Enhancing thermostability and organic solvent tolerance of ω-transaminase through global incorporation of fluorotyrosine

Article abstract of DOI:10.1002/adsc.201300706

Here, we have utilized the incorporation of non-canonical amino acids as a tool kit to improve enzyme properties for organic synthesis applications. The global incorporation of 3-fluorotyrosine (FY) into ω-transaminase (ω-TA) to give ω-TA[FY] enhanced the thermostability and organic solvent tolerance without altering substrate specificity and enantioselectivity. Moreover, ω-TA[FY] was able to completely convert 25 mM of acetophenone into (S)-1-phenylethylamine (ee>99%) in the presence of 20% DMSO (v/v) which is 2-fold higher when compared to wild-type ω-TA.

Full text of DOI:10.1002/adsc.201300706

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DOI: 10.1002/adsc.201300706
Enhancing Thermostability and Organic Solvent Tolerance of
w-Transaminase through Global Incorporation of Fluorotyrosine
a
a
b
Kanagavel Deepankumar, Minsu Shon, Saravanan Prabhu Nadarajan,
a
a
c
d
Giyoung Shin, Sam Mathew, Niraikulam Ayyadurai, Byung-Gee Kim,
Sei-Hyun Choi, Sang-Hyeup Lee, and Hyungdon Yun *
School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, South Korea
Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea
e
f
b,
a
b
E-mail: hyungdonyun@gmail.com
Department of Biotechnology, Central Leather Research Institute, Chennai 600-020, India
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, South Korea
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, CA 92037, USA
c
d
e
f
Department of Life Chemistry, Catholic University of Daegu, Gyeongbuk, 712-749, South Korea
Received: August 15, 2013; Revised: January 7, 2014; Published online: March 11, 2014
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201300706.
Abstract: Here, we have utilized the incorporation
of non-canonical amino acids as a tool kit to im-
prove enzyme properties for organic synthesis ap-
plications. The global incorporation of 3-fluorotyro-
sine (FY) into w-transaminase (w-TA) to give w-
TA[FY] enhanced the thermostability and organic
solvent tolerance without altering substrate specific-
ity and enantioselectivity. Moreover, w-TA[FY] was
able to completely convert 25 mM of acetophenone
into (S)-1-phenylethylamine (ee>99%) in the pres-
ence of 20% DMSO (v/v) which is ~2-fold higher
when compared to wild-type w-TA.
tions, various techniques have been developed and ex-
tensively studied. For example, to remove inhibitory
by-product coupling the reaction system and two-
phase reaction system were well developed. Enzyme
engineering through rational and irrational ap-
proaches is another way to improve the efficiency of
w-TA reactions. Recently, w-TA was successfully engi-
neered for the manufacture of sitagliptin. However,
enzyme engineering using non-canonical amino acids
(NCAA) has not been reported for developing a w-
TA mutant with improved characteristics.
[
3]
Incorporation of NCAA is a fascinating tool in syn-
thetic biology to create proteins with novel function-
ality. Genetic code engineering (residue-specific or
global incorporation), which is the most frequently
used methodology, reassigns the canonical amino acid
Keywords: 3-fluorotyrosine; non-canonical amino
acids; organic solvent tolerance; thermostability; w-
transaminase
[
4]
of the sense codon with NCAA. In this method,
NCAAs are isostructural to canonical amino acids
and therefore recognized by the endogenous host cell
[
5]
machinery. Among NCAAs, fluorinated amino acids
Biocatalysts are increasingly prevalent in the large- (FAA) have gained great interest owing to their
scale synthesis of optically pure amine compounds, unique characteristics. Global incorporation of FAA
which are mainly used for the preparation of pharma- into proteins often yields structures with minimal
ceutical and agrochemical target molecules such as local changes in the interior, while also producing
[
1]
peptides, proteins and many other natural products.
Among the different biocatalysts, w-transaminases because the presence of fluorine can lead to different
w-TAs) have recently gained a lot of attention as bond energies, electron distribution, hydrogen bonds,
large global changes in the properties of the protein
(
[
6]
a promising catalyst due to their ability to produce and steric interactions. Given the significance of in-
a wide range of optically pure amines and unnatural corporation of FAA into proteins, many studies have
amino acids. w-TAs generally show rapid reaction been carried out extensively. For example, global in-
rates, broad substrate specificity, high enantioselectivi- corporation of 4-fluorophenylalanine (FF) into S5
ty and lack of requirement for external cofactor re- phosphotriesterase dimer was found to stabilize the
[2]
[7]
[2]
generation. To improve the efficiency of w-TA reac- protein and lead to enhanced refoldability and im-
Adv. Synth. Catal. 2014, 356, 993 – 998
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993

Kanagavel Deepankumar et al.
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proved activity for substrates at elevated tempera-
[7g]
ACHTUNGTRENNUNGt ures. The goal of the present study is to improve
the biochemical properties of w-TA through global in-
corporation of FAAs such as FF, 3-fluorotyrosine
(
FY), and 5-fluorotryptophan (FW).
For the global incorporation of FAA, w-TA from
[
8]
Vibrio fluvialis JS17, one of the most well character-
ized w-TAs, was selected and incorporation of FAA
[
9]
was carried out as described earlier. The w-TA con-
tains 16 Tyr, 27 Phe and 8 Trp residues. In the SDS-
PAGE analysis, a total cell extract of w-TA incorpo-
rated with FAAs such as FY, FF and FW showed high
yield expression. However, among them only w-
TA[FY] was expressed as a soluble protein (see Fig-
ure S1 in the Supporting Information). Subsequently,
we purified the w-TA and w-TA[FY] using an Ni-
NTA affinity column at 48C (see Figure S2 in the
Supporting Information). Electrospray ionization
mass spectrometric (ESI-MS) analysis of whole-mass
(
see Figure S3 in the Supporting Information) and
tryptic digested fragments (see Tables S1 and S2 in
the Supporting Information) revealed that FY was
successfully incorporated into w-TA with a high incor-
poration efficiency (>95%). The observed molecular
mass of w-TA[FY] (54,579 Da) matched well with the
expected molecular mass (54,578.44 Da).
To examine the effects of incorporation of FY into
w-TA, the purified enzymes were first subjected to an
activity assay, which was conducted using 10 mM (S)-
1
-phenylethylamine (PBA, a typical substrate for w-
TA) and 10 mM pyruvate in 100 mm Tris/HCl buffer
pH 8.0). One unit was defined as the amount of
Figure 1. (A) Residual activity of purified enzyme
(
À1
(
0.05 mgmL ) after 2 h of incubation in 100 mM Tris/HCl
enzyme that catalyzed the formation of 1 mmol of ace- buffer (pH 8.0) containing 50 mM (S)-PBA, 20 mM pyruvate
tophenone per minute. w-TA[FY] (51.6 U/mg) or 100 mM PLP at 608C and 708C. (B) Thermal stability of
showed 20% enhanced activity compared to wild-type w-TA and w-TA[FY] in 100 mM Tris/HCl buffer (pH 8.0)
containing 100 mM PLP at 508C.
w-TA (41.4 U/mg). We expected that incorporation of
FY into w-TA will increase thermal stability of the
enzyme. To validate that, the thermal stability of
enzyme was examined under different conditions be- (7.0 h) was 2.3-fold higher than that of w-TA (3.0 h)
cause (S)-PBA hasa diminishing effect on stability, (Figure 1B). In addition, the results (Figure 1B) were
whereas pyruvate and pyridoxal 5’-phosphate hydrate comparable with those of Figure S5-D showing a simi-
[
8b]
(
PLP) have a beneficial effect on stability. The re- lar residual activity ~63% for w-TA and ~89% for w-
sidual activities of w-TA and w-TA[FY] were exam- TA[FY] at 508C for 2 h in the presence of PLP. A
ined after 2 h of incubation in 100 mM Tris/HCl recent report showed that the replacement of Phe
buffer (pH 8.0) at 608C and 708C in the presence of with FF increased the refoldability and thermoactivity
5
0 mM (S)-PBA, 20 mM pyruvate or 100 mM PLP of S5 phosphotriesterase by stabilizing the interac-
[
7g]
(
Figure 1A, see also Figure S5 in the Supporting In- tions across the dimer interface. w-TA has 16 Tyr
formation). In all the cases, w-TA[FY] showed better residues and is functional as a dimer. The dimer inter-
stability than the wild-type w-TA. For example, the face of w-TA is mediated in part by interactions with
residual activities of w-TA and w-TA[FY] at 708C Y49, Y76, Y82 and Y165 (PDB ID: 3NUI, see Fig-
with 100 mM PLP were 3.3% and 36% of the initial ure S4 in the Supporting Information). The fluorina-
activity, respectively, and the error rate is <5%. Also tion of these residues may help for the enhancement
for w-TA and w-TA[FY] at 708C without additive the of thermostability.
residual activities were 0.4% and 17% of the initial
Since co-solvent (e.g., DMSO) is often added to
activity, respectively. Furthermore, the half-lives of the reaction mixture to dissolve the substrate in w-TA
[
2]
the enzymes were measured in the presence of reactions, the stability of the enzyme was examined
00 mM PLP at 508C. The half-life of w-TA[FY] in the presence of DMSO. The residual activity was
1
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Enhancing Thermostability and Organic Solvent Tolerance
presence of hostile environments such as organic sol-
[
10]
vents and surfactants.
Next, we were curious to know whether the w-
TA[FY] had effects on the basic biochemical proper-
ties such as pH optimum, substrate and product inhib-
ition. Therefore, the basic properties of w-TA and w-
TA[FY] were examined. The optimum pH for both
enzymes is 9.0 (see Figure S6 in the Supporting Infor-
mation). The w-TA[FY] showed slightly higher
enzyme activity than the wild-type w-TA at all tested
pH values. When substrate inhibitions by (S)-PBA
and pyruvate were examined, both enzymes showed
the highest activities with 100 mM (S)-PBA, 10 mM
pyruvate (see Figures S7 and S8 in the Supporting In-
formation). However, w-TA[FY] showed a slightly re-
lieved substrate inhibition. For example, in the pres-
ence of 20 mM pyruvate, the activities of w-TA and
w-TA[FY] with 1M (S)-PBA were 15% and 35% of
their activities with 100 mM (S)-PBA, respectively. In
the presence of 50 mM (S)-PBA, the activities of w-
TA and w-TA[FY] with 100 mM pyruvate were 53%
and 65% of their activities with 10 mM pyruvate, re-
spectively. In the cases of product inhibition by l-Ala
and acetophenone, both enzymes w-TA and w-
TA[FY] were affected similarly (see Figures S9 and
S10 in the Supporting Information).
The substrate specificities of enzymes toward vari-
ous amines were examined with 10 mM amines and
10 mM pyruvate. The incorporation of FY does not
influence the substrate specificity of the enzyme
h of incubation in 100 mM Tris/HCl buffer (pH 8.0), within an error rate <5% (Figure 3B). The enantiose-
00 mM PLP, purified enzyme (0.05 mgmL ) and different lectivity (E) of the enzyme is a crucial factor for the
concentrations of DMSO at 378C. (B) The stability of w-TA application of w-TA[FY] for the production of chiral
Figure 2. (A) Residual activity of w-TA and w-TA[FY] after
2
1
À1
and w-TA[FY] in 100 mM Tris/HCl buffer (pH 8.0) contain-
ing 100 mM PLP and 20% (v/v) DMSO at 508C.
amines. Therefore, E values of the enzymes were
measured through the kinetic resolution of various
amines (1, 3, 4, 7 and 9 in Figure 3A). The enzyme re-
measured after 2 h of incubation in 100 mM Tris/HCl actions were carried out with 10 mM racemic amine,
À1
buffer (pH 8.0) containing 100 mM PLP with different 20 mM pyruvate and enzyme (0.2 mgmL ) for 12 h
concentrations of DMSO (0–50%, v/v) at 378C (Fig- at 378C. The E values of both enzymes toward 1, 3, 7
ure 2A). The residual activity of w-TA and w-TA[FY] and 9 were found to be >100 and E values for w-TA
decreased as the concentration of DMSO increased. and w-TA[FY] towards 4 were 60 and 62, respectively
As expected, w-TA[FY] showed higher stability than (Table 1). These results indicated that incorporation
w-TA. For example, in the presence of 50% (v/v) of FY into w-TA did not affect the substrate specifici-
DMSO, the residual activity of w-TA[FY] was 90% of ty and enantioselectivity of enzyme.
its original activity, whereas the wild-type w-TA re-
Finally, the asymmetric synthesis of (S)-PBA from
tained 51% of its original activity. Also the half-lives acetophenone with w-TA or w-TA[FY] was carried
of the enzymes were measured in the presence of out using l-Ala as an amino donor, in which the in-
100 mM PLP and 20% (v/v) DMSO at 508C. The half- hibitory by-product pyruvate was removed by lactate
lives of w-TA and w-TA[FY] were found to be 7.0 dehydrogenase (LDH). The cofactor NADH required
and 48 h, respectively (Figure 2B). The w-TA[FY] by LDH was recycled employing glucose and glucose
[
11]
showed to be more stable than the wild-type w-TA in dehydrogenase (GDH). w-TA[FY] completely con-
the presence of 20% DMSO. Moreover, the results in verted 10 mM and 25 mM acetophenone into (S)-
Figure 2A were comparable with those of Figure 2B PBA in the presence of 20% DMSO (v/v) (ee>99%).
showing similar residual activity, ~80% for w-TA and However, w-TA gave 70.9% conversion in the 10 mM
~
96% for w-TA[FY] at 20% DMSO for 2 h. Similarly, acetophenone reaction and 50.9% conversion in the
recent reports demonstrate that the incorporation of 25 mM acetophenone reaction after 24 h in the pres-
NCAA enhances the activity of lipase even in the ence of 20% DMSO (v/v) (Figure 4). These results
Adv. Synth. Catal. 2014, 356, 993 – 998
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995

Kanagavel Deepankumar et al.
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Figure 4. The asymmetric synthesis of (S)-PBA from aceto-
phenone in 3 mL of 100 mM Tris/HCl buffer (pH 8.0) con-
taining 100 mM l-Ala, lactate dehydrogenase (62 U/mL),
glucose dehydrogenase (5 U/mL), NADH (1 mM), purified
À1
enzyme (0.5 mgmL
2
for 10 mM acetophenone and
À1
mgmL for 25 mM acetophenone) and 20% (v/v) DMSO
at 378C. Reproducibility was tested by performing duplicate
experiments.
Figure 3. Substrate specificities of w-TA and w-TA[FY]. (A)
Structure of amino donors. (B) Relative reaction rate. Reac-
tion conditions: 10 mM amino donor, 10 mM pyruvate,
1
00 mM Tris/HCl (pH 8.0), and purified enzyme
2
4 h reaction, HPLC analysis showed 93% conver-
À1
(
0.05 mgmL ) at 378C. Initial reaction rate of the enzyme
sion. The (S)-PBA was isolated from this reaction
with 89% yield (see Figures S14 and S15 in the Sup-
porting Information).
towards 1 was taken as 100%.
Table 1. w-TA-catalyzed kinetic resolution of racemic
amines.
In summary, the global incorporation of FY into w-
TA generated an enzyme variant with improved ther-
mal stability and improved DMSO tolerance, while
enantioselectivity, pH optimum, substrate and product
inhibition as well as substrate specificity remained
largely unaltered. Finally, we demonstrated that w-
TA[FY] can serve as a more suitable biocatalyst than
wild-type w-TA for the asymmetric synthesis of chiral
amines. Here, we utilized the incorporation of non-
canonical amino acids as a tool kit to improve
enzyme properties for organic synthesis applications.
One drawback of enzyme engineering with non-can-
onical amino acid mutagenesis is the high cost of the
non-canonical amino acid itself. However, it can be
overcome by the development of a novel and cheap
chemical synthetic route or an in vivo biosynthetic
pathway for the production of non-canonical amino
acids.
[a]
Substrate
w-TA
ee
[%]
w-TA[FY]
Conv.
[%]
[b]
Conv.
%]
E
ee
[%]
E
[
1
3
4
7
9
50.3
43.0
46.5
50.5
35.0
>99 >100 50.6
>99 >100
74.5
80.1
>100 43.5
60 47.5
74.5
83.2
>100
62
>99 >100 50.7
52.5 >100 35.2
>99 >100
52.9 >100
[
a]
The enzyme reactions were carried out in 1 mL of
1
00 mM Tris/HCl buffer (pH 8.0) containing purified
À1
enzyme (0.2 mgmL ), 20 mM pyruvate and 10 mM race-
mic amine for 12 h at 378C. The conversion and ee
values were analyzed using a Crownpak CR column
[12]
(
Daicel Co., Japan).
The enantioselectivity (E) was calculated using the equa-
[
b]
[13]
tion E=ln
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
[(1Àc)
A
H
U
G
R
N
N
(1Àee )]/ln
A
H
U
G
R
N
U
G
ACHTUNGTNERUNNG( 1+ee )].
s
s
clearly demonstrated that w-TA[FY] is a more suita-
ble biocatalyst than wild-type w-TA for the asymmet-
ric synthesis of chiral amines. Finally, a preparative
scale reaction was carried out with 50 mM acetophe-
none (300 mg) using a recombinant whole cell ex-
Experimental Section
Incorporation of Fluorinated Amino Acids into w-TA
pressing w-TA[FY] in 50 mL of 100 mM Tris/HCl Initially, to incorporate FF, FY, and FW into w-TA, we
buffer (pH 8.0) containing 20% DMSO (v/v). After choose polyauxotrophic E. coli strain ME5355 (thi pro arg
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Enhancing Thermostability and Organic Solvent Tolerance
trp tyr phe aroB). Incorporation of fluorinated amino acid
was carried out in the optimized conditions as described ear-
24 h. Then aliquot 0.1 mL of reaction sample was taken for
the conversion analysis by HPLC using a Crownpak CR
column. After 24 h reaction, the product was isolated (see
the Supporting Information).
[9]
lier. Briefly, limiting the concentration of either Tyr
30 mM) or Phe (40 mM) or Trp (7.3 mM) allowed the cells to
(
attain an OD600 0.6–0.8 and the target proteins were induced
with 0.2% l-arabinose followed by simultaneous addition of
non-canonical amino acids (0.5 mM of either FY or FF or
FW) and allowed the expression for 7 h. Lysis of the cells Acknowledgements
was carried out by using BugBuster protein extraction kit
(
Novagen) followed by sonication. Briefly, the collected cell
This research was partially supported by the Basic Science
Research Program through the National Research Founda-
tion of Korea, Ministry of Education, Science and Technolo-
gy, Korea (NRF-2012R1A1A2044222) and also partially sup-
ported by a grant of Global Cosmetics R&D Project, Minis-
try of Health & Welfare, Korea (A10301712131560200).
pellet corresponding to 1 mL of culture was resuspended in
1
1
pernatant was saved as a soluble protein fraction, and pellet
was saved as insoluble fraction. Then the protein expression
level was analyzed by SDS-PAGE (12%).
00 mL of lysis buffer, incubated at room temperature for
0 min, and centrifuged at 9000 g, 48C for 20 min. The su-
Enzyme Assay
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(0.5 mgmL for 10 mM acetophenone and 2 mgmL for
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(
S)-PBA
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(
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buffer (pH 8.0) and 10 mL of DMSO (final concentration,
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Adv. Synth. Catal. 2014, 356, 993 – 998
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asc.wiley-vch.de
997

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