction was added, cultivated at 30 ꢁC,
200 rpm for 10 h.
Fig. 2 Effect of nitrogen source (A) and its concentration (B) on cell
mass (-) and enzyme activity (,). (A). The recombinant E. coli was
cultivated at the medium with different nitrogen source. After inocu-
lation at 37 ꢁC for 2 h, IPTG (0.1 mM) was added, and then cultivated at
30 ꢁC for 12 h. (B). The recombinant E. coli was cultivated at the
medium with different nitrogen concentration. After inoculation at
37 ꢁC for 2 h, IPTG (0.1 mM) was added, and then cultivated at 30 ꢁC for
12 h.
Thus, the IPTG of 0.025 mM was added aer inoculation for 3 h
in the subsequent work.
3.2.4 Effect of pH on cell growth and enzyme production.
The effect of initial pH on cell growth and enzyme production of
recombinant E. coli was examined with different initial pH
ranging from 4.0 to 9.0 (Fig. 4A). The pH of the medium was
adjusted to the desired value at the beginning using phosphoric
acid or NaOH solution. Both the cells mass (5.3 gdcw Lꢀ1) and
specic activity (18.1 U gꢀ1) reached the peak when the initial
pH was adjusted to 6.0. As a result, an initial pH of 6.0 was used
for further study.
organic nitrogen, yeast power is the best choice. It not only
promotes cell growth, but also increases enzyme activity.
The concentration of yeast power was further investigated
and the results showed that the appropriate concentration is
15 g Lꢀ1 with the highest enzyme activity of 105.4 U Lꢀ1
.
Although the amount of cells increased with the increasing of
nitrogen source concentration, the metabolites of amino acids
could inhibit the expression of the target protein when the
concentration >15 g Lꢀ1
.
3.2.5 Effect of induced temperature on cell growth and
enzyme production. Temperature plays a non-negligible role
during the protein expression. On the one hand, the cell growth
is slow at low temperature. On the other hand, protein may be
expressed as inclusion body at high temperate.24 For exploring
the optimal induce temperature, recombinant E. coli was
cultivated at different temperature (20, 25, 30, 35, 40, 45 and 50
ꢁC) aer the addition of IPTG. As shown in Fig. 4B, enzyme
activity is more sensitive to the temperature compared to cell
mass. Both cell mass and enzyme activity reached the peak at
3.2.3 Effect of induction conditions on cell growth and
enzyme production. Both of the recombinant alcohol dehy-
drogenase and glucose dehydrogenase are under the control of
T7 promoter, which can be induced by IPTG. The incubation
time before IPTG added is of signicant to the growth and
enzyme production of recombinant E. coꢁli BL21.22 The
recombinant E. coli BL21 was cultivated at 37 C, and IPTG of
1 mM was added aer incubation for 0–5 h. Aer incubation at
ꢁ
30 C for another 10 h, the enzyme activity and cell mass were
ꢁ
40 C.
determined and the results were shown in Fig. 3A. The highest
3.2.6 Enzymatic synthesis of (S)-N-boc-3-hydroxy piperi-
dine by whole cells cultivated in optimized media and LB
media. Aer optimization, the enzyme activity and cell mass
reached 139.8 U Lꢀ1 and 6.6 gdcw Lꢀ1, which were approximately
11 times and 3.6 times of the original activity and cell mass,
respectively. The performance of converting the substrate 1-boc-
3-piperidinone into S-NBHP by the whole cell cultured in the
initial medium or optimized medium were compared and the
enzyme activity was obtained when IPTG was added aer
inoculation at 37 C for 3 h.
ꢁ
IPTG with exorbitant concentrations might affect the growth
of E. coli BL21 (DE3),23 and the price of IPTG is expansive. Thus,
IPTG with concentrations ranging from 0 to 1 mM were inves-
tigated and the results were shown in Fig. 3B. Between 0.1 to
1 mM, enzyme activity was similar. When IPTG concentration
decreased to 0.025 mM, enzyme activity grows to 90.7 U Lꢀ1
.
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Table 1 Effect of initial pH on the conversion of N-boc-3-piperidonea
Buffer
pH
Conversion (%)
PBS
6
7
8
8
9
9
52.5 ꢄ 2.8
76.3 ꢄ 1.8
86.2 ꢄ 0.1
73.4 ꢄ 0.1
74.6 ꢄ 0.1
70.4 ꢄ 0.2
56.4 ꢄ 0.2
Tris–HCl
Gly-NaOH
10
a
The collected cells were resuspended into the following buffers (nal
concentration, 200 mM): sodium phosphate (pH 6–8) and Tris–HCl
(pH 8–9) and Gly-NaOH (pH 9–10). The suspension was incubated at
30 ꢁC, 1000 rpm in thermo shaker for 15 min, and then added the
substrate and glucose with a nal concentration of 50 mM. The
reaction was carried out at 30 ꢁC for 10 h.
reduction. As shown in Table 1, the highest conversion was
obtained at the pH 8.0 in the PBS buffer. At acid or alkali
solution, the whole cell biocatalysts exhibited lower catalytic
efficiency.
3.3.2 Effects of organic solvent types and the amount of
their addition on the conversion of 1-boc-3-piperidone. The
substrate 1-boc-3-piperidinone is not uniformly distributed in
the aqueous phase. The addition of co-solvent into the reaction
system is an efficient method to increase the solubility of the
substrate. However, organic solvents always destroy the cell
membrane or affect the enzyme activity. In the following work,
different organic solvents with various concentrations were
added into the reaction system and the conversion was
measured to choose optimal co-solvent. Compared to the
conversion (51%) in the reaction without co-solvent, a relative
low conversion (<10%) was observed when the dimethyl sulf-
oxide used as co-solvent (Table 2), indicating the poor incom-
patibility of dimethyl sulfoxide. Similar conversions were
obtained when the methanol and acetonitrile at the concen-
tration of 5% (v/v) were used as co-solvent, 80.1% and 83.1%,
respectively. However, the methanol is obviously superior to
acetonitrile when the concentration >10% (v/v), indicating the
great tolerance of this enzyme towards methanol. Thus, the
methanol was chosen as the co-solvent in the subsequent work.
3.3.3 Effects of glucose on the conversion of 1-boc-3-
piperidone. Glucose acts as co-substrate to regenerate the
Fig. 4 Effect of pH (A) and temperature (B) on cell mass (-) and
enzyme activity (,). (A). Medium adjusted its initial pH (4.0 ꢃ 9.0)
cultivated at 37ꢁC, 200 rpm for 3 h, then 0.025 mM IPTG was added,
cultivated at 30 ꢁC, 200 rpm for 10 h. (B). Medium cultivated at 37 ꢁC,
200 rpm for 3 h, then 0.025 mM IPTG was added, cultivated in various
temperature (20–50 ꢁC), 200 rpm for 10 h, and the enzyme activity
was measured in 200 mM sodium phosphate buffer (pH 6.0).
results were shown in Fig. 5. Using the cells cultivated in opti-
mized medium as catalysts, the substrate 1-boc-3-piperidinone
of 50 mM was almost entirely converted aer 8 h, and the
conversion reached 95%. However, the conversion of reaction
catalyzed by cells cultivated in LB medium is only 56% aer 8 h.
3.3 Optimization of enzymatic asymmetric reduction of 1-
boc-3-piperidinone
3.3.1 Effect of initial pH on the conversion of 1-boc-3-
piperidone. The N-boc-3-piperidone was reduced at various pH,
ranging from 6 to 10, and the conversion aer 10 h was
measured to investigate the effects of pH on asymmetric
Table 2 Effects of organic solvents towards the conversion of N-boc-
3-piperidonea
5%
10%
15%
20%
Methanol
Isopropyl alcohol
Acetonitrile
80.1 ꢄ 0.1 75.2 ꢄ 0.2 68.7 ꢄ 1.9 67.0 ꢄ 2.4
47.8 ꢄ 2.5 21.4 ꢄ 0.8 10.9 ꢄ 0.3
83.1 ꢄ 0.5 46.4 ꢄ 1.1 45.5 ꢄ 0.8
6.3 ꢄ 1.0
2.5 ꢄ 1.4
7.4 ꢄ 0.4
Dimethyl sulfoxide
9.7 ꢄ 0.5
8.1 ꢄ 0.4
7.4 ꢄ 0.4
Fig. 5 Enzymatic reduction of 1-boc-3-piperidinone by whole cells
cultured in the initial medium (C) or optimized medium (B). A reac-
tion mixture consisting dry cell of 30 g Lꢀ1 and 50 mM substrate in
0.2 M phosphate buffer (pH 7.0) was incubated at 30 ꢁC and 200 rpm.
Sample was taken at different time and the conversion was determined
by GC.
a
The collected cells were resuspended into PBS buffer (pH 8.0, 200
mM). The suspension was incubated at 30 ꢁC, 1000 rpm in thermo
shaker for 15 min, and then added the substrate and glucose with
a nal concentration of 50 mM. Various organic solvent were added
into the reaction mixture with different nal concentration (5%, 10%,
15% and 20%). The reaction was carried out at 30 ꢁC for 10 h.
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Table 3 Effects of glucose concentration on the conversion of N-
boc-3-piperidonea
Glucose (mM)
Glucose/substrate (n : n)
Conversion (%)
25
50
75
100
125
0.5
1.0
1.5
2.0
2.5
55.7 ꢄ 4.3
86.4 ꢄ 0.4
91.1 ꢄ 2.3
93.1 ꢄ 0.9
94.4 ꢄ 1.8
a
Reaction condition: the collected cells were resuspended into PBS
buffer (pH 8.0, 200 mM). The suspension was incubated at 30 ꢁC,
1000 rpm in thermo shaker for 15 min, and then added the substrate
(nal concentration 50 mM) and glucose (nal concentration of 25,
50, 75, 100 and 125 mM). Methanol of 5% (v/v) was used as the co-
solvent. The reaction was carried out at 30 ꢁC for 10 h.
Fig. 6 Effect of temperature on the conversion of N-boc-3-piper-
idone. The collected cells were resuspended into PBS buffer (pH 8.0,
200 mM). The suspension was incubated at 30 ꢁC, 1000 rpm in thermo
shaker for 15 min, and then added the substrate (final concentration of
50 mM) and glucose (final concentration of 75 mM). Methanol of 5% (v/
v) was used as the co-solvent. The reaction was carried out at various
temperatures (C 30; O40; - 50 and > 60 ꢁC) for 10 h. Sample was
taken at different time and the conversion was determined by GC.
NADPH by offering a molecule of [H] to NADP+ catalysed by the
GDH. However, the formed gluconic acid would result in the
decrease of pH in reaction system. Various amounts of glucose
were added into the reaction system, and the results showed
that the conversion was increased obviously with the increase of
the glucose amount. When the equivalent ratio was 1.5, the
conversion reached 91%. However, the increase of conversion
was not signicant with further increase of glucose amount
beyond 1.5 times to substrate. The amount of the co-substrate is
determined to be 1.5 equivalents to substrate in the subsequent
study (Table 3).
reported for S-NBHP was 552 g Lꢀ1 dꢀ1 using the commercial
ketone reductase as catalyst and extra glucose dehydrogenase
was added to regenerate the coenzyme in this process.25
3.4 Asymmetric reduction of 1-boc-3-piperidinone in
preparative scale
3.3.4 Effects of temperature on the conversion of 1-boc-3-
piperidone. Enzymatic reactions usually have an optimum
temperature. On the one hand, the reaction speed is faster at
higher temperature. On the other hand, the temperature which
is too high will lead to protein inactivation.
Finally, the asymmetric reduction of N-boc-3-piperidone was
conducted at preparative-scale (see Section 2.8 for details).
Optically pure S-NBHP of 1.67 g was recovered in a total yield of
83% and >99% ee (Fig. S3†). 1H NMR (500 MHz, CDCl3) d 3.78–
3.81 (m, 1H), 3.71 (s, 1H), 3.58 (s, 1H), 3.01–3.06 (m, 1H), 2.99
(dd, J ¼ 12.7, 8.0 Hz, 1H), 2.74 (s, 1H), 1.90 (s, 1H), 1.75–1.77 (m,
1H), 1.45 (s, 11H).
The bioreduction catalyzed by the recombinant E. coli was
ꢁ
conducted at temperatures from 30 to 70 C to investigate the
effect of temperature. As shown in the Fig. 6, the conversion
reached 96% aer 10 h when the temperature was 30 ꢁC. As the
temperature increase, the reaction velocity increased gradually.
ꢁ
At 50 C, the conversion reached to 97.8% aer 2 h, but as the
temperature continues to rising, the reaction speed is reduced,
which may be due to the enzyme inactivation. At 70 ꢁC, the
substrate was not converted (data no shown), and the enzyme
may have been completely inactivated. The reaction was deter-
ꢁ
mined to be conducted at 50 C in the subsequent work.
3.3.5 Asymmetric reduction of 1-boc-3-piperidinone by the
recombinant E. coli with different catalyst loading. The asym-
metric reduction of N-boc-3-piperidone with different cell load
was conducted without the addition of coenzyme, and the
substrate concentration was increased to 500 mM. As shown in
Fig. 7, the conversion reached 29.8%, 95.5%, 98.4% at 24 h
when the cell load was 10, 30, 50 g Lꢀ1, respectively. A faster
bioreduction was achieved when the cell load was increased,
which is attributed to the increase of both catalysts and internal
cofactors NADP+ from the recombinant E. coli. Actually, the
conversion reached 96.2% within merely 3 h when the cell load
was increased to 50 g Lꢀ1, representing a relatively highspace-
time yield (STY, 774 g Lꢀ1 dꢀ1) for S-NBHP. The highest STY
Fig. 7 Asymmetric reduction of 1-boc-3-piperidinone by E. coli BL21
harboring pRSFDuet-bsgdh-tbadh with different cell concentration.
The collected cells were resuspended into PBS buffer (pH 8.0, 200
mM) with various concentration (- 10; C 30; and O 50 g Lꢀ1). The
suspension was incubated at 30 ꢁC, 200 rpm in a shaker for 15 min, and
then added the substrate (final concentration 500 mM) and glucose
(final concentration of 750 mM). Methanol of 5% (v/v) was used as the
co-solvent. The reaction was carried out at 50 ꢁC for 24 h. Ammonia
solution (5 M) was periodical added to the reaction system for
neutralizing the produced acid during the reaction. Samples were
taken at different time and the conversion was determined by GC.
2330 | RSC Adv., 2019, 9, 2325–2331
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Review
RSC Advances
6 G. W. Zheng, H. L. Yu, J. D. Zhang and J. H. Xu, Adv. Synth.
Catal., 2009, 351, 405–414.
4. Conclusions
7 Y. Ni, J. Y. Zhou and Z. H. Sun, Process Biochem., 2012, 47,
1042–1048.
A recombinant E. coli BL21 (DE3) was constructed for co-
expressing alcohol dehydrogenase and glucose dehydrogenase
using the plasmid pRSFduet-1, which contains two multiple
clone sites. The application potential of the constructed
recombinant E. coli harbouring pRSFduet-1-bsgdh-tbadh was
further improved through the optimization of the enzyme
production condition and reaction condition. Finally, the bio-
reduction catalyzed by the recombinant E. coli was conducted at
the substrate concentration of 500 mM without external addi-
tion of cofactors, giving satisfactory conversion (96.2%) and
enantiomeric excesses for S-NBHP (ee value > 99%). The STY
reported in this study (774 g Lꢀ1 dꢀ1) was the highest reported
for S-NBHP preparation so far. S-NBHP was obtained in
a preparative experiment in 83% isolated yield and 99% ee
value. Thus, the recombinant E. coli harbouring pRSFduet-1-
bsgdh-tbadh was competitive and promising compared to other
biocatalysts for asymmetrically reducing 1-boc-3-piperidinone.
Further work about the immobilization of this recombinant E.
coli and optimization of the reaction for large scale preparation
is in the progress.
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Acknowledgements
This work was nancially supported by Shanghai Collaborative
Innovation Center of Fragrance, Flavor and Cosmetics
(1021ZK170004002), the Shanghai Institute of Technology
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