Efficient biosynthesis of uridine diphosphate glucose from maltodextrin by multiple enzymes immobilized on magnetic nanoparticles

Article abstract of DOI:10.1016/j.carres.2010.04.025

Uridine diphosphate glucose (UDP-Glc) serves as a glucosyl donor in many enzymatic glycosylation processes. This paper describes a multiple enzyme, one-pot, biocatalytic system for the synthesis of UDP-Glc from low cost raw materials: maltodextrin and uridine triphosphate. Three enzymes needed for the synthesis of UDP-Glc (maltodextrin phosphorylase, glucose-1-phosphate thymidylytransferase, and pyrophosphatase) were expressed in Escherichia coli and then immobilized individually on amino-functionalized magnetic nanoparticles. The conditions for biocatalysis were optimized and the immobilized multiple-enzyme biocatalyst could be easily recovered and reused up to five times in repeated syntheses of UDP-Glc. After a simple purification, approximately 630 mg of crystallized UDP-Glc was obtained from 1 l of reaction mixture, for a moderate yield of around 50% (UTP conversion) at very low cost.

Full text of DOI:10.1016/j.carres.2010.04.025

Carbohydrate Research 345 (2010) 1622–1626
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Efficient biosynthesis of uridine diphosphate glucose from maltodextrin
by multiple enzymes immobilized on magnetic nanoparticles
Qing Dong, Li-Ming Ouyang, Hui-Lei Yu, Jian-He Xu *
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
130 Meilong Road, 200237 Shanghai, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Uridine diphosphate glucose (UDP-Glc) serves as a glucosyl donor in many enzymatic glycosylation pro-
cesses. This paper describes a multiple enzyme, one-pot, biocatalytic system for the synthesis of UDP-Glc
from low cost raw materials: maltodextrin and uridine triphosphate. Three enzymes needed for the syn-
thesis of UDP-Glc (maltodextrin phosphorylase, glucose-1-phosphate thymidylytransferase, and pyro-
phosphatase) were expressed in Escherichia coli and then immobilized individually on amino-
functionalized magnetic nanoparticles. The conditions for biocatalysis were optimized and the immobi-
lized multiple-enzyme biocatalyst could be easily recovered and reused up to five times in repeated syn-
theses of UDP-Glc. After a simple purification, approximately 630 mg of crystallized UDP-Glc was
obtained from 1 l of reaction mixture, for a moderate yield of around 50% (UTP conversion) at very
low cost.
Received 10 March 2010
Received in revised form 25 April 2010
Accepted 27 April 2010
Available online 4 June 2010
Keywords:
UDP-Glc
Biocatalytic synthesis
One-pot
Magnetic nanoparticles
Immobilized biocatalyst
Ó 2010 Elsevier Ltd. All rights reserved.
Glycosylation is an important reaction in biology. Drug research
now indicates that the actions of many drugs with principal roles
in molecular recognition, transmembrane signaling, and other
physiological or pathological processes involve glycosylation reac-
EC 2.4.1.1), glucose-1-phosphate thymidylytransferase (G1PTTase,
EC 2.7.7.24), and inorganic pyrophosphatase (PPase, EC 3.6.1.1),
were cloned and expressed inEscherichia coli and then immobilized
on amino-functionalized magnetic nanoparticles. In this system,
maltodextrin is first phosphorolyzed to glucose monophosphate
by MalPase. The Glc-1-P is then converted to UDP-Glc by G1PTTase,
generating pyrophosphate (PPi) as a byproduct. PPi is further
hydrolyzed to phosphoric acid (Pi) by PPase, which serves both
to drive the reaction scheme in the UDP-Glc direction and also to
regenerate one starting product for the MalPase reaction.
Although this reaction sequence will very effectively allow the
synthesis of UDP-Glc, all of the reaction components, that is, the
enzymes, their substrates, and their products, are highly water sol-
uble, which makes the purification of UDP-Glc from the reaction
mixture very complicated. Immobilization of enzymes can be help-
ful for isolating the protein biocatalysts from the reaction mixture.
As another benefit, immobilization can also improve the stability
and reusability of the enzymes, thus further reducing the cost of
this type of production process. In the present paper, we exploit
amino-functionalized magnetic nanoparticles for this purpose.
1
tions. Glycosylation is catalyzed almost exclusively by glycosyl-
2
,3
transferases that utilize sugar nucleotides in the uridine form.
In biological systems, uridine diphosphoglucose (UDP-Glc) is the
most commonly encountered sugar nucleotide and is widely used
as a glucosyl donor in glucosyltransferase reactions. Although su-
gar nucleotides such as UDP-Glc can be synthesized by chemical
methods, glycosylation using the enzymes that widely exist in nat-
ure is more attractive due to the strong regioselectivity, the high
conversion rate, and the mild reaction conditions of enzymatic
reactions. For example, biosynthesis of UDP-Glc has been catalyzed
using glucose-1-phosphate thymidylytransferase, with UTP and
glucose-1-phosphate (Glc-1-P) as substrates.4 However, this reac-
tion is not economically feasible for industrial scale production of
UDP-Glc because of the high cost of Glc-1-P. Leloir glycosyltrans-
ferase catalysis is also one of the common ways to synthesize oli-
5
,6
gosaccharides,
but it also depends on the availability of
expensive sugar phosphates.
In this paper, inexpensive raw materials—maltodextrin, UTP,
and phosphate—were utilized for UDP-Glc synthesis with a newly
designed multiple-enzyme system for in vitro synthesis of UDP-Glc
MalPase
+ Pi
G1PTTase
+UTP
Glc
Glc-1-P
PPase
PP + UDP-Glc
i
n
(Scheme 1). Three enzymes, maltodextrin phosphorylase (MalPase,
*
Corresponding author. Fax: +86 21 6425 2250.
E-mail address: jianhexu@ecust.edu.cn (J.-H. Xu).
Scheme 1. Biosynthesis of UDP-Glc by one-pot reaction with multiple enzymes.
0
008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.04.025

Q. Dong et al. / Carbohydrate Research 345 (2010) 1622–1626
1623
Amino-functionalized magnetic nanoparticles are a relatively
Table 2
Induction and expression conditions for recombinant strains
new material for enzyme immobilization, with many advantages
that are now attracting much attention.7 The immobilization of
enzymes onto aldehyde-functionalized magnetic nanoparticles is
a rapid process and these nanoparticles act as very effective sup-
ports for immobilized enzymes. More importantly, the nanoparti-
cle-bound enzymes can easily be separated from the reaction
mixture and recycled simply by using a magnetic field. The low
cost of fabrication of amino-functionalized magnetic nanoparticles
also makes this form of immobilization promising for industrial
scale enzyme processes. Therefore, we used amino-functionalized
magnetic nanoparticles in the present study to co-immobilize Mal-
Pase, GlPTTase, and PPase for the efficient synthesis of UDP-Glc
from maltodextrin and UTP.
–9
Enzyme
IPTG concn
(mM)
Induction temperature
(°C)
Induction time
(h)
MalPase 0.05
15
30
30
20
4
4
G1PTase
PPase
1
1
3
0 °C for 30 min. One unit of MalPase was defined as the amount
of enzyme producing 1
lmol of UDP-Glc within 1 min under the
described conditions.
(
ii) G1PTTase: The activity of G1PTTase was defined as the re-
lease of UDP-Glc. The reaction is as follows: the reaction mixture
contained 50 mM phosphate (pH 7.5), 5 mM MgCl , 5 mM glu-
2
cose-1-phosphate, 2 mM UTP, and 1 mg G1PTTase protein in a total
1
1
. Materials and methods
volume of 1 ml. The reaction mixture was incubated at 30 °C for
1
1
min. One unit of G1PTTase was defined as the activity to produce
l
mol of UDP-Glc in 1 min under the described conditions.1
iii) PPase: The assay mixture contained 50 mM Tris–HCl (pH
7.5), 5 mM MgCl , 10 mM Na P O , and 1 mg PPase crude enzyme
6,17
.1. Materials
(
Taq DNA polymerase, agarose gel DNA Purification Kit Ver.2.0,
2
4 2 7
and T4 DNA ligase were purchased from TaKaRa (Dalian, China).
The plasmid vectors of pET-11a (+) and pET-24a (+) were from
our laboratory. Primers were synthesized by Invitrogen (Shanghai,
China). UTP was purchased from Meiyapharm (Hangzhou, China).
Amino-functionalized magnetic particles7 were from our
laboratory.
powder in a total volume of 1 ml. The reaction mixture lacking the
enzyme was used as a control. The reaction mixture was incubated
at 30 °C for 1 min. After the reaction, 20
withdrawn and transferred to the system which contained 500
fresh phosphorus-determining reagent solution (17% H SO : 2.5%
ll of reaction mixture was
ll
2
4
Na MoO –(NH ) SO : 10%
2
4
4 2
4
L
-ascorbic acid: H O = 1:1:1:2) and
2
4
2
80 ll H O. Then the reaction was incubated at 45 °C for 10 min.
1
.2. Cloning, expression, and preparation of enzymes
The absorbance at 660 nm was recorded. One unit of PPase was de-
fined as the activity to produce 1 mol of inorganic phosphate in
1 min under the described conditions.
l
Genes coding for MalPase,10,11 GlPTTase , and PPase
3
12–14
were
18,19
amplified from E. coli K12 genomic DNA by PCR. The plasmids
and strains used are listed in Table 1. All of the experimental pro-
cedures used were general methods.15 E. coli BL21 (DE3) containing
exogenous recombinant plasmids were grown in LB medium at
1.4. HPLC analysis of UDP-Glc
The chromatography was performed on a Shimadzu system (LC-
3
7 °C, with shaking at 200 rpm. When OD600 of the culture broth
10ADVP) equipped with an Agela SAX NH2 column (150
lm,
4
reached about 0.7, IPTG (isopropyl-1-thio-b- -galactopyranoside)
D
4.6 ꢀ 150 mm) using an isocratic elution with 67 mM Na HPO –
2
was added and the cultivation was continued at a suitable temper-
ature to induce the expression of each target enzyme protein.
Induction and expression conditions for different recombinant
strains are listed in Table 2. The cells were then harvested by cen-
trifugation at 12,000g for 10 min at 4 °C, followed by disruption by
ultrasonication (400 W). The whole process of ultrasonication
lasted for 100 cycles. Each cycle included 4 s of sonication and
2 4
KH PO (pH 5.3) at 30 °C. The flow rate was 1.5 ml/min and UV
detection was performed at 260 nm.
1.5. Immobilization of enzyme
Amino-functionalized magnetic particles (10 mg/ml) were sus-
pended in phosphate buffer or Tris–HCl buffer (50 mM, pH 7.5)
and then ultrasonicated for 20 min. Thereafter, 25% glutaraldehyde
6
s of pause. The supernatant was collected and lyophilized to ob-
tain a crude enzyme powder.
(20 ll/ml) was added and shaken for 12 h at 30 °C, 1100 rpm. The
nanoparticles were then separated by magnetic decantation and
1
.3. Activity assays
i) MalPase: The activity of MalPase was defined as the release of
washed five times with phosphate buffer or Tris–HCl buffer
(
50 mM, pH 7.5) to eliminate the excess unreacted glutaraldehyde.
(
Each enzyme solution was mixed separately with aldehyde-func-
tionalized magnetic nanoparticles and shaken at 30 °C for 30 min,
1100 rpm. After this reaction period, each immobilized enzyme
was removed by magnetic decantation and washed five times with
phosphate buffer or Tris–HCl buffer (50 mM, pH 7.5) to remove any
unbound enzyme.
UDP-Glc, which can be measured using a coupled assay containing
MalPase proteins, G1PTTase, and PPase. The reaction mixture con-
tained 100 mM phosphate (pH 7.5), 5% maltodextrin, 5 mM MgCl
2
,
5
mM UTP, 0.25 U G1PTTase, 1 U PPase, and 1 mg MalPase protein
in a total volume of 1 ml. The reaction mixture was incubated at
Table 1
Endonucleases, vectors, and hosts for different genes
0
0
Gene
Endonucleases
Vector
Host
Primers (5 ?3 )
malpase
BamHI/NotI
pET24a (+)
E. coli BL21 (DE3)
MP1: ctggatccatgtcacaacctatttttaacg
MP2: cttgcggccgcttagcgttttgcctgccag
GP1: ctgggaattccatatgatgaaaatgcgtaaag
GP2: cttgcggatccttaaacttaggaagc
PP1: ctgggaattccatatgatgagcttactcaacg
PP2: cttgcggatccttatttattctttgcgcgctcg
g1pttase
ppase
BamHI/NdeI
BamHI/NdeI
pET11a (+)
pET11a (+)
E. coli BL21 (DE3)
E. coli BL21 (DE3)

1
624
Q. Dong et al. / Carbohydrate Research 345 (2010) 1622–1626
1
.6. Reuse of immobilized enzymes in repeated synthesis of
Table 3
Protein loading and activity recovery for different enzymes
UDP-Glc
MalPase
G1PTase
PPase
A reaction mixture (200 ml in a 500 ml narrow neck flask), mal-
todextrin 5% (m/v), UTP 2 mM, and MgCl 10 mM, in 100 mM so-
Protein loadinga (mg/g support)
Activity recovery (%)
47.5
85.8
65
71
68.4
93.2
2
dium phosphate buffer (pH 7.5) was combined with immobilized
enzymes for the one-pot synthesis. The product concentration
was analyzed periodically by HPLC. When the UDP-Glc concentra-
tion showed no further increases, the reaction was terminated and
the immobilized enzymes were recovered by magnetic separation.
The recovered immobilized enzymes were washed three times
with phosphate buffer (100 mM, pH 7.5) and then employed again
in the next batch of fresh reaction mixture.
a
The amount of protein was determined by the Bradford method using BSA as a
standard.
1
00
A
80
60
40
20
0
1
.7. Purification of UDP-Glc
To isolate UDP-Glc, the immobilized enzymes were removed
with a magnet and an equal volume of methanol was added to
the remaining reaction mixture containing the UDP-Glc product.
This mixture was cooled at 4 °C for 15 min, then centrifuged at
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
pH
1
2,000g for 5 min at 4 °C. The supernatant was concentrated by ro-
1
00
B
tary evaporation at 50 °C and the concentrated solution was ap-
plied to SAX (strong anion exchange) ion-exchanger column
8
0
(
40
l
m, 25 ꢀ 300 mm) and eluted with 67 mM Na
2
HPO
4
–KH
2
PO
4
60
(
pH 5.3) at a flow rate of 2 ml/min. The fractions containing UDP-
4
0
0
0
Glc were identified by HPLC, pooled, and desalted on an ODS (octa-
decylsilyl) column (40
with 70% methanol (aq) at a flow rate of 2 ml/min. The purified
2
lm, 25 ꢀ 300 mm) using isocratic elution
5
.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
pH
product was lyophilized and stored at 4 °C.
2
2
. Results and discussion
1
00
80
C
.1. Expression of enzymes
6
4
0
0
On SDS–PAGE gels, the three enzymes expressed in E. coli had
molecular weights identical to the predicted values (MalPase,
8 kDa; G1PTTase, 32 kDa; PPase, 19 kDa; Fig. 1). G1PTTase and
PPase were mainly expressed as soluble forms, while Malpase
was mainly expressed as an insoluble form.
8
20
0
5
.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
pH
2
.2. Immobilized enzymes
Figure 2. Effect of initial pH on activities of free enzymes (Ç) and immobilized
enzymes (j). (A) MalPase; (B) GlPTTase; (C) PPase.
The enzymes were immobilized on amino-functionalized mag-
netic nanoparticles by the method represented in Section 1.5. The
protein loading and activity recovery are shown in Table 3. After
immobilization, more than 70% of the original activity of the re-
combinant enzymes was retained. The specific activities of the
immobilized enzymes were MalPase 5.66 U/g, G1PTTase
2.3. Properties of the free and immobilized enzymes
Figure 2 shows a comparison of reactions of the free and the
immobilized enzymes in response to external pH. The overall
trends of activity were similar for both the free and the immobilized
1
02.49 U/g, and PPase 506.23 U/g.
Figure 1. SDS–PAGE analysis of the recombinant enzymes expressed in E. coli which were used for one-pot biosynthesis of UDP-Glc. For each enzyme, lane M was the low-
range molecular weight marker proteins; lane 1 was for the total protein of BL21 (DE3) host cells (control); lane 2 was the total protein of recombinant cells; lane 3 was the
supernatant of recombinant cell culture; lane 4 was the soluble fraction of intracellular proteins of the recombinant cells; lane 5 was the insoluble fraction (pellet) of the
recombinant cell lysate.

Q. Dong et al. / Carbohydrate Research 345 (2010) 1622–1626
1625
100
Table 4
Kinetic parameters of individual enzyme
80
Enzyme
Substrate
K
m
(mM)
V
max
(lmol L min )
ꢁ
1
ꢁ1
60
40
20
0
Free
Immobilized Free
Immobilized
MalPase Maltodextrin
Sodium
39.5
23.2
59.5
40.0
7.2
9.0
23.0
30.9
A
phosphate
0
6
12
18
24
G1PTase Glu-1-P
UTP
2.72
0.27
7.47
1.69
0.28
8.35
420
420
410
580
MalPase (mU/ml)
PPase
PPi
28,200 33,600
100
enzymes, with the exception of G1PTTase. Immobilized MalPase
appeared to be more stable to pH changes than the free MalPase.
This might be correlated with a prevention of hydrogen ion distri-
bution between the surface and the solution by the amino groups
on the nanoparticles.
8
6
0
0
40
2
0
B
The kinetic behavior of every enzyme was examined prior to and
0
following immobilization. The apparent Michaelis constant (K
the maximal reaction rate (Vmax) are presented in Table 4. After
immobilization, K value and Vmax values of every enzyme for the
major substrates increased. For example, the K value of MalPase to-
m
) and
0
50
100
150
200
G1PTTase (mU/ml)
m
m
100
ward maltodextrin increased 1.5 times and that toward phosphate
increased 1.7 times as compared with the free enzyme, indicating
some resistance of the carrier against the interaction between the
substrate and the enzyme. The Vmax values of the immobilized Mal-
Pase were almost 3.2-fold (toward maltodextrin) and 3.4-fold (to-
ward phosphate) higher than those of the free enzyme. It is well
known that the immobilization of enzyme may cause the variation
of enzymes kinetic behavior. This suggests an improvement in the
efficiency of UDP-Glc production when it is catalyzed by the immobi-
lized enzymes, rather than by free enzyme in homogeneous solution.
8
0
6
0
40
C
2
0
0
0
50
100
150
200
250
PPase (mU/ml)
Figure 4. Optimization of immobilized enzymes load for the UDP-Glc production.
Reaction conditions: maltodextrin, 5% (m/v); UTP, 2.5 mM; MgCl , 10 mM; buffer,
2
.4. Optimization of reaction conditions
2
sodium phosphate, pH 7.5, 100 mM; 30 °C, 4 h. (A) MalPase, 0ꢁ24 mU/ml;
GlPTTase, 50 mU/ml; PPase, 50 mU/ml; (B) MalPase, 18 mU/ml; GlPTTase,
The substrate concentration was optimized in 1.5 ml tubes
0
ꢁ200 mU /ml; PPase, 50 mU/ml; (C) MalPase, 18 mU/ml; GlPTTase, 100 mU/ml;
(
Eppendorf SafeLock). Figure 3A shows that increasing the
PPase, 0ꢁ250 mU/ml.
1
00
100
80
60
40
20
0
A
B
8
6
4
2
0
0
0
0
0
0
50
100
150
200
0
5
10
15
20
Maltodextrin (m/v,%)
Phosphate (mM)
1
00
D
1
00
80
60
40
20
0
C
8
0
0
0
0
0
6
4
2
0
5
10
Mg2+ (mM)
15
0
5
10
15
UTP (mM)
Figure 3. Effect of substrate concentration on the UDP-Glc production. Common reaction conditions: MalPase, 6 mU/ml; GlPTTase, 50 mU/ml; PPase, 50 mU/ml; 30 °C, 4 h.
, 2 mM; buffer, sodium phosphate, pH 7.5, 100 mM; (B) maltodextrin, 5% (m/v); UTP, 2 mM; MgCl
mM; buffer, sodium phosphate, pH 7.5, 0ꢁ200 mM; (C) maltodextrin, 5% (m/v); UTP, 0ꢁ15 mM; MgCl , 2 mM; buffer, sodium phosphate, pH 7.5, 100 mM; (D) maltodextrin,
Other conditions: (A) maltodextrin, 0ꢁ20% (m/v); UTP, 2 mM; MgCl
2
2
,
2
5
C
2
*
M
C ꢁC
0
CM
% (m/v); UTP, 2.5 mM; MgCl
2
, 0ꢁ15 mM; buffer, sodium phosphate, pH 7.5, 100 mM. The relative activity is defined as: Rð%Þ ¼
is the yield of certain conditions for UDP-Glc.
ꢀ 100 where R is the relative activity,
M
is the yield of optimal condition for UDP-Glc; C
0

1
626
Q. Dong et al. / Carbohydrate Research 345 (2010) 1622–1626
2
1
1
0
0
.0
.5
.0
.5
.0
were retrieved with a magnet and reused five times. As shown in
Figure 5, the reaction rate and the UDP-Glc yield decreased as
the number of reuses increased, so the reaction time for each batch
was extended to obtain a higher yield. After five consecutive reac-
tions (about 80 h totally), the immobilized enzymes still retained
some activity. This suggested that the covalently immobilized en-
zymes were stable on the magnetic nanoparticles.
The supernatants of all five batch reactions were combined and
concentrated and the concentrate was purified by successive SAX
column and ODS column, as described earlier. A white floccule of
about 0.63 g was obtained after lyophilization. The isolated yield
of product was 53% (UTP conversion). The product was character-
ized by HPLC and MS and confirmed to be UDP-Glc. MS results
0
10 20 30 40 50 60 70 80
Time (h)
Figure 5. Time course of the repeated UDP-Glc production using three enzymes
MalPase, GlPTTase, and PPase) immobilized on amino-functionalized magnetic
nanoparticles. Reaction conditions: MalPase, 18 mU/ml; GlPTTase, 100 mU/ml;
PPase, 25 mU/ml; maltodextrin, 5% (m/v); UTP, 2 mM; MgCl , 10 mM; buffer,
sodium phosphate, pH 7.5, 100 mM; 30 °C; total volume, 200 ml.
(
+
+
+
+
were: 605.2 [M+K ]; 634.2 [M+2K ]; 665.3 [M+2K +Na ]; 681.2
+
+
+
[M+3K ]; 682.2 [M+3K +H ] which were consistent with calculated
values.
2
3
. Conclusions
concentration of maltodextrin did not increase the rate of produc-
tion of UDP-Glc. This was because a high concentration of malto-
dextrin made the reaction system too viscous and caused caking
of the immobilized enzymes. Suitable concentrations of phos-
phate and UTP in this system were 100 mM and 2.5 mM, respec-
tively (Fig. 3B and C). A too high salt concentration of salt may
inactivate the enzymes by destroying their hydration layer. The
Mg concentration required to activate G1PTTase and PPase also
modulated the synthesis of UDP-Glc. A fivefold increase in UDP-
Glc production resulted from an increase in Mg2+ from 2 mM to
We have established a one-pot system to synthesize the UDP-
Glc from cheap substrates, maltodextrin, UTP, and phosphate, thus
greatly reducing the cost compared to reactions that depend on
expensive glucose-1-P. Three enzymes were cloned and expressed
in E. coli and individually immobilized onto amino-functionalized
magnetic nanoparticles. These immobilized enzymes were opera-
tionally stable and reusable and retained efficient catalytic activity.
A gram-scale synthesis of UDP-Glc by reuse of the immobilized en-
zymes in repeated batch reactions was achieved. The reaction sys-
tem described here has great potential for production of NDP-
nucleotide sugars.
2
+
1
0 mM, but there were no further increases beyond 10 mM
2+
(Fig. 3D). A 1:5 ratio of UTP and Mg was the most suitable for
UDP-Glc synthesis.
Figure 4A and B shows the effects of manipulation of immobi-
lized enzyme concentrations. Optimal UDP-Glc production was ob-
tained with 18 mU/ml of MalPase and 100 mU/ml of G1PTTase. If
the load of immobilized enzymes was increased further, the prod-
uct yield became saturated. We speculate that too great an addi-
tion of amino-functionalized magnetic nanoparticles in the
system may hamper the free movement of enzymes.
PPase played an auxiliary role in the synthesis of UDP-Glc and
UDP-Glc could be synthesized even in the absence of added immo-
bilized PPase (Fig. 4C). The addition of PPase to a certain level im-
proved the yield of UDP-Glc, indicating a pivotal role for PPase in
maximizing the reaction, but additions beyond this level had no
further effect.
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5
1
The time course of UDP-Glc production also was studied. We
found that 2 mM UTP was completely transformed by the immobi-
lized enzymes in about 10 h.
The optimal reaction conditions for UDP-Glc synthesis by multi-
ple immobilized enzymes were: MalPase, 18 mU/ml; G1PTTase,
4
11. Bae, J.; Lee, D. H.; Kim, D.; Cho, S. J.; Park, J. E.; Koh, S.; Kim, J.; Park, B. H.; Choi,
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1
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8
1
1
2
00 mU/ml; PPase, 25 mU/ml; maltodextrin, 5% (m/v); UTP,
mM; and MgCl , 10 mM; in 100 mM sodium phosphate buffer,
1
1
4. Hajirezaei, M.; Sonnewald, U. Potato Res. 1999, 42, 353–372.
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pH 7.5; at 30 °C for 10 h.
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2
.5. Preparation and characterization of UDP-Glc
1
After the optimization of reaction conditions, a gram-scale reac-
tion was performed in 200 ml. The same immobilized enzymes
19. Lahti, R.; Pitkaranta, T.; Valve, E.; Ira, I.; Kalske, E. K.; Heinonen, J. Bacteriology
988, 170, 5901–5907.
1