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.)