Báo cáo Y học: Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium pptx

Purification and characterization of novel chondroitin ABC and AC
lyases from
Bacteroides stercoris
HJ-15, a human intestinal anaerobic
bacterium
Sung-Woon Hong
1
, Byung-Taek Kim
1
, Ho-Young Shin
1
, Wan-Suk Kim
2
, Keun-Sook Lee
1
, Yeong-Shik Kim
2
and Dong-Hyun Kim
1
1
College of Pharmacy, Kyung Hee University, Seoul, Korea;
2
Natural Products Research Institute, Seoul National University,
Seoul, Korea
Two novel chondroitinases, chondroitin ABC lyase
(EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have
been purified from Bacteroides stercoris HJ-15, which was
isolated from human intestinal bacteria with glycosami-
noglycan degrading enzymes. Chondroitin ABC lyase was
purified to apparent homogeneity by a combination of
QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and

Sephacryl S-300 column chromatography with a final spe-
cific activity of 45.7 lmolÆmin
)1
Æmg
)1
. Chondroitin AC
lyase was purified to apparent homogeneity by a combina-
tion of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite
and phosphocellulose column chromatography with a final
specific activity of 57.03 lmolÆmin
)1
Æmg
)1
. Chondroitin
ABC lyase is a single subunit of 116 kDa by SDS/PAGE and
gel filtration. Chondroitin AC lyase is composed of two
identical subunits of 84 kDa by SDS/PAGE and gel filtra-
tion. Chondroitin ABC and AC lyases showed optimal
activity at pH 7.0 and 40 °C, and 5.7–6.0 and 45–50 °C,
respectively. Both chondroitin lyases were potently inhibited
by Cu
2+
,Zn
2+
,andp-chloromercuriphenyl sulfonic acid.
The purified Bacteroidal chondroitin ABC lyase acted to the
greatest extent on chondroitin sulfate A (chondroitin 4-sul-
fate), to a lesser extent on chondroitin sulfate B (dermatan
sulfate) and C (chondroitin 6-sulfate). The purified chon-
droitin AC lyase acted to the greatest extent on chondroitin

sulfate A, and to a lesser extent on chondroitin C and
hyaluronic acid. They did not act on heparin and heparan
sulfate. These findings suggest that the biochemical proper-
ties of these purified chondroitin lyases are different from
those of the previously purified chondroitin lyases.
Keywords: Bacteroides sterocirs HJ-15; chondroitin ABC
lyase; chondroitin AC lyase; chondroitin sulfate; purifica-
tion.
Enzymes degrading glycosaminoglycans (GAGs) have been
become increasingly important in understanding of the
GAGs and proteoglycans, which are involved in the
regulation of various cellular processes such as adhesion,
differentiation, migration and proliferation [1–5]. The
design and preparation of GAG-based therapeutic agents
becomes possible using these enzymes [6]. Chondroitin
sulfates are the most common type of GAG chains found in
proteoglycans [6,7]. They are sulfated linear polysaccharides
with alternating 1–3 and 1–4 linkages. The major classes are
chondroitin sulfate A, dermatan sulfate (chondroitin sul-
fate B) and chondroitin sulfate C. The biological roles of
chondroitin sulfate GAGs are poorly understood and their
exact chemical structures have not been determined.
Enzyme methods are preferable to chemical methods when
determining polysaccharide structures [8,9]. Enzymes are
often very specific and act under mild conditions giving
oligosaccharide products. Two classes of enzyme that act on
GAGs are polysaccharide lyases and hydrolases. Prokar-
yotic polysaccharide lyases depolymerize GAGs through an
eliminative mechanism, whereas enzymes from eukaryotic
sources act through a hydrolytic mechanism [6].

Bacterial degradation of GAGs has been studied using
enzymes produced from Flavobacterium heparinum [10–13]
and Bacteroides thetaiotaomicron [14,15]. Recently, the
acharan sulfate degrading bacterium was isolated from
human intestine and identified as Bacteroides stercoris
HJ-15 [16]. This organism also cleaved heparin, heparan
sulfate, chondroitin sulfate A, chondroitin sulfate C and
even dermatan sulfate [16–18].
We report here reproducible schemes for the purification
of chondroitin ABC lyase and chondroitin AC lyase, which
have not been purified from Bacteroides sp., to apparent
homogeneity and the determination of their physical
properties, kinetic properties, optimal catalytic conditions
and specificity.
MATERIALS AND METHODS
Materials
Chondroitin sulfate A (chondroitin 4-sulfate from bovine
trachea), chondroitin sulfate B (dermatan sulfate from
bovine mucosa), chondroitin sulfate C (chondroitin
Correspondence to D H. Kim, College of Pharmacy,
Kyung Hee University, 1, Hoegi-dong, Dongdaemun-ku,
Seoul 130-701, South Korea.
Fax: + 82 2 957 5030, Tel.: + 82 2 961 0374,
E-mail:
Abbreviations: GAG, glycosaminoglycan; IEF, isoelectric focusing.
Enzymes: chondroitin ABC lyase (EC 4.2.2.4); chondroitin AC lyase
(EC 4.2.2.5).
(Received 22 January 2002, revised 23 April 2002,
accepted 30 April 2002)
Eur. J. Biochem. 269, 2934–2940 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02967.x

6-sulfate from shark cartilage), hyaluronic acid (rooster
coomb), heparin (porcine intestinal mucosa), porcine
heparan sulfate (porcine intestinal mucosa), thioglycolic
acid (sodium salt), QAE-cellulose fastflow, HA Ultrogel
(microcrystalline hydroxyapatite, 4% beaded in agarose),
phosphocellulose (coarse mesh) and low molecular mass
markers for gel filtration were purchased from Sigma
Chemical Co. CM-Sephadex C-50, Sephacryl S-300 HR
resins, high molecular mass markers for gel-filtration and
low molecular mass markers for protein electrophoresis
were from Amersham Pharmacia Biotech. DEAE-cellulose
resin was obtained from Wako Pure Chemical Industries.
Protein Assay Reagent, SDS and Coomassie Brilliant Blue
R-250 were supplied by Bio-Rad laboratories. Tryptic soy
broth was provided by Difco Co. Acharan sulfate, which
has a uniform repeating disaccharide structure of fi 4)-a-
D
-GlcNAc(1 fi 4)-a-
L
-IdoA2S(1 fi , was prepared from
the giant African snail, Achatina fulica, according to the
previous method [17]. All other chemicals were of the
highest grade available.
Bacterial strains and cultivation
B. stercoris HJ-15 was isolated and cultivated as described
previously [16]. It was cultured anaerobically under an
atomosphere of 90% nitrogen and 10% carbon dioxide at
37 °C in 10 L of tryptic soy broth (pH 7.2) containing
chondroitin sulfate A (0.15 gÆL
)1

) instead of glucose,
0.01% (w/v) sodium thioglycolate and 0.1% (w/v)
ascorbic acid.
Purification procedure of chondroitin ABC lyase
The cultured cells in 10-L of the broth described above were
harvested in the late exponential phase (11–12 h) by
centrifugation at 3000 g for 30 min at 4 °C and the resulting
cell pellet was washed twice with cold 0.89% NaCl. The cell
pellet was suspended in 150 mL of 50 m
M
sodium phos-
phate buffer, pH 7.0. Cell suspension (30 mL at a time) was
placed into a 50-mL centrifuge tube and disrupted by
15-min periods of sonication at 1-s intervals on an ultrasonic
processor (Eyela Co.) at a 40% output with cooling. Cell
debris was removed by centrifugation at 21 000 g for
60 min at 4 °C. All operations were carried out at 4 °C
unless otherwise noted. One hundred and fifty milliliters of
cell extract (or 150 mL) was passed through a QAE-
cellulose column (2.8 · 38 cm) which had been preequili-
bratedwith200mLof50m
M
sodium phosphate buffer,
pH 7.0. The column was washed with the same buffer until
no further lyase activities were detectable in the effluent. The
noninteracting fluid (350 mL) passed through the column
and was loaded onto a CM-Sephadex C-50 column
(2.8 · 38 cm) equilibrated with 50 m
M
sodium phosphate

buffer, pH 7.0 and the column was washed with 300 mL of
the same buffer (the fraction passing through the column
without binding was used for the purification of chondroitin
AC lyase). Then CM-Sephadex C-50 column binding
chondroitin ABC lyase was eluted with a total 300 mL
linear gradient of KCl from 0 to 0.6
M
in 50 m
M
sodium
phosphate buffer, pH 7.0 (Fig. 1). The fractions containing
chondroitin ABC lyase activity were pooled and dialyzed
against 3 L of 50 m
M
sodium phosphate buffer, pH 7.0 for
12 h for next step. The dialyzed enzyme preparation was
applied to a hydroxyapatite column (2.5 · 6 cm) previously
equilibrated with 50 m
M
sodium phosphate buffer, pH 7.0.
The column was washed with 500 mL of the same 50 m
M
sodium phosphate buffer, pH 7.0 and then eluted with an
800-mL linear gradient of potassium chloride from 0 to
0.5
M
. Chondroitin lyase-positive fractions were pooled and
concentrated to approximately 2 mL by a ultrafiltration
unit (Advantec Co.). The concentrated enzyme preparation
was loaded onto a Sephacryl S-300 HR column

(3.5 · 70 cm) and eluted with 50 m
M
sodium phosphate
buffer, pH 7.0 at a flow rate of 1 mLÆmin
)1
. Fractions
containing chondroitin ABC lyase activity were tested for
purity by electrophoresis.
Purification procedure of chondroitin AC lyase
from
B. stercoris
HJ-15
In the procedure of the purification of chondroitin ABC
lyase, the fluid (390 mL) passing through CM-Sephadex
C-50 column (2.8 · 38 cm) without binding was pooled and
its activity was measured (Fig. 1). The crude chondroitinase
AC lyase was applied to a hydroxyapatite column
(3 · 10 cm) previously equilibrated with 50 m
M
sodium
phosphate buffer, pH 7.0. The column was washed with
500mLofthesame50m
M
sodium phosphate buffer,
pH 7.0 and then eluted with a total 800 mL linear gradient
of potassium chloride from 0 to 0.5
M
. Chondroitin AC
lyase-positive fractions were pooled and dialyzed against
3 L of the same buffer. The desalted enzyme preparation

was loaded onto a phosphocellulose column (3 · 10 cm)
preequilibrated with 50 m
M
sodium phosphate buffer,
pH 7.0 and unbound proteins were removed by a 500-mL
wash of 50 m
M
sodium phosphate buffer, pH 7.0. The
column was eluted with total 400 mL linear gradient of 50–
400 m
M
sodium phosphate and chondroitin AC lyase
activity containing fractions were tested for homogeneity
by electrophoresis.
Enzyme activity assays
Chondroitin lyase activity was measured according to the
following method. The spectrophotometer (Jasco V-530)
was adjusted to 40 °C and a 1-mL quartz cuvette containing
Fig. 1. Elution profile of chondroitin lyases on CM-Sephadex C-50 ion
exchange chromatography. Solid circle, chondroitin sulfate A-degra-
ding activity; open circle, dermatan sulfate-degrading activity; solid
triangle, chondroitin sulfate C-degrading activity; simple line,
absorbance at 280 nm.
Ó FEBS 2002 New bacteroidal chondroitin ABC and AC lyases (Eur. J. Biochem. 269) 2935
1 mg of substrate in 650 lLof50m
M
sodium acetate
buffer, pH 5.8 for chondrotinase AC (or 50 m
M
sodium

phosphate buffer, pH 7.0 for chondroitinase ABC) was
thermally equilibrated. Fifty microliters of enzyme solution
was added and the cuvette was gently inverted twice to mix
the contents. It was then immediately placed in the warmed
holder of spectrophotometer and the change of absorbance
at 232 nm was measured at 1-s intervals over 5 min. The
activity was calculated from the change of absorbance per
minute using an extinction coefficient of 3800
M
)1
for
products (1 U ¼ 1 lmol of uronic acid containing product
formed per min) [19]. The specific activity was calculated by
dividing the micromoles of product produced per minute by
the milligrams of protein in the cuvette.
Protein determination
Protein concentration was measured by a Bradford assay
based on a bovine serum albumin standard curve [20].
Characterization of chondroitin lyases
SDS/PAGE was performed for the determination of
molecular mass according to Laemmli’s procedure [21].
The gels were stained with Coomassie Brilliant Blue
R-250 solution and further stained with silver. The pI
values of chondroitin lyases were determined by IEF
electrophoresis using Model 111 Mini IEF Cell (Bio-Rad)
according to the manufacturer’s instructions. The molecu-
larmassofthenativeenzymewasestimatedbygel-
filtration using Sephacryl S-300 HR column (1.6 · 70 cm)
calibrated with gel filtration low molecular mass calibra-
tion kit (from Sigma Co.) and high molecular calibration

kit (from Amersham Pharmacia Biotech). The pH opti-
mum of chondroitin lyases were determined using 50 m
M
sodium phosphate buffer (pH 5.0–8.5). The temperature
dependence of the enzyme was investigated by measuring
enzyme activity at different temperatures (20–60 °C). To
investigate the effect of divalent metal ions and KCl on
the lyase activity, divalent metal ion (final concentration,
100 l
M
) and KCl (0–500 m
M
) were added into the
reaction mixture (acetate buffer was used instead of
phosphate buffer). Kinetic constants of chondrotin lyases
were determined by measuring the initial rates at various
substrate concentrations (200, 400, 600, 1000, 2000,
3000 lg) under the standard reaction conditions. These
lyase activities on other sulfated polysaccharides were also
measured. One milligram of each substrate was added to
the reaction mixture.
Amino-acid composition analysis was performed on an
Applied Biosystem model 420/130 Derivatizer/Amino Acid
Analyzer, using phenylisothiocyanate precolumn derivati-
zation chemistry. Hydrolysis was performed using using 6
M
hydrochloric acid, 0.1% phenol at 155 °Cfor1h.
Internal amino-acid sequences of two purified chondro-
itin lyases were analyzed by an Applied Biosystem protein
sequencer model 492.

RESULTS
Purification of two chondroitin lyases from
B. stercoris
HJ-15
B. stercoris HJ-15, which degrades a variety of GAGs
including chondroitin sulfates, heparin and heparan sulfate
[16], constitutively produced chondroitin lyase activity.
However, when induced with chondroitin sulfate A, total
chondroitin lyase activity increased  fivefold (data not
shown). Following ultrasonic disruption of B. stercoris
HJ-15, the crude extract was subjected to a combination
of QAE-cellulose and DEAE-cellulose column chromatog-
raphy to remove interacting proteins. Chondroitin ABC
lyase activity passed through these columns without binding
to the matrices. The effluent was further purified to
homogeneity with yield of 2.37% by a series of CM-Sepha-
dex C-50 column chromatography (Fig. 1), hydroxyapatite
column and Sephacryl S-300 gel filtration chromatography,
and its final specific activity was 45.7 lmolÆmL
)1
Æmg
)1
(Table 1). Chondroitin AC lyase, which passed through
CM-Sephadex C-50 resin without binding, was purified to
single band on SDS/PAGE with a yield of 6.28% by a
combination of hydroxyapatite column and phosphocellu-
lose column chromatography. The specific activity of
purified chondroitin AC lyase was 57.03 lmolÆmL
)1
Æmg

)1
(Table 1). Discontinuous SDS/PAGE illustrated that chon-
droitin ABC lyase and chondroitin AC lyase were appar-
ently homogeneous and their molecular mass values were
estimated to be 116 and 84 kDa, respectively (Fig. 2).
Table 1. Purification summary of chondroitin ABC and AC lyases from B. sterocoris HJ-15. One unit (U) is the activity forming 1 lmol disac-
charides per min. The activity was assayed in 50 m
M
sodium phosphate buffer, pH 7.0.
Stage Total activity (U) Total protein (mg) Specific activity (UÆmg
)1
)
Chondroitin ABC lyase
Crude extract 418.53 1000 0.42
QAE-cellulose column chromatography 193.86 243.5 0.8
CM Sephadex C-50 column chromatography 218.03 14.94 14.59
Hydroxyapatite column chromatography 88.34 2.15 41.09
Sephacryl S-300 column chromatography 9.94 0.22 45.7
Chondroitin AC lyase
Crude extract 418.53
b
1000 0.42
QAE-cellulose column chromatography 193.86 243.5 0.8
CM-Sephadex C-50 column chromatography 137.14 265.9 0.52
Hydroxyapatite column chromatography 115.74 23 5.03
Phosphocellulose column chromatography 26.3 0.461 57.03
2936 S W. Hong et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Characterization of two chondroitin lyases
When the molecular masses of chondroitin ABC and AC
lyases under nondenaturing conditions were determined by

gel filtration, chondroitin ABC and AC lyases were
estimated to be 116 and 170 kDa, respectively. It suggests
that chondroitin ABC lyase is composed of one subunit and
chondroitin AC lyase is composed of two identical subunits.
The optimal pH values of chondroitin ABC and AC
lyases were determined to be 7.0 and 5.7–6.0 for chondroitin
sulfate A, respectively, and the optimum temperatures for
the maximal activity were 40 °C and 45–50 °C, respectively
(data not shown).
The activity of chondroitin ABC lyase was inhibited by
addition of Ni
2+
,Mg
2+
,Zn
2+
,Cu
2+
and Co
2+
.Partic-
ularly, Cu
2+
and Zn
2+
potently inhibited chondroitin ABC
lyase and chondroitin AC lyase.
Heparin competitively inhibited only chondroitin AC
lyase, not chondroitin ABC lyase. Its inhibition was
reversible (IC

50
was 18 l
M
) (Fig. 3). The addition of
50 m
M
KCl in 50 m
M
sodium phosphate buffer slightly
increased the activity of both chondroitin ABC and AC
lyases. However, the addition of more than 50 m
M
salt
inhibited the activity of chondroitin lyases.
Both enzymes were inhibited by p-chloromercuriphenyl
sulfonic acid; chondroitin AC lyase was inhibited by
iodoacetic acid as well as p-chloromercuriphenyl sulfonic
acid. However, both enzymes were little inhibited by the
other chemical modifying agents (data not shown).
Amino-acid composition analysis revealed that the both
chondroitin lyases contain a large proportion of lysine (data
not shown), consistent with their pI values of 7.9–8.3. The
amino-acid compositions of the chondroitin lyases were
similar, but not identical. The pI values of the purified
chondroitin ABC and AC lyases were 7.9 and 8.3,
respectively, slightly higher than those of the previously
purified chondroitin ABC lyase of B. thetaiotaomicron (pI
values 7.9 to 8.0). We analyzed the internal sequences of a
peptide obtained by digestion of each enzyme with trypsin
(Table 3). The internal sequences of chondroitin ABC and

AC lyase show significantly greater homology of 59
and 80% to Flavobacterial chondroitin AC lyase, and 57
and 33% to B. thetaiotaomicron chondroitin ABC lyase
previously reported [22–24], respectively. However, internal
sequences of the present chondroitin lyases did not signi-
ficantly greater homology to P. vulgaris chondroitin ABC
lyase.
Substrate specificity of two purified chondroitin lyases
Chondroitin ABC lyase depolymerized chondroitin sul-
fate A, C and dermatan sulfate (Table 4). When chondro-
itin sulfate ABC lyase for chondroitin sulfate A was taken
as 100%, this enzymes activity for chondroitin sulfate C
anddermatansulfatewas40and32%,respectively.
Chondroitin lyase ABC did not act on hyaluronic acid,
heparin and heparan sulfate.
Chondroitin AC lyase depolymerized chondroitin sul-
fate A and C (Table 4). When chondroitin AC lyase for
chondritin sulfate A was taken as 100%, this enzymes
activity for chondroitin sulfate C and hyaluronic acid was
Fig. 2. SDS/PAGE of the purified chondroitin ABC (A) and AC (B)
lyases at various steps of purification. (A) Lane 1, preparation after
crude extract; lane 2, preparation after QAE-cellulose column chro-
matography; lane 3, preparation after CM-Sephadex C-25 column
chromatography; lane 4, preparation after hydroxyapatite ultragel
column chromatography; lane 5, Sephacryl S-300 column chroma-
tography; lane M, marker. (B) Lane 1, preparation after CM-Sephadex
C-25 column chromatography; lane 2, preparation after hydroxyapa-
tite ultragel column chromatography; lane 3, preparation after phos-
phocellulose column chromatography; lane M, markers.
Table 2. Effect of divalent metal ions on the activity of chondroitin

lyases. Final concentration of divalent ion, 1 m
M
.0.03Uofthe
homogenously purified enzyme activity was taken as 100%.
Metal ion
Relative activity (%)
Chondroitin ABC lyase Chondroitin AC lyase
Control 100 100
Co
2+
89 91
Ni
2+
42 76
Cu
2+
76
Mg
2+
41 102
Mn
2+
95 103
Ca
2+
97 105
Zn
2+
313
EDTA 100 100

Fig. 3. Inhibitory effect of heparin on chondroitin ABC and AC lyases.
Solid circle, chondroitin sulfate ABC; solid square, chondroitin AC.
Ó FEBS 2002 New bacteroidal chondroitin ABC and AC lyases (Eur. J. Biochem. 269) 2937
46 and 67%, respectively. However, dermatan sulfate,
heparin, heparan sulfate and acharan sulfate were not
substrates for this enzyme.
Kinetic constants of two purified chondroitin lyases
Michaelis–Menten constants were determined using the
optimal reaction conditions in experiments designed to
calculate reaction velocities at each substrate concentration .
The K
m
and V
max
of chondroitin ABC and AC lyases
towards chondroitin sulfate A, dermatan sulfate (chondro-
itin sulfate B), and chondroitin sulfate C were determined
(Table 5).
DISCUSSION
B. stercoris HJ-15 isolated from human intestine is capable
of producing the GAG degrading enzymes. In the present
report, we have purified two chondroitin lyases. CM-
Sephadex C-50 chromatography efficiently resolved chon-
droitin sulfate degrading lyases of B. stercoris HJ-15. As
Fr-a showed a higher specificity to chondroitin sulfates A
and C, and Fr-b fractions showed a higher specificity to
chondroitin sulfates A, B and C, they were considered to be
chondroitin AC and ABC lyases, respectively (Fig. 1).
Chondroitin ABC lyases have been purified previously
from three other Gram-negative species, P. vulgaris [25],

F. heparinum [10–13] and B. thetaiotaomicron [14,15]. These
chondroitin ABC lyases were equally active against chon-
droitin sulfates A and C. The activity against chondroitin
sulfate B was 13–40% of the activity against chondroitin
sulfate A. However, the purified chondroitin ABC lyase
activity for chondroitin sulfate B and C was 32 and 40%
against chondroitin sulfate A, respectively. Hyaluronic acid
was not a substrate for this enzyme. The present chondroitin
ABC lyase substrate specificity was different to the enzymes
previously purified from B. thetaiotaomicron, Bacillus sp.
[6], F. heparinum and P. vulgaris.
Chondroitin AC lyases have also been purified previously
from some bacterial species, Arthrobacter aurescens [26],
F. heparinum and Aeromonas quefaciens [27]. These chon-
droitin AC lyases were equally active against chondroitin
sulfate A and C. The activity against chondroitin sulfate B
was not. However, the purified chondroitin AC lyase
Table 3. Internal amino-acid sequences of chondroitin lyases from Bacteroides stercoris HJ-15.
Enzyme Internal amino acid sequence Homology (%)
B. stercoris chondroitin ABC lyase
YEYAVLPR
B. thetaiotaomicron chondroitin ABC lyase 521YEYMVLIQ 57
P. vulgaris chondroitin AC lyase 873YEYMVFLD 40
F. heparinum chondroitin ABC lyase
578YAYIVLPG 59
B. stercoris chondroitin AC lyase
PGINHPEQ
F. heparinum chondroitin AC lyase 584PGINKPEE 80
B. thetaiotaomicron chondroitin ABC lyase
509PGLNMATP 33

P. vulgaris chondroitin ABC lyase
125PTIDFGEK 28
Table 4. Substrate specificity of chondroitin lyases. Activity on chondroitinase A as the substrate was set at 100%.
Relative activity (%)
B. stercoris F. heparinum
a
B. thetaiotaomicron
a
P. vulgaris
a
Substrate ABC AC ABC AC ABC ABC
Chondroitin sulfate A 100 100 100 100 100 100
Chondroitin sulfate C 40 46 100 110 80–130 100
Dermatan sulfate 32 0 100 0 13–16 34
Hyaluronic acid 0 67 0 107 10–30 60
Heparin 0 0 – – – –
Heparan sulfate (Porcine) 0 0 – – – –
Heparan sulfate (bovine) 0 0 – – – –
a
Data from [6,9–14].
Table 5. K
m
and V
max
values of chondroitin lyases.
Chondrotin ABC lyase Chondroitin AC lyase
Substrate K
m
(lgÆmL
)1

) V
max
(UÆmg
)1
) K
m
(lgÆmL
)1
) V
max
(UÆmg
)1
)
Chondroitin sulfate A 150.1 49.6 388.0 76.7
Dermatan sulfate 42.9 15.9 – –
Chondroitin sulfate C 34.6 22.9 155.6 29.4
2938 S W. Hong et al. (Eur. J. Biochem. 269) Ó FEBS 2002
activity for chondroitin sulfate C and hyaluronic acid was
46% and 67% against chondroitin sulfate A, respectively.
The present substrate specificity of chondroitin AC lyase
was also different to the previously purified enzymes.
Particularly, B. thetaiotaomicron belonging to the same
species with B. stercoris HJ-15 also produces two chondro-
itin ABC lyases, but did not show the chondroitin AC lyase
activity. The chondroitin lyases from B. stercoris HJ-15
were different to the previous purified enzymes from
B. thetaiotaomicron.
When these chondroitin sulfate ABC and AC lyases were
incubated with chondroitin sulfate A, these enzymes mainly
produced disaccharide and tetrasaccharide/hexasaccharide,

respectively (data not shown). These results suggest that
chondroitin ABC and AC lyases have exolytic and endolytic
action patterns.
These chondroitin ABC and AC lyases showed optimal
activity at pH 7.0 and 5.7–6.0 respectively, like most of
previously reported chondroitin lyases which have optimal
pH values in the range 6.0 to 8.0. Most reported
chondroitin lyases had molecular masses between 53 and
118 kDa. The molecular masses of chondroitin ABC and
AC lyases were calculated at 83 and 170 kDa by gel
filtration and by SDS/PAGE, respectively. These results
suggest that chondroitin ABC lyase is composed of a single
subunit and chondroitin AC lyase is composed of two
identical subunits. Amino-acid composition analysis
revealed that the chondroitin ABC and AC lyases contain
a large proportion of lysine, consistent with their pI values
of 7.9–8.3. The pI values of the purified chondroitin lyases
were slightly higher than the previously purified
chondroitin lyases from P. vulgaris and B. thetaiotaomi-
cron. Several attempts at N-terminal analysis failed to
yield sequence information suggesting that the
N-terminus is blocked. Therefore, we analyzed the internal
sequences of a peptide obtained by a tryptic digestion. The
internal sequences of the chondroitin AC lyase reported
here show significantly greater homology to Flavobacterial
chondroitin AC lyase than to B. thetaiotaomicron chon-
droitin ABC lyase. Those of the chondroitin ABC lyase
reported here do not show significantly greater homology
to the previously purified chondroitin ABC lyases from
F. heparinum, B. thetaiotaomicron and P. vulgaris than

those of chondroitin AC lyase. These results suggest that
the chondroitin AC lyase reported here belongs to the
family of previously purified chondroitin AC lyases, but
the present chondroitin ABC lyase is slightly different from
previously identified chondroitin ABC lyases. Molecular
masses, amino-acid composition data and internal amino-
acid sequence homologies of the present chondroitin ABC
lyases were different to the previously reported chondroitin
ABC lyase. There were not any divalent metal ions that
activated the enzymes significantly. Instead, most of
divalent metal ions except for Mn
2+
and Ca
2+
inhibited
the activity of chondroitin lyases. Both chondroitin lyases
were potently inhibited by Cu
2+
and Zn
2+
. Both enzymes
were potently inhibited by p-chloromercuriphenyl sulfonic
acid. This suggests that the active site of these enzymes
may contain a cysteine residue. The chondroitin ABC and
AC lyases reported here showed optimal activity at 40 °C
and 45–50 °C, respectively. The F. heparinum chondroitin
ABC and AC lyases had the lowest optimal temperatures,
30 °Cand40°C, respectively, and the chondroitin ABC
lyases from P. vulgaris and B. thetaiotaomicron showed
optimal activity at 37 °C. The chondroitin lyases reported

here were completely inactivated above 60 °C; i.e. they
seem to be more stable than the previously purified
chondroitin lyases.
In conclusion, this is the first report on the purification
and characterization of chondroitin ABC and AC lyases,
particulary chondroitin AC lyase, from an anaerobic
bacterium (B. stercoris) in human intestine. The substrate
specificity and other characteristics of the two chondroitin
lyases reported here are different from the previous reported
chondroitin lyases.
ACKNOWLEDGEMENT
This work was supported by KOSEF grant 1999-2-209-010-5
(D. H. K. and Y. S. K.) and the BK 21 grant from the Ministry of
Education (D. H. K. and Y. S. K.).
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