Highly selective synthesis of d-amino acids from readily available l-amino acids by a one-pot biocatalytic stereoinversion cascade

Article abstract of DOI:10.1039/c9ra06301c

d-Amino acids are key intermediates required for the synthesis of important pharmaceuticals. However, establishing a universal enzymatic method for the general synthesis of d-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we constructed and optimized a cascade enzymatic route involving l-amino acid deaminase and d-amino acid dehydrogenase for the biocatalytic stereoinversions of l-amino acids into d-amino acids. Using l-phenylalanine (l-Phe) as a model substrate, this artificial biocatalytic cascade stereoinversion route first deaminates l-Phe to phenylpyruvic acid (PPA) through catalysis involving recombinant Escherichia coli cells that express l-amino acid deaminase from Proteus mirabilis (PmLAAD), followed by stereoselective reductive amination with recombinant meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) to produce d-phenylalanine (d-Phe). By incorporating a formate dehydrogenase-based NADPH-recycling system, d-Phe was obtained in quantitative yield with an enantiomeric excess greater than 99%. In addition, the cascade reaction system was also used to stereoinvert a variety of aromatic and aliphatic l-amino acids to the corresponding d-amino acids by combining the PmLAAD whole-cell biocatalyst with the StDAPDH variant. Hence, this method represents a concise and efficient route for the asymmetric synthesis of d-amino acids from the corresponding l-amino acids.

Full text of DOI:10.1039/c9ra06301c

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PAPER
Highly selective synthesis of D-amino acids from
readily available L-amino acids by a one-pot
biocatalytic stereoinversion cascade†
Cite this: RSC Adv., 2019, 9, 29927
Danping Zhang, Xiaoran Jing, Wenli Zhang, Yao Nie *ac and Yan Xu*ab
a
a
a
D-Amino acids are key intermediates required for the synthesis of important pharmaceuticals. However,
establishing a universal enzymatic method for the general synthesis of D-amino acids from cheap and
readily available precursors with few by-products is challenging. In this study, we constructed and
optimized
a cascade enzymatic route involving L-amino acid deaminase and D-amino acid
dehydrogenase for the biocatalytic stereoinversions of L-amino acids into D-amino acids. Using L-
phenylalanine (L-Phe) as a model substrate, this artificial biocatalytic cascade stereoinversion route first
deaminates L-Phe to phenylpyruvic acid (PPA) through catalysis involving recombinant Escherichia coli
cells that express L-amino acid deaminase from Proteus mirabilis (PmLAAD), followed by stereoselective
reductive amination with recombinant meso-diaminopimelate dehydrogenase from Symbiobacterium
thermophilum (StDAPDH) to produce D-phenylalanine (D-Phe). By incorporating
a
formate
dehydrogenase-based NADPH-recycling system, D-Phe was obtained in quantitative yield with an
enantiomeric excess greater than 99%. In addition, the cascade reaction system was also used to
stereoinvert a variety of aromatic and aliphatic L-amino acids to the corresponding D-amino acids by
combining the PmLAAD whole-cell biocatalyst with the StDAPDH variant. Hence, this method represents
a concise and efficient route for the asymmetric synthesis of D-amino acids from the corresponding L-
amino acids.
Received 13th August 2019
Accepted 16th September 2019
DOI: 10.1039/c9ra06301c
rsc.li/rsc-advances
different routes exist, namely those that involve chemical
Introduction
9–12
methods and those that are biocatalytic in nature.
Chemical
D-Amino acids, as chiral directing auxiliaries and chiral syn- methods generally synthesize D-amino acids through the chiral
thons in organic synthesis, play important roles in the resolution of racemic D,L-amino acids or by asymmetric proto-
1,2
production of pharmaceuticals and ne chemicals. Current cols from chiral or prochiral starting materials. High costs and
important applications of D-amino acids include their use as key low yields due to D-amino acid racemization are the major
1
0,12
components in b-lactam antibiotics, fertility drugs, anticoagu- disadvantages of chemical methods.
lants, and pesticides.
However, the develop-
1,3–5
D-Arylalanines, such as D-phenylala- ment of biocatalysis methods has signicantly progressed in
nine (D-Phe), are useful intermediates for the production of the past decade. With enzymes as biocatalysts, D-amino acids
pharmaceuticals, including b-lactam antibiotics, small peptide can be produced under mild reaction conditions with high
4
12,13
hormones, and pesticides. The production of D-Phe is of great enantioselectivities, conversions, and space-time yields.
interest since pharmaceuticals that include analgesics, anti- Enzymatic approaches for the synthesis of D-amino acids can be
stress agents, antidiabetics (e.g., nateglinide), and anticoagu- divided into the following ve types: (1) processes involving D-
6
–8
4,14
lants are synthesized from D-arylalanines.
Many methods for the synthesis of D-amino acids and their aminotransferase-promoted transfer of the amino group of a D-
hydantoinase and D-carbamoylase,
(2) the D-amino acid
15
derivatives have been developed. In general, two fundamentally amino acid to an a-keto acid, (3) hydrolysis of an N-acyl-D-
16,17
amino acid by N-acyl-D-amino acid amidohydrolase,
(4)
a
kinetic resolution of a racemic mixture by L-amino acid
School of Biotechnology, Key Laboratory of Industrial Biotechnology, Ministry of
Education, Jiangnan University, Wuxi 214122, China. E-mail: ynie@jiangnan.edu. oxidase, and (5) the asymmetric reductive amination of an a-
4
4,12
cn; yxu@jiangnan.edu.cn
b
keto acid by D-amino acid dehydrogenase.
Liu et al. used D-
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi
carbamoylase from Arthrobacter crystallopoietes coupled with D-
hydantoinase from Agrobacterium tumefaciens for the enantio-
selective resolution of L-indolylmethylhydantoin into D-trypto-
phan in 99.4% yield and greater than 99% enantiomeric excess
2
14122, China
c
Suqian Industrial Technology Research Institute of Jiangnan University, Suqian
2
23814, China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9ra06301c
14
(
ee). D-Amino acid aminotransferase from Bacillus subtilis
1
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WB600 was used to produce D-Phe at a nal product concen- into the D-amino acid in the reductive amination module. This
ꢀ
1
18
tration of 1.72 g L
,
while Isobe et al. used L-amino acid stereoinverting biocatalysis-based cascade reaction route was
oxidase from Rhodococcus sp. AIU Z-35-1 to produce D-citrulline, designed with the following basic principles in mind: (1) the
19
D-glutamine, D-homoserine, and D-arginine. Although these reaction conditions in both reaction modules need to be
21,29
methods were used to synthesize a variety of D-amino acids, they compatible;
(2) the reductive amination module must
usually require specic substrates that are generally expensive contain only irreversible reactions in order to direct the balance
1,4,20
and commercially unavailable.
of the entire reaction and accumulate the nal product; and (3)
Cascade biocatalysis has been developed in recent years as the 2-keto acid intermediate must not accumulate and no other
30
a promising method for the efficient synthesis of pharmaceu- intermediates can be generated. According to the above prin-
21–24
tical intermediates and ne chemicals.
Cascade biocatalysis ciples, L-amino acid deaminase (LAAD), which catalyzes the
comprises multiple biocatalytic reactions in a single reaction stereospecic oxidative deamination of L-amino acids to the
vessel without the isolation of any intermediate, and starting corresponding 2-keto acids and ammonia, is better suited for
21,23,25
from cheap and available substrates.
Because L-amino the construction of the oxidative deamination module,
acids are mostly available through fermentation from inex- compared to L-amino acid oxidase or L-amino acid dehydroge-
31,32
pensive and renewable natural sources, multi-enzyme cascades nase.
D-Amino acid dehydrogenase (DAADH) is the desired
for the biocatalytic stereoinversions of L-amino acids into D- biocatalyst for use in the reductive amination module, since it
amino acids represent economically effective and environmen- directly catalyzes the asymmetric reductive amination of an a-
8,26–28
4,10
tally benign methods.
keto acid to the corresponding D-amino acid. Although LAAD
Herein, based on the reaction route involving L-amino acid and DAADH can potentially to be used to construct our articial
8
deaminase and D-amino acid dehydrogenase, we constructed multi-enzyme cascade, coordinating these two enzyme-
and optimized the biocatalytic cascade route for the synthesis of catalyzed reactions involving necessary cofactor-recycling
12,21,33–35
D-amino acids. The reaction route is assembled using two system is challenging.
modules, namely an oxidative deamination module and
a reductive amination module. L-Amino acid deaminase from
Oxidative deamination of L-Phe to phenylpyruvic acid
Proteus mirabilis (PmLAAD) and meso-diaminopimelate dehy-
drogenases (DAPDHs) from various microorganisms were
cloned and expressed separately in recombinant Escherichia coli
BL21(DE3) and used in the biocatalytic cascade route. Aer
characterization of the enzymes involved, recombinant E. coli
cells that express PmLAAD and a suitable DAPDH were used in
the cascade route for the synthesis of D-amino acids from L-
amino acids (Scheme 1). As a model reaction, this cascade route
efficiently and completely transformed L-phenylalanine (L-Phe)
into D-Phe with high optical purity. In particular, the articial
biocatalytic cascade also transforms a variety of aromatic and
aliphatic L-amino acids into the corresponding D-amino acids.
In order to construct the catalytic multi-enzyme cascade system,
we used L-amino acid deaminase from Proteus mirabilis
(
PmLAAD) in the oxidative deamination module owing to its high
36
activity toward L-Phe. During the oxidative deamination reac-
tion, PmLAAD catalyzes the deamination of L-Phe to the corre-
sponding a-imino acid with oxygen as the co-substrate, aer
which spontaneous hydrolysis produces phenylpyruvic acid (PPA)
37–39
(Scheme 2).
Because PmLAAD is a membrane-bound
40
protein, different forms of the biocatalyst were prepared in
order to investigate their effects on the yield of PPA from L-Phe. As
shown in Table S1,† the yield of PPA was higher when the bio-
catalyst was in whole-cell form compared to other biocatalyst
types. Therefore, the whole-cell biocatalyst was adopted as the
PmLAAD that catalyzes the oxidative deamination reaction.
In addition, the reaction conditions for the catalytic PmLAAD
whole-cell oxidative deamination were optimized. As shown in
Fig. S1,† the PmLAAD whole-cell biocatalyst was more active in
Results and discussion
Constructing biocatalytic reaction route for stereoinversion of
L-amino acids
As shown in Scheme 1, an L-amino acid is rst converted into
the corresponding 2-keto acid in the oxidative deamination
module, aer which the 2-keto acid is asymmetrically converted
ꢁ
Tris–HCl buffer (50 mM, pH 9.0) at 45 C and the optimum
ꢀ1
biocatalyst concentration was found to be 80 mg mL . Under the
optimized reaction conditions, the catalytic activities of PmLAAD
whole cells toward L-Phe, D-Phe, and PPA, were further evaluated,
with the concentrations of L-Phe, D-Phe, and PPA determined for
each reaction (Fig. 1). Only PPA was produced aer 30 min when
50 mM L-Phe was used as the substrate. On the other hand, the
concentration of D-Phe was unchanged and no PPA or L-Phe was
Scheme 1 Depicting the one-pot biocatalytic cascade route for the
synthesis of enantiomerically pure D-amino acids by L-amino-acid
stereoinversion.
Scheme 2 PmLAAD-catalyzed oxidative deamination of L-Phe.
29928 | RSC Adv., 2019, 9, 29927–29935
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and DAPDH from Symbiobacterium thermophilum (StDAPDH)
and its H227V variant bearing a mutation at the substrate-
binding pocket (StDAPDH/H227V) were employed to catalyze
7,43,44
reductive amination.
As summarized in Table 1, the DAPDHs examined in this
study were all active toward PPA, with the activity of StDAPDH/
ꢀ
1
ꢀ1
H227V (specic activity of 0.46 mmol min mg ) the highest
among the four DAPDHs. Michaelis–Menten kinetic data for the
m
DAPDHs towards PPA were also determined; K and kcat values
were calculated by non-linear tting and are also summarized
in Table 1. Although the affinities of StDAPDH and StDAPDH/
H227V toward PPA as the substrate were similar, with compa-
rable K
rate (kcat ¼ 1.13 s ) among the DAPDHs studied, which led to
StDAPDH/H227V exhibiting higher catalytic efficiency
m
values, StDAPDH/H227V exhibited the highest reaction
ꢀ1
a
compared to the other DAPDHs. The variation at H227 in
StDAPDH enlarges the substrate binding pocket allowing bulky
substrates, such as PPA, easier access to the enzyme active site.
Fig. 1 Catalytic PmLAAD whole-cell oxidative deamination processes
with: (A) L-Phe, (B) D-Phe, and (C) PPA as substrates. (D) PPA formation
7
at different concentrations of L-Phe. Reactions were carried out in
ꢁ
Tris–HCl buffer (50 mM, pH 9.0) at 45 C and a PmLAAD whole-cell Therefore, StDAPDH/H227V was selected for the construction of
concentration of 80 mg mL . The values were averaged from tripli- the reductive amination module.
ꢀ1
cate measurements and the standard deviations are indicated as error
bars.
To further conrm the feasibility of the stereoinverting
multi-enzyme cascade constructed with the above-mentioned
reaction route, the catalytic activities of StDAPDH/H227V
toward PPA, L-Phe, and D-Phe were further evaluated, with the
concentrations of PPA, L-Phe, and D-Phe determined in each
case (Fig. 2). With PPA as the substrate, D-Phe was produced
with an optical purity (ee) greater than 99% and a quantitative
yield aer 4 h. However, no reaction was observed with L-Phe or
D-Phe as the substrate, nor was PPA produced, which indicates
that the target product (D-Phe) cannot be converted into PPA,
the reaction intermediate in the overall reaction process, and
the reaction equilibrium in the cascade reaction system is
directed toward the production of D-Phe.
detected during the reaction process when D-Phe was used as the
substrate. In a similar manner, PPA was also not a substrate in
this system. Clearly, the PmLAAD whole-cell biocatalyst was only
active towards L-Phe and the expected PPA was the only product
formed in the oxidative-deamination reaction process, which is
consistent with requirements of the designed stereoinverting
cascade reaction route.
We also investigated the effect of substrate concentration on
the catalytic PmLAAD whole-cell oxidative deamination process.
L-Phe was rapidly converted in the early stages of the reaction,
aer which its concentration plateaued. A PPA yield of 58.7%
was obtained at an L-Phe concentration of 100 mM, while higher
concentrations of L-Phe led to lower PPA yields. Clearly,
PmLAAD whole cells mainly catalyze the oxidative deamination
of L-Phe to PPA, which is favorable for use in the biocatalytic
stereoinverting cascade system.
Considering that the optimum reaction temperature of the
ꢁ
PmLAAD whole-cell biocatalyst is 45 C, the inuence of reac-
tion temperature on the catalytic activity of StDAPDH/H227V
was investigated in order to coordinate the reaction condi-
tions of the two biocatalysts. Fig. 2D reveals that little difference
in the StDAPDH/H227V-catalyzed reaction efficiency was
ꢁ
ꢁ
observed at 45 C compared to that at 37 C, providing D-Phe in
a quantitative yield aer 4 h. Therefore, the optimum reaction
Reductive amination of PPA to D-Phe
temperature in the stereoinverting biocatalytic linear cascade
ꢁ
Meso-diaminopimelate dehydrogenases (DAPDHs) that belong was determined to be 45 C for both the whole-cell oxidative
to the D-amino acid dehydrogenase family are candidates for the deamination catalyzed by PmLAAD and the StDAPDH/H227V-
catalytic conversion of a 2-keto acid to the corresponding D- catalyzed reductive amination.
4,41,42
amino acid.
DAPDHs catalyze the reductive amination of 2-
keto acids using NADPH as the electron donor (Scheme 3). In
this work, DAPDH from Ureibacillus thermosphaericus
Assembling the L-Phe to D-Phe stereoinverting cascade system
(
UtDAPDH), DAPDH from Lysinibacillus fusiformis (LfDAPDH), Aer analyzing the catalytic characteristics of PmLAAD whole-
cells and StDAPDH/H227V in each reaction step, the two bio-
catalysts were used to construct the stereoinverting cascade
setup. The theoretical ratio of the PmLAAD whole-cell bio-
catalyst to StDAPDH/H227V in the cascade-reaction system
should be 1 U : 1 U. Because the activities of the PmLAAD whole-
cell biocatalyst and StDAPDH/H227V were 0.01 and 0.46
ꢀ
1
ꢀ1
mmol min mg , respectively, the theoretical concentration of
Scheme 3 DAPDH-catalyzed phenylpyruvic acid reductive amination. the PmLAAD whole-cell biocatalyst and StDAPDH/H227V were
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a
Table 1 Specific activities and Michaelis–Menten kinetic data for the DAPDHs toward PPA
Specic activity
(mmol min mg
ꢀ
1
ꢀ1
ꢀ1
cat (s
ꢀ1
ꢀ1
Enzyme
)
K
m
(mM)
k
)
m
kcat/K (s mM )
StDAPDH/H227V
StDAPDH
UtDAPDH
0.460 ꢂ 0.030
0.014 ꢂ 0.002
0.042 ꢂ 0.004
0.035 ꢂ 0.001
12.48 ꢂ 0.23
15.21 ꢂ 0.31
3.07 ꢂ 0.18
7.61 ꢂ 0.14
1.13 ꢂ 0.06
0.13 ꢂ 0.03
0.18 ꢂ 0.02
0.14 ꢂ 0.05
0.091 ꢂ 0.003
0.009 ꢂ 0.002
0.059 ꢂ 0.006
0.018 ꢂ 0.004
LfDAPDH
a
ꢁ
Kinetic data were acquired in 50 mM Na
2
CO
3
/NaHCO
3
buffer (pH 9.0) at 30 C at NH
4
Cl and NADPH concentrations of 200 mM and 0.5 mM,
respectively. The concentration of PPA varied between 2 and 40 mM.
ꢀ1
required to be 100 and 2 mg mL , respectively. However,
Due to the NADPH dependence of DAPDH catalyzing the
considering that the reductive amination module is critical for reductive amination reaction, meanwhile, the cofactor-recycling
the production of D-Phe, the concentration of StDAPDH/H227V system involving Burkholderia stabilis formate dehydrogenase
ꢀ
1
in the cascade reaction system was increased to 4 mg mL to (BsFDH) was applied to couple the StDAPDH/H227V-catalyzed
further improve the efficiency of the reductive amination reductive amination for further enhancement of efficiency of
module.
the one-pot biocatalytic stereoinverting cascade. The BsFDH
ꢀ
1
exhibited the specic activity of 2.88 U mg in Tris–HCl buffer
ꢁ
(
50 mM, pH 9.0) at 45 C (Fig. S2†). By using BsFDH for NADPH
regeneration, the yield of D-Phe was maintained at the same
level as that with excessive loading of NADPH for the reductive
+
amination module (10 mM PPA), even when only 1 mM NADP
was added to initiate the cofactor regeneration (Fig. S3†). Then
ꢀ1
the PmLAAD whole-cell biocatalyst (100 mg mL ), StDAPDH/
ꢀ
1
ꢀ1
H227V (4 mg mL ), and BsFDH (0.35 mg mL , 1 U) were
assembled to construct the one-pot stereoinverting cascade.
Subsequently, the D-Phe was obtained in a yield of 76.2% by the
addition of 30 mM L-Phe, 90 mM NH Cl, 60 mM sodium
4
+
formate, and 3 mM NADP to the 50 mM Tris–HCl (pH 9.0)
reaction buffer during the cascade reaction at 45 C (Fig. S4†).
ꢁ
The concentrations of the PmLAAD whole-cell biocatalyst,
StDAPDH/H227V, and BsFDH in the cascade reaction system
were next optimized in order to further reduce the amount of
residual L-Phe substrate in the reaction system, which would
Fig. 2 DAPDH-catalyzed reductive aminations with: (A) PPA, (B) L-Phe, lower the optical purity of the nal D-Phe product. As shown in
ꢀ
1
(
C) D-Phe as substrates. (D) D-Phe formation under different reaction
Table 2, when 40 mg mL of the PmLAAD whole cells, 4.5–
1
temperatures. The reaction mixture contained StDAPDH/H227V (4 mg
mL ), NADPH (20 mM), NH
pH 9.0). Unless otherwise noted, the reaction temperature was 37 C
for the StDAPDH/H227V-catalyzed reactions. The values were aver-
ꢀ1
ꢀ1
2 mg mL StDAPDH/H227V, and 0.35–0.7 mg mL BsFDH
were added to the reaction system, 30 mM D-Phe was produced
entirely from the L-Phe substrate and no PPA intermediate was
ꢀ1
4
Cl (30 mM), and Tris–HCl buffer (50 mM,
ꢁ
aged from triplicate measurements and the standard deviations are accumulated. To a certain extent, increasing the amount of the
indicated as error bars.
a
Table 2 Influence of biocatalyst concentration on the cascade stereoinversion of L-Phe
StDAPDH/H227V
BsFDH (mg
L-Phe
(mM)
D-Phe
(mM)
ꢀ1
ꢀ1
ꢀ1
b
PmLAAD (mg mL
)
(mg mL
)
mL
)
Optical purity of D-Phe (% ee)
4
4
4
6
6
6
8
8
8
0
0
0
0
0
0
0
0
0
4.5
9
12
4.5
9
12
4.5
9
0.35
0.7
0.7
0.35
0.7
0.7
0.35
0.7
0.7
0
0
0
4.8
1.8
0
6.4
3.9
0
30.0
30.0
30.0
25.2
28.2
30.0
23.6
26.1
30.0
>99
>99
>99
68
88
>99
57
74
79.6
12
a
+
The reaction mixture consisted of 30 mM L-Phe, 3 mM NADP , 90 mM NH
4
Cl, 60 mM sodium formate, and 50 mM Tris–HCl buffer (pH 9.0). The
ꢁ
b
reactions were carried out at 45 C and 220 rpm for 24 h. Optical purity of D-Phe (% ee) was determined by HPLC aer FDAA derivatization.
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catalyst for reductive amination and reducing the amount of the concentrations of the biocatalysts, enantiomerically pure D-Phe
ꢀ
1
catalyst for oxidative deamination enhanced the catalytic effi- (>99% ee) was produced in quantitative yield with 40 mg mL
ꢀ1
ciency of the one-pot stereoinverting cascade, leading to high PmLAAD whole cells, 4.5 mg mL
StDAPDH/H227V, and
ꢀ1
yield and optical purity of the D-Phe. Consistent with the 0.35 mg mL BsFDH.
observation of PmLAAD whole-cell oxidative deamination Using the optimum biocatalyst composition in the catalytic
process towards L-Phe, rapid accumulation of PPA in early stage cascade system, the stereoinversion of L-Phe into D-Phe was
of the reaction would inhibit continuous conversion of L-Phe, tracked by monitoring the changes in the concentrations of L-
and the reductive amination reaction would be the rate-limiting Phe, D-Phe, and PPA. As shown in Fig. 3, quick consumption of
step of the cascade process. Consequently, by optimizing the L-Phe in the early stages of the cascade reaction was observed for
the oxidative deamination reaction and simultaneously the PPA
intermediate was detected in the cascade reaction system.
Meanwhile, D-Phe was generated and subsequently accumu-
lated from the PPA intermediate by the reductive amination
reaction. For the increased substrate concentration up to
8
6
0 mM of L-Phe, the cascade reaction was almost complete aer
h, and both the optical purity and the conversion of the
product were close to 100%. Therefore, coordinating the two
reaction modules of the oxidative deamination and the reduc-
tive amination by applying appropriate proportion of the cor-
responding catalysts to regulate the reaction rates in the
cascade would be important to improve the substrate concen-
tration and reaction efficiency. In an enlarged reaction system,
optically pure D-Phe was produced with 91% isolation yield and
>
99.9% ee. The product was further conrmed to be D-Phe by
1
13
MS, H-NMR, and C-NMR (Fig. S5–S7†). Consequently, we
constructed a biocatalytic cascade system for the asymmetric
synthesis of D-Phe through stereoinversion involving oxidative
deamination and reductive amination.
Substrate scope of the biocatalytic cascade system
The applicability of the biocatalytic cascade system for the
stereoinversion of L-Phe, and its construction strategy, were
further evaluated for the asymmetric conversions of a variety of
natural and noncanonical L-amino acids into the corresponding
D-amino acids. As shown in Table 3, the L-amino acids examined
were mostly converted into the desired D-amino acids. For
Table
3 Stereoinversions of various L-amino acids using the
a
PmLAAD–DAPDH cascade system
PmLAAD–StDAPDH/H227V
b
Substrate
Conversion (%)
Optical purity (% ee)
L-Leucine
L-Glutamic acid
L-Norvaline
L-Tyrosine
70.1
100
75
45.3
100
100
100
100
76.3
68.3
>99
>99
>99
>99
>99
>99
>99
52.1
Fig. 3 Profiles for the stereoinversion of L-Phe into D-Phe. (A) The L-Phenylalanine
cascade reaction system contained 30 mM L-Phe, 3 mM NADP , L-Homophenylalanine
Cl, and 60 mM sodium formate; (B) the cascade reaction 2-Chloro-L-phenylalanine
Cl, and 3-Chloro-L-phenylalanine
00 mM sodium formate; (C) the cascade reaction system contained 4-Chloro-L-phenylalanine
+
9
0 mM NH
4
+
system contained 50 mM L-Phe, 5 mM NADP , 150 mM NH
1
4
+
8
0 mM L-Phe, 8 mM NADP , 240 mM NH
4
Cl, and 160 mM sodium
formate. All the reactions were carried out in 2 mL Tris–HCl buffer
50 mM, pH 9.0) comprising 40 mg mL PmLAAD wet cells, 4.5 mg
mL StDAPDH/H227V, and 0.35 mg mL BsFDH at 220 rpm and
a
+
Reaction conditions: 10 mM substrate, 1 mM NADP , 20 mM sodium
formate, and 30 mM NH
4
Cl were added to 50 mM Tris–HCl buffer (pH
ꢀ1
(
ꢀ1
9.0). The cascade biocatalysts were PmLAAD whole cells (40 mg mL ),
ꢀ1
ꢀ1
ꢀ1
ꢀ1
StDAPDH/H227V (4.5 mg mL ), and BsFDH (0.35 mg mL ). The
ꢁ
ꢁ
b
4
5 C for 12 h. The values were averaged from triplicate measurements reactions were carried out at 45 C. Optical purity (% ee) was
and the standard deviations are indicated as error bars.
determined by HPLC aer FDAA derivatization.
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substrates with bulky groups, such as L-Phe, L-homo- Cloning the gene-encoding PmLAAD, DAPDHs and BsFDH
phenylalanine, 2-chloro-L-phenylalanine, and 3-chloro-L-
phenylalanine, which are aromatic amino acids, the PmLAAD–
StDAPDH/H227V cascade system produced the corresponding
D-amino acids in high conversion (100%) and optical purity
The gene-encoding PmLAAD (GenBank accession no.
EU669819.1), UtDAPDH (GenBank accession no. AB636161.1),
LfDAPDH (GenBank accession no. WP_069481537.1), StDAPDH
(GenBank accession no. BAD40410.1), StDAPDH/H227V, and
(>99% ee). In addition, the constructed PmLAAD–StDAPDH/
BsFDH (GenBank accession no. ACF35003.1) were cloned and
ligated into the pET-28a(+) vector. The pET-28a(+)-PmLAAD,
H227V cascade system efficiently converted aliphatic amino
acids, such as L-leucine, L-norvaline, and L-glutamic acid to the
corresponding D-amino acids, with D-glutamic acid and D-nor-
valine even obtained in quantitative conversion and >99% ee.
Therefore, the constructed biocatalytic cascade system is effi-
cient and applicable to the asymmetric synthesis of D-amino
acids through stereoinversion involving oxidative deamination
and reductive amination.
pET-28a(+)-UtDAPDH,
pET-28a(+)-LfDAPDH,
pET-28a(+)-
StDAPDH, pET-28a(+)-StDAPDH/H227V, and pET-28a(+)-BsFDH
recombinant plasmids were transformed into E. coli BL21(DE3)
cells.
Enzyme expression and purication
Recombinant E. coli BL21(DE3) was propagated in 1 L of Luria–
ꢀ1
ꢁ
Bertani medium containing 100 mg mL kanamycin at 37 C.
The culture was induced by the addition of isopropyl b-D-1-thi-
ogalactopyranoside (IPTG) to a nal concentration of 0.1 mM
when the optical density (l ¼ 600 nm) was 0.6–0.8, aer which it
Conclusions
A versatile and highly efficient biocatalytic cascade system was
constructed for the asymmetric synthesis of D-amino acids
through the stereoinversions of L-amino acids using a combi-
nation of PmLAAD whole cells (oxidative deamination
module), DAPDHs (reductive amination module), and BsFDH
ꢁ
was incubated for an additional 16 h at 17 C at 200 rpm. Aer
ꢁ
centrifugation at 6000 ꢃ g and 4 C for 20 min, the recombinant
cells were washed with potassium phosphate buffer. The
ꢁ
PmLAAD whole cells were cryopreserved at ꢀ20 C for further
(
cofactor regeneration). To understand the basic principles
experiments.
required for constructing the biocatalytic stereoinverting
cascade, the catalytic characteristics of the oxidative deami-
nation module and the reductive amination module were rst
analyzed. Using L-Phe as the model substrate, the catalytic
activities of PmLAAD whole cells and the adopted DAPDHs
toward L-Phe, D-Phe, and PPA were shown to guarantee the
feasibility of the cascade stereoinversion process. The cascade
reaction system was then constructed by assembling the two
modules involving necessary cofactor recycling. By optimizing
the concentrations of the involved biocatalysts and the reac-
tion conditions such that both biocatalytic modules are
compatible, L-Phe was quantitatively converted into D-Phe with
greater than 99% ee using the one-pot cascade process. In
particular, the cascade biocatalytic system also efficiently
converted a variety of readily available and inexpensive
aromatic and aliphatic L-amino acids into the corresponding
D-amino acids. The developed stereoinverting catalytic system
is a promising, sustainable, and cost-efficient alternative for
the synthesis of a broad range of D-amino acids.
To purify UtDAPDH, LfDAPDH, StDAPDH, StDAPDH/H227V,
and BsFDH, the recombinant E. coli BL21(DE3) cells were
resuspended in a lysis buffer containing 20 mM Tris–HCl and
150 mM NaCl (pH 7.5). Cell lysates were produced with a high-
pressure homogenizer (APV, Germany) and then centrifuged at
ꢁ
1
2 000 ꢃ g for 30 min at 4 C to remove cell debris. Proteins
¨
were puried by Ni affinity chromatography with an Akta Puri-
er 10 (GE Healthcare, USA). The gradient elution buffer for the
Ni-NTA column was composed of 20 mM Tris–HCl (pH 7.5),
50 mM NaCl, and 0–500 mM imidazole. The imidazole in the
protein solutions was removed using a PD-10 desalting column
GE Healthcare, USA). Protein concentrations were determined

1
(
using a bicinchoninic acid assay kit (CWBIO, China). The
resulting enzyme solutions were used for activity assays and
catalytic reactions.
Activity assays and kinetics
PmLAAD whole cells were incubated in 50 mM Tris–HCl buffer
(
3
1
pH 9.0) comprising 100 mM L-Phe in a nal volume of 5.0 mL at
ꢁ
7 C. Aer reaction for 60 min, the mixture was centrifuged at
2 000 ꢃ g for 5 min, aer which a 15 mL aliquot of the super-
Experimental
Materials
natant was added to 1 mL of ferric chloride solution and incu-
Enzymes, vectors, oligonucleotides, and other reagents for bation was continued for 2 min at room temperature. The
DNA cloning and amplication were obtained from the concentration of PPA was determined by measuring the absor-
45
Takara-Bio Co., Japan and the Novagen Co., USA. The L-amino bance at 640 nm. One unit of PmLAAD-whole-cell catalytic
acids, 2-keto acids, and D-amino acids were purchased from activity is dened as 1 mmol of PPA generated per minute under
+
31
the Sinopharm Chemical Reagent Co., Ltd. NADPH and NADP
were purchased from Sigma-Aldrich (USA). Acetonitrile and cates and signicant differences (p < 0.05) were recorded.
methanol used for high performance liquid chromatography The activity assaying system for DAPDH-catalyzed reductive
the assay conditions. All data were averaged over three repli-
(
HPLC) were of chromatographic grade (Honeywell Co., USA). amination consisted of 20 mM 2-keto acid, 200 mM NH Cl,
4
All other chemicals were of analytical grade and commercially 0.5 mM NADPH, and 50 mM Na
2
CO
3
–NaHCO
3
buffer (pH 9.0) at
1,43
available.
a nal volume of 200 mL.
Aer incubation, reactions were
29932 | RSC Adv., 2019, 9, 29927–29935
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performed in 96-well plates using a Spectramax M2e microplate three biocatalysts at various concentrations in 2 mL Tris–HCl
reader (Molecular Devices, USA) and initiated by the addition of buffer (50 mM, pH 9.0). Reactions were carried out at 220 rpm
ꢁ
ꢁ
a 20 mL enzyme solution. The OD340 was monitored at 30 C and 45 C, and were terminated by the addition of an equal
ꢀ1
ꢀ1
using a molar extinction coefficient of 6.22 mM cm . One volume of 10% (w/v) trichloroacetic acid solution. The amount
unit of enzyme activity is dened as 1 mmol NADPH consumed of PPA generated in the reaction process was determined using
per minute under the assay conditions. All data were averaged ferric chloride solution, and the amounts of L-Phe and D-Phe
45
over three replicates for each substrate and signicant differ- were determined by HPLC aer derivation with 1-uoro-2,4-
ences (p < 0.05) were determined.
dinitrophenyl-5-L-alanine amide (FDAA).
Kinetic data for the DAPDHs toward PPA were determined by
measuring the initial rates at various concentrations of the Asymmetric synthesis of D-amino acids from L-amino acids
substrate. The reductive amination reactions were carried out
Various L-amino acids, including L-leucine, L-glutamic acid, L-
tyrosine, L-norvaline, L-Phe, L-homophenylalanine, 2-chloro-L-
with various concentrations of PPA, 200 mM NH Cl, and
4
0
3
.5 mM NADPH in 50 mM Na
0 C. The concentration of PPA varied between 2 and 40 mM.
2
CO
3
–NaHCO
3
buffer (pH 9.0) at
phenylalanine, 3-chloro-L-phenylalanine, and 4-chloro-L-
phenylalanine were used as substrates for the asymmetric
synthesis of the corresponding D-amino acids using the cascade
reaction system involving PmLAAD whole cells, StDAPDH/
H227V, and BsFDH. The reaction mixture in 2 mL Tris–HCl
ꢁ
The Michaelis–Menten kinetic parameters were obtained from
a non-linear least-squares t of the Michaelis–Menten equation
to the experimental data and were further validated using
double reciprocal Lineweaver–Burk plots. All data were aver-
aged over three replicates for each substrate concentration and
signicant differences (p < 0.05) were determined.
buffer (50 mM, pH 9.0) contained 10 mM substrate, 1 mM
+
NADP , 30 mM NH
4
Cl, 20 mM sodium formate, and the
combined PmLAAD whole-cell, StDAPDH/H227V, and BsFDH
biocatalysts. The cascade biocatalysts were composed of
The activity assaying system for BsFDH consisted of 50 mM
+
sodium formate, 2 mM NADP , and 50 mM Tris–HCl buffer (pH
ꢀ1
PmLAAD wet cells (40 mg mL ), StDAPDH/H227V (4.5 mg
7.5) at a nal volume of 200 mL. Aer incubation, reactions were
ꢀ1
ꢀ1
mL ), and BsFDH (0.35 mg mL ). The cascade reactions were
performed in 96-well plates using a Spectramax M2e microplate
reader (Molecular Devices, USA) and initiated by the addition of
ꢁ
carried out at 220 rpm and 45 C. The reactions were terminated
by adding equal volumes of 10% (w/v) trichloroacetic acid
solution. The concentrations of the L-amino acids and D-amino
acids were determined by HPLC following FDAA derivation.
ꢁ
a 20 mL enzyme solution. The OD340 was monitored at 30 C
ꢀ1
ꢀ1
using a molar extinction coefficient of 6.22 mM cm . One
unit of the enzyme was dened as the amount that catalyzed the
formation of 1 mmol of NADPH per minute under the assay
conditions. All data were averaged over three replicates for each
substrate and signicant differences (p < 0.05) were determined.
Determining the amount of PPA
The amount of PPA in the reaction process was determined
37
using ferric chloride solution, which was prepared as follows:
.1 M ferric chloride was dissolved in 6 mL dimethyl sulfoxide,
0
Single step reaction
followed by the addition of 4 mL deionized water and 200 mL
acetic acid, aer which the solution was cooled in an ice bath.
To determine the amount of PPA, a 15 mL aliquot of a reaction
sample was added to 1 mL of the ferric chloride solution, and
the mixture was incubated at room temperature for 2 min. The
amount of PPA was immediately determined by measuring the
absorbance at 640 nm using the above-mentioned microplate
To examine the catalytic activity of PmLAAD whole cells toward
different components relevant to oxidative deamination, 50 mM
L-Phe, 50 mM PPA, or 50 mM D-Phe substrate was separately
added to a reaction mixture containing PmLAAD wet cells
ꢀ
1
(
80 mg mL ) and Tris–HCl buffer (50 mM, pH 9.0). The
ꢁ
oxidative deamination reactions were carried out at 45 C for
6
h.
To examine the catalytic activity of StDAPDH/H227V toward
45
reader.
different components relevant to reductive amination, 10 mM L-
Phe, 10 mM PPA, or 10 mM D-Phe substrate was separately
added to a reaction mixture containing StDAPDH/H227V (4 mg
Determining L- and D-amino acids
The FDAA reagent (Sigma-Aldrich, USA) was used to produce
diastereomeric derivatives of the amino acids. A 10 mL sample of
the amino acid, 8 mL of 1 M NaHCO , and 40 mL of 1% (w/v)
FDAA in acetone were mixed and heated for 1 h at 40 C.
ꢀ1
mL ), NADPH (20 mM), NH
4
Cl (30 mM), and Tris–HCl buffer
3
(
50 mM, pH 9.0). The reductive amination reactions were
ꢁ
ꢁ
carried out at 37 C for 10 h.
When the sample was cooled to room temperature, 8 mL of 1 N
HCl and 934 mL of 40% (v/v) aqueous acetonitrile were added to
the mixture, aer which it was vortexed and ltered (0.22 mm)
Combination of biocatalysts for cascade reaction
46
To construct the stereoinverting cascade, PmLAAD wet cells for HPLC. Samples were analyzed using a Develosil ODS-UG-5
ꢀ
1
(
40–80 mg mL
)
that catalyze oxidative deamination, column (150 mm ꢃ 4.6 mm). The HPLC conditions for
ꢀ1
StDAPDH/H227V (4.5–12 mg mL ) that catalyze reductive analyzing phenylalanine were: mobile phase A water (0.05%
ꢀ1
amination, and BsFDH (0.35–0.7 mg mL ) that catalyze triuoroacetic acid), mobile phase B acetonitrile (0.05% tri-
NADPH regeneration were combined in the one-pot biocatalytic uoroacetic acid), with gradients from 20% B to 40% B over
system. The reaction mixture was composed of 30 mM L-Phe, 40 min, and from 40% B to 50% B over 5 min at a ow rate of 1
+
ꢀ1
ꢁ
3
mM NADP , 90 mM NH Cl, 60 mM sodium formate, and the mL min , detection at 340 nm, 40 C, and injection volume of
4
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2
0 mL (Fig. S8†). The HPLC conditions for analyzing other
amino acids were: mobile phase A 5% acetonitrile (0.05% tri-
uoroacetic acid, 1% methanol), mobile phase B 60% acetoni-
trile (0.05% triuoroacetic acid, 1% methanol), linear gradient
4 X. Z. Gao, Q. Y. Ma and H. L. Zhu, Appl. Microbiol. Biotechnol.,
2015, 99, 3341–3349.
5 F.-R. Alexandre, D. P. Pantaleone, P. P. Taylor,
I. G. Fotheringham, D. J. Ager and N. J. Turner,
Tetrahedron Lett., 2002, 43, 707–710.

ꢀ1
from 0% B to 100% B over 45 min at a ow rate 1 mL min
injection volume 20 mL (Fig. S9–S16†).
,
47
6 F. Khorsand, C. D. Murphy, A. J. Whitehead and P. C. Engel,
Green Chem., 2017, 19, 503–510.
7
8
9
X. Z. Gao, F. Huang, J. H. Feng, X. Chen, H. L. Zhang,
Z. X. Wang, Q. Q. Wu and D. M. Zhu, Appl. Environ.
Microbiol., 2013, 79, 5078–5081.
F. Parmeggiani, S. T. Ahmed, M. P. Thompson, N. J. Weise,
J. L. Galman, D. Gahloth, M. S. Dunstan, D. Leys and
N. J. Turner, Adv. Synth. Catal., 2016, 358, 3298–3306.
M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Kesseler,
R. Sturmer and T. Zelinski, Angew. Chem., Int. Ed., 2004,
Preparation, isolation, and characterization of D-Phe
ꢀ1
In a 50 mL reaction system, 80 mM L-Phe, 40 mg mL PmLAAD
ꢀ
1
ꢀ1
whole cells, 4.5 mg mL
StDAPDH/H227V, 0.35 mg mL
+
BsFDH, 8 mM NADP , 240 mM NH
4
Cl, and 160 mM sodium
formate were added in the reaction buffer (50 mM Tris–HCl, pH
ꢁ
9
.0). Reactions were carried out at 220 rpm and 45 C. Aer 6 h,
pH of the reaction mixture was adjusted by diluted HCl and the
precipitated enzymes were removed by centrifugation. The
product was isolated by a cation-exchange resin (732) column
4
3, 788–824.
0 C. N ´a jera and J. M. Sansano, Chem. Rev., 2007, 107, 4584–
671.
1
1
1
1
1
1
20
3 2
and eluted by 6% NH $H O. The product was characterized by
4
1
13
1
MS, H-NMR and C-NMR as follows (Fig. S5–S7†): H-NMR
400 MHz, D O), d/ppm: 3.11 (m, 1H), 3.28 (m, 1H), 3.98 (m,
1 Organic Synthesis Using Biocatalysis, ed. S. K. Au, J. Groover,
B. D. Feske and A. S. Bommarius, Elsevier, 2016.
(
1
5
2
1
3
H), 7.38 (m, 5H); C-NMR (100 MHz, D
6.03, 129.11, 129.37, 135.11, 173.89.
2
O), d/ppm: 36.34,
2 Y. P. Xue, C. H. Cao and Y. G. Zheng, Chem. Soc. Rev., 2018,
4
7, 1516–1561.
3 P. J. Almhjell, C. E. Boville and F. H. Arnold, Chem. Soc. Rev.,
018, 47, 8980–8997.
2
Conflicts of interest
4 Y. F. Liu, G. C. Xu, R. Z. Han, J. J. Dong and Y. Ni, Bioresour.
Technol., 2018, 249, 720–728.
There are no conicts of interest to declare.
5 E. S. Park and J. S. Shin, Adv. Synth. Catal., 2015, 356, 3505–
3
509.
16 R. Mor ´a n-Ramallal, R. Liz and V. Gotor, Org. Lett., 2008, 10,
935–1938.
Acknowledgements
1
This work was nancially supported by the National Natural
Science Foundation of China (21676120, 31872891), the
Program of Introducing Talents of Discipline to Universities
1
7 S. Baxter, S. Royer, G. Grogan, F. Brown, K. E. Holt-Tiffin,
I. N. Taylor, I. G. Fotheringham and D. J. Campopiano, J.
Am. Chem. Soc., 2012, 134, 19310–19313.
(
(
111-2-06), the High-End Foreign Experts Recruitment Program
G20190010083), the Program for Advanced Talents within Six
1
1
2
2
2
2
2
2
8 R. X. Liu, S. P. Liu, S. Cheng, L. Zhang, Z. Y. Ding, Z. H. Gu
and G. Y. Shi, Appl. Biochem. Microbiol., 2015, 51, 695–703.
9 K. Isobe, H. Tamauchi, K. Fuhshuku, S. Nagasawa and
Y. Asano, Enzyme Res., 2010, 2010, 1–6.
0 X. Chen, Y. F. Cui, X. K. Cheng, J. H. Feng, Q. Q. Wu and
D. M. Zhu, ChemistryOpen, 2017, 6, 534–540.
1 J. H. Schrittwieser, S. Velikogne, M. Hall and W. Kroutil,
Chem. Rev., 2017, 118, 270–348.
2 S. Wu, Y. Zhou, T. Wang, H. P. Too, D. I. Wang and Z. Li, Nat.
Commun., 2016, 7, 11917–11930.
3 W. Song, J. H. Wang, J. Wu, J. Liu, X. L. Chen and L. M. Liu,
Nat. Commun., 2018, 9, 1–9.
4 J. A. Houwman, T. Knaus, M. Costa and F. G. Mutti, Green
Chem., 2019, 21, 1–12.
5 A. Al-Shameri, N. Borlinghaus, L. Weinmann, P. N. Scheller,
B. M. Nestl and L. Lauterbach, Green Chem., 2019, 21, 1396–
Industries of Jiangsu Province (2015-NY-007), the National
Program for Support of Top-notch Young Professionals, Post-
graduate Research & Practice Innovation Program of Jiangsu
Province (KYCX18_1794), the Fundamental Research Funds for
the Central Universities (JUSRP51504), the Project Funded by
the Priority Academic Program Development of Jiangsu Higher
Education Institutions, Top-notch Academic Programs Project
of Jiangsu Higher Education Institutions, the Jiangsu Province
“Collaborative Innovation Center for Advanced Industrial
Fermentation” Industry Development Program, the Program for
the Key Laboratory of Enzymes of Suqian (M201803), and the
National First-Class Discipline Program of Light Industry
Technology and Engineering (LITE2018-09).
1
400.
Notes and references
2
6 Y. P. Xue, Y. G. Zheng, Z. Q. Liu, X. Liu, J. F. Huang and
1
2
3
K. Vedha-Peters, M. Gunawardana, J. D. Rozzell and
S. J. Novick, J. Am. Chem. Soc., 2006, 128, 10923–10929.
F. Paradisi, P. A. Conway, A. R. Maguire and P. C. Engel, Org.
Biomol. Chem., 2008, 6, 3611–3615.
P. Merviel, S. Najas, H. Campy, S. Floret and F. Brasseur,
Minerva Ginecol., 2005, 57, 29–43.
Y. C. Shen, ACS Catal., 2014, 4, 3051–3058.
27 F. Parmeggiani, S. L. Lovelock, N. J. Weise, S. T. Ahmed and
N. J. Turner, Angew. Chem., Int. Ed., 2015, 54, 4608–4611.
28 C. J. W. Walton, F. Parmeggiani, J. E. B. Barber, J. L. McCann,
N. J. Turner and R. A. Chica, ChemCatChem, 2018, 10, 470–
474.
29934 | RSC Adv., 2019, 9, 29927–29935
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
2
3
3
3
3
9 T. K. F. G. Mutti, N. S. Scrutton, M. Breuer and N. J. Turner, 40 Y. Hou, G. S. Hossain, J. H. Li, H. D. Shin, G. C. Du and
Science, 2015, 349, 1525–1529. L. Liu, Appl. Microbiol. Biotechnol., 2016, 100, 2183–2191.
0 S. P. France, L. J. Hepworth, N. J. Turner and S. L. Flitsch, 41 H. Akita, Y. Fujino, K. Doi and T. Ohshima, AMB Express,
ACS Catal., 2017, 7, 710–724. 2011, 1, 1–8.
1 L. Liu, G. S. Hossain, H. D. Shin, J. H. Li, G. C. Du and 42 J. Hayashi, T. Seto, H. Akita, M. Watanabe, T. Hoshino,
J. Chen, J. Biotechnol., 2013, 164, 97–104.
2 Y. Song, J. Li, H. D. Shin, G. Du, L. Liu and J. Chen, Sci. Rep.,
K. Yoneda, T. Ohshima and H. Sakuraba, Appl. Environ.
Microbiol., 2017, 83, 1–13.
2
015, 5, 12614–12625.
43 X. Z. Gao, X. Chen, W. D. Liu, J. H. Feng, Q. Q. Wu, L. Hua
and D. M. Zhu, Appl. Environ. Microbiol., 2012, 78, 8595–
8600.
3 M. B. Quin, K. K. Wallin, G. Zhang and C. Schmidt-Dannert,
Org. Biomol. Chem., 2017, 15, 4260–4271.
3
3
3
4 F. Aalbers and M. Fraaije, ChemBioChem, 2019, 20, 20–28.
5 J. M. Sperl and V. Sieber, ACS Catal., 2018, 8, 2385–2396.
6 J. O. Baek, J. W. Seo, O. Kwon, S. I. Seong, I. H. Kim and 45 Y. Hou, G. S. Hossain, J. Li, H. D. Shin, L. Liu and G. Du,
C. H. Kim, J. Basic Microbiol., 2011, 51, 129–135. Appl. Microbiol. Biotechnol., 2015, 99, 8391–8402.
7 G. Molla, R. Melis and L. Pollegioni, Biotechnol. Adv., 2017, 46 R. L. Hanson, B. L. Davis, S. L. Goldberg, R. M. Johnston,
44 X. Z. Gao, Z. Zhang, Y. N. Zhang, Y. Li, H. Zhu, S. Wang and
C. Li, Appl. Environ. Microbiol., 2017, 83, 476–486.
3
3
3
35, 657–668.
W. L. Parker, T. P. Tully, M. A. Montana and R. N. Patel,
Org. Process Res. Dev., 2008, 12, 1119–1129.
47 R. Bhushan and H. Bruckner, Amino Acids, 2004, 27, 231–
247.
8 E. Rosini, R. Melis, G. Molla, D. Tessaro and L. Pollegioni,
Adv. Synth. Catal., 2017, 359, 3773–3781.
9 G. S. Hossain, H. D. Shin, J. H. Li, G. C. Du, J. Chen and
L. Liu, RSC Adv., 2016, 6, 82676–82684.
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