Enzymatic production of 5′-inosinic acid by a newly synthesised acid phosphatase/phosphotransferase

Article abstract of DOI:10.1016/j.foodchem.2012.02.213

5′-Nucleotides including 5′-inosinic acid have characteristic taste and important application in various foods as flavour potentiators. The selective nucleoside acid phosphatase/phosphotransferase (AP/PTase) can catalyse the synthesis of 5′-nucleotides by transfer of phosphate groups. In this study, a 747-bp gene encoding AP/PTase from Escherichia blattae was synthesised. After expression, the recombinant AP/PTase was purified using nickel-NTA. The optimal temperature and pH of this enzyme were 30°C and 5.0, respectively. The activity was partially inhibited by metal ions such as Hg²⁺, Ag⁺ and Cu²⁺, but not by chelating reagents such as EDTA. The values of K_m and V_max for inosine were 40 mM and 3.5 U/mg, respectively. Using this purified enzyme, 16.83 mM of 5′-IMP was synthesised from 37 mM of inosine and the molar yield reached 45.5%. Homology modelling and docking simulation were discussed.

Full text of DOI:10.1016/j.foodchem.2012.02.213

Food Chemistry 134 (2012) 948–956
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Enzymatic production of 50-inosinic acid by a newly synthesised acid
phosphatase/phosphotransferase
⇑
Zhi-Qiang Liu, Ling Zhang, Li-Hui Sun, Xiao-Jun Li, Nan-Wei Wan, Yu-Guo Zheng
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
50-Nucleotides including 50-inosinic acid have characteristic taste and important application in various
foods as flavour potentiators. The selective nucleoside acid phosphatase/phosphotransferase (AP/PTase)
can catalyse the synthesis of 50-nucleotides by transfer of phosphate groups. In this study, a 747-bp gene
encoding AP/PTase from Escherichia blattae was synthesised. After expression, the recombinant AP/PTase
was purified using nickel–NTA. The optimal temperature and pH of this enzyme were 30 °C and 5.0,
respectively. The activity was partially inhibited by metal ions such as Hg2+, Ag+ and Cu2+, but not by che-
lating reagents such as EDTA. The values of Km and Vmax for inosine were 40 mM and 3.5 U/mg, respec-
tively. Using this purified enzyme, 16.83 mM of 50-IMP was synthesised from 37 mM of inosine and the
molar yield reached 45.5%. Homology modelling and docking simulation were discussed.
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 26 November 2011
Received in revised form 13 February 2012
Accepted 29 February 2012
Available online 8 March 2012
Keywords:
Nucleoside
Acid phosphatase/phosphotransferase
50-Inosinic acid
Pyrophosphate
Biotransformation
1. Introduction
et al., 2004) synthesis. It is reported that several microorganisms
including Salmonella typhimurium (Kasahara, Nakata, & Shinagawa,
Nucleotides have been widely used as food additives and phar-
maceutical intermediates (Asano, Mihara, & Yamada, 1999a).
Among the nucleotides, 50-inosinic acid (50-IMP) and 50-guanylic
acid (50-GMP) have received increased attention, because of their
characteristic taste and important application in various foods as
flavour potentiators (Mihara, Ishikawa, Suzuki, & Asano, 2004). Re-
cently, the demand for nucleotides in the food additives market is
increasing, and the production of nucleotides has been well stud-
ied. Nucleotides can be produced by both chemical and biological
methods, including enzymatic reaction and fermentation (Hiroshi,
Katsuaki, & Hitoshi, 1982). The enzymatic phosphorylation process
using acid phosphatase/phosphotransferase (AP/PTase) as biocata-
lyst requires mild reaction conditions (Mori, Iida, Fujio, & Teshiba,
1997; Mori, Iida, Teshiba, & Fujio, 1995), and has potential applica-
tion in industrial production of nucleotides. Currently, screening
and exploration of AP/PTase and its industrial application for the
preparation of 50-nucleotides are hot topics (Mihara et al., 2004).
AP/PTs are classified into two major categories, including non-
specific and specific AP/PTase, according to the substrate specificity
(Mihara, Utagawa, Yamada, & Asano, 2001; Tanaka, Hasan, Hartog,
van Herk, & Wever, 2003). As opposed to the specific AP/PTase,
nonspecific enzymes have a board substrate spectrum, which can
catalyse nucleotide (Asano, Mihara, & Yamada, 1999b), glucose 6-
phosphate (Tanaka et al., 2003), and dihydroxyacetone (Mihara
1991), Zymomonas mobilis, Morganella morganii (Thaller, Berlutti,
Schippa, Lombardi, & Rossolini, 1994), Shigella flexneri (Uchiya,
Tohsuji, Nikai, Sugihara, & Sasakawa, 1996) and Prevotella interme-
dia (Chen et al., 1999) could produce AP/PTase. Mihara et al. puri-
fied an acid phosphatase with regioselective pyrophosphate
nucleoside phosphotransferase activity from M. morganii NCIMB
10466, and cloned its gene, subsequently improving its phospho-
transferase activity by random mutation (Mihara, Utagawa,
Yamada, & Asano, 2000).
In the present study, to explore the production of 50-IMP by
enzymatic methods, the gene encoding AP/PTase from Escherichia
blattae was synthesised and expressed. After purification, the de-
tailed characteristics of this recombinant acid phosphotransferase
were investigated. Nucleoside phosphorylation reaction was stud-
ied as well, using the pyrophosphate as the phosphate donor. This
study showed that this recombinant AP/PTase could possess poten-
tial for the large scale production of nucleotides.
2. Materials and methods
2.1. Bacterial strains, plasmids, and culture conditions
Escherichia coli BL21 (DE3) (Invitrogen, Karlsruhe, Germany)
was used as host for expression. The plasmid pET28b(+) (Novagen,
Darmstadt, Germany) was used for expression of AP/PTase. Luria–
Bertani (LB) (yeast extract 5 g/L, Tryptone 10 g/L, NaCl 5 g/L) was
used for the culture of E. coli and recombinant E. coli strains grown
⇑
Corresponding author. Tel.: +86 571 88320614; fax: +86 571 88320630.
E-mail address: zhengyg@zjut.edu.cn (Y.-G. Zheng).
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.02.213

Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
949
aerobically at 37 °C. To maintain the presence of the plasmids, the
corresponding antibiotics were added to the media for E. coli har-
bouring plasmids. All the chemicals used were of analytical grade
and commercially available.
adding 0.2 ml of 2 M HCl, then centrifuged at 12,000g for 5 min at
4 °C. Quantitative determination of inosine and 50-IMP was carried
out by an LC-20AD prominence liquid chromatography instrument
(Shimadzu, Kyoto, Japan) equipped with an SPD-2A prominence UV/
vis detector using a C18 column (5
l
m ꢀ 250 nm ꢀ 4.6 nm, Elite
2.2. Construction of expression plasmid for AP/PTase gene
Analytical Instruments Co., Ltd., Dalian, China) with detection at
245 nm. The mobile phase was 10 mM potassium phosphate buffer
(pH 2.8):methanol = 90:10 (v/v) and the flow rate was 1 ml/min.
The retention times of 50-IMP and inosine were 3.7 and 8.3 min,
respectively.
A 747-bp nucleotide sequence of AP/PTase from E. blattae
(Genbank Accession No.: E12610.1) (Ishikawa, Mihara, Gondoh,
Suzuki, & Asano, 2000) with mutations of S72F/G74D/I153T was
synthesised using PCR assembly method (Liu, Zhang, Cheng, Ruan,
& Zheng, 2011; Rydzanicz, Zhao, & Johnson, 2005) after optimisa-
tion of the codons by gene designer software against E. coli as host
(Villalobos, Ness, Gustafsson, Minshull, & Govindarajan, 2006). The
synthesised gene with 6ꢀ His-tag was inserted into expression
vector pET28b(+) between NcoI and XhoI restriction endonuclease
sites. The ligated plasmid pET28b(+)-AP/PT was transformed into
E. coli BL21 (DE3) by heat shock method (Chung, Niemela, & Miller,
1989). For the selection of E. coli transformants, Kanamycin (Kan)
with appropriate concentration was added to the medium. DNA
manipulation, plasmid isolation, and agarose gel electrophoresis
were operated according to standard protocol (Sambrook & Russell,
2001) unless additionally stated.
One unit of acid phosphotransferase activity was defined as the
amount of enzyme that produces 1 l
mol of 50-IMP per min under
the assay conditions. Accordingly, one unit of acid phosphatase
activity was defined as the amount of enzyme that produces
1 lmol of inosine per min under the assay conditions. Protein
quantitative analysis was determined by the Bradford method
(Bradford, 1976) with BSA as a standard. Specific enzyme activities
were defined as units per mg enzyme.
2.6. Characterisation of acid phosphotransferase
2.6.1. Effects of temperature and pH
The effect of temperature on acid phosphotransferase activity
was determined in a range from 20 to 60 °C under the standard as-
say conditions mentioned above. Thermal stability was investi-
gated by incubating enzymes at temperatures of 30, 40, 50 and
60 °C for the indicated time intervals from 5 min to 7 h in 10 ml
of 100 mM sodium acetate buffer (pH 5.0) in water bath. The
half-life of acid phosphotransferase at each temperature was calcu-
lated by plotting the natural logarithm of residual activity (ln RA)
at each temperature against time. The effect of pH on acid phos-
photransferase activity was measured in a range from 3.6 to 9.0.
The following buffers with a final concentration of 100 mM were
used in testing the effects of pH on enzymatic activity: sodium ace-
tate buffer (pH 3.6–5.8), potassium phosphate buffer (pH 5.8–8.0)
and Tris–HCl buffer (pH 8.0–9.0). The pH stability of the enzyme
was examined under various pH conditions. After the enzyme
was incubated at 30 °C for 1 h in different buffers ranging from
3.6 to 9.0, the relative acid phosphotransferase activity was as-
sayed under standard reaction conditions (Balqis & Rosma, 2011).
All experiments were performed in triplicate.
2.3. Expression and purification of recombinant AP/PTase
E. coli BL21 (DE3) harbouring the recombinant plasmid was
grown at 37 °C in 50 mL LB media containing 50 lg/mL of Kan until
the absorbance of the culture suspension reached an optimal den-
sity of 0.6–0.8 at 600 nm. The expression of recombinant AP/PTase
was induced by 0.1 mM isopropyl-b-D-thiogalactoside (IPTG) and
the growth was incubated at 28 °C for 10 h. The bacterial cells were
harvested by centrifugation at 10,000g for 10 min at 4 °C and
washed twice with physiological saline solution (NaCl 0.9%). Then
the cells were resuspended in 30 mL 100 mM Tris–HCl (pH 7.6) and
destroyed by sonication with a Vibra-Cell VC 505 ultrasonic pro-
cessor (Sonics and Materials Inc., Newtown, CT) at 300 W for
30 min. Cell debris were removed by centrifugation at 12,000g
for 20 min at 4 °C. The supernatant was used as the crude extract.
The recombinant proteins were purified under the native condi-
tion using a nickel–NTA resin, and the detailed enzyme purification
procedure was carried out according to a previous report (Liu et al.,
2011). The purity of the preparations was confirmed by 12%
sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS–PAGE) and the protein concentration (Tripathi, Tomar, &
Jagannadham, 2011) was determined by the Bradford method with
bovine serum albumin (BSA) as the standard (Bradford, 1976).
2.6.2. Effects of metal ions and chemicals on enzymatic activity
The effects of metal ions on acid phosphotransferase activity
were determined by the addition of metal ions, including CuCl2,
CoCl2, Al2(SO4)3, NiCl2, MnCl2, CaCl2, LiCl, MgCl2, BaCl2, ZnSO4,
AgNO3, HgCl2, FeCl3 and FeSO4 to the reaction mixture at a final
concentration of 2 mM. The effects of chemicals, including
ethylenediaminetetraacetie acid (EDTA), dimethyl sulfoxide
(DMSO), tetrahydrofuran (THF), Tween-20, Tween-80, dithiothrei-
tol (DTT) and Triton X-100, on the enzyme activity were also
2.4. SDS–PAGE analysis
Fifteen microlitres of crude extracts and elution fractions were
analysed by SDS–PAGE performed using a Mini-gel system (Bio-
Rad, Hercules, CA). The gels were cast with 0.75-mm spacers
(Bio-Rad). SDS–PAGE was performed as described by Laemmli
(1970). Polyacrylamide gels were 5% acrylamide stacking gel (pH
6.8) and 12% separating gel (pH 8.8). Proteins in the gel were
stained with Coomassie Brilliant Blue R-250.
investigated (Ye
& Ng, 2010). Activities were measured as
described above and were presented as a percentage of the activity
obtained.
2.6.3. Circular dichroism (CD) measurements
CD spectra of acid phosphotransferase were recorded on a
JASCO J-815 Spectropolarimeter (JASCO Inc., Easton, MD) using
Spectra Manager 228 software. Temperature was controlled using
a Fisher Isotemp Model 3016S water bath (Fisher Scientific, Wal-
tham, MA). A lyophilised powder (purity >95% w/w) was prepared.
Far-UV scans were performed at 0.10 mg/ml protein in a 10-mm
cuvette. The spectra were recorded from 200 to 250 nm with a scan
speed of 20 nm/min. Results are expressed in terms of mean resi-
due ellipticity ([h]mrw,k) (in deg cm2 d molꢁ1) determined according
to Eq. (2) (Fasman, 1996, chap. 16):
2.5. Enzymatic activity assay
Acid phosphotransferase activity was assayed in a standard
mixture containing 100 mM of sodium acetate buffer (pH 5.0),
10 mg/ml of inosine, 250 mg/ml of disodium hydrogen pyrophos-
phate, and 2.12 mg of the enzyme in a total volume of 10 ml. The
reaction mixture was incubated for 10 min at 30 °C. One millilitre
of sample was taken from the reaction mixture and stopped by

950
Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
½hꢂmrw;k ¼ MRWðhkÞ=10lc;
2.1. The geometry of loop regions was corrected using Refine Loop/
MODELER. Finally, the best quality model was chosen for further
calculations, molecular modelling, and docking studies by Autodock
4.0 (Morris et al., 1998).
where MRW is the mean residue weight of the acid phosphotrans-
ferase, hk is the observed ellipticity (mdeg), l is the path length (cm),
and c is the protein concentration (lM).
3. Results and discussion
2.7. Kinetic parameters
3.1. Expression of acid AP/PTase gene
Because the solubility of inosine was limited, the steady-state
kinetic parameters of the recombinant acid phosphotransferase
were measured for inosine using concentrations ranging from 3
to 45 mM. The initial velocities were measured under the standard
reaction conditions described above and kinetic parameters were
calculated from Lineweaver–Burk plots.
Acid phosphatase activity was assayed in a standard mixture
containing 100 mM of sodium acetate buffer (pH 5.0), 0.1 to
1 mM 50-IMP, and the enzyme solution, in a total volume of
10 ml. After incubation at 30 °C for 10 min, 1 ml of sample was ta-
ken from the reaction mixture and stopped by adding 0.2 ml of 2 M
HCl. Quantitative determination of inosine was carried out by
HPLC. Kinetic parameters were calculated from Lineweaver–Burk
plots.
Based on the reported nucleotide sequence, the E. blattae AP/
PTase gene was synthesised after optimisation of the codons. The
synthesised DNA fragment was subcloned into an expression vec-
tor pET28b(+), to construct the recombinant plasmid pET28b(+)-
AP/PT. Subsequently, the recombinant plasmids were introduced
into competent cells of E. coli BL21 (DE3) and several clones were
picked up. The positive transformant containing recombinant
pET28b(+)-AP/PT was identified by colony PCR and double enzy-
matic digestion. The recombinant E. coli harbouring pET28b(+)-
AP/PT was induced by 0.1 mM of IPTG at 28 °C for 10 h when the
OD600 reached the value of 0.6. SDS–PAGE showed protein bands
were visualised in gels by staining with Coomassie Brilliant Blue.
The SDS–PAGE analysis (Fig. 1) shows a band with molecular mass
of about 25 kDa in Lane 5 is obtained, which is in agreement with
the predicted value (24.8 kDa) based on the amino acid sequence of
AP/PTase. The purified protein was observed as a single band on an
SDS–PAGE, indicating that the protein was quite pure.
2.8. Production and isolation of 50-IMP
2.8.1. Time course of 50-IMP synthesis
The time course of 50-IMP synthesis was examined using 100,
150 and 200 mg of inosine, 250 mg of pyrophosphate and
2.12 mg of the purified enzyme in 10 ml sodium acetate buffer.
The reaction was carried out at 30 °C with moderate shaking and
terminated by adding 1 ml of 2 M HCl. Quantitative determination
of 50-IMP was carried out by HPLC as mentioned above.
3.2. Enzyme characterisation
3.2.1. Optimal temperature and thermostability
To test the optimal temperature for the enzymatic activity of the
recombinant acid phosphotransferase, 10 mg/ml of inosine was
used as the substrate and the reaction mixture was incubated at
20, 25, 30, 35, 40, 45, 50, 55 and 60 °C. The enzyme remains active
at temperatures ranging from 20 to 50 °C and had maximal activity
at around 30 °C (Fig. 2a). Mihara et al. (2001) reported that 30 °C
was the optimal temperature of this enzyme from Providencia
stuartii and M. morganii, whereas acid phosphotransferase from
Klebsiella planticola had maximal activity at approximately 35 °C.
After this apex point, the activity decreased and negligible acid
phosphotransferase activity was observed at 60 °C. To test the
2.8.2. Isolation and identification of 50-IMP
Reaction mixture (50 ml) containing 50-IMP was centrifuged at
12,000g for 10 min at 4 °C to remove the cells for purification of
50-IMP. Later, the supernatant was applied onto a column of
201 ꢀ 7 Strong-Base Anion-Exchange Resin (Clꢁ form). The elution
procedure was used as follows. The supernatant was equilibrated
with water and then the impurities were eluted from the column
using a solution containing 0.03 M HCl and 0.1 M NaCl; subse-
quently, the expected product was washed out with an elution buf-
fer (0.1 M HCl and 0.1 M NaCl). Additionally, the fractions
containing 50-IMP were collected, neutralised with NaOH, and then
distilled and dried under vacuum. The crystals of 50-IMP obtained
were studied further.
The 13C and 1H nuclear magnetic resonance (NMR) spectra of 50-
IMP were obtained on an NMR spectrometer (AVANCE 500 MHz,
Bruker, Fällanden, Switzerland) with D2O as the solvent, using
500 and 125 MHz for carbon and proton determinations, respec-
tively. The FTIR spectrum for the 50-IMP was recorded on a Fourier
transform infrared spectrometer (Nicolet 6700; Thermo, Waltham,
MA) with Contimulm FTIR Micro system in the region of 4000–
400 cmꢁ1 using KBr pellets.
2.9. Homology modelling and docking
The three-dimensional homology models of wild-type and mu-
tant AP/PTase were generated using Build Homology Models (MOD-
ELER) in Discovery Studio 2.1 (Accelrys Software, San Diego, CA)
(Morris et al., 1998), using crystal structures of AP/PTase from E.
blattae at 2.4 Å resolution (PDB Accession Code 1EOI) as the tem-
plate (Ishikawa et al., 2000). The generated structures were im-
proved by subsequent refinement of the loop conformations by
assessing the compatibility of an amino acid sequence to known
PDB structures using the Protein Health module in Discovery Studio
Fig. 1. SDS–polyacrylamide gel electrophoresis of the purified enzyme. Lane M:
Marker proteins; lane 1: E. coli BL21 (DE3); lane 2: uninduced E. coli BL21 (DE3)/
pET28b(+)-AP/PT; lane 3: E. coli BL21 (DE3)/pET28b(+)-AP/PT induced by IPTG; lane
4: crude extract; lane 5: the purified enzyme.

Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
951
a
b
110
100
90
80
70
60
50
40
30
20
10
0
7
30oC
40oC
50oC
60oC
6
5
4
3
2
1
0
20
30
40
50
60
0
100
200
Time (min)
300
400
500
Temperature (oC)
20(oC)
30(oC)
40(oC)
50(oC)
60(oC)
110
100
90
80
70
60
50
40
30
20
10
0
c
d
20
10
0
sodium acetate buffer
potassium phosphate buffer
Tris-HCl buffer
-10
-20
200
220
240
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
pH
Wavelength (nm)
70
e
sodium acetate buffer
potassium phosphate buffer
Tris-HCl buffer
60
50
40
30
20
10
0
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH
Fig. 2. Enzyme characterisation. (a) Effect of temperature on the acid phosphotransferase activity. Reactions were performed at 150g for 10 min at various temperatures (20–
60 °C) with 100 mM sodium acetate buffer. (b) The thermostability of the purified enzyme. By incubating enzymes at temperature settings of 30, 40, 50 and 60 °C for the
indicated time intervals from 5 min to 7 h. j 30 °C; d 40 °C; N 50 °C; . 60 °C. (c) Spectroscopic structural analysis. (d) Effect of pH on the acid phosphotransferase activity.
The enzyme activity was measured at different pH values with 100 mM sodium acetate buffer, pH 3.6–5.8 (j), potassium phosphate buffer, pH 5.8–8.0 (d), Tris–HCl buffer,
pH 8.0–9.0 (N). (e) pH stability of acid phosphotransferase. The purified acid phosphotransferase was pretreated in different buffers with pH ranging from 3.6 to 9.0 at 30 °C
for 1 h and the residual activity was tested.
thermostability, the purified enzyme was incubated at 30–60 °C
from 5 min to 7 h at the indicated time intervals and residual activ-
ity was determined according to the standard enzyme assay meth-
od. The residual activity was obtained in accordance with the
activity at the 0th h and its natural logarithm value (Ln E0/E) was
plotted against time. Fig. 2b shows the results of the thermostability
of acid phosphotransferase. The Kd of the enzyme at 30, 40, 50 and
60 °C were 0.0036, 0.0128, 0.05 and 0.1518 minꢁ1, respectively.

952
Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
Accordingly, the half-lives (t1/2) of the purified enzyme at 30, 40, 50
and 60 °C were calculated to be 3.2, 0.90, 0.23 and 0.076 h, respec-
tively. Indeed, the enzyme seemed to be still sensitive to heat, which
can be corroborated by thermostability measurements by CD
(Fig. 2c), which demonstrates that this recombinant enzyme was
not thermostable and needs further improvements.
activity of acid phosphotransferase, which showed that thiol
groups may play an important role in the function of the active site.
3.3. Determination of kinetic parameters
The kinetic data for the recombinant enzyme obtained by vary-
ing the concentration of inosine (3–45 mM) at 30 °C were plotted
according to the Lineweaver–Burk equation and the Michaelis con-
stant Km and Vmax values were estimated as well. The values of
apparent Km and Vmax are 40 mM and 3.5 U/mg for inosine, respec-
tively, while the values of Km and Vmax are 0.32 mM and 24.4 U/mg
for 50-IMP, respectively (Table 2). It is reported that the acid phos-
photransferase activity of M. morganii was much improved, the Km
value for inosine was decreased and the Vmax value for inosine was
increased by introducing mutations G92D/I171T and S72F (Mihara
et al., 2001; Suzuki, Ishikawa, Mihara, Shimba, & Asano, 2007). In
the present study, we found that after three mutations S72F/
G74D/I153T were introduced into the enzyme, the acid phospho-
transferase activity was enhanced. One key distinction of the mu-
tant was a reduced Km value from 202 to 40 mM, compared to the
wild type, for inosine in the transphosphorylation reaction (Suzuki
et al., 2007). Another distinction was a decrease of Vmax value in the
dephosphorylation reaction, and almost the same Km value was
exhibited for inosine as that of the M. morganii G74D/I153T mutant
(43 mM) (Mihara et al., 2000). In addition, the Km value for inosine
in the transphosphorylation reaction was much higher than that
for 50-IMP (0.32 mM) in the dephosphorylation reaction. The Vmax
value for dephosphorylation reaction appeared to be higher than
that for transphosphorylation reaction. Due to these changes in
catalytic properties, the productivity of the synthesised 50-IMP
may have been improved.
3.2.2. Optimal pH and pH stability
To test the optimal pH of the enzyme activity, the reactions
were carried out in the following buffers including sodium acetate
buffer (pH 3.6–5.8), potassium phosphate buffer (pH 5.8–8.0) and
Tris–HCl buffer (pH 8.0–9.0) with
a final concentration of
100 mM at 30 °C. The result showed that the optimal pH for the
acid phosphotransferase was 5.0 with sodium acetate buffer
(Fig. 2d). The pH stability of enzyme was examined by assaying
the residual activity under the standard assay conditions. The re-
sults indicated that the acid phosphotransferase was more stable
at pH 5.8 with about 61% residual activity. In addition, pH higher
than 8.5 or lower than 4.0 resulted in a significant decrease in
the residual activity of acid phosphotransferase (Fig. 2e), which
was similar to the purified enzymes from P. stuartii, M. morganii
and K. planticola (Mihara et al., 2001). On the basis of these results,
further investigations of the phosphorylation reaction were per-
formed at pH 5.0 and 30 °C.
3.2.3. Effect of metal ions and chemicals on enzyme activity
The effects of various metal ions at a final concentration of
2 mM and other reagents, such as EDTA, DMSO, THF, DTT,
Tween-20, Tween-80 and Triton X-100 with concentration of 2%,
on the enzyme activity of the acid phosphotransferase were tested
(Table 1). The activity assayed in the absence of metal ions and
other reagents was considered as control. The results indicated
that the activity was partially inhibited by metal ions, such as
Hg2+, Ag+ and Cu2+. However, no inhibition was observed in the
presence of chelating agent EDTA, which illustrated that the acid
phosphotransferase obtained in the current study did not required
metal ions for its catalysis. It was consistent with acid phospho-
transferase production by M. morganii (Asano et al., 1999b; Mihara
et al., 2000). In addition, the results showed that addition of
Tween-20, Tween-80 and Triton X-100 could slightly activate the
activity of acid phosphotransferase. However, DTT inhibited the
3.4. Production and isolation of 50-IMP
3.4.1. Production of 50-IMP by the purified enzyme
Using the purified enzyme, the time course of 50-IMP synthesis
from different concentrations of inosine and pyrophosphate was
examined (Fig. 3). The data in Fig. 3 showed that as the reaction
time was extended, the dephosphorylation of 50-IMP occurred
and the synthesised 50-IMP was hydrolysed to inosine. In addition,
as the concentration of inosine increased, the rate of hydrolysis of
50-IMP decreased and the amount of 50-IMP synthesised increased.
When 37.3 mM of inosine and 560 mM of disodium hydrogen
pyrophosphate was added to the reaction mixture, 16.83 mM of
50-IMP were produced from 37 mM of inosine in 40 min, and the
molar yield of 50-IMP from inosine was 45.5%, while M. morganii
exhibited lower yield from higher concentration of inosine
(80 mM) (Asano et al., 1999b). Additionally, the enzyme from
Enterobacter aerogenes IAM 1183 synthesised 5.85 mg/ml of 50-
IMP, which was lower than the yield in this study. Mihara reported
that by introducing 11 mutations, the AP/PTase activity was much
improved and 156 g/L of 50-IMP was obtained (Mihara et al., 2004).
As the purified nucleoside phosphorylating enzyme was used in
the reaction, dephosphorylation of synthesised 50-IMP appeared
to be catalysed by the same enzyme. In order to enhance the pro-
ductivity of 50-IMP in the transphosphorylation reaction, it is nec-
essary to suppress the phosphatase activity. In addition, to achieve
efficient phosphorylation, pyrophosphate from phosphate should
be recycled.
Table 1
Effect of metal ions and other reagents on acid phos-
photransferase activity.
Reagent
Relative activity (100%)
Control
CuCl2
CoCl2
Al2(SO4)3
NiCl2
MnCl2
CaCl2
LiCl
MgCl2
BaCl2
ZnSO4
AgNO3
HgCl2
FeCl3
FeSO4
EDTA
DMSO
DTT
THF
Tween-20
Tween-80
Triton X-100
100 1.56
77 2.14
80 1.36
84 3.37
68 2.18
66 0.21
94 1.10
79 2.08
92 1.42
97 0.16
88 2.12
6
0.56
38 1.56
82 2.43
85 2.16
96 1.56
96 0.89
34 1.21
82 1.05
120 1.46
120 1.26
144 1.01
3.4.2. Isolation and identification of 50-IMP
After isolation and purification according to the method de-
scribed in materials and methods (Section 2.8.2), 50-IMP with
98.6% of purity and 39.8% of total yield was obtained. The FTIR
spectrum of the 50-IMP separated is presented in Fig. 4a. The sharp
peaks present at 1679 cmꢁ1 can be assigned to P = O stretching

Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
953
Table 2
Kinetic constants for the transphosphorylation reactions.
Activity
Substrate
Km (mM)
Vmax (U/mg)
Wild type
S72F/G74D/I153T
Wild type
Acid phosphotransferase
Acid phosphotransferase
Acid phosphatase
Acid phosphatase
Inosine
Inosine
50-I MP
50-I MP
202
40
0.18
0.32
1.83
3.5
49.4
24.4
S72F/G74D/I153T
Note: The enzyme activities were assayed as described in Section 2. Initial velocities were determined, and steady-state kinetic constants were calculated from Lineweaver–
Burk plots.
Simultaneously, the conformation of the residues His 150, Arg
183, Arg 122 around His 189 was changed. Direct hydrogen bonds
8
10 mg/ml
15 mg/ml
20 mg/ml
are formed between HZ1 of Lys 115 and O1 of phosphate, HH22
of Arg 122 and O1 of phosphate, HE of Arg 183 and O2, O3 of phos-
phate, HN of His 150 and O2 of phosphate, HD1 of His 150 and O4 of
phosphate. The hydrogen bonds could stabilise the phosphoenzyme
catalytic intermediate. When phosphate acceptor inosine attacks
the binding pocket of AP/PTase, two hydrogen bonds were formed
between 20,30-hydroxyl group of the ribose ring and the carboxyl
group of Glu 104. It leads to the distance between O1 of inosine
and O4 of phosphate group being nearer by 3.1 Å. In the last step
of the catalytic reaction, a water attacks the phosphoenzyme cata-
lytic intermediate, in order to cleave the covalent bond between His
189 and the phosphate; consequently, phosphate group is released
from the enzyme. Later the released phosphate attacks the inosine
which is close to it and replaces the water by inosine; subsequently,
50-IMP was produced.
A binding mode of inosine to the mutated phosphoenzyme
intermediate could be modelled (Fig. 5b). The result in Fig. 5b
showed that after mutation, the interaction between some resi-
dues near His 189, such as Lys 115, Arg 122, His 150, Arg 183
and phosphate, have little changes. However, the structure of
S72F/G74D/I153T altered the interaction between inosine and
phosphoenzyme intermediate. Besides the previously formed
hydrogen bonds between the carboxyl group of Glu 104 and the
hydroxyl group of the ribose, NH of Leu 70, NH of Ser 71, NH of
Asp 108, and OH of Asn 143 form hydrogen bonds with inosine.
These newly-formed hydrogen bonds may enhance the affinity of
enzyme to inosine, accordingly reducing the Km value for inosine.
In addition, the distance between inosine and phosphate was from
3.1 to 2.5 Å, which could help the released phosphate to attack ino-
sine more easily and enhance the catalytic activity. As the structure
of mutated protein S72F/G74D/I153T was constructed, the result
revealed that the overall structure of mutant is identical to that
of the wild type, which indicated that the difference in the Km be-
tween mutant and wild type can be ascribed to the changes in the
binding pocket (Mihara et al., 2001).
7
6
5
4
3
2
1
0
0
2
4
6
8
10
12
Time (h)
Fig. 3. Time course of 50-IMP synthesis from inosine and pyrophosphate by the
purified enzyme. Reaction was carried out at different concentrations of inosine;
enzyme activities were determined according to the standard enzyme assay
method. j, 10 mg/ml inosine; d,15 mg/ml inosine; N, 20 mg/ml inosine.
vibration. The strong and broad absorption band between 3200
and 3400 cmꢁ1 is due to N–H stretching vibration and the peak
appearing at 2875 cmꢁ1 is due to O–H stretching vibration of the
phosphate group. The 13C and 1H NMR spectra were used to char-
acterise 50-IMP dissolved in D2O solution. When 13C NMR was per-
formed, the isolated product gave double signals at the C40 position
which resulted from 13C–31P coupling, which demonstrated that
isolated IMP was phosphorylated at the C50 position (Fig. 4b). Fig
4c shows seven different kinds of H atoms, which correspond to
the structure of 50-IMP, such as two peaks at chemical shifts
d = 8.1877, 8.5563 ppm, represent two H atoms of purine ring at
C2 and C8 position. Accordingly, it was concluded that the isolated
product was 50-IMP.
3.5. Homology modelling and molecular docking
The secondary and tertiary structures analysis showed that AP/
In the inosine binding model, it is found that Thr 153 is situated
near the inosine binding site (Fig. 5a and b). Mutation at position
153 may cause a subtle structural change in the inosine binding
site; the reduced Km could be ascribed to a difference in the inter-
action between the inosine and the inosine binding site. On the
other hand, after mutation at position Asp 74, the flexibility of
the region ranging from Asn 69 to Val 75 is increased in the
G74D/I153T structure, compared to the wild type. The increased
flexibility of this region may enhance the tightness of binding
and the affinity of the enzyme to inosine Moreover, the G74D/
I153T mutations can reduce the Km without either a significant
structural change or a new direct interaction, which suggests that
amino acid substitution around the inosine binding site can reduce
the Km. The S72F mutation can lead to the formation of aromatic–
PTase obtained in this study consisted of 13
a-helices, which is
different from some reported AP/PTases (Strater, Klabunde, Tucker,
Witzel, & Krebs, 1995). Crystal structure of E. blattae AP/PTase (PDB
Accession Code 1EOI, resolution 2.4 Å) was used as the template.
After the transition-state analogue molybdate of the template
was replaced by phosphate, a hypothetical binding mode of inosine
to the phosphoenzyme intermediate could be modelled (Fig. 5a). As
shown in Fig. 5a, the active site contains Lys 115, Arg 122, Ser 148,
Gly 149, His 150, Arg 183, His 189 and Asp 193, which is the same as
previous reports (Hemrika & Wever 1997; Neuwald 1997; Stukey &
Carman, 1997). These residues can be found in the conserved motif
KX6RP-(X12–54)-PSGH-(X31–54)-SRX5HX3D (Stukey & Carman, 1997;
Thaller, Schippa, & Rossolini, 1998). The reaction mechanism can be
proposed as follows: the conserved residue His 189 in motif 3
(SRX5HX3D) was predicted to attack the phosphate group, NE2
of His 189 to form a covalent bond with phosphate, subsequently
aromatic interaction between the base of
a nucleoside and
the side-chain of an aromatic residue of the enzyme. The loop
containing Phe 72 has a notable conformational change on the for-
mation of phosphoenzyme intermediate; therefore, a much better
a
phosphoenzyme catalytic intermediate was formed.

954
Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
Fig. 4. Identification of 50-IMP. (a) Infrared spectroscopy of 50-IMP. (b) Identification of the isolated product performed on 13C NMR. (c) Identification of the isolated product
performed on 1H NMR.

Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
955
Fig. 4 (continued)
Fig. 5. A stereo view of the putative intermediate. (a) Inosine (NOS) docking into the active site of native acid phosphatase/phosphotransferase and its catalytic triad with
inosine. (b) Inosine docking into the active site of the mutative acid phosphatase/phosphotransferase.
aromatic–aromatic interaction is formed and results in lower Km
and higher activities (Suzuki et al., 2007).
expressed and evaluated. Characterisation of this recombinant
enzyme revealed that this enzyme has an excellent function on
the transphosphorylation of inosine. The investigation of the char-
acteristics of the purified acid phosphotransferase showed that
the optimal temperature and pH of this enzyme were 30 °C and
5.0, respectively. The enzyme activity could not be activated by
the metal ions tested in this study, and was partially inhibited
by Hg2+, Ag+ Cu2+, but slightly activated by Tween-20, Tween-80
4. Conclusions
In this current study, an acid phosphatase with strictly
regioselective nucleoside phosphotransferase from E. blattae was

956
Z.-Q. Liu et al. / Food Chemistry 134 (2012) 948–956
Mihara, Y., Ishikawa, K., Suzuki, E. I.,
&
Asano, Y. (2004). Improving the
and Triton X-100. The values of Km and Vmax for inosine were 40 mM
and 3.5 U/mg, respectively. The time course showed that 16.83 mM
of 50-IMP was synthesised from 37 mM of inosine and the molar
yield reached 45.5% after 40 min reaction. The 50-IMP obtained in
this study was further isolated and characterised. Further investiga-
tions on improvement of phosphotransferase activity for industrial
production of 50-IMP are in progress in our laboratory.
pyrophosphate–inosine phosphotransferase activity of Escherichia blattae acid
phosphatase by sequential site-directed mutagenesis. Bioscience Biotechnology
and Biochemistry, 68(5), 1046–1050.
Mihara, Y., Utagawa, T., Yamada, H.,
& Asano, Y. (2000). Phosphorylation of
nucleosides by the mutated acid phosphatase from Morganella morganii. Applied
and Environmental Microbiology, 66(7), 2811–2816.
Mihara, Y., Utagawa, T., Yamada, H.,
& Asano, Y. (2001). Acid phosphatase/
phosphotransferases from enteric bacteria. Journal of Bioscience and
Bioengineering, 92(1), 50–54.
Mori, H., Iida, A., Fujio, T., & Teshiba, S. (1997). A novel process of inosine 50-
monophosphate production using overexpressed guanosine/inosine kinase.
Applied Microbiology and Biotechnology, 48(6), 693–698.
Acknowledgements
Mori, H., Iida, A., Teshiba, S., & Fujio, T. (1995). Cloning of guanosine–inosine kinase
gene of Escherichia coli and characterization of the purified gene-product.
Journal of Bacteriology, 177(17), 4921–4926.
The authors gratefully acknowledge the financial supports of
the National Basic Research Program of China (973 Program) (No.
2011CB710800), National Natural Science Foundation of China
(No. 31101351), Natural Science Foundation of Zhejiang Province
(No. R3110155) and Qianjiang Talent Project of Zhejiang Province.
Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., et al.
(1998). Automated docking using
a Lamarckian genetic algorithm and an
empirical binding free energy function. Journal of Computational Chemistry,
19(14), 1639–1662.
Neuwald, A. F. (1997). An unexpected structural relationship between integral
membrane phosphatases and soluble haloperoxidases. Protein Science, 6(8),
1764–1767.
References
Rydzanicz, R., Zhao, X. S., & Johnson, P. E. (2005). Assembly PCR oligo maker: A tool
for designing oligodeoxynucleotides for constructing long DNA molecules for
RNA production. Nucleic Acids Research, 33, W521–W525.
Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual: Cold
Spring Harbor Laboratory Press.
Strater, N., Klabunde, T., Tucker, P., Witzel, H., & Krebs, B. (1995). Crystal-structure
of a purple acid-phosphatase containing a dinuclear Fe(III)–Zn(II) active-site.
Science, 268, 1489–1492.
Stukey, J., & Carman, G. M. (1997). Identification of a novel phosphatase sequence
motif. Protein Science, 6(2), 469–472.
Suzuki, E., Ishikawa, K., Mihara, Y., Shimba, N., & Asano, Y. (2007). Structural-based
engineering for transferases to improve the industrial production of 50-
nucleotides. Bulletin of the Chemical Society of Japan, 80(2), 276–286.
Tanaka, N., Hasan, Z., Hartog, A. F., van Herk, T., & Wever, R. (2003). Phosphorylation
and dephosphorylation of polyhydroxy compounds by class A bacterial acid
phosphatases. Organic & Biomolecular Chemistry, 1(16), 2833–2839.
Thaller, M. C., Berlutti, F., Schippa, S., Lombardi, G., & Rossolini, G. M. (1994).
Characterization and sequence of PhoC, the principal phosphate-irrepressible
acid-phosphatase of Morganella morganii. Microbiology-UK, 140, 1341–1350.
Thaller, M. C., Schippa, S., & Rossolini, G. M. (1998). Conserved sequence motifs
among bacterial, eukaryotic, and archaeal phosphatases that define a new
phosphohydrolase superfamily. Protein Science, 7(7), 1647–1652.
Tripathi, P., Tomar, R., & Jagannadham, M. V. (2011). Purification and biochemical
Asano, Y., Mihara, Y., & Yamada, H. (1999a). A new enzymatic method of selective
phosphorylation of nucleosides. Journal of Molecular Catalysis B-Enzymatic, 6(3),
271–277.
Asano, Y., Mihara, Y.,
& Yamada, H. (1999b). A novel selective nucleoside
phosphorylating enzyme from Morganella morganii. Journal of Bioscience and
Bioengineering, 87(6), 732–738.
Balqis, Z. S., & Rosma, A. (2011). Artocarpus integer leaf protease: Purification and
characterisation. Food Chemistry, 129(4), 1523–1529.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry, 72, 248–254.
Chen, X. C., Ansai, T., Awano, S., Ilda, T., Barik, S., & Takehara, T. (1999). Isolation,
cloning, and expression of an acid phosphatase containing phosphotyrosyl
phosphatase activity from Prevotella intermedia. Journal of Bacteriology, 181(22),
7107–7114.
Chung, C. T., Niemela, S. L., & Miller, Roger H. (1989). One-step preparation of
competent Escherichia coli: Transformation and storage of bacterial cells in the
same solution. Biochemistry, 86(7), 2172–2175.
Fasman, G. D. (1996). Circular dichroism and the conformational analysis of
biomolecules (1st ed.). New York: Plenum Press.
Hemrika, W., & Wever, R. (1997). A new model for the membrane topology of
glucose-6-phosphatase: The enzyme involved in von Gierke disease. Febs
Letters, 409(3), 317–319.
Hiroshi, M., Katsuaki, S., & Hitoshi, E. (1982). 5-Nucleotidase activity in improved
inosine-producing mutants of Bacillus subtilis. Agricultural and Biological
Chemistry, 46(9), 2347–2352.
characterisation of
1005–1012.
a novel protease streblin. Food Chemistry, 125(3),
Uchiya, K. I., Tohsuji, M., Nikai, T., Sugihara, H., & Sasakawa, C. (1996). Identification
and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri
Ishikawa, K., Mihara, Y., Gondoh, K., Suzuki, E., & Asano, Y. (2000). X-ray structures
of a novel acid phosphatase from Escherichia blattae and its complex with the
transition-state analog molybdate. Embo Journal, 19(11), 2412–2423.
2a encoding
4548–4554.
a nonspecific phosphatase. Journal of Bacteriology, 178(15),
Villalobos, A., Ness, J. E., Gustafsson, C., Minshull, J., & Govindarajan, S. (2006). Gene
designer: A synthetic biology tool for constructing artificial DNA segments. BMC
Bioinformatics, 7, 2–3.
Kasahara, M., Nakata, A.,
& Shinagawa, H. (1991). Molecular analysis of the
Salmonella-typhimurium phon gene, which encodes nonspecific acid-
phosphatase. Journal of Bacteriology, 173(21), 6760–6765.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature, 227(5259), 680–685.
Ye, X. J.,
& Ng, T. B. (2010). Purification and characterisation of an alanine
aminopeptidase from bovine skeletal muscle. Food Chemistry, 124(2), 634–639.
Liu, Z. Q., Zhang, L. P., Cheng, F., Ruan, L. T., & Zheng, Y. G. (2011). Characterization of
a newly synthesized epoxide hydrolase and its application in racemic resolution
of (R, S)-epichlorohydrin. Catalysis Communications, 16, 133–139.