Transglycosylation properties of maltodextrin glucosidase (MalZ) from Escherichia coli and its application for synthesis of a nigerose-containing oligosaccharide

Article abstract of DOI:10.1016/j.bbrc.2010.05.073

The transglycosylation reaction of maltodextrin glucosidase (MalZ) cloned and purified from Escherichia coli K12 was characterized and applied to the synthesis of branched oligosaccharides. Purified MalZ preferentially catalyzed the hydrolysis of maltodextrin, γ-cyclodextrin (CD), and cycloamylose (CA). In addition, when the enzyme was incubated with 5% maltotriose (G3), a series of transfer products were produced. The resulting major transfer products, annotated as T1, T2, and T3, were purified and their structures were determined by TLC, MALDI-TOF/MS, ¹³C NMR, and enzymatic analysis. T1 was identified as a novel compound, maltosyl α-1,3-maltose, whereas T2 and T3 were determined to be isopanose and maltosyl-α-1,6-maltose, respectively. These results indicated that MalZ transferred sugar moiety mainly to C-3 or C-6-OH of glucose of the acceptor molecule. To obtain highly concentrated transfer products, the enzyme was reacted with 10% liquefied cornstarch, and then glucose and maltose were removed by immobilized yeast. The T1 content of the resulting reaction mixture reached 9.0%. The mixture of T1 containing a nigerose moiety can have an immunopotentiating effect on the human body and may be a potential functional sugar stuff.

Full text of DOI:10.1016/j.bbrc.2010.05.073

Biochemical and Biophysical Research Communications 397 (2010) 87–92
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Transglycosylation properties of maltodextrin glucosidase (MalZ) from
Escherichia coli and its application for synthesis of a nigerose-containing
oligosaccharide
Kyung-Mo Song a,1, Jae-Hoon Shim b,1, Jong-Tae Park a, Sung-Hee Kim a, Young-Wan Kim c, Winfried Boos d,
Kwan-Hwa Park b,
*
a Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea
b Department of Biology, University of Incheon, Incheon 406-772, Republic of Korea
c Department of Food and Biotechnology, Korea University, Jochiwon 339-700, Republic of Korea
d Department of Biology, University of Konstanz, Konstanz 78457, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2010
Available online 20 May 2010
The transglycosylation reaction of maltodextrin glucosidase (MalZ) cloned and purified from Escherichia
coli K12 was characterized and applied to the synthesis of branched oligosaccharides. Purified MalZ pref-
erentially catalyzed the hydrolysis of maltodextrin, c-cyclodextrin (CD), and cycloamylose (CA). In addi-
tion, when the enzyme was incubated with 5% maltotriose (G3), a series of transfer products were
produced. The resulting major transfer products, annotated as T1, T2, and T3, were purified and their
structures were determined by TLC, MALDI-TOF/MS, 13C NMR, and enzymatic analysis. T1 was identified
Keywords:
Escherichia coli
MalZ
Maltodextrin glucosidase
Transglycosylation
Nigerose
as a novel compound, maltosyl
a-1,3-maltose, whereas T2 and T3 were determined to be isopanose and
maltosyl- -1,6-maltose, respectively. These results indicated that MalZ transferred sugar moiety mainly
a
to C-3 or C-6–OH of glucose of the acceptor molecule. To obtain highly concentrated transfer products,
the enzyme was reacted with 10% liquefied cornstarch, and then glucose and maltose were removed
by immobilized yeast. The T1 content of the resulting reaction mixture reached 9.0%. The mixture of
T1 containing a nigerose moiety can have an immunopotentiating effect on the human body and may
be a potential functional sugar stuff.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
maltotriose to maltose, which is not the inducer of the maltose uti-
lization system [5]. Notably, the MalZ isolated from the malQ
a
-Glucosidase is distributed widely in microorganisms, plants,
knockout mutant exhibited a similar transferring activity to amylo-
maltase, MalQ [6]. The malZ gene has been found and characterized
as a typical mal gene, but the biological function of the enzyme in
cells remains unclear [6]. In addition, the formation of an unknown
oligosaccharide compound whose chromatographic migration in
thin layer chromatography was situated between maltotriose and
maltotetraose was observed, indicating that MalZ exhibits transfer
and animals [1] and catalyzes the hydrolysis of
linkage in
end of the substrate.
a-1,4-glycosidic
a-glucan, releasing
a-D-glucose from the nonreducing
a
-Glucosidase also has been known as trans-
glycosidase because it catalyzes transglycosylation reactions [2,3].
In contrast, the maltodextrin glucosidase (MalZ) from Escherichia
coli K12 is described as an enzyme that removes glucose residue
from the reducing end of maltodextrin [4]. The cloning and
sequencing of the gene encoding MalZ and the isolation and bio-
chemical characterization of the encoded protein revealed an en-
zyme that hydrolyzed maltopentaose and the smaller
maltodextrin to glucose and maltose, releasing glucose consecu-
tively from the reducing end of the maltodextrin. The smallest sub-
strate is maltotriose [4]. MalZ is involved in the maltose utilization
system in E. coli whereby the physiological action is to degrade
activity [4]. Many bacterial saccharifying amylases catalyze
a
a
-1,3-
-1,4-
or
or
a
a
-1,6-transglycosylation in addition to the hydrolysis of
-1,6-linkages [7,8]. The transglycosylation products used in-
-1,6- or -1,3-glucosidic link-
clude all glucosyl saccharides with
a
a
ages such as isomaltose, panose, isopanose, branched products
with a high degree of polymerization (DP; DP > 4), and nigerosyl
oligosaccharides, when maltodextrins are incubated with bacterial
enzymes. A transglycosylated mixture containing nigerose and
nigerooligosaccharides was produced by using the enzyme from
Acremonium sp. [9]. Physicochemical properties and physiological
function of nigerose-containing oligosaccharides have been inten-
sively investigated, and nigerosyl moiety is known to have an
* Corresponding author. Fax: +82 32 835 0763.
E-mail address: parkkh@incheon.ac.kr (K.-H. Park).
These authors contributed equally.
1
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.05.073

88
K.-M. Song et al. / Biochemical and Biophysical Research Communications 397 (2010) 87–92
immunopotentiating effect on the human body [10–12]. In this
study, we characterize MalZ from E. coli and report on the struc-
tural determination of the novel compound of the transfer product
produced by the enzyme. We also discuss the potential bio-indus-
trial applications of the transfer products.
The products were analyzed by TLC and high-performance anion ex-
change chromatography (HPAEC). HPAEC was performed using a
CarboPac PA1 column (0.4 ꢀ 25 cm; Dionex, Sunnyvale, CA, USA)
and an electrochemical detector (ED40; Dionex). Buffers
A
(150 mM NaOH) and B (600 mM sodium acetate in buffer A) were
used for the elution with a 0–30% (v/v) gradient of buffer B at a flow
rate of 1.0 ml/min.
2. Materials and methods
2.5. Mass spectrometry and NMR spectrometry
2.1. Cloning of the malZ gene
The MALDI-TOF mass spectrum was collected using a Voyager
TM-DE system (Perceptive Biosystems, Framingham, MA, USA)
Based on the genome sequence of E. coli K12 (Accession No. NC
000,913), two primers, MalZ-NdeI (50-AGGGGAATTCATATGT-
TAAATGCA-30) and MalZ-PstI (50-AAACTGCAGGCCACGTTTTATCAA-
30), were synthesized to amplify the malZ gene. A 1.8-kb DNA frag-
ment was PCR-amplified from the chromosomal DNA of E. coli K12
using two primers, and the nucleotide sequence of the resulting
DNA fragment was confirmed using an ABI377 PRISM DNA sequen-
cer (PerkinElmer, Carlsbad, CA, USA). The DNA fragment was di-
gested with NdeI and PstI, and ligated into expression vector
p6ꢀHis119 [8] at the corresponding sites such that the open read-
ing frame of the gene could be fused to six histamine residues in-
frame at the C-terminus. The resulting plasmid, p6ꢀHmalZ, was
transformed into E. coli strain MC1061 [Fꢁ, araD139, recA13, (ara-
ABC-leu)7696, galU, galK, lacX74, rpsL, thi, hsdR2, mcrB] for expres-
sion of 6ꢀHis-tagged MalZ.
with
USA) as the matrix. Next, 1
-cyano-4-hydroxycinnamic acid were dropped on a sample appli-
a
-cyano-4-hydroxycinnamic acid (Sigma, St. Louis, MO,
l
l of the purified sample and 1 l of
l
a
cator and dried thoroughly. The sample plate was operated with a
24-kV acceleration voltage. The 13C nuclear magnetic resonance
(NMR) spectrum was recorded using a JEOL LA-400 FTNMR spec-
trometer (JEOL, Tokyo, Japan). The sample was dissolved in H2O/
[D6] at 24.9 °C with tetramethyl silane as the internal reference.
2.6. Enzymatic analysis of the transfer products, T1, T2, and T3
Glucoamylase (AMG 300 L; Novozyme, Bagsværd, Denmark)
was use to investigate the hydrolytic action pattern of transfer
product T1. Glucoamylase (0.05 U/mg of substrate) was incubated
with 0.25% (w/v) T1, T2, and T3 in 50 mM sodium acetate buffer
(pH 4.5) at 55 °C for 4 h. The resulting hydrolysis products were
analyzed by HPAEC.
2.2. Purification of the enzyme
E. coli cells carrying the p6ꢀHmalZ gene were cultured in 1 L of
LB medium [1% (w/v) bacto-tryptone, 0.5% (w/v) yeast extract, 0.5%
(w/v) NaCl] with ampicillin (100 g/ml), and the cells were collected
by centrifugation. The cells were resuspended in 100 ml of lysis
buffer [50 mM Tris–HCl (pH 7.5), 300 mM NaCl, 10 mM imidazole]
and sonicated over an ice bath (VC-600; Sonics & Materials Inc.,
Newtown, CT, USA; output 4, 5 min ꢀ 3 times). The crude cell ex-
tract was centrifuged (10,000g) at 4 °C for 15 min. After sonication,
the cell lysates were subjected to Ni–NTA affinity chromatography
(Qiagen, Valencia, CA, USA), as described previously [8].
2.7. Production and analysis of the transfer products
To produce transfer products, MalZ (1 U/mg of substrate) was
added to 10% (w/v) liquefied cornstarch (Genedex; Samyang Gen-
ex, Seoul, Korea) in 50 mM phosphate buffer (pH 7.0) and incu-
bated at 37 °C for 30 h. Glucose and maltose were removed by
yeast fermentation at 30 °C for 24 h to produce a highly concen-
trated transfer mixture. Immobilized yeast was prepared using
the procedure described by Yoo et al. [15]. The structures and puri-
ties of reaction products were analyzed by TLC, HPAEC, MALDI/
TOF/MS, and NMR, as described previously [8,15,16].
2.3. Enzyme assay
The hydrolytic activity of MalZ was assayed at 37 °C in 50 mM
sodium phosphate buffer (pH 7.0) with 0.5% (w/v) of substrates
3. Results and discussion
such as maltotriose, a-, b-, c-cyclodextrins (CDs) and cycloamylose
(CA) with DP25 by determining the amount of released glucose
using the glucose oxidase/peroxidase method (Asan set glucose;
Asan Pharmaceutical Co., Ltd., Seoul, Korea) [13]. One unit (U) of
hydrolyzing activity was defined as the amount of enzyme produc-
ing 1 mM of glucose per minute. The protein concentration was
determined using bovine serum albumin (Sigma, St. Louis, MO,
USA) as the standard [14].
3.1. Overexpression and properties of MalZ
To overexpress MalZ using E. coli, the malZ gene was cloned into
the p6ꢀHis119 vector [8]. The resulting recombinant was trans-
formed into E. coli. The enzyme was harvested with a purification
fold of 2.5 and yield of 58% via a one-step purification process
using Ni–NTA affinity chromatography. Based on SDS–PAGE analy-
sis, the estimated molecular mass was 69 kDa (data not shown),
which was correlated with the molecular mass deduced from the
predicted amino acid sequence of MalZ. The optimal reaction tem-
perature of MalZ was 37 °C. Activity of the enzyme decreased dras-
tically at temperatures of 40 °C and above, which is typical of most
mesophilic enzymes. The optimal pH of MalZ was 7.0 when 50 mM
sodium phosphate buffer was used.
2.4. Purification of the transfer products
The reaction was carried out by incubation with MalZ (0.5 U/mg
maltotriose) and 5% (w/v) maltotriose in 50 mM sodium phosphate
buffer (pH 7.0) at 37 °C for 2 h in a water bath. The major transfer
products were purified by preparative thin layer chromatography
(TLC) on Whatman K5F silica gel plates (Whatman, Maidstone, Kent,
UK) with ethyl acetate/methanol/acetic acid/water (12:3:3:1,
respectively, v/v/v/v) as the solvent system followed by paper chro-
matography to remove contaminating silica and binder compounds.
Finally, impurities were removed by recycling preparative high-per-
formance liquid chromatography (RP-HPLC; LC-918; JAI Co., Ltd., To-
kyo, Japan) using a JAIGEL W251 column (20 ꢀ 50 cm; JAI Co., Ltd.).
3.2. Action pattern of MalZ on various substrates
To investigate the hydrolytic action pattern of MalZ, maltooligo-
saccharides (G3–G7),
a-CD, b-CD, c-CD, and CA with DP25 (0.5%
each), were reacted with the enzyme in 50 mM sodium phosphate
buffer (pH 7.0) at 37 °C for 12 h, and the reaction products were

K.-M. Song et al. / Biochemical and Biophysical Research Communications 397 (2010) 87–92
89
analyzed by TLC. MalZ hydrolyzed maltooligosaccharides, ranging
from maltotriose (G3) to maltoheptaose (G7), mainly to glucose
rated into three distinct spots, T1, T2, and T3, on TLC developed
with ethyl acetate/methanol/acetic acid/water (12:3:3:1, respec-
tively). These results indicated that MalZ transferred the sugar
moiety to various positions at C–OH of the acceptor molecules.
and maltose (Fig. 1A). MalZ did not hydrolyze either
a- or b-CD,
but efficiently hydrolyzed -CD, to glucose and maltose (Fig. 1C).
c
Moreover, cycloamylose with DP25 was hydrolyzed to maltose
and glucose by MalZ (Fig. 1). These results indicated that the en-
zyme preferred larger cyclized substrates to smaller ones. MalZ
hydrolyzed G3 readily to maltose and glucose at a low concentra-
tion (0.5%; w/v) of substrate. However, several transfer products
including T1, T2, T3 and other unidentified compounds with
DP P 5 were produced when the enzyme was reacted at 5% (w/
v) of G3 (Fig. 1B). In the TLC analysis of the MalZ reaction products,
one spot of the unknown compound, whose migration distance
was similar to that of b-CD [4], appeared on TLC developed in n-
butanol/ethanol/water at a ratio of 5:3:2 (v/v/v), respectively. In
contrast, as shown in Fig. 1B, the unknown compound was sepa-
3.3. Structural determination of the transfer products
To identify the major transfer products, T1, T2, and T3, they
were purified and analyzed by TLC, HPAEC, and 13C NMR and MAL-
DI/TOF-MS (Table 1, Figs. 1–3). With
a molecular mass of
527.17 Da, T2 was identified as a branched product with DP3,
and with a molecular masses of 689.24 Da, T1 and T3 were identi-
fied as branched products with DP4 (Fig. 3A and C). When T1 was
treated with glucoamylase (AMG 300 L; Novozyme) the T1 peak
partially decreased, whereas peaks of glucose and the unknown
compound appeared, indicating that glucoamylase hydrolyzed an
Fig. 1. Action patterns of MalZ on various substrates. (A) MalZ reaction with 0.5% (w/v) linear maltodextrins (G2 and G3); (B) MalZ reaction with 5% (w/v) G3. L1, reaction
product of MalZ; a and b, unidentified compounds P DP5; L2, T1; L3, T2; L4, T3; (C) MalZ reaction with cyclodextrins (CD) and cycloamylose (CA; DP25).
Table 1
13C NMR analysis of the compound T1 (units: ppm).
Carbon atoms
Maltotetraose
T1
92.19
73.59
71.57
77.23
74.27
60.96
Differences
Ring A
Ring B
Ring C
Ring D
1
2
3
4
5
6
92.17
73.62
71.57
77.19
74.28
60.95
0.02
ꢁ0.03
0.00
0.04
ꢁ0.01
0.01
1
2
3
4
5
6
100.00
73.13
71.48
77.04
74.81
60.81
100.14
72.79
79.65
70.60
74.88
60.74
0.14
ꢁ0.34
8.17
ꢁ6.44
0.07
ꢁ0.07
1
2
3
4
5
6
99.90
73.13
71.83
76.95
74.81
60.74
99.86
73.15
71.75
77.03
74.88
60.74
ꢁ0.04
0.02
ꢁ0.08
0.08
0.07
0.00
1
2
3
4
5
6
99.66
72.97
72.00
69.59
76.46
60.69
99.16
72.92
72.01
69.61
76.42
60.54
0.50
ꢁ0.05
0.01
0.02
ꢁ0.04
ꢁ0.15

90
K.-M. Song et al. / Biochemical and Biophysical Research Communications 397 (2010) 87–92
Fig. 2. Enzymatic analysis of transfer products. Each transfer product [(A), T1; (B), T2; (C), T3] was treated with glucoamylase (AMG, Novozyme) and the reaction products
were analyzed using HPAEC. G1, glucose; IM, isomaltose; IP, isopanose; P, panose.
Fig. 3. MALDI-TOF/MS analyses of T1, T2, and T3.

K.-M. Song et al. / Biochemical and Biophysical Research Communications 397 (2010) 87–92
91
Fig. 4. Proposed mechanism of the MalZ transfer reaction.
a
-1,4-linkage of T1 at the nonreducing end to glucose (G1), but the
rat jejunum, where only a small amount of nigerose is absorbed
by the small intestine into the body [17]. The mixture contained
17.7% isopanose, a proportion higher than that in other commercial
linkage of the resulting triglycoside could not be hydrolyzed
(Fig. 2A). T2 had the same elution time in HPAEC as isopanose
and was easily hydrolyzed, giving two peaks that corresponded
to glucose and isomaltose. These results indicated that transfer
product T2 is isopanose (Fig. 2B). Upon treatment with glucoamy-
lase, the hydrolysis of T3 resulted in glucose and panose. Moreover,
products. Isopanose is known to be hardly hydrolyzed by
sidase. Therefore, this transfer mixture containing a high propor-
tion of both isopanose and maltosyl -1,3-maltose has a high
a-gluco-
a
potential as a substitute sugar for the prevention of diabetes, obes-
T3 had the same elution time in HPAEC as maltosyl-(
a-1,6)-malt-
ity, or cardiovascular disease.
ose [16]. Therefore, T3 was confirmed to be maltosyl-
a-1,6-malt-
ose (Fig. 2C). 13C NMR analysis was performed to determine the
structure of T1. Chemical shifts in the 13C NMR spectrum were
compared to those of maltotetraose (Table 1). The shifts occurred
at position C-3 of the second glucose molecules from the reducing
end, that is, from 71.48 to 79.65 ppm, implying that the maltosyl
unit was attached to C-3 in the glucose of maltose. Therefore, the
molecular structure of T1 was a novel compound named malto-
Acknowledgments
This study was supported in part by Basic Science Research Pro-
gram (2009-0087146) through the National Research Foundation
and in part by the Rural Development Administration through the
Cooperative Research Program for Agricultural Science and Technol-
ogy Development. Authors would like to thank Prof. Chang-Kiu Lee,
Kangwon National University for his help with analysis of 13C NMR
data.
syl-(a-1,3)-maltose. These results show that MalZ transfers sugar
moiety mainly to C-3 or C-6–OH of glucose of the acceptor mole-
cule. As proposed in Fig. 4, the maltosyl moiety of maltotriose (do-
nor molecule) was transferred to either C-3–OH or C-6–OH of the
nonreducing glucose of maltotriose (acceptor), thereby producing
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