Utilization of commercial non-chitinase enzymes from fungi for preparation of 2-acetamido-2-deoxy-D-glucose from β-chitin

Article abstract of DOI:10.1016/S0008-6215(01)00298-1

Commercial non-chitinase enzymes from Aspergilus niger, Acremonium cellulolyticus and Trichoderma viride were investigated for potential utilization in the preparation of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine, GlcNAc) from chitin. Among th

Full text of DOI:10.1016/S0008-6215(01)00298-1

Carbohydrate Research 337 (2002) 133–137
www.elsevier.com/locate/carres
Utilization of commercial non-chitinase enzymes from fungi for
preparation of 2-acetamido-2-deoxy- -glucose from b-chitin
D
a,
b
b
b,
Mongkol Sukwattanasinitt, * Hong Zhu, Hitoshi Sashiwa, Sei-ichi Aiba *
a
Centre for Bioacti6e Compounds, Department of Chemistry, Faculty of Science, Chulalongkorn Uni6ersity, Bangkok 10330, Thailand
b
Green Biotechnology Research Group, The Special Di6ision for Human Life Technology,
National Institute of Ad6anced Industrial Science and Technology, Osaka 563-8577, Japan
Received 9 October 2001; accepted 6 November 2001
Abstract
Commercial non-chitinase enzymes from Aspergilus niger, Acremonium cellulolyticus and Trichoderma 6iride were investigated
for potential utilization in the preparation of 2-acetamido-2-deoxy- -glucose (N-acetyl- -glucosamine, GlcNAc) from chitin.
D
D
Among the tested enzymes, cellulase A. cellulolyticus exhibited highest chitinolytic activity per weight toward amorphous chitin
and b-chitin from squid pen. The optimum pH of the enzyme was 3 where it produced two major hydrolytic products, GlcNAc
and N,N%-diacetylchitobiose ([GlcNAc] ). The product ratio, GlcNAc:[GlcNAc] , increased while the total yield decreased as the
2
2
pH was raised from 3. All of the [GlcNAc]2 produced at pH 3 can be converted in situ to GlcNAc by mixing cellulase A.
cellulolyticus with one of several other enzymes from A. niger resulting in a higher yield of GlcNAc. An appropriate mixing ratio
of cellulase A. cellulolyticus to another enzyme was 9:1 (w/w) and an optimum substrate concentration was 20 mg/mL. © 2002
Elsevier Science Ltd. All rights reserved.
Keywords: N-Acetyl-D-glucosamine; Chitin; Chitinase; Acremonium cellulolyticus; Aspergillus niger; Trichoderma 6iride
under much milder conditions with less toxic reagents.
A continuous two-step process for industrial produc-
1. Introduction
tion of GlcNAc using chitinase from Serratia marcesens
has been developed. Recently, we, however, relied on
2
-Acetamido-2-deoxy-D-glucose (N-acetyl-D-glucos-
5
amine, GlcNAc) is a basic unit in many biologically
important oligosaccharides, glycoproteins and glycol-
ipids. GlcNAc has also been reported to have therapeu-
tic potential in the treatments of various diseases
including osteoarthristis, inflammatory bowel disease
and gastritis. The preparation of GlcNAc is currently
another approach using inexpensive, commercially
available, non-chitinase enzymes for the preparation of
6
GlcNAc. A high yield of GlcNAc can be produced
7
8
1
2
cleanly by hydrolysis of amorphous or b-chitin with
cellulase T6 (Trichoderma 6iride). The present drawback
of this approach is a relatively low chitinolytic activity
of this enzyme. We report here a systematic study
centering on another commercial enzyme, cellulase Ac
3
carried out by reacetylation of D-glucosamine produced
4
from an acid hydrolysis of chitin. The acid hydrolysis
is usually performed by heating chitin in concentrated
hydrochloric acid, which poses technical and environ-
mental concerns. It is also virtually impossible to avoid
decomposition of the desired product without having
an uncompleted hydrolysis under such harsh condi-
tions. The reaction thus usually gives a limited yield of
(
Acremonium cellulolyticus), which has led to higher
efficiency of GlcNAc production from b-chitin by com-
mercial crude enzymes.
2
. Results and discussion
D
-glucosamine. Enzymatic hydrolyses usually proceed
Six commercial non-chitinase enzymes from three
*
Corresponding authors. Fax: +66-2-2541309 (M.S.); fax:
species of fungi, Aspergilus niger (An), A. cellulolyticus
Ac) and T. 6iride (T6), were tested for hydrolytic
activity of amorphous chitin at pH 4.5. Cellulase Ac
+
81-727-519628 (S.-i.A).
E-mail addresses: mongkol@chula.ac.th (M. Sukwat-
tanasinitt), aiba-seiichi@aist.go.jp (S.-i. Aiba).
(
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00298-1

134
M. Sukwattanasinitt et al. / Carbohydrate Research 337 (2002) 133–137
exhibited the highest chitinolytic activity among the six
enzymes evaluated (Table 1). This enzyme, however,
produced two major hydrolytic products, GlcNAc and
Table 3
b-N-Acetylhexosaminidase activity of some commercial non-
chitinase enzymes
a
[
GlcNAc] . Thus this enzyme was not suitable for the
2
b
Enzyme
% Yield
GlcNAc
Relative activity
preparation of GlcNAc in one step. For a one-step
hydrolysis of chitin to GlcNAc, the enzyme preparation
must contain both chitinase (EC 3.2.1.14) and b-N-
acetylhexosaminidase (EC 3.2.1.52) activities. The chiti-
nase is responsible for hydrolyzing polymeric chitin into
smaller oligosaccharides, which are in turn further hy-
drolyzed by b-N-acetylhexosaminidase to GlcNAc. To
our delight, cellulase Ac also showed chitinolytic activ-
ity on b-chitin comparable to that observed on amor-
phous chitin. The use of b-chitin in place of amorphous
chitin can simplify substrate preparation since amor-
phous chitin must be prepared from a highly basic
solution of chitin. Further experiments were thus per-
formed on b-chitin. Our pH dependence study revealed
Cellulase An
Hemicellulase An
Lipase An
Pectinase An
Cellulase Ac
Cellulase T6
100
96
95
100
4
25
24
24
25
1
13
3
a
N,N%-Diacetylchitobiose was used as a substrate, [S]=1
mg/mL; [E]=1 mg/mL in citrate–phosphate buffer pH 3.
b
Percentage yields were determined by HPLC after incuba-
tion at 37 °C for 6 h.
that significant yield of [GlcNAc] was observed at pH
2
3
(Table 2), which was the optimum pH, suggesting
Table 1
that cellulase Ac contained only a limited amount of
b-N-acetylhexosaminidase. The results also indicated
that the chitinase activity in cellulase Ac was highly
acidophilic. The total yields of GlcNAc and [GlcNAc]2
dropped sharply as the pH increased from 3 to 4, but
decreased only slightly as the pH lowered to 2. The
b-N-acetylhexosaminidase activity in this enzyme was,
however, less acidophilic as the yield of GlcNAc de-
creased only slightly as the pH increased to pH 4.
To improve the production of GlcNAc in a one-step
hydrolysis, cellulase Ac should be combined with an-
other enzyme possessing high b-N-acetylhexo-
saminidase activity. All six enzymes were tested again
Chitinolytic activity of some commercial enzymes on amor-
phous chitin
a
b
c
Enzyme
% Yield of GlcNAc
Cellulase An
Hemicellulase An
Lipase An
Pectinase An
Cellulase Ac
Cellulase T6
less than 1%
less than 1%
less than 1%
less than 1%
13.1
8.6
a
[
S]=1.4 mg/mL; [E]=1.5 mg/mL in acetate buffer pH
4
.5.
b
for b-N-acetylhexosaminidase activity using [GlcNAc]
All enzymes were obtained from Amano Enzyme, Inc.
2
except cellulase Ac which was obtained from Meiji Seika Co.
Percentage yields were determined by HPLC after incuba-
tion at 37 °C for 3 days.
as a substrate. Despite possessing lower chitinolytic
activity (Table 1), the enzymes from An and T6 dis-
played higher b-N-acetylhexosaminidase activity than
cellulase Ac (Table 3). Furthermore, the enzymes from
An showed considerably higher b-N-acetylhexo-
saminidase activity than that of the enzyme from T6.
c
Table 2
pH dependence of chitinolytic activity of cellulase Ac on
a
b-chitin
The slow hydrolysis of [GlcNAc] with cellulase Ac also
2
b
c
confirmed that this crude enzyme contained a limited
amount of b-N-acetylhexosaminidase.
pH
% Yield
GlcNAc
The yield of GlcNAc was significantly increased
when the enzymes from T6 and An were mixed with
cellulase Ac (Table 4). The mixed enzymes from cellu-
lase T6 nonetheless gave a significant amount of [Glc-
NAc]2 as a hydrolytic product owing to lower
b-N-acetylhexosaminidase activity of cellulase T6 com-
pared to those of enzymes from An (Table 3). There
(GlcNAc)2
Total
2
3
4
5
6
7
25
28
24
18
9
25
28
11
3
0
0
50
56
35
21
9
0
0
was no [GlcNAc] observed up to the enzyme mixing
2
ratio of 9:1 cellulase Ac–another enzyme, when each of
the enzymes from An was used in the mixing. The yield
of GlcNAc was also increased significantly when the
mixing ratio was changed from 1:1 to 9:1. As the
mixing ratio increased further to 19:1, trace amounts of
a
[
S]=10 mg/mL; [E]=10 mg/mL.
b
Citrate buffer was used for pH 2 and citrate–phosphate
buffer was used for pH 3–7.
c
Percentage yields were determined by HPLC after incuba-
tion at 37 °C for 4 days.

M. Sukwattanasinitt et al. / Carbohydrate Research 337 (2002) 133–137
135
Table 4
Table 6
a
a
Effect of mixing ratio on GlcNAc preparation
Time dependence of rate of GlcNAc production
c
Enzyme
Mixing
ratio
% Yield
Time (day)
Yield of GlcNAc
(mg/mL)
Relative production
rate
b
b
GlcNAc [GlcNAc]2
1
2
4
8
0.50
0.66
1.20
2.32
3.46
100
86
66
58
58
Cellulase Ac
28
28
Cellulase Ac:
Cellulase T6
1
9
55
37
7
24
12
a
Cellulase Ac:
Cellulase An
1
9
43
61
62
0
0
The reaction was carried out by using 9:1 cellulase
Ac:pectinase An and b-chitin in citrate–phosphate buffer pH
3. [E]=2 mg/mL [S]=20 mg/ml at 37 °C.
1
1
1
1
9
trace
b
The relative production rate on the n date was calculated
Cellulase Ac:
Hemicellulase An
1
9
9
45
61
64
0
0
0
from 100×Yield /(Yield ×time).
n
1
The effects of substrate and enzyme concentrations
were investigated by using the mixed enzyme, 9:1 cellu-
lase Ac and lipase An. The increase of substrate concen-
tration while keeping the enzyme concentration
constant resulted in higher GlcNAc yield but lower
percentage yield (Table 5). When both substrate and
enzyme concentrations were raised from 10 to 20 mg/
mL, the GlcNAc yield doubled and the percentage yield
was not altered. The percentage yield was decreased
significantly when both substrate and enzyme concen-
trations were further increased to 40 mg/mL. The re-
duction of the percentage yield of GlcNAc at high
concentration of substrate and enzyme conceivably at-
tributed to a poor mixing as the reaction media became
very thick at this concentration.
Under conditions of excess substrate, the rate of
GlcNAc production by a mixed enzyme, cellulase Ac–
Pectinase An, decreased by over 40% during 12 days of
incubation (Table 6). In most of the enzymatic reac-
tions, there are two major causes for a decrease in the
reaction rate: enzyme denaturation and product inhibi-
tion. The extent of the product inhibition was investi-
Cellulase Ac:
Lipase An
1
9
9
48
61
60
0
0
trace
Cellulase Ac:
Pectinase An
1
9
9
44
61
64
0
0
0
a
b-chitin was used as a substrate; [S]=10 mg/mL; [E]=10
mg/mL in citrate–phosphate buffer pH 3.
b
Weight by weight ratio.
Percentage yields were determined by HPLC after incuba-
c
tion at 37 °C for 4 days.
[GlcNAc]2 were observed in some cases, but the
changes in GlcNAc yields were insignificant. These
results were reasonable since the amount of cellulase
Ac, which possessed most of the chitinase activity essen-
tial for the first slow step in the hydrolysis of insoluble
polymeric chitin, increased by 80% when the mixing
ratio was changed from 1:1 to 9:1. On the other hand,
the amount of cellulase Ac increased by only a mere 5%
upon changing of the mixing ratio from 9:1 to 19:1.
Table 5
a
Effect of concentrations of enzyme and substrate on GlcNAc production
b
Concentration of enzyme
mg/mL)
Concentration of b-chitin
(mg/mL)
% Yield
Yield of GlcNAc (mg/mL)
(
GlcNAc [GlcNAc]2
1
1
1
2
4
0
0
0
0
0
10
20
40
20
40
61
48
28
62
33
0
6.1
9.6
11.2
12.4
12.2
trace
2
trace
4
a
The reaction was carried out by using 9:1 cellulase Ac:lipase An mixed enzymes and b-chitin in citrate–phosphate buffer pH
Percentage yields were determined by HPLC after incubation at 37 °C for 4 days.
3
.
b

136
M. Sukwattanasinitt et al. / Carbohydrate Research 337 (2002) 133–137
Table 7
sis with six commercial enzymes (cellulase An, hemicel-
a
Effect of GlcNAc concentration on the production rate
lulase An, lipase An, pectinase An, cellulase Ac and
cellulase T6). The amorphous chitin and b-chitin con-
tained less than 1% ash content. The degrees of acetyla-
tion of the chitins determined from elemental analysis
Initial concentration of Yield of
GlcNAc (mg/mL)
Relative
production of
GlcNAc
GlcNAc
mg/mL)
b
(
were over 90%. The enzymes had protein (M \
W
0
4.9
4.6
4.1
4.0
100
94
84
10,000) content of around 10%. A typical reaction was
carried out by incubating a mixture of a known concen-
tration of enzyme and the substrate in a suitable buffer
2
5
8
.0
.0
.0
82
solution in the presence of NaN (500 ppm) preserva-
3
tive at 37 °C. The exact set of conditions for each
experiment was specified in the footnote of the table. At
each time point, a portion of the reaction mixture was
a
The reaction was carried out by using 9:1 cellulase
A:pectinase An mixed enzymes and b-chitin in citrate–phos-
phate buffer pH 3. [E]=10 mg/mL [S]=10 mg/mL at 37 °C.
b
sampled, diluted with water, then mixed with CH CN
The relative production of GlcNAc in the presence of x
3
mg/mL GlcNAc was calculated from 100×Yield /Yield .
(at the ratio of 30:70, v/v), filtered, and analyzed by
x
0
HPLC (column: Shodex Asahipak NH P-50; flow rate:
2
1
mL/min; mobile phase: 3:7 water–CH CN; detection:
3
gated by carrying out the reaction in the presence of
GlcNAc intentionally added in various concentrations
at the beginning of the reaction. The results showed
that the production rate decreased by less than 5%
when the initial concentration of GlcNAc was 2 mg/mL
UV at 210 nm). The amounts of GlcNAc and [Glc-
NAc] in the reaction mixtures were determined from
the calibration lines of the corresponding standards.
The percentage yields of GlcNAc or [GlcNAc] were
calculated based on the number of moles of the prod-
ucts produced against the number of moles of such
units presented in the starting chitin.
2
2
(
Table 7), which was the average concentration range in
the previous experiment. The results clearly indicated
that the product inhibition was the only minor cause of
the reduction in the production rate. However, enzyme
denaturation should not be the only reason accounting
for the rest of the rate decline since the rate looked
rather constant after 8 days (Table 6). We speculated
that the chitin substrate itself maybe the main reason
for the decrease in the GlcNAc production rate. The
substrate, b-chitin from squid pen, must contain both
amorphous and crystalline domains integrated in the
materials. The amorphous domains of the b-chitin par-
ticle are far more susceptible to enzymatic hydrolysis
than the crystalline domains. The hydrolysis rate was
thus faster at the beginning and slowed down as less of
the amorphous volume remained in the substrate.
Nonetheless, the b-chitin used mostly in this study
showed hydrolytic susceptibility to cellulase Ac notably
Acknowledgements
This work was sponsored by the Thailand–Japan
Technology Transfer Project (TJTTP) under the Over-
sea Economy Cooperation Fund (OECF), Agency of
Industrial Science and Technology (AIST) under the
Institute for Transfer of Industrial Technology (ITIT)
Fellowship Program, and Japan International Science
and Technology Exchange Center (JISTEC) under the
Science and Technology Agency (STA) Fellowship. We
also would like to thank Sunfive Co. for providing
b-chitin powder, Meiji Seika Co. for providing cellulase
Ac, and Amano Enzyme, Inc. for providing all other
enzymes used in this work.
7
higher than the more natural abundant a-chitin, but
comparable to the amorphous base-regenerated chitin.
In summary, the studies described here have shown the
potential use of some commercial non-chitinase en-
zymes from fungi for a convenient preparation of Glc-
NAc from b-chitin. We are currently developing a
simple and low-cost process for preparation of GlcNAc
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