Enzymatic synthesis of chiral phenylalanine derivatives by a dynamic kinetic resolution of corresponding amide and nitrile substrates with a multi-enzyme system

Article abstract of DOI:10.1002/adsc.201100923

Mutant α-amino-ε-caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D-amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co-expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)-phenylalanine and non-natural (R)-phenylalanine derivatives (4-OH, 4-F, 3-F, and 2-F-Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)-phenylalanine with 84% yield and 99% ee from (RS)-phenylalaninamide (400 mM) in 22 h. (R)-Tyrosine and 4-fluoro-(R)-phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co-expresed two genes encoding mutant ACL racemase and L-amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)-phenylalanine derivatives. Moreover, 2-aminophenylpropionitrile was converted to (R)-phenylalanine by DKR using a combination of the non-stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes. Copyright

Full text of DOI:10.1002/adsc.201100923

UPDATE
DOI: 10.1002/adsc.201100923
Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a
Dynamic Kinetic Resolution of Corresponding Amide and Nitrile
Substrates with a Multi-Enzyme System
Kazuyuki Yasukawaa,b and Yasuhisa Asanoa,b,*
a
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa,
Imizu, Toyama 939-0398, Japan
Fax : (+81)-766-56-2498; phone: (+81)-766-56-7500
address:; e-mail: asano@pu-toyama.ac.jp
Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
b
Received: November 28, 2011; Revised: August 3, 2012; Published online: November 8, 2012
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201100923.
Abstract: Mutant a-amino-e-caprolactam (ACL)
racemase (L19V/L78T) from Achromobacter obae
with improved substrate specificity toward phenyl-
Introduction
Natural and unnatural chiral a-amino acids are exten-
sively used for pharmaceutical agents, animal feeds,
and artificial sweeteners. Fermentation methods have
been successful in the production of (S)-amino acids
such as lysine, phenylalanine, tryptophan, threonine,
arginine, histidine, isoleucine, serine and valine on the
industrial scale.[1] On the other hand, a few proteino-
genic (S)-amino acids and non-natural amino acids
are produced by enzymatic methods. Enzymatic syn-
thesis of chiral a-amino acid involves the following
methodologies. (i) Asymmetric reductive amination of
a-keto acids, for example, the synthesis of (S)-amino
acids from a-keto acids with NAD(P)-dependent l-
amino acid dehydrogenase with cofactor regenera-
tion,[2,3] and the synthesis of (R)-amino acids with d-
amino acid dehydrogenase created by directed evolu-
tion from meso-2,6-d-diaminopimelic acid dehydro-
genase.[4] (ii) Asymmetric addition of ammonia to a,
b-unsaturated carboxylic acids; (R)-amino acids can
be synthesized using d-amino acid aminotransferase
from other amino acids and a-keto acids, although
this gives a mixture of two amino acids and two keto
acids. Ammonia lyase and aminomutase were mainly
used to produce (S)-a-amino acids and (R)-b-amino
acids, respectively.[5] Especially, aspartase and phenyl-
alanine ammonia lyase catalyzed the addition of am-
monia to fumaric acid and cinnamic acid, and directly
converted the substrates to (S)-asparate and (S)-phe-
nylalanine, respectively, which were required in large
quantities for aspartame production. Methylaspartase
catalyzed the synthesis of methylaspartates as report-
ed by us.[6] (iii) Kinetic resolution (KR) by hydrolysis
of racemic amino acid derivatives using stereoselec-
tive lipase, acylase, amidase, hydantoinase and carba-
moylase has been traditionally carried out. However,
ACHTUNGTRENNUNGalaninamide was obtained by directed evolution.
The mutant ACL racemase and thermostable
mutant d-amino acid amidase (DaaA) from Ochro-
bactrum anthropi SV3 co-expressed in Escherichia
coli (pACLmut/pDBFB40) were utilized for synthe-
sis of (R)-phenylalanine and non-natural (R)-phe-
nylalanine derivatives (4-OH, 4-F, 3-F, and 2-F-Phe)
by dynamic kinetic resolution (DKR). Recombinant
E. coli with DaaA and mutant ACL racemase
genes catalyzed the synthesis of (R)-phenylalanine
with 84% yield and 99% ee from (RS)-
phenylalaninACHTUNGTRENNUNGamide (400 mM) in 22 h. (R)-Tyrosine
and 4-fluoro-(R)-phenylalanine were also efficiently
synthesized from the corresponding amide com-
pounds. We also co-expresed two genes encoding
mutant ACL racemase and l-amino acid amidase
from Brevundimonas diminuta in E. coli and per-
formed the efficient production of various (S)-phe-
nylalanine derivatives. Moreover, 2-aminophenyl-
propionitrile was converted to (R)-phenylalanine by
DKR using a combination of the non-stereoselec-
tive nitrile hydratase from recombinamt E. coli and
mutant ACL racemase and DaaA from E. coli en-
coding mutant ACL racemase and DaaA genes.
Keywords: amidases; amino acids; a-amino-e-cap-
rolactam racemase; biotransformation; directed
evolution; dynamic kinetic resolution
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Kazuyuki Yasukawa and Yasuhisa Asano
UPDATE
the theoretical yield of product is limited to 50% in
the KR.[7] The KR of a-aminonitriles using nitri-
lase[8–12] and dynamic kinetic resolution (DKR) are
the examples of the third category.[13] DKR would be
one of the most powerful and elegant methods for ef-
ficient synthesis of one enantiomer from racemic
starting materials. Therefore, DKR has attracted in-
creasing interest from both the industrial and the aca-
demic sides. For instance, optically pure a-amino
acids were produced from a-amino-e-caprolactam
(ACL), N-acyl-a-amino acid, and 5-monosubstitued
hydantoin compounds by racemization and hydrolysis
by racemase and stereoselective hydrolase(s) in
a one-pot reaction.[13] We focused on a-amino acid
amides as starting compounds for chiral a-amino acid
Scheme 1. Dynamic kinetic resolution of (RS)-phenylalanin-
AHCTUNGTRENGNaUN mide to form (R)-phenylalanine.
synthesis by DKR, because the substrate can be easily Results and Discussion
obtained via hydrolysis of an a-amino nitrile which is
prepared by the Strecker synthesis. We have been Co-expression of the Mutant ACL Racemase and d-
studying many (R)- or (S)-stereoselective amino acid Amino Acid Amidase Genes in E. coli Cells
amide hydrolases since the information had been lim-
ited on enzymes acting on a-amino acid amides. We The E. coli transformant co-expressing mutant ACL
have utilized (R)-stereoselective amino acid amidases racemase and DaaA was constructed (see Supporting
such as d-aminopeptidase, d-amino acid amidase, al- Information, Table S1) and used as biocatalyst for
kaline d-peptidase, and R-amidase in the KR of DKR of a-amino acid amides to (R)-a-amino acids.
amino acid amides.[7] (S)-Stereoselective amino acid Both enzymes were efficiently expressed such that
amide hydrolases such as l-amino acid amidase from enzyme activity of mutant ACL racemase and DaaA
Pseudomonas azotoformans[14] and Brevundimonas di- from this recombinant E. coli were 0.210 and
minuta[15] have been characterized by our group. Soda 25.9 UmLÀ1 culture, in the optimum cultivation condi-
et al. reported that ACL racemase acts only on neu- tions (378C for 24 h without IPTG), respectively. The
tral cyclic amides such as ACL, a-amino-d-valerolac- amount of both enzymes in recombinant E. coli were
tam, and a-amino-b-thio-e-caprolactam[16,17], although estimated to be 5% of the total soluble protein, re-
Asano et al. newly discovered that the enzyme can spectively, however, theses enzyme activities in 1 mL
catalyze the racemization of a-amino acid amides.[17,18] culture were not balanced because the specific activity
Moreover, we succeeded in the production of chiral of DaaA (approximately 1,100 UmgÀ1) was about 100
a-amino acids such as (R) and (S) isomers of alanine, times higher than that of the mutant ACL racemase
a-aminobutyric acid, methionine and (R)-serine by (approximately 10 UmgÀ1).
DKR of the corresponding a-amino acid amide using
Cell-free extracts of E. coli co-expressing the
(R)- or (S)-stereoselective amidase and ACL race- mutant ACL racemase and DaaA catalyzed the trans-
mase.[18–20] However, ACL racemase has a narrow sub- formation of (S)-phenylalaninamide HCl (100 mM) as
strate specificity, preferentially acting on small a- a substrate with higher yield and ee % compared with
amino acid amides such as alaninamide, a-aminobu- E. coli co-expressing natural ACL racemase and
tyramide and leucinamide, but hardly racemizes a- DaaA (Figure 1). The latter one caused only a slight
amino acid amides with bulky side chains such as phe- increase of (R)-phenylalanine and achieved accumula-
nylalaninamide.
tion of only 7.3 mM for 24 h, while natural l-amino
Recently, directed evolution has become one of the acid amidase from E. coli had a great influence on an
methods for improving biocatalysts without a detailed unfavorable production of 13.0 mM (S)-phenylala-
knowledge of enzyme structure and catalytic mecha- nine. E. coli co-expressing mutant ACL racemase and
nism.[21] In our earlier study, we obtained a mutant DaaA quickly converted (S)-phenylalaninamide
ACL racemase (L19V/L78T) with high phenylalanin- (100 mM) into (R)-phenylalanine (99.4 mM) with
ACHTUNGTRENNUNG
published results).
E. coli hardly affected the enantiomeric purity.
In this report, we have developed an efficient syn-
thetic method for (R)-phenylalanine and (R)-phenyl-
ACHTUNGTRENNUNGalanine derivatives by DKR using mutant ACL race-
mase and (R)-stereoselective amino acid amidase
(Scheme 1).
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Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution
Figure 1. Time course for the production of (R)-phenylala-
nine from (S)-phenylalaninamide using E. coli pACL60/
pDBFB40 (A) or E. coli pACLmut/pDBFB40 (B). Symbols:
*
*
(R)-phenylalanine ( ), (S)-phenylalanine ( ), (R)-phenyla-
&
laninamide ( ), (S)-phenylalaninamide (&).
Figure 2. Time course for the enzymatic synthesis of (R)-
phenylalanine using E. coli pACLmut/pDBFB40. Symbols:
&
*
*
(R)-acid ( ), (S)-acid ( ), (R)-amide ( ), (S)-amide (&).
Enzymatic Conversion of Several (R)-Amino Acids
from the Corresponding (RS)-Amino Acid Amides
because the mutant ACL racemase showed difficulty
The yields and enantiomeric excesses were measured in racemizing these halogen-containing substrates. To
for (R)-amino acids from several substrates (Table 1, inhibit natural l-amidase from E. coli, cell-free ex-
Figure 2). (Æ)-1e, (Æ)-1f, (Æ)-1g, (Æ)-1h, and (Æ)-1i tract was treated at 508C for 10 min and used for the
were efficiently converted to the corresponding (R)- DKR of (Æ)-1c, (Æ)-1j, and (Æ)-1k. The yield and op-
a-amino acids. (Æ)-1l (50 mM) was quickly converted tical purity of (R)-2j were dramatically increased up
to (R)-2l (47.9 mM) in 4 h at 408C but (Æ)-1l to 99% and 95% ee, whereas the optical purities of
(100 mM) inhibited the activity of E. coli co-express- (R)-2c and (R)-2k were rather decreased. On the
ing mutant ACL racemase and DaaA so that (R)-2l other hand, the substrate specificity of d-amino acid
(87.8 mM) was produced after 12 h and with an opti- amidase purified with a His-tag column from E. coli
cal purity of 77% ee. (R)-2j and (R)-2k were hardly was examined. The reaction mixture contained
produced with high yield and optical purity by DKR 100 mM Tris-HCl buffer, pH 8.0, 40 mM racemic sub-
strate, and an appropriate amount of the purified
enzyme. The specific activities towards (Æ)-1b, (Æ)-1c,
Table 1. Conversion of (RS)-a-amino acid amides to (R)-a-
(Æ)-1g, (Æ)-1h, (Æ)-1i, (Æ)-1j, and (Æ)-1k were 2.30,
amino acids with E. coli pACLmut/pDBFB40.[c]
0.47, 4.58, 6.26, 3.69, 21.1, and 24.0% of the activity
towards phenylalaninamide (1520 UmgÀ1), respective-
ly. While an analog with an electron-donating sub-
stituent such as (Æ)-1d was not a substrate for the re-
combinant enzyme and it was accepted by the natural
amidase from E. coli with a higher formation of (S)-
2d than (R)-2d. DaaA activity was not only influ-
enced by the size of the side chain of the substrate,
but also by the position of the substituent of the
phenyl ring, while the strict (R)-stereoselectivity was
always maintained by these substitutions.
Enzymatic Synthesis of (R)-2e, (R)-2f and (R)-2g
using E. coli Co-expressing Mutant ACL Racemase
and DaaA (pACLmut/pDBFB40)
[a]
The yields were determined by chiral HPLC.
The enantiomeric excesses were determined by chiral
(R)-2e, (R)-2f and (R)-2g were synthesized by the E.
[b]
coli transformant co-expressing mutant ACL race-
mase and DaaA in the optimum conditions (100 mM
Tris-HCl buffer, pH 8.0, 408C) (see Supporting Infor-
HPLC.
[c]
The reaction (volume: 1 mL) was carried out at 408C by
using cell-free extract (5 mL culture cell) of E. coli
pACLmut/pDBFB40 containing 100 mM Tris-HCl buffer,
pH 8.0, 2 mM PLP, and substrate. Reactions were not op-
timized except for (Æ)-5.
mation, Figure S1, A and B). (Æ)-1e was added to
the reaction mixture intermittently in portions of 0.2 g
for four times, because mutant ACL racemase was in-
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Kazuyuki Yasukawa and Yasuhisa Asano
UPDATE
sion of mutant ACL racemase and l-amino acid ami-
dase by E. coli pACLmut/pLaaABd (0.95 UmgÀ1 for
mutant ACL racemase activity and 8.41 UmgÀ1 for
LaaABd activity) were at the same level of the homo-
geneous expression of ACL racemase in E. coli
pACLmut (1.0 UmgÀ1), but lower than the LaaABd in
E. coli pBD3 (25.9 UmgÀ1)[15], respectively. Optically
pure (S)-2e with 61% isolation yield was synthesized
in 24 h from (Æ)-1e (400 mM) using cell-free extract
of 25 mL culture (see Supporting Information, Fig-
ure S13). LaaABd acted on broad range of (S)-a-
amino acid amides as substrates,[15] preferentially on
aromatic substrates. On the other hand, the enzyme
showed a small amount of (R)-phenylalaninamide hy-
drolyzing activity forming (R)-phenylalanine. Howev-
er, (R)-phenylalanine was synthesized only at
10.5 mM and easily removed by recrystallization. This
result indicated that E. coli pACLmut/pLaaABd effi-
ciently produced optically pure (S)-2e from (Æ)-1e.
The substrate specificity of the biocatalyst, E. coli
pACLmut/pLaaABd, is summarized in Table S2 in
the Supporting Information. (Æ)-1f–1i were rapidly
and efficiently transformed into (S)-2f–2i with an op-
Figure 3. Time course for the enzymatic synthesis of (R)-
phenylalanine from (RS)-2-aminophenylpropionitrile by
DKR. Reaction mixture contained 100 mM Tris-HCl buffer,
pH 8.0, 2 mM PLP, 50 mmol (RS)-2-aminophenylpropioni-
trile, cell-free extract of recombinant E. coli containing non-
stereoselective NHase gene (34 U) and E. coli pACLmut/
pDBFB40 (1.1 U of mutant ACL racemase and 130 U of
DaaA) in a total volume of 1 mL, and the reaction was per-
formed at 308C. After 30 min, 50 mmol (RS)-2-aminophenyl-
propionitrile were added to the reaction mixture again.
~
~
*
Symbols: (R)-nitrile ( ), (S)-nitrile ( ), (R)-acid ( ), (S)-
&
*
acid ( ), (R)-amide ( ), (S)-amide (&).
dicated to be subject of substrate inhibition at more tical purity of 90–98% ee and 99% yield. The non-nat-
than 100 mM substrate concentration (see Supporting ural amino acid known as (S)-2l is an important chiral
Information). Finally, the concentration of (R)-2e intermediate to synthesize a variety of pharmaceuti-
reached 395 mM in the reaction mixture with an opti- cals including angiotensin-converting enzyme inhibi-
cal purity of 99% ee (Figure 3). (R)-2f and (R)-2i tors, b-lactam antibiotics, acetylcholine esterase inhib-
showed very low solubility in the reaction mixture at itors and neutral endopeptidase inhibitors. (S)-2l was
neutral pH, so that the product was quickly precipitat- also continuously precipitated in the reaction mixture
ed thus efficiently shifting the reaction equilibrium and efficiently synthesized with >99% yield and 98%
toward the product formation. After 24 h, 356 mM of ee in 12 h.
(R)-2f with 96% optical purity were produced. As
a result, (R)-2f (isolation yield 74% and 99% ee) was
synthesized from 0.4 g of (Æ)-1f (369 mM) (see Sup- Synthesis of (R)-2e from (RS)-2-Aminophenyl-
porting Information, Figure S5). Moreover, non-natu- propionitrile using Nitrile Hydratase and E. coli
ral a-amino acid (R)-2i, is a raw material for peptido- pACLmut/pDBFB40
mimetic research,[24] and can also be synthesized from
0.44 g (Æ)-1i (400 mM) in a similar procedure (isola- In the previous report, we have successfully synthe-
tion yield 73% and>99% ee) (see Supporting Infor- sized aliphatic (R)- and (S)-a-amino acids such as a-
mation, Figure S9).
aminobutyric acid from (RS)-a-aminobutyronitrile by
DKR using purified non-stereoselective nitrile hydra-
tase (NHase), (R)- or (S)-stereoselective amino acid
amidase and ACL racemase.[25] When (RS)-2-amino-
phenylpropionitrile (10 mM) was used as aromatic
substrate, (S)-phenylalanine was produced in 12 h
(yield: 99%, 97% ee), although it was difficult to pro-
Enzymatic Synthesis of (S)-2e using E. coli
Transformant Co-expressing Mutant ACL Racemase
and Recombinant l-Amino Acid Amidase
In this study, we synthesized a high concentration of duce (R)-phenylalanine. In this study, sequential con-
(S)-2e from (Æ)-1e by DKR using recombinant E. version of (RS)-2-aminophenylpropionitrile to (R)-
coli co-expressing mutant ACL racemase and re- phenylalanine was performed. Since NHase and ACL
combinant l-amino acid amidase in optimum condi- racemase were inhibited by the a-amino nitrile, the
tions (100 mM KPB, pH 7.0, 408C).
substrate needed to be quickly and completely con-
E. coli JM109 harboring pACLmut and pLaaABd verted to the a-amino acid amide by the NHase. Fi-
(see Supporting Information, Table S1) was cultivated nally, we successfully carried out a fed-batch reaction.
in LB medium supplemented with ampicilin In the initial 5 min, (RS)-2-aminophenylpropionitrile
(80 mgmLÀ1), and chloramphenicol (30 mgmLÀ1) at (50 mmol) was quickly converted to racemic phenyl-
378C for 24 h under optimum conditions. The expres-
ACHTUNGTRENaNGNU laninamide, and then (R)-phenylalaninamide was si-
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Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution
tonics, Bremen, Germany). Data evaluation was performed
multaneously hydrolyzed to (R)-phenylalanine by
DaaA. After 30 min, further 50 mmol of the substrate
were added to the reaction mixture and quickly con-
verted to (R)-phenylalaninamide and ultimately to
(R)-phenylalanine. Remaining (S)-phenylalaninamide
was racemized by the mutant ACL racemase, and
after 4 h, (RS)-2-aminophenylpropionitrile (100 mM)
was completely converted to (R)-phenylalanine with
95% yield and 90% ee (Figure 3). This one-pot syn-
thesis also represents a new synthetic approach to
phenylalanine derivatives, which serve as interesting
intermediate compounds for the synthesis of chiral
compounds.
using the Generate Molecular Formula software suite within
Bruker Daltonics micrOTOF DataAnalysis version 3.4.
Concentration and enantiopurity of all a-amino acid amides
and a-amino acids were determined by HPLC equipped
with a Crownpak CR (+) column (Daicel Chemical Indus-
tries, Ltd., Osaka, Japan) using a solvent system of 60 mM
HClO4/10% methanol.
Construction of pACLmut and pDBFB40
Plasmid pET15ACL[22] containing the gene coding for the
ACL racemase was used as a template in mutagenesis. We
targeted the mutagenesis to residues that interact with 4
ꢁngstrom in the ligand, e-caprolactam of active site, based
on the crystal structure.[22] These residues have been identi-
fied as L19, W49, L78, M293, T295 and W436. A mutation
library was constructed to contain possible amino acid
changes at these residues. This library was screened against
d-phenylalaninamide as substrate. After screening, L78T,
L78S, and L78V single mutants were having a higher activity
than the native enzyme. These mutants were used as the
parent for the second round of mutagenesis and screening.
As in the second screening, the mutagenesis was performed
on residues within the 6 ꢁngstrom in the ligand. Mutant
ACL racemase (L19V/L78T) having both L19V and L78T
mutations was found to have the higher activity than the
parent. The details of gene mutation and the enzymatic
properties of the mutant ACL racemase will be described
elsewhere. Mutant ACL racemase gene was inserted down-
stream of the lac promoter in high-copy-number plasmid
pUC18, yielding pACLmut.
Conclusions
The mutant ACL racemase used in this study showed
a higher catalytic efficiency toward phenylalanin-
ACHTUNGTRENNUNGamide as compared with the natural one. The details
of the design of the mutant and its properties will be
reported elsewhere.
In this study, we have successfully synthesized natu-
ral and non-natural (R)-phenylalanine derivatives of
commercial interest with excellent enantiomeric puri-
ties in high yields from the corresponding (RS)-a-
amino acid amides by DKR using cell-free extract of
E. coli co-expressing both d-amino acid amidase and
mutant ACL racemase. (S)-Phenylalanine was also
synthesized from (RS)-phenylalaninamide by cell-free
extract of E. coli co-expressing the two enzymes l-
amino acid amidase and mutant ACL racemase. Opti-
cally pure (R)-phenylalanine was obtained from (RS)-
2-aminophenylpropionitrile via (RS)-phenylalanin-
d-amino acid amidase gene from Ochrobactrum anthropi
SV3 was amplified with oligonucleotides primer 5’-GAAA
TAAGCTTTAaggaggAATAGCCGATGAGTG-3’
and
5’-
TTGAAGGTACCCTAACTGCGGGAG-3’ (restriction site, Shine-
Dalgarno sequence, and initiation codon are shown in italics,
lowercase and bold letters, respectively) from plasmid
pDAA-BFB40[22], digested with HindIII and KpnI and in-
serted into low-copy-number plasmid pSTV29 vector to give
pDBFB40. Plasmid pACLmut and pDBFB40 were intro-
duced into E. coli JM109, and the resulting transformant (E.
coli pACLmut/pDBFB40) was cultivated in LB medium
supplemented with ampicilin (80 mgmLÀ1), and chloramphe-
nicol (30 mgmLÀ1).
ACHTUNGTRENNUNGamide in one step by a combination of the cell-free
extracts from recombinant E. coli encoding NHase as
well as E. coli co-expressing d-amino acid amidase
and mutant ACL racemase. This new DKR method
could be very useful in the production of optically
pure phenylalanine derivatives.
Preparation of Enzyme from Recombinant E. coli
Experimental Section
Recombinant E. coli transformants harboring pACLmut and
pDBFB40 was grown in LB medium containing ampicilin
and chloramphenicol at 378C for 24 h. Cells were harvested
by centrifugation at 9,500ꢂg for 5 min at 48C, and washed
with 0.85% NaCl. The washed cells from 2 L culture were
resuspended in 20 mMK2HPO4/KH2PO4buffer (KPB) and
disrupted by sonication for 20 min (19 kHz, Insonator model
201m; Kubota, Tokyo, Japan). Cell-free extract was pre-
pared by centrifugation of the lysate at 15,000 x g for 20 min
at 48C.
Chemicals
Restriction enzymes, DNA modifying enzymes, pUC18, and
pSTV29 vectors were from Takara Shuzo Co., Ltd. (Kyoto,
Japan). All other chemicals were from commercial sources.
Analytical Methods
Optical rotations were measured on a SEPA-300 (Horiba,
Ltd., Kyoto, Japan). The H NMR spectra were recorded on
1
a Bruker Biospin AVANCE II 400 (Bruker Biospin, Rhein-
stetten, Germany) system. The reaction products were char-
acterized by MS using a Bruker-Daltonics micrOTOF instru-
ment with an ESI source in the positive mode (Bruker-Dal-
Definition of ACL Racemase Activity
Activity of ACL racemase was measured by detecting the
formation of (R)-phenylalanine via (R)-phenylalaninamide
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Kazuyuki Yasukawa and Yasuhisa Asano
UPDATE
from (S)-phenylalaninamide at 308C. The standard assay so- References
lution contained 100 mM KPB (pH 7.0), 2 mM PLP, and
20 mM (S)-phenylalaninamide. One unit of enzyme activity
was defined as the amount of enzyme catalyzing the conver-
sion of substrate to the product at a rate of 1 mmol/min.
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Definition of d-Amino Acid Amide Amidase Activity
d-Amino acid amide amidase (DaaA) activity was measured
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mixture contained 100 mM Tris-HCl buffer (pH 8.0), 20 mM
(R)-phenylalaninamide and appropriate amount of the
enzyme. One unit of enzyme activity was defined as the
amount of enzyme catalyzing the conversion of substrate to
the product at a rate of 1 mmol/min.
Enzymatic Synthesis of (R)-Phenylalanine from (RS)-
Phenylalaninamide
The reaction mixture contained 1 mmol Tris-HCl buffer,
pH 8.0 (100 mM), 20 nmol PLP (2 mM), the cell-free extract
of E. coli pACLmut/pDBFB40 (11 U of mutant ACL race-
mase and 1300 U of DaaA) from 50 mL culture, and 0.8 g
(RS)-phenylalaninamide HCl (400 mM) in a total volume of
10 mL. After the reaction mixture had been incubated
under stirring at 408C for 22 h, it was adjusted to pH 1.0
using concentrated HCl and then, the solution was filtered
and neutralized with 6N NaOH. The reaction mixture was
evaporated under vacuum and recrystallized from water-
methanol. Finally, (R)-phenylalanine was obtained with 99%
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1
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3.0 (dd, 1H, J=14.5, 8.0 Hz); HR-MS m/z=166.0884, calcd
for C9H12N1O2 [M+H]+ 166.0863. HPLC elution profile,
1H NMR, and mass spectra of the isolated (R)-phenylala-
nine are described in the Supporting Information.
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Supporting Information
1
Experimental details of HPLC elution profile, H NMR, and
mass spectra of isolated products are described in the Sup-
porting Information file.
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Acknowledgements
We thank Prof. ZTakashi Yamane (Nagoya University) for
performing the X-ray structure analysis of ACL racemase.
This work was supported in part by Grants-in-Aid for scien-
tific research A (23248015) and B (20380053) from Japan So-
ciety for the Promotion of Science to Yasuhisa Asano.
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Adv. Synth. Catal. 2012, 354, 3327 – 3332