Purification and characterization of novel salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15 Sung-Woon Hong 1 , Ho-Young Shin 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 Salt-active acharan sulfate lyase (no EC number) has been purified from Bacteroides stercoris HJ-15, which was iso- lated from human intestinal bacteria with GAG degrading enzymes. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, diethylaminoethyl (DEAE)-cellulose, CM-Sephadex C-50, HA ultrogel and phosphocellulose column chromatography with the final specific activity of 81.33 lmolÆmin )1 Æmg )1 . The purified salt- active acharan sulfate lyase was activated to 5.3-fold by salts (KCl and NaCl). The molecular weight of salt-active acha- ran sulfate lyase was 94 kDa by SDS/PAGE and gel filtra- tion. The salt-active acharan sulfate lyase showed optimal activity at pH 7.2 and 40 °C. Salt-active acharan sulfate lyase activity was potently inhibited by Cu 2+ , Ni 2+ and Zn 2+ . This enzyme was inhibited by some agents, butanediol and p-chloromercuric sulfonic acid, which modify arginine and cysteine residues. The purified Bacteroidal salt-active acharan sulfate lyase acted to the greatest extent on acharan sulfate, to a lesser extent on heparan sulfate and heparin. The biochemical properties of the purified salt-active acharan sulfate lyase are different from those of the previously puri- fied heparin lyases. However, these findings suggest that the purified salt-active acharan sulfate lyase may belong to heparin lyase II. Keywords: Bacteroides stercoris HJ-15; salt-active acharan sulfate lyase; acharan sulfate lyase; acharan sulfate; heparin. Heparin, heparan sulfate and acharan sulfate glycosamino- glycans (GAGs) are comprised of alternating 1–4-linked glucosamine and uronic acid residues. Heparan sulfate is composed primarily of monosulfated disaccharides of N-acetyl- D -glucosamine and D -glucuronic acid while hep- arin is composed mainly of trisulfated disaccharides of N-sulfonyl- D -glucosamine and L -iduronic acid [1,2]. Acha- ran sulfate, isolated from the giant African snail Achatina fulica, has a structure closely related to heparin and heparan sulfate, with a uniform repeating disaccharide structure of fi4)-a- D -GlcNAc(1fi 4)-a- L -IdoA2S (1fi[3]. Acharan sulfate exclusively contains N-acetyl- D -glucosamine instead of N-sulfonyl- D -glucosamine in GAGs. Related to the degradation of these GAGs, some heparin lyases that can eliminatively cleave polysaccharides (heparin or heparan sulfate GAGs) have been reported [4–6]. These enzymes are classified as: (a) heparin lyase I (heparinase I, EC 4.2.2.7), acting primarily at the fi 4)-a- D -GlcNS(6S or OH)(1fi4)-a- L -IdoA2S(1fi linkages present in heparin; (b) heparin lyase II (heparinase II or heparitinase II), acting at the fi4)-a- D -GlcNS(6S or OH)(1fi 4)-a- L -IdoA(2S or OH) or -b- D -GlcA(1fi linkages present in both heparin and heparan sulfate; and (c) heparin lyase III (heparinase III or heparitinase, EC 4.2.2.8), acting on the fi4)-a- D -GlcNS(or Ac) (1fi 4)-b- D -GlcA (or IdoA) (1fi linkages found exclu- sively in heparan sulfate. The heparin lyases have become increasingly important in understanding the biological roles and structure of the glycoaminoglycans (and proteoglycan), which are involved in the well known anticoagulant activity [7] and the regulation of various cellular processes such as the potentiation of angiogenesis [8] and the modulation of cellular proliferation [9]. Several heparin lyases of bacterial origin have been purified and characterized from various species including Flavobacterium heparinum [4,10], Bacillus sp. BH 100 [11], Prevotella heparinolyticus [12], and Bacter- oides stercoris HJ-15 [13,14]. Bacteroides stercoris HJ-15 has been recently isolated from human intestine and it contains several kinds of GAG degrading enzymes including heparin, heparan sulfate, acharan sulfate and chondroitin sulfate [13–16]. We purified two kinds of novel heparin lyases, heparin lyase II-1 (acharan sulfate lyase 1), and heparin lyase II-2 (acharan sulfate lyase 2) and III, from this B. stercoris HJ-15 [15,16]. The Bacteroidal heparin lyase III cleaved heparin as well as heparan sulfate, but did not cleave acharan sulfate. The Bacteroidal acharan sulfate lyase, which potently cleaved acharan sulfate as well as heparin, are highly specific to acharan sulfate compared to the previously reported heparin lyases [16]. The purified acharan sulfate lyases 1 and 2 (no EC number) were not activated by salts such as KCl. However, when Bacteroidal acharan sulfate lyase-active fraction isolated from B. stercoris HJ-15 was incubated with salts (KCl), the enzyme fraction was 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: CM, carboxymethyl; DEAE, diethylaminoethyl; DUA, 4-deoxy-a- L -threo-hex-4-enopyranosyl uronic acid; GAG, glycosaminoglycan; GlcA, glucuronic acid; GlcN, glucosamine; HA, hydroxyapatite; IdoA, iduronic acid; IEF, isoelectric focusing; QAE, quaternary amino ethyl; PCMS, p-chloromercurisulfonic acid. (Received 3 April 2003, revised 15 May 2003, accepted 30 May 2003) Eur. J. Biochem. 270, 3168–3173 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03696.x activated by salts. Therefore, we tried to purify salt-active acharan sulfate lyase from B. sterocoris HJ-15 that acts predominantly on acharan sulfate. Materials and methods Materials Heparin (porcine intestinal mucosa), heparan sulfate (por- cine intestinal mucosa), chondroitin sulfate A (bovine trachea), chondroitin sufate B (porcine intestinal mucosa), chondroitin sufate C (shark cartilage), thioglycolic acid (sodium salt), QAE cellulose Fastflow, and HA Ultrogel (microcrystalline hydroxyapatite, 4% beaded in agarose) were supplied by Sigma Chemical Co. Sodium dodecyl sulfate, CM-Sephadex C-50, phosphocellulose, Sephacryl S-300 HR resins and molecular weight markers for gel filtration and protein electrophoresis were obtained from Pharmacia Biotech Co. (Uppsala, Sweden). Diethylamino- ethyl (DEAE)-cellulose resin was purchased from Wako Pure Chemical Industries (Tokyo, Japan). Protein assay kit and Coomassie Brilliant Blue R-250 were from Bio-Rad (Hercules, CA, USA). Tryptic soy broth was provided by Difco Co. Acharan sulfate was prepared as described by Kim et al. [3]. All other chemicals were of the highest grade available. Bacterial strains and purification of salt-active acharan sulfate lyase B. stercoris HJ-15 was isolated and cultivated as described previously [14,16]. It was cultured anaerobically under an atmosphere of 90% (v/v) nitrogen and 10% (v/v) carbon dioxide at 37 °C in 100 L of tryptic soy broth (pH 7.2) containing heparin (0.15 gÆL )1 ) instead of glucose, 0.01% (w/v) sodium thioglycolate and 0.1% (w/v) ascorbic acid. The cultured cells were harvested in the late exponential phase (11–12 h) by centrifugation at 4000 g 1 for 30 min at 4 °C and the resulting cell pellet was washed twice with saline containing 50 m M sodium phosphate (pH 7.0). The cell pellet was suspended in 600 mL of Buffer A (50 m M sodium phosphate buffer, pH 7.0). Cell suspension (30 mL at a time) was placed into a 50-mL centrifuge tube and disrupted by 30-min periods of sonication at 1-s intervals on an ultrasonic processor (Eyela Co.) at an 80% output with cooling. Cell debris was removed by centrifugation at 25 000 g 2 for 60 min at 4 °C. All operations were carried out at 4 °C unless otherwise noted. The cell extract (600 mL) was passed through a QAE cellulose column (5 · 40 cm) which had been pre-equilibrated with Buffer A. The column was washed with the same buffer until no acharan sulfate lyase activity was detectable in the effluent. The fractions which passed through the column were applied to a DEAE-cellulose column (5 · 30 cm) equili- brated with Buffer A. The column was then eluted with the same buffer until any ASL activity could not be detected. The noninteracting fluid passed through the column was collected. The total volume of the flow through was 1800 mL. The eluate was loaded onto a CM-Sephadex C- 50 column (3 · 30 cm) previously equilibrated with Buffer A. The column was washed with 1000 mL (1 L) of the same buffer and then eluted with a 2-L linear gradient of KCl from 0 to 0.6 M in Buffer A at a flow rate of 105 mLÆh )1 .All fractions obtained were assayed for heparin lyase and acharan sulfate lyase activities. Four fractions (Fr-s, Fr-a, Fr-b and Fr-c) containing the activity of these enzymes were collected separately and assayed for the activities degrading acharan sulfate and heparan sulfate. Fr-s had acharan sulfate lyase activity, which was activated by salts, was dialyzed against Buffer A for the further purification. The dialyzed enzyme preparation (210 mL) was applied to a HA Ultrogel column (2.5 · 9 cm) equilibrated with Buffer A. Being washed with 500 mL of the same buffer, the column was eluted with a 800-mL linear gradient, from 50 m M sodium phosphate buffer (pH 7.0) to 400 m M sodium phosphate buffer (pH 7.0) at a flow rate of 120 mLÆh )1 . The active fractions were pooled and dialyzed twice against 2 L of Buffer A. The dialyzed enzyme (78 mL) was loaded onto a phosphocellulose column (3 · 25 cm) equilibrated with Buffer A. After washing the nonabsorbed proteins with 300 mL of the same buffer, the column was eluted with an 800-mL linear gradient, from 50 m M sodium phosphate buffer (pH 7.0) to 400 m M sodium phosphate buffer (pH 7.0) at a flow rate of 100 mLÆh )1 . The active fractions (salt-active acharan sulfate lyase, fraction numbers 17–25) were investigated for homogeneity by SDS/PAGE. Enzyme activity assay The activity of acharan sulfate lyase was measured accord- ing to the previously published procedure [17]. 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 micromole of DUA containing product formed per minute) [17]. The specific activity was calculated by dividing the micromoles of product produced per minute by the milligrams of protein in the cuvette. Protein concentra- tion was measured by a Bradford assay using bovine serum albumin as a standard [18]. Characterization of salt-active acharan sulfate lyase SDS/PAGE was performed for the determination of molecular mass according to Laemmli’s procedure [19]. The gel was stained with Coomassie Brilliant Blue R-250 solution and further stained with silver. The pI value of heparin lyase was determined by IEF electrophoresis using Model 111 Mini IEF Cell (from Bio-Rad) according to the manufacturer’s instructions. The molecular weight of the native enzyme was estimated by gel filtration using Seph- acryl S-300 HR column (1.6 · 70 cm) calibrated with gel filtration low molecular weight calibration kit (from Sigma Co.) and high molecular calibration kit (from Amersham Pharmacia Biotech). The pH optimum of acharan sulfate lyase was determined using 50 m M sodium phosphate buffer (pH 6.0–8.5). Temperature dependency of the enzyme was investigated by measuring enzyme activity at different temperatures (25–60 °C). To investigate the effect of divalent metal ions and KCl (or NaCl) on the lyase activity, divalent metal ion (final concentration, 100 l M ), chemical modifying agents (50 l M ) and KCl (0–500 m M ) were added into the reaction mixture. Kinetic constant of acharan sulfate lyase was determined by measuring the initial rates at Ó FEBS 2003 Bacteroidal salt-active acharan sulfate lyase (Eur. J. Biochem. 270) 3169 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. Because of their low solubility, 100 lg of acharan sulfate were used in this assay. The internal amino acid sequence of purified salt-active acharan sulfate lyase was analyzed by an Applied Biosystem protein sequencer model 492 in Korea Basic Science Institute. Results Purification of salt-active acharan sulfate lyase Bacteroides stercoris HJ-15, which degrades a variety of GAGs including heparin, heparan sulfate, acharan sulfate and chondroitin sulfates [13], constitutively produced hep- arin lyase and acharan sulfate lyase activities. However, when induced with acharan sulfate or heparin, total acharan sulfate activity increased by about 3.5-fold (data not shown). Furthermore, total acharan sulfate lyase activity was activated 5.7-fold by salts, KCl and NaCl. However, the previously purified enzymes, acharan sulfate lyases and heparinase III, from B. sterocoris were not activated by salts. Therefore, to purify salt-active heparin lyase, B. stercoris HJ-15 cells were disrupted by ultrasonic, and the super- natant, the crude extract, was subjected to a combination of QAE-cellulose and DEAE-cellulose column chromato- graphy to remove interacting proteins. Acharan sulfate lyase activity passed through these columns without binding to the matrices. The effluent was applied to CM-Sephadex C-50 column chromatography (Fig. 1). The salt-active acharan sulfate lyase activity fraction was then further purified to homogeneity by a series of hydroxyapatite Ultrogel chromatography and finally phosphocelluose col- umn chromatography (Fig. 2). The specific activity and total activity at each purification step are summarized in Table 1. The specific activity of the purified acharan sulfate lyase activity had 80.33 UÆmg )1 proteinwithayieldof7.4%.The purified acharan sulfate lyase was apparently homogeneous by SDS/PAGE and its molecular mass was identically estimatedtobe94000Da(Fig.2). Characterization of salt-active acharan sulfate lyase The molecular weight of salt-active acharan sulfate lyase under nondenaturing conditions was determined by gel filtration (data not shown). Acharan sulfate lyase was estimated to be about 94 000 Da. It suggests that acharan sulfate lyase is composed of one subunit. The optimal pH of acharan sulfate lyase was determined to be 7.2–7.3 for acharan sulfate, heparin and heparan sulfate (Fig. 3), and the optimum temperature for the maximal activity was shown at 40 °C (Fig. 4). Fig. 1. Elution profile of CM-Sephadex C-50 ion exchange (A), hydroxyapatite ultrogel (B) and phosphocellulose (C) column chromato- graphies. s, acharan sulfate lyase activity without KCl; m, acharan sulfate lyase activity with 50 m M KCl; 8 n, heparin lyase activity; simple line, absorbance at 280 nm. Fig. 2. SDS/PAGE of the purified salt-active acharan sulfate lyase at various steps of purification. Lanes 1 and 8, marker; lane 2, preparation after crude extract; lane 3, preparation after QAE column chroma- tography; lane 4, preparation after DEAE-cellulose column chroma- tography; lane 5, preparation after CM-Sephadex C-25 column chromatography; lane 6, preparation after hydroxyapatite ultragel column chromatography; and lane 7, purified salt-active heparin lyase II after phosphocellulose column chromatography. 3170 S W. Hong et al. (Eur. J. Biochem. 270) Ó FEBS 2003 The purified acharan sulfate lyase was activated 5.3-fold by the strength of salts, such as KCl and NaCl (Fig. 5). However, divalent cations CaCl 2 , and MgCl 2 , did not activate this enzyme compared to KCl and NaCl. The salt- active acharan sulfate lyase activity was slightly increased by addition of Mn 2+ , whereas they were severely inhibited by Cu 2+ , Ni 2+ and Zn 2+ (Table 2). The purified enzyme was inhibited by PCMS 3 and butane-1,3-diol. Particularly, PCMS potently inhibited salt-active acharan sulfate lyase 4 , but little inhibited by the other agents that modify histidine and cysteine residues (Table 3). Amino acid composition analysis revealed that the salt- active acharan sulfate lyase contains a large proportion of lysine (data not shown). The pI value of the purified salt- active acharan sulfate lyase was 8.5, but slightly lower than those of the previously purified Flavobacterial heparin lyases range from 8.9 to 10.1. We analyzed the internal sequences of a peptide obtained by digestion of each enzyme with trypsin (Table 4). The internal sequence of the salt- active acharan sulfate lyase showed homology of 50% to Flavobacterial heparin lyases I and II previously reported, but did not showed homology to Flavobacterial heparin lyase III [10,20]. Fig. 3. Effect of pH on the activity of salt-active acharan sulfate lyase. The enzyme activity was assayed in 50 m M sodium phosphate buffer at the indicated pH. d, activity for acharan sulfate; j, activity for hep- aran sulfate; m, activity for heparin. Fig. 4. Effect of temperature on the activity of salt-active acharan sul- fate lyase. The enzyme activity was assayed in 50 m M sodium phos- phate. Fig. 5. Effect of KCl on the activity of salt-active acharan sulfate lyase. d, salt-active acharan sulfate lyase II purified from Fr-s in Fig. 1; m, heparin lyase II-1 (acharan sulfate lyase 1) purified from Fr-a in Fig. 1 [16]; j, heparin lyase II-2 (acharan sulfate lyase 2) purified from Fr-a in Fig. 1 [16]. Table 1. Purification summary of salt-active acharan sulfate lyase. One unit (U) is the activity forming 1 lmol disaccharides from acharan sulfate per minute. Numbers in parentheses indicate the activities for heparin used as a substrate. Stage Total activity (U) Total protein (mg) Specific activity (UÆmg )1 ) Crude extract 165.4 5250.12 0.03 QAE cellulose column chromatography 65.2 1407.18 0.05 DEAE-cellulose column chromatography 90.3 747.04 0.12 CM Sephadex C-50 column chromatography 37.2 13.67 2.72 Hydroxyapatite ultrogel column chromatography 34.5 1.82 18.96 Phosphocellulose column chromatography 12.2 (1.3) 0.15 81.33 (8.67) Table 2. Effect of divalent metal ions on salt-active acharan sulfate lyase activity. Metal ion a Residual activity b (%) Control 100 Mg 2+ 94.4 Cu 2+ 0 Ni 2+ 7.7 Co 2+ 67.3 Mn 2+ 102.6 Ca 2+ 85.4 Zn 2+ 14.5 Pb 2+ 93.3 EDTA 107 a Final concentration, 1 m M . b 0.03 U of homogenously purified enzyme activity was taken as 100%. 5 Ó FEBS 2003 Bacteroidal salt-active acharan sulfate lyase (Eur. J. Biochem. 270) 3171 Substrate specificity of purified salt-active acharan sulfate lyase The purified salt-active acharan sulfate lyase degraded heparin and heparan sulfate as well as acharan sulfate (Table 5). The salt-active acharan sulfate lyase was the most active to acharan sulfate. When the salt-active acharan sulfate lyase activity for heparan was taken as 100%, the enzyme activities for acharan sulfate and heparan sulfate were 952.3 and 149.5%, respectively. However, all types of chondroitin sulfates were not also substrates for the enzyme. Kinetic constants of purified salt-active acharan sulfate lyase Michaelis–Menten constants were determined under the optimum reaction conditions in experiments designed to calculate reaction velocities at each substrate concentration (Table 6). K m and V max of acharan sulfate on salt-active acharan sulfate lyase were estimated at 65.4 lgÆmL )1 and 131.2 lmolÆmin )1 Æmg )1 , respectively. As for heparin on salt- active acharan sulfate lyase, K m and V max values of heparin and heparan sulfate were calculated at 18.5 lgÆmL )1 , 8.7 lmolÆmin )1 Æmg )1 and 40.7 lgÆmL )1 , 13.1 lmolÆ min )1 Æmg )1 , respectively. Discussion In the present report, we have purified salt-active acharan sulfate lyase specifically acting on acharan sulfate from Fr-s fraction of CM-Sephadex C-50 chromatography, which efficiently resolved GAGs degrading lyases of B. stercoris HJ-15. As Fr-b and Fr-c fractions showed a higher specificity to heparan sulfate and heparin, they were considered to be similar to heparin lyase III and heparin Table 3. Effect of divalent metal ions on salt-active acharan sulfate lyase activity. Homogenously purified enzyme activity (0.03 U) was taken as 100%. Chemical modifying agent IC 50 (m M ) Control >1 N-Tosyl- L -phenylalanine chloromethyl ketone >1 Butane-1,4-diol 0.4 Paraoxon >1 2-Mercaptoethanol >1 dl-Dithiothreitol >1 Phenylmethylsulfonyl fluoride >1 Iodoacetic acid >1 Sodium-p-tosyl- L -lysine chloromethyl ketone >1 p-Chloromercuric sulfonic acid 0.02 Table 4. Internal amino acid sequence of salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15. Enzyme Internal amino acid sequence Homology (%) Salt-active acharan sulfate lyase …GTIQYG… Flavobacterial heparin lyase I a 213 50 …GKITYV… Flavobacterial heparin lyase II a 157 50 …GAIVYD… Flavobacterial heparin lyase III a 567 33 …LMIQSL… a Data from [10,20]. Table 5. Substrate specificity of acharan sulfate lyase and the previously reported heparin lyases. Activity on heparin (or heparan sulfate in Flavobacterial heparin lyase III) as the substrate was set at 100%. 6 Hep., heparin lyase. Substrate Activity (%) Bacteroidal Favobacterial a Salt-active Hep II Hep II-1 (ASL1) b HepII-2 b (ASL2) Hep III b Hep I Hep II Hep III Heparin 100 100 100 100 100 100 0 Heparan sulfate (porcine) 149.5 128 121.7 610 30 172 100 Acharan sulfate 952.3 549.5 339 0 0 100 c 0 Chondroitin sulfate A 0 0 0 0 0 0 0 Chondroitin sulfate B 0 0 0 0 0 0 0 Chondroitin sulfate C 0 0 0 0 0 0 0 a Data from [1,4,5,13,21]. b Data from [15,16]. c Unpublished data. Table 6. 7 K m and V max values of salt-active acharan sulfate lyase. Enzyme K m (lgÆmL )1 ) V max (UÆmg protein )1 ) Acharan sulfate Heparin Heparin sulfate Acharan sulfate Heparin Heparin sulfate Salt-active acharan sulfate lyase 65.4 18.5 40.7 131.2 8.7 13.1 Acharan sulfate lyase 1 a 28.1 8.8 7.5 65.0 11.6 14.3 Acharan sulfate lyase 2 a 42.2 20.6 16.4 107.6 22.9 31.3 a Data from [16]. 3172 S W. Hong et al. (Eur. J. Biochem. 270) Ó FEBS 2003 lyase I prepared from F. heparinum, respectively [15,16]. Two acharan sulfate lyases previously purified from Fr-a fraction showed the different substrate specificity compared to those of Fr-b, Fr-c and the previously reported heparin lyases [16]. These enzymes were highly specific to acharan sulfate compared to heparin and heparan sulfate. The salt- active acharan sulfate lyase purified from Fr-s fraction showed a different substrate compared heparin lyase I and III, but exhibited a similar substrate specificity of two acharan sulfate lyase previously purified from Fr-a. Acha- ran sulfate was the best substrate for the purified present enzyme. Particularly, the present enzyme was significantly activated by salts, KCl and NaCl, although two acharan sulfate lyases were not activated by salts. Several attempts at N-terminal analysis failed to yield sequence information suggesting the N-terminus to be blocked. Therefore, we analyzed the internal sequences of a peptide obtained by digestion with trypsin. The internal sequence of the peptide showed poor homology to Flavobacterial heparin lyases I and II. Although the substrate specificities of heparin lyase I and III are well understood, the structural requirement for the cleavage of heparin and heparan sulfate by heparin lyase II is not well characterized. This could be due to a structural complexity of heparin and heparan sulfate and a lack of homogeneous polysaccharide. A recently characterized GAG, acharan sulfate, from African giant snail Achatina fulica can provide the criteria to classify the heparin lyase II among the specificity of heparin lyases. In conclusion, the substrate specificity as well as the characterization of salt-active acharan sulfate lyase are different from those of the previous reported heparin lyases (heparin lyase I and III prepared from F. heparinum), but is similar to those of Flavobacterial heparin lyase II. There- fore, we suggest that the salt-active acharan sulfate lyase may belong to heparin lyase II. Acknowledgements This work was supported by KOSEF grant 1999-2-209-010-5. References 1. Jackson, R.L., Busch, S.J. & Cardin, A.D. (1991) Glycosamino- glycans: molecular properties, protein interactions, and role in physiological processes. Physiol. Rev. 71, 481–539. 2. Griffin, C.C., Linhardt, R.J., Van Gorp, C.L., Toida, T., Hileman, R.E., Schubert, R.L. & Brown, S.E. (1995) Isolation and char- acterization of heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin. Carbohydr. Res. 276, 183–197. 3. Kim, Y.S., Jo, Y.Y., Chang, I.M., Toida, T., Park, Y. & Linhardt, R.J. (1996) A new glycosaminoglycan from the giant African snail Achatina fulica. J. Biol. Chem. 271, 11750–11755. 4. Lohse, D. & Linhardt, R. (1992) Purification and characterization of heparin lyases from Flavobacterium heparinum. J. Biol. Chem. 267, 24347–24355. 5. Desai, U.R., Wang, H.M. & Linhardt, R.J. (1993) Substrate specificity of the heparin lyases from Flavobacterium heparinum. Arch. Biochem. Biophys. 306, 461–468. 6. Desai, U.R., Wang, H.M. & Linhardt, R.J. (1993) Specificity studies on the heparin lyases from Flavobacterium heparinum. Biochemistry 32, 8140–8145. 7. Bourin, M C. & Lindahl, U. (1993) Glycosaminoglycans and the regulation of blood coagulation. Biochem. J. 289, 313–330. 8. Folkman, J. & Shing, Y. (1992) Control of angiogenesis by heparin and other sulfated polysaccharides. Adv. Exp. Med. Biol. 313, 355–364. 9. Castellot, J.J. Jr, Choay, J., Lormeau, J.C., Petitou, M., Sache, E. & Karnovsky, M.J. (1986) Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-O-sulfate group. J. Cell. Biol. 102, 1979–1984. 10. Godavarti, R., Davis, M., Venkataraman, G., Cooney, C., Langer, R. & Sasisekharan, R. (1996) A comparative analysis of the primary sequences and characteristics of heparinases I, II, and III from Flavobacterium heparinum. Biochem. Biophy. Res. Com- mun. 225, 751–758. 11. Bellamy, R.W. & Horikoshi, K. (1992) Heparinase Produced by Microorganism Belonging to the Genus Bacillus in US Patent. Research Development Corporation of Japan. 12. Watanabe, M., Tsuda, H., Yamada, S., Shibata, Y., Nakamura, T. & Sugahara, K. (1998) Characterization of heparinase from an oral bacterium Prevotella heparinolytica. J. Biochem. (Tokyo) 123, 283–288. 13. Ahn,M.Y.,Shin,K.H.,Kim,D.H.,Jung,E.A.,Toida,T.,Lin- hardt, R.J. & Kim, Y.S. (1998) Characterization of a Bacteroides species from human intestine that degrades glycosaminoglycans. Can. J. Microbiol. 44, 423–429. 14. 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. 15. Hong, S H., Kim, B T., Shin, H Y., Kim, W S., Lee, K S., Kim, Y S. & Kim, D H. (2002) Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides ster- coris HJ-15, a human intestinal anaerobic bacterium. Eur. J. Biochem. 269, 2934–2940. 16. Kim, B.T., Hong, S.H., Kim, W.S., Kim, Y.S. & Kim, D H. (2001) Purificatio and characterization of acharan sulfate lyases, two novel heparinases, from Bacteroides stercoris HJ-15. Eur. J. Biochem. 268, 2635–2641. 17. 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. 18. 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. 19. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227, 680–685. 20. Su, H., Blain, F., Musil, R.A., Zimmermann, J.J.F., Gu, K. & Bennett, D.C. (1996) Isolation and expression in Escherichia coli of hepB and hep C, genes coding for the glycosaminoglycan- degrading enzymes heparinase II and heparinase III, respectively, from Flavobacterium heparinum. Appl. Environ. Microbiol. 62, 2723–2734. 21. Riley, T.V. & Mee, B.J. (1984) Heparinase production by Bac- teroides species. Microbios Lett. 25, 141–149. Ó FEBS 2003 Bacteroidal salt-active acharan sulfate lyase (Eur. J. Biochem. 270) 3173 . lyase II. Keywords: Bacteroides stercoris HJ-15; salt-active acharan sulfate lyase; acharan sulfate lyase; acharan sulfate; heparin. Heparin, heparan sulfate. Purification and characterization of novel salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15 Sung-Woon Hong 1 ,