Efficient synthesis of α-D-glucose-1-phosphate by maltodextrin phosphorylase immobilized on amino-functionalized magnetic nanoparticles

Article abstract of DOI:10.1016/S1872-2067(10)60116-7

α-D-Glucose-1-phosphate (Glc-1-P) is an expensive intermediate in the biosynthesis of nucleotide glucose. This paper describes a biocatalytic system for the efficient synthesis of Glc-1-P from the low cost raw materials: maltodextrin and phosphate at ordinary temperatures. After molecular cloning and the expression of a maltodextrin phosphorylase (MalPase) gene from E. coli (Escherichia coli) K12, the resultant recombinant enzyme was immobilized on amino-functionalized magnetic nanoparticles for recycling and repeated use. Conditions for the biocatalytic reaction were optimized and the immobilized MalPase could be easily recovered and reused over eight cycles in the repeated synthesis of Glc-1-P. After simple purification steps approximately 440 mg of crude product was obtained with a moderate isolation yield of 70.5.

Full text of DOI:10.1016/S1872-2067(10)60116-7

CHINESE JOURNAL OF CATALYSIS
Volume 31, Issue 10, 2010
Online English edition of the Chinese language journal
RESEARCH PAPER
Cite this article as: Chin J Catal, 2010, 31: 1227–1232.
Efficient Synthesis of α-D-Glucose-1-Phosphate by
Maltodextrin Phosphorylase Immobilized on
Amino-functionalized Magnetic Nanoparticles
DONG Qing, OUYANG Liming, LIU Jianwen, XU Jianhe*
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
Abstract: α-D-Glucose-1-phosphate (Glc-1-P) is an expensive intermediate in the biosynthesis of nucleotide glucose. This paper describes
a biocatalytic system for the efficient synthesis of Glc-1-P from the low cost raw materials: maltodextrin and phosphate at ordinary tem-
peratures. After molecular cloning and the expression of a maltodextrin phosphorylase (MalPase) gene from E. coli (Escherichia coli) K12,
the resultant recombinant enzyme was immobilized on amino-functionalized magnetic nanoparticles for recycling and repeated use. Con-
ditions for the biocatalytic reaction were optimized and the immobilized MalPase could be easily recovered and reused over eight cycles in
the repeated synthesis of Glc-1-P. After simple purification steps approximately 440 mg of crude product was obtained with a moderate
isolation yield of 70.5%.
Key words: α-D-Glucose-1-phosphate; maltodextrin phosphorylase; maltodextrin; magnetic nanoparticle; immobilized biocatalyst
Glycoconjugates have important roles in various types of
cellular behavior including energy storage, cell wall structure,
signaling, and cell-to-cell interactions [1] and, therefore, gly-
cosylation is an important biological reaction. Glycosylation
may also alter the biological activities of glycoside drugs sig-
nificantly [2]. Carbohydrate-based drug discovery is currently
a subject of great interest [3]. Glycosyl phosphates are impor-
tant building blocks for the biosynthesis of nucleotide sugars,
which can be used directly as glycosyl donors for glycosylation
reactions catalyzed with different glycosyltransferases [4,5].
Glucose-1-phosphate is the most common phosphoactivated
sugar and it can be synthesized through the isomerization of
glucose-6-phosphate or galactose-1-phosphate in vivo. How-
ever, if isomerization is selected as the technique of choice for
the synthesis of glucose-1-phosphate in vitro, the separation
and purification of the product becomes rather difficult.
Moreover, the use of glucose-6-phosphate or galactose-1-
phosphate as a substrate for the production of the less expen-
sive Glc-1-P is not economically feasible. Maltodextrin
phosphorylase or α-glucan phosphorylase (EC 2.4.1.1) cata-
lyzes the synthesis of glucose-1-phosphate from α-1,4-glucans
such as starch and glycogen derived from bacteria, yeasts,
animals, or plants [6]. Maltodextrin is a kind of soluble starch
and is a cheap source of sugars, which can be used to derive
proteins and produce glycoproteins [7]. The degradation of
maltodextrin, which is cheaper than glucogen from animals has
already been reported. The degradation involves the use of
maltodextrin phosphorylase (MalPase) to produce phosphoac-
tivated glucose (Scheme 1) [8–10]. This can provide a more
effective and economical method for producing high
value-added Glc-1-P.
Although a MalPase-catalyzed reaction effectively catalyzes
the synthesis of Glc-1-P from maltodextrin, all the components
of the reaction system including the enzyme, substrates, and
products are very water-soluble making product purification
very complicated and not cost effective [11]. Immobilized
enzymes are considered to be helpful in isolating protein bio-
catalysts from reaction mixtures. Thus, the overall cost of
Received date: 26 March 2010.
*Corresponding author. Tel: +86-21-6425-2498; Fax: +86-21-6425-0840; E-mail: jianhexu@ecust.edu.cn
Foundation item: Supported by the National Natural Science Foundation of China (20773038, 20902023), the National Basic Research Program of China (973
Program, 2009CB724706, 2009ZX09501-016), the China National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204), and the Shanghai
Leading Academic Discipline Project (B505).
Copyright © 2010, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier BV. All rights reserved.
DOI: 10.1016/S1872-2067(10)60116-7

DONG Qing et al. / Chinese Journal of Catalysis, 2010, 31: 1227–1232
OH
OH
OH
OH
OH
Maltodextrin
phosphorylase
O
O
HO
HO
HO
HO
O
+ Pi
+
O
O
O
HO
HO
O
HO
-
OH
OH
HO
2
OPO
OH
OH
3
OH
OH
-
OH
n
n 1
Scheme 1. Biosynthesis of α-D-glucose-1-phosphate by maltodextrin phosphorylase from E. coli K12 immobilized on amino-functionalized magnetic
nanoparticles.
Glutaraldehyde
Fe O
+
Fe O
3 4
3
4
-SiO (CH ) NH
2
Maltodextrin phosphorylase
H N-
2
3
2 3
Scheme 2. Cross-linking between Fe3O4 nanoparticles and the enzyme.
production may be reduced further because enzyme reusability
improves after immobilization. We used amino-functionalized
magnetic nanoparticles for this purpose in this study.
1 Experimental
1.1 Cloning, expression, and preparation of the enzyme
Amino-functionalized magnetic nanoparticles are a rela-
tively new material for enzyme immobilization. They have
several advantages that have attracted the attention of many
researchers [12–15]. Treating Fe3O4 particles with NaOH
results in the formation of a number of hydroxyl groups on
its surface, which allows amine-functionalized magnetic
particles to be produced by the chemical coating of
3-aminopropyltriethoxysilane. With glutaraldehyde as the
coupling reagent, amino groups on magnetic nanoparticles
can react directly to generate aldehyde groups, which in turn
will form Schiff bases with MalPase. The immobilization of
enzymes onto amino-functionalized magnetic nanoparticles
(Scheme 2) is a rapid process. These nanoparticles act as ef-
fective supports for immobilizing enzymes. More importantly,
the immobilized enzyme can be easily separated from the
reaction mixture and recycled through the simple application of
The malpase gene was amplified from E. coli K12 (Es-
cherichia coli) genomic DNA in a 20 μl PCR reaction using
forward 5′-ctggatccatgtcacaacctatttttaacg-3′ and reverse 5′-
cttgcggccgcttagcgttttgcctgccag-3′ primers [18]. After purifica-
tion, the target gene was inserted into the linearized pET24a(+)
vector with T4 DNA polymerase. Standard methods for mo-
lecular cloning were used [19]. E. coli BL21 (DE3) containing
exogenous recombinant plasmids were grown in LB medium
(tryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L) at 37 °C
with shaking at 200 r/min. When the OD600 of the culture broth
reached about 0.7, 0.05 mol/L IPTG (isopropyl-β-D-Thioga-
lactoside) was added and the cultivation temperature was re-
duced and kept at 15 °C with shaking at 120 r/min for 20 h to
induce the expression of the target enzyme protein. The cells
were then harvested by centrifugation at 12 000 × g for 10 min
at 4 °C followed by disruption using ultrasonication (400 W).
The ultrasonication process lasted for 100 cycles. Each cycle
included 4 s of sonication and a 6 s pause. After centrifugation
the supernatant was collected and lyophilized forming a crude
enzyme powder.
a
magnetic field. The low cost of fabrication of
amino-functionalized magnetic nanoparticles (0.06 US$/g)
also makes this form of immobilization support promising for
industrial-scale processes requiring enzymes. Although other
materials have been used for immobilizing α-glucan phos-
phorylase [16,17], there have been no reports regarding im-
mobilization of MalPase onto amino-functionalized magnetic
nanoparticles for the synthesis of glucose-1-phosphate.
Therefore, amino-functionalized magnetic nanoparticles were
used in the present study to immobilize MalPase for the effi-
cient synthesis of glucose-1-phosphate from maltodextrin and
phosphate.
1.2 Activity assays
The diluted sample was mixed with 1 ml of a solution con-
taining 5% maltodextrin, 2 mmol/L UTP, 10 mmol/L MgCl2, 1
mg maltodextrin phosphorylase, 0.25 U glucose-1-phosphate
thymidylytransferase, and 0.5 U pyrophosphatase in 100

DONG Qing et al. / Chinese Journal of Catalysis, 2010, 31: 1227–1232
mmol/L PBS (sodium phosphate buffer) (pH 7.5). The reaction
1/V = Km/Vmax·Cs+ 1/Vmax
was allowed to proceed for 60 min and then the samples were
frozen at –20 °C. The yield of UDP-glucose (uridine diphos-
phate glucose) was measured by HPLC. Chromatography was
performed on a Shimadzu system (LC-10ADVP) equipped
with an Agela SAX NH2 column (150 μm, 4.6 × 150 mm) using
isocratic elution with 67 mmol/L Na2HPO4-KH2PO4 (pH 5.3).
The flow rate was 1.5 ml/min and UV detection was carried out
at 260 nm [8]. One unit of MalPase activity was defined as the
amount of enzyme producing 1 μmol of UDP-glucose within 1
min under the described conditions.
where V is the initial rate of reaction at different maltodextrin
concentrations or phosphate concentrations CS, Km is the
Michaelis constant, and Vmax is the maximum reaction rate.
1.5 Reuse of immobilized MalPases and purification of
Glc-1-P
A 200 ml reaction mixture in a 500-ml narrow neck flask
containing 5 g/L maltodextrin in 100 mmol/L PBS (pH 7.5)
was combined with immobilized MalPase for the synthesis
reaction. The product concentration was analyzed periodically
by HPLC. After one batch of reactions, the immobilized en-
zyme was recovered through magnetic separation. The recov-
ered immobilized enzyme was washed thrice with PBS (100
mmol/L, pH 7.5) and reused in a new reaction with fresh sub-
strate. To isolate Glc-1-P, the reaction mixture was collected
after removing the immobilized enzymes. The pH of the mix-
ture was adjusted to 4.2 with H3PO4 and an equivalent volume
of methanol was added. The mixture was deposited at 4 °C for
15 min. The supernatant was then collected after centrifugation
at 9 000 × g for 5 min at 4 °C and it was concentrated by rotary
evaporation at 50 °C. After the pH of the concentrated solution
was adjusted to 8 using NaOH, Glc-1-P was precipitated by the
addition of three times the volume of ethanol. The precipitates
were collected after centrifugation at 9 000 × g for 20 min at 4
°C and vacuum dried at 30 °C. The purity of Glc-1-P was
analyzed by HPLC using the method described above and the
product was characterized by mass spectrometry (MS).
Glc-1-P was identified and quantified by ion chromatogra-
phy (IC) using an ICS-1500 ion chromatography system with a
Ion Pac AS11-HC (4 mm×250 mm) analytical column. Elution
was carried out with a 12 mmol/L NaOH solution at a flow rate
of 1 ml/min at 30 °C.
1.3 Immobilization of the enzyme
Amino-functionalized magnetic particles (10 mg/ml) were
suspended in PBS (50 mmol/L, pH 7.5) and then ultrasonicated
for 20 min. Thereafter, 25% glutaraldehyde (20 μl/ml) was
added and shaken for 12 h at 30 °C and 1 100 r/min. The
nanoparticles were then separated by magnetic decantation and
washed five times with PBS (50 mM, pH 7.5) to eliminate the
excessive unreacted glutaraldehyde. The MalPase solution was
mixed with the amino-functionalized magnetic nanoparticles
and shaken at 30 °C for 30 min at 1 100 r/min. After this reac-
tion period, the nanoparticles were separated by magnetic
decantation and washed five times with PBS (50 mmol/L, pH
7.5) to remove any unbound enzymes. The MalPase binding
efficiency is defined as:
2 Results and discussion
E = (C1 – C0)/C1
Recombinant MalPase was expressed after IPTG induction.
Although the conditions of induction were optimized,
SDS-PAGE revealed that the insoluble fraction was more than
80% of the whole cell recombinant MalPase. It showed a pro-
tein with a molecular weight identical to the predicted value of
88 kDa (kilodalton) [20]. After immobilization on the
amino-functionalized magnetic nanoparticles, 85.8% of the
original activity of the recombinant MalPase was retained. The
MalPase binding efficiency was 47.5% and the enzyme loading
was 47.5 mg/g. The specific activity of the immobilized Mal-
Pase was 5.66 U/g.
where E is the MalPase binding efficiency, and C1 and C0 are
the amounts of MalPase protein in the solution before and after
immobilization, respectively. The activity recovery of the
immobilized MalPase was calculated as follows:
R = A/(A1 – A0)
where R is the activity recovery of the immobilized MalPase, A
is the activity of the immobilized MalPase, and A1 and A0 are
the activity of the free MalPase in solution before and after
immobilization respectively.
1.4 Comparison between the free and immobilized
MalPases
Figure 1 shows a comparison between the free and immo-
bilized MalPase in response to the external pH. The overall
trends of activity were similar for both the free and immobi-
lized MalPase. However, the immobilized MalPase appeared to
be more stable against pH changes than the free MalPase.
The kinetic behavior of the free and immobilized MalPase
was examined at different maltodextrin and phosphate con-
centrations. The apparent Michaelis constant Km and the
maximum reaction rate Vmax are presented in Table 1. After
immobilization, the Km value of MalPase toward maltodextrin
The effects of pH on the initial reaction rate of free and
immobilized MalPase were determined at various pH (ranging
from 6.5 to 8.5). The apparent kinetic parameters Km and Vmax
of the free and immobilized MalPase were determined at 30 °C
by measuring the initial reaction rate by the method described
above. Km and Vmax were calculated from Lineweaver-Burk
plots according to the following formula:

DONG Qing et al. / Chinese Journal of Catalysis, 2010, 31: 1227–1232
100
80
60
40
20
0
2.0
1.5
1.0
Free
Immobilized
0.5
0.0
6.0
6.5
7.0
7.5
pH
8.0
8.5
0
2
4
6
8
10 12 14 16 18
Time (h)
Fig. 1. Effect of pH on initial activity of free and immobilized MalPase.
Fig. 3. The time-course for Glc-1-P synthesis by immobilized MalPase.
Reaction conditions: MalPase 6 mU/ml, maltodextrin 5 g/L, PBS (pH 7.5)
50 mmol/L, 30 °C.
Table 1 Kinetic parameters of the free and immobilized MalPases
toward maltodextrin and phosphate
phosphate in this reaction system was found to be 100 mmol/L
(Fig. 2). We assumed that an excessively high concentration of
salt might cause enzyme deactivation by destroying its hydra-
tion layer.
Km/(mmol/L)
Vmax/(μmol/(min·mg))
Enzyme
Maltodextrin Phosphate Maltodextrin Phosphate
Free
39.5
59.5
23.2
40.0
7.2
9.0
Immobilized
23.0
30.9
Figure 2 also shows the effect of immobilized enzyme
loading. An optimal Glc-1-P production was achieved with 24
mU/ml of immobilized MalPase. When the load of immobi-
lized enzyme was increased further, the product concentration
became saturated. The excess heavy support in the system may
hamper the free movement of the immobilized enzyme.
The time course of Glc-1-P production was examined and is
shown in Fig. 3. The concentration of Glc-1-P did not markedly
increase after transformation by the immobilized enzymes over
about 8 h.
increased 1.5 times whereas that toward phosphate increased
1.7 times compared with the free enzyme. This indicated some
resistance from the carrier toward the interaction between the
substrate and the enzyme. The Vmax values of the immobilized
MalPase were almost 3.2-times (toward maltodextrin) and
3.4-times (toward phosphate) higher than those of the free
enzyme. The immobilization of the enzyme might have caused
a variation in its enzyme kinetic behavior. This suggests an
improvement in the efficiency of Glc-1-P production when it is
catalyzed using immobilized MalPase rather than using the free
enzyme in a homogeneous solution.
The optimal reaction conditions for Glc-1-P synthesis by the
immobilized MalPase were: MalPase, 24 mU/ml; maltodextrin,
5 g/L; phosphate buffer, 100 mmol/L, pH 7.5; temperature 30
°C and duration 8 h.
The substrate concentration was optimized in 1.5 ml tubes
(Eppendorf SafeLock). Figure 2 shows that an increase in the
maltodextrin concentration did not increase the production rate
of Glc-1-P. This was due to a high concentration of maltodex-
trin leading to a viscous reaction system and to aggregation of
the immobilized enzyme. The most suitable concentration of
After optimization of the reaction conditions, the reaction
was carried out in a 200 ml system for the gram-scale prepa-
ration of Glc-1-P. After the first batch of reactions, the immo-
bilized enzyme was recovered with a magnet and reused over
100
80
60
40
20
0
(a)
(b)
(c)
0
5
10
15
20
0
50
100
150
200
0
6
12
18
24
30
Maltodextrin (g/L)
Phosphate (mmol/L)
MalPase (U/ml)
Fig. 2. System optimization for Glc-1-P production. (a) Effect of maltodextrin; (b) Effect of phosphate; (c) Optimization of the immobilized MalPase
loaing. Reaction conditions: (a) MalPase 6 mU/ml, PBS (pH 7.5) 50 mmol/L, 30 °C, 4 h; (b) MalPase 6 mU/ml, maltodextrin 5 g/L, PBS (pH 7.5) 200
mmol/L, 30 °C, 4 h; (c) maltodextrin 5 g/L, PBS (pH 7.5) 100 mmol/L, 30 °C, 4 h. The relative activity is defined as R = (CM–C0)/CM, where R is relative
activity, CM is the optimal condition yield for Glc-1-P, and C0 is the yield of specific conditions for Glc-1-P.

DONG Qing et al. / Chinese Journal of Catalysis, 2010, 31: 1227–1232
was purified by simple precipitation and centrifugation. The
1.6
1.2
0.8
0.4
0.0
free and immobilized enzyme as well as the unspent substrates
and organic solvent for purification were recovered efficiently.
Therefore, this method of generating Glc-1-P is extremely
cost-effective. The reaction system described in this study has
great potential for the practical production of high-value-added
Glc-1-P or other glucose monophosphates.
References
1
2
3
4
5
Goedl C, Schwarz A, Minani A, Nidetzky B. J Biotechnol,
2007, 129: 77
1
2
3
4
5
6
7
8
Batch
Biggins J B, Onwueme K C, Thorson J S. Science, 2003, 301:
1537
Fig. 4. Repeated batch production of Glc-1-P with recycled immobi-
lized MalPase. Reaction conditions: MalPase 24 mU/ml, maltodextrin 5
g/L, PBS (pH 7.5) 100 mmol/L, 30 °C, total volume 200 ml.
Wong C H. Carbohydrate-Based Drug Discovery. Weinhein:
Wiley-VCH. 2003
Thorson J S, Barton W A, Hoffmeister D, Albermann C, Ni-
kolov D B. ChemBioChem, 2004, 5: 16
eight cycles. As shown in Fig. 4, the Glc-1-P yield decreased
gradually with an increase in the number of repeat reactions
(1−4 batches). Subsequently, we extended the time for each run
to 12 h to obtain higher yields (5−8 batches). However, in the
eighth reaction the yield was not at the same level as that of the
previous batches. After eight consecutive reactions (about 96 h
in total) the immobilized MalPase still retained some activity.
This indicates that the covalently immobilized enzyme was
quite stable on the magnetic nanoparticles.
Griffith B R, Langenhan J M, Thorson J S. Curr Opin Bio-
technol, 2004, 16: 622
6
7
Schinzel R, Nidetzky B. FEMS Microbiol Lett, 1999, 171: 73
Nahálka J, Shao J, Gemeiner P. Biotechnol Appl Biochem,
2007, 46: 1
8
9
Nahálka J. J Ind Microbiol Biotechnol, 2007, 35: 219
Chen S Q, Liu J F, Pei H D, Li J, Zhou J, Xiang H. Enzyme
Microb Technol, 2007, 41: 390
10 Hong Y, Wu L, Liu B, Peng C, Sheng D H, Ni J F, Shen Y L.
J Mol Catal B, 2008, 54: 27
The supernatants of the eight batches of reactions were
combined and concentrated, and the concentrate was purified
as described earlier. A white solid of about 440 mg was ob-
tained after vacuum drying. The isolated yield of the product
was 70.5%. The final product was characterized by LC-MS.
The MS results showed peaks at 263.21 [M+Na+H]+, 299.16
[M+Ka+H]+, and 305.27 [M+2Na+H]+, which were consistent
with the calculated values.
11 Bae J, Lee D H, Kim D, Cho S J, Park J E, Koh S, Kim J, Park
B H, Choi Y, Shin H J, Hong S I, Lee D S. Process Biochem,
2005, 40: 3707
12 Hu B, Pan J, Yu H L, Liu J W, Xu J H. Process Biochem, 2009,
44: 1019
13 Li Y G, Gao H S, Li W L, Xing J M, Liu H Z. Bioresour
Technol, 2009, 100: 5092
14 Yong Y, Bai Y X, Li Y F, Lin L. Process Biochem, 2008, 43:
1179
15 Zhu H, Pan J, Hu B, Yu H L, Xu J H. J Mol Catal B, 2009, 61:
174
3 Conclusions
16 Garg S, Kumar A. Afr J Biotechnol, 2007, 6: 2715
17 Garg N, Kumar A. J Braz. Chem Eng, 2008, 25: 229
18 Nora A C, David D, Alejandro M V, Francisco J M, Edurne B
F, Marĺa T M Z, Gustavo E, Steven B, Javier P R. J Bacteriol,
2006, 188: 77
A gram-scale synthesis of glucose-1-phosphate from
low-cost maltodextrin was achieved through repeat batch re-
actions using a recyclable biocatalyst. The biocatalyst was
prepared using a recombinant maltodextrin phosphorylase
cloned from E. coli K12, which was covalently immobilized
onto amino-functionalized magnetic nanoparticles. The im-
mobilized MalPase was operationally stable and reusable,
retaining high catalytic activity in repeat reactions. The product
19 Sambrook J, Russell D. Molecular Cloning 3. Woodbury: Cold
Spring Harbor Laboratory Press, 2001
20 Dong Q, Ouyang L M, Yu H L, Xu J H, Lin G Q. Tetrahedron
Lett, 2009, 51: 1603