Bioproduction of l-2-Aminobutyric Acid by a Newly-Isolated Strain of Aspergillus tamarii ZJUT ZQ013

Article abstract of DOI:10.1007/s10562-017-1999-3

Abstract: l-2-Aminobutyric acid (l-ABA), an unnatural amino acid, is a key intermediate of several important drugs. Although some methods have been developed to prepare pure chiral l-ABA, there are still many drawbacks, including low catalytic efficiency, cumbersome steps and high cost due to the addition of some expensive catalysts or coenzymes. Herein, with chemical and biological approaches together, we discovered a newly isolated Aspergillus tamarii ZJUT ZQ013 strain containing a microbial lipase which could be employed to resolve racemic methyl N-Boc-2-aminobutyrate to produce l-ABA with high enantioselectivity (e.e._s?>?99.9%, E = 257). Moreover, the subsequent gram scale experiment confrimed that A. tamarii ZJUT ZQ013 could be an attractive biocatalyst for the efficient preparation of optically pure acid. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10562-017-1999-3

Catal Lett (2017) 147:837–844
DOI 10.1007/s10562-017-1999-3
Bioproduction of l-2-Aminobutyric Acid by a Newly-Isolated
Strain of Aspergillus tamarii ZJUT ZQ013
Zhengfang An1 · Xiaoxu Gu1 · Yue Liu1 · Jingyan Ge1 · Qing Zhu1
Received: 5 January 2017 / Accepted: 10 February 2017 / Published online: 28 February 2017
© Springer Science+Business Media New York 2017
Abstract l-2-Aminobutyric acid (l-ABA), an unnatu-
ral amino acid, is a key intermediate of several important
drugs. Although some methods have been developed to
prepare pure chiral l-ABA, there are still many draw-
backs, including low catalytic efficiency, cumbersome
steps and high cost due to the addition of some expensive
catalysts or coenzymes. Herein, with chemical and bio-
logical approaches together, we discovered a newly isolated
Aspergillus tamarii ZJUT ZQ013 strain containing a micro-
bial lipase which could be employed to resolve racemic
methyl N-Boc-2-aminobutyrate to produce l-ABA with
high enantioselectivity (e.e.s > 99.9%, E=257). Moreover,
the subsequent gram scale experiment confrimed that A.
tamarii ZJUT ZQ013 could be an attractive biocatalyst for
the efficient preparation of optically pure acid.
Graphical Abstract
NHBoc
O
NHBoc
O
NHBoc
OH
+
O
A. tamarii ZJUT
O
ZQ013
O
rac
N-
-methyl Boc-2-aminobutyrate
D
-isomer
L-ABA
Keywords L-2-Aminobutyric acid · Methyl N-Boc-2-
aminobutyrate · Aspergillus tamarii · Enantioselective
hydrolysis · Kinetic resolution
1 Introduction
l-2-Aminobutyric acid (l-ABA), an unnatural amino acid,
has been used as a precursor for synthesis of many chiral
drugs,such as anti-epileptic Levetiracetam, anti-tubercu-
lotic Ethambutol and Brivaracetam [1–4]. For example, (S)-
2-amino butanol, a key intermediate of ethambutol, can be
synthesized by esterification and hydrogenation reduction
starting from l-ABA [5]. Hence, due to the great market
demand for l-ABA in both pharmaceutical and chemical
Electronic supplementary material The online version of this
article (doi:10.1007/s10562-017-1999-3) contains supplementary
material, which is available to authorized users.
* Qing Zhu
zhuq@zjut.edu.cn
1
Key Laboratory of Bioorganic Synthesis of Zhejiang
Province, College of Biotechnology and Bioengineering,
Hangzhou 310014, China
Vol.:(0123456789)
1 3

838
Z. An et al.
industries, the preparation of optically pure l-ABA with
high efficiency has attracted much attention.
2 Materials and Methods
2.1 Materials
Up to now, the preparation of l-ABA is mainly achieved
by chemical synthesis or enzymatic catalyst. Among the
chemical methods, synthesis of l-ABA has been exten-
sively reported including ammonolysis of α-Halogen acid
[6], Reduction reaction [7], ammoniation hydrolysis reac-
tion and butanone acid reduction [8]. But these chemical
methods had many shortcomings, including poor selectiv-
ity, harsh reaction conditions, various byproducts, difficulty
in separation and purification.
DL-2-aminobutyric acid (purity 99%), Di-tert-butyl dicarbo-
nate (98% purity) and other chemicals were purchased from
Aladdin Industrial Corporation.
2.2 Synthesis of Methyl rac-N-Boc-2-aminobutyric Acid
Synthesis of N-Boc-2-aminobutyric acid [12]: 10.3 g
(0.1 mol) DL-2-aminobutyric acid was dissolved in the solu-
tion of 95mL 1 M NaOH solution and 65mL of methanol,
and 27.5mL Di-tert-butyl dicarbonate(0.12 mol) was added
in ice bath. Then the solution was warmed to room tempera-
ture and stirred for 12 h. Solvent methanol was removed on a
rotary evaporator. The pH of residue was adjusted to1–2 with
1 M hydrochloric acid. The product was extracted with ethyl
acetate, and then washed with saturated sodium chloride
solution (50mL×3), dried over anhydrous sodium sulfate and
concentrated under vacuum to obtain the DL-2-aminobutyric
acid (compound a).
Compared with chemical synthesis, the biosynthetic
methods aroused many interests due to their high selec-
tivity and mild conditions. For example, l-ABA can be
biochemically synthesized from the chemical l-threonine
catalyzed by l-threonine deaminase and l-leucine dehy-
drogenase. But it needs formate as a co-substrate for
NADH regeneration catalyzed by formate dehydrogenase
[9]. Seo developed a fusion protein catalyst (VHb-DAAO)
through binding to Vitreoscilla hemoglobin (VHb) on
D-amino acid oxidase (DAAO), which can selectively
catalyze D-ABA to ketoacid, while l-ABA was kept
unchanged [10]. However these processes were compli-
cated and required co-substrate or several enzymes. l-ABA
could also be produced in a transamination reaction from
l-threonine and l-aspartic acid catalyzed by threonine
deaminase and aromatic aminotransferase. 2-Oxobutyric
acid and benzylamine using ω-transaminase purified from
Vibrio fluvialis JS17 was reported to produce l-ABA,
but benzaldehyde, a harmful chemical to the microorgan-
isms was also detected in the reaction [3]. Clearly, these
methods have many weaknesses, such as low catalytic effi-
ciency, cumbersome catalytic process, and the demand for
expensive catalysts or coenzymes, which could not satisfy
mass-production requirements [11].
DL-N-Boc-2-aminobutyric acid was dissolved in DMF,
and NH4HCO3 and CH3I were added at room temperature.
The mixture was stirred for 12 h in room temperature. The
reaction solution (compound b) was extracted with ethyl ace-
tate, and then washed with saturated sodium chloride solution
(50mL×3), dried over anhydrous sodium sulfate and concen-
trated under vacuum [13] (Scheme 1).
a White solid (93.4% yield). 1H NMR (500 MHz, CDCl3)
δ 4.31 (s, 1H), 1.91 (s, 1H), 1.73 (s, 1H), 1.46 (s, 9H), 0.99 (s,
3H). 13C NMR (126 MHz, CDCl3) ä 177.34, 155.64, 80.14,
54.46,27.81,25.63, 9.48.
b Light yellow liquid (84.3% yield). 1H NMR (500 MHz,
CDCl3) δ4.28 (s, 1H), 3.75 (s, 3H), 1.65 (s, 2H), 1.45 (s,
9H), 0.92 (s, 3H). 13C NMR (126 MHz, CDCl3) ä 172.87,
155.09, 79.50, 54.19, 52.07, 27.81, 25.85, 9.54.
To overcome the drawbacks of chemical or enzymatic
methods, herein, we report a simple method to obtain the
optical pure l-ABA by combining both chemical and bio-
logical methods. First, methyl rac-Boc-2-aminobutyric
ester was synthesized from rac-2-aminobutyric acid.
Then, a microbe named Aspergillus tamarii ZJUT ZQ013
was successfully screened out among hundreds strains
to resolve methyl rac-Boc-2-aminobutyric ester and (S)-
isomer was obtained in a high yield. Its substrate spec-
trum of rac-N-Boc-2-aminobutyric esters was further
determined.
2.3 Screening of Strains and Enantioselective
Hydrolysis to Methyl rac-N-Boc-2-aminobutyrate
The soil samples were collected from various locations in
China, and then the strains were screened by utilizing N-Boc-
2-aminobutyrate as a sole carbon source and selective plate
with bromocresol purple indicator. Isolated strains were
transferred to 150 mL fermentation medium and cultivated
at 200 rpm, 30°C for 48 h. After the cells were harvested,
Scheme 1 Synthesis of rac-
NHBoc
NHBoc
NH
2
N-Boc-2-aminobutyric acid and
methyl rac-N-Boc-2-aminobu-
tyric acid
(Boc) O
MeI,KHCO
2
3
O
COOH
COOH
DMF
NaOH,CH OH
3
O
a
b
1 3

Bioproduction of l-2-Aminobutyric Acid by a Newly-Isolated Strain of Aspergillus tamarii…
839
1 g wet cell were washed and suspended in 5 mL 0.1 M,
pH 7.0 potassium phosphate buffer (KPB). Cell suspen-
sions were mixed with 50 μL substrate solution (N-Boc-2-
aminobutyrate:acetone=1:4,V/V) and kept at 200 rpm, 30°C
for 6 h reaction, the enantioselective hydrolysis of the sub-
strate was determined by GC-MS. The screening medium
contained (per liter, pH 7) 0.5 g KCl, 0.5 g MgSO4∙7H2O,
1 g K2HPO4∙3H2O, 3 g NaNO3, 0.01 g FeSO4, 0.25 g Bro-
mocresol purple, 20 g agar. Fermentation medium con-
tained (per liter, pH 7) 0.5 g KCl, 0.5 g MgSO4∙7H2O, 1 g
K2HPO4∙3H2O, 3 g NaNO3, 0.01 g FeSO4, 20 g sucrose.
1 H), 1.67 (dt, J=14.2, 7.3 Hz, 1 H), 1.44 (s, 9 H), 0.92 (t,
J=7.5 Hz, 3 H).
Compound 2: Light yellow liquid (67.1% yield),1 H
NMR (500 MHz, CDCl3) δ 5.06 (d, J = 7.0 Hz, 1 H),
4.27–4.19 (m, 2 H), 1.94–1.79 (m, 1 H), 1.70 (dd,
J = 14.2, 7.1 Hz, 1 H), 1.28 (dd, J = 13.8, 6.6 Hz, 10 H),
0.98–0.81 (m, 6 H).
Compound 3: Light yellow liquid (73.4% yield),1 H
NMR (500 MHz, CDCl3) δ 5.09 (d, J = 7.6 Hz, 1 H),
4.23 (d, J = 13.1 Hz, 1 H), 4.11–4.01 (m, 3 H), 2.23 (s,
1 H), 1.81 (dd, J = 17.2, 10.1 Hz, 2 H), 1.65 (dt, J = 14.2,
7.0 Hz, 4 H), 1.42 (s, 9 H), 1.22 (s, 2 H).
Compound 4: Light yellow liquid (71.8% yield),1 H
NMR (500 MHz, CDCl3) δ5.10 (d, J = 7.7 Hz, 1 H),
4.14–4.09 (m, 2 H), 3.62 (t, J = 6.7 Hz, 2 H), 2.99 (s,
3 H), 2.18 (d, J = 6.0 Hz, 2 H), 1.64 (dq, J = 13.8, 6.8 Hz,
2 H), 1.55 (d, J = 4.3 Hz, 2 H), 1.44 (s, 9 H), 1.34 (d,
J = 3.3 Hz, 3 H).
2.4 Identification of the Aspergillus tamarii ZJUT
ZQ013
The isolated strain was preliminarily identified by mor-
phological and microscopic observation. Furthermore, its
18 S-internal transcribed spacer (ITS) regions of the rDNA
were obtained through gene sequencing. The 18 S-ITS
region sequence was deposited in the GenBank database.
Related sequences were obtained from GenBank database
(National Center for Biotechnology Information) using
the BLAST system. The 18 S-ITS regions determined
were aligned with the reference sequences obtained from
GenBank databases using ClustalW ver.1.81 [14]. MEGA
ver.5.1 was applied for the calculation of evolutionary dis-
tance and finally a phylogenetic tree was constructed using
the neighbor-joining method [15, 16].
Compound 5: Light yellow liquid (64.2% yield),1 H
NMR (500 MHz, CDCl3) δ 4.13–4.08 (m, 1 H), 3.61
(t, J = 6.7 Hz, 3 H), 2.03 (d, J = 6.4 Hz, 2 H), 1.63 (t,
J = 11.0 Hz, 1 H), 1.58–1.50 (m, 3 H), 1.43 (s, 4 H),
1.37–1.33 (m, 1 H), 1.28 (d, J = 6.9 Hz, 9 H), 0.98–0.78
(m, 7 H).
Compound 6: Light yellow liquid (76.8% yield),1 H
NMR (500 MHz, CDCl3) ä 5.10 (s, 1 H), 4.38–4.25 (m,
1 H), 3.73 (s, 3 H), 1.43 (s, 9 H), 1.37 (d, J = 7.2 Hz, 3 H).
Compound 7: Light yellow liquid (74.2% yield),1 H
NMR (500 MHz, CDCl3) ä 5.03 (d, J = 7.5 Hz, 1 H),
4.29 (d, J = 5.5 Hz, 1 H), 4.11 (q, J = 7.1 Hz, 1 H), 3.73
(m, 4 H), 3.70 (s, 3 H), 1.44 (s, 9 H), 0.93 (d, J = 7.3 Hz,
3 H).
Compound 8: Light yellow liquid (79.1% yield),1 H
NMR (500 MHz, CDCl3) ä 5.04 (d, J = 8.3 Hz, 1 H),
4.21 (dd, J = 9.0, 4.8 Hz, 1 H), 3.73 (s, 3 H), 2.12 (dd,
J = 12.2, 6.6 Hz, 1 H), 1.44 (s, 9 H), 0.94 (s, 3 H), 0.88
(d, J = 6.9 Hz, 3 H).
2.5 Preparation of Derivatives of Methyl
rac-N-Boc-2-aminobutyrate
As described in synthesis of methyl rac-N-Boc-2-aminobu-
tyric acid, compounds 1, 6, 7, 8 were synthesized. Com-
pounds 9–12 are purchased from Aladdin. The structure of
compounds 1–12 were showed in Table 1.
2.6 Analytical Methods
The synthesis of compounds 2–5 was shown in
Scheme 2. 5 mmol (1 eq.) N-Boc-2-amino butyric acid was
dissolved in 30 mL dry dichloromethane, adding 0.5 mmol
4-DMAP (0.1 eq.), 6 mmol alcohols (R–OH, 1.2 eq.) and
6 mol DCC (1.2 eq.) at 0 °C. Then the reaction was allowed
to warm slowly to room temperature and stirred overnight.
The precipitate was removed by filtration, then washed with
saturated sodium chloride solution (30mL×2), dried over
anhydrous sodium sulfate and concentrated under vacuum
to get the product. Products have been purified by thin layer
chromatography.
N-Boc-2-aminobutyric acid and its ester were detected
and separated by GC (6890 N of Agilent) with chiral cap-
illary column BGB-175. The initial temperature of oven
was 120 °C for 2 min, 4 °C/min to 195 °C for 2 min, and
the flow rate was 1mL/min. The value of e.e.s is expressed
as enantiomeric excess of the remaining ester, which was
calculated using the following equation: e.e.s=([R]−[S])/
([R]+[S]). E value was enantiomeric ratios of esters,
which was calculated using the following equation:
E = ln[(1−c)(1−e.e.s)]/ln[(1−c)(1 + e.e.s)] (c conversion,
e.e.s ee of the remaining epoxide) [17].
Compound 1: Light yellow solid (78.7% yield),1 H
NMR (500 MHz, CDCl3) δ 5.06 (d, J=6.9 Hz, 1 H), 4.26
(dd, J=13.3, 7.2 Hz, 1 H), 3.73 (s, 3 H), 1.83 (q, J=6.6 Hz,
1 3

840
Z. An et al.
Table 1 Enantioselective
hydrolysis of methyl rac-N-
Boc-2-aminobutyrate and its
analogues
No Substrate
Time (h) Configuration e.e.s (%) e.e.p (%) c (%)
E
O
1
6
6
6
6
S
S
S
S
99.98
96.90
53.25
48.75
99.99
99.96
99.96
99.93
50.10 >200
O
NHBoc
O
2
3
4
52.24 68.59
38.36 21.82
37.37 16.04
O
NHBoc
O
O
NHBoc
O
O
NHBoc
O
5
6
6
6
S
S
10.43
85.91
N.P.
N.P.
19.97 2.7
O
NHBoc
51.65 25.17
O
O
NHBoc
O
7
8
9
6
6
6
S
S
S
64.46
19.55
57.11
N.P.
42.64 27.24
25.72 4.33
69.61 2.76
O
NHBoc
O
99.98
N.P.
O
NHBoc
O
O
Br
O
10
11
6
6
No reaction
No reaction
0.21
1.90
N.P.
N.P.
23.44 1.02
45.36 1.06
O
NHBoc
O
O
Br
12
O
6
No reaction
1.74
N.P.
34.28 1.09
O
N
3
Reaction conditions 0.1 g Fungus powder, 50 µL methyl N-Boc-2-aminobutyrate, 200 µL DMF, 5 mL
0.1 M KPB (pH 7.2), 200 rpm, 30 ºC, 6 h
Scheme 2 Synthesis of deriva-
tives of methyl rac-N-boc-2-am-
inobutyric acid
NHBoc
O
NH
NHBoc
COOH
2
(Boc) O
DCC,4-DMAP
CH Cl
2
R-OH
R
NaOH,CH OH
COOH
3
2
2
O
1 3

Bioproduction of l-2-Aminobutyric Acid by a Newly-Isolated Strain of Aspergillus tamarii…
841
on PDA agar plate, the colonies were flat, velvety, spore
dense and violet-black sclerotia in the incubator. In the pro-
cess, the white colony turned to yellowish green gradually
till a deep kelly or brown, with a light brown in the reverse
simultaneously. The conidiophores showed a spherical
shape, and spore stems are separated under the microscopic
observation (Fig. 1).
3 Results and Discussion
3.1 Screening and Identification of a Strain Producing
l-ABA
We have successfully isolated more than 60 strains from
soil samples using methyl N-Boc-2-aminobutyrate as the
sole carbon source. The entioselectivity of the isolated
microorgansims towards methyl N-Boc-2-aminobutyrate-
were checked by chiral GC-MS. More than 30 strains
showed (S)-configuration, whereas, 14 of them showed
moderate and good enantioselectivity towards R configura-
tion. After the D7 strain was inoculated at 30°C for 3 days
The ITS sequence analysis of the ZJUT ZQ013 was
further carried out [18]. The sequence data had been sub-
mitted to GenBank under the accession NO. KJ470706.
A phylogenetic tree (Fig. 2) was further constructed, and
this strain was closely clustered with Aspergillus tamari
(GenBankaccession No. D63701.1), having 99% sequence
Fig. 1 The colony morphol-
ogy of Strain D7 (left) and
Microscopic observation of
strain ZJUTZQ013 cultured on
PDA agar plate for 48 h at 30°C
(right)
Fig. 2 The phylogenetic tree
based on 18 S-ITS rDNA
sequences, constructed by the
neighbor-joining method, show-
ing the relationship between
strain ZJUTZQ013 and repre-
sentatives of some related taxa
1 3

842
Z. An et al.
identity. Based on the results of phylogenetic analysis and
phenotypic tests, the isolated strain ZJUT ZQ013 was des-
ignated as Aspergillus tamarii ZJUT ZQ013 and deposited
in the China Center for Type Culture Collection (CCTCC
M 2,014,046).
50
40
30
20
10
0
100
80
60
40
20
0
3.2 Optimization of Biotransformation Conditions
e.e.
e.e.
Yield
s
p
To obtain the optimum reaction conditions for asymmetric
hydrolysis of methyl N-Boc-2-aminobutyrate by Aspergil-
lus tamarii ZJUT ZQ013, different factors, such as buffer
solution, pH, reaction time, reaction temperature, were
investigated [19]. It is well known that buffer solutions
and pH values can influence both of structures of enzymes
and substrates, resulting in the alternation of the complex
between substrates and the active site of enzymes [20–22].
As shown in Fig. 3, the rate of reaction was increased mar-
ginally with the increscent of pH when pH was below 5.6,
at the cost of the activity and stereo selectivity of enzymes.
Interestingly, even at the same pH, the selective catalytic
activity in KPB buffer system was significantly better than
Tris–HCl. Therefore, 0.1 M KPB (pH 7.2) was selected as
the best enzyme catalytic reaction meidum.
15 20 25 30 35 40 45 50
Temperature (
)
Fig. 4 Effects of temperature on the catalytic reaction. Temperature
varying from 15 to 50°C; Reaction conditions 0.1 g Fungus powder,
50 µL methyl N-Boc-2-aminobutyrate, 200 µL DMF, 5 mL 0.1 M
KPB (pH 7.2), substrate/acetone=1:8, 200 rpm, 6 h
3.3 Substrate Specificity of Chiral Resolution
by Aspergillus tamarii ZJUT ZQ013
Enzymes are known to have strict selection towards their
substrates [23, 24]. To explore the scopes of substrates and
to better understand the interaction between the enzyme and
substrate, the substrate spectrum was determined by check-
ing the length of carbon chain on ester and types of amino
acids etc. As shown in Table 1, with the extension of the
carbon chain of the alcohols (1–5), the entioselectivity of
Aspergillus tamarii ZJUT ZQ013 was gradually decreased.
The bulky groups on carbon chain of the amino acids also
have great impact on the catalytic activity and stereoselec-
tivity of the organism. When a longer and branched carbon
chain (7 and 8 respectively) was introduced into the sub-
strate, the selective catalytic ability of Aspergillus tamarii
ZJUT ZQ013 was cut down sharply. When the Boc group
The optimization of temperature was analyzed from 15
to 50°C. As shown in Fig. 4, the e.e.s of methyl N-Boc-
2-aminobutyrate reached the maximum 99.999% at 30°C,
and the yield of l-N-Boc −2-amino butyric acid was up to
45.4%.
Under the above optimized condition, the catalytic
reaction time was further studied. According to Fig. 5,
the e.e.s of methyl N-Boc-2-aminobutyrate gradually
increased, it reached higher than 99% at 5.5 h. After 1 h,
optical pure N-Boc-L-2- amino butyric acid was detected
(e.e.p > 99.9%), and the yield was gradually increased,
reaching a maximum of 45.7% at 6 h.
60
50
100
100
50
40
30
80
60
40
80
60
40
30
20
20
10
0
40
20
0
e.e.
e.e.
Yield
s
p
e.e.
S
20
0
10
0
e.e.
P
Yield
4.0
4.8
5.6
6.4
7.2
8.0
8.8
0
1
2
3
4
5
6
7
8
pH(HAc/AcNa(4.0-5.6), KPB(6.0-8.0), Tris-HCl(7.2-8.8))
Time (h)
Fig. 3 Effects of buffer and pH on the catalytic reaction. Reaction
conditions: 5 mL different buffer solutions (pH 4.0–5.6 HAc–NaAc,
pH 6.0–8.0 Tris–HCl, pH 7.2–8.8 KPB), 0.1 g Fungus powder, 50 µL
methyl N-Boc-2-aminobutyrate, 200 µL DMF, 5 mL 0.1 M KPB (pH
7.2), substrate/acetone=1:8, 200 rpm, 6 h
Fig. 5 Effects of reaction time on the catalytic reaction. Time vary-
ing from 0 to 8 h; Reaction conditions 0.1 g Fungus powder, 50 µL
methyl N-Boc-2-aminobutyrate, 200 µL DMF, 5 mL 0.1 M KPB (pH
7.2), substrate/acetone=1:8, 200 rpm, 6 h
1 3

Bioproduction of l-2-Aminobutyric Acid by a Newly-Isolated Strain of Aspergillus tamarii…
843
unnatural amino acids unnatural amino acids using transami-
nases. Trends Biotechnol 16:412–418
was replaced with bromide (9), the enantioselectivity of
Aspergillus tamari ZJUT ZQ013 decreased significantly
(high conversion rate, low E value). The results also show
that there is no enantioselectivity for γ-butyrolactone com-
pounds (10–12), indicating that entioselectivity of Asper-
gillus tamarii ZJUT ZQ013 has strict substrate selectivity.
2. Zhu L, Tao RS, Wang Y et al (2011) Removal of L-alanine
from the production of l-2-aminobutyric acid by introduction
of alanine racemase and d-amino acid oxidase. Appl Microbiol
Biotechnol 90(3):903–910
3. Shin JS, Kim BG (2009) Transaminase-catalyzed asymmet-
ric synthesis of L-2-aminobutyric acid from achiral reactants.
Biotechnol Lett 31(10):1595–1599
4. Futagawa T, Canvat JP, Cavoy E, et al. Process for the prepara-
tion of levetiracetam. US Patent 6,107,492, 22 Aug 2000
5. Ragonese R, Macka M, Hughes J et al (2002) The use of the
Box-Behnken experimental design in the optimisation and
robustness testing of a capillary electrophoresis method for the
analysis of ethambutol hydrochloride in a pharmaceutical for-
mulation. J Pharm Biomed Anal 27(6):995–1007
3.4 Preparative Scale Production of l-2-Aminobutyric
Acid
To further evaluate the practical application of Aspergillus
tamarii ZJUT ZQ013 in industry, a preparative scale reso-
lution of rac-N-Boc -2-amino butyric acid was carried out
in gram scale. Under the optimized resolution conditions,
the enantioselective hydrolysis reaction was successfully
performed at a concentration of 50 mM of rac-N-Boc -2-
amino butyrate. Up to 2.5 g of optically pure N-Boc-l-2-
amino butyric acid (99.9% e.e.p) was successfully obtained,
and the protecting group Boc was removed in 20% TFA
solution affording l-2-aminobutyric acid in 42% total yield.
This result indicates that Aspergillus tamarii ZJUT ZQ013
is a powerful biocatalyst which could be employed for the
efficient preparation of optically pure l-ABA.
6. Jefery E A, Meisters A (1978) Electrochemical synthesis of
amino acids by reductive amination of keto acids. I. Reduction
at mercury electrodes. Aust J Chem 31(1):79–84
7. Babievskii KK, Belikov VM, Tikhonova NA (1965) Syn-
thesis of D, L—threonine and D, L-2-aminobutyrie acid
based on condensations of nitroacetie ester. Russ Chem Bull,
14(1):76–81
8. Chiyuki F. Process for production of (+)-2-amino-1-butanol. US
Patent 3,979,457, 07 Sept 1976
9. Tao RS, Jiang Y, Zhu FY, Yang S (2014) A one-pot system
for production of L-2-aminobutyric acid from L-threonine by
L-threonine deaminase and a NADHregeneration system based
on L-leucine dehydrogenase and formate dehydrogenase. Bio-
technol Lett 36:835–841
10. Seo YM, Mathew S, Bea HS, Khang YH, Lee SH, Kim BG, Yun
H (2012) Deracemization of unnatural amino acid: homoalanine
using D-amino acid oxidase and omega-transaminase. Org Bio-
mol Chem 10(12):2482–2485
4 Conclusion
11. Fotheringham IG, Grinter N, Pantaleone DP, Senkpeil RF, Tay-
lor PP (1999) Engineering of a novel biochemical pathway for
the biosynthesis of L-2-aminobutyric acid in Escherichia coli
K12. Bioorg Med Chem 7:2209–2213
l-ABA was an important chiral material and pharmaceu-
tical intermediates. In this work, a newly isolated strain,
Aspergillus tamarii ZJUT ZQ013 capable of resolving
methyl rac-N-Boc-2-aminobutyrate to l-ABA with high
enantioselectivity was reported herein. By single-factor
experiment, the conditions of the kinetic resolution, such
as pH, reaction time, reaction temperature were opti-
mized. The results showed that Aspergillus tamarii ZJUT
ZQ013 could afford an excellent enantio pure l-ABA with
e.e.s > 99.9%, conversion rate of 45.7%, and E value of
257, which are, to our best knowledge, the highest values.
It was demonstrated that Aspergillus tamarii ZJUT ZQ013
was an efficient biocatalyst and has promising potential for
industry application.
12. Alvaro G, Decor A, Fontana S, Hamprecht D, Large C, Marasco
A. Imidazolidinedione derivatives. WO 2011069951 A1, 16 July
2011
13. Brenner M, Vecchia LL, Leutert T, Seebach
D (2003)
(4 S)-4- (1-methylethyl)-5,5- diphenyl- 2-oxazolidinone[(2-
oxazolidinone,4-(1-methylethyl)-5,5- diphenyl-, (4 S)-)]. Organ
Synth 80:57–65.
14. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, positions-specific
gap penalties and weight matrix choice. Nucleic Acids Res
22(22):4673–4680
15. Xu F, Deng G, Cheng S et al (2012) Molecular cloning, char-
acterization and expression of the phenylalanine ammonia-lyase
gene from Juglans Regia. Molecules 17(7):7810–7823
16. Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molec-
ular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Mol
Biol Evol 28(10):2731–2739
Acknowledgements This work was supported by National Science
Foundation of China (Nos. 21272212, 21472172) and Zhejiang Natu-
ral Science Fund (Nos. LY17B060009, LY12B02018), Project of Sci-
ence Technology Department of Zhejiang Province (2014C33141),
and Project of Department of Science Technology Jinhua City
(2013-3-003).
17. Chen CH, Sih CJ (1989) General aspects and optimization of
enantioselective biocatalysis in organic solvents: the use of
lipases. Angew Chem Int Ed 28(6):695–707
18. Baldwin BG (1992) Phylogenetic utility of the internal tran-
scribed spacers of nuclear ribosomal DNA in plants: an example
from the compositae. Mol Phylogenet Evol 1(1):3–16
19. Dosanjh NS, Kaur J (2002) Immobilization, stability and ester-
ification studies of a lipase from a Bacillus sp. Appl Biochem
36(1):7–12
References
1. Taylor PP, Pantaleone DP, Senkpeil RF, Fotheringham IG
(1998) Novel biosynthetic approaches to the production of
1 3

844
Z. An et al.
20. Dong HP, Wang YJ, Zheng YG (2010) Enantioselective hydroly-
sis of diethyl 3-hydroxyglutarate to ethyl (S)-3-hydroxyglutar-
ate by immobilized Candida antarctica lipase B. J Mol Catal B
Enzym 66(1):90–94
asymmetric synthesis of (R)-methyl mandelate. Bioproc Biosyst
Eng 33(7):797–804
23. Zhang Z, Sheng Y, Jiang K (2010) Bio-resolution of glycidyl
(o,m,p)-methylphenyl ethers by Bacillus megaterium. Biotechnol
Lett 32:513–516
21. Long WS, Kow PC, Kamaruddin AH (2005) Comparison of
kinetic resolution between two racemic ibuprofen esters in an
enzymic membrane reactor. Process Biochem 40(7):2417–2425
22. Guo JL, Mu XQ, Xu Y (2010) Integration of newly isolated bio-
catalyst and resin-based in situ product removal technique for the
24. Chang CS, Ho SC (2011) Enantioselective esterification of (R,
S)-2-methylalkanoic acid with Carica papaya lipase in organic
solvents. Biotechnol Lett 33(11):2247–2253
1 3