Accepted Manuscript

Title: Structural diversity of fungal glucans

Authors: Andriy Synytsya, Miroslav Novák

PII: S0144-8617(12)00995-2
DOI: doi:10.1016/j.carbpol.2012.09.077
Reference: CARP 7057

To appear in:

Received date: 19-4-2012

Revised date: 27-9-2012
Accepted date: 27-9-2012

Please cite this article as: Synytsya, A., & Novák, M., Structural diversity of fungal
glucans, Carbohydrate Polymers (2010), doi:10.1016/j.carbpol.2012.09.077

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 1 Structural diversity of fungal glucans

3 Andriy Synytsya,* Miroslav Novák

t
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5 Department of Carbohydrates and Cereals, Institute of Chemical Technology in Prague, Technická 5, 166 28

6 Prague 6 Dejvice, Czech Republic

cr
7

us
8 Abstract

9 Fungal glucans represent various structurally different D-glucose polymers with a large diversity of molecular

an
10 mass and configuration. According to glucose anomeric structure, it is possible to distinguish α-D-glucans, β-D-

11 glucans and mixed α,β-D-glucans. Further discrimination could be made on the basis of glycosidic bond position

12 in a pyranoid ring, distribution of specific glycosidic bonds along a chain, branching and molecular mass. Fungal
M
13 glucans can be chemically modified to obtain various derivatives of potential industrial or medicinal importance.

14 NMR spectroscopy is a powerful tool in structural analysis of fungal glucans. Together with chemolytic methods
d

15 like methylation analysis and periodate oxidation, NMR is able to determine exact structure of these
te

16 polysaccharides. Fungal glucans or their derivatives exert various biological activities, which are usually linked

17 to structure, molecular weight and substitution degree.

p

18
ce

19 Keywords: Fungal glucans, Structural diversity, Nuclear magnetic resonance, Chemical modification, Structure-

20 activity relationship
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21

22

23 1. Introduction

24

25 Fungal glucans are structirally variable polymers of D-glucopyranose (D-Glcp). They

*
Corresponding author. Address: Department of Analytical Chemistry and Technology, Institute of Chemical
Technology in Prague, Technická 5, 166 28 Prague 6, Czech Republic. Tel.: +420-220-443-116; fax: +420-220-
445-130. E-mail: sinicaa@vscht.cz (A. Synytsya).

1
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26 constitute an obligatory part of cell walls in mycelia, fruiting bodies or other parts of different

27 micro- and macromycetes. Some exopolysaccharides of microscopic fungi were also defined

28 as glucans. Basic structure of common fungal glucans is demonstrated in Figure 1. Despite of

29 simplicity of their monosaccharide composition (according to definition they should contain

t
30 only glucose), large diversity can be found concerning number and anomeric configuration of

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31 D-Glcp units, position and sequence of glycosidic bonds along a chain, branching degree and

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32 chain conformation. Branched glucans may contain various side chains, with one or more

33 monosaccharide units, attached to the backbone at different positions.

us
34 According to anomeric structure of D-Glcp units, it is possible to distinguish three main

35 groups of these fungal polysaccharides: α-D-glucans, β-D-glucans and mixed α,β-D-glucans.

36
an
Further discrimination can be made on the basis of glycoside bond positions or molecular
M
37 mass, i.e., linear or branched glucans, (1→3)-, (1→4)- and/or (1→6)-linked glucans, high-,

38 medium- or low-Mw glucans, etc. Besides (1→3)-β-D-glucans known for their

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39 immunomodulation and antitumour activities, a wide range of fungal glucans of different

te

40 structure have been described (Wasser, 2002; Singh, Saini, & Kennedy, 2008; Wiater, Paduch,

41 Pleszczyńska, Próchniak, Choma, Kandefer-Szerszeń, et al., 2011; Synytsya, Míčková,

p

42 Synytsya, Jablonský, Spěváček, Erban, V., et al., 2009; Chakraborty, Mondal, Pramanik, Rout,
ce

43 & Islam, 2004; and many others). Some fungal glucans were chemically modified to obtain

44 water-soluble derivatives of potential industrial or medicinal importance.

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45 To determine exact structure of fungal glucans many analytical methods have been

46 applied. First of all it is chemolytic methods including methylation anylysis, oxidation

47 methods (periodate, lead(IV) acetate), Smith's degradation, and other. NMR spectroscopy is

48 the most effective up-to-date non-destructive method of structural analysis. It has been widely

49 used in characterisation of fungal glucans. Chemical shifts and coupling constants of 1H and
13
50 C identify anomeric forms of D-Glcp residues and indicate positions of glycosidic linkages

2
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51 or substituents (Mulloy, 1984). The H-1 and C-1 signals at δ 4.9–5.1 (4.3–4.6) and δ 98–100

52 (103–104), respectively, indicate α- (β-) anomeric form of D-Glcp (effect of anomeric

53 configuration). Furthermore, a downfield shift of carbon signal (δ ~4.5–8) confirms that this

54 carbon participate in glycosidic bond formation (α-glycosylation effect), while a less

t
55 pronounced upfield shift takes place for the neighbour carbon signals (β-glycosylation effect)

ip
56 (Goffin, Bystricky, Shashkov, Lynch, Hanon, Paquot, et al., 2009). Correlation homo- and

cr
57 heteronuclear NMR experiments are commonly used to help in the signal assignment (Mulloy,

58 1984; Kim, Kim, Cheong, Williams, Kim, & Lim, 2000; Lukondeh, Ashbolt, Rogers, & Hook,

us
59 2003; Yalin, Cuirong, & Yuanjiang, 2006). Homonuclear 1H, 1H correlated spectroscopy

60 (COSY) detects interaction between neighbour protons in a D-Glcp unit, while total

61
an
correlation spectroscopy (TOCSY) may assign all the protons based on their interaction with
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62 one of them (commonly H-1). Heteronuclear single/multiple-quantum correlation

63 spectroscopy (HSQC, HMQC) is used to assign C-H signals. Finally, both heteronuclear
d

64 single- or multiple-bond correlation spectroscopy (HSBC, HMBC) and nuclear or rotating-

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65 frame Overhauser effect spectroscopy (NOESY, ROESY) are able to determine inter-unit

66 connections in the glucan macromolecule.

p

67
ce

68

69 2. Fungal α-D-glucans
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70

71 2.1. Linear α-D-glucans

72

73 Linear α-D-glucans were found in many yeasts and higher fungi. Chemical structure of these

74 polysaccharides is variable, depending on a fungal source. Some of linear α-D-glucans are

75 composed of (1→3)-, (1→4)- or (1→6)-linked α-D-Glcp units, while others contain a

3
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76 combination of the mentioned glycosidic bonds (Grün, 2003).

77

78 2.1.1. Linear α-D-glucans having one type of linkage

79

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80 Linear (1→3)-α-D-glucans (1, Figure 1a) are common cell wall polysaccharides of various

ip
81 fungi. For example, this structure was established for pseudonigeran isolated from mycelium

cr
82 of Aspergillus niger (Horisberger, Lewis, & Smith, 1972). Kiho, Yoshida, Nagai, Ukai, &

83 Hara (1989) described (1→3)-α-D-glucan (Mw = 560 kDa) obtained from Agrocybe

us
84 cylinducea by alkali extraction. James & Cherniak (1990) extracted very pure linear (1→3)-α-

85 D-glucan (Mw ~9 kDa) from trama of Piptoporus betulinus using a 4-methylmorpholine-N-

86

87
oxide – water (3:2) mixture and Me2SO.
an
M
88 [3)-α-D-Glcp-(1→]n (1)

89
d

90 Similar α-D-glucans were isolated from mycelia of Penicillium chrysogenum (Wang,

te

91 Deng, Li, & Tan, 2007) and Poria cocos (Jin, Zhang, Tao, Zeng, Chen, & Cheung, 2004),
p

92 fruiting bodies (basidiocarps) and mycelia of Ganoderma tsugae (Peng, Zhang, Zhang, Xu, &
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93 Kennedy, 2005; Chen, Zhou, Zhang, Nakamura, & Norisuye, 1998) and Ganoderma lucidum

94 (Chen, Zhang, Nakamura, & Norisuye, 1998), basidiocarps of Pleurotus ostreatus (Wiater,
Ac

95 Paduch, Pleszczyńska, et al., 2011; Synytsya, Míčková, Synytsya, et al., 2009), Pleurotus

96 eryngii (Synytsya, Míčková, Synytsya, et al., 2009), Lentinula edodes (Wiater, Paduch,

97 Pleszczyńska, et al., 2011; Unursaikhan, Xu, Zeng, & Zhang, 2006; Zhang, Zhang, & Cheng,

98 2000, 2002), Laetiporus sulphurous (Wiater, Paduch, Pleszczyńska, et al., 2011), Piptoporus
13
99 betulinus (Wiater, Paduch, Pleszczyńska, et al., 2011) and many other sources. C NMR

100 spectrum of pseudonigeran from lichenised basidiomycete Dictyonema glabratum (Carbonero,

101 Sassaki, Gorin & Iacomini, 2002) showed the signals at δ 100.9 (C-1) and 83.8 (C-3)

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102 indicated (1→3)-α-glycosidic linkages (Table 1).

103 Palacios, García-Lafuente, Guillamón, & Villares (2012) described α-D-glucan PH

104 isolated by hot water extraction from basidiocarps of Pleurotus ostreatus. This polysaccharide

105 was almost totally hydrolysed by α-glucosidase. Methylation analysis and 1H NMR confirmed

t
106 that it is amylose-like linear (1→4)-α-D-glucan (2, Figure 1b). Gonzaga, Ricardo, Heatley, &

ip
107 Soares (2005) isolated water soluble polysaccharides from basidiocarps of Agaricus blazei.

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108 Authors reported the presence of (1→4)-α-D-glucan together with (1→6)-! -D-glucan in the

109 fractions that was confirmed by 13C NMR.

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110

an
111 [4)-α-D-Glcp-(1→]n (2)

112

113 Luo, Xu, Yu, Yang, & Zheng (2008) isolated biologically active α-D-glucan IPS-B2 (Mw
M
114 = 49.5 kDa) from a hot aqueous extract of Armillariella tabescens mycelia. Han, Wu, Chai,
d

115 Chen, Dai, Dong, et al. (2011) described a water-soluble α-D-glucan HCP (Mw = 670 kDa)
te

116 isolated from basidiocarps of Sarcodon aspratus. Mondal, Chakraborty, Pramanik, Rout, &

117 Islam (2004) reported a structure of α-D-glucan, named PS-II (Mw ~6 kDa), isolated from a
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118 hot aqueous extract of Termitomyces eurhizus basidiocarps. All these polysaccharides were
ce

119 characterized as simple linear (1→6)-α-D-glucans (3, Figure 1c).

120
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121 [6)-α-D-Glcp-(1→]n (3)

122

123 This configuration was confirmed by NMR analysis (Table 1). Single α-anomeric proton and

124 carbon signals were observed at δ 4.82–4.92 and 97.6–100.2, respectively. These signals

125 showed strong correlation in HSQC experiment. The downfield chemical shift at δ 65.5–68.1

126 (C-6) indicated substitution at O-6. The glycosidic (1→6)-linkage was also confirmed by key

127 HMBC correlation from H-1 (δ 4.82) to C-6 (δ 65.5) (Luo, Xu, Yu, Yang, & Zheng, 2008).

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128

129 2.1.2. Linear mixed-linkage α-D-glucans

130

131 Nigeran (4, Figure 1d), a cold-water insoluble polysaccharide originated from mycelia of

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132 Aspergillus niger and some other species of Aspergillus and Penicillium genera, consists of

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133 alternating (1→3)- and (1→4)-linked α-D-Glcp residues (Bobbitt & Nordin, 1978; Bock,

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134 Gagnaire, Vignon, & Vincendon, 1983). Its 13C NMR spectrum showed carbon resonances of

135 both types of the units (Table 1). The low-field signals at δ 79.8 (C-4) and 83.2 (C-3)

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136 indicated substitution of α-D-Glcp units at the corresponding positions, i.e., respective (1→4)-

137 and (1→3)-α-glycosidic linkages. However, Painter (1990) reported that only 78% of the

138
an
(1→4)-linked α-D-Glcp residues of the Aspergillus niger nigeran were present as isolated
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139 singlets having (1→3)-linked neighbour residues, while the rest 22 % were present as isolated

140 doublets (2), and sequences of more than two contiguous (1→4)-linked units were not found.
d

141 Tsumuraya, Misaki, Takaya, & Torii (1978) described α-D-glucan, named elsinan isolated
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142 from culture filtrates of Elsinoe leucospila grown on a potato extract-sucrose medium. This

143 glucan is an essentially linear polymer containing both (1→4)-α- and (1→3)-α- linkages and
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144 is mainly composed of (1→3)-α-linked maltotriose residues.

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145

146 {3)-α-D-Glcp-(1→[4)-α-D-Glcp-(1→]m}n m = 1 or 2 (4)

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147

148 Isolichenan (5) is a cold-water soluble α-D-glucan (Mw vary from ~6-8 to 2000 kD) of the

149 lichen Cetraria islandica consisting of (l→3)- and ( 1→4)- linked residues in the ratio of 3:1,

150 3:2 or 2:1 (Olafsdottir, Ingolfsdortir, Barsett, Smestad Paulsen, Jurcic, & Wagner, 1999).

151
152 {[3)-α-D-Glcp-(1→]m4)-α-D-Glcp-(1→}n m = 1, 2 or 3 (5)

153

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154 Nigeran- or isolichenan-type α-D-glucans with alternating (1→3)- and (1→4)-linkages in

155 various ratios were isolated from lichenised fungi of Cladina, Cladonia, Parmotrema,

156 Ramalina, Rimelia and other genera (Carbonero, Montai, Mellinger, Eliasaro, Sassaki, Gorin,

157 & Iacomini, 2005; Cordeiro, Stocker-Wörgöotter, Gorin, & Iacomini, 2003; 2004;

t
158 Woranovicz-Barreira, Gorin, Sassaki, Tischer, Ahti, & Iacomini, 1999; Stuelp, Carneiro-Leão,

ip
159 Gorin, & Iacomini, 1999; Carbonero, Montai, Woranovicz-Barreira, Gorin, & Iacomini, 2002;

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160 Carbonero, Sassaki, Stuelp, Gorin, Woranovicz-Barreira, & Iacomini, 2001; Baron, Gorin, &

161 Iacomini, 1988). Both these linear mixed linkage (l→3),(1→4)-α-D-glucans were found in the

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162 thalli of lichen Ramalina peruviana (Cordeiro, Stocker-Wörgöotter, Gorin, et al., 2004), but

163 only nigeran was also found in free micobiont. Authors suggested fungal origin for both

164
an
polysaccharides, but isolichenan should be produced by the mycobiont in the presence of
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165 Trebouxia photobiont only.

166 Water insoluble α-D-glucan (Mw ~850 kDa) was isolated from Aspergillus wentii (Choma,
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167 Wiater, Komaniecka, Paduch, Pleszczyńska, & Szczodrak, 2012). It was a linear polymer
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168 composed almost exclusively of (1→3)-linked α-D-Glcp, but the chain was divided into

169 blocks of 200 units separated by a short spacers (1-3 units) of (1→4)-linked α-D-Glcp.
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170 Grün et al. (2003; 2005) described cell wall α-D-glucans isolated from wild-type and
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171 mutant yeasts Schizosaccharomyces pombe. The wild-type polysaccharide was a polymer (DP

172 = 260) with two interconnected linear chains, each consisting of about 120 (1→3)-linked α-D-
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173 Glcp and some (1→4)-linked α-D-Glcp residues at the reducing end. By contrast, α-D-glucan

174 of the mutant yeast consisted of a single chain only. Authors proposed that coupling of two α-

175 D-glucan chains is necessary for creation of the mature polysaccharide essential for yeast

176 morphogenesis. An alkali-soluble α-D-glucan was isolated from basidiocarps of Lentinula

177 edodes (Shida, Uchida, & Matsuda, 1978). This glucan was partially degraded by amylolytic

178 enzymes. It was shown to have a slightly branched structure composed of (1→3)- and (1→4)-

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179 linked α-D-Glcp units in the ratio 5.3:1. The latter residues are present in the regions near non-

180 reducing ends.

181 Mondal, Chakraborty, Pramanik, Rout, & Islam (2004) reported a structure of α-D-glucan

182 PS-I (Mw ~9 kDa) isolated from the hot aqueous extract of Termitomyces eurhizus

t
183 basidiocarps. The polysaccharide was identified as a linear mixed linkage (1→3),(1→6)-α-D-

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184 glucan with blocking distribution of these two types of glycosidic bonds (6).

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185

186 [6)-α-D-Glcp-(1→]m[3)-α-D-Glcp-(1→]n (6)

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187

188 The 1H NMR spectrum of PS-I had two H-1 signals at δ 5.33 and 5.08 assigned to (1→3)-

189

190 an
and (1→6)-linked α-D-Glcp units, respectively, in a molar ratio of ~1: 2.5 (Mondal,

Chakraborty, Pramanik, et al. 2004). Corresponding C-1 signals were found at δ 97.01 and
M
191 99.18. The C-3 and C-6 signals showed characteristic downfield shifts due to the α-

192 glycosylation effect (Table 1). The sequence of glycosidic linkages in PS-I was confirmed by
d

193 the specific inter-unit NOE contacts observed in NOESY experiment.

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194 Pullulan (7) is water-soluble α-D-glucan originated from Aureobasidium pullulans (Singh,
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195 Saini, & Kennedy, 2008), Tremella mesenterica (Jennings, & Smith, 1973) or Cyttaria harioti
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196 (Waksman, de Lederkremer, & Cerezo, 1977). This linear polysaccharide contains both

197 (1→4)-α- and (1→6)-α-linkages in a ~2:1 molar ratio. It is a copolymer of regularly repeating
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198 maltotriose fragments or, in some strains of Aureobasidium pullulans, 5–7 % of maltotetraose

199 fragments connected by (1→6)-α-glycosidic bonds (Cartley & Whelan, 1971). Reis, Tischer,

200 Gorin, & Iacomini (2002) described pullulan-like α-D-glucan from the lichenised ascomycete

201 Teloschistes flavicans. It consisted of equal amount of alternating (1→4)- and (1→6)- linked

202 α-D-Glcp units.

203

204 {6)-α-D-Glcp-(1→[4)-α-D-Glcp-(1→]m}n m = 1, 2 or 3 (7)

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205
13
206 C NMR data of pullulans (Table 1) suggest that the C-4 carbon resonance signal of (1→4)-

207 linked α-D-Glcp residues shifted downfield when one or two neighbour units are (1→6)-

208 linked (Mclntyre & Vogel, 1993; Reis, Tischer, Gorin, & Iacomini, 2002). Thus this signal

t
209 can be used for evaluation of glycosidic bond distribution in pullulan. Linear (1→4),(1→6)-α-

ip
210 D-glucan, a polysaccharidic part of biologically active proteoglycan, was found as the main

cr
211 polysaccharide component in a hot water extract obtained from the fruiting bodies of

212 Agaricus blazei (Mizuno, Morimoto, Minato, & Tsuchida, 1998).

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213

214

215 2.2. Branched α-D-glucans

an
M
216

217 Two fractions of branched (1→4),(1→6)-α-D-glucan (glycogen), soluble and insoluble, were
d

218 obtained from baker's yeast Saccharomyces cerevisiae (Gunja-Smith, & Smith, 1974). The
te

219 water soluble fraction is intracellular, while the insoluble one is integrated with cell wall β-D-

220 glucan and can be solubilised by acetic acid extraction or β-D-glucanase (Arvindekar, & Patil,
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221 2002). Yeast glycogen consists of linear fragments of 10–14 (1→4)-linked α-D-Glcp units;
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222 these fragments are joined by (1→6)-linkages (Kwiatkowski, Thielen, Glenney, & Moran,

223 2009; Aklujkar, Sankh, & Arvindekar, 2008). Qiu, Tang, Tong, Ding, & Zuo (2007) deduced
Ac

224 structures of two glycogen-like polysaccharides isolated from Gastrodia elata Bl. as (1→4)-

225 α-D-glucans with (1→4)-linked α-D-Glcp side chains attached to O-6 with different branching

226 degrees (8).

227
228 [4)-α-D-Glcp-(1→]m 4)-α-D-Glcp (8)
229 1
230 ↓
231 6
232 {[4)-α-D-Glcp-(1→]m4)-α-D-Glcp-(1→}n

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233

234 Branched (1→4),(1→6)-α-D-glucans with single α-D-Glcp in side chains (9, Figure 1e)

235 have been described for various fungi. Smiderle, Sassaki, Van Arkel, Iacomini, Wichers, &

236 Van Griensven (2010) isolated branched α-D-glucan from a hot water extract of Agaricus

t
237 bisporus basidiocarps. This polysaccharide was completely degraded by α-amylase. Similar

ip
238 polysaccharide has been isolated from mycelium of Flammulina velutipes (Pang, Yao, Yang,

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239 Xie, Liu, Zhang, et al. 2007). Yalin, Cuirong, & Yuanjiang (2006) reported that mycelium of

240 a Chinese edible fungus Cordyceps sinensis contained similar branched α-D-glucan, which

us
241 gave with iodine a faint blue colour complex (λmax = 564 nm) and thus indicated (1→4)-α-D-

242 glucan with short side chains. NMR analysis of these polysaccharides confirmed the α-

243
an
configuration of the D-Glcp residues by the position of H-1 (δ 4.74–5.47) and C-1 (δ 98.7–
M
244 102.8) resonances (Table 2). The C-4 carbon signal at δ 78.9–79.8 indicated the (1→4)-

245 linkage as the major one. Methylene carbon signals were found at δ 60.3–63.4 (non-
d

246 substituted) and 66.2–72.2 (O-substituted). Therefore, these three polysaccharides consisted
te

247 of (1→4)-α-D-glucan backbone substituted at O-6 by single α-D-Glcp units in the ratio 1:7

248 (Flammulina velutipes), 1:8 (Agaricus bisporus) or 1:10 (Cordyceps sinensis).

p

249
ce

250 α-D-Glcp (9)

251 1
252 ↓
253 6
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254 {[4)-α-D-Glcp-(1→]m4)-α-D-Glcp-(1→}n
255

256 Li, Dobruchowska, Gerwig, Dijkhuizen, and Kamerling (2013) isolated water-soluble α-D-

257 glucan (Mw = 1267 kDa) from fruiting bodies of Coprinus comatus. This polysaccharide had a

258 (1→4)-linked α-D-Glcp backbone with ~10% branching at O-6 by terminal α-D-Glcp or α-D-

259 Glcp-(1→6)-α-D-Glcp disaccharide (7:3) (10).

260

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261 α-D-Glcp α-D-Glcp-(1→6)-α-D-Glcp (10)
262 1 1
263 ↓ ↓
264 6 6
265 {[4)-α-D-Glcp-(1→]m4)-α-D-Glcp-(1→[4)-α-D-Glcp-(1→]m4)-α-D-Glcp-(1→}n
266

267 Hoshi, Yagi, Iijima, Matsunaga, Ishihara, & Yasunara (2005) isolated a bioactive α-D-

t
268 glucan-protein complex from mycelia of Tricholoma matsutake. 1H and 13
C NMR spectra

ip
269 confirmed presence of α-anomeric sugars (H-1 at δ ~5.4 and C-1 at δ ~100), and methylation

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270 analysis detected O-4 linked and much less O-2 and O-6 linked D-Glcp units. Thus, it was

271 suggested that the polysaccharide part of this complex consisted of (1→4)-linked α-D-Glcp

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272 backbone with small amount of α-D-Glcp side chains attached to the O-2 and O-6 positions of

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273 some backbone units (11).

274
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275 α-D-Glcp
276 1
277 ↑
278 6
279 {[4)-α-D-Glcp-(1→]m4)-α-D-Glcp-(1→[4)-α-D-Glcp-(1→]m4)-α-D-Glcp-(1→}n (11)
d

280 2
281 ↑
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282 1
283 α-D-Glcp

284
p

285
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286 3. Fungal β-D-glucans

Ac

287

288 3.1. Linear β-D-glucans

289

290 Linear β-D-glucans were found in many fungal sources including lichens. These

291 polysaccharides are composed of (1→3)- or (1→6)-linked β-D-Glcp units. Cellulose-like

292 (1→4)-β-D-glucans were not found in fungal sources. However, combination of (1→4)- and

293 (1→3)-linkages is possible for linear mixed linkage β-D-glucans (lichenans) of some

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294 lichenised fungi.

295

296 3.1.1. Linear β-D-glucans having one type of linkage

297

t
298 Pachyman (12, Figure 1f) is simple linear (1→3)-β-D-glucan derived from sclerotia of Poria

ip
299 cocos. This polysaccharide is insoluble in water at room temperature and can be hydrolyzed

cr
300 by endo-(1→3)-β-D-glucanase (Hoffman Simson, & Timell, 1971). Similar β-D-glucans were

301 isolated from baker’s yeast Saccharomyces cerevisiae (Medeiros, Cordeiro, Cavalcanti,

us
302 Melchuna, da Silva Lima, Filho, et al. 2012; Freimund, Sauter, Käppeli, & Dutler, 2003) and

303 from basidiocarps of Termitomyces eurhizus (Chakraborty, Mondal, Rout, & Islam, 2006) and

304
an
Ganoderma lucidum (Wang & Zhang, 2009) by alkali extraction. Lichenized and some free-
M
305 living fungi contain linear (1→3)-β-D-glucan, often defined as laminaran (Alquini,

306 Carbonero, Rosado, Cosentino, & Iacomini, 2004; Baron, Gorin, & Iacomini, 1988).
d

307 Previously algal origin of this polysaccharide was assumed in some reports (Carbonero,
te

308 Montai, Woranovicz-Barreira, et al., 2002; Carbonero, Sassaki, Stuelp, et al., 2001; Stuelp,

309 Carneiro-Leão, Gorin, et al., 1999; Baron, Gorin, & Iacomini, 1988) because of its low
p

310 content in lichen thalli and its structure similarity with algal laminarans. However, isolation of
ce

311 (1→3)-β-D-glucan from the aposymbiotically cultured mycobiont finally confirmed its fungal

312 origin (Cordeiro, Stocker-Wörgöotter, Gorin, & Iacomini, 2003; 2004).

Ac

313

314 [3)-β-D-Glcp-(1→]n (12)

315

316 A high-field H-1 signal at δ 4.55 and low-field C-1 signal at δ 102.7 indicated β-configuration

317 of water-insoluble D-glucan from basidiocarps of Laetiporus sulphureus (Alquini, Carbonero,

318 Rosado, et al., 2004). The13C spectrum of this β-D-glucan contained six signals, as expected

319 for a hexose homopolysaccharide (Table 3). Low field signal at δ 86.0 indicated glycosylation

12
Page 12 of 71
320 at O-3, while an inverted DEPT signal at δ 60.9 is typical for unsubstituted C-6 (CH2OH)

321 carbons. The other signals at δ 76.3, 72.7, and 68.3 corresponded to unsubstituted C-5, C-2

322 and C-4, respectively. These results confirmed that this polysaccharide is linear (1→3)-β-D-
13
323 glucan (laminaran). Similarly, C NMR spectra of the mentioned β-D-glucans from baker’s

t
324 yeast and Termitomyces eurhizus confirmed their structure (Table 3).

ip
325 Pustulan (13, Figure 1f) is water-soluble linear (1→6)-β-D-glucan isolated from the

cr
326 lichen Lasallia pustulata (Pereyra, Prieto, Bernabé, & Leal, 2003) and reported to be a

327 taxonomic marker for lichens and lichenised fungi of the family Umbilicariaceae (Narui,

us
328 Sawada, Culberson, Culberson, & Shibata, 1999). In the native state this polysaccharide is

329 partially O-acetylated (DS ~10 %).

330

331 an
M
[6)-β-D-Glcp-(1→]n (13)

332

333 In addition to alkali-soluble yeast (1→3)-β-D-glucan mentioned above, Saccharomyces

d

334 cerevisiae also contains acid-soluble (1→6)-β-D-glucan (Manners, Masson, Patterson,

te

335 Bjørndal, & Lindberg, 1973; Fleet & Manners, 1976). Two linear β-D-glucans, i.e., (1→3)-β-
p

336 D-glucan (laminaran) and slightly O-acetylated (1→6)-β-D-glucan (pustulan), were isolated
ce

337 from the lichenized fungus Umbilicaria mammulata (Carbonero, Smiderle, Gracher,
13
338 Mellinger, Torri, Ahti, et al., 2006). C NMR spectra confirmed the structure of these
Ac

339 polysaccharides (Table 3). Minor carbon signals of pustulan at δ 20.8 (CH3), 175.0 (C=O),

340 77.7 and 71.2 (not shown) arose from O-acetyls and O-acetylated carbons. Sassaki, Ferreira,

341 Glienke-Blanco, Torric, Toni, Gorin, et al. (2002) described a pustulan-like

342 exopolysaccharide (Mr = 200 kD) produced by phytopathogenic fungus Guignardia citricarpa.

343 Kruppa, Lowman, Chen, Selander, Scheynius, Monteiro, et al. (2009) reported that linear

344 (1→6)-β-D-glucan is the major polysaccharide isolated from Malassezia sympodialis cell wall.

345 Similar β-D-glucans were isolated from basidiocarps of Agaricus bitorquis (Nandan, Patra,

13
Page 13 of 71
346 Bhanja, Adhikari, Sarkar, Mandal, et al., 2008), Agaricus blazei (Kawagishi, Inagaki, Kanao,

347 & Mizuno, 1989; Kawagishi, Inagaki, Kano, Shimura, Ito, Hagiwara, et al., 1990), Bulgaria

348 inquinans (Bi, Ni, Liu, Iteku, Tai, Zhou, et al., 2009) and somatic hybrid Pflo Vv5 FB,

349 obtained through protoplast fusion between Pleurotus florida and Volvariella volvacea strains

t
350 (Das, Mondal, Roy, Maiti, Bhunia, Maiti, et al., 2010). Glucans of the somatic hybrid and

ip
351 mushrooms Agaricus bitorquis were water-soluble and were isolated from hot water extracts,

cr
352 while the polysaccharide of Agaricus blazei formed a water-insoluble complex with protein.

353 NMR spectra of polysaccharide ABPS from Agaricus bitorquis showed anomeric CH

us
354 signals (H-1 at δ 4.50, C-1 at δ 103.4) and coupling constants (JH-1,H-2 ~8.5 Hz, JH-1,C-1 ~160

355 Hz) typical for β-anomeric form of D-Glcp. The C-6 signal is shifted downfield to δ 69.2 due

356
an
to glycosylation at O-6 position (Table 3). Inter-unit 13
C-1H correlations obtained from the
M
357 HMBC experiment also confirmed (1→6)-connection between β-D-Glcp units. Cross peaks

358 were found between H-1 (δ 4.50) and C-6 (δ 69.2), and between C-1 (δ 103.4) and H-6 (δ
d

359 4.20 and 3.84) of neighbour residues (Nandan et al., 2008).

te

360 Ukawa, Ito, & Hisamatsu (2000) described three polysaccharide fractions isolated from a

361 hot-water extract of Lyophyllum decastes basidiocarps. The highest Mw (305 kDa) and lowest
p

362 Mw (14 kDa) fractions were identified as (1→3)-β-D-glucan and (1→6)-β-D-glucan,

ce

363 respectively; the fraction of Mw = 130 kDa was defined as a mixture of these two

364 polysaccharides or branched (1→3),(1→6)-β-D-glucan.

Ac

365

366 3.1.2. Linear mixed linkage β-D-glucans

367

368 Lichenan (14, Fig. 1h) is a linear mixed-linkage (1→3),(1→4)-β-D-glucan of the lichen

369 Cetraria islandica, having a closely related structure to storage cereal β-D-glucans. Despite

370 this similarity, Honegger and Haisch (2001) suggested based on SEM and

14
Page 14 of 71
371 immunocytochemical analyses that it is primary a structural element of the fungal cell wall

372 rather than a storage component of lichenised fungi. Lichenan consists of cellotriosyl (78%),

373 cellotetraosyl (4%) and longer cellulose-like (18%) segments (Wood, Weisz, & Blackwell,

374 1994). Following lichenase digestion of lichenan, the molar ratio of tri- to tetrasaccharides

t
375 (DP3/DP4) was found to be much higher (24.5) than the corresponding values of cereal β-D-

ip
376 glucans (3.7–2.1) (Lazaridou, Biliaderis, Micha-Screttas, & Steele, 2004).

cr
377

378 {3)-β-D-Glcp-(1→[4)-β-D-Glcp-(1→]m}n (14)

us
379

380 Lichenan-type polysaccharides have been also described for lichenised fungi of genera

381

382 an
Parmotrema and Rimelia (Carbonero, Montai, Mellinger, et al., 2005). The β-configuration

was of these glucans was confirmed by high-field resonances at of δ 4.38 and 4.28 (H-1)
M
383 assigned to (1→3)- and (1→4)-linked β-D-Glcp units, respectively. The ratio of glycosidic

384 linkages (1:3.1) determined by comparing the areas of these peaks was identical to the value
d

385 obtained by methylation analys is. The low-field carbon signals at δ 87.0 (C-3) and 80.0–80.1
te

386 (C-4) confirmed glycosylation at O-3 and O-4, while the signal at δ 60.4 (C-6) arised from
p

387 non-substituted CH2OH groups. HPLC analysis of Smith degraded products confirm that all
ce

388 the (1→3)-linked units are interspersed between the (1→4)-linked ones.

389
Ac

390

391 3.2 Branched β-D-glucans

392

393 Branched β-D-glucans containing (1→3)- and (1→6)- glycosidic linkages are the main

394 common constituents of fungal cell walls. There are many reports about isolation, structure

395 and biological effects of these polysaccharides (Wasser, 2002; Chen, & Seviour, 2007). Most

396 of branched β-D-glucans have a (1→3)-linked β-D-Glcp backbone and side-chains of (1→6)-

15
Page 15 of 71
397 linked β-D-Glcp units (Chen, & Seviour, 2007), while in some cases the arrangement is

398 opposite, i.e., the main chain is formed by (1→6)-β-linked units with (1→3)-β-linked

399 branches (Figure 1h) (Dong, Yao, Yang, & Fang, 2002; Sun, Yu, Zhang, Yue, Yang, Liu, et

400 al., 2012; Ge, Zhang, & Sun, 2009). A degree of branching (DB) as well as possible

t
401 branching at O-4 and even at O-2 depends on a fungal source and a way of isolation and

ip
402 purification. The DB values of some fungal β-D-glucans are given in Table 4 (Novak, &

cr
403 Vetvicka, 2008; Chen, & Seviour, 2007).

404

us
405 3.2.1. Branched β-D-glucans with (1→3)-β-D-glucan backbone

406

407
an
As it was mentioned above, many branched β-D-glucans from various sources have
M
408 (1→3)-linked β-D-Glcp backbone with single β-D-Glcp side chains attached at O-6 position

409 (15, Figure 1i). Great variability in DB and branching distribution as well as complexation
d

410 with other cell wall polysaccharides, mainly with chitin, are common for these
te

411 polysaccharides.

412
p

413 β-D-Glcp (15)

414
ce

1
415 ↓
416 6
417 {[3)-β-D-Glcp-(1→] m3)-β-D-Glcp-(1→}n
418
Ac

419

420 Some of these (1→3),(1→6)-β-D-glucans have own names derived from the fungal source.

421 Lentinan (DB ~0.5 – 0.33), a polysaccharide from Lentinula (Lentinus) edodes, is one of the

422 best known among them (Zhang, Li, Wang, Zhang, & Cheung, 2011; Saitô, Ohki, Takasuka,

423 & Sasaki, 1977; Saitô, Ohki, & Sasaki, 1979; Sasaki & Takasuka, 1976). Similar highly

424 branched β-D-glucan was isolated from Aureobasidium pullulans (Tada, Tanioka, Iwasawa,

16
Page 16 of 71
425 Hatashima, Shoji, Ishibashi, et al., 2008). Its molecule was shown to comprise a mixture of a

426 (1→3)-β-D-glucan backbone with (1→6)-linked β-D-Glcp in side chains attached to the every

427 second (major structure) or the every third (minor structure) residue. Following three

428 polysaccharides having trivial named were also defined as (1→3),(1→6)-β-D-glucans but

t
429 something less branched (DB ~0.33 – 0.25). These are grifolan extracted from fungus Grifola

ip
430 frondosa (Ohno, Adachi, Suzuki, Sato, Oikawa, & Yadomae, 1986; Tada, Adachi, Ishibashi,

cr
431 & Ohno, 2009) and two extracellular polysaccharides schizophyllan and scleroglucan

432 produced by fungi Schizophyllan commune (Tabata, Ito, Kojima, Kawabata, & Misaki, 1981)

us
433 and Sclerotium sp. (Coviello, Palleschi, Grassi, Matricardi, Bocchinfuso, & Alhaique, 2005),

434 respectively. NMR data of grifolan LE were presented by Tada, Adachi, Ishibashi, et al.

435
an
(2009). The H-1 (δ 4.2–4.7) and C-1 (δ 103.0–103.1) resonance signals indicated β-anomeric
M
436 configuration. These signals were assigned to four types of β-D-Glcp units (Table 5).

437 Observed four inter-unite 1H, 13

C HMBC cross-peaks indicated specific glycosidic linkages
d

438 between these units. The ratio of the main chain to the side chain units calculated from the
te

439 areas of anomeric protons was approximately 3:1.

440 Many other (1→3),(1→6)-β-D-glucans of similar structure were isolated from

p

441 basidiocarps of Boletus erythropus (Chauveau, Talaga, Wieruszeski, Strecker, & Chavant,
ce

442 1996), Dictyophora indusiata (Wang, Xu, Zheng, Li, Deng, Xu, et al., 2009), Hericium

443 erinaceum (Dong, Jia, & Fang, 2006), Pleurotus florida (Rout, Mondal, Chakraborty,
Ac

444 Pramanik, & Islam, 2005; Rout, Mondal, Chakraborty, & Islam, 2008), Pleurotus

445 pulmonarius (Smiderle, Carbonero, Mellinger, Sassaki, Gorin, & Iacomini, 2006; Smiderle,

446 Olsen, Carbonero, Baggio, Freitas, Marcon, et al., 2008; Smiderle, Sassaki, Van Arkel, et al.,

447 2010), Pleurotus eryngii (Synytsya, Míčková, Synytsya, et al., 2009; Carbonero, Gracher,

448 Smiderle, Rosado, Sassaki, Gorin, et al. 2006), Pleurotus ostreatoroseus (Carbonero, Gracher,

449 Smiderle, et al. 2006), Pleurotus ostreatus (Palacios, García-Lafuente, Guillamón, et al. 2012;

17
Page 17 of 71
450 Synytsya, Míčková, Synytsya, et al., 2009; Yoshioka, Tabeta, Saitô, Uehara, & Fukuoka,

451 1985), Pleurotus tuber-regium (Chenghua, Xiangliang, Xiaoman, Yan, Jingyan, & Huibi,

452 2000; Zhang, Zhang, Dong, Guo, Song, & Cheung, 2001), Pleurotus sajor-chaju (Carbonero,

453 Ruthes, Freitas, Utrilla, Gálvez, da Silva, et al., 2012), Sparassis crispa (Tada, Harada, Nagi-

t
454 Miura, Adachi, Nakajima, Yadomae, et al., 2007) and many other sources. Branched

ip
455 (1→3),(1→6)-β-D-glucans were isolated from basidiocarps (stems) of Pleurotus ostreatus and

cr
456 Pleurotus eryngii (Synytsya, Míčková, Synytsya, et al., 2009). These polysaccharides,

457 partially complexed with proteins, predominated in the hot water extracts of these mushrooms.

us
458 In the alkali extracts β-D-glucans were found in lesser amounts together with more

459 pronounced (1→3)-α-D-glucans, and the alkali-insoluble solids were defined as chitin-glucan

460
an
complexes with the prevalence of (1→3),(1→6)-β-D-glucans. Šandula, Kogan, Kačuráková,
M
461 & Machová (1999) described two water-insoluble (1→3),(1→6)-β-D-glucans isolated from S.

462 cerevisiae and A. niger. The former glucan has a low-branched structure (DB = 0.125), while
d

463 the latter one was complexed with chitin. Lukondeh, Ashbolt, Rogers, & Hook (2003)
te

464 isolated low branched alkali-insoluble β-D-glucan (Mw ~300 kDa) containing mostly (1→3)-

465 β- linkages from yeast cells of Kluyveromyces marxianus.

p

466 Various branched (1→3),(1→6)-β-D-glucans were isolated from fruiting bodies (Hung,
ce

467 Wang, Chen, & Yang, 2008; Chang & Lu, 2004; Bao, Wang, Dong, Fang, & Li, 2002) and

468 submerged culture mycelia (Sone, Okuda, Wada, Kishida, & Misaki, 1985) of medicinal
Ac

469 mushroom Ganoderma lucidum, as well as from fruiting bodies of other Ganoderma species,

470 i.e., G. appalantum (Usui, Iwasaki, & Mizuno, 1983), G. japonicum (Ukai, Yokoyama, Hara,

471 & Kiho, 1982) and G. resinaceum (Amaral, Carbonero, Simão, Kadowaki, Sassaki, Osaku, et

472 al., 2008). Structure of these polysaccharides depended on a source and a way of isolation.

473 Polysaccharide from Ganoderma japonicum (Ukai, Yokoyama, Hara, et al., 1982) was

474 defined as extremely low-branched water-insoluble (1→3),(1→6)-β-D-glucan (Mw = 82 kDa,

18
Page 18 of 71
475 DB ~0.03). By contrast, glucan GLG from fruiting bodies of Ganoderma lucidum (Chang &

476 Lu, 2004) is highly branched polysaccharide. The 13C NMR resonance signals (Table 5) at δ

477 86.1 (C-3) and 103.1 (C-1) indicated that it has the (1→3)-β-D-glucan backbone, but evident

478 splitting of C-3 (δ 85.8–86.7) and C-2 (δ 73.0–73.8) regions into three signals of each kind

t
479 confirmed pronounced difference between the units. Two peaks at δ 70.3 (C-4) and 75.0 (C-5)

ip
480 arose from branching →3,6)-β-D-Glcp-(1→ and side chain terminal β-D-Glcp-(1→ units,

cr
481 respectively. The DB of GLG (0.35) was obtained from the ratio between integrated peak

482 areas of proton resonance signals H-1 at δ 4.24 (side chains) and 4.54 (internal units); for

us
483 comparison, the DB of more branched lentinan was 0.42 (Chang & Lu, 2004).

484 Amaral, Carbonero, Simão, et al. (2008) described water-soluble β-D-glucan from

485
an
basidiocarps of Ganoderma resinaceum further purified by alkaline extraction. Structure of
13
M
486 this polysaccharide was analysed by C NMR (Table 5). The downfield shifted carbon

487 signals at δ 84.8 (C-3), 79.1 (C-4) and 69.0 (C-6) arose from O-3, O-4 and O-6 glycosylated
d

488 β-D-Glcp units, respectively; signals of non-substituted C-6 were found at δ 61.0 and 60.4.
te

489 The backbone structure was identified by a controlled Smith degradation, which gave linear

490 (1→3)-β-D-glucan. Thus the native polysaccharide was defined as a highly branched glucan
p

491 (DB ~0.5) containing a (1→3)-linked β-D-Glcp backbone partially substituted at O-6 by side
ce

492 chains of (1→4)-linked β-D-Glcp on the every second backbone residue (16).

493
Ac

494 [4)-β-D-Glcp-(1→]m4)-β-D-Glcp (16)

495 1
496 ↓
497 6
498 [3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→]n

499

500 Several branched (1→3)-β-D-glucan obtained from basidiocarps and submerged culture

501 mycelia of Ganoderma lucidum (Sone, Okuda, Wada, et al., 1985) had different DB values

502 (1/3 – 1/23) and two types of side chains, i.e., mainly single β-D-Glcp attached at O-6 and a

19
Page 19 of 71
503 few short (1→4)-β-D-glucan residues at the O-2 positions (17).

504

505 β-D-Glcp
506 1
507 ↓
508 6
509 {[3)-β-D-Glcp-(1→]m3)-β-D-Glcp-(1→[3)-β-D-Glcp-(1→]m3)-β-D-Glcp-(1→}n (17)

t
510 2

ip
511 ↑
512 1
513 [4)-β-D-Glcp-(1→]k4)-β-D-Glcp

cr
514

515

us
516 Barbosa, Steluti, Dekker, Cardoso, & Corradi da Silva (2003) described botryosphaeran,

517 an exopolysaccharide produced by lignolytic fungus Botryosphaeria sp. It was suggested to

518

519 an
be (1→3)-β-D-glucan with about 22% of side chains at O-6 consisted of β-D-Glcp and

gentiobiosyl residues (18). The 13C NMR signals attributed to a (1→3)-β-D-glucan backbone
M
520 were similar to those of a residual linear polysaccharide obtained after Smith degradation

521 (Table 5). The downfield shifted C-3 carbon signals at δ 85.5–86.3 confirmed (1→3)-
d

522 linkages. Among them, the signal of non-substituted backbone units at δ 86.3 was more
te

523 intense than the others; lesser C-3 signals at δ 86.0 and 85.5 were attributed to branching
p

524 point units carrying O-6 linked β-D-Glcp and gentiobiosyl residues, respectively. Resonance
ce

525 signals at δ 70.1 arose from the O-substituted C-6 carbons and disappeared after Smith

526 degradation. Non-substituted C-6 carbons showed signals at δ 60.7–61.1, and the main signal
Ac

527 at δ 61.1 was comparable to that obtained after Smith degradation and thus attributed to

528 internal units inside linear fragments of the backbone. Other signals at δ 60.9 and 60.7 arose

529 from non-substituted backbone units attached to the branching point units. Authors proposed

530 random distribution of branching along a chain based on relative intensities of C-3 substituted

531 and free C-6 regions. Methacanona, Madla, Kirtikara, & Prasitsil (2005) described the

532 structure of several fungal exopolysaccharides isolated from three strains of fungi,

533 Akanthomyces pistillariiformis BCC2694, Cordyceps dipterigena BCC2073 and

20
Page 20 of 71
534 Phytocordyceps sp. BCC2744. These polysaccharides were irregularly branched

535 (1→3),(1→6)-β-D-glucan having a (1→3)-β-D-glucan backbone substituted at O-6 with single

536 or (1→6)-liked two or three β-D-Glcp units in side chains (18). The highest DB was observed

537 for the former strain polysaccharide, followed subsequently by the second and third ones.

t
538

ip
539 [6)-β-D-Glcp-1→]k6)-β-D-Glcp k = 0, 1 or 2 (18)
540 6

cr
541 ↑
542 1
543 {[3)-β-D-Glcp-(1→]m3)-β-D-Glcp-(1→}n
544

us
545 Mandal, Maity, Bhunia, Dey, Patra, Sikdar et al. (2010) described calocyban (Mw ~200

an
546 kDa), a new water-insoluble (1→3),(1→4)-β-glucan isolated from an alkaline extract of
13
547 Calocybe indica basidiocarps. A C NMR spectrum of this polysaccharide confirmed β-
M
548 anomeric structure, presence of terminal β-D-Glcp and the lack of O-6 linkage (Table 5). The

549 downfield shifted signals at δ 87.0 (C-3) and 79.9 (C-4) indicated (1→3,4)-linked β-D-Glcp as
d

550 a branching point; two other signals of C-3 at δ 87.3 and 87.7 belonged to backbone (1→3)-
te

551 linked β-D-Glcp units. The former signal was upfield shifted due to neighboring effect of rigid

552 branching point units, whereas the latter appearing in downfield region indicated backbone
p

553 units aside from branching. Therefore, calocyban consists of a (1→3)-β-D-glucan backbone
ce

554 with (1→4)-linked β-D-Glcp units as side chains bound to the every fourth unit (19). Wu, Sun,

555 & Pan (2005) described an extracellular polysaccharide isolated from a hot water extract of
Ac

556 Cordyceps sinensis mycelia. It also consisted of a (1→3)-β-D-glucan backbone carried

557 individual (1→4)-linked β-D-Glcp residues.

558

559 {[3)-β-D-Glcp-(1→]33)-β-D-Glcp-(1→}n (19)

560 4
561 ↑
562 1
563 β-D-Glcp
564

21
Page 21 of 71
565

566 3.2.2. Branched β-D-glucans with β-(1→6)-D-glucan backbone

567

568 Maji, Sen, Behera, Maiti, Mallick, Sikdar, et al. (2012) described a water soluble β-D-

t
569 glucan PS-I isolated from a hot aqueous extract of basidiocarps of an edible hybrid mushroom

ip
570 Pfle1r of Pleurotus florida and Lentinula edodes. A 13C NMR spectrum of PS-I showed three

cr
571 anomeric carbon signals at δ 103.0, 102.9 and 102.7 (Table 5); the former one was almost

572 three times more intense than each of the latter ones. The downfield shifts of C-3 at δ 84.2

us
573 and C-6 at δ 68.7 with respect to the standard values of methyl glycosides indicated the

574 presence of a branching unit →3,6)-β-D-Glcp(1→. This residue is the most rigid part of the

575
an
backbone of this glucan, so it's C-6 signal appeared at the upfield region in comparison to that
M
576 of the other (1→6)-β-linked residues. Among the three latter residues, one was linked to the

577 rigid branching point unit, hence, it's C-6 signal (δ 69) showed δ 0.2 downfield shift with
d

578 respect to that of another two residues (δ 68.8) due to the neighbouring effect. Finally, one
te

579 residue showed resonance signals similar to the standard values of methyl glycoside of β-D-

580 Glcp and was established as a terminal unit. Therefore, this polysaccharide consists of a
p

581 (1→6)-β-D-glucan backbone with single β-D-Glcp side chains at the O-3 position of the every
ce

582 four residue of the main chain (20, Fig. 1j).

583
Ac

584 {[6)-β-D-Glcp-(1→]36)-β-D-Glcp-(1→}n (20)

585 3
586 ↑
587 1
588 β-D-Glcp
589

590 Medicinal mushroom Agaricus brasiliensis (=Agaricus blazei) is a source of various α-

591 and β-D-glucans forming complexes with proteins (Gonzaga, Ricardo, Heatley, & Soares,

592 2005). Mizuno, Hagiwara, T., Nakamura, Ito, Shimura, Sumiya, et al. (1990) identified water

22
Page 22 of 71
593 soluble (1→6),(1→3)-β-D-glucan from this mushroom. According to Dong, Yao, Yang, et al.

594 (2002), this polysaccharide, named Ab2-2N, has a (1→6)-β-D-glucan backbone with β-D-

595 Glcp-(1→3)-β-D-Glcp-1→ disaccharide side chains attached at O-3 of every third backbone

596 residue (21). Three C-1 resonance signals at δ 105.1 (internal O-6 or O-3 substituted units),

t
597 104.7 (branching O-6 and O-3 disubstituted units) and 104.4 (terminal units) indicated β-

ip
598 anomeric configuration (Table 5). The signals of substituted C-6 and C-3 were downfield

cr
599 shifted respectively to δ 71.0 and 86.5 in comparison with those of non-substituted carbons

600 found at δ 62.9 (C-6) and 78.1 (C-3). Comparing of 13C NMR signal intensities led authors to

us
601 the conclusion that glycosylation at O-3 (side chains) is less pronounced than at O-6

602 (backbone). Similar structure was reported for water-soluble β-D-glucans originated from

603
an
Pleurotus citrinopileatus (PCP-W1, Mw = 45 kDa) (Sun, Yu, Zhang, et al., 2012) and
M
604 Phellinus baumii (Mw = 1920 kDa) (Ge, Zhang, & Sun, 2009).

605

606
d
{[6)-β-D-Glcp-(1→]26)-β-D-Glcp-(1→}n (21)
607 3
608 ↑
te

609 1
610 β-D-Glcp-(1→3)-β-D-Glcp
611
p

612 As it was mentioned above, Kawagishi, Inagaki, Kanao, et al. (1989) and Kawagishi,
ce

613 Inagaki, Kano, et al. (1990) described alkali soluble linear (1→6)-β-D-glucan of A. blazei

614 isolated as a part of a protein-glucan complex. Contrary to this, Ohno, Furukawa, Miura,
Ac

615 Adachi, Motoi, & Yadomae (2001) detected a small but sufficient amount of (1→3)-linkages

616 in such β-D-glucan. According to Camelini, Maraschin, de Mendonça, Zucco, Ferreira, &

617 Tavares (2005), β-D-glucan preparations from Agaricus brasiliensis basidiocarps in different

618 stages of maturity contained greater amounts of (1→6)-β-D-glucans and the (1→3)-β-D-

619 glucan content increased with the fruiting bodies maturation. Thus highly branched structures

620 predominated in hot water extracts of fungal glucans, while alkali-soluble fractions mostly

23
Page 23 of 71
621 were linear polysaccharides with no or small branching.

622 Insoluble cell wall glucan isolated from fungi Candida albicans (Iorio, Torosantucci,

623 Bromuro, Chiani, Ferretti, Giannini, et al., 2008) consisted of (1→6)-β-D-glucan backbones

624 carrying O-3 linked shorter (1→3)-β-D-glucan side chains (22). The 1H and 13
C resonances

t
625 typical of linear (1→6)-β-D-glucan and (1→3)-β-D-glucan fragments were observed in the

ip
626 water soluble derivatives of this polysaccharide obtained by partial enzymatic hydrolysis with

cr
627 endo-(1→3)-β-D-glucanase. A high molecular fraction (46.3 %) consisted of long (1→6)-β-D-

628 glucan fragments with short (1→3)-β-D-glucan side chains (DB = 0.14), whereas a low

us
629 molecular fraction contained glucose and short linear (1→3)-β-D-glucan fragments. Further

630 digestion of the high molecular fraction by (1→6)-β-D-glucanase yielded glucose and short

631
an
linear or O-3 branched (1→6)-β-D-glucan fragments.
M
632

633 {[6)-β-D-Glcp-(1→]k6)-β-D-Glcp-(1→}n (22)

634 3
635 ↑
d

636 1
637 [3)-β-D-Glcp-(1→]m3)-β-D-Glcp
te

638

639 Han, Chai, Jia, Han, & Tu (2010) described water-soluble polysaccharide HBP (Mw = 430
p

640 kDa) isolated from basidiocarps of Sarcodon aspratus. Five H-1 resonance signals were
ce

641 found in the region of δ 4.52–4.78, while only one C-1 signal was observed around δ 103.7;

642 all of them confirmed β-anomeric configuration. The downfield shifted carbon signals at δ
Ac

643 85.0 (C-3) and 81.0 (C-4) confirmed glycosylation at these positions (Table 5). Similarly,

644 downshifted C-6 signals of at δ 69.6 and 68.8 confirmed O-6 substitution. The sequence of

645 glycosyl residues was determined from NOESY and HMBC experiments. Authors concluded

646 that HBP is a glucan with a (1→6)-linked β-D-Glcp backbone randomly substituted at O-3

647 position by tetrasaccharide side chains composed of two (1→3)-linked β-D-Glcp residues and

648 a terminal (1→4)-β-D-Glcp residue (23).

24
Page 24 of 71
649
650 {[6)-β-D-Glcp-(1→]m 6)-β-D-Glcp-(1→6}n (23)
651 3
652 ↑
653 1
654 β-D-Glcp-(1→4)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp
655

t
656

ip
657 3.2.3. Branched β-D-glucans with mixed linkage backbone

cr
658

659 Karácsonyi & Kuniak (1994) described pleuran, an alkali-insoluble cell wall β-D-glucan

us
660 isolated from basidiocarps of Pleurotus ostreatus. This polysaccharide having a (1→3)-β-D-

661 glucan backbone substituted at O-6 of the every fourth unit with single β-D-Glcp also

662

663 an
contained a small proportion (7 %) of (1→6)- and (1→4)-linked residues in the backbone

chain. Bhunia, Dey, Maity, Patra, Mandal, Maiti, et al. (2011) described unusual branched
M
664 (1→3),(1→6)-β-D-glucan (Mw ~198 kDa) from basidiocarps of Lentinula (Lentinus)

665 squarrosulus using 2D NMR. HMQC data confirmed correlations between anomeric proton
d

666 signal at δ 4.52 and two anomeric carbon signals at δ 103.4 and 102.9, and, similarly, between
te

667 H-1 signal at δ 4.50 and two C-1 signals at δ 103.2 and 102.7 (Table 5). The signal at δ 102.9
p

668 was almost double intense than the other C-1 signals. The HMBC and NOESY data supported
ce

669 that (1→6)-linkages were present both in the backbone and side chains of this polysaccharide

670 (24).
Ac

671

672 β-D-Glcp (24)

673 1
674 ↓
675 6
676 [3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→6)-β-D-Glcp-(1→]n
677

678

679 Liu & Wang (2007) described a water-soluble β-D-glucan PRP isolated from a hot water

25
Page 25 of 71
680 extract of fruiting bodies of Phellinus ribis. A 13C NMR spectrum contained three anomeric

681 signals at δ 107.0, 105.6 and 105.3, assigned to terminal, (1→4)- and (1→6)-linked β-D-Glcp

682 residues, respectively (Table 5). The C-6 signals (inverted in DEPT spectrum) were found at δ

683 63.4, 65.2 and 71.4; the latter indicated O-6 substitution. The C-3 signal of branching (1

t
684 →3),(1→6)-β-D-Glcp residues showed a downfield shift to δ 87.2 because of O-3 substitution.

ip
685 Resonance signals of branching and terminal units disappeared after mild hydrolysis of PRP,

cr
686 yielding linear (1→4),(1→6)-β-D-glucan. Authors concluded that this polysaccharide consists

687 of a mixed (1→4),(1→6)-linked β-D-glucan backbone with single β-D-Glcp side chains at the

us
688 O-3 position of the (1→6)-linked backbone units the every eights residue of the main chain

689 (25).

690

691 an
M
{[6)-β-D-Glcp-(1→]2 [4)-β-D-Glcp-(1→]3 [6)-β-D-Glcp-(1→]2 6)-β-D-Glcp-(1→}n (25)
692 3
693 ↑
694 β-D-Glcp
695
d

696 Krestin (PSK, polysaccharopeptide Krestin) prepared from medicinal mushroom

te

697 Trametes (Coriolus) versicolor is a mixture of polysaccharide–peptide complexes (Mw ~100

p

698 kD) consisting of β-D-glucans covalently linked to various peptides (25–38 %) (Tsukagoshi,
ce

699 Hashimoto, Fujii, Kobayashi, Nomoto, & Orita, 1984). Proposed structure of the

700 polysaccharide part of PSK is branched β-D-glucan having a (1→4)-β-D-glucan backbone

Ac

701 with (1→6)-β- and (1→3)-β-linked side chains attached to approximately the every fourth

702 backbone unit (Tsukagoshi, Hashimoto, Fujii, Kobayashi, Nomoto, & Orita, 1984; Ooi & Liu,

703 2000). Jeong, Yang, Ra, Wilson, Cho, Gua, et al. (2004). Polysaccharopeptide (PSP),

704 another biologically active glucan–peptide complex from the same source, probably is (1→3)-

705 β-D-glucan branched at O-4 and O-6 positions and also covalently linked to peptide moieties

706 (Chu, Ho, & Chow, 2002). However, other sources reported more complex character of the

707 PSK and PSP polysaccharides (Cui & Chisti, 2003). According to Ng, (1998), both

26
Page 26 of 71
708 preparations contain (1→4)-α- and (1→3)-β-linked D-Glcp. While D-glucose is the major

709 monosaccharide unit, other sugars are also present in significant amounts (Wang, Ng, Liu,

710 Ooi, & Chang, 1996; Cheng, Wu, Zhou, & Cheng, 1998).

711

t
712

ip
713 4. Fungal mixed-linkage α, β-D-glucans

cr
714

715 4.1. Linear mixed-linkage α, β-D-glucans

us
716

717 There are two reports about fungal glucans having alternating α- and β-glycosidic linkages

718
an
along the chain. Chakraborty, Mondal, Pramanik, et al. (2004) described such linear α,β-D-

glucan AQS-I from fruiting bodies of Astraeus hygrometricus. 1H NMR spectrum of this
M
719

720 polysaccharide showed two H-1 signals at δ 4.83 (JH-1,H-2 3.9 Hz) and 4.39 (JH-1,H-2 8.5 Hz)

assigned to (1→4)-α- and (1→6)-β-linked D-Glcp units, respectively (1:1 mol mol-1). The
d

721
te

722 corresponding C-1 signals were found at δ 98.4 and 103.4 as evident from HSQC experiments

723 (Table 6). The downfield shifted carbon signals at δ 66.0 (C-6) and 75.3 (C-4) arose from O-6
p

724 and O-4 substituted units, respectively. The alternating sequence of the units along a chain
ce

725 was confirmed by NOESY experiment. Therefore, in AQS-I the D-Glcp units are linked by

726 repeating (1→4)-α- and (1→6)-β-glycosidic bonds (26).

Ac

727

728 [4)-α-D-Glcp-(1→6)-β-D-Glcp-(1→]n (26)

729

730 Chandra, Ghosh, Roy, Mondal, Maiti, Ojha, et al. (2007) described another linear α,β-D-

731 glucan PS-I isolated from an edible mushroom Termitomyces microcarpus. A 1H NMR

732 spectrum of this polysaccharide showed two H-1 signals at δ 5.14 (JH-1,H-2 ~3.5 Hz) and 4.55

733 ppm (JH-1,H-2 ~6.5 Hz) assigned to α- and β-D-Glcp units (1:1 mol mol-1), respectively.

27
Page 27 of 71
734 Corresponding C-1 signals were observed at δ 98 and 102.4 (Table 6). The downfield shifted

735 carbon signals at δ 75.7 (C-4) and 85.1 (C-3) confirmed the presence of (1→4)-α- and (1→3)-

736 β- linkages (α-glycosylation effect). In addition, in both cases the signals of neighbour

737 carbons showed an upfield shift due to the β-glycosylation effect. NOESY experiment

t
738 confirmed the alternating sequence of these glycosidic bonds in PS-I (27).

ip
739

cr
740 [4)-α-D-Glcp-(1→3)-β-D-Glcp-(1→]n (27)

741

us
742 Both these linear mixed-linkage α,β-D-glucans are well soluble in water.

743

744

745 4.2. Branched mixed-linkage α, β-D-glucans an

M
746

747 Several reports are devoted to mixed-linkage α,β-D-branched fungal glucans, i.e., those
d

748 containing both α- and β-glycosidic linkages of various positions and configurations
te

749 depending on a fungal source and a way of isolation. Following structural variants of such
p

750 polysaccharides are defined.

ce

751

752 4.2.1. α-D-Glucan backbone with β-linked side chains

Ac

753

754 Olennikov, Agafonova, Rokhin, Penzina, & Borovskii et al. (2012) described branched

755 α,β-D-glucan, named piptoporan I (Mw ~270 kDa), isolated from the fruiting bodies of wood-

756 decaying fungus Piptoporus betulinus. It was branched polysaccharide with a (1→3)-α-D-

757 glucan backbone substituted at the O-6 position by single β-D-Glcp residues (DB = 17.3 %)

758 (28).

759

28
Page 28 of 71
760 β-D-Glcp (28)
761 1
762 ↓
763 6
764 {[3)-α-D-Glcp-(1→]n 3)-α-D-Glcp-(1→}n
765

766

t
767 Three C-1 carbon signals at δ 101.5, 101.8 and 106.3 indicated unsubstituted and

ip
768 substituted backbone α-D-Glcp and side chain β-D-Glcp units, respectively (Table 7). The C-3

cr
769 carbon signals of the unsubstituted and substituted backbone units at δ 85.3 and 86.3,

770 respectively, and the C-6 signal of the substituted moieties at δ 68.1 confirmed the

us
771 substitution at these positions. By contrast, the C-5 signal of the substituted backbone units

an
772 was upfield shifted to δ 73.5, which also supported substitution at O-6. The carbon signals of

773 side chain units were close to those of free β-D-Glcp. Smith-degraded piptoporan I (Mw = 225
M
774 kDa) was identified by 13C NMR as linear (1→3)-α-D-glucan.

775 Santos-Neves, Pereira, Carbonero, Gracher, Alquini, Gorin, et al. (2008) identified
d

776 branched α,β-D-glucan MRFS–HW (Mw ~1100 kDa) isolated from fruiting bodies of P.
te

13
777 florida. C NMR spectra of this polysaccharide showed several C-1 signals at δ 102.8

778 (terminal β-D-Glcp), 102.9 (O-3 substituted β-D-Glcp), 99.4 (O-3 substituted α-D-Glcp) and
p

779 99.5 (3,6-substituted α-D-Glcp) (Table 7). In addition, high and low field H-1 signals at δ 5.43
ce

780 and 4.59 confirmed the presence of α- and β-D-Glcp. The (1→3)- and (1→6)-glycosidic

781 linkages were indicated by the downfield shifted C-3 (81.4, 80.9 and 80.7) and C-6 (δ 69.0,
Ac

782 68.6 and 68.5) signals, respectively. The backbone structure of MRFS–HW was confirmed

783 by controlled triple Smith degradations, which gave linear (1→3)-α-D-glucan as the final

784 product (Table 7). Authors concluded that this polysaccharide contained a (1→3)-α-D-glucan

785 backbone partially substituted at O-6 by single β-D-Glcp, and a smaller amount of

786 trisaccharide side chains with (1→3)-linked β-D-Glcp units (29).

787

29
Page 29 of 71
788 β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp β-D-Glcp (29)
789 1 1
790 ↓ ↓
791 6 6
792 {[3)-α-D-Glcp-(1→]m3)-α-D-Glcp-(1→[3)-α-D-Glcp-(1→]m3)-α-D-Glcp-(1→}n

793

794

t
ip
795 4.2.2. β-D-Glucan backbone with α-linked side chains

796

cr
797 Mandal, Maity, Bhunia, et al. (2010) described water-soluble branched α,β-D-glucan PS-I

us
798 (Mw ~187 kDa) isolated from an alkaline extract of Calocybe indica basidiocarps. A 1H NMR

799 spectrum contained three H-1 signals at δ 5.38, 4.52 and 4.50 (1:1:2); corresponding C-1

an
800 signals appeared at δ 100.0, 103.2 and 103.4. The downfield shifts of C-4 (δ 76.4) and C-6 (δ

801 69.2 and 69.3) indicated O-4 and O-6 substitution (Table 7). According to NOESY and
M
802 HMBC experiments, this polysaccharide consisted of a (1→6)-β-D-glucan backbone with a

803 side chain of (1→4)-linked α-D-Glcp at the every third backbone unit (30).
d

804
te

805 {[6)-β-D-Glcp-(1→]26)-β-D-Glcp-(1→}n (30)

806 4
807 ↑
p

808 1
809 α-D-Glcp
ce

810

811
Ac

812 4.2.3. Mixed-linkage α,β-D-glucan backbone with α-linked side chains

813

814 Rout, Mondal, Chakraborty, et al. (2005) described the structure of water soluble

815 branched α,β-D-glucan from an aqueous extract of the fruiting bodies of Pleurotus florida.

816 Three H-1 signals were found at δ 5.09, 4.95 and 4.44 (2:1:1); corresponding C-1 resonances

817 appear at δ 100.1, 99.8 and 103.1. Thus this glucan was composed of a tetrasaccharide

818 repeating fragment consisting of three α-D-Glcp and one β-D-Glcp units. Smith degradation

30
Page 30 of 71
 13
819 led to insoluble product giving twelve C NMR signals; two of them at δ 103.4 and 100.2

820 (1:2) arose from (1→3)-β- and (1→3)-α- linked D-Glcp units, respectively (Table 7). This was

821 confirmed by downfield shifted C-3 signals of these units at δ 86.6 and 83.2 ppm, respectively,

822 as a result of the α-glycosylation effect. Therefore, the backbone of the native polysaccharide

t
823 was mixed (1→3)-α,β-D-glucan, and α-D-Glcp side chains were attached at O-6 to all the β-

ip
824 anomeric backbone units (31).

cr
825

826 α-D-Glcp (31)

us
827 1
828 ↓
829 6
830 {[3)-α-D-Glcp-(1→]2 3)-β-D-Glcp-(1→}n

an
831

832
M
833 Maity, Kar (Mandal), Maity, Gantait, Das, Maiti, et al. (2011) isolated water-soluble

834 branched α,β-D-glucan (~198 kDa) from an alkaline extract of the fruiting bodies of a somatic
d

835 hybrid PCH9FB of Pleurotus florida and Calocybe indica var. APK2 strains. The 1H NMR
te

836 spectrum showed two H-1α signals at δ 5.38 and 4.95, and two H-1β signals at δ 4.51 and

837 4.50 (1:1:1:3). Corresponding C-1 signals were found in the anomeric region at δ 98.3, 100.2,
p

838 103.3 and 103.4 (Table 7). The downfield shift of C-4 (δ 76.4) and C-6 (δ 69.2) confirmed O-
ce

839 4 and/or O-6 glycosylation of some units. The unit sequence was determined from ROESY,

840 NOESY and HMBC experiments. Structure of the repeating unit of this glucan was
Ac

841 demonstrating as (32).

842

843 {[6)-β-D-Glcp-(1→]3→3)-β-D-Glcp-(1→4)-α-D-Glcp-(1→}n (32)

844 4
845 ↑
846 1
847 α-D-Glcp
848

849 Roy, Das, Mondal, Maiti, Bhunia, Maiti, et al. (2009) described another mixed α,β-D-

31
Page 31 of 71
850 glucan isolated from a hot water extract of the fruiting bodies of Pleurotus florida, cultivar

851 Assam Florida. NMR spectra had five H-1 signals at δ 5.11, 4.97, 4.50, 4.49 and 4.48 and

852 corresponding five C-1 signals at δ 99.4, 98.5, 103.3, 103.1 and 102.0 (1:1:1:1:1). All these

853 five residues were identified by correlation NMR experiments (Table 7). Smith degradation

t
854 led to formation of oligosaccharide containing (1→3)-α- and (1→3)-β-glycosidic bonds. Thus

ip
855 the native polysaccharide consisted of a (1→3)-α,β-(1→6)-β-D-glucan backbone with O-6

cr
856 linked α-D-Glcp side chains at the every fourth (1→3)-β-linked backbone unit (33).

857

us
858 α-D-Glcp (33)
859 1
860 ↓

an
861 6
862 [3)-α-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→6)-β-D-Glcp-(1→]n

863
M
864 Mandal, Maity, Maity, Gantait, Behera, Maiti, et al. (2012) described water-soluble

865 glucan isolated from a hot aqueous extract of the fruiting bodies of Calocybe indica. A 1H
d

866 NMR spectrum of this polysaccharide showed four anomeric proton signals at δ 5.37, 4.93,
te

867 4.51 and 4.50 in a ratio of nearly 2:1:1:2; corresponding carbon signals were found at δ 100.0,
p

868 98.3, 103.2, and 103.4 (Table 7). First two residues were assigned as α-D-Glcp, the second
ce

869 two as β-D-Glcp. The downfield shifted C-4 signal at δ 76.0 indicated presence of (1→4)-

870 linked α-D-Glcp; another α-anomeric unit is a terminal one. Similarly, the downfield shifts of
Ac

871 C-4 (δ 76.0) and C-6 (δ 69.2 and 69.3) signals indicated (1→4,6)- and (1→6)-linked β-D-Glcp

872 units. Two dimensional correlation NMR experiments confirmed following structure of this

873 polysaccharide (34).

874

875 α-D-Glcp (34)

876 1
877 ↓
878 6
879 {[4)-α-D-Glcp-(1→]2[6)-β-D-Glcp-(1→]2 4)-β-D-Glcp-(1→}n
880

32
Page 32 of 71
881

882 4.2.4. Mixed-linkage α,β-D-glucan backbone with β-linked side chains

883

884 Pramanik, Chakraborty, Mondal, & Islam (2007) obtained water-soluble mixed α,β-D-

t
885 glucan from the fruiting bodies of Pleurotus sajor-caju. Four H-1 signals of equile intensity

ip
886 were found at δ 5.08, 4.94, 4.47, and 4.46; corresponding C-1 signals arose at δ 98.8, 98.4,

cr
887 102.7 and 102.4 (Table 7). Free and linking C-6 carbon signals appeared at δ 60.6 and 68.6,

888 respectively. The downfield shift of C-2 to δ 77.0 indicated (1→2)- glycosidic bonds, unusual

us
889 for fungal glucans. According to correlation NMR analysis, the structure of the

890 tetrasaccharide repeating fragment of this glucan was determined as (35).

891
892
893
β-D-Glcp
1 an (35)
M
894 ↓
895 6
896 [6)-α-D-Glcp-(1→2)-α-D-Glcp-(1→3)-β-D-Glcp-(1→]n
897
d

898
te

899 Branched α,β-D-glucan (~70 kDa) described by Ghosh, Chandra, Ojha, & Islam (2008)
p

900 was isolated from a hot aqueous extract of the fruiting bodies of Volvariella diplasia. Four
ce

901 H-1 signals were found at δ 5.05, 4.91, 4.42, and 4.40 (1:1:1:1), while three corresponding

902 C-1 signals appeared at δ 99.0, 98.5 and 103.4 (1:1:2) (Table 7). The sequences of glycosyl
Ac

903 residues were confirmed by HMBC experiment. The structure of this polysaccharide was

904 described as (36).

905

906 β-D-Glcp (36)

907 1
908 ↓
909 6
910 [6)-β-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→]n
911

912

33
Page 33 of 71
913 5. Structure – activity relationship

914

915 Many of fungal glucans exert a biological activity, which is usually linked to their

916 structure and molecular weight, in particular. Among the most studied fungal glucans

t
917 showing notable physiological effects belong linear and branched β-D-glucans described

ip
918 above. These effects are their most important quality and the reason why so much attention

cr
919 has been devoted to them. Fungal β-D-glucans belong to a group of physiologically active

920 compounds, collectively termed biological response modifiers (BRMs) (Bohn & BeMiller,

us
921 1995; Novak & Vetvicka, 2008). Due to their BRM activity these glucans serve as remedies

922 or adjuvants in many pathological conditions, such as bacterial, viral or protozoal infections,

923
an
and they are also potent antitumour drugs (Vetvicka & Novak, 2011). Contradictory data exist
M
924 on the influence of molecular weight (Mw), degree of branching (DB), conformation and

925 intermolecular associations of β-glucans on antitumour activity and on the mechanism(s) of

d

926 their action as BRM (Wasser, 2002; Novak & Vetvicka, 2008; Chen, & Seviour, 2007).
te

927 Until recently, biologically efficient β-D-glucans were supposed to have similar structure –

928 the main chain of (1→3)-β-linked D-Glcp moieties to which some D-Glcp units are randomly
p

929 connected by (1→6)-β-linkages (Chen, & Seviour, 2007; Novak & Vetvicka, 2008). Range of
ce

930 DB about 0.2–0.3 is probably responsible for the highest antitumour activity, represented by

931 lentinan, schizophyllan or yeast β-D-glucan; β-D-glucans with high or very low DB are
Ac

932 substantially less active (Misaki, Kakuta, Sasaki, Tanaka, & Miyaji, 1981; Misaki,

933 Kawaguchi, Miyaji, Nagae, Hokkoku, Kakuta, & Sasaki, 1984). In native β-D-glucans, their

934 fibrils are composed from organized parts in which the main chain is coiled to triple helix

935 (Sletmoen & Stokke, 2008) and these regions are combined with single or double filaments

936 (Saitô, Tabeta, Yoshioka, et al., 1987; Ohno, Kurachi, & Yadomae, 1988; Okobira, Miyoshi,

937 Uezu, Sakurai, & Shinkai, 2008). However, the detailed structure of β-D-glucans from

34
Page 34 of 71
938 dissimilar sources differs and so does their biological activity. For example, antitumour

939 activity of schizophyllan is supposedly conditioned by triple helix presence and Mw higher

940 than 100 kDa (Kojima, Tabata, Itoh, & Yanaki, 1986). The triple helix, formed by three H-

941 bonds in C-2 position and stabilized by side chains, is probably present only in high-

t
942 molecular β-D-glucans: minimal Mw for stable triple helix of schizophyllan is 25–40 kDa, and

ip
943 below this value only single strands exist in aqueous solution. Moreover, the triple helix

cr
944 structure most likely should not be a solely effective form of β-D-glucan because alkali

945 treatment, commonly used in isolation procedures, destroys this structure (Young, & Jacobs,

us
946 1998). Certain recent opinions do not confirm established ideas of the necessity of high Mw

947 and/or branching of biologically active β-D-glucans. In a series of papers (Jamois, Ferrieres,

948
an
Guegan, Yvin, Plusquellec, & Vetvicka, 2005; Descroix, Vetvicka, Laurent, Jamois, Yvin, &
M
949 Ferrières, 2010; Saraswat-Ohri, Vashishta, Vetvicka, Descroix, Jamois, Yvin, Ferrieres, 2011;

950 Vetvicka, Saraswat-Ohri, Vashishta, Descroix, Jamois, Yvin, Ferrieres, 2011) significant
d

951 antitumour effects of non-branched and small-molecule oligosaccharides with (1→3)-β-

te

952 frame are described.

953 These results imply a conclusion that effects of molecular size and structure of β-D-
p

954 glucans on their biological, especially antitumour, activities will need further investigation.
ce

955 This conclusion can be undoubtedly applied to the other biologically active fungal glucans

956 and their derivatives as well.

Ac

957

958

959 6. Chemically modified fungal glucans

960

961 Some medicinal applications, especially immunomodulation ones, need fungal glucans

962 readily water-soluble, while many glucan preparations from fungal raw materials comprise

35
Page 35 of 71
963 mostly insoluble polysaccharides or their complexes. To improve the solubility of such

964 preparations specific chemical modifications can be used. Carboxymethylation, sulphation,

965 phosphorylation as well as some other modifications are common ways to prepare water-

966 soluble derivatives of fungal glucans. Introduction of carboxymethyl and/or sulphate groups

t
967 into β-D-glucan improved its water solubility significantly and enhanced the stiffness of the

ip
968 chains (Wang & Zhang, 2006). Moreover, such modifications of fungal glucans may induce

cr
969 or significantly enhance specific biological activities. The effectiveness of polysaccharide

970 modification is usually monitored by NMR spectroscopy and by determination of the

us
971 substitution degree (DS).

972 Carboxymethylation of glucans is usually made by the reaction of glucan suspension with

973
an
chloroacetic acid at alkaline conditions. Ukai, Yoshida, Honda, et al. (1992) described the
M
974 preparation and analysis of the carboxymethyl derivatives of scleroglucan and (1→3)-α-D-

975 glucan from Agrocybe cylindracea. Based on GC-MS analysis authors concluded that
d

976 distribution of the carboxymethyl substituents depends on the type of glucan and its
te

977 conformation. Wiater, Paduch, Pleszczyńska et al. (2011) prepared carboxymethylated

978 derivatives of (1→3)-α-D-glucans from various fungal sources (Lentinula edodes, Pleurotus
p

979 ostreatus, Piptoporus betulinus and Laetiporus sulphureus) and described their biological
ce

980 activities. Water-soluble carboxymethylated derivatives of insoluble (1→3)-β-D-glucan from

981 the sclerotium of P. cocos (Wang & Zhang, 2006), branched (1→3),(1→6)-β-D-glucan from S.
Ac

982 cerevisiae (Šolteš, Alföldi, & Šandula, 1993) and chitin–glucan from mycelium of A. niger

983 (Dergunova et al., 2009) were also prepared and described. Carboxymethylation causes

984 downfield shift of the substituted carbon signals by δ 8–9. The downshifted signals at δ 83.6

985 (C-2), 80.5 (C-4) and 71.5–72.3 (CH2 of carboxymethyl) found in the 13C NMR spectrum of

986 modified S. cerevisiae β-D-glucan confirmed partial carboxymethylation at O-2, O-4 and O-6

987 positions (Šolteš, Alföldi, & Šandula, 1993).

36
Page 36 of 71
988 Sulphation of fungal glucans is commonly achieved by the reaction with chlorosulphonic

989 acid – pyridine or sulphur trioxide – pyridine complexes in dimethyl sulphoxide medium

990 (Zhang, Chen, Xu, Lin, Cheung, & Kennedy, 2005; Zhang, Zhang, & Cheng, 2002). Reaction

991 takes place preferably at O-6, but is also possible at O-2, O-3 and O-4 (Zhang, Zhang, Wang,

t
992 & Cheung, 2003; Zhang, Zhang & Cheung, 2002). Water-soluble sulphated derivatives were

ip
993 obtained from various fungal glucans: (i) (1→3)-α-D-glucans originating from mycelia of P.

cr
994 cocos (Zhang, Chen, Xu, et al., 2005; Lin, Zhang, Chen, Jin, Zeng, Jin, et al. 2004; Huang,

995 Zhang, Cheung, & Tan, 2006) and from basidiocarps of Lentinula edodes (Zhang, Zhang, &

us
996 Cheng, 2002); (ii) (1→4),(1→6)-α-D-glucans from Gastrodia elata Bl. (Qiu et al. 2007);

997 (iii) various branched (1→3),(1→6)-β-D-glucans, i.e., those from Pleurotus tuber-regium

998
an
(Tao, Zhang, & Cheung, 2006; Zhang, Zhang, Wang et al., 2003; Zhang, Cheung, Ooi, &
M
999 Zhang, 2004), Russula virescens (Sun, He, Liang, Zhou, & Niu, 2009), Sacharomyces

1000 cerevisiae (Williams, Pretus, McNamee, Jones, Ensley, Browder, et al., 1991),
d

1001 botryosphaeran (Mendes, Dos Santos, Barbosa, Vasconcelos, Aranda-Selverio, Monteiro, et

te

1002 al., 2009), grifolan (Nie, Shi, B., Ding, & Tao (2006) and lentinan (Wang & Zhang, 2009).

1003 Depending on the polysaccharide structure and reaction conditions, the products showed DS
p

1004 in the range of 0.17–1.74. Sulphation causes strong downfield shift (δ 7–10) of the substituted
ce

1005 carbon signals (Table 8, bold). Zhang, Zhang & Cheung (2002) analysed 13C NMR spectra of

1006 native (1→3)-α-D-glucan L-FV-II originated from Lentinula edodes and its sulphated
Ac

1007 derivative SL-FV-II. The downfield shifted resonance signals at δ 87.5 (C-2), 79.8 (C-2),

1008 77.4 (C-4) and 66.5 (C-6) were assigned to sulphated carbons. Similarly, Zhang, Zhang,
13
1009 Wang et al. (2003) compared C NMR data of native (TM8) and sulphated (S-TM8)

1010 (1→3),(1→6)-β-D-glucans from Pleurotus tuber-regium and assigned the resonance signals at

1011 δ 80.3 (C-2), 79.4 (C-2), 77.4 (C-4) and 67.7 (C-6) to carbons sulphated at the corresponding

1012 positions.

37
Page 37 of 71
1013 Huang & Zhang (2011) described phosphorylation of water-insoluble (1→3)-α-D-glucan

1014 from mycelia of Poria cocos with H3PO4 in solution of LiCl and urea in dimethylsulfoxide. A
31
1015 P NMR spectrum of the phosphated glucan exhibited several intense signals in the region of

1016 δ 0.4–1.3 ppm confirming that phosphate groups were bound to the polysaccharide at

t
1017 different positions. Water-solubility and chain stiffness of the phosphated derivative increased

ip
1018 in comparison with the original α-D-glucan.

cr
1019 Several reports are devoted to comparing physical properties and biological activities of

1020 differently functionalized fungal glucans. Bao, Duan, Fang, & Fang (2001) reported

us
1021 aminopropylated, hydroxyethylated, sulphated, carboxymethylated, carboxymethylated-

1022 sulphated, and benzylamidated-carboxymethylated derivatives of linear (1→3)-α-D-glucan

1023
an
isolated from spores of Ganoderma lucidum (Fr.) Karst. Chen, Zhang, & Cheung (2010)
M
1024 prepared a carboxymethylated-sulphated derivative of (1→3)-β-D-glucan extracted from

1025 Poria cocos. The modified polysaccharide contained carboxymethyl and sulphate groups with
d

1026 DS of 1.05 and 0.36, respectively. Wang, Zhang, Li, Hou, & Zeng (2004) described
te

1027 preparation and analyses of five derivatives (sulphated, carboxymethylated, methylated,

1028 hydroxyethylated, and hydroxypropylated) of water-insoluble (1→3)-β-D-glucan isolated

p

1029 from fresh sclerotium of Poria cocos. Tao, Zhang, & Zhang (2009) reported sulphated and
ce

1030 carboxymethylated derivatives of two water-soluble polysaccharide-protein complexes

1031 extracted from sclerotia of Pleurotus tuber-regium. Using ultrasonic treatment and subsequent
Ac

1032 chemical derivatisation, water-soluble carboxymethyl and sulphoethyl derivatives of insoluble

1033 baker’s yeast (1→3),(1! 6)-β-D-glucan were obtained with high yield by Šandula, Kogan,

1034 Kačuráková et al. (1999). By contrast, carboxymethylation of chitin-glucan complex isolated

1035 from mycelium of Aspergillus niger was less successful (DS = 0.3).

1036

1037

38
Page 38 of 71
1038 7. Conclusions

1039

1040 Many reports reviewed here clearly illustrate large structural variability of fungal glucans

1041 depending on raw materials as well as ways of isolation, purification and possible chemical

t
1042 modification. Among fungal glucans and their derivatives, large diversity in configuration,

ip
1043 position and sequence of glycosidic bonds, molecular weight, branching and specific

cr
1044 substitution were reported. Some of these polysaccharides are simple linear polymers, the

1045 other are more or less branched ones containing mono- and/or oligosaccharide side chains.

us
1046 Different anomeric structures, i.e., α-, β- and combination of both these forms, were described

1047 for various fungal glucans. Glycoside bond positions, branching and molecular mass of these

1048
an
polysaccharides significantly varied as it was shown in many structural investigations. It is
M
1049 interesting to note that the common structural motifs, for example (1→3)- and (1→6)-linked

1050 β-D-Glcp, may have opposite location (in a backbone or side chains) for different fungal
d

1051 glucans. Evidently, structure parameters of fungal glucans are prerequisites of their
te

1052 physiological role in the fungal cell walls as well as of their physical, chemical and biological

1053 properties. Chemical modifications are often used to change these properties, first of all to
p

1054 obtain derivatives soluble in aqueous media. Structural diversity of fungal glucans permits to
ce

1055 search novel macromolecular agents with potential qualities for various industrial and

1056 medicinal applications.

Ac

1057

1058

1059 Acknowledgements

1060

1061 This work was supported by the grant of the Czech Science Foundation No. 525/09/1133 and

1062 by the project No. CEZ: MSM6046137305 of the Ministry of Education of the Czech

39
Page 39 of 71
1063 Republic.

1064

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1486 glucan sulfate derived from Saccharomyces cerevisiae. Immunopharm, 22, 139-156.

1487 Wood, P. J., Weisz, J., & Blackwell, B. A. (1994). Structural studies of (1→3)(1→4)-β-D-glucans by 13C-nuclear

us
1488 magnetic resonance spectroscopy and by rapid analysis of cellulose-like regions using highperformance

1489 anion-exchange chromatography of oligosaccharides released by lichenase. Cereal Chemistry, 71, 301–307.

an
1490 Woranovicz-Barreira, S. M., Gorin, P. A. J., Sassaki, G. L., Tischer, C. A., Ahti, T., Iacomini, M., (1999).

1491 Chemotyping glucans from lichens of the genus Cladonia. Phytochemistry, 52, 1069–1074.

1492 Wu, Y., Sun, C., & Pan, Y. (2005). Structural analysis of a neutral (1→3),(1→4)-β-D-glucan from the mycelia of
M
1493 Cordyceps sinensis. Journal of Natural Products, 68, 812-814.

1494 Yalin, W, Cuirong, S, & Yuanjiang, P. (2006). Studies on isolation and structural features of a polysaccharide
d

1495 from the mycelium of a Chinese edible fungus (Cordyceps sinensis). Carbohydrate Polymers, 63, 251-256.
te

1496 Yoshioka, Y., Tabeta, R., Saitô, H., Uehara, N., & Fukuoka, F. (1985). Antitumor polysaccharides from P.

1497 ostreatus (Fr.) Quél.: Isolation and structure of a β-glucan. Carbohydrate Research, 140, 93-100.
p

1498 Young, S. H., & Jacobs, R. R. (1998). Sodium hydroxide-induced conformational change in schizophyllan
ce

1499 detected by the fluorescence dye, aniline blue. Carbohydrate Research, 310, 91-99.

1500 Zhang, L., Chen, L., Xu, X., Lin, Y., Cheung, P. C. K., & Kennedy, J. F. (2005). Comparison on chain stiffness
Ac

1501 of a water-insoluble (1→3)-α-D-glucan isolated from Poria cocos mycelia and its sulfated derivative.

1502 Carbohydrate Polymers, 59, 257-263.

1503 Zhang, L., Zhang, M., Dong, J., Guo, J., Song, Y., & Cheung, P.C. (2001). Chemical structure and chain

1504 conformation of the water-insoluble glucan isolated from Pleurotus tuber-regium. Biopolymers, 59, 457-464.

1505 Zhang, M., Cheung, P. C. K., Ooi, V. E. C., & Zhang, L. (2004). Evaluation of sulfated fungal β-glucans from

1506 the sclerotium of Pleurotus tuber-regium as a potential water-soluble anti-viral agent. Carbohydrate

1507 Research, 339, 2297-2301.

1508 Zhang, M., Zhang, L., Wang, Y., & Cheung, P. C. K. (2003). Chain conformation of sulfated derivatives of β-

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1509 glucan from sclerotia of Pleurotus tuber-regium. Carbohydrate Research, 338, 2863-2870.

1510 Zhang, P., Zhang, L., & Cheng, S. (2000). Effects of urea and sodium hydroxide on the molecular weight and

1511 conformation of α-(1→3)-D-glucan from Lentinus edodes in aqueous solution. Carbohydrate Research, 327,

1512 431-438.

1513 Zhang, P., Zhang, L., & Cheng, S. (2002). Solution properties of an α-(1→3)-D-glucan from Lentinus edodes and

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1514 its sulfated derivatives. Carbohydrate Research, 337, 155-160.

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1515 Zhang, Y., Li, S., Wang, X., Zhang, L., & Cheung, P. C. K. (2011). Advances in lentinan: isolation, structure,

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1516 chain conformation and bioactivities. Food Hydrocolloids, 25, 196-206.

1517

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1517 Highlights

1518 !! Fungal glucans represent a number of structurally variable polysaccharides.

1519 !! There are various linear or branched α-, β- and mixed α,β-D-glucans.

t
1520 !! Structure of fungal glucans depends on the source and the way of isolation

ip
1521 !! Molecular masses and glycoside bond positions are also highly variable.

cr
1522 !! Chemical modifications of fungal glucans improve their properties for medicinal use.

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1523

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Table 1. Chemical shifts of the 13C resonance signals for linear fungal α-D-glucans

us
Source (name) Sugar residue C-1 C-2 C-3 C-4 C-5 C-6 Reference

Aspergillus niger →4)-α-D-Glcp-(1→ 100.0 72.7 73.9 79.8 71.4 60.9 Bock, Gagnaire, Vignon, et al., 1983

an
(nigeran) →3)-α-D-Glcp-(1→ 101.0 71.4 83.2 70.3 73.5 61.3

(pseudonigeran) →3)-α-D-Glcp-(1→ 100.9 71.3 83.8 70.8 73.1 61.2 Carbonero, Sassaki, Gorin, et al., 2002

M
Agaricus blazei →4)-α-D-Glcp-(1→ 99.8 71.5 75.3 78.7 71.2 60.1 Gonzaga, Ricardo, Heatley, et al., 2005

Armillariella tabescens (HCP) →6)-α-D-Glcp-(1→ 97.6 73.3 73.4 69.5 70.1 65.5 Han et al., 2011

ed
Sarcodon aspratus (IPS-B2) →6)-α-D-Glcp-(1→ 100.2 72.0 72.7 73.9 75.9 68.1 Luo, Xu, Yu, et al., 2008

Termitomyces eurhizus →3)-α-D-Glcp-(1→ 97.0 70.3 80.1 69.8 73.4 61.8 Mondal, Chakraborty, Pramanik, et al.,
pt
(PS-I) →6)-α-D-Glcp-(1→ 99.2 72.9 74.9 71.0 71.7 67.1 2004

(PS-II) →6)-α-D-Glcp-(1→ 98.8 71.3 74.6 70.8 72.5 66.7

ce

Aureobasidium pullulans →6)-α-D-Glcp-(1→ 101.1 72.1 74.0 70.6 71.3 67.6 McIntyre & Vogel, 1993

(pullulan) G6-G4-G4 →4)-α-D-Glcp-(1→ 98.8 72.1 74.2 72.1 61.8

Ac

78.7

G4-G4-G6 →4)-α-D-Glcp-(1→ 100.7 72.1 74.2 78.2 72.1 61.5

G4-G4-G4 →4)-α-D-Glcp-(1→ 100.7 72.1 74.2 77.7 72.1 61.5

Teloschistes flavicans →6)-α-D-Glcp-(1→ 98.1 70.6 72.1 71.3 73.3 66.8 Reis, Tischer, Gorin, et al., 2002

Page 57 of 71
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(pullulan) G6-G4-G6 →4)-α-D-Glcp-(1→ 100.5 71.5 71.4 79.5 73.6 60.7

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Table 2. Chemical shifts of the 13C resonance signals for branched fungal α-D-glucans

us
Source (name) Sugar residue C-1 C-2 C-3 C-4 C-5 C-6 Reference

Agaricus bisporus →4)-α-D-Glcp-(1→ 100.3 78.9 60.3 Smiderle, Sassaki, Van

an
→4,6)-α-D-Glcp-(1→ 100.1 66.2 Arkel, et al., 2010

α-D-Glcp-(1→ 99.7 71.8 72.8 70.0 71.3 60.8

M
Flammulina velutipes →4)-α-D-Glcp-(1→ 102.8 74.0 76.1 79.8 73.2 63.4 Pang, Yao, Yang, et al.,

(FVP2) →4,6)-α-D-Glcp-(1→ 102.4 74.2 76.1 79.8 73.2 72.2 2007

ed
α-D-Glcp-(1→ 101.6 75.7 76.1 74.4 75.6 63.4

Cordyceps sinensis →4)-α-D-Glcp-(1→ 99.9 71.8 73.3 78.6 70.8 61.0 Yalin, Cuirong, &
pt
→4,6)-α-D-Glcp-(1→ 78.6 71.3 Yuanjiang, 2006

α-D-Glcp-(1→ 98.7 72.1 74.0 69.7 72.7

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Table 3. Chemical shifts of the 13C resonance signals for linear fungal β-D-glucans

us
Source Sugar residue C-1 C-2 C-3 C-4 C-5 C-6 Reference

Termitomyces eurhizus →3)-β-D-Glcp-(1→ 103.3 73.9 86.3 68.7 76.2 61.1 Chakraborty, Mondal, Rout, et al., 2006

an
Laetiporus sulphureus →3)-β-D-Glcp-(1→ 102.7 72.7 86.0 68.3 76.3 60.9 Alquini, Carbonero, Rosado, et al., 2004

Saccharomyces cerevisiae →3)-β-D-Glcp-(1→ 102.9 72.9 86.0 68.4 76.3 60.9 Medeiros, Cordeiro, Cavalcanti, et al. 2012

M
→3)-β-D-Glcp-(1→ 102.5 72.5 85.8 68.2 76.0 60.7 Freimund, Sauter, Käppeli, et al., 2003

Umbilicaria mammulata →3)-β-D-Glcp-(1→ 102.8 72.7 86.0 68.3 76.2 60.7 Carbonero, Smiderle, Gracher, et al., 2006

ed
(two linear β-D-glucans) →6)-β-D-Glcp-(1→ 103.1 73.3 76.4 69.9 75.4 68.4

Agaricus bitorquis →6)-β-D-Glcp-(1→ 103.4 73.5 76.0 69.9 75.3 69.2 Nandan et al., 2008
pt
Agaricus blazei →6)-β-D-Glcp-(1→ 103.0 73.1 75.6 69.5 74.9 68.8 Gonzaga, Ricardo, Heatley, et al., 2005

Guignardia citricarpa →6)-β-D-Glcp-(1→ 103.3 73.3 76.6 69.9 75.4 67.2 Sassaki, Ferreira, Glienke-Blanco, et al., 2002
ce

Bulgaria inquinans →6)-β-D-Glcp-(1→ 104.7 74.7 76.6 71.1 77.3 70.5 Bi, Ni, Liu, et al., 2009

→3)-β-D-Glcp-(1→ 103.5 68.4 76.3 60.8 Carbonero, Montai, Mellinger, et al., 2005
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Parmotrema austrosinense 87.0

(lichenan) →4)-β-D-Glcp-(1→ 102.6 80.1 60.4

Page 60 of 71
Table 4. Degree of branching (DB) of fungal ! -D-glucans

Name Source DB (mol mol-1)

pachymaran (pachyman) Poria cocos 0.015 – 0.02

yeast glucan Saccharomyces cerevisiae 0.03 – 0.2

t
yeast glucan Candida albicans 0.14

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lentinan Lentinula edodes 0.23 – 0.42

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pleuran Pleurotus ostreatus 0.25

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grifolan Grifola frondosa 0.31 – 0.36

scleroglucan Sclerotium glucanicum 0.30 – 0.33

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schizophyllan Schizophyllum commune 0.33

GLG Ganoderma lucidum 0.35

M
SSG Sclerotinia sclerotiorum 0.50

pestalotan Pestalotia sp. 815 0.60

d

epiglucan Epicoccum nigrum 0.67

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Table 5. Chemical shifts of the 13C resonance signals for branched fungal β-D-glucans

us
Source (name) Sugar residue C-1 C-2 C-3 C-4 C-5 C-6 Reference

Grifola frondosa →3)-β-D-Glcp-(1→ 103.0 72.8 86.6 68.6 76.2 60.9 Tada, Adachi,

an
(Grifolan LE) →3)-β-D-Glcp-(1→ 103.0 73.1 86.1 68.6 76.4 61.0 Ishibashi, et al.,

→3,6)-β-D-Glcp-(1→ 103.0 72.9 85.8 68.7 75.0 68.6 2009

M
β-D-Glcp-(1→ 103.1 73.7 76.3 70.2 76.6 61.2

Ganoderma lucidum →3)-β-D-Glcp-(1→ 103.1 73.2 86.1, 86.7 68.7 76.5 61.2 Chang & Lu,

ed
(GLG) →3,6)-β-D-Glcp-(1→ 103.0 73.0 85.8 68.7 75.0 68.7 2004

β-D-Glcp-(1→ 103.1 73.8 76.5 70.3 76.3 61.0

pt
Lentinula squarrosulus →3)-β-D-Glcp-(1→ 102.9 73.5 84.8, 85.0 69.9 75.9 61.1 Bhunia et al.,

→6)-β-D-Glcp-(1→ 103.2 73.5 75.9 70.0 75.3 69.3 2011

ce

→3,6)-β-D-Glcp-(1→ 103.4 73.3 84.4 69.9 75.1 69.0

β-D-Glcp-(1→ 102.7 74.4 76.2 70.0 75.9 61.1

Ac

Botryosphaeria sp. →3)-β-D-Glcp-(1→ 103.1 73.0 86.3 68.7 76.7 61.1, 60.9, 60.7 Barbosa, Steluti,

(botryosphaeran) →3)-β-D-Glcp-(1→* 103.1 73.0 86.3 68.7 76.7 61.1 Dekker, et al.,

→6)-β-D-Glcp-(1→ 102.9 73.0 74.8 68.7 76.3 70.1 2003

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→3,6)-β-D-Glcp-(1→ 102.9 73.0 86.0, 85.5 68.7 76.3 70.1 2003

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β-D-Glcp-(1→ 103.3 73.8 75.5 68.8 76.7 61.1

Calocybe indica →3)-β-D-Glcp-(1→ 103.9 73.8, 73.6 87.3, 87.7 69.5 77.4 61.9 Mandal et al.,

an
(calocyban) →3,4)-β-D-Glcp-(1→ 103.9 73.6 87.0 79.9 75.7 61.9 2010

β-D-Glcp-(1→ 103.9 74.7 77.6 71.2 77.1 62.1

M
Pfle1r (Pleurotus →6)-β-D-Glcp-(1→ 103.0 73.0 74.9 69.6 75.5 68.8; 69.0 Maji, Sen,

florida × Lentinula →3,6)-β-D-Glcp-(1→ 102.7 72.8 84.2 69.5 75.5 68.7 Behera, et al.,

ed
edodes) (PS-I) β-D-Glcp-(1→ 102.9 73.0 75.9 69.6 75.9 60.7 2012

Agaricus brasiliensis →6)-β-D-Glcp-(1→ 105.1 75.2 77.8 71.8 77.1 71.0 Dong, Yao,
pt
(Ab2-2N) →3)-β-D-Glcp-(1→ 105.1 75.2 86.5 71.8 78.1 62.9 Yang, et al., 2002

→3,6)-β-D-Glcp-(1→ 104.7 75.2 86.5 71.8 77.1 70.4

ce

β-D-Glcp-(1→ 104.4 75.2 77.8 71.8 78.1 62.9

Phellinus ribis (PRP) →4)-β-D-Glcp-(1→ 105.6 75.8 78.3 74.7 65.2 Liu & Wang,
Ac

78.6

→6)-β-D-Glcp-(1→ 105.3 76.0 77.6 72.3 72 7 71.4 2007

→3,6)-β-D-Glcp-(1→ 87.2

β-D-Glcp-(1→ 107.0 74.1 78.2 70.9 74.7 63.4

Page 63 of 71
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Ganoderma →3)-β-D-Glcp-(1→ 102.8 84.8 Amaral et al.,

→3)-β-D-Glcp-(1→*

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resinaceum 102.9 72.8 86.2 68.5 76.4 60.9 2008

→4)-β-D-Glcp-(1→ 102.4 79.1

an
→3,6)-β-D-Glcp-(1→ 102.6 69.0

β-D-Glcp-(1→ 103.0

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*
product of Smith degradation

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Table 6. Chemical shifts of the 13C resonance signals for linear fungal α,β-D-glucans

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Source (name) Sugar residue C-1 C-2 C-3 C-4 C-5 C-6 Reference

Astraeus hygrometricus →4)-α-D-Glcp-(1→ 98.4 71.8 70.6 75.3 69.3 61.2 Chakraborty, Mondal,

an
(AQS-I) →6)-β-D-Glcp-(1→ 103.4 73.5 76.0 69.9 73.8 66.0 Pramanik, et al., 2004

Termitomyces →4)-α-D-Glcp-(1→ 98.0 71.4 71.7 75.7 69.6 60.6 Chandra et al., 2007

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microcarpus (PS-I) →3)-β-D-Glcp-(1→ 102.4 72.3 85.1 68.9 75.9 60.7

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Table 7. Chemical shifts of the 13C resonance signals for branched fungal α,β-D-glucans

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Source (name) Sugar residue C-1 C-2 C-3 C-4 C-5 C-6 Reference

an
→3)-α-D-Glcp-(1→ 101.5 72.6 85.3 70.1 73.8 61.2

Piptoporus betulinus →3)-α-D-Glcp-(1→* 100.1 71.4 84.9 69.8 73.4 60.9

Olennikov et al., 2012

M
(Piptoporan I) →3,6)-α-D-Glcp-(1→ 101.8 72.6 86.3 70.1 73.5 68.1

β-D-Glcp-(1→ 106.3 73.1 76.1 71.3 75.8 61.8

ed
→3)-α-D-Glcp-(1→ 99.4 69.9 60.7

→3)-α-D-Glcp-(1→* 99.6 70.9 83.0 69.7 72.1 60.7

Pleurotus florida
pt
→3,6)-α-D-Glcp-(1→ 99.5 69.0 Santos-Neves et al., 2008
(MRFS–HW)
→3)-β-D-Glcp-(1→ 102.9 73.2
ce

β-D-Glcp-(1→ 102.8 73.3

→6)-β-D-Glcp-(1→ 103.4 73.3 76.0 69.9 75.3

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69.3

Calocybe indica (PS-I) →4,6)-β-D-Glcp-(1→ 103.2 73.4 75.3 76.4 73.7 69.2 Mandal et al., 2010

α-D-Glcp-(1→ 100.0 71.6 73.4 70.0 71.9 61.1

Pleurotus florida →3)-α-D-Glcp-(1→ 100.1 70.0 80.4 69.3 72.5 61.1

10

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α-D-Glcp-(1→ 99.8 72.1 74.0 71.0 72.5 60.6 Rout, Mondal,

→3)-α-D-Glcp-(1→*

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100.2 71.3 83.2 69.8 72.3 60.5 Chakraborty, et al., 2005

→3,6)-β-D-Glcp-(1→ 103.1 73.0 85.2 68.8 76.4 67.0

an
→3)-β-D-Glcp-(1→* 103.4 73.2 86.6 68.8 76.7 61.2

somatic hybrid →4)-α-D-Glcp-(1→ 98.3 71.9 73.5 76.4 70.0 61.6

M
PCH9FB of Pleurotus α-D-Glcp-(1→ 100.2 71.9 73.5 69.9 71.5 61.6
Maity et al., 2011a
florida and Calocybe →6)-β-D-Glcp-(1→ 103.4 73.4 75.9 69.9 70.4 69.2

ed
indica var. APK2 →4,6)-β-D-Glcp-(1→ 103.3 73.4 75.3 76.4 69.9 69.2

→3)-α-D-Glcp-(1→ 99.4 69.8 80.1 67.2 71.6 61.1

pt
α-D-Glcp-(1→ 98.5 71.6 73.4 70.0 71.8 61.4
Pleurotus florida,
→3)-β-D-Glcp-(1→ 103.1 73.4 85.2 69.8 76.6 61.1 Roy et al., 2009
ce

cultivar Assam Florida

→6)-β-D-Glcp-(1→ 102.0 73.5 76.4 70.0 75.2 69.0

→3,6)-β-D-Glcp-(1→ 103.3 73.2 69.8 75.9

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85.2 68.8

Calocybe indica →4)-α-D-Glcp-(1→ 100.0 71.9 73.2 76.0 72.2 60.8 Mandal, Maity, Maity, et

α-D-Glcp-(1→ 98.3 71.7 73.5 69.9 72.9 61.1 al., 2012

→6)-β-D-Glcp-(1→ 103.4 74.4 76.0 69.9 75.3 69.3

11

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→4,6)-β-D-Glcp-(1→ 103.2 74.5 75.3 76.0 75.0 69.2

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→6)-α-D-Glcp-(1→ 98.4 69.4 66.7 70.3 69.7 68.6

→2,6)-α-D-Glcp-(1→ 98.8 77.0 73.3 70.3 69.7 68.6 Pramanik, Chakraborty,

Pleurotus sajor-caju

an
→3)-β-D-Glcp-(1→ 102.7 73.0 83.8 76.1 73.3 60.6 Mondal, & Islam , 2007

β-D-Glcp-(1→ 102.4 73.3 74.8 76.1 76.1 60.6

M
→4)-α-D-Glcp-(1→ 99.0 72.4 73.1 76.6 70.8 61.4

→4,6)-α-D-Glcp-(1→ 98.5 72.4 73.1 77.4 69.9 68.7 Ghosh, Chandra, Ojha, &
Volvariella diplasia

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→6)-β-D-Glcp-(1→ 103.4 73.8 76.0 70.8 75.1 69.2 Islam, 2008

β-D-Glcp-(1→ 103.4 73.4 76.6 70.8 76.0 61.2

pt
*
product of Smith degradation
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Table 8. Chemical shifts of the 13C resonance signals for native and sulphated fungal glucans

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Source (name) Structure C-1 C-2 C-3 C-4 C-5 C-6 Reference

Lentinus edodes (L-FV-II) 101.7, 100.6 71.1 83.2 70.6 72.8 61.1
Zhang &

an
(1→3)-α-D-glucan 100.7, 103.8 71.9 84.0 70.4 70.3 61.3
(SL-FV-II)* Cheung, 2002
79.8, 87.5 77.4 66.5

M
Pleurotus tuber-regium 103.0 72.5, 73.7 86.0–87.0 68.5 76.1 60.9 Zhang,

(TM8) 76.8 Zhang,

(1→3),(1→6)-β-D-glucan

ed
103.1 72.4, 73.7 85.7– 86.9 68.4 76.2 Wang, &
(S-TM8)*
101.5 79.4, 80.3 76.8 77.4 67.7 Cheung, 2003
pt
*
sulphated glucans
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Figure captions

Figure 1. Structure of fungal glucans: (a) (1→3)-α-D-glucan; (b) (1→4)-α-D-glucan; (c)

(1→6)-α-D-glucan; (d) mixed-linkage (1→3),(1→4)-α-D-glucan; (e) branched (1→4),(1→6)-

t
α-D-glucan; (f) (1→3)-β-D-glucan; (g) (1→6)-β-D-glucan; (h) mixed-linkage (1→3),(1→4)-

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β-D-glucan; (i) branched (1→3),(1→6)-β-D-glucan; (j) branched (1→6),(1→3)-β-D-glucan.

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Figure(s)

i
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