Selective N-deacetylation of p-nitrophenyl N,N'-diacetyl-β-chitobioside and its use to differentiate the action of two types of chitinases

Article abstract of DOI:10.1016/S0008-6215(99)00002-6

We report the synthesis of a novel compound for chitinase assays, p-nitrophenyl 2-acetamido-4-O-(2-amino-2-deoxy-β-D-glucopyranosyl)-2-deoxy-β-D-glucopyranoside [GlcNGlcNAc-pNP] by selective N-deacetylation of p-nitrophenyl 2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-β-D-glucopyranoside [(GlcNAc)₂-pNP] using a purified chitin deacetylase isolated from Colletotrichum lindemuthianum ATCC 56676. FABMS, ¹H NMR, and ¹³C NMR analyses confirmed the structure of this new compound. This disaccharide derivative can be used to distinguish special chitinases that effectively remove partially deacetylated parts of substrates within a mixture of chitinases which degrades (GlcNAc)₂-pNP. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00002-6

Carbohydrate Research 316 (1999) 173–178
Selective N-deacetylation of p-nitrophenyl
N,N%-diacetyl-b-chitobioside and its use to differentiate
the action of two types of chitinases
Ken Tokuyasu a,*, Hiroshi Ono a, Yuki Kitagawa b, Mayumi Ohnishi-Kameyama a,
Kiyoshi Hayashi a, Yutaka Mori a
a National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba,
Ibaraki 305-8642, Japan
b Graduate School of Engineering, Nagoya Uni6ersity, Chikusa, Nagoya 464-0814, Japan
Received 5 May 1998; accepted 1 November 1998
Abstract
We report the synthesis of a novel compound for chitinase assays, p-nitrophenyl 2-acetamido-4-O-(2-amino-2-de-
oxy-b- -glucopyranosyl)-2-deoxy-b- -glucopyranoside [GlcNGlcNAc-pNP] by selective N-deacetylation of p-nitro-
phenyl 2-acetamido-4-O-(2-acetamido-2-deoxy-b- -glucopyranosyl)-2-deoxy-b- -glucopyranoside [(GlcNAc)2-pNP]
D
D
D
D
1
using a purified chitin deacetylase isolated from Colletotrichum lindemuthianum ATCC 56676. FABMS, H NMR,
and 13C NMR analyses confirmed the structure of this new compound. This disaccharide derivative can be used to
distinguish special chitinases that effectively remove partially deacetylated parts of substrates within a mixture of
chitinases which degrades (GlcNAc)2-pNP. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Chitin deacetylase; Colletotrichum lindemuthianum; p-Nitrophenyl 2-acetamido-4-O-(2-amino-2-deoxy-b-
nosyl)-2-deoxy-b- -glucopyranoside; b-N-Acetylhexosaminidase; Chitinase
D-glucopyra-
D
glycosyl hydrolases which hydrolyse the N-
acetyl-b- -glucosaminide bonds of chitin and
1. Introduction
D
its oligomers, and are responsible for a variety
of phenomena such as (1) defense from inva-
sion [2,3], (2) penetration of the cell wall for
invasion [4,5], (3) control of cell surface struc-
tures [6], and (4) metabolism of chitin for
utilization as energy, carbon and nitrogen
sources [7]. It is known that chitinases greatly
differ in their properties [8], and in order to
elucidate their biological functions, research
on the properties of chitinases has been per-
formed intensively [8–10].
Chitin is among the most abundant bio-
polymers on earth and is a linear polymer of
N-acetyl- -glucosamine residues. It is found
D
on the cell surface of fungal cell walls and is a
component of arthropod integuments. Some
of the GlcNAc residues in chitin are deacety-
lated in its natural form [1]. Chitinases are
Abbre6iations: GlcN, 2-amino-2-deoxy-
2-acetamido-2-deoxy- -glucose; (GlcNAc)2, N,N%-diacetylchi-
D-glucose; GlcNAc,
D
tobiose; GlcNGlcNAc, (GlcNAc)2 derivative which is N-
deacetylated at the sugar residue on the non-reducing end;
GlcNAcGlcN, (GlcNAc)2 derivative which is N-deacetylated
at the sugar residue at the reducing end; GlcNAc-pNP,
Chitinase activity has been estimated by
methods such as the turbidimetric [11], visco-
metric [12], and reducing sugar assays [13]. In
addition, the colorimetric method using the
p-nitrophenyl 2-acetamido-2-deoxy-b-
D-glucopyranoside.
* Corresponding author. Fax: +81-298-387996.
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00002-6

174
K. Tokuyasu et al. / Carbohydrate Research 316 (1999) 173–178
chitobioside derivative, (GlcNAc)2-pNP, was
found to be useful for the facile and sensitive
estimation of chitinase activity, especially for
bacterial chitinases [13,14]. Herein, we have
modified (GlcNAc)2-pNP into a novel com-
pound, GlcNGlcNAc-pNP, to remove back-
ground activity due to contaminating
b-N-acetylhexosaminidases, which may de-
grade (GlcNAc)2-pNP stepwise from its non-
8020, Tosoh Co., Ltd.), an integrator (Power-
Chrom 2.0.7, ADInstruments), and
a
prepacked column of Lichrospher 100 RP-18
(e) (Cica-Merck). The mobile phase was
methanol/0.1% trifluoroacetic acid (7:93), the
flow rate was 0.5 mL/min, and the operating
temperature was 25 °C. Detection of each
compound was performed by measuring ab-
sorbance at 300 nm. After the reaction was
complete, stirring of the reaction mixture was
stopped and the supernatant fraction was re-
covered. A part of the supernatant (5 mL) was
then applied to a reverse-phase column (Sep-
Pak plus C-18 (1 mL), Waters Corporation)
equilibrated with water, and the column was
eluted with 3 mL of methanol. The eluted
fraction (3 mL) was evaporated, and the white
solid residue (6.19 mg) was used in further
studies.
reducing
end,
finally
liberating
the
yellow-colored p-nitrophenol. Furthermore,
we found that apparently identical chitinases
from one microorganism can be distinguished
according to their specificities with this novel
substrate.
2. Experimental
Enzyme assays of glycosidases using pNP-
deri6ati6es as the substrates.—GlcNAc-pNP,
(GlcNAc)2-pNP, or GlcNGlcNAc-pNP was
used as the substrate for chitinase and b-N-
acetylhexosaminidase assays at the final con-
centration of 2 mM. The reaction mixture (20
mL) composed of the substrate (2 mM), 20
mM NaH2PO4 –Na2HPO4 buffer (pH 6.0),
and an aliquot of the enzyme was incubated
for 60 min at 30 °C, and then 80 mL of 1 M
Na2CO3 was added to stop the reaction and to
increase the pH. Absorbance of the liberated
p-nitrophenol was measured at 405 nm using
a spectrophotometer (DU-600, Beckman) or a
microplate reader (model 3550, Bio Rad). Es-
timation of the liberated sugar was carried out
by HPAEC on a Dionex DX-300 equipped
with a pulsed amperometric detector (Dionex
Co.). The pre-packed column was a Dionex
CarboPac™ PA1 analytical column (4×250
mm) accompanied by a CarboPac™ PA1
guard column (4×50 mm). The mobile phase
was 15 mM NaOH, and the column was
washed with 100 mM NaOH and a mixture of
1 M NaOAc and 100 mM NaOH after each
analysis. The operating temperature was
25 °C, the flow rate was 1.0 mL/min, and the
injection volume was 10 mL.
Materials.—(GlcNAc)2-pNP and GlcNAc-
pNP were purchased from Seikagaku Kogyo
Co., Japan. Chitinase preparation from Strep-
tomyces griseus and b-N-acetylhexosaminidase
from Penicillium oxalicum were purchased
from Sigma and Seikagaku Kogyo Co., re-
spectively. GlcNAcGlcN was kindly donated
by Dr K. Ohishi in the Numazu Industrial
Research Institute of Shizuoka Prefecture [15].
All other chemicals were reagent grade.
Deacetylation of (GlcNAc)2-pNP.—Chitin
deacetylase (E.C.3.5.1.41) from Colletotrichum
lindemuthianum (ATCC56676) was purified by
the method of Tokuyasu and co-workers [16].
Deacetylation of (GlcNAc)2-pNP was carried
out by the method of Tokuyasu and co-work-
ers [17] with slight modification. The reaction
mixture (10 mL) contained (GlcNAc)2-pNP as
the substrate (0.2%, w/v), 20 mM (final)
sodium tetraborate/HCl buffer (pH 8.5),
purified chitin deacetylase solution (0.15 U/
mL), and 0.15 mL/mL (wet volume, equili-
brated with the same buffer) of anion-
exchange resin (Q Sepharose Fast Flow, Phar-
macia), which was added in order to remove
chitin deacetylase from the reaction mixture
for reuse. The deacetylation reaction was per-
formed at 30 °C with stirring by a magnetic
stirrer, and aliquots were sampled for moni-
toring by HPLC. Monitoring of the reaction
was carried out on an HPLC system (Tosoh
Co., Ltd.) equipped with a UV monitor (UV-
Separation of the chitinase preparation by gel
filtration.—Chitinase preparation from S.
griseus was loaded on a gel filtration column
(Superose HR 12 10/30, Pharmacia Biotech.)

K. Tokuyasu et al. / Carbohydrate Research 316 (1999) 173–178
175
equilibrated with 20 mM NaH2PO4–
Na2HPO4 buffer (pH 6.0) containing 150 mM
NaCl, and eluted with the same solution (flow
rate: 0.2 mL/min). The amounts of eluted
proteins were estimated by measuring the ab-
sorbance at 280 nm. Fractions of 0.5 mL were
collected and p-nitrophenol-liberating activi-
ties using GlcNAc-pNP, (GlcNAc)2-pNP, and
GlcNGlcNAc-pNP as substrates were mea-
sured in aliquots of the collected fractions.
Analytical methods.—The FAB mass spec-
trum of the reaction product was obtained
using a JEOL JMS-SX102A mass spectrome-
ter. The positive-ion mode was used for the
analysis and glycerol was used as the matrix.
NMR spectra were recorded on a Bruker
DRX-600 spectrometer at 303 K using inverse
5 mm QXI (1H/13C, 15N, 31P; xyz-gradient)
and QNP (X (13C, 15N, 31P)/1H; z-gradient)
probeheads. DQF-COSY, TOCSY, HMQC
and HMBC experiments were carried out to
the possibility that the reaction product was
GlcNAcGlcN was eliminated.
The structure of the deacetylation product
was determined by FABMS and NMR spec-
troscopy: FABMS (glycerol) m/z 526 [M+
1
Na]+, 504 [M+H]+, 365 [M−pNP]+; H
NMR (600.13 MHz, D2O) l 1.230 (3 H, s,
acetyl), 2.688 (1 H, dd, J 9.7, 8.2 Hz, H-2%),
3.382 (1 H, dd, J 9.7, 8.5 Hz, H-3%), 3.410 (1
H, dd, J 9.4, 8.5 Hz, H-4%), 3.503 (1 H, ddd, J
9.4, 5.9, 2.2 Hz, H-5%), 3.741 (1 H, dd, J 12.4,
5.9 Hz, H-6%), 3.823-3.879 (3 H, m, H-3, H-4,
H-5), 3.918 (1 H, dd, J 12.4, 2.2 Hz, H-6),
3.976 (1 H, br d, J 12.0 Hz, H-6), 4.045–4.081
(1 H, m, H-2), 4.515 (1 H, d, J 8.2 Hz, H-1%),
5.330 (1 H, d, J 8.5 Hz, H-1), 7.172–7.199 (2
H, m (A2X2 type), H-pNP-3), 8.230–8.257 (2
H, m (A2X2 type), H-pNP-2); 13C NMR
(150.90 MHz, D2O) l 23.78 (acetyl methyl),
56.68 (C-2), 58.40 (C-2%), 61.71 (C-6), 62.35
1
13
assign H and C signals, and all NMR data
were reported in ppm (l) downfield from
Me4Si. The purified sample (2 mg) was dis-
solved in D2O (450 mL) for NMR analyses,
and 2-methyl-2-propanol was used as the in-
ternal standard.
3. Results and discussion
Previously we found that the chitin deacety-
lase from C. lindemuthianum deacetylated only
the N-acetyl group of the GlcNAc residue at
the non-reducing end of (GlcNAc)2 to give
GlcNGlcNAc [17]. Here we focussed on using
(GlcNAc)2-pNP as the substrate for enzymatic
deacetylation, expecting to convert it into a
novel compound, GlcNGlcNAc-pNP. Indeed,
the chitin deacetylase quantitatively converted
the substrate into a new compound giving a
single HPLC peak, which differed from the
peak of (GlcNAc)2-pNP (data not shown).
The reaction product was degraded by a chit-
inase preparation from Streptomyces griseus,
and the liberated sugar was estimated using
HPAEC (Fig. 1). Under the HPAEC condi-
tions, a single peak having the same retention
time as GlcNGlcNAc was detected (Fig. 1(b)).
GlcNAcGlcN eluted at around 23 min (data
not shown) under the HPAEC conditions, so
Fig. 1. (a) HPAEC profiles of standard sugars and (b) the
compound formed by chitinase treatment of the novel sub-
strate prepared from (GlcNAc)2-pNP.

176
K. Tokuyasu et al. / Carbohydrate Research 316 (1999) 173–178
graded by exo-type enzymes. For example,
Usui and co-workers [20] enzymatically syn-
thesized a novel compound, p-nitrophenyl 45-
O-b-D
-galactosyl-a-maltopentaoside, as
a
substrate for human alpha amylases in which
a galactose residue was introduced at its non-
reducing end to protect the structure from
attack by exo-type glucosidases. The same
effect can be expected when GlcNGlcNAc-
pNP is used as a substrate for b-N-acetyl-
hexosaminidases.
Fig. 2. Structure of the deacetylation product from (Glc-
NAc)2-pNP.
(C-6%), 71.28 (C-4%), 73.60 (C-3), 76.71 (C-5),
77.29 (C-3%), 77.90 (C-5%), 79.66 (C-4), 100.16
(C-1), 104.44 (C-1%), 118.24 (C-pNP-3), 127.80
(C-pNP-2), 144.42 (C-pNP-4), 163.36 (C-pNP-
Conversely, we expected chitinases which
degraded (GlcNAc)2-pNP would degrade Glc-
NGlcNAc-pNP as well. When the chitinase
preparation from S. griseus was used, it de-
graded GlcNGlcNAc-pNP to liberate Glc-
NGlcNAc and p-nitrophenol (data not
shown). Ohtakara and co-workers [21] re-
ported the degradation of partially N-acetyl-
ated chitosan by the same chitinase
preparation after a partial purification, and
reported the structures of the reaction prod-
ucts in terms of the distribution of GlcNAc
residues and GlcN residues. In their report, it
was shown that this enzyme liberated Glc-
NGlcNAc from polymeric chitinous com-
pounds. In our case, the enzyme activity using
GlcNGlcNAc-pNP as the substrate was esti-
mated to be one-eighth the activity using (Glc-
NAc)2-pNP as the substrate. To elucidate the
reasons for the difference in the enzyme activ-
ity, further studies were performed using gel
filtration chromatography.
1
1), 176.60 (acetyl carbonyl); H–13C HMBC
experiment, H-1/C-5, H-1/C-pNP-1, H-2/C-3,
H-6/C-5, H-6/C-4, H-1%/C-4, H-2%/C-1%, H-2%/
C-3%, H-pNP-3/C-pNP-1. Thus, the compound
was p-nitrophenyl 2-acetamido-4-O-(2-amino-
2-deoxy-b-
D
-glucopyranosyl)-2-deoxy-b- -
D
glucopyranoside (Fig. 2).
The first application of GlcNGlcNAc-pNP
was as a novel substrate that could not be
hydrolysed by b-N-acetylhexosaminidases but
could be hydrolysed by chitinases. Drouillard
and co-workers [18] reported GlcNGlcNAc
acted as an inhibitor of a chitobiase from
Serratia marcescens (characterized as a b-N-
acetylhexosaminidase), mainly because it
lacked the ability to remove a GlcN residue at
the non-reducing end of the substrate, and
because the remaining GlcNAc residue bound
at the active site of the enzyme. As GlcNGlc-
NAc-pNP has a deacetylated
D
-glucosamine
residue at its non-reducing end, b-N-acetyl-
hexosaminidases are probably unable to de-
grade it to liberate p-nitrophenol. Indeed, we
determined that b-N-acetylhexosaminidase
from Penicillium oxalicum did not liberate p-
nitrophenol from GlcNGlcNAc-pNP. In con-
trast, when (GlcNAc)2-pNP was used as the
substrate with the same enzyme, p-nitrophenol
was slowly liberated to give a yellow color in
the reaction solution. As this exo-type enzyme
is known as the one which can degrade (Glc-
NAc)2 and (GlcNAc)3 [19], it is reasonable to
conclude that p-nitrophenol was liberated by
the sequential removal of the saccharides from
the non-reducing end of the substrate.
Fig. 3 shows the chromatograms after sepa-
ration of the enzyme preparation from S.
griseus by gel filtration. According to the
profile of the colorimetric assay of (GlcNAc)2-
pNP degrading activity (the line with circles),
it was found that the chitinase preparation
was composed of a mixture of one broad
group of chitinases and two smaller groups
that eluted later. On the other hand, the
profile of the colorimetric assay of GlcNGlc-
NAc-pNP degrading activity (the line with
triangles) was different. In Fractions 12–23,
there were only two sharp peaks of chitinases
(Fractions 14 and 22) that degraded GlcNGlc-
NAc-pNP, which contrasted the broad peak
of chitinases in the fractions that degraded
(GlcNAc)2-pNP. Chitinases that eluted be-
tween Fractions 16 and 21 could not degrade
Substitution of the sugar residue at the non-
reducing end of the oligomer can be used to
design novel substrates that cannot be de-

K. Tokuyasu et al. / Carbohydrate Research 316 (1999) 173–178
177
Fig. 3. Superose 12 column chromatography of chitinase preparation from Streptomyces griseus (Sigma). p-Nitrophenol liberating
activity (estimated as the absorbance at 405 nm) of each fraction using GlcNAc-pNP, (GlcNAc)2-pNP, or GlcNGlcNAc-pNP as
the substrate was measured as well as the absorbance at 280 nm.
GlcNGlcNAc-pNP in spite of the fact that
they effectively degraded (GlcNAc)2-pNP. As
for the two small peaks of chitinases (Frac-
tions 25 and 30), the production of p-nitro-
phenol seemed to be more active from
GlcNGlcNAc-pNP than from (GlcNAc)2-
pNP. The data indicate that the recognition
of these substrates differs for the group of
chitinases produced by this microorganism.
Although it is reported that some of the
GlcNAc residues in chitin are deacetylated in
residues at the non-reducing end of the sub-
strate, and provide proof that chitinases can
be classified in two types according to their
ability to remove (GlcNAc)2 and GlcNGlc-
NAc residues at the non-reducing ends of
substrates. The chitinases that can effectively
degrade GlcNGlcNAc-pNP may be responsi-
ble for special functions, such as removing
the N-deacetylated GlcN residues in chitin.
This would facilitate the complete degrada-
tion of chitin by other chitinases produced by
the organism.
Thus, the novel substrate, GlcNGlcNAc-
pNP, has enabled us to observe a new aspect
of chitinase action, and will offer a novel
method for the classification of these en-
zymes.
its natural form [1], b- -glucosaminidases,
D
which remove the GlcN residue at the non-re-
ducing end of the substrate, have been rarely
reported [22,23]. It is suggested that another
mode of removing N-deacetylated parts of
chitin may be working instead of removing
GlcN residues by b- -glucosaminidases. Mit-
D
sutomi and co-workers [24] analyzed the reac-
tion products of partially N-acetylated
chitosan by two chitinases, chitinase A1 and
chitinase D from Bacillus circulans WL-12. In
their report, they found chitinase A1 de-
graded the substrate to produce GlcNGlc-
NAc, which was not detected in the reaction
products after degradation by chitinase D.
Their results suggested that the two chitinases
can be distinguished according to their ability
to remove GlcNGlcNAc residues at the non-
reducing end of the substrate.
Acknowledgements
This work was partly supported by a grant
(Leading Research Utilizing Potential of Re-
gional Science and Technology) of the Sci-
ence and Technology Agency of the Japanese
Government. We are grateful to Dr Kazukiyo
Kobayashi, Dr Yoshihiro Nishida, Dr Shioka
Hamamatsu and Dr Randhir S. Makkar for
helpful discussions and advice. We also thank
Ms Chika Aoyagi, Ms Kazumi Kano and Ms
Masako Tamura for excellent technical
assistance.
In this report, we provide a novel method
to detect the removal of GlcNGlcNAc

178
K. Tokuyasu et al. / Carbohydrate Research 316 (1999) 173–178
[13] A. Ohtakara, Methods Enzymol., 161 (1988) 462–470.
References
[14] W.K. Roberts, C.P. Selitrennikoff, J. Gen. Microbiol.,
134 (1988) 169176.
[15] K. Ohnishi, M. Yamagishi, T. Ohta, M. Motosugi, H.
Izumida, H. Sano, K. Adachi, T. Miwa, Biosci. Biotech.
Biochem., 61 (1997) 1113–1117.
[16] K. Tokuyasu, M. Ohnishi-Kameyama, K. Hayashi,
Biosci. Biotech. Biochem., 60 (1996) 1598–1603.
[17] K. Tokuyasu, H. Ono, M. Ohnishi-Kameyama, K.
Hayashi, Y. Mori, Carbohydr. Res., 303 (1997) 353–358.
[18] S. Drouillard, S. Armand, G.J. Davies, C.E. Vorgias, B.
Henrissat, Biochem. J., 328 (1997) 945–949.
[19] K. Yamamoto, K.M. Lee, H. Kumagai, T. Tochikura,
Agric. Biol. Chem., 49 (1985) 611–619.
[20] T. Usui, K. Ogawa, H. Nagai, H. Matsui, Anal.
Biochem., 202 (1992) 61–67.
[21] A. Ohtakara, H. Matsunaga, M. Mitsutomi, Agric. Biol.
Chem., 54 (1990) 3191–3199.
[22] F. Nanjo, R. Katsumi, K. Sakai, J. Biol. Chem., 265
(1990) 10088–10094.
[23] M. Nogawa, H. Takahashi, A. Kashiwagi, K. Ohshima,
H. Okada, Y. Morikawa, Appl. En6iron. Microbiol., 64
(1998) 890–895.
[1] R.A.A. Muzzarelli (Ed.), Natural Chelating Polymer,
Pergamon Press, New York, 1973, p. 85.
[2] S.A. Hedrick, J.N. Bell, T. Boller, C.J. Lamb, Plant
Physiol., 86 (1988) 182–186.
[3] D. Koga, T. Hirata, N. Sueshige, S. Tanaka, A. Ide,
Biosci. Biotech. Biochem., 56 (1992) 280–285.
[4] T.P. Mommsen, Comp. Biochem. Physiol., 60A (1978)
371–375.
[5] F. Ishikawa, K. Oishi, K. Aida, Agric. Biol. Chem., 45
(1981) 2361–2362.
[6] S.E. Reynolds, R.I. Samuels, Ad6. Insect Physiol., 26
(1996) 157–232.
[7] G.W. Gooday, Ad6. Microbiol. Ecol., 11 (1989) 387–430.
[8] D. Koga, A. Isogai, S. Sakuda, S. Matsumoto, A.
Suzuki, S. Kimura, A. Ide, Agric. Biol. Chem., 51 (1987)
471–476.
[9] B. Iseli, T. Boller, J.-M. Neuhaus, Plant Physiol., 103
(1993) 221–226.
[10] T. Watanabe, W. Oyanagi, K. Suzuki, H. Tanaka, J.
Bacteriol., 172 (1990) 4017–4022.
[11] C. Jeuniaux, Methods Enzymol., 8 (1966) 644–650.
[12] A. Ohtakara, Agric. Biol. Chem., 25 (1961) 50–54.
[24] M. Mitsutomi, H. Kidoh, H. Tomita, T. Watanabe,
Biosci. Biotech. Biochem., 59 (1995) 529–531.
.
.