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Purification and characterization of novel chondroitin ABC and AC


Eur. J. Biochem. 269, 2934–2940 (2002) ? FEBS 2002

doi:10.1046/j.1432-1033.2002.02967.x

Puri?cation and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic 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 puri?ed from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was puri?ed to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a ?nal speci?c activity of 45.7 lmol?min)1?mg)1. Chondroitin AC lyase was puri?ed to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a ?nal speci?c activity of 57.03 lmol?min)1?mg)1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel ?ltration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel ?ltration. 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 Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The puri?ed Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The puri?ed chondroitin 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 ?ndings suggest that the biochemical properties of these puri?ed chondroitin lyases are di?erent from those of the previously puri?ed chondroitin lyases. Keywords: Bacteroides sterocirs HJ-15; chondroitin ABC lyase; chondroitin AC lyase; chondroitin sulfate; puri?cation.

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 sulfate 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

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)

often very speci?c and act under mild conditions giving oligosaccharide products. Two classes of enzyme that act on GAGs are polysaccharide lyases and hydrolases. Prokaryotic 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 identi?ed 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 puri?cation of chondroitin ABC lyase and chondroitin AC lyase, which have not been puri?ed from Bacteroides sp., to apparent homogeneity and the determination of their physical properties, kinetic properties, optimal catalytic conditions and speci?city.

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

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New bacteroidal chondroitin ABC and AC lyases (Eur. J. Biochem. 269) 2935

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 fast?ow, HA Ultrogel (microcrystalline hydroxyapatite, 4% beaded in agarose), phosphocellulose (coarse mesh) and low molecular mass markers for gel ?ltration were purchased from Sigma Chemical Co. CM-Sephadex C-50, Sephacryl S-300 HR resins, high molecular mass markers for gel-?ltration 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 ? 4)-aD-GlcNAc(1 ? 4)-a-L-IdoA2S(1 ? , 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 g?L)1) instead of glucose, 0.01% (w/v) sodium thioglycolate and 0.1% (w/v) ascorbic acid. Puri?cation 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 mM sodium phosphate 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 ?fty milliliters of cell extract (or 150 mL) was passed through a QAEcellulose column (2.8 · 38 cm) which had been preequilibrated 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 ef?uent. The noninteracting ?uid (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 puri?cation 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 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 mM sodium phosphate buffer, pH 7.0 for 12 h for next step. The dialyzed enzyme preparation was

Fig. 1. Elution pro?le of chondroitin lyases on CM-Sephadex C-50 ion exchange chromatography. Solid circle, chondroitin sulfate A-degrading activity; open circle, dermatan sulfate-degrading activity; solid triangle, chondroitin sulfate C-degrading activity; simple line, absorbance at 280 nm.

applied to a hydroxyapatite column (2.5 · 6 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 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 ultra?ltration 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 ?ow rate of 1 mL?min)1. Fractions containing chondroitin ABC lyase activity were tested for purity by electrophoresis. Puri?cation procedure of chondroitin AC lyase from B. stercoris HJ-15 In the procedure of the puri?cation of chondroitin ABC lyase, the ?uid (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 0.5 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 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 40 °C and a 1-mL quartz cuvette containing

2936 S.-W. Hong et al. (Eur. J. Biochem. 269)

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1 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 coef?cient of 3800 M)1 for products (1 U ? 1 lmol of uronic acid containing product formed per min) [19]. The speci?c 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 molecular mass of the native enzyme was estimated by gel?ltration using Sephacryl S-300 HR column (1.6 · 70 cm) calibrated with gel ?ltration low molecular mass calibration kit (from Sigma Co.) and high molecular calibration kit (from Amersham Pharmacia Biotech). The pH optimum 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 (20–60 °C). To investigate the effect of divalent metal ions and KCl on the lyase activity, divalent metal ion (?nal 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 derivatization chemistry. Hydrolysis was performed using using 6 M hydrochloric acid, 0.1% phenol at 155 °C for 1 h. Internal amino-acid sequences of two puri?ed chondroitin lyases were analyzed by an Applied Biosystem protein sequencer model 492.

RESULTS
Puri?cation 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  ?vefold (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 chromatography to remove interacting proteins. Chondroitin ABC lyase activity passed through these columns without binding to the matrices. The ef?uent was further puri?ed to homogeneity with yield of 2.37% by a series of CM-Sephadex C-50 column chromatography (Fig. 1), hydroxyapatite column and Sephacryl S-300 gel ?ltration chromatography, and its ?nal speci?c activity was 45.7 lmol?mL)1?mg)1 (Table 1). Chondroitin AC lyase, which passed through CM-Sephadex C-50 resin without binding, was puri?ed to single band on SDS/PAGE with a yield of 6.28% by a combination of hydroxyapatite column and phosphocellulose column chromatography. The speci?c activity of puri?ed chondroitin AC lyase was 57.03 lmol?mL)1?mg)1 (Table 1). Discontinuous SDS/PAGE illustrated that chondroitin ABC lyase and chondroitin AC lyase were apparently homogeneous and their molecular mass values were estimated to be 116 and 84 kDa, respectively (Fig. 2).

Table 1. Puri?cation summary of chondroitin ABC and AC lyases from B. sterocoris HJ-15. One unit (U) is the activity forming 1 lmol disaccharides per min. The activity was assayed in 50 mM sodium phosphate bu?er, pH 7.0. Stage Chondroitin ABC lyase Crude extract QAE-cellulose column chromatography CM Sephadex C-50 column chromatography Hydroxyapatite column chromatography Sephacryl S-300 column chromatography Chondroitin AC lyase Crude extract QAE-cellulose column chromatography CM-Sephadex C-50 column chromatography Hydroxyapatite column chromatography Phosphocellulose column chromatography Total activity (U) Total protein (mg) Speci?c activity (U?mg)1)

418.53 193.86 218.03 88.34 9.94 418.53b 193.86 137.14 115.74 26.3

1000 243.5 14.94 2.15 0.22 1000 243.5 265.9 23 0.461

0.42 0.8 14.59 41.09 45.7 0.42 0.8 0.52 5.03 57.03

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Fig. 3. Inhibitory e?ect of heparin on chondroitin ABC and AC lyases. Solid circle, chondroitin sulfate ABC; solid square, chondroitin AC. Fig. 2. SDS/PAGE of the puri?ed chondroitin ABC (A) and AC (B) lyases at various steps of puri?cation. (A) Lane 1, preparation after crude extract; lane 2, preparation after QAE-cellulose column chromatography; lane 3, preparation after CM-Sephadex C-25 column chromatography; lane 4, preparation after hydroxyapatite ultragel column chromatography; lane 5, Sephacryl S-300 column chromatography; lane M, marker. (B) Lane 1, preparation after CM-Sephadex C-25 column chromatography; lane 2, preparation after hydroxyapatite ultragel column chromatography; lane 3, preparation after phosphocellulose column chromatography; lane M, markers.

Characterization of two chondroitin lyases When the molecular masses of chondroitin ABC and AC lyases under nondenaturing conditions were determined by gel ?ltration, 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 Ni2+, Mg2+, Zn2+, Cu2+ and Co2+. Particularly, 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 puri?ed chondroitin ABC and AC lyases were 7.9 and 8.3,

respectively, slightly higher than those of the previously puri?ed 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 signi?cantly 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?cantly greater homology to P. vulgaris chondroitin ABC lyase. Substrate speci?city of two puri?ed chondroitin lyases Chondroitin ABC lyase depolymerized chondroitin sulfate A, C and dermatan sulfate (Table 4). When chondroitin sulfate ABC lyase for chondroitin 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 sulfate 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
Table 2. E?ect of divalent metal ions on the activity of chondroitin lyases. Final concentration of divalent ion, 1 mM. 0.03 U of the homogenously puri?ed enzyme activity was taken as 100%. Relative activity (%) Metal ion Control Co2+ Ni2+ Cu2+ Mg2+ Mn2+ Ca2+ Zn2+ EDTA Chondroitin ABC lyase 100 89 42 7 41 95 97 3 100 Chondroitin AC lyase 100 91 76 6 102 103 105 13 100

2938 S.-W. Hong et al. (Eur. J. Biochem. 269)
Table 3. Internal amino-acid sequences of chondroitin lyases from Bacteroides stercoris HJ-15. Enzyme B. stercoris chondroitin ABC lyase B. thetaiotaomicron chondroitin ABC lyase P. vulgaris chondroitin AC lyase F. heparinum chondroitin ABC lyase B. stercoris chondroitin AC lyase F. heparinum chondroitin AC lyase B. thetaiotaomicron chondroitin ABC lyase P. vulgaris chondroitin ABC lyase Internal amino acid sequence
YEYAVLPR 521YEYMVLIQ 873YEYMVFLD 578YAYIVLPG PGINHPEQ 584PGINKPEE 509PGLNMATP 125PTIDFGEK

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Homology (%)

57 40 59 80 33 28

Table 4. Substrate speci?city of chondroitin lyases. Activity on chondroitinase A as the substrate was set at 100%. Relative activity (%) B. stercoris Substrate Chondroitin sulfate A Chondroitin sulfate C Dermatan sulfate Hyaluronic acid Heparin Heparan sulfate (Porcine) Heparan sulfate (bovine)
a

F. heparinuma AC 100 46 0 67 0 0 0 ABC 100 100 100 0 – – – AC 100 110 0 107 – – –

B. thetaiotaomicrona ABC 100 80–130 13–16 10–30 – – –

P. vulgarisa ABC 100 100 34 60 – – –

ABC 100 40 32 0 0 0 0

Data from [6,9–14].

46 and 67%, respectively. However, dermatan sulfate, heparin, heparan sulfate and acharan sulfate were not substrates for this enzyme. Kinetic constants of two puri?ed 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 (chondroitin 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 puri?ed two chondroitin lyases. CMSephadex C-50 chromatography ef?ciently resolved chondroitin sulfate degrading lyases of B. stercoris HJ-15. As Fr-a showed a higher speci?city to chondroitin sulfates A
Table 5. Km and Vmax values of chondroitin lyases. Chondrotin ABC lyase Substrate Chondroitin sulfate A Dermatan sulfate Chondroitin sulfate C Km (lg?mL)1) 150.1 42.9 34.6

and C, and Fr-b fractions showed a higher speci?city 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 puri?ed 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 chondroitin sulfates A and C. The activity against chondroitin sulfate B was 13–40% of the activity against chondroitin sulfate A. However, the puri?ed 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 speci?city was different to the enzymes previously puri?ed from B. thetaiotaomicron, Bacillus sp. [6], F. heparinum and P. vulgaris. Chondroitin AC lyases have also been puri?ed previously from some bacterial species, Arthrobacter aurescens [26], F. heparinum and Aeromonas quefaciens [27]. These chondroitin AC lyases were equally active against chondroitin sulfate A and C. The activity against chondroitin sulfate B was not. However, the puri?ed chondroitin AC lyase

Chondroitin AC lyase Vmax (U?mg)1) 49.6 15.9 22.9 Km (lg?mL)1) 388.0 – 155.6 Vmax (U?mg)1) 76.7 – 29.4

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activity for chondroitin sulfate C and hyaluronic acid was 46% and 67% against chondroitin sulfate A, respectively. The present substrate speci?city of chondroitin AC lyase was also different to the previously puri?ed enzymes. Particularly, B. thetaiotaomicron belonging to the same species with B. stercoris HJ-15 also produces two chondroitin ABC lyases, but did not show the chondroitin AC lyase activity. The chondroitin lyases from B. stercoris HJ-15 were different to the previous puri?ed 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 ?ltration 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 puri?ed chondroitin lyases were slightly higher than the previously puri?ed chondroitin lyases from P. vulgaris and B. thetaiotaomicron. 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 signi?cantly greater homology to Flavobacterial chondroitin AC lyase than to B. thetaiotaomicron chondroitin ABC lyase. Those of the chondroitin ABC lyase reported here do not show signi?cantly greater homology to the previously puri?ed 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 puri?ed chondroitin AC lyases, but the present chondroitin ABC lyase is slightly different from previously identi?ed chondroitin ABC lyases. Molecular masses, amino-acid composition data and internal aminoacid 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 signi?cantly. 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 40 °C and 45–50 °C, respectively. The F. heparinum chondroitin ABC and AC lyases had the lowest optimal temperatures, 30 °C and 40 °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 puri?ed chondroitin lyases. In conclusion, this is the ?rst report on the puri?cation and characterization of chondroitin ABC and AC lyases, particulary chondroitin AC lyase, from an anaerobic bacterium (B. stercoris) in human intestine. The substrate speci?city 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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