Effects of polyhydroxy compounds on enzymatic synthesis of L-tryptophan catalyzed by tryptophan synthase

Article abstract of DOI:10.1007/s10562-011-0743-7

The effects of polyethylene glycol (PEG) of different molecular (1000, 2000), glycerol, ethylene glycol on the catalytic activity of tryptophan synthase were studied. The results indicated that the addition of PEG 2000 increased the enzymatic activity of tryptophan synthase. The enzymatic activity of tryptophan synthase was enhanced 25.2% by 10 g L^-1 PEG 2000. Reaction conditions were optimized by using 10 g L^-1 PEG 2000 at pH 9 and 40 °C. L-Serine conversion rate reach 89.9% under the optimal conditions. The kinetic parameters indicated the specificity of TSase to substrate was improved. Graphical Abstract: The mechanism of reaction catalyzed by tryptophan synthase [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10562-011-0743-7

Catal Lett (2012) 142:282–286
DOI 10.1007/s10562-011-0743-7
Effects of Polyhydroxy Compounds on Enzymatic Synthesis
of L-Tryptophan Catalyzed by Tryptophan Synthase
•
Lisheng Xu Zhiyuan Wang Junzhong Liu
•
•
•
Pingting Mao Hongjuan Zhang Ji Gao
•
•
•
Qian Liu Qingcai Jiao
Received: 1 October 2011 / Accepted: 11 November 2011 / Published online: 29 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The effects of polyethylene glycol (PEG) of
different molecular (1000, 2000), glycerol, ethylene glycol
on the catalytic activity of tryptophan synthase were
studied. The results indicated that the addition of PEG
2000 increased the enzymatic activity of tryptophan syn-
thase. The enzymatic activity of tryptophan synthase was
enhanced 25.2% by 10 g L-1 PEG 2000. Reaction condi-
tions were optimized by using 10 g L-1 PEG 2000 at pH 9
and 40 °C. L-Serine conversion rate reach 89.9% under the
optimal conditions. The kinetic parameters indicated the
specificity of TSase to substrate was improved.
fermentation method, direct fermentation method, and
enzymatic method. The enzymatic method using trypto-
phan synthase (TSase; EC 4.2.1.20) or tryptophanase
(Tpase; EC 4.1.99.1) has merits in terms of high purity of
the product. Tryptophan synthase, which is mainly found in
bacteria and plants, catalyzes L-tryptophan synthesis from
indole and L-serine [3–5]. Enzymatic synthesis of L-ha-
lotryptophans catalyzed by tryptophan synthase [6].
Structures of tryptophan synthase catalysis were studied
[7–9]. Enzymatic conversion method for production of
L-tryptophan from indole and L-serine was developed by
tryptophan synthase in our laboratory. The mechanism of
L-tryptophan from L-serine and indole catalyzed by tryp-
tophan synthase is suggested in Scheme 1. We found that
the activity of tryptophan synthase was not high in our
previous study. We hope to improve the enzymatic activity
of tryptophan synthase by adding polyhydroxy compounds.
The effects of polyhydroxy compounds on enzymatic
synthesis of L-tryptophan catalyzed by tryptophan synthase
were poorly studied.
Keywords Tryptophan synthase ꢀ Polyethylene glycol ꢀ
Additives ꢀ Kinetic parameters
1 Introduction
L-Tryptophan is an essential amino acid, required for
metabolism in mammals, which depend on plants and
microorganisms for its supply [1]. L-Tryptophan has a
potential market as an additive for animal feed, with esti-
mates indicating a potential annual consumption of
11,000 t [2]. There are three methods for microbial pro-
duction of L-tryptophan, i.e., the precursor-conversion
The native conformation of many proteins and enzymes
can be stabilized by surfactants and polyhydroxy molecules
such as sugar, polyethylene glycols (PEGs), and glycerol,
but there are also experimental results that indicate the
direct binding of PEGs to proteins [10–12]. PEGs present
attractive physical, chemical, and biological properties
such as very low toxicity, good solubility in water and
many organic solvents, and low melting point. Furthermore
PEGs are economical and easily available in a variety of
molecular weights [13]. In the present study, PEG of dif-
ferent molecular (1000, 2000), glycerol, ethylene glycol on
the catalytic activity of tryptophan synthase were investi-
gated. The kinetic parameters, fluorescence spectra and
other factors such as pH, temperature, substrate mole
concentration and reaction time were studied.
L. Xu ꢀ Z. Wang ꢀ J. Liu ꢀ P. Mao ꢀ H. Zhang ꢀ J. Gao ꢀ
Q. Liu ꢀ Q. Jiao (&)
State Key Laboratory of Pharmaceutical Biotechnology,
School of Life Science, Nanjing University, Nanjing 210093,
People’s Republic of China
e-mail: jiaoqc@yahoo.com.cn
L. Xu
Department of Chemistry and Life Science, Suzhou University,
Suzhou 234000, People’s Republic of China
123

Effects of Polyhydroxy Compounds
283
NH2
OH
3 Results
OH
H2O
O
O
+
NH2
N
3.1 Effect of Polyhydroxy Compounds on TSase
Activity
HO
Tryptophan synthase
H
N
H
Indole
L-tryptophan
L-serine
The effect of additives including Glycerol, ethylene glycol,
PEG 1000, PEG 2000 were examined at 30 °C. Among the
above additives, PEG 2000 has positive effect. As show in
Fig. 1, TSase activity was enhanced to a maximum 25.2%
compared to that in pure buffer at L-serine concentration of
100 mmol L-1. TSase activity increased with the increas-
ing PEG 2000 concentration supply, peaking at PEG 2000
concentration of 10 g L-1, and decreased as the PEG
concentration increased from 15 to 20 g L-1. We can
classify the four polyols in terms of their protective
effect as follows: ethylene glycol \ glycerol \ PEG
1000 \ PEG 2000.
Scheme 1 The mechanism of reaction catalyzed by tryptophan
synthase
2 Experimental
2.1 Materials and Reagents
Tryptophan synthase and L-serine were purchased from
Sigma (St. Louis, Mo, USA). Glycerol, ethylene glycol,
PEG 1000, PEG 2000 and indole were purchased from
Shanghai Chemical and Medicine Co. Ltd. All chemicals
are of analytical grade.
3.2 Effect of pH and Temperature on TSase Activity
2.2 TSase Activity Assay
Enzyme activity can be affected by environmental factors
such as pH and temperature. In this study, using L-serine
and indole as substrate, a pH range from 4 to 10 was used
to determine the optimal initial pH. As shown in Fig. 2,
with the initial pH increasing from 4 to 9, TSase activity
increased significantly and reached its maximum at pH 9
when PEG 2000 concentration was 10 g L-1. TSase
activity reached its maximum at pH 8 without PEG 2000.
The optimal temperature was about 40 °C at pH 8.0 when
PEG 2000 concentration was 10 g L-1 (Fig. 3). The opti-
mal temperature was about 35 °C at pH 8.0 without PEG
2000 (Fig. 3).
By measuring the tryptophan synthesized from L-serine
and indole, TSase activity was determined with amino
acid analyzer (Hitachi L-8900, Japan). The reaction was
carried out in 50 mL containing 5 mmol L-serine,
5 mmol indole, additives of polyhydroxy compounds and
0.5 g TSase at different temperature and pH. One enzyme
unit (U) was defined as the amount of TSase that syn-
thesize 1 lmol of L-tryptophan per min from L-serine
and indole. The specific activity is defined as unit/g of
TSase.
2.3 Determination of Kinetic Parameters
The kinetic parameters, Km and Vmax values of TSase were
determined by measuring initial rates of the reactions with
L-serine as substrate. Km and Vmax values were calculated
from Lineweaver–Burk plots by linear regression analysis.
130
PEG2000
PEG1000
Glycerol
Ethylene glycol
120
110
Vꢁꢁ1 ¼ fKm=Vmaxg½Sꢂꢁ1þVꢁ1
Where [S] was the concentration of substrate, v and Vmax
represented the initial and maximum rate of reaction,
respectively.
max
100
90
80
2.4 Fluorescence Spectra Measurement
70
The fluorescence spectra of TSase at various concentra-
tions of PEG 2000 were recorded on Shimadzu RF-5300PC
spectrofluorometer (Kyoto). The fluorescence spectra of
TSase were measured in the presence of PEG 2000 at
25 °C in a 1 cm path length cell. TSase was excited at
295 nm, and the emission spectra were recorded from 300
to 400 nm.
60
0
5
10
15
20
-1
Additive concentration (g L )
Fig. 1 Effect of polyhydroxy compounds concentration on the TSase
activity. The reaction was carried out in 50 mL containing 5 mmol
L-serine, 5 mmol indole, additives and TSase at 30 °C, pH = 7. The
concentrations of L-tryptophan were measured after 2 hours
123

284
L. Xu et al.
With PEG 2000
Without PEG2000
with PEG2000
without PEG2000
100
90
80
70
60
50
40
100
90
80
70
60
50
40
0
100
200
300
400
500
4
5
6
7
8
9
10
Substrate concentration (mmol/l)
pH
Fig. 4 Effect of substrate concentration on the TSase activity. The
reaction was carried out in 0.5 g PEG and TSase at pH = 9 (filled
square). The reaction was carried out in 0 g PEG and TSase at
pH = 8 (filled circle). The concentrations of L-tryptophan were
measured after 2 hours
Fig. 2 Effect of pH on the TSase activity. The reaction was carried
out in 50 mL containing 5 mmol L-serine, 5 mmol indole, 0.5 g PEG
and TSase at 30 °C (filled square). The reaction was carried out in
50 mL containing 5 mmol L-serine, 5 mmol indole, 0 g PEG and
TSase at 30 °C (filled circle). The concentrations of L-tryptophan
were measured after 2 hours
3.3 Effect of Substrate Concentration and Reaction
Time
was 2 hours when PEG 2000 concentration was 10 g L-1
.
L-Serine conversion rate reach 89.9% under the optimal
conditions. The optimal reaction time was 3.5 hours with
PEG 2000 (Fig. 5).
The relationship between synthesis of TSase activity and
substrate (L-serine) is shown in Fig. 4. The highest TSase
activity occurred at L-serine concentration 100 mmol L-1
.
3.4 Effect of Kinetic Parameters and Fluorescence
Spectra of TSase
The results indicated that TSase activity increased with
L-serine concentration increased. TSase activity inhibition
was observed at a high substrate concentration. The opti-
mal substrate concentration would be 100 mmol L-1 when
PEG 2000 concentration was 10 g L-1. The reaction time
The kinetic parameters of TSase activity was presented
in Table 1. The Km, Vmax, Kcat and Kcat/Km of the
enzyme in the absence of PEG 2000 were 4.309 mM,
105
Without PEG2000
with PEG2000
without PEG2000
With PEG2000
100
95
90
85
80
75
70
65
60
90
80
70
60
50
40
30
10
20
30
40
50
60
0
1
2
3
4
5
Temperature (°C)
Time (h)
Fig. 3 Effect of temperature on the TSase activity. The reaction was
carried out in 50 mL containing 5 mmol L-serine, 5 mmol indole,
0.5 g PEG and TSase at pH = 9 (filled square). The reaction was
carried out in 50 mL containing 5 mmol L-serine, 5 mmol indole, 0 g
PEG and TSase at pH = 8 (filled circle). The concentrations of L-
tryptophan were measured after 2 hours
Fig. 5 Effect of reaction time on the TSase activity. The reaction was
carried out in 50 mL containing 5 mmol L-serine, 5 mmol indole,
0.5 g PEG and TSase at 40 °C, pH = 9 (filled square). The reaction
was carried out in 50 mL containing 5 mmol L-serine, 5 mmol
indole, 0 g PEG and TSase at 35 °C, pH = 8 (filled circle)
123

Effects of Polyhydroxy Compounds
285
4 Discussion
Table 1 Kinetic parameters of TSase in the presence of PEG 2000 at
40 °C, pH = 9
From these experiments, we can classify the four polyols
in terms of their protective effect as follows: ethylene
glycol \ glycerol \ PEG 1000 \ PEG 2000. The hydro-
xyl groups of polyhydroxy compounds improved enzy-
matic stabilization. Enzymatic stabilization is related with
the number of hydroxyl of polyhydroxy compounds. It is
noticeable that this classification follows the increase in
the polyol molecular masses. These results are in
accordance with previous studies on the effect of poly-
hydric alcohols on enzyme stability [14–16]. Some
studies have shown that polyhydric alcohol behavior is
much different. Bull and Breese reported that n-alcohols
bound strongly to protein molecules and dehydrate them
which lead to their denaturation [17]. However, Nurul
Alam et al. [18] found that n-alcohols act as activators of
thermolysin-catalyzed peptide synthesis. The mechanism
by which polyhydric alcohols stabilize biological mac-
romolecules is generally well documented. Polyhydric
alcohols do not denature proteins and contribute to
enzymatic stabilization [19]. In the present study, the
effect of additives including Glycerol, ethylene glycol,
PEG 1000, PEG 2000 were examined. The result showed
that PEG 2000 has positive effect, but ethylene glycol
has negative effect. The reason may be PEG 2000 has
been used to improve the TSase stabilization. The speed
of reaction, catalyzed by TSase is significantly affected
by pH and temperature. Generally, a high pH and tem-
perature inhibit reaction. The TSase activity reached its
maximum at pH 9.0 and 40 °C when PEG 2000 con-
centration was 10 g L-1, which shifted 1 pH Units by
PEG 2000 and was beneficial to the stability of TSase.
The speed of reaction was improved by PEG 2000. The
entails that the enzymatic efficiency expressed as Kcat/Km
is raised to about six times that obtained in buffer. The
higher ratio of Kcat/Km increase indicated TSase has a
better specificity to the substrate due to the changes of
environment around L-serine. The kinetic parameters
indicated the specificity of TSase to substrate was
improved. PEG could dehydrate availability and reduce
hydrolyze effect of protein so as to stabilize the protein
structure [13]. Fluorescence spectra of TSase indicated
that the structure of TSase was not changed with adding
10 g L-1 of PEG 2000. The microenvironment of TSase
amino acid residues was changed with an increase of the
PEG 2000. PEG 2000 can form hydrogen bonds and be
helpful to form a solvent layer of the protein surface.
PEG 2000 can draw away water from their environment
for their own hydration, generating a solvent layer of
TSase that increases the surface tension and solution
viscosity. Furthermore, PEG 2000 is a nonpolar polyol
and can bind to the hydrophobic sites of proteins. PEG
PEG
2000
Vmax
(mmol
min-1
Km
(mM)
Kcat
(s-1
Kcat/Km
(mM-1 s-1
)
)
(g L-1
)
per-1 g-1
)
0
0.163
0.169
0.314
0.236
0.209
4.309
3.369
1.386
2.317
3.316
12.3
12.7
23.6
17.7
15.7
2.85
3.77
17.0
7.64
4.73
5
10
15
20
0.163 mmol min-1 per-1 g-1
,
12.3 s-1and 2.85 mM-1
s-1, respectively. When the concentration of PEG 2000
was 10 g L-1, the value of Km, Vmax, Kcat and Kcat/Km of
the enzyme were 1.386 mM, 0.314 mmol min-1
per-1 g-1
, , respectively.
23.6 s-1and 17.0 mM-1 s-1
Fluorescence spectra of TSase was measured in the pres-
ence of PEG 2000. The maximal intensity was observed at
330 nm. No change in the spectra was observed at the
concentration 10 g L-1 of PEG 2000, suggesting that
10 g L-1 of PEG 2000 did not affect the structure of TSase
(Fig. 6). The emission peak at 330 nm was not shifted, but
the shape of the spectra between 310 and 350 nm changed
with the increase in PEG 2000 concentration. This indi-
cated that the microenvironment of TSase amino acid
residues was changed with an increase of the PEG 2000
from 20 to 30 g L-1
.
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
300
320
340
360
380
400
Wavelength (nm)
Fig. 6 Fluorescence spectra of TSase in the presence of PEG 2000.
(Red line: 10 g L-1 PEG 2000, Black line: 0 g L-1 PEG 2000, Green
line: 20 g L-1 PEG 2000, Blue line: 30 g L-1 PEG 2000). The
fluorescence spectra of TSase were measured in the presence of PEG
2000 at 25 °C in a 1 cm path length cell. TSase was excited at
295 nm, and the emission spectra were recorded from 300 to 400 nm
123

286
L. Xu et al.
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5 Conclusion
In summary, PEG 2000 was found to be efficient addi-
tive in the TSase-catalyzed reactions. The enzymatic
activity of tryptophan synthase was enhanced 25.2% by
10 g L-1 PEG 2000 at pH 9 and 40 °C. L-Serine con-
version rate reach 89.9% under the optimal conditions.
No change in the fluorescence spectra was observed at
the concentration 10 g L-1 of PEG 2000, suggesting that
10 g L-1 of PEG 2000 did not affect the structure of
TSase. The entails that the enzymatic efficiency expres-
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Acknowledgment This work was supported by the National
Technology-Innovation Fund (02CJ-13-01-16) and the Open Fund of
State Key Laboratory of Pharmaceutical Biotechnology of Nanjing
University, P.R. China.
123