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308
Chemistry Letters 2001
Production of N-Acetyl-D-glucosamine from β-Chitin by Enzymatic Hydrolysis
Hitoshi Sashiwa, Shizu Fujishima, Naoko Yamano, Norioki Kawasaki, Atsuyoshi Nakayama,
Einosuke Muraki, and Sei-ichi Aiba*
Special Division of Human Life Technology, AIST Kansai, National Institute of Advanced Industrial Science and Technology, 1-8-31
Midorigaoka, Ikeda, Osaka 563-8577
(Received January 29, 2001; CL-010082)
N-Acetyl-D-glucosamine, which is a focusing material for
the improvement of osteoarthritis, was obtained from β-chitin
by enzymatic hydrolysis in high yield (76%).
taken out, diluted with H2O (0.4 mL) and CH3CN (1.0 mL), fil-
tered, and directly analyzed by HPLC.6 The amount of GlcNAc
in the reaction mixture was estimated from the calibration curve
of commercial GlcNAc.
N-Acetyl-D-glucosamine (GlcNAc) has been a focusing
material for the improvement of osteoarthritis as well as D-glu-
cosamine (GlcN) hydrochloride or sulfate, which are already
commercialized for this disease.1 These GlcN salts, however,
are not suitable for oral administration owing to their bitter
tastes. In contrast, GlcNAc will be able to apply for oral
administrated drug, because of its sweet taste. Until now,
GlcNAc is mainly produced by acid (concd HCl) hydrolysis of
chitin, which is a mucopolysaccharide composed of repeating
GlcNAc unit and exists in crab shell (α-chitin) or squid pen (β-
chitin). This procedure, however, has some problems such as
high cost, low yield (below 65 %),2 and acidic wastes by use of
concd HCl, etc. Although N-acetylation of GlcN is also possi-
ble to produce GlcNAc, this product is not approval as a natural
type material owing to its chemical modification process (N-
acetylation). Therefore, more effective and milder process to
produce natural type of GlcNAc is required, although there is
no report for these processes. We report herein the enzymatic
production of GlcNAc from β-chitin by use of crude enzymes
like cellulase (Scheme 1).3
In general, β-chitin has more swelling property in water
than α-chitin. Moreover crude enzymes, which well degrade
partially N-acetylated chitosans,3 have some advantage to pro-
duce GlcNAc owing to their low cost and including both endo-
and exo-type of chitinases. So we selected β-chitin and crude
enzymes to produce GlcNAc. Table 1 shows the production of
GlcNAc from β-chitin by various enzymes. Among these crude
enzymes, cellulase Tricoderma viride (T. v.) and cellulase
Acremonium (A.) were effective for the production of GlcNAc
(runs 1 and 2). Hemicellulase, papain, lipase, and pectinase
were not very effective to produce GlcNAc under these condi-
tions. Although the crude preparations of cellulase T. v. and
cellulase A. essentially degrade cellulose, they also degrade
chitin7 or partially N-acetylated chitosans3 owing to including
chitinase, and produce monomer or oligomers. Since the yield
of GlcNAc by cellulase T. v. was almost the same degree as
that by cellulase A., we selected cellulase T. v. for the next
experiment. The yield of GlcNAc by cellulase T. v. was
increased with increasing reaction time or the amount of
enzyme (Table 2). The highest yields (74–76%) of GlcNAc
were shown by 8-day hydrolysis with 2 or 4 folds of cellulase
T. v. against chitin (runs 5 and 7). Instead of the above advan-
tage in crude enzyme, a large amount of enzyme is required for
the production of GlcNAc within short period (ca. 8 days).
Typical procedure is as follows: β-chitin4 (100 mg) was
suspended in 10 mL of 0.1 M AcOH buffer (pH = 4.8). To the
suspension, enzyme5 (100 mg) was added and shaken at 37 °C
for 4 or 8 days. A part (0.1 mL) of the reaction mixture was
From the H and 13C NMR (in D2O) analyses, purified
1
GlcNAc8 produced by enzymatic hydrolysis showed the same
1H and 13C signals as those of a commercial GlcNAc, which
suggests that the chemical structure of the enzymatic
Copyright © 2001 The Chemical Society of Japan
Sashiwa, Hitoshi
