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 a
Trang 1Purification and characterization of novel chondroitin ABC and AC
bacterium
Sung-Woon Hong1, Byung-Taek Kim1, Ho-Young Shin1, Wan-Suk Kim2, Keun-Sook Lee1, Yeong-Shik Kim2 and Dong-Hyun Kim1
1
College of Pharmacy, Kyung Hee University, Seoul, Korea;2Natural 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)1Æmg)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)1Æmg)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 40C, and 5.7–6.0 and 45–50 C, respectively Both chondroitin lyases were potently inhibited
by Cu2+, Zn2+, and p-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 chonchon-droitin 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 sulsul-fate 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
M A T E R I A L S A N D M E T H O D S
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: dhkim@khu.ac.kr
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)
Trang 26-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(1fi 4)-a-L-IdoA2S(1fi , 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 stercorisHJ-15 was isolated and cultivated as described
previously [16] It was cultured anaerobically under an
atomosphere of 90% nitrogen and 10% carbon dioxide at
37C 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 4C and the resulting
cell pellet was washed twice with cold 0.89% NaCl The cell
pellet was suspended in 150 mL of 50 mM 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 4C 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-brated with 200 mL of 50 mM 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 mM 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 0.6M in 50 mM sodium
phosphate buffer, pH 7.0 (Fig 1) The fractions containing
chondroitin ABC lyase activity were pooled and dialyzed
against 3 L of 50 mMsodium 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 mMsodium phosphate buffer, pH 7.0 The column was washed with 500 mL of the same 50 mM sodium phosphate buffer, pH 7.0 and then eluted with an 800-mL linear gradient of potassium chloride from 0 to 0.5M 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 mM 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 fromB 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 mM sodium phosphate buffer, pH 7.0 The column was washed with
500 mL of the same 50 mM sodium phosphate buffer,
pH 7.0 and then eluted with a total 800 mL linear gradient
of potassium chloride from 0 to 0.5M 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 mM sodium phosphate buffer,
pH 7.0 and unbound proteins were removed by a 500-mL wash of 50 mM sodium phosphate buffer, pH 7.0 The column was eluted with total 400 mL linear gradient of 50–
400 mM 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 40C and a 1-mL quartz cuvette containing
Fig 1 Elution profile of chondroitin lyases on CM-Sephadex C-50ion exchange chromatography Solid circle, chondroitin sulfate A-degra-ding activity; open circle, dermatan sulfate-degraA-degra-ding activity; solid triangle, chondroitin sulfate C-degrading activity; simple line, absorbance at 280 nm.
Trang 31 mg of substrate in 650 lL of 50 mM sodium acetate
buffer, pH 5.8 for chondrotinase AC (or 50 mM 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 3800M )1 for
products (1 U¼ 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-lar mass of the native enzyme was estimated by
gel-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 calibracalibra-tion
kit (from Amersham Pharmacia Biotech) The pH
opti-mum of chondroitin lyases were determined using 50 mM
sodium phosphate buffer (pH 5.0–8.5) The temperature
dependence of the enzyme was investigated by measuring
enzyme activity at different temperatures (20–60C) To
investigate the effect of divalent metal ions and KCl on
the lyase activity, divalent metal ion (final concentration,
100 lM) and KCl (0–500 mM) 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 6M hydrochloric acid, 0.1% phenol at 155C for 1 h
Internal amino-acid sequences of two purified chondro-itin lyases were analyzed by an Applied Biosystem protein sequencer model 492
R E S U L T S
Purification of two chondroitin lyases fromB 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)1Æmg)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)1Æmg)1 (Table 1) Discontinuous SDS/PAGE illustrated that chon-droitin ABC lyase and chonchon-droitin 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
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
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
Trang 4Characterization 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 40C and 45–50 C, respectively
(data not shown)
The activity of chondroitin ABC lyase was inhibited by
addition of Ni2+, Mg2+, Zn2+, Cu2+and Co2+
Partic-ularly, Cu2+and Zn2+potently inhibited chondroitin ABC
lyase and chondroitin AC lyase
Heparin competitively inhibited only chondroitin AC
lyase, not chondroitin ABC lyase Its inhibition was
reversible (IC50 was 18 lM) (Fig 3) The addition of
50 mM KCl in 50 mM sodium phosphate buffer slightly
increased the activity of both chondroitin ABC and AC
lyases However, the addition of more than 50 mM 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 sulsul-fate (Table 4) When chondro-itin sulfate ABC lyase for chondrochondro-itin sulfate A was taken
as 100%, this enzymes activity for chondroitin sulfate C and dermatan sulfate was 40 and 32%, 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 0.03 U of the homogenously purified enzyme activity was taken as 100%.
Metal ion
Relative activity (%) Chondroitin ABC lyase Chondroitin AC lyase
Fig 3 Inhibitory effect of heparin on chondroitin ABC and AC lyases Solid circle, chondroitin sulfate ABC; solid square, chondroitin AC.
Trang 546 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 Km and Vmax of chondroitin ABC and AC lyases
towards chondroitin sulfate A, dermatan sulfate
(chondro-itin sulfate B), and chondro(chondro-itin sulfate C were determined
(Table 5)
D I S C U S S I O N
B stercorisHJ-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 chonchon-droitin 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 chonchon-droitin 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.
B stercoris chondroitin ABC lyase YEYAVLPR
B thetaiotaomicron chondroitin ABC lyase 521YEYMVLIQ 57
B stercoris chondroitin AC lyase PGINHPEQ
B thetaiotaomicron chondroitin ABC lyase 509PGLNMATP 33
Table 4 Substrate specificity of chondroitin lyases Activity on chondroitinase A as the substrate was set at 100%.
Relative activity (%)
B stercoris F heparinuma B thetaiotaomicrona P vulgarisa
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)
Trang 6activity 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 chondrochondro-itin 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 chonchon-droitin 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, 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 Mn2+and Ca2+inhibited
the activity of chondroitin lyases Both chondroitin lyases
were potently inhibited by Cu2+and Zn2+ 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 40C
and 45–50C, respectively The F heparinum chondroitin
ABC and AC lyases had the lowest optimal temperatures,
30C and 40 C, respectively, and the chondroitin ABC
lyases from P vulgaris and B thetaiotaomicron showed optimal activity at 37C The chondroitin lyases reported here were completely inactivated above 60C; 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
A C K N O W L E D G E M E N T 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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