Synthesis of α(1→4)-linked non-natural mannoglucans by α-glucan phosphorylase-catalyzed enzymatic copolymerization

Article abstract of DOI:10.1016/j.carbpol.2016.06.057

α-Glucan phosphorylase catalyzes enzymatic polymerization of α-D-glucose 1-phosphate (Glc-1-P) as a monomer from a maltooligosaccharide primer to produce α(1→4)-glucan, i.e., amylose, with liberating inorganic phosphate (Pi). Because of quite weak specificity for the recognition of substrates by thermostable α-glucan phosphorylase (from Aquifex aeolicus VF5), in this study, we investigated the enzymatic copolymerization of Glc-1-P with its analogue monomer, α-D-mannose 1-phosphate (Man-1-P) under the conditions for removal of Pi as the precipitate with ammonium and magnesium in ammonia buffer containing Mg²⁺ ion to produce α(1→4)-linked non-natural mannoglucans composed of Glc/Man units. The reaction was conducted in different feed ratios using the maltotriose primer at 40?°C for 7?days. The MALDI-TOF mass and ¹H NMR spectra of the products fully supported the mannoglucan structures.

Full text of DOI:10.1016/j.carbpol.2016.06.057

Carbohydrate Polymers 151 (2016) 1034–1039
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Synthesis of ˛(1→4)-linked non-natural mannoglucans by ˛-glucan
phosphorylase-catalyzed enzymatic copolymerization
Ryotaro Baba, Kazuya Yamamoto, Jun-ichi Kadokawa∗
Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 April 2016
Received in revised form 10 May 2016
Accepted 14 June 2016
Available online 16 June 2016
˛-Glucan phosphorylase catalyzes enzymatic polymerization of ˛-d-glucose 1-phosphate (Glc-1-P) as
a monomer from a maltooligosaccharide primer to produce ␣(1→4)-glucan, i.e., amylose, with liber-
ating inorganic phosphate (Pi). Because of quite weak specificity for the recognition of substrates by
thermostable ˛-glucan phosphorylase (from Aquifex aeolicus VF5), in this study, we investigated the
enzymatic copolymerization of Glc-1-P with its analogue monomer, ˛-d-mannose 1-phosphate (Man-1-
P) under the conditions for removal of Pi as the precipitate with ammonium and magnesium in ammonia
buffer containing Mg2+ ion to produce ˛(1→4)-linked non-natural mannoglucans composed of Glc/Man
units. The reaction was conducted in different feed ratios using the maltotriose primer at 40 ◦C for 7 days.
The MALDI-TOF mass and 1H NMR spectra of the products fully supported the mannoglucan structures.
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
Enzymatic copolymerization
˛-Glucan phosphorylase
Mannoglucan
Non-natural polysaccharide
1. Introduction
Because KGM is a high molecular weight, water-soluble, and non-
ionic polysaccharide and shows film and gel formation properties,
Polysaccharides are widely distributed in nature and have
been regarded as structural materials, as suppliers of energy,
and as key materials for specific biological and vital functions
(Berg, Tymoczko, & Stryer, 2012). Natural polysaccharides exhibit
a great variety of chemical structures owing to many kinds of
monosaccharide residues and different stereo- and regio-types of
glycosidic linkages, which contribute to serving a wide range of
their in vivo functions (Schuerch, 1986). Besides homopolysac-
charides composed of a kind of monosaccharide repeating units,
such as cellulose, starch (a mixture of mostly linear amylose
and highly branched amylopectin), and chitin, there are a vari-
ety of heteropolysaccharides in nature, which are consisting of
multiple kinds of monosaccharides units (Stephen, Phillips, &
Williams, 2006). Mannose (Man)-containing heteropolysaccha-
rides are widespread in nature representatively as glucomannans
are found in softwoods, tubers, and bulbs (Tester & Al-Ghazzewi,
2013). For example, konjac glucomannan (KGM) is found in tubers
of the Amorphophallus konjac plant. It is ˇ(1→4)-linked het-
eropolysaccahride composed of Man and glucose (Glc) residues (ca.
1.6:1) with a low degree of acetyl groups (approximately1 acetyl
group per 17 residues) at C-6 position (Dave & McCarthy, 1997).
it has been developed into biomaterials with different application
focuses (Wang, Liu, Li, Wang, & Wang, 2015; Zhang, Chen, & Yang,
2014). Accordingly, synthesis of non-natural polysaccharides com-
posed of Man and Glc residues with well-defined structure has
attracted much attention for applications as new environmentally
benign functional materials in the research fields of biomedical and
tissue engineering.
Because it has been well accepted that an enzymatic method
is a powerful approach to synthesize well-defined polysaccha-
rides (Kadokawa, 2011b; Kobayashi & Makino, 2009; Kobayashi,
Uyama, & Kimura, 2001; Shoda, Izumi, & Fujita, 2003; Shoda,
Uyama, Kadokawa, Kimura, & Kobayashi, 2016), we have inves-
tigated ␣-glucan phosphorylase-catalyzed enzymatic reactions
using several monosaccharide 1-phosphate substrates to obtain
non-natural polysaccharides (Kadokawa, 2015; Shoda, Uyama,
Kadokawa, Kimura & Kobayashi, 2016). ␣-Glucan phosphorylase
is the enzyme that induces polymerization of an ␣-d-glucose
1-phosphate (Glc-1-P) monomer from the maltooligosaccharide
primer according to the reaction manner of successive glu-
cosylations to produce ˛(1→4)-glucan, that is, amylose, with
liberating inorganic phosphate (Pi) (Fujii et al., 2003; Kitaoka
& Hayashi, 2002; Nakai, Kitaoka, Svensson, & Ohtsubo, 2013;
Seibel, Jordening, & Buchholz, 2006; Yanase, Takaha, & Kuriki,
2006; Ziegast & Pfannemüller, 1987). The ␣-glucan phosphorylase-
catalyzed enzymatic polymerization is one of the efficient methods
∗
Corresponding author.
E-mail address: kadokawa@eng.kagoshima-u.ac.jp (J.-i. Kadokawa).
http://dx.doi.org/10.1016/j.carbpol.2016.06.057
0144-8617/© 2016 Elsevier Ltd. All rights reserved.

R. Baba et al. / Carbohydrate Polymers 151 (2016) 1034–1039
1035
HO
HO
HO
HO
HO
O
O
O
OH
HO
HO
O
HO
O
HO
O
O
n
m
+
+
HO
OH
OH
HO
OH
O
O
O P O-
O-
HO
HO
HO
OH
O P OH
Maltotriose (Glc3)
OH
-D-Mannose 1-phosphate
(Man-1-P)
-D-Glucose 1-phosphate
(Glc-1-P)
HO
O
Thermostable -glucan phosphorylase
(Aquifex aeolicus VF5)
HO
O
HO
OH
O
HO
OH
Ammonia buffer
H
O
HO
(0.5 mol/L, pH 8.5) / Mg2+
p
Fig. 1. Thermostable ˛-glucan phosphorylase-catalyzed enzymatic copolymerization of Glc-1-P with Man-1-P from Glc3 to produce ˛(1→4)-linked non-natural mannoglu-
cans.
to obtain a pure amylose composed linear ˛(1→4)-glucan struc-
ture because the complete separation of natural amylose from
starch is difficult, and moreover, which often accompanied with a
slightly branched structure (Kim & Willett, 2004; Majzoobi, Rowe,
Connock, Hill, & Harding, 2003; Shibanuma, Takeda, Hizukuri, &
Shibata, 1994). Because this enzyme shows weak specificity for
the recognition of substrates (Evers, Mischnick, & Thiem, 1994;
Evers & Thiem, 1997; Kadokawa, 2011a; Kawazoe, Izawa, Nawaji,
Kaneko, & Kadokawa, 2010; Nawaji, Izawa, Kaneko, & Kadokawa,
2008a,2008b), and furthermore, ␣-glucan phosphorylase isolated
from thermophilic bacterium (thermostable ␣-glucan phospho-
rylase) exhibits the more tolerance for the specificity than the
most well-known ␣-glucan phosphorylase (potato ␣-glucan phos-
phorylase) (Kadokawa, 2013; Takata, Shimohigoshi, Yamamoto, &
Kadokawa, 2014; Takata, Yamamoto, & Kadokawa, 2015; Takemoto
et al., 2013; Umegatani et al., 2012), we have found that ␣(1→4)-
linked Man and glucosamine (GlcN) oligosaccharides are obtained
by thermostable ␣-glucan phosphorylase (from Aquifex aeolicus
VF5)-catalyzed successive mannosylations and glucosaminylations
of maltotriose (Glc3) as a glycosyl acceptor with ␣-d-mannose
1-phosphate (Man-1-P) and ␣-d-glucosamine 1-phosphate (GlcN-
1-P) as glycosyl donors, respectively (Shimohigoshi, Takemoto,
Yamamoto, & Kadokawa, 2013). Furthermore, thermostable ␣-
glucan phosphorylase-catalyzed reaction of GlcN-1-P was acceler-
ated with removal of the produced Pi as the ammonium magnesium
phosphate precipitate in ammonia buffer containing Mg2+ ion, on
the basis of the fact that Pi formed an insoluble salt with ammo-
nium and magnesium (Borgerding, 1972), leading to the progress
of the enzymatic polymerization. Consequently, this reaction
system gives an ˛(1→4)-linked GlcN polysaccharide as the enzy-
matic polymerization product, which corresponds to a chitosan
stereoisomer (Kadokawa, Shimohigoshi, Yamashita, & Yamamoto,
2015). Under the similar conditions, we successfully synthesized
an ˛(1→4)-linked non-natural heteroaminopolysaccharide com-
posed of Glc/GlcN units (˛(1→4)-linked glucosaminoglucan) by
the thermostable ␣-glucan phosphorylase-catalyzed enzymatic
copolymerization of Glc-1-P/GlcN-1-P as comonomers in ammonia
buffer containing Mg2+ ion (Yamashita, Yamamoto, & Kadokawa,
2015).
2. Experimental part
2.1. Materials and methods
A monomer, Man-1-P with free form, was synthesized according
to the literature procedure (MacDonald, 1962, 1966). A stan-
dard amylose sample (Fig. S1) was prepared by the thermostable
␣-glucan phosphorylase-catalyzed enzymatic polymerization of
Glc-1-P. Thermostable ␣-glucan phosphorylase from Aquifex aeoli-
cus VF5 was kindly supplied from Ezaki Glico, Osaka, Japan
(Bhuiyan, Rus’d, Kitaoka, & Hayashi, 2003; Yanase et al., 2006;
Yanase, Takata, Fujii, Takaha, & Kuriki, 2005). All other reagents
and solvents were used as received from commercial sources. 1H
NMR spectra was recorded on JEOL ECA 600 and ECX 400 spec-
trometers. MALDI-TOF MS measurements were conducted by using
SHIMADZU Voyager Biospectrometry Workstation Ver. 5.1 with
2,5-dihydroxybenzoic acid as a matrix containing 0.05% trifluo-
roacetic acid under positive ion mode.
2.2. ˛-Glucan phosphorylase-catalyzed enzymatic
copolymerization of Glc-1-P with Man-1-P and subsequent
glucoamylase treatment for MALDI-TOF MS measurement
A mixture of Glc-1-P (disodium salt, 12.5 mg, 0.0411 mmol),
Man-1-P (free form, 10.5 mg, 0.0404 mmol), and Glc3 (4.2 mg,
8.33 ␮m) in 0.5 mol/L ammonia buffer (pH 8.5, 0.60 mL) dissolved
MgCl2 (7.6 mg, 0.0798 mmol) was incubated in the presence of ther-
mostable ␣-glucan phosphorylase (20 U) at 40 ◦C for 7 days. After
the precipitate was removed by centrifugation, supernatant was
maintained at 100 ◦C for 1 h to deactivate the enzyme. The deacti-
vated enzyme was removed by centrifugation, and the supernatant
was lyophilized to obtain the crude product (43.5 mg). It was dis-
solved in water (3.0 mL) and the solution was incubated in the
presence of glucoamylase (GA, 20 U) at 40 ◦C for 1 h. The reaction
mixture was maintained at 100 ◦C for 1 h to deactivate the enzyme,
and the deactivated enzyme was removed by centrifugation. The
supernatant was lyophilized to give the GA-treated product. Both
the products before and after the GA treatment were subjected to
MALDI-TOF MS measurement (Fig. 2, Table 1).
On the basis of the above background, in this paper, we report
the synthesis of a novel ˛(1→4)-linked non-natural mannoglu-
can composed of Glc/Man units by the thermostable ␣-glucan
phosphorylase-catalyzed enzymatic copolymerization of Glc-1-
P/Man-1-P as comonomers in ammonia buffer containing Mg2+ ion
(Fig. 1). The present material has a potential for practical applica-
tions as a new functional polysaccharide material in biomedical and
tissue engineering.
2.3. General procedure for the synthesis of ˛(1→4)-linked
mannoglucans by thermostable ˛-glucan
phosphorylase-catalyzed enzymatic copolymerization of Glc-1-P
with Man-1-P
A typical experimental procedure was as follows (run 2). A
mixture of Glc-1-P (disodium salt, 25.4 mg, 0.0836 mmol), Man-1-

1036
R. Baba et al. / Carbohydrate Polymers 151 (2016) 1034–1039
Table 1
MALDI-TOF MS data (Fig. 2) of crude product from thermostable ␣-glucan phosphorylase-catalyzed enzymatic copolymerization (Glc-1-P:Man-1-P:Glc3 = 5:5:1) and product
after glucoamylase (GA) treatment.
Calcd: m/z
Found: m/z (crude product)
Found: m/z (after GA treatment)
[(Glc/Man) + Glc4 + Na]+
[[(Glc/Man)2 + Glc3 + Na]+
[(Glc/Man)3+ Glc3 + Na]+
[(Glc/Man)4 + Glc3 + Na]+
[(Glc/Man)5 + Glc3 + Na]+
[(Glc/Man)6 + Glc3 + Na]+
[(Glc/Man)7 + Glc3 + Na]+
[(Glc/Man)8 + Glc3 + Na]+
[(Glc/Man)9 + Glc3 + Na]+
[(Glc/Man)10-Glc3 + Na]+
[(Glc/Man)11 + Glc3 + Na]+
[(Glc/Man)12 + Glc3 + Na]+
[(Glc/Man)13 + Glc3 + Na]+
[(Glc/Man)14 + Glc3 + Na]+
[(Glc/Man)15 + Glc3 + Na]+
689.5674
851.7080
689.0016
850.8956
689.5661
851.5695
1013.8486
1175.9892
1338.1298
1500.2704
1662.4110
1824.5516
1986.6922
2148.8328
2310.9734
2473.1140
2635.2546
2797.3952
2959.5358
1012.7755
1174.6287
1336.4920
1498.3279
1660.1541
1822.0037
1983.8406
2145.6686
2308.4645
2470.2708
2632.1227
2793.9147
2955.7286
1013.5642
1175.5352
1337.5022
1499.4501
1661.4026
1823.3531
1985.2967
2147.2208
2309.0788
Fig. 2. MALDI-TOF MS of (a) crude product from thermostable ␣-glucan phosphorylase-catalyzed enzymatic copolymerization (Glc-1-P:Man-1-P:Glc3 = 5:5:1) and (b) product
after glucoamylase (GA) treatment.
P (free form, 22.5 mg, 0.865 mmol), and Glc3 (4.2 mg, 8.33 ␮mol)
in 0.5 mol/L ammonia buffer (pH 8.5, 0.80 mL) dissolved MgCl2
(15.6 mg, 0.164 mmol) was incubated in the presence of ther-
mostable ␣-glucan phosphorylase (20 U) at 40 ◦C for 7 days. The

R. Baba et al. / Carbohydrate Polymers 151 (2016) 1034–1039
1037
Table 2
Results in thermostable ␣-glucan phosphorylase-catalyzed enzymatic copolymerization of Glc-1-P with Man-1-P from Glc3.a
d
Run
1
Glc-1-P:Man-1-P:Glc3
5:5:1
Conv.b (%)
51.7
Yieldc (%)
12.2
Average numbers of Glc/Man unitsd (Compositional ratios; Glc:Man)
Mn
14.5/3.7
(1:0.26)
3500
3970
6600
6600
7900
2
3
4
10:10:1
20:20:1
10:30:1
30:10:1
44.5
43.6
44.8
53.1e
40.3
40.1
23.7
44.6
18.1/3.3
(1:0.18)
32.9/4.9
(1:0.15)
28.2/8.5
(1:0.30)
5
41.7/3.9
(1:0.094)
a
Reaction was carried out using Glc3 (8.1–8.3 ␮mol) as the primer in the presence of thermostable ␣-glucan phosphorylase (20 U) in ammonia buffer containing an
equimolar amount of Mg2+ ion with monomers (Glc-1-P and Man-1-P) at 40 ◦C for 7 days.
b
Determined by weight of ammonium magnesium phosphate precipitate from reaction mixture.
c
Fraction insoluble in methanol.
d
Determined by 1H NMR spectra.
e
Determined by weight of insoluble fraction after washing precipitate with DMSO.
Fig. 3. 1H NMR spectrum of the isolated ␣(1→4)-linked mannoglucan (run 2) in D2O.
precipitate was then removed by centrifugation, and the super-
natant was maintained at 100 ◦C for 1 h to deactivate the enzyme.
The deactivated enzyme was removed by centrifugation, and the
supernatant was lyophilized. The solution of the resulting crude
product in water (3.0 mL) was then treated with an anion-exchange
resin (Amberlite IRA-400J Cl) for 1 h, filtered, and concentrated.
Subsequent addition of a large amount methanol to the concen-
trated solution resulted in yielding the precipitate, which was
isolated by filtration and lyophilized to obtain the ˛(1→4)-linked
mannoglucan (12.8 mg) in 40.3% yield based on the total Glc and
Man residues present in the reaction system. 1H NMR (D2O, ppm)
ı 3.26–3.99 (H-3,4,5,6, Glc-H-2), 4.07 (Man-H-2), 4.66 (H-1␤ of
reducing end), 5.23 (H-1␣ of reducing end), 5.30 (Man-H-1 of Man-
Man), 5.32 (Glc-H-1 of Glc-Man), 5.34 (Man-H-1 of Man-Glc), 5.42
(Glc-H-1 of Glc-Glc).
3. Results and discussion
In order to evaluate by the MALDI-TOF MS measurement
(Fig. 2, Table 1) whether the enzymatic copolymerization of Glc-
1-P with Man-1-P is progressed by the thermostable ␣-glucan
phosphorylase catalysis under the conditions for removal of Pi
as the precipitate, the reaction was first demonstrated in the
low monomers/primer feed ratio (Glc-1-P:Ma-1-P:Glc3 = 5:5:1) in
ammonia buffer (0.5 mol/L, pH 8.5) in the presence of an equimolar
amount of MgCl2 with the monomers (Glc-1-P + Ma-1-P) at 40 ◦C
for 7 days. With the progress of reaction times, the precipitate
was gradually formed in the system, speculating the occurrence
of the copolymerization. The precipitate was thus removed by cen-
trifugation and the supernatant was lyophilized to give the crude
product. The MALDI-TOF MS of the product in Fig. 2a observes plu-
ral signals separated by m/z 162, probably corresponding to the

1038
R. Baba et al. / Carbohydrate Polymers 151 (2016) 1034–1039
molecular masses of polysaccharides composed of Glc/Man units
with the DPs = 4–18. This result suggests the occurrence of the
enzymatic polymerization by the thermostable ␣-glucan phospho-
rylase catalysis in the present system, however, the presence of
Man units in the product is not evident by the MS, because the
molecular masses of both the anhydroglucose and anhydroman-
nose units are identical. To confirm the mannoglucan structure
further, the product was treated with GA to be hydrolyzed at the
˛(1→4)-glucan nonreducing end. Because GA catalyzes exo-type
hydrolysis from the nonreducing end of ˛(1→4)-glucan, giving
Glc restudies, the GA-catalyzed exowise hydrolysis is inhibited
at the Man unit position in the produced polysaccharide if it is
present. The MALDI-TOF MS after the GA-treatment in Fig. 2b still
observes plural signals corresponding to the molecular masses of
polysaccharides composed of Glc/Man units although the signals
are detected at the smaller molecular mass area than those before
the treatment, owing to the partial hydrolysis of the ˛(1→4)-glucan
nonreducing end. This result strongly indicates that the enzymatic
copolymerization of Glc-1-P with Man-1-P from Glc3 occurs by
the thermostable ␣-glucan phosphorylase catalysis to produce the
non-natural mannoglucan.
The thermostable ˛-glucan phosphorylase-catalyzed enzymatic
copolymerization of Glc-1-P with Man-1-P from Glc3 was per-
formed in different feed ratios under the same conditions (Table 2).
After removing the precipitated ammonium magnesium phos-
phate by centrifugation, the supernatants were treated with an
anion-exchange resin to adsorb the unreacted monomers, and sub-
sequently the products were precipitated from methanol, which
were isolated by filtration and then lyophilized to obtain the
mannoglucans. The monomer conversions were estimated by
weight of the phosphate precipitates because Pi was stepwise lib-
erated at the each transfer reaction step of Glc/Man residues from
the monomers to the propagating end. The structures of the iso-
lated products were determined by 1H NMR analysis to be the
non-natural mannoglucans. For example, the 1H NMR spectrum of
the produced polysaccharide (run 2) in D2O (Fig. 3) shows the char-
acteristic signals due to H-2 and H-1 of Man residues in addition to
the H-1 signals due to Glc residues and the reducing end and the sig-
nals due to the other protons in the saccharide backbone, strongly
supporting the presence of Man units in the product by the enzy-
matic copolymerization. Furthermore, the detection of two sets of
the anomeric signals at ı 5.32/5.42 and ı 5.30/5.34 due to each of ␣-
glucoside and ␣-mannoside, corresponding to the respective two
sequences, Glc-Man/Glc-Glc and Man-Man/Man-Glc, indicates the
relative random sequence of Glc/Man units in the product. From
the integrated ratio among the anomeric signals of ␣-glucoside, ␣-
mannoside, and the reducing end, the average numbers of Glc/Man
units (the compositional ratio) and the Mn value were calculated to
be 18.1:3.3 (1:0.18) and 3970, respectively.
trum of the material in NaOD/D2O did not indicate the presence of
Man units and was identical with that of amylose (Fig. S1). This
result indicates that under the conditions in such higher Glc-1-
P/Man-1-P feed ratio, the homopolymerization of Glc-1-P takes
place simultaneously with the copolymerization owing to its higher
reactivity to produce the water-insoluble Glc polysaccharide, that
is, amylose.
4. Conclusions
This paper reports the thermostable ˛-glucan phosphorylase-
catalyzed enzymatic copolymerization of Glc-1-P with Man-1-P
from the Glc3 primer under the conditions for removal of Pi as the
precipitate to produce the non-natural mannoglucans. The struc-
tures of the products were confirmed by the MALDI-TOF mass
and 1H NMR spectra. The Mn values were relatively depended on
the monomers/primer feed ratios. However, the Glc/Man compo-
sitional ratios were much higher than the Glc-1-P/Man-1-P feed
ratios because of the higher reactivity of the native substrate, Glc-1-
P, than the analogue one, Man-1-P, for the ␣-glucan phosphorylase
catalysis. The incorporation of Man residues into a natural polysac-
charide, amylose, as demonstrated in this study, is expected to lead
to exhibiting new functions from the present non-natural materi-
als. Accordingly, we are now investigating the further study on the
functions related to the structures of the present polysaccharides
and possible applications as new environmentally benign materi-
als.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carbpol.2016.06.
057.
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