Enantioselective acylation of β-phenylalanine acid and its derivatives catalyzed by penicillin G acylase from alcaligenes faecalis

Article abstract of DOI:10.1080/10826068.2012.719847

This study developed a simple, efficient method for producing racemic β-phenylalanine acid (BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25°C and pH 10 in an aqueous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U/mL enzyme and 0.1 M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.1%, which, however, increased to 42.9% as the pH value and the molar ratio of phenylacetamide to BPA were elevated to 11 and 3:1, respectively. Under the relatively optimum reaction conditions, the maximum conversion efficiencies of BPA derivatives all reached about 50% in a relatively short reaction time (45-90 min). The enantiomeric excess value of product (eep) and enantiomeric excess value of substrate (ees) were all above 98% and 95%, respectively. These results suggest that the method established in this study is practical, effective, and environmentally benign and may be applied to industrial production of enantiomerically pure BPA and its derivatives.

Full text of DOI:10.1080/10826068.2012.719847

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ENANTIOSELECTIVE ACYLATION OF
β-PHENYLALANINE ACID AND ITS
DERIVATIVES CATALYZED BY PENICILLIN G
ACYLASE FROM Alcaligenes faecalis
Dengchao Li a , Lilian Ji a , Xinfeng Wang a & Dongzhi Wei b
a Jiangsu Key Laboratory for Biomass-Based Energy and Enzyme
Technology, Huaiyin Normal University, Huai'an, Jiangsu Province,
P.R. China
b State Key Laboratory of Bioreactor Engineering, New World
Institute of Biotechnology, East China University of Science and
Technology, Shanghai, P.R. China
Accepted author version posted online: 22 Aug 2012.Published
online: 10 Jan 2013.
To cite this article: Dengchao Li , Lilian Ji , Xinfeng Wang & Dongzhi Wei (2013) ENANTIOSELECTIVE
ACYLATION OF β-PHENYLALANINE ACID AND ITS DERIVATIVES CATALYZED BY PENICILLIN G ACYLASE
FROM Alcaligenes faecalis , Preparative Biochemistry and Biotechnology, 43:2, 207-216, DOI:
10.1080/10826068.2012.719847
To link to this article: http://dx.doi.org/10.1080/10826068.2012.719847
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Preparative Biochemistry & Biotechnology, 43:207–216, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2012.719847
ENANTIOSELECTIVE ACYLATION OF b-PHENYLALANINE ACID
AND ITS DERIVATIVES CATALYZED BY PENICILLIN G ACYLASE
FROM Alcaligenes faecalis
Dengchao Li,1 Lilian Ji,1 Xinfeng Wang,1 and Dongzhi Wei2
1Jiangsu Key Laboratory for Biomass-Based Energy and Enzyme Technology,
Huaiyin Normal University, Huai’an, Jiangsu Province, P.R. China
2State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology,
East China University of Science and Technology, Shanghai, P.R. China
& This study developed a simple, efficient method for producing racemic b-phenylalanine acid
(BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase
from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25ꢀC and pH 10 in an aque-
ous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U=mL enzyme and 0.1
M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.1%, which, however,
increased to 42.9 % as the pH value and the molar ratio of phenylacetamide to BPA were elevated to
11 and 3:1, respectively. Under the relatively optimum reaction conditions, the maximum conver-
sion efficiencies of BPA derivatives all reached about 50 % in a relatively short reaction time
(45–90 min). The enantiomeric excess value of product (eep) and enantiomeric excess value of sub-
strate (ees) were all above 98 % and 95 %, respectively. These results suggest that the method estab-
lished in this study is practical, effective, and environmentally benign and may be applied to
industrial production of enantiomerically pure BPA and its derivatives.
Keywords b-phenylalanine acid, Alcaligenes faecalis, derivatives, enantioselective
acylation, penicillin G acylase
INTRODUCTION
b-Amino acids are increasingly attracting interest due to their biological
activities and importance as key compounds in the synthesis of pharmaceu-
ticals.[1] Optically pure b-phenylalanine (3-amino-3-phenylpropionic acid,
BPA) and its derivatives have become an important group of b-amino acids
Address correspondence to Dengchao Li, Department of Bioengineering, School of Life Sciences,
Huaiyin Normal University, 111 Western Changjiang Road, Huai’an, Jiangsu Province, 223300,
P.R. China. E-mail: dcli@hytc.edu.cn

208
D. Li et al.
in producing antibiotics and chiral building blocks. For instance, (R)-BPA
is a component of astins A–C, the antitumor cyclopentapeptides isolated
from the roots of the medicinal plant Aster tataricus,[2] while (S)-BPA can
be used to synthesize pyloricidins A–D, novel anti-Helicobacter pylori antibio-
tics.[3] Compared to the chemical catalyst, the biocatalyst (enzyme) usually
has higher catalytic efficiency and enantioselectivity, and its optimum reac-
tion conditions are also milder. Therefore, the enzyme-catalyzed synthetic
method has been viewed as a potential approach to the production of opti-
cally pure BPA and derivatives, and a number of enzymes have been used in
this field, including lipase,[4] a-chymotrypsin,[5] penicillin G acylase,[6]
amino-acylase,[7] and b-phenylalanine amino transferases.[8] However, the
application of this method to industrial production of optically pure
BPA and its derivatives is still at a preliminary stage and needs to be
developed.
Penicillin G acylase (PGA) has traditionally served as an industrial cata-
lyst for production of 6-aminopenicillanic acid[9] and the synthesis of
b-lactam antibiotics[10] and peptides,[11] and for the resolution of race-
mates.[12,13] The PGA-catalyzed enantioselective resolution of racemic sub-
stances could be an alternative to the existing biocatalytic approaches
because of its fast reaction rate, high enantioselectivity, and simple isolation
procedures. For example, the acylation of amines catalyzed by PGA from
Alcaligenes faecalis (Af-PGA) in an aqueous solution was shown to be surpris-
ingly efficient and highly enantioselective for (R)-amines.[14] The direct
condensation of phenylglycinonitrile with phenylacetic acid catalyzed by
PGA from Escherichia coli (Ec-PGA) was effective and exclusively enantiose-
lective for (S)-phenylglycinonitrile, which led to nearly stoichiometric
acylation.[13]
In our earlier study on Ec-PGA-catalyzed production of enantiomeri-
cally pure b-phenylalanine in an aqueous medium, an alkaline medium
(pH 10) was found to favor Ec-PGA-catalyzed acylation reaction.[15] How-
ever, Af-PGA, which was less studied than Ec-PGA, has been demonstrated
to bear a broader optimum pH range (8–10) and to maintain nearly 80% of
its maximum catalytic activity at pH 11,[14] with a higher thermostability
(due to the presence of a disulfide), catalytic activity, and ratio of synthesis
to hydrolysis.[16] Recently, some researchers indicated that an Af-PGA-cata-
lyzed enantioselective acylation in an aqueous medium resulted in the pro-
duction of pharmacologically interesting a -D-phenylalanine and its p-
substituted derivatives.[17] However, to our knowledge, Af-PGA-catalyzed
enantioselective acylation of b-amino acids has not been reported. Here,
we present the results of the Af-PGA-catalyzed enantioselective acylation
of BPA and its derivatives (Figure 1), as well as optimized reaction
conditions.

Enantioselective Acylation of b-phenylalanine Acid
209
FIGURE 1 The scheme of Af-PGA-catalyzed enantioselective acylation of BPA and its derivatives.
EXPERIMENTAL
Materials
Potassium penicillin G was purchased from Shijiazhuang Pharmaceu-
tical (Shijiazhuang, Hebei Province, China). p-Dimethylaminobenzalde-
hyde (PDAB) was supplied by SSS Reagent Company (Shanghai,
China). Amberzyme oxirane resin, a polymeric support for enzyme immo-
bilization, was a gift kindly provided by Rohm and Haas Company
(Philadelphia, PA). All other reagents and chemicals used were of analyti-
cal grade.
The recombinant Af-PGA was produced by the E. coli strain DH5a that
was transformed with the plasmid pSMLFPGA carrying the A. faecalis pga
gene, whose expression was controlled by the trc promoter.[18] The cell-free
extract of the cultured bacterial cells was subjected to a series of purifi-
cation processes: fractionating the precipitate with a 40–80% saturation
ammonium sulfate solution and then dialyzing in potassium phosphate
buffer (pH 8.0). The enzyme preparation was loaded on a DEAE Sepharose
column chromatograph (2.5 cm diameter  35 cm length, Amersham
Pharmacia Biotech Co.) and then concentrated with PEG 20,000. The
purity of the isolated free enzyme was above 80% and its activity was about
332 U=mL. To immobilize the free enzyme on Amberzyme matrix, 4000 U
free enzyme was mixed with 10 g (wet weight) matrix in 1 M phosphate buf-
fer (pH 8.0) and incubated at room temperature with agitation at 300 rpm
for 24 hr. The resultant immobilized enzyme activity was 260 U=g (wet
weight).

210
D. Li et al.
Synthesis of Racemic b-Amino Acids 1a–1f and
N-Phenylacetyl-b-amino Acids 5a–5f
Racemic b-amino acids 1a–1f were synthesized as described pre-
viously[19] with some modifications. Benzaldehyde or its derivatives
(0.2 mol), maionic acid (0.2 mol), and ammonium acetate (0.6 mol) were
refluxed in EtOH (500 mL) for 6 hr. The reaction mixture was allowed
to cool to room temperature before the white precipitate was collected
by filtration. The precipitated crude amino acids la–1f were washed with
methanol, dried in vacuum, and then used for phenylacetylation.
N-Phenylacetyl-b-amino acids 5a–5f were synthesized according to the
methods previously described.[6] Phenylacetyl chloride (0.13 mol) in
15 mL acetone was added to the homogeneous solution of racemic b-amino
acid (0.10 mol) and triethylamine (0.24 mol) in 60 mL aqueous acetone
(the ratio of H2O to MeCOMe was 3:1) at À5ꢀC with stirring for 0.5 hr.
The mixture was stirred for 2 hr at À5ꢀC and then 3 hr at room tempera-
ture. After the reaction mixture was filtered and acetone was evaporated,
the residue was washed with diethyl ether (3 Â 25 mL) to remove unreacted
phenylacetyl chloride. The aqueous phase was acidified to pH 2.0 with 2 N
HC1 and then extracted with ethyl acetate (3 Â 50 mL). The combined
organic layers were dried with anhydrous sodium sulfate and then the sol-
ution was concentrated under reduced pressure. The residue was recrystal-
lized from ethyl acetate=hexane to give a crystalline product. The yields and
melting points (mp) of 1a–1f and 5a–5f are presented in Tables 1 and 2,
respectively.
Enzymatic Acylation Reactions
The enzymatic acylation reactions were run in a pH-stat at a constant
temperature with continuous stirring. First, 0.1 M 1a–1f was dissolved in
30 mL of an aqueous solution (20 mM dipotassium hydrogen phosphate);
next, 0.2 or 0.3 M acyl donor (phenylacetamide or methyl phenylacetate)
TABLE 1 Yields and Melting Points (mp) of Synthesized b-Amino Acids 1a–1f
mp (ꢀC)
Product
Molar Yield (%)
Found
Reported
1a
1b
1c
1d
1e
1f
74
68
60
76
58
61
218–221
214–219
213–215
233–235
215–217
230–232
218–219[6]
219[19]
no report
237[6]
216–217[19]
228–229[6]

Enantioselective Acylation of b-phenylalanine Acid
211
TABLE 2 Yields and Melting Points (mp) of Synthesized N-Phenylacetic-b-amino Acids
5a–5f
mp (ꢀC)
Product
Molar Yield (%)
Found
Report
5a
5b
5c
5d
5e
5f
70
62
88
70
61
74
136–138
169–171
170–174
191–192
157–160
153–156
134–140[6]
No report
No report
189–193[6]
159–165[6]
155–158[6]
was added into the solution and incubated for 10 min at a constant pH and
25ꢀC. The reaction was started by adding 0.93 g Af-PGA to the reaction mix-
ture and the pH was kept constant by automatic titration of 2 M KOH sol-
ution. To monitor the reaction, aliquots of 100 mL were taken from the
reaction mixture at intervals of 10 or 15 min, diluted about 10 times with
the mobile phase of high-performance liquid chromatography (HPLC),
and assayed using HPLC.
Enzyme Activity Assay
PGA activity was determined by a spectrophotometric assay with PDAB
as a colorimetric substrate.[20] First, a certain amount of Af-PGA (0.10 g
immobilized enzyme or 0.10 mL of diluted free enzyme) was mixed with
5 mL of a penicillin potassium salt solution (prepared by adding 0.4 g peni-
cillin to 10 mL of 0.1 M phosphate buffer, pH 7.8) and incubated at 37ꢀC
for 5 min, and then the reaction was terminated by adding 5 mL of 95%
ethanol to the reaction mixture. Second, the resultant solution was centri-
fuged at 12,000 rpm for 5 min, and then 1 mL of the resultant supernatant
was diluted with distilled water about 10-fold. Third, after 0.5 mL of the
diluted supernatant was mixed with 3.5 mL PDAB, the mixture was settled
at room temperature for 5 min, and its absorbance was then measured at
415 nm. One unit of PGA was defined as the amount of enzyme required
to produce 1 mmol 6-APA per min from 4% (w=v) penicillin G in 0.1 M
phosphate buffer (pH 7.8) at 37ꢀC.
Assay Methods
Samples were analyzed using HPLC with a Zorbax XDB C-18 column
(250 Â 4.6 mm, 5 mm); the mobile phase was 7 mM phosphate (pH 3.0),
containing acetonitrile 35% (v=v) and 2.36 mM sodium dodecyl sulfate
(SDS); the flow rate was set at 1 mL=min and absorbance was measured

212
D. Li et al.
at 210 nm. The enantiomeric excess value (ee) of the 2a–2f was determined
using HPLC with a Chirobiotic T column (250 Â 4.6 mm, 5 mm), the mobile
phase was the mixture of methanol and 20 mM acetic acid buffer (pH 5.5) in
a ratio of 1:4 (v:v), the flow rate was set at 0.5 mL=min, and absorbance was
measured at 210 nm. The ee of 3a–3f was determined using HPLC with a Chir-
obiotic R column (150 Â 4.6 mm, 5 mm); the mobile phase was the mixture of
methanol, acetic acid, and triethylamine in a ratio of 100:0.4:0.1 (v:v:v); the
flow rate was set at 0.4 mL=min; and absorbance was measured at 254 nm.
RESULTS AND DISCUSSION
We previously had demonstrated the Ec-PGA-catalyzed production of
enantiomerically pure (S)-BPA and (R)-BPA in an aqueous medium under
optimum reaction conditions: at 25ꢀC and pH 10 with 0.15 M BPA, 0.3 M
phenylacetamide, and 8 U=mL enzyme.[15] Under these conditions,
Af-PGA-catalyzed acylation of BPA at 40 min only achieved the maximum
BPA conversion efficiency of 36.1% (50% conversion efficiency is the
maximum in the stereoisomer reaction and the following reactions)
(Figure 2A). Therefore, we investigated the effects of pH, acyl donor,
and molar ratio of phenylacetamide to BPA on this acylation reaction.
As shown in Figure 2, the maximum BPA conversion efficiency at
40 min increased to 38.5% when methyl phenylacetate was used as an acyl
donor (Figure 2B), which indicated that methyl phenylacetate was a better
acyl donor than phenylacteamide for this type of reaction. As the molar
ratio of phenylacetamide to BPA was elevated from 2:1 to 3:1, the conver-
sion efficiency increased to 47.8%, but the reaction time at the maximum
conversion was prolonged to 130 min because of the low solubility of phe-
nylacetamide in the aqueous medium (Figure 2A). Compared to Ec-PGA,
Af-PGA is more stable and active in alkaline conditions, so the acylation
reaction was then run under the condition of pH 11; consequently, the con-
version efficiency increased to 42.9% and 46.5% at 60 min and 90 min,
respectively (Figures 2C and 2D). The result also showed that the
maximum BPA conversion efficiency rose as the pH value was elevated from
10 to 11, which indicated that the alkaline condition favored this acylation
reaction. However, it needed a longer time to accomplish the maximum
conversion efficiency, which might be due to the fact that the enzyme
activity and stability at pH 11 were lower than that at pH 10.
In the kinetic acylation reaction, an acyl donor binds to the immobilized
enzyme to form an acyl–enzyme complex, which can either be hydrolyzed to
produce the enzyme and phenylacetic acid, or react with a nucleophilic
amino acid to form 3a–3f. Furthermore, the hydrolysis of the resultant
2a–2f can also occur simultaneously. Therefore, it is necessary to reduce

Enantioselective Acylation of b-phenylalanine Acid
213
FIGURE 2 Effects of molar ratio of acyl donor to BPA, acyl donor, and pH on BPA conversion efficiency.
(A) The effect of the molar ratio of phenylacetamide to BPA on BPA conversion. (B) The effect of acyl
donor on BPA conversion. (C) The effect of pH on BPA conversion using phenylacetamide as acyl
donor. (D) The effect of pH on BPA conversion using methyl phenylacetate as acyl donor. Reaction con-
ditions: (A) 0.1 M BPA, 0.1–0.3 M phenylacetamide and 8 U=mL enzyme at 25ꢀC and pH 10. (B) 0.1 M
BPA, 0.2 M acyl donor and 8 U=mL enzyme at 25ꢀC and pH 10. (C) 0.1 M BPA, 0.2 M phenylacetamide
and 8 U=mL enzyme at 25ꢀC, and pH 10 and 11. (D) 0.1 M BPA, 0.3 M methyl phenylacetate and
8 U=mL enzyme at 25ꢀC, and pH 10 and 11 (color figure available online).
TABLE 3 Af-PGA-Catalyzed Acylation of BPA and Its Derivatives
Entry [1a–1f] Conversiona (%) [1a–1f] Timeb (min) (s=h)0
[2a–2f] eep (%) [3a–3f] eesd (%)
c
a
b
c
d
e
f
46.5
48.6
49.8
49.8
49.9
49.4
90
60
90
45
75
75
0.7
>99
>99
98.4
98.0
>99
>99
96.5
98.3
97.6
95.2
98.5
98.6
0.99
1.71
1.36
1.04
2.88
aConversion—the maximum conversion efficiency of 1a–1f.
bTime—the reaction time for achieving the maximum conversion efficiencies of 1a–1f.
cDetermined as a ratio of initial rates for accumulation of both reaction products.
dDetermined by chiral HPLC.

214
D. Li et al.
ꢀ
~
&
FIGURE 3 The stability of Af-PGA at 25 C, and pH 10 and 11. ( ) pH 10; ( ) pH 11.
the occurrence of hydrolytic side reaction or to improve the ratio of syn-
thesis to hydrolysis (s=h). The preceding scheme was also used to examine
the acylation of several BPA derivatives, and the results are shown in
Table 3. Af-PGA exhibited an excellent enantioselectivity for R-isomer from
the corresponding racemates (eep > 98%), leaving the remaining S-BPA deri-
vatives in moderate to high ees (>95%). The maximum conversion efficien-
cies of 1a–1f all reached about 50% (it means 100% conversion efficiency of
the desired stereoisomer) in a relatively short time (within 45–90 min). Both
the conversion efficiency and the ees of 1b–1f slightly increased in compari-
son with those of 1a. It might be mainly due to that the benzene ring of BPA
was substituted by electron-withdrawing groups substituted, which caused
the hydrolytic rate of 2b–2f to be slower than that of 2a, leading to Af-PGA-
catalyzed BPA derivatives with a higher s=h than BPA.
The optimal pH for the acylation of BPA and its derivatives was 11,
whereas the Af-PGA has an optimum pH range between 8 and 10. The stab-
ility of Af-PGA at pH 10 and pH 11 is indicated in Figure 3. The half lives of
Af-PGA at pH 10 and 11 were about 24 hr and 5 hr, respectively.
CONCLUSIONS
In this study, we developed a method of producing enantiomerically
pure BPA and its derivatives by Af-PGA-catalyzed acylation of racemates.

Enantioselective Acylation of b-phenylalanine Acid
215
Under relatively optimum reaction conditions, the maximum conversion
efficiencies of racemates reached about 50% in a relatively short time
(45–90 min), and the products also had high enantiomeric purity.
Considering the short half-life (5 hr) of Af-PGA at pH 11 and its relatively
high thermostability, in the future we may attempt to improve its efficiency
by appropriately increasing reaction temperature, and to enhance its stab-
ility using gene engineering techniques and enzyme immobilization meth-
ods. All in all, due to its effectiveness and environmentally friendly nature,
this method appears to be a promising approach to the green synthesis of
BPA and its derivatives.
ACKNOWLEDGMENTS
This study was supported by the Huai’an City Industrial Fund for
Science and Technology Support Program (HAG2010020) and the Foun-
dation of Key Disciplines of Shanghai.
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