Highly atom economic synthesis of D-2-aminobutyric acid through an in vitro tri-enzymatic catalytic system

Article abstract of DOI:10.1002/open.201700093

D-2-Aminobutyric acid is an unnatural amino acid serving as an important intermediate in pharmaceutical production. Developing a synthetic method that uses cheaper starting materials and produces less by-product is a pressing demand. A tri-enzymatic catalytic system, which is composed of L-threonine ammonia lyase (L-TAL), D-amino acid dehydrogenase (D-AADH), and formate dehydrogenase (FDH), has thus been developed for the synthesis of D-2-aminobutyric acid with high optical purity. In this cascade reaction, the readily available L-threonine serves as the starting material, carbon dioxide and water are the by-products. D-2-Aminobutyric acid was obtained with >90% yield and >99% enantioselective excess, even without adding external ammonia, demonstrating that the ammonia from the first reaction can serve as the amino donor for the reductive amination step. This multi-enzymatic system provides an attractive method with high atomic economy for the synthesis of D-α-amino acids from the corresponding L-α-amino acids, which are readily produced by fermentation.

Full text of DOI:10.1002/open.201700093

DOI: 10.1002/open.201700093
Highly Atom Economic Synthesis of d-2-Aminobutyric Acid
through an In Vitro Tri-enzymatic Catalytic System
[
a]
[a]
[a, b]
[a]
[a]
Xi Chen, Yunfeng Cui, Xinkuan Cheng,
Jinhui Feng, Qiaqing Wu, and
[a, b]
Dunming Zhu*
d-2-Aminobutyric acid is an unnatural amino acid serving as an
important intermediate in pharmaceutical production. Devel-
oping a synthetic method that uses cheaper starting materials
and produces less by-product is a pressing demand. A tri-enzy-
matic catalytic system, which is composed of l-threonine am-
monia lyase (l-TAL), d-amino acid dehydrogenase (d-AADH),
and formate dehydrogenase (FDH), has thus been developed
for the synthesis of d-2-aminobutyric acid with high optical
purity. In this cascade reaction, the readily available l-threonine
serves as the starting material, carbon dioxide and water are
the by-products. d-2-Aminobutyric acid was obtained with
>90% yield and >99% enantioselective excess, even without
adding external ammonia, demonstrating that the ammonia
from the first reaction can serve as the amino donor for the re-
ductive amination step. This multi-enzymatic system provides
an attractive method with high atomic economy for the syn-
thesis of d-a-amino acids from the corresponding l-a-amino
acids, which are readily produced by fermentation.
1
. Introduction
[
12]
Although d-a-amino acids do not exist in the nature as widely
as their l-counterparts, naturally occurring d-a-amino acids
play an important role in defence mechanisms (cell wall,
tives have been used to realize the separation of l/d-amino
acids. w-Transaminases have been used for the synthesis of d-
2-aminobutyric acid through the amino-transfer reaction, pro-
[
1]
[13]
venoms) of many microorganisms and plants. d-a-Amino
viding an alternative biocatalytic method. d-2-Aminobutyric
acids have also been detected in a variety of peptides synthe-
acid has also been synthesized through asymmetric alkylation
[
2]
[14]
sized by animal cells and in human tissue, appearing as indi-
at low temperature, or de-racemization of l/d-amino acids
[
3]
cators of aging. Several enzymes producing or metabolizing
using l-amino acid oxidase from Proteus myxofaciens in combi-
[
4]
[15]
d-amino acids have been discovered. d-2-Aminobutyric acid,
nation with amine boranes.
which is an unnatural amino acid, has been widely used in the
Compared to the previously reported methods, reductive
amination of 2-oxobutyric acid with ammonia by d-amino acid
dehydrogenase provides an attractive synthetic method for d-
2-aminobutyric acid, owing to the intrinsic atomic economy
and low environmental impact of the reaction. Taking into ac-
count that many l-amino acids are available through fermenta-
tion from inexpensive and renewable natural sources, and l-
amino acid oxidases catalyze their conversion to the 2-oxo
acids, we envisaged that the two-step enzymatic reactions
with l-amino acid oxidase and d-aminoacid dehydrogenase
would offer economically effective and environmentally benign
methods for the synthesis of d-aminoacids from the corre-
sponding l-amino acids. For d-2-aminobutyric acid, although
l-2-aminobutyric acid is an unnatural amino acid and not read-
ily available as the natural counterparts, 2-oxobutyric acid can
be easily obtained from l-threonine by using l-threonine am-
monia lyase (l-TAL, EC 4.3.1.19). The enzyme usually shows
high reactivity and threonine can be readily produced by fer-
[
5]
synthesis of antibiotics, angiotensin-converting enzyme 2 in-
[
6]
hibitors, brain-permeable polo-like kinase-2 (Plk-2) inhibi-
[
7]
[8]
tors, matrix metalloproteinase inhibitors, and antiprolifera-
[
9]
tives. Generally speaking, resolution of the racemate of a-
amino acids is currently the main method to obtain the opti-
cally pure d-amino acids. For d-2-aminobutyric acid, aminoacy-
[
1,10]
[11]
lase,
protease, or the formation of diastereomeric deriva-
[a] Dr. X. Chen, Y. Cui, X. Cheng, Prof. Dr. J. Feng, Prof. Dr. Q. Wu,
Prof. Dr. D. Zhu
National Engineering Laboratory for Industrial Enzymes and
Tianjin Engineering Center for Biocatalytic Technology
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences
Tianjin 300308 (P.R. China)
E-mail: zhu_dm@tib.cas.cn
[b] X. Cheng, Prof. Dr. D. Zhu
University of Chinese Academy of Sciences
Beijing 100049 (P.R. China)
[16]
mentation. Tao et al. reported the enzymatic synthesis of l-
2-aminobutyric acid from threonine by using l-TAL combina-
tion with l-amino acid dehydrogenase (l-AADH) and formate
The ORCID identification number(s) for the author(s) of this article can
be found under https://doi.org/10.1002/open.201700093.
ꢀ
2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
[17]
dehydrogenase (FDH). Therefore, similar multiple enzymatic
reactions should provide an efficient approach for the biosyn-
thesis of d-amino acids by employing a d-amino acid dehydro-
genase. Recently, it has been reported that meso-2,6-diamino-
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pimelic acid dehydrogenase (DAPDH, EC 1.4.1.16) and its
mutant enzymes can catalyze the reductive amination of 2-
[
18]
keto acids to give d-a-amino acids. For example, meso-dia-
minopimelate dehydrogenase from Symbiobacterium thermo-
philum (StDAPDH) can transform 2-keto acids to the corre-
sponding d-a-amino acid, and its excellent thermo-stability
[
18c]
provides one-step heat-treatment purification.
The crystal
structure of StDAPDH revealed the structural basis of its
[
19]
unique catalytic properties, and site saturation mutagenesis
resulted in mutant H227V with 35-fold activity enhancement
over the wild-type enzyme for phenyl pyruvic acid as the
[
20]
substrate.
With this unique d-amino acid dehydrogenase (d-AADH) in
hand, we combined it with l-threonine ammonia lyase (l-TAL)
and formate dehydrogenase (FDH) to establish a tri-enzymatic
catalytic system for the synthesis of d-2-aminobutyric acid
from l-threonine (Scheme 1).
Figure 1. A) pH profile of PFDH; B) pH profile of M-StDAPDH. The buffers
100 mm) were NaOAc-HOAc (pH 5.0, 6.0), Na HPO -NaH PO (pH 6.0, 7.0,
8.0), Tris-HCl (pH 8.0, 9.0), and Na CO -NaHCO (pH 9.0, 10.0).
(
2
4
2
4
Scheme 1. Tri-enzymatic synthesis of d-2-aminobutyric acid from l-
2
3
3
threonine.
buffer and pH value (Figure 1B), and the activity was higher in
basic buffer. In phosphate buffer, M-StDAPDH showed the
lowest enzymatic activity; whereas, in the Na CO -NaHCO
3
2
. Results and Discussion
2
3
2.1. Expression and Purification of l-TAL, d-AADH and FDH
buffer (pH 9.0), the specific activity of M-StDAPDH was
À
The enzymes l-TAL (EcTAL) from Escherichia coli, d-AADH (M-
StDAPDH) from Symbiobacterium thermophilum, FDH (PFDH,
CdFDH) from Pseudomonas sp. 101 and Candida boidinii have
0.55 Umg and Km and k for 2-oxobutyric acid were 12.3Æ
cat
À1
0.4 mm and 1.6Æ0.2 s , respectively.
[
16,18c,21]
all previously been reported and produced in E. coli.
En-
2.3. Influence of the Reaction Components on the Enzyme
zymes were purified by heat treatment or precipitated with
ammonium sulfate, except CbFDH, which was purified by
using ꢁKTA purifier. CbFDH lost >90% enzyme activity when
incubated at 508C for 1 h, whereas 90% activity of PFDH was
retained when heated at 578C for 1 h. PFDH was more heat
tolerant and could be purified by heat treatment. As such,
PFDH was used in the following studies.
Activity
The influences of the reaction components on the enzyme ac-
tivity were studied, and the results are presented in Figure 2
and Figure 3. For EcTAL, the conversion of l-threonine was de-
termined when different compounds were added into Na CO -
2
3
NaHCO (100 mm, pH 9.0) buffer at 308C. When 120 mm 2-oxo-
3
butyric acid, d-2-aminobutyric acid, or 240 mm ammonium for-
mate was added, the conversion of l-threonine was not affect-
ed (Figure 2A). The existence of l-threonine, 2-oxobutyric acid,
and ammonium chloride in the reaction mixture showed little
influence on the enzyme activity of PFDH, except at 480 mm of
d-2-aminobutyric acid (Figure 2B).
For M-StDAPDH, it was found that the enzymatic activity
was affected by addition of d-2-aminobutyric acid or l-threo-
nine (Figure 3A,3B). In particular, the activity of M-StDAPDH
was reduced to nearly 50% when only 10 mm l-threonine was
added (Figure 3B). However, it is interesting that the reductive
amination of 2-oxobutyric acid proceeded smoothly and was
completed in 12 h with 200 mm l-threonine (Figure 3C), al-
though l-threonine greatly affected the specific activity of the
enzyme, as measured by using spectrometric methods. Moni-
2
.2. pH Influence on the Enzyme Activity of the M-StDAPDH
and PFDH
A previous study showed that the activity of EcTAL was high
À
[16]
enough (specific activity >200 Um at pH 7.5) for the trans-
formation, so we chose the enzyme as the biocatalyst for the
ammonia lysis reaction of threonine. Because of the relatively
low specific activity of M-StDAPDH and PFDH, the pH profiles
of their activity were studied. As shown in Figure 1A, PFDH
showed increased activity with pH from 5.0 to 10.0, and the ac-
tivity was also affected by the nature of the buffers. A similar
pH-dependent trend was observed for glucose dehydrogenas-
[
22]
[23]
es and alcohol dehydrogenases for the oxidation reaction.
For M-StDAPDH, the enzyme activity was affected by the
&
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Figure 2. A) Conversion rate of l-threonine catalyzed by EcTAL when a differ-
ent reaction component was added to the reaction mixture (CK: no additive
was added). B) Effects of different reaction components on the enzyme ac-
tivity of PFDH.
toring of the conversion rate of 2-oxobutyric acid showed that,
even when 200 mm l-threonine was added to the reaction
mixture, the yield of d-2-aminobutyric acid did not show much
difference to that of the control reaction without addition of l-
threonine (Figure 3C).
2
.4. Influence of the Biocatalyst Type on the Yield and ee
Figure 3. A) Effects of different reaction components on the activity of M-
StDAPDH. B) Relative activity of M-StDAPDH when different concentrations
of l-threonine were added to the reaction mixture; the specific activity
toward the 2-oxobutyric acid without the addition of l-threonine was de-
fined as 100%. C) Time course for the reductive amination of 2-oxobutyric
acid when 0, 100, or 200 mm l-threonine was added into the reaction
mixture.
Value
In previous studies, cell lysates could be used as the catalyst
[
17]
for the synthesis of l-2-aminobutyric acid. However, for the
d-configured counterpart, the use of whole cells or cell lysates
might reduce the ee value of the product, owing to the endog-
enous l-amino acid dehydrogenases, amino acid racemases, or
amino transferase. As such, the different forms of the biocat-
alyst were employed to examine their effect on the optical
purity of the product.
[
24]
Table 1. Yield and ee values of the product with three different types of
biocatalyst.
The reductive amination of 2-oxobutyric acid was carried
out with same activity units of whole cells, cell-free extract, or
Biocatalyst
Yield [%]
ee [%]
whole cells
cell-free extract
purified enzymes
<5
>95
>98
not measured
99.0
>99.5
purified enzyme in Na CO -NaHCO (100 mm, pH 9.0) buffer.
2
3
3
The yield and ee value of d-2-aminobutyric acid were mea-
sured, and the results are summarized in Table 1. When the
cell-free extract or purified enzyme was used as the catalyst,
the yield of the product was >95%, whereas the yield with
whole cells as the catalyst was <5%. This may be attributed
to the fact that the cell membrane prevents transfer of the
substrate into the cell for the enzymatic reaction. The ee value
of the product for the reaction with cell-free extract was slight-
ly lower than that with purified enzyme. The endogenous
enzyme in the cell-free extract may be responsible for the
slightly reduced ee value of the product.
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or sodium formate. By monitoring the change in the concen-
trations of substrate and product, it was found that the deami-
nation of l-threonine was completed within 4 h, and the quick
consumption of l-threonine reduced the substrate inhibition
effect during the second reaction. More importantly, although
the reaction rate of the second step was decreased with addi-
tion of sodium formate, the transformation was nearly com-
plete in 20 h, and the yield of the product reached 90% after
ion-exchange purification. Therefore, the ammonia, which is
produced in the first reaction catalyzed by EcTAL, can be used
as the amino source in the reductive amination reaction. The
multi-enzymatic transformation proceeded smoothly without
addition of external ammonia, making it more atomically
economic.
Figure 4. Time course for the formation of d-2-aminobutyric acid with differ-
ent amounts of enzymes and varied concentrations of substrates. The reac-
tions were conducted in Na
tored at different time intervals. I: 0.7 U EcTAL, 1 U M-StDAPDH, 2 U PFDH,
00 mm l-threonine, 150 mm ammonium formate; II: 2 U EcTAL, 1 U
2 3 3
CO –NaHCO buffer (100 mm, pH 9.0), and moni-
1
M-StDAPDH, 2 U PFDH, 100 mm l-threonine, 150 mm ammonium formate;
III: 2 U EcTAL, 2 U M-StDAPDH, 4 U PFDH, 100 mm l-threonine, 150 mm am-
monium formate; IV: 2 U EcTAL, 2 U M-StDAPDH, 4 U PFDH, 200 mm l-threo-
nine, 300 mm ammonium formate; V: 2 U EcTAL, 2 U M-StDAPDH, 4 U PFDH,
3
. Conclusions
A new effective enzymatic method has been developed for the
synthesis of d-2-aminobutyric acid from commercially and
readily available l-threonine. In this multi-enzymatic approach,
l-threonine ammonia lyase catalyzes the deamination of l-
threonine to give 2-oxobutyric acid, which is reductively ami-
nated by d-amino acid dehydrogenase with formate dehydro-
genase for co-factor regeneration to produce d-2-aminobutyric
acid. Particularly, this biotransformation can proceed smoothly
at an l-threonine concentration of 200 mm without extra addi-
tion of ammonia, rendering it a highly atom-efficient reaction.
The chosen thermostable enzymes are well suited for industrial
applications, owing to their easy purification by heat-treatment
and robustness, although their specific activity needs to be im-
proved for higher efficiency. The (co-)immobilization of these
enzymes will also increase the economic advantage of this tri-
enzymatic catalytic system and these studies are under way. In
general, a similar approach should be applicable for the prepa-
ration of a wide range of d-amino acids by combining a suita-
ble l-amino acid oxidase with a d-amino acid dehydrogenase,
and this will be further explored in our laboratory.
2
00 mm l-threonine, 300 mm sodium formate; VI: 2 U EcTAL, 2 U M-
StDAPDH, 4 U PFDH, 300 mm l-threonine, 450 mm ammonium formate; VII:
U EcTAL, 2 U M-StDAPDH, 4 U PFDH, 300 mm l-threonine, 450 mm sodium
formate.
2
2.5. Optimization of the Reaction Conditions
The purified enzymes EcTAL, M-STDAPDH, and PFDH were
used as the catalysts for the reactions with different amounts
of the enzymes and varied concentrations of substrates. The
results are summarized in Figure 4. It can be seen that at con-
dition I with 0.7 U EcTAL, 1 U M-StDAPDH, 2 U PFDH, 100 mm
l-threonine, and 150 mm ammonium formate in Na CO -
2
3
NaHCO buffer (pH 9.0), the yield of product was close to 90%
3
in 24 h. Enhancing the amount of EcTAL (Figure 4, I and II) in-
creased the reaction rate, as evidenced by the fact that the
yield of d-2-aminobutyric acid was improved from 52 to 92%
at 8 h when 2.0 U EcTAL was added instead of 0.7 U enzyme.
Further enhancing the amount of EcTAL to 3.0 U did not in-
crease the reaction rate (data not shown). As expected, when
more PFDH and M-StDAPDH were added, the reaction rate
was significantly increased (II and III). The yield of product
reached 90% at 6 h when 2 U M-StDAPDH and 4 U PFDH were
added (condition III), compared to about 60% at condition II
after 6 h. Under these conditions, when the concentration of l-
threonine increased to 200 and 300 mm, the reaction still pro-
ceeded successfully (Figure 4 IV and VI, 95% yield at 8 h and
Experimental Section
General
All chemicals were obtained from commercial sources and the co-
factors (NADH and NAD ) were obtained from Roche. Materials
+
used for culture media including peptone, yeast extract, and agar
were purchased from Becton, Dickinson, and Company (BDX). The
product yield and ee value were determined by HPLC analysis on
an Eclipse plus C18 column (4.6ꢂ250 mm, Agilent, USA) after
being derived with 1-fluoro-2–4-dinitrophenyl-5-l-alanine amide
92% yield at 24 h, respectively). When no extra ammonia was
added, the reaction was completed with 200 mm of l-threo-
nine (Figure 4, V). However, without the addition of extra am-
monia, the transformation at the l-threonine concentration of
(FDAA).
300 mm was not completed within 24 h, and the yield of the
product was less than 80% (Figure 4, VII).
Expression of the EcTAL, M-StDAPDH, and FDH
All of the selected enzymes have previously been reported. EcTAL
gene (Gene ID: 948287) was amplified from the genome of E.
coli. The EcTAL fragment was digested at the sites of BamHI and
HindIII, and ligated into plasmid vector pRSFduetꢃ-1. FDH gene
(Gene ID: 4033692) from Pseudomonas sp. 101 was synthesized by
Shanghai Xuguan Biotechnological Development Co., Ltd. (China)
2
.6. Preparation of d-2-Aminobutyric Acid
[21]
In order to test the applicability of this enzymatic system, the
reaction was conducted in 50 mL reaction solution, which con-
tained 200 mm of l-threonine, 300 mm of ammonium formate,
&
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and cloned into the NdeI/XhoI sites of pET21a. The pET21d-CbFDH
plasmid with FDH gene (Gene ID: 74654561) from Candida boidinii
was available in our laboratory. The mutant (R35E/R36V/Y76V) of
StDAPDH (Gene ID: 2979910) from Symbiobacterium thermophilum
The activity of M-StDAPDH toward the reductive amination of 2-ox-
obutyric acid and the activity of the FDH toward the oxidation of
formate were determined by measuring the oxidation of NADH or
+
À1
À1
reduction of NAD at 340 nm (e=6.22 mm cm ), respectively.
The reductive amination activity was measured at room tempera-
ture in a 96-well plate; each well contained 2-oxobutyric acid
(20 mm), NH Cl (60 mm), and NADH (0.40 mm) in Na CO –NaHCO
(
M-StDAPDH) was preserved in our laboratory, and the cofactor
specificity of M-StDAPDH has been changed from NADP(H) to
NAD(H).
4
2
3
3
(
100 mm, pH 9.0, 190 mL) solution. The reaction was initiated by ad-
The plasmids pRSFduet-EcTAL, pET-32a(+)-M-StDAPDH, and
pET21a-PFDH were transformed into E. coli BL21(DE3) cells. Re-
combinant E. coli BL21(DE3) was propagated in 1 L of Luria–Bertani
medium containing 100 mgmL ampicillin (50 mgmL kanamycin
for pRSFduet-EcTAL) at 378C. The culture was induced by addition
of isopropyl b-d-1-thiogalactopyranoside (IPTG) with a final con-
centration of 0.1 mm when the optical density (l=600 nm)
reached 0.6–0.8 and then incubated for an additional 16 h at 258C
dition of M-StDAPDH (10 mL solution containing appropriate
amount of enzyme).The kinetic parameters of M-StDAPDH towards
2
2
of FDH was measured at room temperature in a 96-well plate, in
which each well contained sodium formate (20 mm) and NAD
-oxobutyric acid were determined with the final concentration of
-oxobutic acid being between 1 and 60 mm. The oxidative activity
À1
À1
+
(0.50 mm) in Na CO -NaHCO (100 mm, pH 9.0, 190 mL) solution.
2 3 3
The reaction was initiated by the addition of the FDH (10 mL solu-
tion containing appropriate amount of enzyme). The specific activi-
ty was defined as the number of micromoles of NAD or NADH
converted by 1 mg of enzyme in 1 min (mmolmin mg ).
(
6
pET21d-CbFDH at 208C) with 200 rpm. After centrifugation at
000ꢂg, 48C for 20 min, the recombinant cells were washed with
+
potassium phosphate buffer and cryopreserved at À208C without
À1
À1
loss of activity over 2 weeks.
The pH Influence on the Enzyme Activity
Purification of the EcTAL, M-StDAPDH, and FDHs
The influences of pH on the enzyme activities were measured at
various pH by activity assay methods mentioned above. The buf-
fers (100 mm) were NaOAc-HOAc (pH 5.0, 6.0), Na HPO -NaH PO
4
The enzymes were purified by following the procedures described
herein. For EcTAL, the recombinant cells were suspended in
2
4
2
1
00 mm potassium phosphate buffer (pH 7.0) and lysed by French
(
pH 6.0, 7.0, 8.0), Tris-HCl (pH 8.0, 9.0) and Na CO -NaHCO (pH 9.0,
2 3 3
press at an operating pressure of 12000 psi. The cell debris was re-
moved by centrifugation at 10000ꢂg for 30 min at 48C. The result-
ing cell-free extract was mixed with 0.025% PEI solution. After cen-
trifugation, the supernatant was precipitated with 40–60% ammo-
nium sulfate. The resulting precipitate was collected after centrifu-
gation and dissolved in potassium phosphate buffer (100 mm,
pH 7.0).
1
0.0).
The Influence of the Reaction Components on the Enzyme
Activity
For EcTAL, a final concentration of 100 mm 2-oxobutyric acid,
1
00 mm d-2-aminobutyric acid, or 200 mm ammonium formate
For M-StDAPDH, PFDH, and CbFDH, The recombinant cells were
suspended in 100 mm potassium phosphate buffer (pH 8.0) and
lysed by French press at an operating pressure of 12000 psi. The
cell debris was removed by centrifugation at 10000ꢂg for 30 min
at 48C. The resulting cell-free extract was mixed with 0.025% PEI
solution. After centrifugation, the supernatant of M-StADPDH was
heat-treated at 708C for 80 min; the supernatant of PFDH was
heat-treated at 578C for 60 min. After centrifugation, the superna-
tant of M-StDAPDH and PFDH was concentrated by ultrafiltration
was added to the reaction mixture, which contained EcTDA (9 mg),
l-threonine (100 mm), and pyridoxal phosphate (10 mm). The activi-
ty of EcTDA was determined through the assay method mentioned
above. For PFDH, a different final concentration (60, 120, 240, and
4
80 mm) of l-threonine, ammonium chloride, 2-oxobutyric acid, or
d-2-aminobutyric acid was added into the reaction mixture, which
contained PFDH (1 mg), sodium formate (20 mm), and NAD
+
(0.50 mm). The activity of PFDH was determined by the assay
method mentioned above. For M-StDAPDH, a different final con-
centration (60, 120, 240, 480 mm) of sodium formate, d-2-amino
butyric acid, or l-threonine was added into the reaction mixture,
which contained M-StDAPDH (1 mg), 2-oxobutyric acid (20 mm),
(
Amicon Ultra 30 kDa, Millipore) and stored in 5% glycerol at
À808C for further use. For CbFDH, after centrifugation, the super-
natant was precipitated with 55% ammonium sulfate and the su-
pernatant was then loaded into a 20 mL Phenyl Sepharose 6 Fast
Flow column (high sub) (GE Healthcare) on ꢁKTA purifier. The
target protein was washed with elution buffer (100 mm potassium
phosphate buffer, pH 8.0) and dialyzed against potassium phos-
phate buffer (100 mm, pH 8.0). Then, the protein was concentrated
by ultrafiltration (Amicon Ultra 30 kDa, Millipore) and stored in 5%
glycerol at À808C.
NH Cl (60 mm), and NADH (0.40 mm). The activity was determined
4
by the assay procedure mentioned above. To further study the
effect of l-threonine on the M-StDAPDH-catalyzed transformation,
1
00 mm l-threonine was added into the reaction mixture (1 mL)
containing 2 U PFDH, 1 U M-StDAPDH, 100 mm 2-oxobytyric acid,
and 200 mm ammonium formate. The reaction was carried out at
3
08C, and the yield of d-2-amino butyric acid was measured by
HPLC analysis after FDAA derivatization every 3 h. The retention
times of d- and l-2-amino butyric acid were 13.0 and 8.1 min,
respectively.
Activity Assay of the EcTAL, M-StDAPDH and FDH
The activity of EcTAL toward l-threonine was determined by meas-
uring the decrease of threonine in the reaction mixture through
HPLC analysis. Into 500 mL reaction solution, which contained
The Influence of the Biocatalyst Type on the ee Value
1
00 mm l-threonine and 10 mm pyridoxal phosphate, an appropri-
ate amount of EcTAL was added. After a certain time, the remain-
ing amount of l-threonine was measured. The specific activity was
defined as the number of micromoles of l-threonine converted by
Wet recombinant cells expressing the PFDH or M-StDAPDH gene
À1
(30 mgmL ) were suspended in Na CO -NaHCO (100 mm, pH 9.0)
2
3
3
and lysed by sonication; the cell debris was removed by centrifu-
gation at 10000ꢂg for 2 min. The specific activity of the resulting
À1
À1
1
mg of enzyme in 1 min (mmolmin mg ).
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À
crude PFDH enzyme solution was 32 Um , corresponding to
the yield and ee value of d-2-amino butyric acid were detected by
HPLC analysis at the time intervals shown in Figure 5. After 24 h,
the pH of the reaction mixture was adjusted to below 2 and the
precipitated enzymes were removed by centrifugation. The prod-
uct was purified on a cation-exchange resin column and eluted by
3% NH ·H O. 0.92 g (99.6% ee, 90% yield for the reaction with
À
1
.6 Um wet whole cells. The specific activity of crude enzymatic
solution of M-StDAPDH was 8.2 Um , corresponding to 0.41 Um
À
À
wet whole cells. The reaction mixture (1 mL) containing 2 U of
PFDH wet whole cells (or enzyme solution or purified enzyme), 1 U
of M-StDAPDH wet whole cells (or enzyme solution or purified
enzyme), 100 mm 2-oxobutyric acid, and 200 mm ammonium for-
mate was shaken at 308C for 16 h. The yield and ee value of d-2-
aminobutyric acid was determined by HPLC after FDAA derivation.
3
2
sodium formate) and 0.95 g (99.6% ee, 92% yield for the reaction
with ammonium formate) d-2-amino butyric acid were obtained,
1
respectively. H NMR (D O, 400 MHz) d (ppm) 3.62 (t, J=6.0 Hz,
2
1
H), 1.80 (m, 2H), 0.89 (m, J=8.4 Hz, 3H).
Optimization of Tri-enzymatic Reaction System
Acknowledgements
In 1 mL Na CO -NaHCO buffer (100 mm, pH 9.0), 0.7 U EcTAL, 1 U
2
3
3
M-StDAPDH, 2 U PFDH, 100 mm l-threonine, and 150 mm ammoni-
um formate were added (I), and the reaction was monitored by
measuring the yield of d-2-aminobutyric acid at 2, 4, 6, 8, 12, and
This work was financially supported by the National Natural Sci-
ence Foundation of China (No. 21472232) and Tianjin Municipal
Science and Technology Commission (No 15PTGCCX00060).
2
4 h. The same procedure was followed for the reactions with dif-
ferent amount of enzymes and substrates, which were as follows.
II: 2 U EcTAL, 1 U M-StDAPDH, 2 U PFDH, 100 mm l-threonine, and
1
50 mm ammonium formate; III: 2 U EcTAL, 2 U M-StDAPDH, 4 U Conflict of Interest
PFDH, 100 mm l-threonine, and 150 mm ammonium formate; IV:
2
3
U EcTAL, 2 U M-StDAPDH, 4 U PFDH, 200 mm l-threonine, and
00 mm ammonium formate; V: 2 U EcTAL, 2 U M-StDAPDH, 4 U
The authors declare no conflict of interest.
PFDH, 200 mm l-threonine, and 300 mm sodium formate; VI: 2 U
EcTAL, 2 U M-StDAPDH, 4 U PFDH, 300 mm l-threonine, and
Keywords: atom economy
· d-2-aminobutyric acid · d-
4
50 mm ammonium formate; VII: 2 U EcTAL, 2 U M-StDAPDH, 4 U
aminoacid dehydrogenase · enzymatic cascade · l-threonine
PFDH, 300 mm l-threonine, and 450 mm sodium formate.
ammonia lyase
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FULL PAPERS
X. Chen, Y. Cui, X. Cheng, J. Feng, Q. Wu,
D. Zhu*
&
& – &&
Highly Atom Economic Synthesis of d-
-Aminobutyric Acid through an In
Vitro Tri-enzymatic Catalytic System
2
Atom-economic synthesis: l-Threonine
is converted to d-2-aminobutyric acid
with high economic efficiency through
an enzymatic cascade reaction catalyzed
by l-threonine ammonia lyase and a d-
amino acid dehydrogenase with for-
mate dehydrogenase for co-factor re-
generation.
&
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ÝÝ These are not the final page numbers!