Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase

Article abstract of DOI:10.1016/S0008-6215(02)00007-1

Finely powdered α- and β-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and β-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and Bacillus

Full text of DOI:10.1016/S0008-6215(02)00007-1

Carbohydrate Research 337 (2002) 557–559
www.elsevier.com/locate/carres
Note
Quantitative production of 2-acetamido-2-deoxy-
D-glucose from
crystalline chitin by bacterial chitinase
Rath Pichyangkura,a,* Sanya Kudan,a Kamontip Kuttiyawong,a
Mongkol Sukwattanasinitt,b Sei-ichi Aibac
aDepartment of Biochemistry, Faculty of Science, Chulalongkorn Uni6ersity, Bangkok 10330, Thailand
bCenter for Bioacti6e Compounds, Department of Chemistry, Faculty of Science, Chulalongkorn Uni6ersity, Bangkok 10330, Thailand
cThe Special Di6ision for Human Life Technology, National Institute of Ad6anced Industrial Science and Technology, Osaka 563-8577, Japan
Received 21 November 2001; accepted 3 January 2002
Abstract
Finely powdered a- and b-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and b-N-acetylhexosaminidase (EC
3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and
Bacillus licheniformis SK-1 were used to digest a- and b-chitin powder. Chitinase from B. cepacia TU09 produced GlcNAc in
greater than 85% yield from b- and a-chitin within 1 and 7 days, respectively. B. licheniformis SK-1 chitinase completely
hydrolyzed b-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose. However, only a 41% yield
of GlcNAc was achieved from digesting a-chitin with B. licheniformis SK-1 chitinase. © 2002 Elsevier Science Ltd. All rights
reserved.
Keywords: Chitin; Chitinase; N-Acetyl-D-glucosamine; 2-Acetamido-2-deoxy-D-glucose; Bacillus licheniformis; Burkholderia cepacia
out.4 Unfortunately, this method added an additional
substrate preparation step into the production of Glc-
NAc. The work on commercially available crude en-
zymes has also been extended to a production of
2-Acetamido-2-deoxy-
cosamine, GlcNAc) and 2-amino-2-deoxy-
D
-glucose
(N-acetyl-
D-glu-
D
-glucose (D-
glucosamine, GlcN) have recently been promoted as a
treatment or as nutriceutical agents for patients with
osteoarthritis and inflammatory bowel disease.1,2 In
contrast to GlcN hydrochloride or sulfate, both of
which have a bitter taste, GlcNAc has a sweet taste that
facilitates its use in daily consumption. However, Glc-
NAc has not been widely commercialized mainly due to
the lack of an economical process for its production
that is acceptable for food and medicine. The current
acidic hydrolysis of chitin using concentrated HCl is
inefficient and poses environmental and technical con-
cerns.3 On the other hand, hydrolysis of chitin with
enzymes can produce GlcNAc under mild and environ-
mentally friendly conditions. An approach whereby
commercially available crude enzymes were used to
hydrolyze amorphous chitin substrate has been carried
6
GlcNAc by direct hydrolysis of b-chitin powder.5
These reports have shown that enzymatic hydrolysis of
chitin can produce GlcNAc in relatively higher yields
than acid hydrolysis. Nevertheless, the remaining major
impediment of an enzymatic hydrolysis process is the
extremely low hydrolytic susceptibility of the natural
chitin substrate, due to its high crystallinity. We show
herein for the first time that crystalline chitin in both
the a- and b-forms can be cleanly hydrolyzed, produc-
ing GlcNAc in virtually quantitative yield.
Powdered a-chitin (14 mm in size) from crab shells
and b-chitin (3 mm in size) from squid pens were used
as substrates for digestion by crude bacterial chitinase
from Burkholderia cepacia TU09 and Bacillus licheni-
formis SK-1. A typical reaction contained 100 mU/mL
(1 unit=the amount of enzyme that produces 1 mmol
of GlcNAc per min from colloidal chitin) of the enzyme
and 10–40 mg/mL of the substrate, unless indicated
otherwise. Digestion reactions were carried out in 3–5
* Corresponding
author.
Tel.:
+66-2-2185416/17;
fax: +66-2-2185418.
E-mail address: rath.p@chula.ac.th (R. Pichyangkura).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00007-1

558
R. Pichyangkura et al. / Carbohydrate Research 337 (2002) 557–559
Table 1
Production of GlcNAc from b-chitin by chitinase from B. licheniformis SK-1
a
b-Chitin/enzyme (mg/U)
Digestion time (day)
% Yield b
GlcNAc
(GlcNAc)2
Total
400
200
100
1
3
6
1
3
6
1
3
6
9
18
25
16
34
46
28
53
75
22
27
22
18
38
29
50
40
20
31
45
47
34
72
75
78
93
95
a [E]=0.1 U/mL in 0.1 M citrate–phosphate buffer, pH 6.0.
b HPLC yield.
speculate that the crystalline domains in a-chitin are
completely resistant to digestion by chitinase from B.
licheniformis SK-1. The GlcNAc produced was proba-
bly liberated from the amorphous regions of the sub-
strate. Chitinase from B. cepacia TU09 showed superior
characteristics in hydrolyzing a-chitin as an 85% yield
mL of 0.1 M citrate–phosphate buffer, pH 6.0, in
10-mL glass vials. The reactions were incubated in a
shaking water bath, with moderate shaking, at 37 and
50 °C, respectively, when the enzyme from B. cepacia
TU09 and B. licheniformis SK-1 were used. At each
time point, a portion of the reaction mixture was
withdrawn, diluted with water and then mixed with
CH3CN (at the ratio 31:69), filtered, and analyzed by
HPLC (column: Shodex Asahipak NH2P-50; flow rate:
1 mL/min; mobile phase: 31:69 water/CH3CN; detec-
tion: UV at 210 nm). The amount of GlcNAc in the
reaction mixture was determined from a calibration
curve of GlcNAc standard.
Table 2
Production of GlcNAc from b-chitin by chitinase from B.
cepacia TU09
a
b-Chitin/enzyme
(mg/U)
Digestion time
(day)
% Yield b
GlcNAc
The percent yield of GlcNAc production increased
with the reduction of substrate/enzyme ratio. Although,
at the substrate/enzyme ratio of 100 mg/U, chitinase
from B. licheniformis SK-1 completely hydrolyzed b-
chitin, it gave a mixture of GlcNAc and N,N%-diacetyl-
chitobiose [(GlcNAc)2] (Table 1). The gradual increase
of the GlcNAc/(GlcNAc)2 product ratio with incuba-
tion time implied the presence of low b-N-acetyl-
hexosaminidase (EC 3.2.1.52) activity in the crude
enzyme from B. licheniformis SK-1 under the reaction
conditions. On the other hand, hydrolysis of b-chitin
with chitinase from B. cepacia TU09 gave mostly Glc-
NAc with a trace amount of chitotriose. At the sub-
strate–enzyme ratio of 100 mg/U, a 90% yield of
GlcNAc was obtained within 1 day, and a quantitative
yield was realized upon prolonged incubation (Table 2).
The tightly packed chitin strands of a-chitin are
known to have low susceptibility to enzymatic hydroly-
sis. We found that when chitinase from B. licheniformis
SK-1 was used, it was unable to completely hydrolyze
a-chitin. Only 41% of a-chitin were hydrolyzed in 6
days, even when the concentration of the enzyme used
in the reaction was tenfold over the amount that was
used to completely hydrolyze b-chitin (Table 3). We
400
200
100
1
3
6
1
3
6
1
3
6
31
57
65
62
81
84
90
96
100
a [E]=0.1 U/mL in 0.1 M citrate–phosphate buffer, pH 6.0.
b HPLC yield.
Table 3
Production of GlcNAc from a-chitin by chitinase from B.
licheniformis SK-1
a
a-Chitin/enzyme
(mg/U)
Digestion time
(day)
% Yield
GlcNAc b
10
1
3
6
32
40
41
a [E]=1 U/mL in 0.1 M citrate–phosphate buffer, pH 6.0.
b HPLC yield.

R. Pichyangkura et al. / Carbohydrate Research 337 (2002) 557–559
559
Table 4
GlcNAc. Despite all these beneficial factors in using
bacterial chitinase, care must be taken in further devel-
opment to ensure food safety and enhance cost effi-
ciency for the industrial production of GlcNAc.
Production of GlcNAc from a-chitin by chitinase from B.
cepacia TU09
a
a-Chitin/enzyme
(mg/U)
Digestion time
(day)
% Yield b
GlcNAc
100
33
1
3
7
1
3
7
37
54
57
41
57
85
Acknowledgements
This work was sponsored by Thailand–Japan Tech-
nology Transfer Project (TJTTP) under the Oversea
Economy Cooperation Fund (OECF), Agency of In-
dustrial Science and Technology (AIST) under the In-
stitute for Transfer of Industrial Technology (ITIT)
Fellowship Program, and the Japan International Sci-
ence and Technology Exchange Center (JISTEC) under
the Science and Technology Agency (STA) Fellowship.
We would also like to thank Sunfive Company for
providing a- and b-chitin powder.
a [E]=0.1 U/mL in 0.1 M citrate–phosphate buffer, pH 6.0.
b HPLC yield.
of GlcNAc was achieved after 7 days of incubation
(Table 4). It is worth noting that the hydrolysis of
a-chitin with chitinase from B. cepacia TU09 consists of
two steps. First, there is a rapid hydrolysis step in the
first 24 h, during which we believe the amorphous
portion (ꢀ40%) of the chitin particle is hydrolyzed.
The second step is a slower step, where the remaining
tightly packed chitin is slowly hydrolyzed. Because of
this slower degradation rate, 300 mU/mL of the enzyme
was used to ensure sufficient amount of active enzyme
present throughout the hydrolysis. The isolation and
characterization of the enzymes used here will be pub-
lished elsewhere.
We have demonstrated here for the first time that
chitinase from certain bacteria can completely hy-
drolyze both powdered a- and b-chitin to give GlcNAc
in very high to quantitative yields. The cleanliness of
the reaction, mild conditions, ease of substrate prepara-
tion, and high-production yield undeniably render the
approach of using enzyme more attractive than the
current acid-hydrolysis process for the production of
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