Available online at www.sciencedirect.

com

Enzyme and Microbial Technology 42 (2008) 242–251

Characterization of chitosan and chitosan–glucan complex extracted

from the cell wall of fungus Gongronella butleri
USDB 0201 by enzymatic method
Nitar Nwe a,b,c , Willem F. Stevens c,d , Seiichi Tokura a , Hiroshi Tamura a,∗
aFaculty of Chemistry, Materials and Bioengineering and HRC, Kansai University, Suita, Osaka 564-8680, Japan
b Japan Society for Promotion of Science (JSPS), Japan
c Food Engineering and Bioprocess Technology, Asian Institute of Technology, P.O Box 4, Klong Luang, Pathumthani 12120, Bangkok, Thailand
d Centre of Excellence for Shrimp Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand

Received 21 October 2006; received in revised form 8 August 2007; accepted 1 October 2007

Abstract
The fungus Gongronella butleri USDB 0201 was grown on sweet potato pieces supplemented with mineral and urea solution. Mycelia were
harvested after 7 days of cultivation. Chitosan in the fungal cell wall exists in two forms, free chitosan and chitosan bounded to ␤-glucan.
The linkage between chitosan and ␤-glucan in the chitosan–glucan complex (CGC) was successfully cleaved using a heat stable ␣-amylase.
The resultant chitosan and ␤-glucan polymeric moieties were characterized. Data from elementary analysis, IR and 13 C NMR spectroscopy
confirmed that all intramolecular glycosidic linkages in either chitosan or glucan polymeric moieties are in the ␤-configuration. Data from specific
enzymatic degradation of the CGC obtained from different treatments and reducing sugar analysis indicate that chitosan and glucan are linked
by an intermolecular ␣-1,4 glycosidic bond. These evidences supported to prove the ␤-linked chitosan and ␤-linked glucan are linked with ␣-1,4
glycosidic bond. However heat stable ␣-amylase enzyme could not cleave the CGC completely. Therefore high yield with pure chitosan could be
obtained from the cell wall of fungus G. butleri USDB 0201 by Termamyl enzyme treatment.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Chitin/chitosan; Chitosan–glucan complex (CGC); Fungal cell wall; Glucan; Glycosidic bond

1. Introduction [9,11]. In Zygomycetes, chitosan is synthesized by deacetyla-

Nowadays, chitin, chitosan and glucan polymers have
increasing interest on many pharmaceutical applications espe-
cially in tissue engineering, medicine and immunology [1–4].
Chitin/chitosan and glucan are the main fungal skeletal polysac-
charides [5–8]. In the fungal cell wall, chitin/chitosan occurs
in two forms, as free aminoglucoside and covalently bound to
␤-glucan.
Chitin biosynthesis starts with the conversion of glu-
cose into N-acetylglucosamine-1-phosphate, which reacts with
UTP to form UDP-N-acetylglucosamine in a reaction cat-
alyzed by glucosamine-6-phosphate synthase [9,10]. The
N-acetylglucosamine moiety of this sugar-dimer is transferred to
the growing chitin polymer chain by the enzyme chitin synthase
∗ Corresponding author. Tel.: +81 6 6368 0871; fax: +81 6 6330 3770.
E-mail address: tamura@ipcku.kansai-u.ac.jp (H. Tamura).
tion of the growing chitin chain through the action of the enzyme
chitin deacetylase [12,13]. The individual chains of chitin bind
by hydrogen bonds to form microfibrils.
Fungi synthesize glucan as extracellular branched ␤-(1-3; 1-
6)-d-glucan and intracellular ␣- and ␤-glucans [14–17]. The
structure of ␣- and ␤-polysaccharides from many fungi has been
characterized [14,15,18]. The (1-3)-␤-glucan and C-6 substi-
tuted (1-3)-␤-glucan are known to form triple helices [19,20].
These glucan chains are bound by hydrogen bonding to each
other.
In 1990, Wessels et al. proposed that initially chitin and ␤-
glucan chains accumulate individually in the fungal cell wall
and thereafter form the interpolymer linkage [17]. The forma-
tion of the chitin/chitosan–glucan complex chains results in a
rigid cross-linked network in the cell wall [7,8,13] and causes
a considerable problem for the extraction of intact chitosan and
glucan. It does not break down easily under mild extraction con-
dition [21,22]. However the free chitosan in the cell wall can

0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2007.10.001
 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251
be easily extracted after treatment with 1 M NaOH [21,23,24].
Therefore most of the researchers are trying to find the link-
age between the chitosan and glucan in the fungal cell wall by
digestion with different enzymes [17,25–31].
Sietsma and Wessels reported that 90% of ␤-glucan could be
collected from the glucan/chitin complex by digestion with (1-
3)-␤-glucanase. N-Acetyl-glucosamine, lysine and/or citrulline
were identified as products after enzymatic hydrolysis of the
residual cell wall material by chitinase [25]. Therefore they
proposed that the bridge linking the glucan chain with the
chitin contains lysine, citrulline, glucose and (N-acetyl) glu-
cosamine. Similar evidence was obtained by Gopal et al. for
the degradation of chitin-glucan complex by (1,3)-␤- and (1,6)-
␤-glucanase, and subsequently by chitinase [26]. Carbohydrate
expressed as glucose and N-acetylglucosamine monomers was
detectable in equivalent amounts in the hydrolysate. The residue
after chitinase was further treated with ␣-amylase but addi-
tional release of glucose could not be detected colorimetrically.
Surarit et al. suggested a glycosidic linkage between position
6 of N-acetylglucosamine in chitin and position 1 of glucose
in ␤-(1-6)-glucan in the cell wall of Candida albicans [27]. In
1990, Wessels et al. proposed that a direct link between free
amino groups in the glucosaminoglycan and the reducing end
of the glucan chains forming the inter-polymer linkages in the
chitin-glucan complex [17]. Up to 1992, no clear evidence of the
identity of the chemical link between the chitosan and glucan
polymer chains had been uncovered [28].
After 1995, Kollar et al. and Fontaine et al. digested the cell
wall of Saccharomyces cerevisae and Aspergillus fumigatus with
1 M NaOH or 1% SDS and ␤-1,3-endoglucanase and chitinase
and analyzed the soluble fractions [29,30]. They concluded that
the terminal reducing residue of a chitin chain is attached to the
non-reducing end of a ␤-1,3-glucan chain by a ␤-1,4 linkage.
An insoluble residue was remained at the end of both extraction
processes. In 2002, we could completely digest the cell wall of
Gongronella butleri and cleave the covalent link between chi-
tosan and glucan with a heat-stable alpha-amylase. We proposed
that chitosan and glucan chains are bound by an ␣-(1,4) glyco-
sidic bond in the cell wall of fungus G. butleri USDB 0201 [31].
However, there are lacks of the characteristic data of resultant
products, the bond between the chitosan and glucan in the cell
wall of fungus G. butleri USDB 0201 could not be proved.
Therefore, fungus G. butleri USDB 0201 was grown on
the solid substrate and chitosan and insoluble and soluble glu-
can were extracted from the fungal cell wall by chemical and
enzymatic extraction methods. The resultant products were
characterized by IR spectroscopy, 13 C NMR spectroscopy and
elementary analysis. Based on these data, purity of resultant chi-
tosan and glucan and the linkage between chitosan and glucan
in the cell wall of fungus G. butleri USDB 0201 were discussed.
2. Materials and methods
2.1. Microorgansism and growth conditions
G. butleri USDB 0201 (class Zygomycetes) was obtained from the Depart-
ment of Biological Sciences, National University of Singapore. The fungus was
243
grown on peeled sweet potato pieces supplied with mineral solution and urea
[32,33]. After 7 days, mycelial mass was harvested, washed with water and dried
at 45 ◦ C.
2.2. Extraction of free chitosan
Free non-glucan-bound chitosan was isolated by treatment of mycelia at
45 ◦ C for 13 h using 1 M NaOH, followed by extraction in 0.35 M acetic acid
at 95 ◦ C for 5 h. The dissolved chitosan was precipitated by neutralization,
collected and dried [21].
2.3. β-Glucanase treatment
Dried mycelia were treated with 11 M NaOH, followed by suspension in
0.35 M acetic acid at 95 ◦ C for 5 h. Chitin as far as present will be converted
by deacetylation into chitosan. The suspension was centrifuged at 16,000 × g
and the supernatant was subjected to vacuum filtration through GF/C filter paper.
The chitosan solution was incubated with ␤-glucanase (Cereflo (Novo Nordisk),
density 1.2 g/ml, activity 200 ␤-glucanase Units per gram of enzyme) at pH 7.5,
30 ◦ C for 5 h with the aim to cleave the bond between the chitosan and the glucan.
Chitosan was recovered as a precipitate from the chitosan solution by adjusting
the pH to 9. The chitosan precipitate was centrifuged, washed and dried [34].
2.4. Extraction of chitosan and glucan from fungal cell wall using
11 M NaOH and Termamyl
In this method the chitin present in the mycelial wall is converted into chi-
tosan. The free (non-glucan-bound) chitosan and chitosan liberated from the
CGC are extracted simultaneously.
Dried mycelia (1 g) were treated with 10 ml of 11 M NaOH per g mycelia
at 45 ◦ C for 13 h. The alkali insoluble material (AIM) was suspended in 0.35 M
acetic acid at 95 ◦ C for 5 h and treated with Termamyl, Type LS (Novo Nordisk,
density 1.2 g/ml, activity 120 Kilo Novo alpha-amylase Units per gram of
enzyme) in a shaking water-bath at optimal conditions: 4% (v/v) enzyme, 65 ◦ C,
200 rpm for 3 h [31]. The enzyme treated suspension was centrifuged at 1600 × g
for 15 min to achieve separation between the dissolved chitosan solution and
the insoluble glucan fraction. Chitosan was precipitated from the solution by
adjusting the pH to 8–9 with 1 M NaOH. The chitosan precipitate was collected
by centrifugation at 1600 × g for 15 min and treated again with 1 M NaOH to
remove the enzyme from the chitosan. After that chitosan precipitate was washed
and freeze-dried.
Glucan precipitated after Termamyl treatment contains ␤-glucan that is insol-
uble in alkali and ␤-glucan that can dissolve in alkali but not as long as it is bound
to alkali insoluble chitosan. Both glucan fractions are not soluble in acetic acid.
The alkaline soluble glucan therefore did not dissolve in 11 M alkali prior to
Termamyl treatment, neither in acetic acid after Termamyl treatment. It could
be extracted from the precipitate after Termamyl treatment by 1 M NaOH. The
glucan insoluble in 1 M NaOH was sedimentated by centrifugation, whereas the
alkaline-soluble glucan was recovered by adjusting the pH of alkaline extract
solution to 4.5 with 0.35 M acetic acid. Alkaline insoluble and alkaline soluble
glucan were washed with distilled water up to neutral pH and freeze-dried.
3. Analytical methods
The amount of reducing ends was measured in the chitosan
solution after centrifugation by the Dygert method. Anhydrous
glucose was used as a standard [35]. The nitrogen content of the
chitosan sample was measured by the Micro-Kjeldahl method.
The degree of deacetylation of chitosan was determined by first-
derivative UV spectrophotometry with the modification of Tan
et al. [36,37]. Relative molecular weight was determined by
gel permeation chromatography (GPC) using a Waters HPLC
equipped with Ultrahydrogel 2000, 1000 and 500 columns and a
WatersTM 410 Differential Refractometer Detector. The solvent
244 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251
used was 0.2 M acetic acid/0.1 M sodium acetate buffer at a
flow rate of 0.6 ml/min. Dextrans of various molecular weights
ranging from 9.9 × 103 to 2 × 106 Da were used as standards.
It should be noted that the GPC analysis of chitosan products
only detects material with a size smaller than 0.45 ␮m (cut-off
of cellulose acetate membrane filter used in the procedure of
GPC analysis). Therefore the molecular weight of these products
might not represent the total polymer population in the sample.
IR-spectra were recorded using an FTIR instrument (model
2000, Perkin Elmer, Norwalk, CT) scanning between 400 and
4000 cm−1 . Solid-state 13 C NMR spectra were recorded with a
Bruker CXP-300 spectrometer.
4. Results
The fungus, G. butleri USDB 0201 was grown on sweet
potato pieces in a solid substrate fermentor at 26 ◦ C. The fungal
biomass was collected at the end of the cultivation period and
dried at 45 ◦ C.
4.1. Isolation of chitosan and glucan from the cell wall of
fungus G. butleri USDB 0201
The fungal biomass was treated with 11 M NaOH and resul-
tant chitosan–glucan complex was suspended in 0.35 M acetic
acid at 95 ◦ C for 5 h. This suspension was filtered through GF/C
filter paper. The insoluble part retained on the filter was mostly
glucan. The filtrate was treated with ␤-glucanase. The reac-
tion mixture was centrifuged and collected the supernatant.
The supernatant was adjusted to alkaline pH and the precipi-
tated product (8.2 g per 100 g mycelia) was labeled product “A”
(Table 1). The product A was dissolved again in dilute acetic
acid and digested with Termamyl, Type LS enzyme, at optimal
conditions. The reducing end in the chitosan solutions was mea-
sured before (A) and after (B) digestion with Termamyl enzyme
(Table 2). Some glucan precipitate could be observed at the end
of the Termamyl treatment. The clear chitosan solution was col-
lected by centrifugation and adjusted to alkaline pH to recover
chitosan product. The amount of resulting product was reduced
to about 40% (product “B”). The amount of reducing ends in the
chitosan B was increased about 40 times compared with product
A (Table 2). Therefore product A was not the result of complete
cleavage of CGC by ␤-glucanase. Product A was still containing
Table 1
The relative number average molecular weight and amount of chitosan obtained
by different extraction procedures from alkaline insoluble material after 11 M
NaOH treatment
Product Amount of product (g/100 g Number average molecular
of mycelia) weight (kDa)
A 8.2 ± 0.4 27 ± 0.1
B 3.2 ± 1.3 32 ± 0.3
C 5.91 ± 0.11 42 ± 0.1
Product A: filtration with GF/C filter paper followed by ␤-(1-3)-glucanase
enzyme.
Product B: product A treated with Termamyl, Type LS enzyme.
Product C: chitosan–glucan complex treated with Termamyl, Type LS enzyme.
Table 2
The measurement of reducing ends in the chitosan solutions, 5 mg/ml (A) and
(B)
Condition Reducing ends concentration (mg/ml)
Before Termamyl treatment (A) 0.043 ± 0.001
After Termamyl treatment (B) 1.85 ± 0.085
CGC with a diameter that could pass the filter paper (diameter
less than 1.2 ␮m).
Chitosan C, 5.9 g per 100 g mycelia was obtained from
the chitosan–glucan suspension treated with Termamyl, Type
LS enzyme. In this case filtration was not applied. This pro-
cess might extract the total chitosan in the fungal cell wall.
The ␤-glucanase treatment has a disadvantage compared with
the Termamyl treatment. The ␤-glucanase can only remove ␤-
glucose residues from the CGC. Moreover particles with a size
of larger than 1.2 ␮m are lost in the filtration step. These parti-
cles contain chitosan, as shown in the treatment with Termamyl
directly applied after acidification of the alkaline insoluble mate-
rial.
The glucan precipitate obtained from the direct digestion of
chitosan–glucan complex with Termamyl Type LS enzyme was
treated with dilute alkaline solution. Alkaline soluble and insol-
uble fractions were separated by centrifugation. Some of the
alkaline soluble glucan could be recovered from the alkaline
soluble fraction.
4.2. Characterization of purified fungal chitosan, soluble
glucan and insoluble glucan
IR spectroscopy, NMR spectroscopy and elementary anal-
ysis were employed to determine the structural characteristic
properties of chitosan and alkaline soluble and insoluble glucan
extracted from fungal cell wall.
4.2.1. IR spectroscopy
Fig. 1A shows the infrared spectra of chitosan and glucan
polymers extracted from the CGC. The reference spectra of
soluble starch (Wako Pure Chemical Industries Ltd.) and P-
cellulose (Wako Pure Chemical Industries Ltd.) are shown in
Fig. 1B. The peaks assignment for glucan, chitosan, cellulose
and starch are presented in Table 3. The absorption bands at
964, 843 and 749 cm−1 were observed in the spectrum of starch
and these bands could not observe in cellulose, glucan and
chitosan spectra. This observation suggesting that there is no
␣-d-glucopyranosyl residues in the polymers of fungal chitosan
and glucan. The characteristic absorption bands of cellulose, glu-
can and chitosan are at 1036–1072 cm−1 and 847–892 cm−1 .
These absorption bands are absent in the spectrum of soluble
starch. These results confirm that the monomers in chitosan are
linked by ␤-d-glycosidic linkages. The same holds for the bonds
between the monomers in the glucan component. The action
of Termamyl can therefore not be explained by hydrolysis of
␣-glycosidic regions in the glucan fraction.
All the spectra of starch, cellulose, glucan and chitosan have
the absorption band at the region of 1640 cm−1 . This absorption
 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251 245

Fig. 1. Infrared spectra of fungal chitosan and alkaline soluble and insoluble glucan (A) and soluble starch and P-cellulose (B).

Table 3
Peak assignments from the infrared spectra of starch, cellulose, glucan and chitosan
Starch Cellulose Soluble glucan Insoluble glucan Chitosan Specification

Wave numbers of absorption bands (cm−1 )

3392 3358 3379 3427 3379 υ(Poly-OH)a
2914 2887 2917 2913 2910 υ(Poly-CH)a
2120 2113 – – – υ(poly–CH)
1642 1636 1653 1630 1642 Amide Ia , δ(H2 O)b
– – 1539 1549 1576 N–H banda
1427 – 1441 – – O-6 H-bondb
– 1393 1383 1371 1387 w(CH2 )b
– 1366 – – 1377 w(CH2 )b
– 1311 – – 1316 w(CH2 )b
– – 1214 – 1249 τ(CH2 )b
– – 1147 – 1151
– 1036 1064 1057 1072
964 – – – – ␣-Bond
– – – – – ␣-Bondb
– 892 847 887 891 ␤-Bondb
843 – – – – ␣-Bondb
749 – – – – ␣-Bondb
a Struszczyk et al. [48].
b Mathlouthi and Koenig [38].

band is due to associated with water or amide band [38]. Absorp-

tion at 1550 cm−1 was observed in the spectra of alkaline soluble
and insoluble glucan and chitosan, but this band was absent in
the spectra of starch and cellulose. The shoulder amide band in
the spectra of alkaline soluble and insoluble glucan might be
contributed from the chitosan polymer.

4.2.2. 13 C NMR spectroscopy

13 C NMR spectrum of fungal chitosan is shown in Fig. 2. The

assigned 13 C NMR signals are shown in Table 4 and compared

with the shrimp and crab shell chitosan [39]. In the 13 C NMR
spectrum of the fungal chitosan the methyl and carbonyl signals
are absent due to it high degree of deacetylation. The absence of
the chemical shift on fungal chitosan spectrum at approximately
δ 68 clearly shows that there is no branching chain in the fun-
gal chitosan and lack of the free non-reducing end in the major
Fig. 2. 13 C NMR spectrum of purified fungal chitosan.
monomer units in the polymers. All the units in the (1, 3; 1,6)
246 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251

␤-d-glucan have free non-reducing ends (see Fig. 3). Therefore
it can be concluded that fungal chitosan is free from contami-
nation of (1, 3; 1,6) ␤-d-glucan. The observed signals from C-1
carbon at ␦107 strongly suggest that only ␤-anomeric carbons
are present in the chitosan polymers.
4.3. Determination of nitrogen content in fungal chitosan
Chitosan, 8 g/100 g of mycelia and glucan, 13 g/100 g of
mycelia could be easily extracted from the CGC obtained from
the fungus, G. butleri USDB 0201. The nitrogen content in the

Fig. 3. Possible molecular structures of chitosan and glucan obtained from the treatment of chitosan–glucan complex by Termamyl, Type LS enzyme: (A) 1-4
aminoglucanyl-␤-glucan structure and (B) 1-4 ␤-glucanyl-aminoglucan structure.
 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251 247

Fig. 3. (Continued ).

Table 4 isolated chitosan was 8%. The relative number average molec-
13 C chemical shifts, ppm of fungal chitosan compared with various sources of ular weight and degree of deacetylation of the chitosan were
chitosan
58 kDa and 89%, respectively.
Source C-1 C-4 C-5, C-3 C-6 C-2

Fungal 107 86–80 75 62 59 5. Discussions

Crab shella 105 86, 81 76 60 56
Shrimp shella 105, 104 84–80 75 61 57 In the fungal mycelia, chitosan can be isolated in two forms:
a Saito et al. [39]. as free chitosan and as chitosan/chitin covalently bound in a
248 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251
chitin/chitosan–glucan complex [11,12,31,40]. Chitosan could
not be extracted directly from the mycelia with 0.35 M acetic
acid. About 2 g of free chitosan could be isolated from the fun-
gal cell wall after treatment of 100 g mycelia (dry base, db)
with dilute alkaline solution (1 M NaOH) followed by extrac-
tion with dilute acetic acid [21]. The ␣-glucan and most of the
unbound ␤-glucan are usually extracted from the fungal cell
wall with alkali [14,18]. The presence of free chitosan suggests
that chitosan can be formed in the fungal cell wall separately
from glucan, and that these two polymers are linked together
afterwards to form the chitosan–glucan complex, CGC. These
observations support the assumption that chitin and ␤-glucan
chains initially accumulate individually in the fungal cell wall
and then form the interpolymer linkage [17]. The residual alkali
insoluble matrix after treatment with 1 M NaOH is very rigid
and cannot be decomposed by treatment with 0.35 M acetic
acid at 95 ◦ C for 5 h. Fontaine et al reported that 11% of the
total cell wall of fungus, Aspergillus fumigatus remained after
subsequent treatments with 1 M NaOH, Quantazyme (recom-
binant endo-␤-1,3-glucanase), 1 M NaOH, chitinase and 1 M
NaOH [30]. Due to its rigidity, enzymes could penetrate the
matrix only to a very limited extend to break the bond between
the chitin and glucan in the cell walls. For this reason, the
knowledge about fungal cell wall biopolymers remained for
many years very limited and their biosynthesis insufficiently
understood.
Importantly, cell wall matrix must be broken down far enough
in order to study the linkage between the chitosan and glucan
in the fungal cell wall. Therefore further treatment of cell wall
matrix was investigated in a randomized experimental design
[41]. It was observed that the alkali insoluble matrix obtained
by treatment with 11 M NaOH at 45 ◦ C for 13 h can be totally
disintegrated in 0.35 M acetic acid during 5 h at 95 ◦ C [21]. Muz-
zarelli et al. concluded that the residue obtained after treatment of
Aspergillus niger mycelia with 40% NaOH consists of a CGC,
insoluble in acetic acid [22]. The treatment in strong alkali is
apparently required to break the intermolecular bonds in the
complex of chitosan and ␤-glucan. Once this complex is suffi-
ciently broken down, at last a part of the CGC can form a quite
turbid but stable suspension in diluted acid.
The chitosan component of the CGC is heavily protonated at
pH 4 and supports the CGC to stay in suspension. In this condi-
tion the glucan does not dissolve but gives rise to the turbidity of
the acidic suspension. Surprisingly, this turbid suspension can
be separated in heavy sediment and a crystal clear supernatant
by raising the pH to 9. At pH 9 the chitosan will have lost its
charge and does not longer sustain the CGC to remain in suspen-
sion. The chitosan biopolymer is apparently very tightly linked
to glucan in the fungal cell wall because due to the link the glucan
component can stay for a long time in suspension and doesn’t
dissociate between pH 4.0 and 9.0.
A large number of enzymes have been tested for their effec-
tiveness to disintegrate remove glucan from the CGC [41]. It
was observed that the enzyme ␣-amylase (Termamyl Type LS
enzyme) effectively cleaves the glucan from the CGC [31]. The
action of Termamyl is very clearly demonstrated by observ-
ing the physical nature of the major polymers involved. During
incubation of the turbid CGC at pH 4.5 with the enzyme, the
suspension does not remain stable. A precipitate and a clear
supernatant are formed. The clear supernatant appears to contain
the chitosan and the precipitate is concluded as ␤-glucan.
Termamyl enzyme is a starch degradation enzyme. There-
fore, increase amount of reducing sugar concentration in the
chitosan/glucan complex and chitosan solutions was determined
before and after treatment with Termamyl enzyme (Table 2). It
was observed that the Termamyl enzyme treatment is effective
and leads to a sharp increase in the concentration of C1 end
groups in the solution. Termamyl does not split the ␤-glycosidic
bonds in chitosan. The glucan remained polymeric and became
insoluble in reaction mixture; therefore it must contain a major-
ity of bonds that are not hydrolyzed by Termamyl like ␤ 1-3 and
1-6. Therefore, the increase must originate from hydrolysis of
1-4 bonds between chitosan and glucan.
Sietsma and Wessels reported that ␤-glucan in the yeast cell
wall is linked to chitin chains, probably through the reducing
ends of the glucans because of their resistance to alkaline degra-
dation [42]. According to the previous authors, most fungal cell
walls contain ␤-1,3 glucans with branches of one or more ␤-1,6
linked glucose residues. Most end groups in the glucan polymers
are non-reducing ends (see Fig. 3).
Prior to enzyme treatment the reducing sugar concentration in
the before enzyme treatment solution was low since the insoluble
CGC might not be included in the Dygert reaction. Therefore
the data do not distinguish between an ␣-glycosidic bonding
between the C1-reducing end of the chitosan chain and the non-
reducing C-4 end of the ␤-glucan (an 1-4 aminoglucanyl-glucan
bond) or vice versa: an ␣-glycosidic bonding between the C1-
reducing end of the ␤-glucan chain and the non reducing C-4 end
of the chitosan (an 1-4 glucanyl-aminoglucan bond) (see Fig. 3A
and B). The action of the Termamyl enzyme resulted in a steep
increase in reducing sugar concentration in the supernatant, but
for either bonding this can be explained by the appearance of
the chitosan chains in the supernatant. The solubilized chitosan
may originate from either the aminoglucanyl-␤-glucan or from
the ␤-glucanyl-aminoglucan structure.
When at the junction of the chitosan and the ␤-glucan the
latter moiety has also three- or six-connections to other parts
of the ␤-glucan molecule, steric hindrance might occur for the
enzyme to act on the 1-4 [aminoglucanyl-␤-glucan] bond. On
that grounds Termamyl might more probably act on the 1-4
[glucanyl-␤-aminoglucan] bond.
There are also arguments in favor of the 1-4 [aminoglucanyl-
␤-glucan] bond. The ␤-glucan, due to the occurrence of 1–6
bonding and the concomitant branching has many C-4 hydroxyl
groups available for the1-4 [aminoglucanyl-␤-glucan] bond, but
relatively few for the 1-4 [glucanyl-␤-aminoglucan] bond (see
Fig. 3A and B). Upon the action of the Termamyl, a large
number of reducing end increase in the supernatant has been
observed. Therefore the bond between the chitosan and glucan
in the cell wall of fungus G. butleri USDB 0201 must be ␣-1-4
[aminoglucanyl-␤-glucan] bond.
Moreover the glucan precipitate obtained after Termamyl
Type LS enzyme treatment was treated with dilute alkaline solu-
tion. Alkaline soluble and insoluble fractions were separated
 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251
by centrifugation. Some of the alkaline soluble glucan could be
recovered from the alkaline soluble fraction. This polymer could
not dissolve during the alkaline treatment of fungal mycelia due
to its linkage with chitosan. After the cleavage of the chitosan
from the complex by Termamyl, this material could not dissolve
in the acid solution, but could be extracted from the precipitate
by diluted alkali. This observation agrees with the consensus
on glucan alkaline solubility in most of fungal cell walls and
yeast. Sietsma and Wessels reported that depolymerization of
the glucosaminoglycan with chitinase resulted in solubilisation
of most of the ␤-glucan from the alkaline-insoluble wall residue
of Schizophyllum commune [25]. In Saccharomyces cerevisiae,
all ␤-glucan become soluble in alkali after a chitinase treatment
[43]. Schmid et al reported that ␤-glucans can be dissolved in
1 M NaOH and precipitated by ethanol [15].
In order to confirm the purity of chitosan and glucan, the
extracted chitosan and soluble and insoluble glucans were fur-
ther analyzed by IR and 13 C NMR spectroscopes and elementary
analysis. Most of the authors reported that the characteristic
absorption at about 758 cm−1 in the IR spectrum is indicative for
the ␣-glycosidic linkage and the ␤-anomer gave an absorption
at 909 cm−1 , 896 cm−1 and 856 cm−1 [15,38,44–46]. The char-
acteristic absorption bands of glucan and chitosan are at 1036 –
1072 cm−1 and 847–892 cm−1 and could not observe the absorp-
tion bands at 749 cm−1 . This observation suggesting that there is
no ␣-d-glucopyranosyl residues in the polymers of chitosan and
soluble and insoluble glucan and confirmed that the monomers
in these polymers are linked by ␤-d-glucosidic. Absorption at
1550 cm−1 was observed in the spectra of alkaline soluble and
insoluble glucan and chitosan, According to the previous stud-
ies, the absorption intensities of amide I and amide II bands of
chitosan appear at 1655 and 1560 cm−1 respectively [47–49].
This result point out that Termamyl enzyme could not access to
all the bond between the chitosan and glucan since amide band
appear in the IR spectra of alkaline soluble and insoluble glucan.
Many (1,3)-(1,6)-␤-d-glucans adopt ordered triple helices con-
formations [17,19,20]. Therefore, it can be assumed that some
chitosan polymer might be entrapped in the glucan triple helices
and heat stable ␣-amylase enzyme could not cleave the chitosan
from the glucan polymers. The spectrum of fungal chitosan has
a trace band at the position of amide I. Wang et al. reported that
amide I band decreased with increased degree of deacetylation
of chitosan [47]. Therefore the chitosan fraction obtained from
the CGC seems to have a high degree of deacetylation. This
data is agreement with the degree of deacetylation of chitosan
data obtained from UV spectrometer. This chitosan spectrum
pattern is similar to that of the IR spectra of chitosan with a high
degree of deacetylation obtained from G. butleri IFO8081 [50],
Absidia coerulea IFO 5301 and Mortierella isabelina IFO 6739
[51]. Moreover the 13 C chemical shifts of the fungal chitosan
spectrum are similar to those of chitosan obtained from shrimp
and crab shell. According to the results obtained from previous
authors, the liquid state 13 C NMR spectrum of extracellular glu-
can, unbranched C-6-(CH2 -OH) resonates at approximately δ
61, whereas branched C-6-(CH2 -R) and free non-reducing end
of C4 resonates at approximately δ 68 [15,52]. Therefore the
absence of the chemical shift on fungal chitosan spectrum at
249
approximately δ 68 clearly shows that there is no branching
chain in the fungal chitosan and lack of the free non-reducing
end in the major monomer units in the polymers (Fig. 2). All the
units in the (1, 3; 1,6) ␤-d-glucan have free non-reducing ends
(see Fig. 3). Therefore it can be concluded that fungal chitosan is
free from contamination of (1, 3; 1,6) ␤-d-glucan. Moreover, the
resonance at 100 ppm was attributed to the ␣-anomeric carbon
of glucopyranose and the resonance at 103 ppm was ␤-anomeric
carbon of glucopyranose [14,15,45]. The observed signals from
C-1 carbon at δ 107 strongly suggest that only ␤-anomeric
carbons are present in the fungal chitosan polymers (Fig. 2).
Therefore the resultant chitosan is free from glucan but pure
glucan could not be extracted by Termamyl enzyme treatment.
The analysis result from determination of nitrogen content in the
chitosan strongly proved that 95% purify chitosan was obtained
from Termamyl treatment.
6. Conclusion
In the fungus G. butleri USDB 0201, chitosan and glucan
are synthesized separately and then form the CGC to serve as
a building block of fungal cell wall. According to the results
obtained from the specific enzymatic digestion, IR and NMR
spectroscopy and nitrogen content analysis, it has been con-
cluded that chitosan and glucan in the fungal cell wall are linked
by ␣-(1-4)-glycosidic bond. Chitosan was obtained from the
CGC with the help of heat stable ␣-amylase enzyme. However
some chitosan could not be cleaved completely from the glucan
by this enzyme.
The results presented here show that the fungus G. butleri
USDB 0201 is a rich source for the isolation of chitosan and
glucan. It has been shown that these materials can be isolated
in a form of CGC that has been claimed for medical and immu-
nization supportive application. The knowledge of the nature of
the bond between both ␤-anomeric polymers, elucidated as an
␣-glycosidic bond opens the road to the isolation of either pure
medium-size fungal chitosan and pure fungal ␤-glucan. This is
an attractive perspective for pharmaceutical and cosmetic indus-
try for the isolation of shrimp protein free chitosan. It is very
attractive as well for the agricultural sector where medium size
chitosan is being applied as promoter of growth and differenti-
ation. Whereas the total amount of shrimp shell material in the
world is huge but limited, fungal supply can take care for the
enormous amounts that will be required in the near future for
the agricultural sector.
Acknowledgements
The authors would like to extend their thanks to the late
Prince Leo de Lignac (The Netherlands), “High-Tech Research
Center” Project for Private Universities: matching fund subsidy
from MEXT (Ministry of Education, Culture, Sports, Science
and Technology, Japan), 2005–2009 and the Japan Society for
Promotion of Science (JSPS), Japan (H19:07068) for provid-
ing funds for this research, to Siam Modified Starch Co. LTD
(Thailand) and Novo Nordisk Bioindustrial (Thailand, Japan
and Demark) for providing Termamyl, Type LS, and to Dr. Ng
250 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251
Chuen How and Mr Egawa Tomoya for valuable instrumental
assistance.
References
[1] Ikeda I, Sugano M, Yoshida K, Sasaki E, Iwamoto Y, Hatano K. Effects
of chitosan hydrolysates on lipid absorption and on serum and liver lipid
concentration in rats. J Agric Food Chem 1993;41:431–5.
[2] Kubala L, Ruzickova J, Nickova K, Sandula J, Ciz M, Lojek A. The effect
of (1-/3)-␤-d-glucans, carboxymethylglucan and schizophyllan on human
leukocytes in vitro. Carbohydr Res 2003;338:2835–40.
[3] Huang Y, Onyeri S, Siewe M, Moshfeghian A, Madihally SV. In vitro
characterization of chitosan–gelatin scaffolds for tissue engineering. Bio-
materials 2005;26:7616–27.
[4] Nwe N, Maeda Y, Stevens WF, Furuike T, Tamura H. Growth of fibroblast
cell NIH/3T3 on chitosan and chitosan–glucan complex scaffolds. Polymer
2007;56:2181 [Preprints, Japan].
[5] Bartnicki-Garcia S. Cell wall chemistry, morphogenesis and taxonomy of
fungi. Annu Rev Microbiol 1968;22:87–108.
[6] Bartnicki-Garcia S. The biochemical cytology of chitin and chitosan syn-
thesis in fungi. In: Chitin and chitosan. London: Elsevier; 1998. p. 23–35.
[7] Gooday GW. Cell walls. In: The growing fungus. Chapman and Hall; 1995.
p. 3–62.
[8] Robson G. Hyphal cell biology. In: Molecular fungal biology. Cambridge:
University Press; 1999. p. 164–84.
[9] Burnett JH. In: Crane R, editor. Fundamentals of mycology. 2nd ed. Lon-
don: Edward Arnold, Ltd; 1976. p. 673.
[10] Milewski S, Janiak A, Wojciechowski M. Structural analogues of reac-
tive intermediates as inhibitors of glucosamine-6-phosphate synthase and
phosphoglucose isomerase. Arch Biochem Biophys 2006;450:39–49.
[11] Gooday GW, Humphreys AM, Mcintosh WH. Roles of chitinases in fungal
growth. In: Chitin in nature and technology. New York: Plenum press; 1986.
p. 83–91.
[12] Davis LL, Bartnicki-Garcia S. The co-ordination of chitosan and chitin
synthesis in Mucor rouxii. J Gen Microbiol 1984;130:2095–102.
[13] Sietsma JH, Vermeulen CA, Wessels JGH. The role of chitin in hyphal
morphogenesis. In: Chitin in nature and technology. Plenum Press; 1986.
p. 63–9.
[14] Carbonero ER, Montai AV, Mellinger CG, Eliasaro S, Sassaki GL, Gorin
PAJ, et al. Glucans of lichenized fungi: significance for taxonomy of the
genera Parmotrema and Rimelia. Phytochemistry 2005;66:929–34.
[15] Schmid F, Stone BA, McDougall BM, Bacic A, Martin KL, Brownlee RTC,
et al. Structure of epiglucan, a highly side-chain/branched (1 → 3; 1 → 6)-
␤-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht.
Carbohydr Res 2001;331:163–71.
[16] Hochstenbach F, Klis FM, Ende Hvd, Donselaar Ev, Peters PJ, Klausner
RD. Identification of a putative alpha-glucan synthase essential for cell
wall construction and morphogenesis in fission yeast. Proc Natl Acad Sci
1998;95:9161–6.
[17] Wessels JGH, Mol PC, Sietsma JH, Vermeulen CA. Wall structure, wall
growth, and fungal cell morphologenesis. In: Biochemistry of cell walls
and Membranes in fungi. Berlin: Springer-Verlag; 1990. p. 1–84.
[18] Wolski EA, Lima C, Agusti R, Daleo GR, Andreu AB, de Lederkremer
RM. An ␣ glucan elicitor from the cell wall of a biocontrol binucleate
Rhizoctonia isolate. Carbohydr Res 2005;340:619–27.
[19] Jeksma J, Kreger DR. Ultrastructural observations on (1-3)-␤-d-glucan
from fungal cell walls. Carbohydr Res 1975;43:200–3.
[20] Marchessault H, Deslandes Y. Fine structure of (1-3)-␤-d-glucan: curdlan
and paramylon. Carbohydr Res 1979;75:231–42.
[21] Nwe N, Stevens WF. Production of fungal chitosan by solid sub-
strate fermentation followed by enzymatic extraction. Biotechnol Lett
2002;24:131–4.
[22] Muzzarelli RAA, Tanfani F, Scarpini G. Chelating, film-forming and coag-
ulating ability of the chitosan–glucan complex from Aspergillus niger
industrial wastes. Biotechnol Bioeng 1980;22:885–96.
[23] Tan SC, Tan TK, Wong SM, Khor E. The chitosan yield of Zygomycetes
at their optimal harvesting time. Carbohydr Polym 1996;30:239–42.
[24] Suntornsuk W, Pochanavanich P, Suntornsuk L. Fungal chitosan production
on food processing by-products. Proc Biochem 2002;37:727–9.
[25] Sietsma JH, Wessels JGH. Evidence for covalent linkages between
chitin and beta-glucan in a fungal wall. J Gen Microbiol 1979;114:
99–108.
[26] Gopal P, Sullivan PA, Shepherd MG. Isolation and structure of glucan
from regenerating spheroplasts of Candida albicans. J Gen Microbiol
1984;130:1217–25.
[27] Surarit R, Gopal PK, Shepherd MG. Evidence for a glycosidic linkage
between chitin and glucan in the cell wall of Candida albicans. J Gen
Microbiol 1988;134:1723–30.
[28] Roberts GAF. Preparation of chitin and chitosan. In: Chitin chemistry. 1st
ed. London: Macmillan Press; 1992. p. 55–83.
[29] Kollar R, Petrakova E, Ashwell G, Robbins PW, Cabib E. Architecture of
the yeast cell wall, the linkage between chitin and ␤-1,3-glucan. J Biol
Chem 1995;270:1170–8.
[30] Fontaine T, Simenel C, Dubreucq G, Adam O, Delepierre M, Lemoine J,
et al. Molecular organization of the alkali-insoluble fraction of Aspergillus
fumigatus cell wall. J Biol chem 2000;275:27594–607.
[31] Nwe N, Stevens WF. Chitosan isolation from the chitosan–glucan com-
plex of fungal cell wall using amylolytic enzymes. Biotechnol Lett
2002;24:1461–4.
[32] Nwe N, Stevens WF. Effect of urea on fungal chitosan production in solid
substrate fermentation. Proc Biochem 2004;39:1639–42.
[33] Nwe N, Stevens WF. Optimization of solid substrate fermentation for the
production of fungal chitosan. J Chitin Chitosan 2006;11:11–5.
[34] Nwe N, Chandrkrachang S, Stevens WF, Maw T, Tan TK, Khor E, et al.
Production of fungal chitosan by solid state and submerged fermentation.
Carbohydr Polym 2002;49:235–7.
[35] Dygert S, Li LG, Floride D, Thomas JA. Determination of reducing sugar
with improved precision. Anal Biochem 1965;13:367–74.
[36] Muzzarelli RAA, Rocchetti R. Determination of the degree of acetylation
of chitosans by first-derivative ultraviolet spectrophotometry. Carbohydr
Polym 1985;5:461–72.
[37] Tan SC, Khor E, Tan TK, Wong SM. The degree of deacetylation of
chitosan: advocating the first-derivative UV-spectrophotometry method of
determination. Talanta 1998;45:713–9.
[38] Mathlouthi M, Koenig J. Vibrational spectra of carbohydrates. Adv Carbo-
hydr Chem Biochem 1986;44:7–86.
[39] Saito H, Tabeta R, Ogawa K. High-resolution solid-state 13 C NMR study of
chitosan and its salts with acids: Conformational characterization of poly-
morphs and helical structures as viewed from the conformation-dependent
13 C chemical shifts. Macromolecules 1987;20:2424–30.
[40] Kogan G, Machova E, Chorvatovicova D, Sandula J. Chitin–glucan com-
plex of Aspergillus niger and its derivatives: antimutagenic and antinfective
activity. In: Advances in chitin science. Keelung; 1998. p. 372–9.
[41] Nwe N, Stevens WF, Montet D, Tokura S, Tamura H. Effect of NaOH
treatment conditions on the nature of fungal chitosan–glucan complex and
chitosan production. In: Advances in chitin science. Montpellier; 2006. p.
57–63.
[42] Sietsma JH, Wessels JGH. Solubility of (1-3)-␤-d/(1-6)-␤-d-glucose in
fungal walls: importance of presumed linkage between glucan and chitin.
J Gen Microbiol 1981;125:209–12.
[43] Mol PC, Wessels JGH. Linkages between glucosaminoglycan and glu-
can determine alkali-insolubility of the glucan in walls of Saccharomyces
cerevisiae. FEMS Microbiol Lett 1987;41:95–9.
[44] Rout D, Mondal S, Chakraborty I, Pramanik M, Islam SS. Chemical anal-
ysis of a new (1-3)-, (1-6)-branched glucan from an edible mushroom,
Pleurotus florida. Carbohydr Res 2005;340:2533–9.
[45] Zhao G, Kan J, Li Z, Chen Z. Characterization and immunostimulatory
activity of an (1 → 6)-␣-d-glucan from the root of Ipomoea batatas. Int
Immunopharmacol 2005;5:1436–45.
[46] Yalin W, Cuirong S, Yuanjiang P. Studies on isolation and structural fea-
tures of a polysaccharide from the mycelium of an Chinese edible fungus
(Cordyceps sinensis). Carbohydr Polym 2006;63:251–6.
[47] Wang W, Bo S, Li S, Qin W. Determination of the Mark–Houwink equa-
tion for chitosans with different degree of deacetylation. J Biol Macromol
1991;13:281–5.
 N. Nwe et al. / Enzyme and Microbial Technology 42 (2008) 242–251
[48] Struszczyk MH, Loth F, Peter MG. Analysis of degree of deacetylation
in chitosans from various sources. In: Advances in chitin science. Lyon:
Jacques Andre Publisher; 1997. p. 71–7.
[49] Matsubara M, Kuroda H. Physiological and biochemical studies on germi-
nation fungal spores. VII. Chemical composition of cell walls in conidia of
Cochliobolus miyabeanus. Chem Pharm Bull 1985;33:1175–80.
[50] Yokoi H, Aratake T, Nishio S, Hirose J, Hayashi S, Takasaki Y. Chi-
tosan production from Shochu distillery wastewater by funguses. J Fermen
Bioeng 1998;85:246–9.
251
[51] Miyoshi H, Shimura K, Watanabe K, Onodera K. Characterization of some
fungal chitosans. Biosci Biotechnol Biochem 1992;56:1901–5.
[52] de Lourdes Corradi da Silva M, Izeli NL, Martinez PF, Silva IR, Constantino
CJL, Cardoso MS, et al. Purification and structural characterization of
(1 → 3; 1 → 6)-␤-d-glucans (botryosphaerans) from Botryosphaeria rho-
dina grown on sucrose and frucrose as carbon sources: a comparative study.
Carbohydr Polym 2005;61:10–7.