Substrate profile of an ω-transaminase from Burkholderia vietnamiensis and its potential for the production of optically pure amines and unnatural amino acids

Article abstract of DOI:10.1016/j.molcatb.2013.11.013

A new (S)-enantioselective ω-transaminase (ω-TA) gene from Burkholderia vietnamiensis G4 was functionally expressed in Escherichia coli BL21 (DE3), and the purified recombinant N-terminal His-tagged ω-TA (HBV-ω-TA) had a dimeric structure with optimum pH and temperature of 8.4 and 40 C, respectively. The enzyme showed higher activities toward aromatic amines than aliphatic amines and (S)-1-methylbenzylamine ((S)-α-MBA) was the most active amino donor. For amino acceptor, keto acids, keto esters and aldehydes were more reactive than ketones with pyruvate ethyl ester being most active. Several chiral amines and unnatural amino acids or esters were synthesized using HBV-ω-TA as the catalyst and isopropylamine or (S)-α-MBA as amino donor. Notably, HBV-ω-TA catalyzed the amino transfer to β-keto esters to give optically pure β-amino acid esters. In addition, glyoxylate was used as amino acceptor for the first time in the kinetic resolution of racemic amines and optically pure amines, such as (R)-1-methylbenzylamine, (R)-1-phenylpropylamine, (R)-2-amino-4-phenylbutane and (R)-1-aminotetraline, were obtained.

Full text of DOI:10.1016/j.molcatb.2013.11.013

Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Substrate profile of an ␻-transaminase from Burkholderia
vietnamiensis and its potential for the production of optically pure
amines and unnatural amino acids
Jinju Jianga,b, Xi Chena, Jinhui Fenga, Qiaqing Wua, Dunming Zhua,∗
a National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
b University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2013
Received in revised form 6 November 2013
Accepted 16 November 2013
Available online 25 November 2013
A new (S)-enantioselective ␻-transaminase (␻-TA) gene from Burkholderia vietnamiensis G4 was func-
tionally expressed in Escherichia coli BL21 (DE3), and the purified recombinant N-terminal His-tagged
␻-TA (HBV-␻-TA) had a dimeric structure with optimum pH and temperature of 8.4 and 40 ◦C,
respectively. The enzyme showed higher activities toward aromatic amines than aliphatic amines and (S)-
1-methylbenzylamine ((S)-␣-MBA) was the most active amino donor. For amino acceptor, keto acids, keto
esters and aldehydes were more reactive than ketones with pyruvate ethyl ester being most active. Sev-
eral chiral amines and unnatural amino acids or esters were synthesized using HBV-␻-TA as the catalyst
and isopropylamine or (S)-␣-MBA as amino donor. Notably, HBV-␻-TA catalyzed the amino transfer to ␤-
keto esters to give optically pure ␤-amino acid esters. In addition, glyoxylate was used as amino acceptor
for the first time in the kinetic resolution of racemic amines and optically pure amines, such as (R)-1-
methylbenzylamine, (R)-1-phenylpropylamine, (R)-2-amino-4-phenylbutane and (R)-1-aminotetraline,
were obtained.
Keywords:
Biocatalysis
␻-Transaminase
Chiral amine
Unnatural amino acid
␤-Keto ester
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
acids [15–18]. Among these methods, ␻-TA-catalyzed amino trans-
fer reaction to carbonyl compounds is one of the most prominent
Chiral amines and unnatural amino acids, including ␤-amino
acids have been widely used in diverse sectors such as the pharma-
ceutical, chemical, cosmetic, food, and agricultural industries [1–3].
For example, ␤-amino acids are key building blocks in many nat-
ural and synthetic drugs such as taxol and cispentacin, which are
used for their antitumor and antifungal activities, respectively [3,4].
The peptides containing unnatural amino acids usually show higher
stability against peptidases than natural peptides and such mixed
␣/␤-peptides often retain their biological activity [5–9], thus pro-
viding useful chemical building blocks for new drugs that are not
degraded or rejected by the human body [10–13].
Given the significance of chiral amines and unnatural amino
acids, efficient synthesis of these compounds in optically pure form
has become an attractive challenge. However, unlike natural amino
acids which are usually produced using fermentation method [14],
chemical and biocatalytic methods have been explored for the
production of enantiomerically pure amines and unnatural amino
approaches because of its superior features compared to other
enzymatic and chemical methods [19–22]. ␻-TAs show high stere-
oselectivity, rapid reaction rate, broad substrate specificity, and
no requirement for external cofactor. Moreover, ␻-TAs can be
applied in both the kinetic resolution of racemic amines [23–26]
and the asymmetric synthesis of optically pure amines from the
corresponding prochiral carbonyl compounds [24,27,28] (Fig. 1).
Although some excellent examples have demonstrated the success
of transaminases in the production of important chiral amines and
unnatural amino acids [25,28,29], the full potential of this group
of enzymes for industrial application is yet to be realized. Present
challenges include the unfavorable thermodynamic equilibrium
and severe product and substrate inhibitions shown by various
transaminases [19,20,24]. In this study, we take the approach of dis-
covering a new omega-transaminase making use of the microbial
genome database as a source of new biocatalysts better known as
genome mining. The new ␻-TA gene was cloned and overexpressed
in Escherichia coli BL21. The recombinant enzyme was then charac-
terized with respect to its enzymatic properties, substrate spectrum
and its potential for the production of optically pure amines and
unnatural amino acids.
∗
Corresponding author. Tel.: +86 22 84861962; fax: +86 22 84861996.
E-mail address: zhu dm@tib.cas.cn (D. Zhu).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.11.013

J. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
33
␤-mercaptoethanol. The purified enzyme solution was then stored
at 4 ◦C for further experiments.
2.4. Protein determination and molecular mass measurement
Protein purity was analyzed by SDS-PAGE according to standard
procedure using 12.5% polyacrylamide gels. The protein bands were
visualized by Coomassie blue staining. Protein concentration was
determined with BCA protein assay kit (CWBIO, China).
The apparent molecular mass of HBV-␻-TA was estimated by
gel filtration on a Superdex 200 HR 10/30 column (GE, USA) equili-
brated and eluted with 50 mM phosphate buffer (pH 7.2) containing
150 mM NaCl at a flow rate of 0.4 ml/min.
2.5. Enzyme assays
Fig. 1. ␻-TA catalyzed reactions. (a) Asymmetric synthesis of chirally pure unnatu-
ral amino acids using (S)-␣-MBA as amino donor. (b) Kinetic resolution of racemic
amines using glyoxylate as amino accepter.
Unless otherwise specified, enzyme assays were carried out at
37 ◦C in phosphate buffer (100 mM, pH 7.4) containing PLP (20 ␮M),
(S)-␣-MBA (10 mM) and pyruvate (10 mM). The typical reaction
volume was 1 ml, and the reaction was initiated by adding puri-
fied enzyme (6.5 × 10−3 mg) to the reaction mixture. After 5 min,
the reaction was stopped by adding 375 ␮l of 16% (v/v) perchloric
acid. The produced acetophenone was analyzed by HPLC according
to Section 2.10.
2. Materials and methods
2.1. Chemicals
To study the effect of temperature on enzyme activity, reac-
tions were carried out at various temperatures from 25 ◦C to 50 ◦C.
To investigate the thermostability of HBV-␻-TA, the enzyme was
incubated in phosphate buffer (100 mM, pH 7.4) at the specific tem-
perature (30, 35, 40, 45, 50, 55, 60 ◦C) for 20 min and the remaining
activity was assayed as described above.
Most of the chemicals were of the highest grade available
and obtained from commercial sources such as Alfa Aesar and
Sigma–Aldrich Chemical Co. 4-Aminovaleric acid was prepared
chemically according to reported methods [30]. Materials used for
culture media including peptone, yeast extract and agar were pur-
chased from Becton, Dickinson and Company (BDX).
To study the effect of pH on enzyme activity, reactions were
carried out at various pH from 5.0 to 10.0. For the effect of pH
on enzyme stability, the enzyme was incubated in the specific pH
buffer (100 mM, pH = 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) at 4 ◦C for 72 h
and then the remaining activity was assayed as described above.
The buffers used were sodium acetate (pH 3.8–5.6), sodium phos-
phate (pH 5.8–7.6), boric acid–borax (pH 7.8–9.2) and borax sodium
hydroxide (pH 9.3–10.1).
2.2. Selection of the ␻-TA gene
The Vibrio fluvialis JS17 ␻-transaminase, which has been
found to use pyruvate methyl ester and pyruvate ethyl ester as
amino acceptor and ethyl ␤-aminobutyrate as amino donor [31],
was used as a template for BLASTP search in NCBI at default
settings (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify new
␻-transaminase. A putative ␻-transaminase from Burkholderia viet-
namiensis G4, which showed 53% identity and 71% similarity to the
V. fluvialis enzyme, was selected (Fig. S1 in Supplemental material).
2.6. Measurement of kinetic parameters
To determine the kinetic parameters, initial rates (i.e. conver-
sion <5%) were measured at varying concentrations of (S)-␣-MBA
and pyruvate by following the activity assay procedure described
above. The produced acetophenone was analyzed by HPLC accord-
ing to Section 2.10. For the kinetic constants of HBV-␻-TA toward
(S)-␣-MBA, pyruvate (50 mM) was used as the amino acceptor and
the concentration of (S)-␣-MBA was as follows: 1, 2, 4, 6, 8, 10, 15,
20, 30 and 50 mM. For the kinetic constants of HBV-␻-TA toward
pyruvate, (S)-␣-MBA (50 mM) was used as the amino donor and
the concentration of pyruvate was as follows: 1, 2, 3, 4, 5, 6, 8
and 10 mM. With the help of software Origin (OriginLab, USA), the
kinetic constants for (S)-␣-MBA and pyruvate were calculated.
2.3. Overexpression and purification of the ␻-transaminase
The putative transaminase gene from B. vietnamiensis G4 (Acces-
sion No. YP 001110355.1; Supplemental material) was codon-
optimized and synthesized with a His-tag coding sequence at N-
terminus by Shanghai Xuguan Biotechnological Development Co.,
Ltd. and ligated into the pET-32a expression vector at Nde I/BamH I
restriction sites. The vector was then transformed into E. coli BL21
(DE3). The transformant was grown at 37 ◦C in 500 ml Luria-Bertani
broth supplemented with ampicillin (100 ␮g/ml). When the OD600
reached approximately 0.6, isopropyl-␤-d-thiogalactopyranoside
(IPTG, 0.1 mM) was added. After induction at 25 ◦C for 12 h, the cells
were harvested and washed twice with 200 ml of phosphate buffer
(50 mM, pH 7.0). The cells were re-suspended in 50 ml of lysis buffer
[50 mM phosphate buffer, pH 7.0, 20 ␮M pyridoxal 5ꢀ-phosphate
(PLP), 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.01% (v/v)
␤-mercaptoethanol, 30 mM imidazole and 500 mM NaCl] and dis-
rupted by high pressure homogenizer. Cell debris was removed by
centrifugation at 14,000 × g for 20 min at 4 ◦C. Supernatant was
then applied on a 10 ml Ni-NTA affinity column and the eluted
solution containing HBV-␻-TA was collected, and dialyzed against
phosphate buffer (50 mM, pH 7.0) containing 20 ␮M PLP and 0.01%
2.7. Substrate specificity and enantioselectivity
Amino donor specificity was assayed by following the simi-
lar procedure of Section 2.5. The amino donor substrates (10 mM)
listed in Table 1 reacted with pyruvate (10 mM) as amino accep-
tor. For racemic amino donor, the concentration was 20 mM. After
the reaction, residual pyruvate was analyzed by HPLC according to
Section 2.10.
Amino acceptor specificity was assayed by following the similar
procedure of Section 2.5. The amino acceptor substrates (10 mM)
listed in Table 2 reacted with (S)-␣-MBA (10 mM) as amino donor.

34
J. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
Table 1
After the reaction, the amount of produced acetophenone was ana-
lyzed and the ee values of the produced amines were determined
Amino donor specificity of HBV-␻-TA.
according to Section 2.10.
Amino donor
Relative
activity
(%)a
2.8. Asymmetric synthesis of enantiomerically pure amine
compounds
(S)-1-Methylbenzylamine (D1)
(R)-1-Methylbenzylamine (D2)
100
0
In a typical procedure, (S)-␣-MBA (10 mM) or isopropylamine
(100 mM) were mixed with the carbonyl compounds (10 mM,
Table 3) in 1 ml of phosphate buffer (100 mM, pH 7.4) containing
PLP (20 ␮M). The reaction was started by adding 0.8 mg/ml of HBV-
␻-TA, and the mixture was incubated at 37 ◦C for 12 h. Then the
residual (S)-␣-MBA, acetophenone and 4-phenyl-2-butanone and
the ee values of the products were detected according to Section
2.10.
Isopropylamine (100 mM) and ethyl propionylacetate (10 mM)
were mixed in 100 ml of phosphate buffer (100 mM, pH 7.4) con-
taining PLP (20 ␮M). The reaction was started by adding 0.8 mg/ml
of HBV-␻-TA, and the mixture was shaken at 37 ◦C for 12 h. After
that, the pH was adjusted to about 4.0 with HCl solution (1 M), and
the reaction mixture was extracted with ethyl acetate (50 ml 3×).
After separation of the organic phase, the pH of the aqueous phase
was adjusted to about 9.0 with saturated sodium carbonate solu-
tion and the mixture was extracted with ethyl acetate (50 ml 3×).
The organic extract was dried over anhydrous sodium sulfate and
removal of the solvent gave the product, 3-aminopentanoic acid
ethyl ester (13 mg, 9% yield). 1H NMR (400 MHz, CDCl3): ı 0.98
(t, 3H, J = 8 Hz), 1.27 (t, 3H, J = 8 Hz), 1.60 (m, 2H), 2.47–2.51 (dd,
J = 16 Hz, 8 Hz, 1H), 2.58–2.62 (dd, J = 16 Hz, 8 Hz, 1H), 3.29 (m, 2H),
4.17 (q, 3H). HRMS: calcd for C7H16NO2 (MH+) 146.1181, found
146.1162.
(S)-1-Phenylpropylamine (D3)
(S)-1-Phenylbutylamine (D4)
25.7
6.2
(R,S)-2-Amino-4-phenylbutane (D5)
(S)-1-Aminoindane (D6)
17.3
32.3
(S)-1-Aminotetraline (D7)
32.9
Benzylamine (D8)
27
2.9. Kinetic resolution of racemic amines
Cyclopentylamine (D9)
5.5
Racemic amine (10 mM, Table 4) was mixed with glyoxylate
(10 mM) in 1 ml of phosphate buffer (100 mM, pH 7.4) contain-
ing PLP (20 ␮M). The reaction was started by adding 0.8 mg/ml of
HBV-␻-TA, and the mixture was incubated at 37 ◦C for 12 h. The
amount and ee value of the residual amino donor were determined
according to Section 2.10.
Cyclohexylamine (D10)
Isopropylamine (D11)
(R,S)-2-Pentanamine (D12)
5.7
4.9
5.6
2.10. Analytical methods
(S)-2-Aminohexane (D13)
dl-3-Aminobutyric acid ethyl ester (D14)
␤-Alanine (D15)
6.5
9.4
2.6
The concentrations of acetophenone, 4-formylbenzoic acid, 4-
phenyl-2-butanone, 1-indanone and amines in the samples were
determined by HPLC analysis on an Eclipse XDB-C18 column
(4.6 mm × 150 mm, Agilent, USA) with isocratic elution of acetoni-
trile/water (50/50, v/v) at 1 ml/min. Similarly, the concentration of
pyruvate was analyzed on an Aminex HPX-87H column (Bio-Rad,
USA) at 45 ◦C. An aqueous solution of H2SO4 (5 mM) was used as
eluent at a flow rate of 0.6 ml/min. All UV detections were carried
out at 205 nm.
dl-␤-Phenylalanine (D16)
0
Chiral analysis of alanine was performed on PIRKLE
(S,S) WHELK-O1 column (REGIS, USA) with isocratic elu-
tion (0.6 ml/min) of isopropanol/hexane (20/80, v/v) and
UV detection at 254 nm after derivatization with N-(9-
l-␣-Leucine (D17)
1.6
a
fluorenylmethoxycarbonyloxy)succinimide
(Fmoc-OSu).
The
The activity for (S)-␣-MBA (34 U/mg) was defined as 100%. Relative activity of
less than 1% was expressed as zero. One unit of the enzyme activity was defined
as the amount of the enzyme that catalyzed the formation of 1 ␮mol acetophenone
from 10 mM (S)-␣-MBA and 10 mM pyruvate in 1 min.
derivatization procedure was as follows. Fmoc-OSu was added
to the reaction mixture at pH 9.0 and incubated at 37 ◦C for 12 h.
After centrifugation, the supernatant was extracted with tert-butyl
methyl ether. The pH of the aqueous phase was adjusted to 3.0
and the mixture was extracted with ethyl acetate. The extract
was analyzed to measure the ee values of the derivatized product.
Chiral analysis of 1-methylbenzylamine, 1-phenylpropylamine,
1-aminoindane and 1-aminotetraline were performed on CROWN-
PAK CR(+) column (Daicel Chemical Industries, Japan) with

J. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
35
Table 2
Amino acceptor specificity of HBV-␻-TA.
Amino acceptor
Pyruvate (A1)
Relative activity (%)a
ee (%)
100
>99 (S)
>99 (S)
Pyruvate ethyl ester (A2)
127.7
Ethyl acetoacetate (A3)
5
>99 (S)
>99 (S)
Ethyl propionylacetate (A4)
1.4
Levulinic acid (A5)
1.1
0
>99 (S)
␣-Ketoglutarate (A6)
n/ab
1-Indanone (A7)
1.7
2.4
1.8
>99 (S)
>99 (S)
>99 (S)
1-Tetralone (A8)
1-Phenylpropanone (A9)
4-Phenyl-2-butanone (A10)
2-Hexanone (A11)
2.7
0
>99 (S)
n/a
Benzaldehyde (A12)
100.6
n/a
4-(Trifluoromethyl)benzaldehyde (A13)
4-Cyanobenzaldehyde (A14)
24.8
89.6
n/a
n/a
4-Formylbenzoic acid (A15)
Salicylaldehyde (A16)
68.1
n/a
n/a
119.3
Caproaldehyde (A17)
1-Nonanal (A18)
30.9
8.1
n/a
n/a
2,6-Dimethyl-5-heptenal (A19)
17.2
15.4
n/a
n/a
(E,E)-2,4-Hexadienal (A20)

36
J. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
Table 2 (Continued)
Amino acceptor
Relative activity (%)a
ee (%)
2-trans-4-trans-Decadienal (A21)
7.1
3.6
n/a
Citral (A22)
n/a
n/a
trans-3-(3-Pyridyl) acrolein (A23)
27.1
11.4
2-Furanacrolein (A24)
n/a
Ethyl trans-4-oxo-2-butenoate (A25)
32.6
98.4
n/a
n/a
Glyoxylate (A26)
a
The activity for pyruvate (34 U/mg) was defined as 100%. The relative activity of less than 1% was expressed as zero. One unit of the enzyme activity was defined as the
amount of the enzyme that catalyzed the formation of 1 ␮mol acetophenone from 10 mM (S)-␣-MBA and 10 mM pyruvate in 1 min.
b
n/a, not applicable.
isocratic elution (0.5 ml/min) of perchloric acid solution (pH 1.5)
containing 10% of methanol at 30 ◦C and UV detection at 200 nm.
Chiral analysis of ␣-alanine ethyl ester, 3-aminobutyric acid ethyl
ester and 3-aminopentanoic acid ethyl ester were performed by
GC on Chirasil-DEX CB column (25 m × 0.25 mm × 0.25 ␮m, Varian,
USA) after acetylation, which was performed by mixing acetic
anhydride, pyridine and the product (3:1.2:1) in tetrahydrofuran.
GC conditions were as follows: for ␣-alanine ethyl ester, column
temperature was 115 ◦C, isothermal, the split ratio was 20:1, flow
rate of the carrier gas (helium) was 2 ml/min, FID detector (220 ◦C);
for 3-aminobutyric acid ethyl ester and 3-aminopentanoic acid
ethyl ester, column temperature was 140 ◦C, isothermal, the
split ratio was 20:1, flow rate of the carrier gas (helium) was
2 ml/min, FID detector (220 ◦C) [32]. Chiral analyses of 2-amino-
4-phenylbutane and 4-aminovaleric acid were performed on C18
column with isocratic elution of phosphoric acid–triethylamine
(50 mM, pH 3.0)/acetonitrile and UV detection at 340 nm after
FDAA derivatization. For 2-amino-4-phenylbutane, the ratio of
phosphoric acid–triethylamine to acetonitrile was 55/45 (v/v) and
the flow rate was 1 ml/min, while those for 4-aminovaleric acid
were 70/30 (v/v) and 0.5 ml/min, respectively. FDAA derivatiza-
tions were performed according to the instruction manual of FDAA
(Thermo Fisher Scientific, USA). The retention times refer to Table
S1 (Supplemental material).
3. Results and discussion
3.1. Overexpression, purification and molecular mass of the
␻-transaminase
A putative ␻-transaminase gene from B. vietnamiensis G4 was
overexpressed in E. coli BL21 (DE3), and the purified HBV-␻-TA
Table 3
Asymmetric synthesis of enantiomerically pure amine compounds by HBV-␻-TA.
Substrate
Concentration (mM)
10
Conversion (%)
16
ee (%)
Acetophenonea
>99 (S)
4-Phenyl-2-butanonea
10
30
>99 (S)
Ethyl propionylacetateb
Levulinic acidb
10
10
10
17
66
89
>99 (S)
>99 (S)
>99 (S)
Ethyl acetoacetateb
a
100 mM isopropylamine was used as the amino donor.
10 mM (S)-␣-MBA was used as the amino donor.
b

J. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
37
Table 4
Kinetic resolution of amines with glyoxylate as amino acceptor.
Substrate
Concentration (mM)
10
Conversion (%)
50
ee value (%)
(R,S)-1-Methylbenzylamine
(R,S)-1-Phenylpropylamine
(R,S)-2-Amino-4-phenylbutane
>99 (R)
>99 (R)
>99 (R)
10
10
50
53
(R,S)-1-Aminotetraline
10
51
>99 (R)
showed a single band of about 45 kDa on SDS-PAGE (Fig. 2), con-
sistent with the predicted value of a 461-amino acid polypeptide
(50,279 Da). The molecular mass of the native HBV-␻-TA was esti-
mated to be 100 kDa by gel filtration chromatography, suggesting
that the enzyme has a dimeric structure of the enzyme.
amino donor that enhances the formation of an external aldimine,
which was also observed for the known transaminases from
Mesorhizobium sp. Strain LUK (pH 7.6) [27], Caulobaceter crescentus
(pH 8.5) [33], Arthrobacter sp. KNK168 (pH 8.0–9.0) [34], Alcaligenes
denitrificans Y2k-2 (pH 9.0) [26], and V. fluvialis JS17 (pH 9.2) [35].
The effects of temperature and pH on the enzyme stability
are shown in Fig. 4. When HBV-␻-TA was incubated in phos-
phate buffer (100 mM, pH 7.4) at various temperatures for 20 min,
the enzyme activity remained nearly unchanged up to 50 ◦C, but
dropped sharply above 50 ◦C. The activity was completely lost at
60 ◦C. The enzyme was incubated in various pH buffers for 72 h and
the remaining activity of the enzyme was measured. HBV-␻-TA
was relatively stable between pH 5.0 and 9.0. Its activity decreased
above pH 9.0 and 60% activity was lost after incubation at pH 10.0
for 72 h.
3.2. Effects of pH, temperature and substrate concentrations
The effects of temperature and pH on the enzyme activity are
shown in Fig. 3. The optimum reaction temperature was between
42 ◦C and 45 ◦C, and the activity of HBV-␻-TA decreased 20% when
the temperature was 50 ◦C. HBV-␻-TA was more active under weak
alkaline conditions with the optimum pH being about pH 8.4. This
weak alkaline pH optimum for the enzyme activity was likely due
to the increase in the effective concentration of the deprotonated
The effects of the substrate concentration of (S)-␣-MBA and
pyruvate on the enzyme activity were examined. Using the
nonlinear fit method of Origin software, the apparent Km for (S)-
␣-MBA and pyruvate were calculated to be 14.67 1.02 mM and
1.65 0.22 mM, respectively. The Vmax for (S)-␣-MBA and pyruvate
were calculated to be 82.98 2.52 U/mg and 77.80 2.90 U/mg,
respectively. The apparent Km values for pyruvate were 2.29, 3.9
and 11 mM for the reported transaminases from Arthrobacter sp.
KNK168, Mesorhizobium sp. Strain LUK and A. denitrificans Y2k-2,
respectively [26,27,34].
3.3. Substrate specificity and enantioselectivity
The amino donor specificity of HBV-␻-TA was examined in
terms of initial rate with pyruvate as the amino acceptor [31]. It
can be seen from Table 1 that HBV-␻-TA was active toward a wide
range of amino donors. Similar to the V. fluvialis transaminase
[31], HBV-␻-TA showed high activity toward aromatic (S)-amines
such as (S)-1-methylbenzylamine (D1, the most effective one),
(S)-1-aminoindane (D6) and (S)-1-aminotetraline (D7), while
no activity was detected for (R)-1-methylbenzylamine (D2). The
high enantioselectivity toward the amino donor was beneficial
for preparation of enantiopure amines via kinetic resolution. The
initial activity decreased in the order of (S)-1-methylbenzylamine
(D1), (S)-1-phenylpropylamine (D3) and (S)-1-phenylbutylamine
(D4). This was consistent with the two-binding pocket model for
the conserved substrate recognition mechanism of ␻-transaminase
[31]. The two substrate binding pockets were located at subunit
interface and constituted active site together with PLP binding
Fig. 2. SDS-PAGE of HBV-␻-TA. Lanes: (1) protein marker (molecular mass 116.0,
66.2, 45.0, 35.0, 25.0, 18.4, 14.4 kDa); (2) purified enzyme; (3) soluble fraction of cell
extract; (4) precipitate fraction of cell extract.

38
J. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
120
100
80
60
40
20
0
120
100
80
60
40
20
0
20 25 30 35 40 45 50 55
4
5
6
7
8
9
10 11
Temperature(
(a)
)
pH
(b)
Fig. 3. Effects of temperature (a) and pH (b) on the enzyme activity. Reactions were carried out at various temperatures from 25 ◦C to 50 ◦C (a) and various pHs from 5.0
to 10.0 (b), respectively. The buffers for different pHs were sodium acetate (pH 3.8–5.6), sodium phosphate (pH 5.8–7.6), boric acid–borax (pH 7.8–9.2) and borax sodium
hydroxide (pH 9.3–10.1).
pocket (including catalytic lysine residue) [36]. Several key active
site residues in the binding pockets were identified using the
crystal structure of ␻-transaminase from Paracoccus denitrificans
[36]. The large pockets suitably accommodated the aryl group,
while the small pocket suitably accommodated methyl group [31].
In addition, the steric constraint in small pocket was studied using
free energy analysis method with structurally similar substrates
such as (S)-1-methylbenzylamine and (S)-1-phenylpropylamine
[37]. In this study, the enzyme activity decreased when the methyl
group was replaced with ethyl and propyl groups because of the
increasing steric constraint in the small binding pocket of HBV-
␻-TA. Most of the aliphatic amines showed relatively low activity
compared with aromatic amines. Short-chain ␤-amino acid esters
showed considerable activity (1.7 U/mg for dl-3-aminobutyric
acid ethyl ester (D14)), whereas aromatic ␤-amino acids such as
dl-␤-phenylalanine (D16) was inert. ␤-alanine (D15), a typical
substrate of ␻-transaminases, showed low activity. Overall, amino
acids were less reactive substrates than amines.
The amino acceptor specificity of HBV-␻-TA was examined by
measuring the initial rate with (S)-␣-MBA as the amino donor [31].
As shown in Table 2, HBV-␻-TA accepted a wide range of amino
acceptors. Pyruvate ethyl ester (A2) was the most active amino
acceptor among the tested carbonyl compounds. In addition,
pyruvate (A1), benzaldehyde (A12), salicylaldehyde (A16) and gly-
oxylate (A26) also showed good activity, whereas ␣-ketoglutarate
(A6) was inert. Glyoxylate is a cheaper amine acceptor than
pyruvate and may be used in the large-scale kinetic resolution
[38]. It is noteworthy that the enzyme showed distinct activity
toward ethyl acetoacetate (1.7 U/mg) (A3), ethyl propionylacetate
(0.5 U/mg) (A4) and levulinic acid (0.4 U/mg) (A5). The ee values
of all the corresponding products exceeded 99%. To the best of
our knowledge, there was no reported method to produce (S)-3-
aminopentanoic acid ethyl ester and (S)-4-aminovaleric acid using
transaminase as catalyst, suggesting that HBV-␻-TA might have
potential for use in the asymmetric synthesis of some valuable
enantiomerically pure unnatural amino acids. Relatively high
activity was observed for most of the tested aldehydes, while all
the ketones showed poor activity. In addition, this enzyme showed
considerable activity toward ␣,␤-unsaturated aldehydes such
as 2,6-dimethyl-5-heptenal (A19), trans-3-(3-pyridyl) acrolein
(A23) and ethyl trans-4-oxo-2-butenoate (A25). These carbonyl
compounds have not been reported as the substrates of ␻-TA
before. Overall, HBV-␻-TA showed higher activity toward keto
acids, keto esters and aldehydes than ketones.
3.4. Asymmetric synthesis of enantiomerically pure amine
compounds
The feasibility of HBV-␻-TA for the synthesis of optically pure
amine compounds by asymmetric amino transfer reaction was
tested with different carbonyl compounds as the amino acceptor
(Fig. 1a). The results are listed in Table 3. All the prochiral carbonyl
compounds were aminated with excellent enantioselectivity.
4-Phenyl-2-butanone and acetophenone were aminated with
low conversions which might be caused by the unfavorable ther-
modynamic equilibrium, poor solubility and severe inhibitions of
ketone substrate [28,39,40].
␻-Transaminase is an attractive alternative biocatalyst for the
production of optically pure unnatural amino acids. Levulinic
acid was aminated by HBV-␻-TA to give (S)-4-aminovaleric acid
with 66% conversion and high ee value (99%). This is the first
report that optically pure (S)-4-aminovaleric acid was produced
with transaminases as biocatalyst. It has been reported that the
transaminase-catalyzed amination of ␤-keto acids giving ␤-amino
acids was hindered by their instability caused by spontaneous
decarboxylation of the carboxyl moiety [21,27]. In this study, HBV-
␻-TA can catalyzed the amino transfer to the stable ␤-keto esters
(ethyl acetoacetate and ethyl propionylacetate) to give optically
pure ␤-amino acid esters, which could be hydrolyzed to the corre-
sponding ␤-amino acids, offering an alternative approach to access
these compounds by transaminase [21]. It was the first report that
optically pure (S)-3-aminopentanoic acid ethyl ester was produced
120
100
80
120
100
80
60
40
20
0
60
40
20
0
25 30 35 40 45 50 55 60 65
4
5
6
7
8
9
10 11
Temperature(
(c)
)
pH
(d)
Fig. 4. Effects of temperature (c) and pH (d) on the enzyme stability. The remaining activity was assayed after 20 min incubation at certain temperatures (c) or 72 h incubation
at 4 ◦C in the specific pH buffers (d).

J. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 32–39
39
with transaminases. The conversion for ethyl acetoacetate was 89%,
Appendix A. Supplementary data
while that for ethyl propionylacetate was only 17%. The relatively
low conversion of ethyl propionylacetate may be due to the lower
activity caused by the steric hindrance in small pocket of the ␻-TA
[31]. Christopher et al. [29] enlarged the substrate binding pockets
of the ␻-TA from Arthrobacter sp. KNK168 by protein engineering
methods and endowed a new enzyme with extraordinary ability
to asymmetrically synthesize sitagliptin using high concentration
of isopropylamine as amino donor. The small pocket amino acid
residues are mainly engaged in the binding step but not signifi-
cantly involved in the catalytic step [37], indicating that the small
pocket of HBV-␻-TA may be enlarged to relieve the steric con-
straint toward bulky substituents by protein engineering in the
future.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.2013.
11.013.
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Acknowledgements
This work was funded by the National Natural Science Foun-
dation of China (Grant No. 21072151), National Basic Research
Program of China (973 Program, No. 2011CB710800) and the CAS
Special Grant for Postgraduate Research, Innovation and Practice
(Grant No. Y2J8041021).