Production of N-acetyl-D-glucosamine from alpha-chitin by crude enzymes from Aeromonas hydrophila H-2330.

Article abstract of DOI:10.1016/S0008-6215(02)00034-4

The selective and efficient production of N-acetyl-D-glucosamine (GlcNAc) was achieved from flake type of alpha-chitin by using crude enzymes derived from Aeromonas hydrophila H-2330.

Full text of DOI:10.1016/S0008-6215(02)00034-4

Carbohydrate Research 337 (2002) 761–763
www.elsevier.com/locate/carres
Note
Production of N-acetyl- -glucosamine from a-chitin by crude
D
enzymes from Aeromonas hydrophila H-2330
Hitoshi Sashiwa,a Shizu Fujishima,a Naoko Yamano,a Norioki Kawasaki,a
Atsuyoshi Nakayama,a Einosuke Muraki,a Kazumi Hiraga,b Kohei Oda,b
Sei-ichi Aibaa,*
aGreen Biotechnology Research Group, The Special Di6ision for Human Life Technology, National Institute of Ad6anced Industrial Science and
Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan
bDepartment of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
Received 5 July 2001; received in revised form 21 November 2001; accepted 6 February 2002
Abstract
The selective and efficient production of N-acetyl- -glucosamine (GlcNAc) was achieved from flake type of a-chitin by using
D
crude enzymes derived from Aeromonas hydrophila H-2330. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Enzymatic hydrolysis; GlcNAc; Osteoarthritis
D
-Glucosamine and N-acetyl-
D
-glucosamine (Glc-
of GlcNAc oligomers (G2–G7: dimer–heptamer) was
negligible. This fact suggests that both endo- and exo-
type chitinases exist in crude enzymes A and produce
GlcNAc selectively by cooperation. To achieve selective
production of GlcNAc, the enzymatic activity (mU/mg)
of endo- and exo-type chitinase was evaluated (Scheme
1). Higher exo-chitinase activity was observed than that
of endo-chitinase, thus demonstrating that chitin was
hydrolyzed slowly by endo-chitinase and produced Glc-
NAc oligomers.
NAc) have attracted much attention owing to their
therapeutic activity in osteoarthritis. They have also
been evaluated as a food supplement.1–3 GlcNAc is
more suitable than
D-glucosamine for oral administra-
tion because of its sweet taste. GlcNAc is mainly pro-
duced by acid (concd HCl) hydrolysis of chitin. This
procedure, however, has some problems such as low
yield (below 65%)4 or acidic wastes. Most recently, we
have reported the production of GlcNAc in good yield
from b-chitin derived from squid pen by use of cellu-
lases.5 These crude enzymes, however, did not effi-
ciently hydrolyze a-chitin derived from crab or shrimp
shell, which is more available, at low cost and more
widely distributed in nature compared with b-chitin.
The present work describes the effective production of
GlcNAc from a-chitin by crude enzymes derived from
Aeromonas hydrophila (A).6 Fig. 1 shows a typical
HPLC chromatogram of the hydrolyzate. Since the
activity of the crude enzyme preparation tends to de-
crease at higher temperature, the hydrolysis was carried
out at 17 °C. The selective production of GlcNAc was
observed during continuous hydrolysis. The production
Fig. 2 shows the time courses on the production of
GlcNAc from various a-chitins by crude enzymes A. In
all cases, similar behavior was observed on the hydroly-
sis of a-chitin and GlcNAc was produced in 66–77%
(mol/mol) yield from these a-chitins after 10 days
(Table 1). The Mw of a-chitin measured by GPC with
hexafluoro-2-propanol (HFP) as eluent was independent
of the degradability. To confirm the chemical structure
of hydrolyzate, crude enzymes A was dialyzed before
hydrolysis to remove low Mw of components. From the
1H NMR analysis, the hydrolyzate by crude enzymes A
1
showed the good agreement in H NMR signals with
authentic GlcNAc.
Most recently, we found out that three kinds of
enzyme (Mw=90, 72, and 62 kDa from SDS-PAGE
analysis) were included in crude enzymes A. The de-
* Corresponding author. Fax: +81-727-519628.
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00034-4

762
H. Sashiwa et al. / Carbohydrate Research 337 (2002) 761–763
Fig. 1. HPLC chromatogram of the hydrolyzate of a-chitin by crude enzymes A. Condition, see Section 1.
tailed study on the enzymatic property of separated
three enzymes will be published in near future. The
selective production of GlcNAc from a-chitin could be
achieved by crude enzymatic hydrolysis in good yield
(77%). This process will be useful for the efficient
production of GlcNAc.
and Koyo Chemical Co., Ltd., respectively. Powdered
a-chitin (pulverized to 3.8 mm, crab shell) was supplied
from Sunfive Co., Ltd. Crude enzymes derived from A.
hydrophila H-2330 was prepared in our laboratory
(Kyoto Institute of Technology) according to the
previous report.6 Chitin (20 mg) was suspended in 4 mL
of 20 mM phosphate buffer (pH 7.0) containing crude
enzymes A (7.4 mg) and EDTA 2 Na (5 mM). The
suspension was shaken at 17 °C. After the prescribed
time, a part of the reaction mixture (10–20 mL) was
taken out, diluted with water (0.48–0.49 mL) and
CH3CN (1.0 mL), filtered, and analyzed by HPLC. The
amount of GlcNAc in the reaction mixture was
estimated from the calibration curve of standard
GlcNAc. Yield of GlcNAc was calculated as follows.
1. Experimental
Materials.—a-Chitin (derived from crab shell) of
flake A (\50 mm,) and flake B (\50 mm) were
purchased from Wako Pure Chemical Industries Ltd.
Fig. 2. Time courses on the production of GlcNAc from
a-chitin by crude enzymes A. ꢀ, flake-A (\50 mm); ꢁ,
flake-B (\50 mm); ꢂ, fine powder (3.8 mm). a-Chitin: [S]=5
mg/mL; crude enzymes A: [E]=1.85 mg/mL; pH 7.0; 17 °C.
Scheme 1.

H. Sashiwa et al. / Carbohydrate Research 337 (2002) 761–763
763
estimated by HPLC from the production of GlcNAc
using (GlcNAc)2 as substrate. One unit means 1 mmol
of GlcNAc produced after 1 min of hydrolysis per 1 mg
of protein.
Table 1
Production of GlcNAc from a-chitin by crude enzymes A a
b
Entry a-Chitin
(mg/mL)
Mn
Mw
Yield (%)
Chemical analysis of hydrolyzate from h-chitin.—
Crude enzymes A containing EDTA 2 Na (5 mM) in 20
mM phosphate buffer (25 mL) was dialyzed using
Vivapore dialysis kit (Mw cut off=7500 of polyether-
sulfone membrane, Vivascience Co.) to remove low Mw
of components. Remained high Mw of fraction (2 mL)
was diluted to 25 mL with water. To a solution was
added a-chitin (500 mg) and shaken at 17 °C. After 10
days, the hydrolyzate was dialyzed by cellulose mem-
brane (Mw cut off=12,000) and low Mw of fraction
was collected, evaporated, and dried (350 mg, 70%
(w/w) from chitin). Finally, the hydrolyzate was dis-
1
2
3
flake-A (5)
flake-A (20)
flake-B (5)
flake-B (20)
flake-B (20)
powder (5)
powder (20)
180,000 1,200,000
180,000 1,200,000
77
77
69
66
64
66
64
130,000
130,000
130,000
50,000
390,000
390,000
390,000
180,000
180,000
4
5 c
6
7
50,000
a Enzymes A, 1.85 mg/mL; pH 7.0 (4 mL); 17 °C; 10 days.
b Flake-A, Wako; flake-B, Koyo TCL; powder, fine powder
(3.8 mm).
c Enzymes A was dialyzed before hydrolysis (see Section 1).
1
solved in D2O and the H NMR spectrum taken (JEOL
Alpha-500 NMR spectrophotometer).
Yield (%)=GlcNAc produced (mol)/
repeating unit (mol) of chitin added×100
Acknowledgements
Measurements.—HPLC analysis was performed on a
Tosoh LC-8020 apparatus (column, Shodex Asahipak
NH2P-50; rt; 7:3 CH3CN–water; flow rate=1.0 mL/
min; injection, 0.1 mL; detection, UV at 210 nm). GPC
analysis of a-chitin was performed as follows. Chitin
(10 mg) was suspended in hexafuluoro-2-propanol
(HFP: 2 mL) containing 0.1 mM CF3CO2Na. After 3
days, the solution was filtered (0.45 mm) and applied for
GPC (apparatus, Tosho HLC-8220; column, Tosho
TSK-gel super H-RC and HM-N; eluent, HFP contain-
ing 0.1 mM CF3CO2Na; standard, polymethyl
methacrylate; flow rate, 0.2 mL/min; temp, 40 °C; de-
tector IR).
Analysis of enzymatic acti6ity.-.—endo-Chitinase ac-
tivity was estimated from increasing the reducing-end
groups by the modification of Schale’s procedure.7
Since chitin was insoluble in aqueous solvent, 70%
deacetylated chitin which was soluble in acidic water
(pH 4) was used as substrate. One unit means 1 mmol of
reducing-end group produced after 1 min of hydrolysis
per 1 mg of protein. The exo-chitinase activity was
We are indebted to Sunfive Co., Ltd. for supplying
a-chitin fine powder.
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