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: 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) 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.). REFERENCES 1. Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130. 2. Folkman, J. & Klagsbrun, M. (1987) Angiogenic factors. Science 235, 442–447. 3. Ingber, D.E. (1989) Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro:roleofextracellularmatrix.J. Cell. Biol. 109, 317–330. 4. D’Amore, P.A. (1990) Modes of FGF release in vivo and in vitro. Cancer Metastasis Rev. 9, 227–238. 5. Vlodavsky, I., Fuks, Z., Isai-Michaeli, R., Bashkin, P., Levi, E., Korner, G., Bar-Shavit, R. & Klagsburn, M. (1991) Extracellular matrix resident basic fibroblast growth factor: Implication for the control of angiogenesis. J. Cell. Biochem. 45, 167–176. 6. Jackson, R.L., Busch, S.J. & Cardin, A.D. (1991) Glycosami- noglycans: molecular properties, protein interactions, and role in physiological processes. Physiol. Rev. 71, 481–539. 7. Kjell, L. & Lindahl, U. (1991) Proteoglycans: structures and interactions. Ann. Rev. Biochem. 60, 443–475. 8. Ernst, S., Langer, R., Cooney, C.L. & Sasisekharan, R. (1995) Enzymatic degradation of glycosaminoglycans. Crit.Rev.Bio- chem. Mol. Biology 30, 387–444. 9. Linhardt, R.J., Galliher, P.M. & Cooney, C.L. (1986) Poly- saccharide lyases. Appl. Biochem. Biotechn 12, 135–175. 10. Gu,K.,Liu,J.,Pervin,A.&Linhardt,R.J.(1993)Comparisonof the activity of two chondroitin AC lyases on dermatan sulfate. Carbohydr. Res. 244, 369–377. 11. Michelacci, Y.M. & Dietrich, C.P. (1975) A comparative study between a chondroitinase B and a chondroitinase AC from Fla- vobacterium heparinum. Biochem. J. 151, 121–129. 12. Michelacci, Y.M., Horton, D.S.P.Q. & Poblacion, C.A. (1987) Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum. Biochim. Biophys. Acta 923, 291– 301. 13. Gu, K., Linhardt, R.J., Laliberte, M. & Zimmermann, J. (1995) Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum. Biochem. J. 133, 383–386. 14. Linn, S., Chan, T., Lipeski, L. & Salyers, A.A. (1983) Isolation and characterization of two chondroitin lyases from Bacteroides thetaiotaomicron. J. Bacteriol. 156, 859–866. 15. Salyers, A.A., Vercellotti, J.R., West, S.E.H. & Wilkins, T.D. (1977) Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl. Environ. Microbiol. 33, 319–322. Ó FEBS 2002 New bacteroidal chondroitin ABC and AC lyases (Eur. J. Biochem. 269) 2939 16. Ahn, M.Y., Shin, K.H., Kim, D.H., Jung, E.A., Toida, T., Linhardt, R.J. & Kim, Y.S. (1998) Characterization of a Bacter- oides species from human intestine that degrades glycosami- noglycans. Can. J. Microbiol. 44, 423–429. 17. Kim, B T., Kim, W S., Kim, Y S., Linhardt, R.J. & Kim, D H. (2000) Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15. J. Biochem. 128, 323–328. 18. Kim, B T., Hong, S H., Kim, W S., Kim, Y S. & Kim, D H. (2001) Purification and characterization of acharan sulfate lyases, two novel heparinases, from Bacteroides stercoris HJ-15. Euro. J. Biochem. 268, 2635–2641. 19. Linhardt, R.J. (1994) Analysis of glycosaminoglycans with poly- saccharide lyase In Curr. Prot. Mol. Biol. (Varki, A., ed.), pp. 17.13.17–17.13.32. Wiley Interscience, New York. 20. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. 21. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 22. Tkalec, A.L., Fink, D., Blain, F., Zhang-Sun, G., Laliberte, M., Bennett, D.C., Gu, K., Zimmermann, J.J.F. & Su, H. (2000) Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chon- droitinase AC and chondroitinase B, respectively, from Flavo- bacterium heparinum. Appl. Environ. Microbiol. 66, 29–35. 23. Sato,N.,Shimada,M.,Nakajima,H.,Oda,H.&Kimura,S. (1994) Cloning and expression in Escherichia coli of the gene encoding the Proteus vulgaris chondroitin ABC lyase. Appl. Microbiol. Biotechnol. 41, 39–46. 24. Cheng, Q., YuM.C., Reeves, A.R. & Salyers, A.A. (1995) Iden- tification and characterization of a Bacteroides gene, csuF, which encodes an outer membrane protein that is essential for growth on chondroitin sulfate. J. Bacteriol. 177, 3721–3727. 25. Sato, N., Murata, K. & Kimura, A. (1986) Subunit structure of chondroitinase ABC from Proteus vulgaris. Agric. Biol. Chem. 50, 1057–1059. 26. Hiyama, K. & Okada, S. (1975) Crystallization and some prop- erties of chondroitinase form Arthrobacter aurescens. J. Biol. Chem. 250, 1824–1828. 27. Takegawa, K., Iwahara, K. & Iwahara, S. (1991) Purification and properties of chondroitinase produced by a bacterium isolated from soil. J. Ferm. Bioeng. 72, 128–131. 2940 S W. Hong et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . 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. 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. 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