Effect of sub- and supercritical water pretreatment on enzymatic degradation of chitin

Article abstract of DOI:10.1016/j.carbpol.2011.12.007

We examined the effect of sub- and supercritical water pretreatment (300-400 °C, 0.5-15 min) on enzymatic degradation of chitin to N,N′-diacetylchitobiose (GlcNAc)₂. The yield of (GlcNAc) ₂ by enzymatic degradation of supercritical water pretreated chitin at 400 °C for 1.0 min was up to 37%, compared to 5% without the pretreatment. X-ray diffraction (XRD) analysis revealed that the d-spacing and the crystallite size increased by sub- and supercritical water pretreatment, which is indicative of swelling of the chitin. The swelling of the chitin crystal structure improved enzymatic degradation by allowing the enzymes easy access to the chitin.

Full text of DOI:10.1016/j.carbpol.2011.12.007

Carbohydrate Polymers 88 (2012) 308–312
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Effect of sub- and supercritical water pretreatment on enzymatic degradation
of chitin
Mitsumasa Osada∗, Chika Miura, Yuko S. Nakagawa, Mikio Kaihara, Mitsuru Nikaido, Kazuhide Totani
Department of Chemical Engineering, Ichinoseki National College of Technology, Ichinoseki, Iwate 021-8511, Japan
a r t i c l e i n f o
a b s t r a c t
We examined the effect of sub- and supercritical water pretreatment (300–400 ◦C, 0.5–15 min) on enzy-
matic degradation of chitin to N,Nꢀ-diacetylchitobiose (GlcNAc)2. The yield of (GlcNAc)2 by enzymatic
degradation of supercritical water pretreated chitin at 400 ◦C for 1.0 min was up to 37%, compared to 5%
without the pretreatment. X-ray diffraction (XRD) analysis revealed that the d-spacing and the crystallite
size increased by sub- and supercritical water pretreatment, which is indicative of swelling of the chitin.
The swelling of the chitin crystal structure improved enzymatic degradation by allowing the enzymes
easy access to the chitin.
Article history:
Received 3 November 2011
Received in revised form 7 December 2011
Accepted 8 December 2011
Available online 17 December 2011
Keywords:
Supercritical water
Chitin
© 2011 Elsevier Ltd. All rights reserved.
Enzymatic degradation
N-Acetylglucosamine
1. Introduction
The aim of this study is the production of (GlcNAc)2 from chitin
by a combination of sub- and supercritical water pretreatment
N,Nꢀ-Diacetylchitobiose (GlcNAc)2, is
a
dimer of N-
(Tc = 374.3 ◦C, Pc = 22.1 MPa) and enzymatic degradation. There are
some reports that sub- and supercritical water treatment on its
own is not sufficient to obtain (GlcNAc)2 and GlcNAc from chitin
because, under these conditions, they decompose at the same time
as the chitin is hydrolyzed (Sakanishi, Ikeyama, Sakaki, Shibata, &
Miki, 1999; Quitain, Sato, Daimon, & Fujie, 2001; Yoshida, Ehara,
& Saka, 2004; Sato, 2004). In this study, we used pretreatment of
chitin in sub- and supercritical water for enzymatic degradation
and investigated conditions that promote enzymatic degradation
without including decomposition of (GlcNAc)2 and GlcNAc.
acetylglucosamine (GlcNAc). The GlcNAc from crustacean chitin
(␣-chitin) is a versatile functional compound used in skin moistur-
izers, analgesics for joint pain, and as antitumoral and antimicrobial
agents (Liang, Chen, Yen, & Wang, 2007; Liu et al., 2011; Muzzarelli
et al., 2012; Muzzarelli, 2011; Suzuki et al., 1986; Wang, Lin,
Yen, Liao, & Chen, 2006; Wang et al., 2008). (GlcNAc)2 has milder
and additional physiological activities compared to GlcNAc and
it is an attractive building block for the production of oligomers
(Nanjo, Sakai, Ishikawa, Isobe, & Usui, 1989; Usui, Matsui, & Isobe,
1990). Although the market for (GlcNAc)2 and GlcNAc is rapidly
expanding, their production from crab shells involves numerous
steps and requires strong acid because of the crystallinity and the
insolubility of ␣-chitin. In light of the difficulties associated with
the traditional production process for production of (GlcNAc)2
and GlcNAc, environmentally compatible and reproducible alter-
natives for enzymatic degradation are desired. An enzymatic
process that depolymerizes chitin completely would be environ-
mentally acceptable since it would avoid the use of deleterious
substances and the generation of large amounts of wastewater.
However, ␣-chitin is insoluble in water under ambient conditions
and its crystallinity is possibly too high to permit enzymatic
depolymerization.
2. Materials and methods
2.1. Materials and enzymes
Crab chitin (Yaizu Suisankagaku Industry) was used as the
starting material. The size of the flaked chitin was about
3 mm × 3 mm × 0.5 mm. For the enzymatic degradation, a crude
enzyme extract from Streptomyces griseus (Eikon CHL from Rakuto
Kasei Industrial) was used as chitinase. For each substrate, we
used 90 [U] of enzyme. 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. The enzyme produces (GlcNAc)2 as a major
product from chitin, which was then hydrolyzed to GlcNAc by
chitobiase.
∗
Corresponding author. Tel.: +81 191 24 4767; fax: +81 191 24 2146.
E-mail address: osada@ichinoseki.ac.jp (M. Osada).
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.12.007

M. Osada et al. / Carbohydrate Polymers 88 (2012) 308–312
309
50
400 ºC
350 ºC
400
300
200
100
0
Untreated chitin
(GlcNAc)2
GlcNAc
40
30
20
10
0
300 ºC
Supercritical water
treated chitin
(GlcNAc)2
GlcNAc
0
20
40
60
80
Enzyme Reaction Time [h]
0
1
2
3
4
Treatment Time [min]
Fig. 2. Reaction time profile for enzymatic degradation of untreated and supercrit-
ical water treated chitin at 400 ◦C for 1 min (n = 3, bars indicate SDs).
Fig. 1. Internal temperature profile of the reactor (quenched in a water bath at
2 min).
2.5. Characterization of the chitin by X-ray diffraction (XRD) and
infrared red (IR)
2.2. Sub- and supercritical water pretreatment
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 the
Pretreatment of chitin in sub- and supercritical water was con-
ducted in a stainless steel 316 tube reactor of 6 cm3 volume. Chitin
flakes (0.2 g) and water (3 g) were loaded in the reactor, and the
reactor was submerged into a molten-salt bath (KNO3–NaNO3)
kept at 300, 350, and 400 ◦C reaction temperatures. Fig. 1 shows
the internal temperature profile of the reactor at each temperature
setting. The reaction start point was set at the moment when the
reactor was submerged into the molten-salt bath; therefore, the
reaction times include the initial heating period. After a given reac-
tion time, the reactor was removed from the molten-salt bath and
rapidly quenched in a water bath to cool down to room temper-
ature. After cooling, the products were collected from the reactor
and divided into the water-soluble and solid fractions (chitin) using
a membrane filter (pore size: 0.20 ␮m, Millipore). After sub- and
supercritical water pretreatment, the chitin was dried at 90 ◦C for
24 h.
equation 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 the
crystallinity index (Lavall, Assis, & Campana-Filho, 2007).
(I110 − Iam
)
Crystallinity index (%) =
× 100
(2)
I110
The d-spacing of the peaks at [1 1 0] lattice was calculated using
Bragg’s equation
2d sin ꢀ = ꢁ
(3)
where d is the spacing between the planes in the lattice; ꢀ is the
Bragg angle; and ꢁ is the X-ray wavelength.
The crystallite size of chitin at [1 1 0] lattice was calculated using
the Scherrer equation
0.9ꢁ
(H cos ꢀ)
2.3. Enzymatic degradation of chitin
L =
(4)
Enzymatic degradation of chitin was conducted as follows.
20 mg of chitin (1% of final concentration) was mixed with 1.8 mL
of 10 mmol/L phosphate buffer (pH 6.0) and 0.2 mL of 10 mg/mL
enzyme diluted with 10 mmol/L phosphate buffer (0.1% of final con-
centration, approximately 100 U). The reaction mixture was shaken
at 1400 rpm at 40 ◦C, and 0.4 mL of the mixture was harvested at the
appropriate time. Theharvested reactionsolutionwas filtered(pore
size 0.45 ␮m, ADVANTEC) after boiling for 10 min, and centrifuged.
where L is the crystallite size perpendicular to the plane; and H is
the full-width at half-maximum in radians.
The FT-IR spectroscopy of the chitin was measured with a Nico-
let iS10 spectrometer (Thermo Fisher Scientific Inc.).
3. Results
3.1. Effect of the sub- and supercritical water treatment on
enzymatic degradation of chitin
2.4. High-performance liquid chromatography (HPLC)
Fig. 2 shows the yield of (GlcNAc)2 and GlcNAc by enzymatic
degradation of untreated and supercritical water treated chitin
at 400 ◦C for 1 min. For untreated chitin, the yield of (GlcNAc)2
at 72 h was only 5%. The yield of (GlcNAc)2 after treatment with
supercritical water was 37% at 72 h. Fig. 2 indicates that the enzy-
matic degradation was completed by 48 h. The yields of GlcNAc
by enzymatic degradation at 72 h of untreated and supercritical
water treated chitin were only 0 and 1.7%, respectively, therefore
we focused our analysis on (GlcNAc)2. We also analyzed the aque-
ous solution recovered after supercritical water treatment without
subsequent enzymatic treatment; however (GlcNAc)2, and GlcNAc
were not obtained under these conditions.
The HPLC system consisted of a Model 300S ELSD detector
(SofTA), and L-2000 system (Hitachi). Products were separated on a
Shodex SUGAR KS-802 column (Ø 0.8 cm × 30 cm) using ultrapure
water (Millipore) as the mobile phase, at a flow rate of 0.6 mL/min
at 60 ◦C. Yield was calculated from the area of the peak of the enzy-
matic reaction mixture and two standards of (GlcNAc)2 and GlcNAc.
The product yield is defined as given below
weight of (GlcNAc)2or GlcNAc
Product yield (%) =
× 100.
(1)
weight of chitin(20 mg)

310
M. Osada et al. / Carbohydrate Polymers 88 (2012) 308–312
at 400 ◦C. After 2 min of supercritical water treatment at 400 ◦C, the
50
40
30
20
10
0
(a)
chitin flakes crumbled and became a powder. At 350 and 300 ◦C,
the chitin flakes crumbled after 5 and 15 min of subcritical water
treatment, respectively.
400 ºC
The results shown in Fig. 4 were obtained by XRD analysis.
Fig. 4(a) and (b) shows the effect of supercritical and subcrit-
ical water treatment temperature and time on the crystallinity
index of chitin flakes. At 400 ◦C, the crystallinity index decreased
slightly up to 1.5 min and decreased drastically by 2 min. The crys-
tallinity index at 350 ◦C also decreased slightly up to 3 min and then
decreased drastically. In contrast, the crystallinity index was almost
unchanged after treatment at 300 ◦C
Fig. 4(c) and (d) shows the effect of supercritical and subcrit-
ical water treatment on the d-spacing of chitin flakes. At 400 ◦C,
the d-spacing increased markedly during the first 1.5 min, indi-
cating that the lattice spacing became wider. The d-spacing then
decreased slightly by 2 min. The d-spacing at 350 ◦C also increased
during the first 3 min and then decreased by 5 min. At 300 ◦C, the
d-spacing gradually increased for the first 5 min but did not change
significantly after that time.
Fig. 4(e) and (f) shows the effect of supercritical and subcritical
water treatment on the crystallite size of chitin flakes. At 400 ◦C, the
crystallite size increased with treatment time, indicating that the
crystal grows in response to supercritical water treatment, how-
ever, crystallite size started to decrease at 2 min. The crystallite
size also increased up to 3 min at 350 ◦C, after which it decreased.
At 300 ◦C, the crystallite size gradually increased for the first 5 min
but did not change significantly after that time.
Fig. 5 illustrates the FT-IR spectra of chitin before and after the
supercritical water treatment. The FT-IR spectra were not changed
for the first 1.5 min. At 2 min, the hydrogen bonds in the chitin
around 3200–3400 cm−1 retracted and the cleavage of the cyclic
ether linkage around 1000 cm−1 in the glucosamine skeleton unit
was enhanced. The ether linkages around 1400 cm−1 between the
N-acetylglucosamine skeleton unit were hydrolyzed at 2 min of
supercritical water treatment. The deacetylation reaction of the N-
acetyl group (C O at 1700 cm−1) at C-2 of the N-acetyl glucosamine
unit occurred at 2 min of supercritical water treatment. FT-IR spec-
troscopy showed that the chitin structure was stable up to 1.5 min
at 400 ◦C.
0
1
2
Supercritical Water Treatment Time
[min]
50
40
30
20
10
0
(b)
350 ºC
300 ºC
0
5
10
15
Subcritical Water Treatment Time
[min]
Fig. 3. Effect of (a) supercritical and (b) subcritical water pretreatment time on
enzymatic degradation of chitin, using an enzyme reaction time of 48 h (n = 3, bars
indicate SDs).
4. Discussion
Fig. 3 shows the temperature effect of (a) supercritical and
(b) subcritical water treatment on the yield of (GlcNAc)2 after
enzymatic degradation for 48 h. At 400 ◦C, the yield of (GlcNAc)2
increased with treatment time and reached 37% at 1 min, after
which it decreased to approximately 2% at 2 min. At 350 ◦C, the
peak of (GlcNAc)2 yield was at about 3 min, and at 300 ◦C, the yield
of (GlcNAc)2 peaked at around 10 min. Therefore, the time at which
the maximal yield occurred after treatment in sub- and supercriti-
cal water increased with decreasing temperature. Interestingly, the
maximum yields of (GlcNAc)2 were similar at each temperature,
with values in the 30–40% range.
The conversion of microcrystalline cellulose to cellooligosac-
charides and glucose in supercritical water has been reported to
proceed through the following steps (1) swelling, (2) dissolution,
and (3) hydrolysis (Sasaki, Adschiri, & Arai, 2004). In this study, we
used a batch type reactor, in which the treatment times included
heat-up time (Fig. 1). The chitin was stable and was not dena-
tured up to 0.5 min at 400 ◦C; therefore, the yield of (GlcNAc)2 was
not significantly higher than that for untreated chitin. Most chitin
swelling occurred between 1.0 and 1.5 min, and the swelling of the
chitin was consistent with the shape of the chitin flakes in this
time interval. The swollen chitin in supercritical water underwent
rapidly cooling. Therefore, the crystallinity index was maintained,
although the d-spacing increased slightly due to the swelling. The
increase of d-spacing causes an increase of the crystallite size. After
2 min at 400 ◦C, dissolution and hydrolysis of the chitin occurred,
as indicated by XRD analysis (Fig. 4(a), (c), and (e)). The FT-IR
spectra were also changed (Fig. 5), and this was associated with
chitin flake crumbling. We analyzed the solution recovered after
2 min of supercritical water treatment; however, (GlcNAc)2 and
GlcNAc were not detected, indicating that these compounds imme-
diately decomposed in supercritical water, as reported in literature
(Sakanishi et al., 1999; Quitain et al., 2001). At 350 and 300 ◦C, chitin
In absence of water, treatment at 400 ◦C for 1 min resulted in
the chitin flacks becoming a black solid resembling char. The yield
of (GlcNAc)2 by enzymatic degradation of the black solid was 0%,
indicating that the substrate might be denatured. Therefore, these
results confirmed the requirement for water in the pretreatment of
chitin.
3.2. Properties of the sub- and supercritical water treated chitin
The
size
of
untreated
chitin
flakes
was
about
3 mm × 3 mm × 0.5 mm and no significant difference in size
was observed until after 1.5 min of supercritical water treatment

M. Osada et al. / Carbohydrate Polymers 88 (2012) 308–312
311
100
90
80
70
60
50
40
100
(a)
(b)
90
80
70
60
350 ºC
300 ºC
400 ºC
50
40
0
1
2
0
5
10
15
Supercritical Water Treatment Time
[min]
Subcritical Water Treatment Time
[min]
0.468
0.464
0.460
0.456
0.452
0.468
0.464
0.460
0.456
0.452
(c)
(d)
350 ºC
300 ºC
400 ºC
0
1
2
0
5
10
15
Subcritical Water Treatment Time
Supercritical Water Treatment Time
[min]
[min]
15
15
(e)
(f)
14
13
12
11
10
14
13
12
11
10
350 ºC
300 ºC
400 ºC
0
1
2
0
5
10
15
Supercritical Water Treatment Time
[min]
Subcritical Water Treatment Time
[min]
Fig. 4. Effect of sub- and supercritical water pretreatment time on (a-b) crystallinity index, (c-d) d-spacing, and (e-f) crystallite size of chitin.
swelling occurred up to 3 and 10 min, respectively, followed by dis-
solution and hydrolysis of the chitin. These results indicate that
there is an optimum pretreatment time at each temperature for
chitin swelling without dissolution and hydrolysis.
of chitin with phosphoric acid or hydrochloric acid at ambient
temperature resulted in a high enzymatic degradation ratio, but
these pretreatments did not affect the chitin (Ilankovan, Hein, Ng,
Trung, & Stevens, 2006). These results indicate that a decrease of
the crystallinity index is not essential for promoting the enzymatic
degradation of chitin.
The d-spacing of the chitin increased after sub- and super-
critical water treatment, leading to an increase in crystallite size.
This indicated that hydrogen bonds between hydroxyl groups of
the chitin chains had become weak and the treated chitin had
become swollen. Therefore, there was an increase in the number
of the hydroxyl groups without forming hydrogen bonds, which
was supported by the gradual decrease in the FT-IR spectra at
3200–3400 cm−1 at 1.0 and 1.5 min (Fig. 5). The free hydroxyl
We previously found that the crystallinity index had a major
influence on enzymatic degradation of chitin (Nakagawa et al.,
2011). Mechanochemical grinding with a ball mill resulted in a
decrease in the crystallinity index of chitin and a corresponding
increase in the enzymatic degradation ratio. In this study, the crys-
tallinity index did not change significantly up to 1.5 min (at 400 ◦C),
3 min (at 350 ◦C), and 15 min (at 300 ◦C) as shown in Fig. 4(a) and
(b); however, the yield of (GlcNAc)2 showed peaks at earlier treat-
ment times of 1.0 min (at 400 ◦C), 3 min (at 350 ◦C), and 10 min
(at 300 ◦C) as shown in Fig. 3. In a separate study, pretreatment

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M. Osada et al. / Carbohydrate Polymers 88 (2012) 308–312
2.0 min
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We have demonstrated for the first time that sub- and super-
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This work was supported by the Program for Promotion of Basic
and Applied Researches for Innovations in Bio-oriented Industry.