Development of innovative technologies to decrease the environmental burdens associated with using chitin as a biomass resource: Mechanochemical grinding and enzymatic degradation

Article abstract of DOI:10.1016/j.carbpol.2010.10.050

The production of N-acetylglucosamine (GlcNAc, chitin monosaccharide) from crab or shrimp shells generally requires numerous steps and the use of deleterious substances. One goal for researchers is the direct production of GlcNAc and NN′-diacetylchitobiose [(GlcNAc)₂, chitin structural dimeric unit] from both chitin and crab shells. The present study reports the development of an intensive ball "converge" mill for the rapid mechanochemical conversion of chitin or crab shells into amorphous, chitinase-sensitive microparticles. Optimal crab shell grinding parameters were determined, and close to 100% direct degradation of chitin from crab shell to GlcNAc was achieved. This is the first report of using a mechanochemical process with enzymatic degradation to decrease the environmental burdens associated with GlcNAc production from chitin.

Full text of DOI:10.1016/j.carbpol.2010.10.050

Carbohydrate Polymers 83 (2011) 1843–1849
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Development of innovative technologies to decrease the environmental burdens
associated with using chitin as a biomass resource: Mechanochemical grinding
and enzymatic degradation
Yuko S. Nakagawaa, Yasuhiro Oyamaa, Nobuko Kona, Mitsuru Nikaidoa, Koichi Tannoa,
Jun Kogawab, Shoji Inomatab, Ayano Masuic, Akihiro Yamamurac, Mitsuaki Kawaguchic,
Yoshiharu Matahirac, Kazuhide Totania,∗
a Department of Chemical Engineering, Ichinoseki National College of Technology, Ichinoseki 021-8511, Japan
b Machinery Engineering Department, Earthtechnica Co. Ltd., Yachiyo 276-0022, Japan
c R&D of Functional Food Div., Yaizu Suisankagaku Industry Co. Ltd., Yaizu 425-8570, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The production of N-acetylglucosamine (GlcNAc, chitin monosaccharide) from crab or shrimp shells gen-
erally requires numerous steps and the use of deleterious substances. One goal for researchers is the
direct production of GlcNAc and NNꢀ-diacetylchitobiose [(GlcNAc)2, chitin structural dimeric unit] from
both chitin and crab shells. The present study reports the development of an intensive ball “converge” mill
for the rapid mechanochemical conversion of chitin or crab shells into amorphous, chitinase-sensitive
microparticles. Optimal crab shell grinding parameters were determined, and close to 100% direct degra-
dation of chitin from crab shell to GlcNAc was achieved. This is the first report of using a mechanochemical
process with enzymatic degradation to decrease the environmental burdens associated with GlcNAc
production from chitin.
Received 2 September 2010
Received in revised form 21 October 2010
Accepted 21 October 2010
Available online 29 October 2010
Keywords:
Converge mill
Mechanochemical effect
Chitin
Enzymatic degradation
N-acetylglucosamine
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
as a donor substrate of transglycosylation by chitinolytic enzyme
such as lysozyme for synthesis of chitin oligomers that have higher
Chitin is a highly abundant biomass present in crab, shrimp, and
insect shells, as well as fungal cell walls, and is synthesized in nature
at a rate of 1011 tons per year. Chitin is a weakly cation polysac-
charide composed of GlcNAc units covalently connected by ␤-1,4
linkages. Native chitins in crustacean shells are highly crystalline
with strong hydrogen bonding. Chitins have attracted particular
interest for their potential conversion to GlcNAc and oligomers,
which possess versatile functional properties as skin moisturiz-
ers, joint-pain relievers, and antitumoral and antimicrobial agents
(Liang, Chen, Yen, & Wang, 2007; Suzuki et al., 1986; Wang, Lin, Yen,
Liao, & Chen, 2006; Wang et al., 2008). Compared to glucosamine
(GlcN), GlcNAc is chemically stable and has a refreshing, sweet
taste. GlcNAc therefore has tremendous potential as a functional
food or usage for cosmetics.
degree of polymerization or synthesis of novel glycosides (Usui,
Matsui, & Isobe, 1990). Chitin oligomers are known for their elicitor
activities in plants and their immunoenhancing, intestinal regulat-
ing, and bifidobacteria growth-stimulating activities. It is expected
that chitin oligomers could also be used in many applications,
including biopesticides or foods (Hirano, 2004).
In light of the difficulties associated with traditional GlcNAc pro-
duction processes, environmentally compatible and reproducible
enzymatic degradation alternatives are desired. If chitin could be
completely degraded by enzymes, it would not be necessary to
use deleterious substances or to produce excessive amounts of
waste water, allowing the establishment of an environmentally
conscious process. However, crustacean shells are not soluble in
general medium and their crystallinity is potentially too high to
be degraded enzymatically. Furthermore, enzymes are expensive.
To solve these problems, shellfish waste pretreatment to loose the
chitin crystal structure is widely considered to be important. The
direct degradation and separation of ␣-chitin from crab or shrimp
shells are a significant challenge, due to the difficulty in separating
GlcNAc from the crude degradation products derived from shell
proteins.
The chitin product NNꢀ-diacetylchitobiose [(GlcNAc)2] has
potentially milder or other physiological activities compared to the
monosaccharide. (GlcNAc)2 is especially useful when it was used
∗
Corresponding author. Tel.: +81 191 24 4675; fax: +81 191 24 2146.
E-mail address: ktotani@ichinoseki.ac.jp (K. Totani).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.10.050

1844
Y.S. Nakagawa et al. / Carbohydrate Polymers 83 (2011) 1843–1849
Takara Bio, enzyme Y) were used as chitinase, and commercially
Stroke of
powder and
medium ball
available enzyme preparation from Bacillus subtilis (Umamizyme
from Amano enzyme, peptidase U) which is known hyperactive
chitobiase was used. For degradation each substrate, we used
90 U of enzyme E and 108 U of enzyme Y. Enzyme Y has high
chitobiase activity (703 U/g) and produces monosaccharide from
chitin, enzyme E produces (GlcNAc)2 as a major product from
chitin, and peptidase U produces GlcNAc from (GlcNAc)2 (140 U/g).
To obtain monosaccharide after degradation with Enzyme E, we
used peptidase U.
Impact
point
Fixed guide
vane
2.2. Mechanochemical grinding
The grinding equipment comprised a converge mill made by
Makabe Giken (internal capacity, 1000 cm3). The mill structure,
made of two flat zirconian drums, contains a fixed guide vane
within it (Fig. 2). Twenty grams of the three materials and 271 g
of zirconian balls (10 mm dia.) were ground at 500–800 rpm for
30–120 min (Table 1).
Medium ball
2.3. Determination of average particle size
High speed
revolving vessel
The average particle size (median size D50) was determined by a
particle size distributer (Nikkiso, HRA [X-100]). For disperse media,
methanol was used.
Thin powder layer
Fig. 1. Simplified schematic of converge mill. The converge mill has larger amount of
grinding capacity than the planetary ball mill (Tanno et al., 2006; Sato et al., 2006).
Rounding revolving vessel (outer circle) and fixed guide vane made thin powder
layer of substances (inner black circle) onto the inner wall. Fixed guide vane change
the direction of medium balls (small open circles) and crystal structure of substance
is destroyed at a defined focal point as compared to general tumbling ball mill.
2.4. Crystallinity determination by X-ray diffraction (XRD)
Equatorial diffraction profiles were obtained with Cu-K␣ from
a powder X-ray generator (Japan Electronic Organization Co. Ltd.,
JDX-3530) operating at 30 kV and 30 mA. The crystallinity index
was calculated from normalized diffractograms according to equa-
tion below. The intensities of the peaks at [1 1 0] lattice (I110, at
2ꢀ = 20 corresponding to the maximum intensity of chitin) and
Iam at 2ꢀ = 16 (amorphous diffraction) were used to calculate ICR
(Lavall, Assis, & Campana-Filho, 2007).
Recently, we developed a novel “converge” mill (Sato, Asada,
Takeda, & Tanno, 2006; Tanno, Sato, Maruyama, Yamaya, & Fujitaka,
2006) that is a derivative of the medium ball mill. A powder layer
forms on the inner wall of a revolving vessel, which undergoes
intensive impact from a medium ball (Fig. 1). The target materi-
als are most impacted by the medium ball at a defined focal point,
and the generated mechanochemical effect changes the crystal
structure and properties of the materials. This causes the crushed
materials to become amorphous or potentially activated (Takeda
et al., 2009; Van Craeyveld, Delcour, & Courtin, 2008; Van Craeyveld
et al., 2009; Venkataraman & Narayanan, 1998). There are several
types of ball mills using medium balls, such as the conventional
tumbling mill or planetary ball mill. However, the milling time is
much shorter and the amount of sample loaded is larger in the
converge mill.
I1 1 0 − Iam
Crystallinity index (%) =
× 100
I1 1 0
2.5. Scanning electron microscopy (SEM)
The morphology of the fine particles of the ground chitin sam-
ples was observed using SEM (Keyence VE-7800).
2.6. Assay for hydrolytic activity
Fujimoto et al. (2008) and Takeda et al. (2009) previ-
ously developed a successful degradation process combining
mechanochemical pretreatment and enzymatic degradation. We
also greatly enhanced the degradation of cider sawdust by grind-
ing using a converge mill with cellulase. In this study, suitable
conditions for the mechanochemical treatment of chitin were
investigated, resulting in the successful direct degradation of crus-
tacean shells. Using our process, the steps to product GlcNAc
decreased to less than 1/3 (Fig. 2).
Chitinase activity was measured as follows. Reaction mixture
was composed of 0.05% ethylene glycol chitin, 10 mM sodium phos-
phate buffer (pH 6.0) and enzyme solution in total volume of 1.1 ml.
Table 1
Grinding conditions and average particle size of each material.
Materials
Crab shell
Grinding
Average particle
size (D50) (␮m)
rpm
500
Time (min)
30
60
30
60
30
60
30
60
30
60
30
60
15.1
12.0
10.1
10.7
30.5
23.0
22.9
19.5
–
2. Materials and methods
700
500
700
500
700
Crab chitin
2.1. Materials and enzymes
Crab chitin, crab shell, and shrimp chitosan (Yaizu suisankagaku
industry) were used for materials. For the enzymatic degrada-
tion, crude enzyme from Streptomyces griseus (Eikon CHL from
Rakuto kasei industrial, enzyme E) and commercially available
enzyme preparation from Corynebacterium sp. (Yatalase from
Shrimp chitin
90.1
99.8
36.4

Y.S. Nakagawa et al. / Carbohydrate Polymers 83 (2011) 1843–1849
1845
Fig. 2. The traditional method of manufacturing N-acetylglucosamine and decrease the environmental burdens by the innovative technology Left side shows industrially
applied process to make chitin, chitin oligomers and GlcNAc from crustacean shells. Using converge mill for pre-treatment, it is possible to decrease 2/3 steps from traditional
method.
After incubation at 40 ◦C for 30 min, the reaction of the enzyme was
terminated byheatingat 100 ◦Cfor 10 minand the amountofreduc-
ing sugar was determined by the method of Imoto and Yagishita
(1971) using N-acetylglucosamine as a reference compound. One
unit of the enzyme activity was defined as the amount of enzyme
required to release 1 ␮mol of N-acetylglucosamine from ethylene
glycol chitin solution in 1 min at 40 ◦C, pH 6.0.
with 1.8 ml of 10 mM phosphate buffer (pH 4.0–8.0) and 0.2 ml of
10 mg/ml enzyme diluted with 10 mM phosphate buffer (0.1% of
final concentration, about 100 U). The reaction mixture was shaken
at 1400 rpm at 25–60 ◦C, and 0.4 ml of the mixture was harvested
at the appropriate time.
2.8. High-performance liquid chromatography (HPLC)
Chitobiase activity was measured by determining p-nitrophenol
released from p-nitropheny-N-acetyl-␤-d-glucosaminide calori-
metrically at 405 nm. One unit of the enzyme activity was defined as
the amount of enzyme required to release 1 ␮mol of p-nitrophenol
from 2 mM substrate solution in 1 min at 37 ◦C, pH 6.0.
The harvested reaction solution was filtered (pore size 0.45 ␮m,
Advantec) after boiling for 10 min, and centrifuged. The HPLC sys-
tem consisted of an SPD-10A UV detector (Shimadzu), RI-101 RI
detector (Shodex), and LC-10Avp system (Shimadzu). Sugars were
separated on a Shodex SUGAR KS-802 column (Ø 0.8 × 30 cm) using
milliQ water (Millipore) as the mobile phase, at a flow rate of
0.6 ml min−1 at 60 ◦C. GlcNAc and (GlcNAc)2 were detected by mon-
itoring the absorbance at 210 nm. Degradation ratio was calculated
from the square of the peak of the enzymatic reaction mixture and
2.7. Enzymatic degradation of chitin
The reaction mixture was prepared as follows. Twenty micro-
grams of each substrate (1% of final concentration) was mixed

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Y.S. Nakagawa et al. / Carbohydrate Polymers 83 (2011) 1843–1849
Table 2
Yields of GlcNAc and (GlcNAc)2 using activated charcoal–celite column chromatography.
Substrates
Enzymes
GlcNAc (mg)
(GlcNAc)2 (mg)
Total yield (mg)
Yield constant (%)
Degradation ratio (%)
Chitin (1%, 1 g)
E
E + P
E
38
291
10
380
–
180
418
291
190
42
43
29
37
Crab shell (1%, 1 g)
84a
97a
aCalculated by chitin content of crab shell tested: 22.5%.
two standards of mono- and disaccharides. The product ratio was
divided by the substrate concentration and reported as a percent.
but obviously decreased with an increasing number of rotations or
grinding time (Fig. 3(a and d)).
The XRD patterns of untreated and mechanochemical-ground
crab chitin and crab shell are shown in Fig. 3(a and d), respec-
tively. The untreated crab chitin and shell had the same two specific
peaks at 2ꢀ = 9.4◦ and 19.2◦. Peaks in the XRD patterns of materi-
als ground with the converge mill became smaller and smoother,
until eventually no obvious peaks were apparent, especially under
severe conditions. The SEM images confirmed these results (Fig. 3(c
and f)). These ground materials were digested as substrates by the
enzymes.
2.9. Nuclear magnetic resonance (NMR) analysis
Digested products were pretreated as for HPLC, and the
monosaccaride and (GlcNAc)2 peaks were isolated. These fractions
were vacuum concentrated, freeze-dried, and analyzed by 1H or 13
NMR (400-MR, Varian Inc.) in D2O at 25 ◦C.
C
2.10. Isolation and purification of enzymatically degraded
products
3.3. Analysis of the degraded products
The total volume of the enzymatic degradation was scaled to
100 ml (50×), and used the following combination of substrate and
enzymes: (1) chitin and enzyme E, (2) chitin, enzyme E, and pepti-
dase U, (3) crab shell and enzyme E, and (4) crab shell, enzyme E,
and peptidase U. The weight ratio of enzyme E and peptidase U in
combination 2 was 1:2.
Chromatography on an activated charcoal–celite column (Ø
2 × 30 cm, charcoal: celite = 1:1, Wako Pure Chemical Industries)
was performed to purify the digested products. The sample was
loaded onto the column, which had been washed with 50% ethanol,
equilibrated with milliQ water, and eluted with a 0–50% linear gra-
dient of ethanol. The product fractions were vacuum-concentrated
and freeze-dried. Products were analyzed by HPLC to determine
the purity and yield (%) = [(dry weight of product)/(weight of
chitin) × 100]. The extracted chitin content of this crab shell is
22.5%.
The products of enzymatic digestion were analyzed by HPLC.
Fig. 3(b and e) displays the degradation ratios calculated with the
total concentrations of GlcNAc and (GlcNAc)2. The degradation
ratio of ground crab chitin using the converge mill was 10 times
greater than that of untreated substrate (Fig. 3(b)). Increased ves-
sel revolving speeds and grinding times gave higher degradation
ratios. Crab shell showed the same trend (i.e., more severe grind-
ing conditions increased the degradation ratio) (Fig. 3(e)), although
excessive grinding reduced the degradation ratio due to micropar-
ticle aggregation (data not shown).
The HPLC chromatograms from crab chitin and crab shell
degraded by different enzymes are shown in Fig. 4. All enzymes
showed two major peaks (Fig. 4(c–h)), one of which had the same
pattern as the GlcNAc (Fig. 4(a)) or (GlcNAc)2 (Fig. 4(b)) standard.
Enzyme Y mainly produced GlcNAc (Fig. 4(c–d)), enzyme E pro-
duced (GlcNAc)2 (Fig. 4(e–f)), and peptidase U degraded (GlcNAc)2
to GlcNAc (Fig. 4(g–h)). These results indicate that mono- and dis-
accharides were successfully obtained by enzymatic degradation
from ground chitin or crab shells. The corresponding peaks for
mono- and disaccharides were then isolated and analyzed by NMR.
The spectra of both sugars corresponded with the standard prepa-
rations of GlcNAc and (GlcNAc)2, which were confirmed as the
degradation products of chitin and crab shell, respectively (data
not shown).
3. Results
3.1. Determination of suitable conditions for degradation of
chitin materials
All three enzymes could degrade substrates under mildly acidic
(pH 4.0–5.0) conditions. When the substrate concentration was
fixed at 1% (w/v), the enzyme E concentration achieved 50% degra-
dation over a 24-h reaction period. A 0.1% (w/v) enzyme E (around
100 U in 2 ml) was also necessary for degradation. We used an equal
unit of the enzyme Y. The Peptidase U was used combined with
enzyme E and more than 70 U was necessary to obtain GlcNAc from
the degraded product of enzyme E. Since the sodium acetate buffer
(pH 5.8) affected HPLC analysis, chitinases with sodium phosphate
buffer at pH 6.0 and 40 ◦C were used for enzymatic degradation.
3.4. Isolation and purification of the enzymatically degraded
products
GlcNAc resulting from the enzymatic degradation of chitin or
crab shells was isolated from the flow-through of the activated
carbon and celite mixed column. (GlcNAc)2 was isolated from the
binding fraction eluted by a linear ethanol concentration gradient
(Fig. 5(a and c)). The product from fraction 2 (F-2 in Fig. 5(a and
c)) was confirmed by HPLC (Fig. 5(b and d)). Table 2 shows the
yields and purity by HPLC analysis of GlcNAc and (GlcNAc)2 from
chitin and crab shell. We successfully purified 190 mg of GlcNAc
and (GlcNAc)2 from 1 g of crab shell.
3.2. Properties of the grinding materials and enzymatic
degradation ratio
Table 1 displays the grinding conditions and median diame-
ters of the grinding materials. The best combination for grinding
␣-chitin was determined when chromium balls and a stainless mill
were used compared to zirconia (data not shown). The median
diameters were decreased after grinding with the converge mill
(Fig. 1). The particle diameter was immediately decreased by grind-
ing, and crab shell showed the smallest median diameter (10.1 ␮m)
(Table 1). Compared to the particle diameter, crystallinity gradually
4. Discussion
4.1. Degradation and mechanochemical effects
This is the first report of a chitinolytic system utilizing combi-
nations of mechanochemical grinding and enzymatic degradation.

Y.S. Nakagawa et al. / Carbohydrate Polymers 83 (2011) 1843–1849
1847
Fig. 3. Relationship between grinding parameters and degradation ratio of crab chitin or shell. (a–c) Represented crab chitin and (d–f) crab shell. (a) Peaks in the XRD patterns
of chitin ground with different milling. 20 g of crab chitin was mixed with 271 g of 10 mm dia. zirconia ball in 1-l size zirconia pot, and ground at 500–800 rpm for 30–120 min.
(b) Degradation ratio of crab chitin with enzyme E under various grinding parameters. (c) SEM images of micro-particle crab shell chitin grinding with converge mill at
700 rpm. White bar in untreated material represented 200 ␮m and the other bars represented 40 ␮m. (d) Peaks in the XRD patterns of crab shell ground for different milling
condition. 20 g of crab shell was mixed with 271 g of 10 mm dia. zirconia ball in 1-l size zirconia pot, and ground at 500–700 rpm for 30–120 min. (e) Degradation ratio of crab
shell with enzyme E under various grinding parameters. The ratio of crab shell is weight percent of GlcNAc and (GlcNAc)2 toward not chitin but crab shell which contains
chitin in 23%. (f) SEM of micro-particle crab shell grinding with converge mill at 700 rpm. Automatic mortar-40 min corresponds to “Untreated”. White bar in untreated
material represented 200 ␮m and the other bars represented 40 ␮m.

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Y.S. Nakagawa et al. / Carbohydrate Polymers 83 (2011) 1843–1849
The direct enzymatic degradation ratios of untreated crab chitin
(7.1%) and crab shell (4.2%) were 7-fold and 3.5-fold increased,
respectively, by mechanochemical grinding (Fig. 3(b and e)). After
mechanochemical grinding, the crab shell showed the smallest
median particle diameter (10.1 ␮m, Table 1) due to its hard cal-
ciferous materials. Hard materials were ground easily, while soft
and fluffy materials were unsuitable to be made small by the con-
verge mill. We also enzymatically degraded chitosan by enzyme
Y to allow comparison with the other materials. Chitosan is origi-
nally soluble and grinding had no effect on this material (data not
shown). From these results, it can be concluded that the converge
mill is suitable for grinding hard crystal materials, such as chitin
and crab shell.
4.2. Direct degradation of chitin and crab shell
The degradation ratio was calculated using 100% for the weight
of the crab shell (Fig. 3(e)). The direct degradation ratio of the chitin
in crab shell was ∼100%, since the chitin content of the used crab
shell was 22.5%. Direct degradation of both ground chitin and crab
shell to GlcNAc or (GlcNAc)2 was enabled by mechanochemical
grinding. Ilankovan, Hein, Ng, Trung, and Stevens (2006) pro-
duced (GlcNAc)2 from 0.5 vol.% of chitin using 0.1% of commercial
enzymes (pepsin) and the yield was around 10%. Thus, grinding
with the converge mill is very effective for the pretreatment of
chitin or crab shell before enzymatic treatment.
4.3. Degradation products
The degradation products were analyzed by HPLC and NMR,
revealing that the produced monosaccharide and biose were
Fig. 4. HPLC chromatogram of enzymatic degraded product from each substrate.
(a) GlcNAc and (b) (GlcNAc)2 are the chromatogram of standard. From (c) to (h), left
panels represented patterns of crab chitin and right panels represented crab shell.
(c and d) Enzyme Y, (e) and (f) enzyme E and (g and h) enzyme E + peptidase U were
used for degradation.
Breaking down the chitin crystal structure reduced the crys-
tallinity and improved enzymatic degradation (Fig. 3(a, b, d,
and e)), allowing the enzymes to easily access and exert the
catalytic action. Wu and Miao (2008) previously showed that
mechanochemical treatment markedly increases the glucose yield
from enzymatic corn flour hydrolysis. Similar results were observed
by Fujimoto et al. (2008) for lignocellulosic biomass and Van
Craeyveld et al. (2008) showed improvement of extractability and
affects molecular properties of Psyllium seed husk arabinoxy-
lan by ball milling. Crystallinity not only affects the enzymatic
degradation, but also serves as one component of the degradation
ratio.
Fig. 5. Spectrum of charcoal–celite column chromatography and HPLC analysis. (a)
Elution pattern of enzymatic degraded product of crab chitin from the column.
Absorbance (ABS) was observed at 210 nm. (b) HPLC analysis of the product from
crab chitin that separated products at 210 nm and by RI. (c) Elution pattern of crab
shell. (d) HPLC analysis of the product from crab shell.
Mechanochemical grinding with the converge mill was
extremely effective for pretreatment of chitin and crab shell before
enzymatic digestion same as plant cell walls (Takeda et al., 2009).

Y.S. Nakagawa et al. / Carbohydrate Polymers 83 (2011) 1843–1849
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GlcNAc and (GlcNAc)2, respectively. These products were easily
purified using activated charcoal–celite column chromatography
in one step, with a 40% yield and 95% purity. Using hydrochloric
acid, it is hard to control the length of the generated oligomers and
to avoid deacetylation from the C-2 amide residue.
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We have clearly demonstrated for, to the extent of our knowl-
edge, the first time that the mechanochemical pretreatment of
chitin and crustacean shell carries with it significant advantages
for their direct enzymatic degradation. Ground chitin and crab shell
showed significant reduction of crystallinity. With mechanochem-
ical grinding, direct degradation ratio of crab shell became about
100%. The products from enzymatic degradation were determined
as GlcNAc and (GlcNAc)2. We successfully purified 190 mg of Glc-
NAc and (GlcNAc)2 from 1 g of crab shell using activated carbon
and celite mixed column by one-step. To dissolve many problems
such as low oligosaccharide yields, environmental pollution with
massive amounts of discharged water and avoid complicated pro-
duction processes result in an expensive cost of GlcNAc product
from the traditional method, our novel method is effective and
implementable.
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Acknowledgements
This work was supported by grants in 2007 and 2008 from
the Japan Science and Technology Agency (JST), and was partly
supported by the Program for Promotion of Basic and Applied
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