ISSN 0003-6838, Applied Biochemistry and Microbiology, 2008, Vol. 44, No. 3, pp. 251–255. © Pleiades Publishing, Inc.

, 2008.
Original Russian Text © K.S. Rysakova, V.Yu. Novikov, V.A. Mukhin, E. M. Serafimchik, 2008, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 3,
pp. 281–286.

Glycolytic Activity of Enzyme Preparation from the Red King

Crab (Paralithodes camtschaticus) Hepatopancreas
K. S. Rysakovaa, V. Yu. Novikova, V. A. Mukhina, and E. M. Serafimchikb
a Knipovich Polar Research Institute of Marine Fisheries and Oceanography, Murmansk, 183038 Russia
b Murmansk State Technical University, Murmansk, 183010 Russia

Received October 4, 2006

Abstract—Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with

N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas.
The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation.
HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is
indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the depen-
dence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chiti-
nase and protease activities are exhibited by different enzymes.
DOI: 10.1134/S0003683808030046

Currently, considerable efforts of biochemists in dif-
ferent countries are aimed at the development and
improvement of enzymatic processes that are used in
the production and modification of natural polysaccha-
rides. Special attention to this direction is determined
by the high specificity of enzymatic reactions, which
makes it possible to obtain unique compounds of a cer-
tain structure, and environmental friendliness of bio-
chemical manufacture, in contrast to purely chemical
manufacture, which is expressed in renunciation of
large volumes of aggressive chemicals and absence of
contaminated chemical discharges. Of special interest
are chitooligosaccharides, whose biological activity is
higher than that of natural chitin and chitosan or their
monomers. Published data on the properties of chiti-
nases of marine organisms (such as molecular weight
and temperature and pH optima) are highly discrepant.
It is known that chitinase activity varies depending on
the physiological state of the organism.
Obtaining of chitinases from the processing waste
of marine organisms, in turn, may help to solve the
problem of multipurpose use of marine life.
In this study, we described the results of the investi-
gation of chitinolytic activity of enzyme systems of
invertebrates, in particular, the enzyme preparation
obtained from the red king crab (Paralithodes
camtschaticus) hepatopancreas.
The goal of this study was to discover a new source
of chitinolytic enzymes intended for chitin modifica-
tion during the processing of crustaceans.
MATERIALS AND METHODS
Obtaining enzyme preparation. In this study, we
used the enzyme preparation obtained from the red king
crab hepatopancreas by the conventional procedure [1]
including washing the material with acetone and
n-butanol. The enzyme preparation was a mixture of
proteins exhibiting primarily proteolytic and col-
lagenolytic activities. Earlier, we detected chitinolytic
activity in this preparation [2].
Determination of chitinolytic activity. Chiti-
nolytic (exochitinase) activity was determined by the
formation of N-acetyl-D-glucosamine as a result of
chitin hydrolysis; total glycolytic activity, by the total
content of N-acetyl-D-glucosamine and D(+)-glu-
cosamine in hydrolysis products.
Colloidal chitin and chitosans with different
deacetylation degrees were used as substrates.
Obtaining colloidal substrates. Colloidal chitin
was prepared from shrimp chitin as described in [3] by
reprecipitation in a concentrated hydrochloric acid.
Colloidal chitosan was obtained by reprecipitation
from 1 M hydrochloric acid. The concentration of
working solutions of colloidal chitin and chitosan was
approximately 20 mg/ml.
Preparing enzyme preparation suspension. To
obtain 1% suspension, the enzyme preparation (1 g)
was thoroughly ground in a mortar in a small volume of
water and then mixed with the remaining amount of
water (total volume of water, 99 ml). The final suspen-
sion was passed through a paper filter.
Hydrolysis. Colloidal substrates were hydrolyzed at
37°C for 30 min. Briefly, the substrate (4 ml) was mixed
with 1% enzyme preparation (1 ml). When hydrolysis
was ended, the reaction mixture was cooled to 4°C and
centrifuged at 20 000 g for 5 min. Aliquots (1 ml) of
supernatants of each specimen were taken for analysis.

251
252 RYSAKOVA et al.
Determination of exochitinase activity. The con-
centration of N-acetyl-D-glucosamine was determined
by the reaction with 4-dimethylaminobenzaldehyde
[4]. For this purpose, 10% 4-dimethylaminobenzalde-
hyde was prepared in a mixture of glacial acetic acid
and 11.5 M hydrochloric acid (87.5 : 12.5, v/v). Before
use, this solution was diluted with glacial acetic acid in
the ratio 1 : 1 (v/v).
Water and working solution (0.5 ml each) were
poured into the control and experimental tube, respec-
tively. Then, 0.1 ml of saturated sodium tetraborate was
added to each tube; the latter were heated in a boiling
water bath for 3 min and cooled to 4°C. Thereafter,
each tube was supplemented with 3 ml of 4-dimethy-
laminobenzaldehyde, heated in a thermostat at 37°C for
20 min, and cooled to 4°C. The optical density of the
experimental solution was measured at 585 nm relative
to the control. The concentration of N-acetyl-D-glu-
cosamine was calculated using a calibration curve.
Determination of total glycolytic activity. The
total concentration of reducing sugars was determined
by the reaction with potassium hexacyanoferrate [5].
Briefly, water and working solution (1.5 ml each) were
poured into the control and experimental tube, respec-
tively. Both tubes were supplemented with 2 ml of
0.05% potassium hexacyanoferrate (III) (K3[Fe(CN)6]),
heated in a boiling water bath for 15 min, and cooled.
The optical density of the control samples was mea-
sured at 420 nm relative to the experimental solution.
The concentration of reducing sugars was calculated
using a calibration curve.
The calibration curves used for calculating the con-
centration of N-acetyl-D-glucosamine and the total
concentration of N-acetyl-D-glucosamine and D(+)-
glucosamine were plotted using the results of reactions
of glucose, N-acetyl-D-glucosamine, and D(+)-glu-
cosamine solutions of known concentrations ranging
from 0.01 to 0.02 M with 4-dimethylaminobenzalde-
hyde and potassium hexacyanoferrate (III).
The optical density of solutions was recorded in the
ultraviolet and visible regions of the spectrum with a
UV-3101PC Shimadzu spectrophotometer (Shimadzu,
Japan) at constant temperature (20°C).
The pH of solutions was measured with an Anion
4151 ionomer (Infraspak-Analit, Russia).
Chromatographic determination of D(+)-glu-
cosamine. The hydrolysis products of chitin and chito-
san were separated chromatographically by the reverse-
phase HPLC on a SupelcosilTM LC-18 column (30 cm ×
0.4 mm, 5 µm; Supelco, United States) in an LC-10AVP
Shimadzu chromatograph (Shimadzu, Japan). The elu-
ent flow rate was 1 ml/min, and the column temperature
was 35°C. The compounds containing free amino
groups were converted into fluorescent derivatives
using the reaction with o-phthalaldehyde [6]. The reac-
tion products were recorded with an RF-10AXL Shi-
madzu fluorometric detector at excitation and emission
wavelengths 340 and 450 nm, respectively.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
RESULTS AND DISCUSSION
Kinetics of formation of reducing sugars and
n-acetyl-D-glucosamine. In contrast to N-acetyl-D-
glucosamine formation kinetics, which was determined
during the study of the exochitinase activity [7, 8], we
observed no decrease in the hydrolysis rate, which
might be caused by the enzyme inactivation (Fig. 1a).
The dependence of the hydrolysis rate on the enzyme con-
centration remained linear for at least 6 h of incubation.
The distinctions in the kinetic curves for reducing
sugars and N-acetyl-D-glucosamine can be explained
by different concentrations of enzyme preparations
used for hydrolysis. At higher concentrations (starting
with 2 g/l), autohydrolysis of the enzyme took place,
which decreased the rate of formation of chitin hydrol-
ysis products (N-acetyl-D-glucosamine). At low
enzyme concentrations, the rate of enzymatic hydroly-
sis of the substrate decreased and remained nearly con-
stant, which determined the linear mode of the kinetic
curve of formation of the chitin hydrolysis products.
Probably, the enzyme present in the enzyme–substrate
complex is not accessible for hydrolysis by free
enzyme molecules and, hence, is not digested during
interaction with chitin.
A comparison of the kinetic curves of formation of
reducing sugars and N-acetyl-D-glucosamine shows
that, although the concentration of the enzyme prepara-
tion was considerably higher in the second case, the
concentration of reducing sugars was higher than that
of N-acetyl-D-glucosamine. This phenomenon can be
explained by the fact that, in addition to N-acetyl-D-
glucosamine, hydrolysis of chitin may yield other prod-
ucts (D(+)-glucosamine and chitooligosaccharides)
exhibiting reducing properties. Since the molecules of
chitin and its oligosaccharides are strictly linear, each
molecule can have only one reducing end. Therefore,
the molar concentration of reducing sugars serves as a
measure of the total amount of molecules in a solution
(in our case, it reflects the degree of enzymatic hydrol-
ysis of chitin, including exo- and endohydrolysis).
Effect of pH. Curves illustrating the experimental
dependences of glycolytic, exochitinase [7, 8], and pro-
teolytic [2] activities of the enzyme preparation on the
pH of the reaction medium are shown in Fig. 1b. The
optimum pH values for exochitinase and total glyco-
lytic activities coincided and were found to be 4.5. The
second maximum of exochitinase activity, which was
absent in the case of glycolytic activity, was recorded at
pH 8.0.
The pH value at which the proteolytic activity of the
enzyme preparation was maximum (pH 8.5) did not
coincide with the pH corresponding to the maximum
glycolytic activity but was close to the pH at which the
exochitinase activity was maximum. The phenomenon
when one animal organ contains different enzymes
capable of cleaving the same substrate at different pH
values is widespread in nature and is apparently a bio-
chemical adaptation formed in the course of evolution
Vol. 44 No. 3 2008
 GLYCOLYTIC ACTIVITY OF ENZYME PREPARATION FROM THE RED KING CRAB 253

mM (‡) mM (b) %
5 2.0 100
4 1.5 80
1
3 1 4 60
1.0
2 3
40
1
2 0.5 20
3
0
0 50 100 150 200 250 300 350 400 0 2 4 6 8 10 12
min pH
mM (c) % mM
2.0 100 4 (d)
1
1
1.5 80 3
5
4 60
1.0 2
40
3
0.5 3 20 1
0
0 10 20 30 40 50 60 70 80 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
°C g/l

Fig. 1. Dependence of the concentration of reducing sugars, N-acetyl-D-glucosamine, and the proteolytic activity of the enzyme
preparation (EP) on (a) the duration of enzymatic hydrolysis of chitin at 37°C and pH 4.5, (b) the pH of medium at 37°C, (c) the
temperature of incubation medium at pH 4.5, and (d) the concentration of the enzyme preparation (g/l) during incubation at 37°C
for 30 min at pH 4.5. Designations: 1, reducing sugars, CEP = 0.055 g/l; 2, reducing sugars, CEP = 0.011 g/l; 3, N-acetyl-D-glu-
cosamine, CEP = 2 g/l; 4, proteolytic activity (substrate, 1% hemoglobin); 5, reducing sugars without N-acetyl-D-glucosamine.
Chitin concentration, 2 g/l.

[9]. Our data suggest that glycolytic and proteolytic
activities of the enzyme preparation from the red king
crab hepatopancreas are exhibited by different
enzymes.
The coincidence of pH values at which exochitinase
and proteolytic activities are maximum (pH 8.0–8.5)
can be regarded as indirect evidence confirming the
ability of proteolytic enzymes to exhibit chitinolytic
activity. This is not a generally accepted point of view;
however, some authors reported chitinolytic activity of
some enzymes that have another substrate specificity.
For example, it was shown that lipase [10] and papain
[11] can efficiently hydrolyze chitosan.
Effect of temperature. Figure 1c shows the depen-
dence of glycolytic, exochitinase [7, 8], and proteolytic
[2] activities of the enzyme preparation on incubation
temperature. It can be seen that the optimal temperature
for manifestation of the glycolytic (45°C) and exochiti-
nase (35°C) activities are shifted towards lower temper-
atures relative to the optimum for the proteolytic activ-
ity (55°C).
This finding confirms the conclusion that glycolytic
and proteolytic activities are exhibited by different
enzymes contained in the enzyme preparation from the
red king crab hepatopancreas.
Data obtained in this study suggest that glycolytic
activity is also exhibited by different enzymes with dif-
ferent temperature optima. Therefore, the total temper-
ature optimum (Fig. 1c, curve 1) was shifted relative to
the temperature optimum of the enzyme responsible for
the exochitinase activity (Fig. 1c, curve 3).
Effect of the concentration of the enzyme prepa-
ration. The dependences of glycolytic and exochitinase
activities of the enzyme preparation on its concentra-
tion are similar: the stage of rapid accumulation of
hydrolysis products (observed at ~0.5 g/l enzyme prep-
aration) is followed by a slower stage (Fig. 1d). Appar-
ently, the observed dependence can be explained by
competition between the hydrolysis of the substrate by
the enzyme and the hydrolysis of the enzyme itself as a
protein substrate. Chitin is hydrolyzed primarily at the
first stage. At the second stage, the concentration of the
enzyme preparation decreases, which leads to a
decrease in the hydrolysis rate of chitin. The distinc-
tions between the total glycolytic activity and the exo-
chitinase activity of the enzyme preparation at the same
concentration of the latter can be clearly seen in Fig. 1d.
The reducing sugars, in addition to N-acetyl-D-glu-
cosamine, may also contain chitin and chitosan oligo-
mers as well as D(+)-glucosamine. The average ratio
between the experimentally determined concentrations
of reducing sugars and N-acetyl-D-glucosamine was
2.3. The D(+)-glucosamine/N-acetyl-D-glucosamine
ratio, which can be calculated using the deacetylation
degree of chitin (12%), is 0.136. This value is consider-
ably smaller than the experimental value. The observed
excess of reducing sugars cannot be explained by for-
mation of only monomers. Apparently, these results

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 44 No. 3 2008

254 RYSAKOVA et al.
mM
0.6
0.5
0.4
0.3
0.2
0.1
0 5 10 15
Fig. 2. Dependence of the total concentration of reducing
sugars (mM) on the substrate concentration (g/l) during
enzymatic hydrolysis of chitin. Incubation with the enzyme
preparation (0.055 g/l) was performed at 37°C for 120 min
at pH 4.5.
confirm our assumption on the formation of chitin oli-
gosaccharides.
Effect of substrate concentration. The curve
showing the dependence of the glycolytic activity of the
enzyme preparation on the substrate concentration
(Fig. 2) reaches a plateau at chitin concentrations
higher than 2 g/l.
Thus, the above experiments were performed in the
presence of excess substrate, and all kinetic character-
istics were determined solely by the concentration of
the enzyme preparation and characterize the properties
of the given preparation.
Chromatographic determination of D(+)-glu-
cosamine. It should be taken into account that chitin, its
oligomers, and N-acetyl-D-glucosamine may undergo
enzymatic deacetylation. In this case, D(+)-glu-
cosamine should be produced in larger amounts than it
was predicted theoretically proceeding from the sto-
chastic cleavage of glycosidic bonds in the chitin mol-
ecule.
To assess the amount of D(+)-glucosamine and
other chitosan oligomers containing free amino groups,
the hydrolysis products of chitin and chitosan were sub-
jected to chromatographic analysis.
Chitin samples with a deacetylation degree of 12%
and chitosan samples with deacetylation degrees of 67,
82, and 95% were subjected to enzymatic hydrolysis.
HPLC revealed no D(+)-glucosamine in hydrolysates.
Figure 3 shows the chromatogram of the products of
enzymatic hydrolysis of chitosan with a deacetylation
degree of 67% (curve 1), on which the peak corre-
sponding to D(+)-glucosamine (curve 2) is absent
(9.6 ml). We identified some chromatographic fractions
belonging to amino acids: serine (6.0 ml), histidine
(7.8 ml), unidentified peak (8.9 ml), and glycine
(10.3 ml). These amino acids were bound to the hydrol-
ysis products of proteins containing the enzyme prepa-
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
mV
1600 2
1200
1
800
400
0 5 10 15
20 min
g/l
Fig. 3. HPLC of the products of enzymatic hydrolysis of (1)
chitosan with a deacetylation degree of 67% and
(2) D(+)-glucosamine. Incubation of the substrate (chito-
san, 2 g/l) with the enzyme preparation (0.055 g/l) was per-
formed at 37°C for 120 min at pH 4.5.
ration rather than to the hydrolysis products of chitosan.
Since the method used makes it possible to detect only
free amino groups, we concluded that low-molecular-
weight deacetylated chitosan derivative were absent
among the hydrolysis products.
On the basis of experimental data, it can be con-
cluded that the enzyme preparation exhibits only chiti-
nase activity since it does not cleave single glu-
cosamine molecules. To cleave the glycosidic bond, the
presence of acetylated units is apparently required,
which allows the glycolysis enzymes contained in the
enzyme preparation to be classified into the group of
chitinases (EC 3.2.1.14) [12]. Possibly, the enzyme
preparation contains enzymes exhibiting β-N-acetylhex-
osaminidase activity (EC 3.2.1.52), which cleave chitin oli-
gomers (primarily dimers) to form N-acetyl-D-glucosamine
but not D(+)-glucosamine [12].
The absence of D(+)-glucosamine in the hydrolysis
products is also corroborated by the fact that deacetyla-
tion of N-acetyl-D-glucosamine by N-acetylglu-
cosamine deacetylase (EC 3.5.1.33) does not occur.
The exochitinase activity, leading to cleavage of termi-
nal deacetylated units, has not been detected either.
Thus, the results obtained in this study showed that
the enzyme preparation yields chitin and chitosan oli-
gomers with a low deacetylation degree. Apparently,
the enzyme preparation exhibits a pronounced chitinase
activity. Our earlier results showed the presence of exo-
chitinase activity in the preparation (formation of
N-acetyl-D-glucosamine) [7, 8]. Data obtained in this
work confirmed the presence of endochitinase (and
possibly endochitisanase) activities and the absence of
exochitosanase and N-acetylglucosamine deacetylase
activities.
The experimental results made it possible to make
certain conclusions regarding the composition and
mechanism of action of the enzyme preparation
obtained from the red king crab hepatopancreas. We
Vol. 44 No. 3 2008
 GLYCOLYTIC ACTIVITY OF ENZYME PREPARATION FROM THE RED KING CRAB
have confirmed the presence and quantified the endoch-
itinase activity of the enzyme preparation as well as
demonstrated the absence of exochitosanase and
N-acetylglucosamine deacetylase activities. We
assumed that the chitinase and protease activities of the
enzyme preparation are apparently exhibited by differ-
ent enzymes.
The practical significance of our results is that they
demonstrated that crab hepatopancreas can be used as a
source of enzymes exhibiting chitinolytic and chito-
sanolytic activities for industrial modification of chitin
and chitosan. This enables maximum utilization of all
components of commercial aquatic organisms and
increases the efficiency of sea fishery owing to utiliza-
tion of its processing waste. The study of the glycolytic
activity of the enzyme preparation from the red king
crab hepatopancreas is of practical interest in terms of
designing chitinase preparations for fighting against
pests and treating fungal infections.
ACKNOWLEDGMENTS
This study was supported by the grant of the Presi-
dent of the Russian Federation no. MD-1010.2005.4.
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