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Abstract: This review describes a new enzymatic method for in vitro glycogen synthesis and its structure and properties. In
this method, short-chain amylose is used as the substrate for branching enzymes (BE, EC 2.4.1.18). Although a kidney bean
BE and Bacillus cereus BE could not synthesize high-molecular weight glucan, BEs from 6 other bacterial sources produced
enzymatically synthesized glycogen (ESG). The BE from Aquifex aeolicus was the most suitable for the production of
glycogen with a weight-average molecular weight (Mw ) of 3,000–30,000 k. The molecular weight of the ESG is controllable
by changing the concentration of the substrate amylose. Furthermore, the addition of amylomaltase (AM, EC 2.4.1.25)
significantly enhanced the efficiency of this process, and the yield of ESG reached approximately 65%. Typical preparations
of ESG obtained by this method were subjected to structural analyses. The average chain length, interior chain length, and
exterior chain length of the ESGs were 8.2–11.6, 2.0–3.3, and 4.2–7.6, respectively. Transmission electron microscopy and
intrinsic viscosity measurement showed that the ESG molecules formed spherical particles. Unlike starch, the ESGs were
barely degraded by pullulanase. Solutions of ESG were opalescent (milky-white and slightly bluish), and gave a reddish-
brown color on the addition of iodine. These analyses revealed that ESG shares similar molecular shapes and solution
properties with natural-source glycogen. Moreover, ESG had macrophage-stimulating activity and its activity depends on
the molecular weight of ESG. We successfully achieved large scale production of ESG. ESG could lead to new industrial
applications, such as in the food, chemical, and pharmaceutical fields.
Key words: glycogen; branching enzyme; enzymatic synthesis; α-glucan; polysaccharide; structure.
Abbreviations: AM, amylomaltase; BE, branching enzyme; DP, degree of polymerization; ESG, enzymatically synthesized
glycogen; GP, α-glucan phosphorylase; HPSEC-MALLS-RI, high-performance size-exclusion chromatography with a multi-
angle laser light scattering photometer and a differential refractive index detector; IAM, isoamylase; NSG, natural-source
glycogen; SP, sucrose phosphorylase; TEM, transmission electron microscopy.
Glycogen has also been isolated from several higher plants. In an-
imal tissues, glycogen is synthesized from UDP-glucose
Glycogen is the storage form of glucose in animals, via the cooperative action of glycogenin (EC 2.4.1.186),
fungi, bacteria, yeasts, and archaea. The polysaccharide glycogen synthase (EC 2.4.1.11), and branching en-
is a highly branched (1→4)(1→6)-linked α-d-glucan zyme (BE, 1,4-α-d-glucan:1,4-α-d-glucan 6-α-d-(1,4-α-
with a high molecular weight (106 –109) (Geddes 1986). glucano)-transferase, EC 2.4.1.18).
Based on the results of electron microscopy, it has been Glycogen isolated from animal tissues or shellfish
shown that glycogen consists of spherical particles with has been used to stimulate the exudation of phagocytic
diameters of 20–40 nm (β-particles), and that the β- cells into the peritoneal cavities of experimental ani-
particles often associate into much larger α-particles mals in immunological studies (Thorpe & Marcus 1967;
(∼200 nm). The molecular weight of an individual Mantovani 1981). It has also been reported that glyco-
β-particle has been shown to be approximately 107 gen has an antitumor effect, probably through its im-
(Geddes 1986). Glycogen aqueous solution is opalescent munomodulating activity, and that this activity seems
(milky-white and slightly bluish), and gives a reddish- to depend on the fine structure of glycogen (Takaya et
brown color on the addition of iodine. Amylopectin, a al. 1998; Takaya 2000). Glycogen has long been consid-
major component of starch, is also a highly branched ered to have health benefits as a food ingredient. As re-
(1→4)(1→6)-linked α-d-glucan. However, the degree of vealed below, we demonstrated that enzymatically syn-
branching, namely the number of (1→6)-α-d-glucosidic thesized glycogen (ESG) has a stimulating activity on
linkages, in glycogen is about twice that in amylopectin. immunocompetent cells, such as macrophages. Glyco-
A glycogen-type polymer, referred to as phytoglycogen, gen has also been used as a raw material in the cosmetic
c 2011 Institute of Molecular Biology, Slovak Academy of Sciences
388 H. Kajiura et al.
industry and as a carrier to enhance the yield of DNA vivo synthesis of glycogen by glycogen synthase and BE.
during precipitation with organic solvent (Heath et al. Then, a question has come up. Can we synthesize glyco-
2001). gen simply using amylose as a substrate? The published
results to date have all been negative: BE can synthe-
In vitro synthesis of glycogen size branched glucans but their molecular weights are
much lower than that of glycogen. For example, Praznik
Extraction of glycogen from natural resources such as et al. (1992) reported that the action of potato BE on
animal tissues or shellfishes is laborious and costly pro- amylose with a weight-average molecular weight (Mw )
cess. Therefore, in vitro synthesis may be possible an- of 67.6 k resulted in a branched product with a Mw of
swer to obtain glycogen in industrial scale. Actually, 33.5 k. Boyer et al. (1982) analyzed the action of maize
the in vitro synthesis of glycogen has been success- BE isozyme I by gel filtration chromatography and ob-
fully achieved by the combined action of α-glucan phos- served that the product was eluted more slowly than the
phorylase (GP, EC 2.4.1.1) and BE using glucose-1- substrate. Kitamura (1996), using light-scattering anal-
phosphate (G-1-P) as a substrate (GP-BE method) yses, also reported that potato BE brought about a de-
about 70 years ago. (Cori & Cori 1943). At that time, crease in the molecular weight of the substrate amylose.
enzymes were extracted from animal tissues and costly. We have reported that Bacillus cereus BE produced
Additionally, GP from animal tissues is allosteric en- branched glucan with a Mw of 50 k from amylose with
zyme and need to be phosphorylated or added an acti- Mw of 320 k (Takata et al. 2005). The decreases in the
vator (glucose-6-phosphate) (Fukui et al. 1982). How- molecular weight of the substrate can be attributable to
ever, microbial or plant enzymes have been developed a cyclization reaction by BE; it has been demonstrated
and used for the in vitro synthesis by many researchers that BE catalyzes a cyclization reaction in addition to
(e.g., Tolmasky & Krisman 1987; Fujii et al. 2003; a branching reaction in vitro. As a result of cycliza-
van der Vlist et al. 2008). The ESG produced by tion, the molecular weight of the substrate should be
the GP-BE method has properties similar to those of decreased. By contrast, an inter-chain branching reac-
the natural-source glycogen (NSG) (Kitahata & Okada tion results in both larger and smaller molecules if two
1988). UDP-glucose, a natural substrate for in vivo molecules of the same size are used as a substrate. An
glycogen synthesis, was also used as substrate by us- intra-chain branching reaction should not change the
ing glycogen synthase and BE as catalysts (Parodi et molecular weight. However, most studies have used BE
al. 1969). Although both G-1-P and UDP-glucose are from plant sources and relatively long-chain amyloses
expensive for industrial applications, the former could were used as the substrate. To evaluate the possibility
be obtained from the inexpensive substrate sucrose by of glycogen synthesis from amylose, we tested the ac-
using sucrose phosphorylase (SP, EC 2.4.1.7) (SP-GP- tivity of eight types of BE from various sources using
BE method) (Ohdan et al. 2006). Furthermore, we have relatively short-chain amyloses as the substrates. We
demonstrated that ESG produced by the SP-GP-BE found that some BEs from bacterial sources can syn-
method has immunomodulating activity (Ryoyama et thesize glycogen (ESG). We then compared some of the
al. 2004). However, the yield of glycogen from sucrose properties of the ESG with those of NSG. Furthermore,
was less than 40%, and a more efficient method is re- we found that ESG has physiological function.
quired to meet the demand for glycogen for industrial
applications. Actions of BEs from several sources on amyloses
In the GP-BE and SP-GP-BE methods, GP elon-
gates a chain of α-(1→4)-glucan and BE introduces To evaluate the possibility of producing glycogen from
branch points in the growing chains, mimicking the in amylose, we tested the actions of BEs from eight sources
Table 1. Weight-average molecular weight and yield of α-glucans produced by the action of several BEs on two amyloses.a
Substrate
Source of BE
Amylose A Amylose AS10
Effect of amylomaltase
Fig. 2. Comparison of aqueous solutions of α-glucans. (a) Waxy corn starch (0.1 g) was gelatinized in 10 mL of distilled water by
heating at 100 ◦C for 30 min. The same amounts of maltodextrin (DE 8–9.5), NSG from rabbit liver and ESG-F were each dissolved
in 10 mL of distilled water. (b) These solutions (1 mL) were added 10 µL of iodine reagent (52 mg of I2 and 520 mg of KI in 10 mL
of water).
Sample name
or NSG source Mw (k) CLa ECLb ICLc
Digestibility of ESG
Reaction conditions
Fig. 6. Susceptibility of α-glucan to pullulanase. ESG-B (), NSG to this role, some reports have suggested that glyco-
from oyster (), and gelatinized waxy corn starch ( ) were treated gen has an immunological activity (Thorpe & Marcus
with pullulanase. Mw values of hydrolyzates were analyzed by 1967; Mantovani 1981; Takaya et al. 1998). However,
HPSEC-MALLS-RI.
strong scientific evidence has not been obtained for
the immunomodulating activity. Indeed, many scien-
amylase hydrolysis of ESGs, we detected much larger tists have been annoyed by the lack of reproducibility
macrodextrins (molecular weight >1,000 k). In contrast of their experimental results. This lack of reproducibil-
to NSG, oligosaccharides with DP 4-7 could not be de- ity may be because their samples of glycogen have been
tected in the ESG hydrolysates. These results suggest extracted from natural resources, as (1) the effect of
that the α-1,6 linkages in ESG molecules are more reg- trace amounts of other materials cannot be ruled out,
ularly distributed than those in NSG molecules. ESG is and (2) important characteristics of each glycogen sam-
synthesized in much simpler circumstances than NSG, ple, such as the average molecular mass and the chain
in which no trimming reactions occur during the syn- length, are quite different depending on the source and
thetic process. This simplicity can result in a regular in- purification procedures. We have clarified the immuno-
ternal structure without long spans between α-1,6 link- logical activity of glycogen by using completely pure
ages. ESG with very uniform characteristics (Kakutani et
al. 2007, 2008). First, we examined the immunestim-
Physiological function of ESG ulating effect of ESG on NO (nitric oxide) produc-
tion using RAW264.7, a murine macrophage cell line.
Glycogen is well known as the energy and carbon re- The result showed glycogen enhanced NO production
serves in animal cells and microorganisms. In addition of RAW264.7 (Fig. 7). The immunostimulatory activity
In vitro synthesis of glycogen 393
of glycogen relates closely to its molecular weight and Boyer C.D., Simpson E.K. G. & Damewood P.A. 1982. The pos-
reaches to the highest at around 5,000 k-6,500 k. Next, sible relationship of starch and phytoglycogen in sweet corn.
II. The role of branching enzyme I. Starch/Stärke 34: 81–85.
the peritoneal-exudate cells collected from C3H/HeJ
Cori G.T. & Cori C.F. 1943. Crystalline muscle phosphorylase.
mice, Toll-like receptor 4 mutants, were also activated IV. Formation of glycogen. J. Biol. Chem. 151: 57–63.
by ESGs with similar profiles as RAW264.7 (Kakutani Fujii K., Takata H., Yanase M., Terada Y., Ohdan K., Takaha T.,
et al. 2007). Furthermore, we demonstrated by flow cy- Okada S. & Kuriki T. 2003. Bioengineering and application of
novel glucose polymers. Biocatal. Biotransform. 21: 167–172.
tometry that biotinylated ESGs bound the macrophage
Fukui T., Shimomura S. & Nakano K. 1982. Potato and rabbit
cell line (Kakutani et al. 2007, 2008). These results muscle phosphorylases: comparative studies on the structure,
strongly suggested that glycogen functions not only as function and regulation of regulatory and nonregulatory en-
a fuel reservoir, but also as a signaling factor in vivo. zymes. Mol. Cell. Biochem. 42: 129–144.
Geddes R. 1986. Glycogen: a metabolic viewpoint. Biosci. Rep.
6: 415–428.
Conclusion and future prospects Hashimoto T., Kurose M., Oku K., Nishimoto T., Chaen H.,
Fukuda S. & Tsujisaka Y. 2006. Digestibility and suppressive
We have developed a novel method (IAM-BE-AM effect on rats’ body fat accumulation of cyclic tetrasaccharide.
method) for producing glycogen from short-chain amy- J. Appl. Glycosci. 53: 233–239.
Hata K., Hata M., Hata M. & Matsuda K. 1983. The structures
lose by using BE. This method has two advantages com- of shellfish glycogens I. J. Jpn. Soc. Starch Sci. 30: 88–94.
pared with the SP-GP-BE method, in which sucrose is Hata K., Hata M., Hata M. & Matsuda K. 1984. A proposed
used as a starting material. First, the yield of glyco- model of glycogen particle. J. Jpn. Soc. Starch Sci. 31: 146–
gen is higher. The yield of glycogen by the SP-GP-BE 155.
Heath E.M., Morken N.W., Campbell K.A., Tkach D., Boyd E.A.
method was less than 40%, whereas that by the IAM- & Strom D.A. 2001. Use of buccal cells collected in mouth-
BE-AM method reached 65% under the optimal condi- wash as a source of DNA for clinical testing. Arch. Pathol.
tions. Second, the molecular weight of glycogen is con- Lab. Med. 125: 127–133.
trollable within the range of 3,000–30,000 k by changing Kajiura H., Kakutani R., Akiyama T., Takata H. & Kuriki T.
2008. A novel enzymatic process for glycogen production. Bio-
the concentration of substrate.
catal. Biotransform. 26: 133–140.
The structures and properties of the ESGs were Kajiura H., Takata H., Kuriki T. & Kitamura S. 2010. Structure
quite similar to those of NSGs. Despite these simi- and solution properties of enzymatically synthesized glyco-
larities, there were two differences (the unit-chain dis- gen. Carbohydr. Res. 345: 817–824.
tribution and final products by α-amylase hydrolysis) Kakutani R., Adachi Y., Kajiura H., Takata H., Kuriki T. & Ohno
N. 2007. Relationship between structure and immunostimu-
between the ESG and NSG. Furthermore, we have lating activity of enzymatically synthesized glycogen. Carbo-
demonstrated that ESG has immunomodulating activ- hydr Res. 342: 2371–2379.
ity. We hypothesize that the macromolecule fraction af- Kakutani R., Adachi Y., Kajiura H., Takata H., Ohno N. &
ter partial hydrolysis of ESG that reaches the intesti- Kuriki T. 2008. Stimulation of macrophage by enzymatically
synthesized glycogen: the relationship between structure and
nal tract stimulates immunocompetent cells resulting biological activity. Biocatal. Biotransform. 26: 152–160.
in the health benefits. Investigations in vivo are now in Kitahata S. 1995. Debranching enzymes (isoamylase, pullu-
progress to gain a further understanding of the effect of lanase), pp. 18–27. In: The Amylase Research Society of
orally administered glycogen. Japan (ed.), Enzyme Chemistry and Molecular Biology of
Amylases and Related Enzymes, CRC Press, Boca Raton.
Kitahata S. & Okada S. 1988. Branching enzymes, pp. 143–154.
Acknowledgements In: The Amylase Research Society of Japan (ed.), Handbook
of Amylase and Related Enzymes, Pergamon Press, Oxford.
Kitamura S. 1996. Starch polymers, natural and synthetic, pp.
We are deeply grateful to Dr. S. Kitamura of Osaka Pre-
7915–7922. In: Salamone J.C. (ed.), Polymeric Materials En-
fecture University for his kind help in performing structural cyclopedia, CRC Press, Boca Raton.
analyses and testing the solution properties of ESG. We are Kitamura S., Kobayashi K., Tanahashi H., Ozaki T. & Kuge T.
also indebted to Drs. H. Matsui and H. Ito of Hokkaido Uni- 1989. On the Mark-Houwink-Sakurada equation for amylose
versity for providing the plasmid used to produce the BE of in aqueous solvents. (Dilute solution properties of starch re-
Phaseolus vulgaris L. We also thank Drs. N. Ohno and Y. lated polysaccharides. Part 1) Denpun Kagaku 36: 303–309.
Adachi of Tokyo University of Pharmacy and Life Science Kjoelberg O., Manners D.J. & Wright A. 1963. α-1,4–Glucosans.
for their advice and valuable discussions during the course of XVII. The molecular structure of some glycogens. Comp.
the immunological studies. This work was supported in part Biochem. Physiol. 34: 353–365.
Manners D.J. 1991. Recent developments in our understanding
by grants from the “Program to develop new technology to
of glycogen structure. Carbohydr. Polym. 16: 37–82.
promote the agriculture, forestry, fisheries and food indus- Mantovani B. 1981. Phagocytosis of immune complexes mediated
tries through collaboration among industry, academia and by IgM and C3 receptors by macrophages from mice treated
the government” and “Research and development projects with glycogen. J Immunol. 126: 127–130.
to promote new policies in agriculture, forestry and fish- Ohdan K., Fujii K., Yanase M., Takaha T. & Kuriki, T. 2006.
eries” of the Ministry of Agriculture, Forestry and Fisheries, Enzymatic synthesis of amylose. Biocatal. Biotransform. 24:
Japan. 77–81.
Okada K., Yoneyama M., Mandai T., Aga H., Sakai S. & Ichikawa
T. 1990. Digestion and fermentation of pullulan. J. Jpn. Soc.
References Nutr. Food Sci. 43: 23–29. (in Japanese)
Parodi A.J., Krisman C.R., Leloir L.F. & Mordoh J. 1967. Prop-
Boyer C. & Preiss J. 1977. Biosynthesis of bacterial glycogen: erties of synthetic and native liver glycogen. Arch. Biochem.
purification and properties of the Escherichia coli B α-1,4– Biophys. 121: 769–778.
glucan: α-1,4–glucan 6–glycosyltransferase. Biochemistry 16: Praznik W., Rammesmayer G. & Spies T. 1992. Characterization
3693–3699. of the (1→4)-α-D-glucan-branching 6-glycosyltransferase by
394 H. Kajiura et al.
in vitro synthesis of branched starch polysaccharides. Carbo- Thorpe B. D. & Marcus S. 1967. Phagocytosis and intracellu-
hydr. Res. 227: 171–182. lar fate of Pasteurella tularensis: in vitro effects of exudate
Ryoyama K., Kidachi Y., Yamaguchi H., Kajiura H. & Takata stimulants and streptomycin on phagocytic cells. J Reticu-
H. 2004. Anti-tumor activity of an enzymatically synthesized loendothel Soc. 4: 10–23.
α-1,6 branched α-1,4-glucan, glycogen. Biosci. Biotechnol. Tolmasky D. S. & Krisman C. R. 1987. The degree of branching in
Biochem. 68: 2332–2340. (α1,4)-(α1,6)-linked glucopolysaccharides is dependent on in-
Takata H., Kajiura H., Furuyashiki T., Kakutani R. & Kuriki trinsic properties of the branching enzymes. Eur. J. Biochem.
T. 2009. Fine structural properties of natural and synthetic 168: 393–397.
glycogens. Carbohydr. Res. 344: 654–659. van der Vlist J., Palomo Reixach M., van der Maarel M., Di-
Takata H., Kato T., Takagi M. & Imanaka T. 2005. Cyclization jkhuizen L., Schouten A.J. & Loos K. 2008. Synthesis of
reaction catalyzed by Bacillus cereus branching enzyme, and branched polyglucans by the tandem action of potato phos-
the structure of cyclic glucan produced by the enzyme from phorylase and Deinococcus geothermalis glycogen branching
amylose. J. Appl. Glycosci. 52: 359–365. enzyme. Macromol. Rapid Commun. 29: 1293–1297.
Takaya Y. 2000. Biological activities of natural resources around
us are now in the limelight. Yakugaku Zasshi 120: 1075–1089. Received December 29, 2011
Takaya Y., Uchisawa H., Ichinohe H., Sasaki J., Ishida K. & Mat- Accepted March 11, 2011
sue H. 1998. Antitumor glycogen from scallops and the in-
terrelationship of structure and antitumor activity. J. Mar.
Biotechnol. 6: 208–213.