Purification and characterization of acharan sulfate lyases, two novel heparinases, from Bacteroides stercoris HJ-15 Byung-Taek Kim1, Sung-Woon

Two novel acharan sulfate lyases (ASL1 and ASL2: no EC
number) have been purified from Bacteroides stercoris HJ-15 which was isolated from human intestinal bacteria with glycosaminoglycan (GAG) degrading enzymes. These enzymes were purified to apparent homogeneity by a com-
bination of QAE-cellulose, DEAE-cellulose, carboxy-
methyl±Sephadex C-50, hydroxyapatite and HiTrap SP Sephadex C-25 column chromatography with the final
specific activity of 50.5 and 76.7 mmol´min21´mg21,
respectively. Both acharan sulfate lyases are single subunits 
of 83 kDa by SDS/PAGE and gel filtration. ASL1 showed 
optimal activity at pH 7.2 and 45 8C. ASL1 activity was 
inhibited by Cu21, Ni21 and Co21, but ASL2 activity was 
inhibited by Cu21, Ni21and Pb2. Both enzymes were
 

slightly inhibited by some agents that modify histidine and
cysteine residues, but activated by reducing agents such as dl-dithiothreitol and 2-mercaptoethanol. Both purified
bacteroidal acharan sulfate lyases acted to the greatest 
extent on acharan sulfate, and to a lesser extents on heparan 
sulfate and heparin. They did not act on de-O-sulfated 
acharan sulfate. These findings suggest that the biochemical 
properties of these purified acharan sulfate lyases are 
different from those of the previously purified heparin 
lyases, but these enzymes belong to heparinase II.
Keywords: Bacteroides stercoris HJ-15; acharan sulfate lyase; heparinase; acharan sulfate; purification.
 



Acharan sulfate, a glycosaminoglycan (GAG) 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 !4)-a-d-
GlcNAc (1!4)-a-l-IdoA2S(1! where GlcN is glucosa-
mine and IdoA is iduronic acid [1]. Heparin and heparan 
sulfate 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 heparin is composed mainly of trisulfated disaccharides of N-sulfonyl-d-glucosamine and l-iduronic acid [2,3].
Related to 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 !4)-a-
d-GlcNS(6S or OH)(1!4)-a-l-IdoA2S(1! linkages pre-
sent in heparin; (b) heparin lyase II (heparinase II or



Correspondence to D.-H. Kim, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-ku, Seoul 130-701, South Korea. Fax: 1 82 2 957 5030, Tel.: 1 82 2 961 0374, E-mail: dhkim@khu.ac.kr
Abbreviations: DUA, 4-deoxy-a-l-threo-hex-4-enopyranosyl uronic acid; IdoA, iduronic acid; GlcA, glucuronic acid; GlcN, glucosamine; GAG, glycosaminoglycan; CM, carboxymethyl; IEF, isoelectric
focusing; TLCK, N-a-tosyl-l-lysylchloromethane; TPCK, N-a-tosyl-l-phenylalanylchloromethane.
(Received 2 January 2001, revised 12 March 2001, accepted 12 March 
2001)
 



heparitinase II), acting at the !4)-a-d-GlcNS(6S or
OH)(1!4)-a-l-IdoA(2S or OH) or -b-d-GlcA(1! linkages 
present in both heparin and heparan sulfate, where GlcA is 
glucuronic acid; and (c) heparin lyase III (heparinase III or 
heparitinase, EC 4.2.2.8), acting on the !4)-a-d-GlcNS(or 
Ac)(1!4)-b-d-GlcA(or IdoA)(1! linkages found exclu-
sively in heparan sulfate. The heparin lyases have become 
increasingly important in understanding the biological roles 
and structure of the GAGs (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], Bacil-
lus sp. BH 100 [11], Prevotella heparinolyticus (formerly 
known as Bacteroides heparinolyticus) [12], and Bacter-
oides stercoris HJ-15 [13]. B. 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].
Recently, we purified a novel heparin lyase, which is close 
to Flavobacterial heparin lyase III, from this B. stercoris 
HJ-15 [14]. The previously purified enzyme from Bacter-
oides stercoris HJ-15 cleaved heparin as well as heparan 
sulfate, although heparin lyase III from Flavobacterium 
heparinum was highly specific for heparan sulfate. However, 
this B. stercoris HJ-15 potently cleaved acharan sulfate as 
well as heparin compared to Flavobacterium heparinum. 
Therefore, we tried to purified acharan sulfate lyases 
from B. stercoris HJ-15 that acts predominantly on acharan 
sulfate. 
 

 

2636 B.-T. Kim et al. (Eur. J. Biochem. 268)

MATERIALS AND METHODS
Materials
Acharan sulfate was prepared as described by Kim et al. [1]. Heparin (porcine intestinal mucosa), heparan sulfate (porcine 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. SDS, carboxymethyl 
(CM)±Sephadex C-50, SP±Sephadex C-25, Sephacryl 
S-300 HR resins and molecular mass markers for gel
filtration and protein electrophoresis were obtained from Pharmacia Biotech Co (Uppsala, Sweden). 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. All other chemicals were of the highest grade available.


Bacterial strains and purification of two acharan sulfate lyases
B. stercoris HJ-15 was isolated and cultivated as described previously [14]. It was cultured anaerobically under an
atmosphere of 90% nitrogen and 10% carbon dioxide at 378C in 100 L of tryptic soy broth (pH 7.2) containing heparin (0.15 g´L21) 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 for 30 min at 4 8C and the
resulting cell pellet was washed twice with NaCl/Pi. The cell pellet was suspended in 600 mL of 50 mm sodium phosphate buffer, pH 7.0. The 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., Tokyo, Japan) at 
80% output with cooling. Cell debris was removed by 
centrifugation at 25 000 g for 60 min at 4 8C. All operations 
were performed at 4 8C unless otherwise noted. The cell 
extract (600 mL) was passed through a QAE-cellulose
column (5 40 cm) which had been pre-equilibrated with
50 mm sodium phosphate buffer, pH 7.0. 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) equilibrated with 50 mm
sodium phosphate buffer, pH 7.0. The column was then 
eluted with the same buffer until any acharan sulfate lyase 
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) pre-
viously equilibrated with 50 mm sodium phosphate buffer, pH 7.0. The column was washed with 1 L of the same buffer and then eluted with a 2-L linear gradient of KCl of 0±0.6 m in 50 mm sodium phosphate buffer, pH 7.0 at a flow rate of 105 mL´h21. All fractions obtained were
assayed for heparin lyase and acharan sulfate lyase 
activities. Three fractions (Fr-a, Fr-b and Fr-c) containing
 

q FEBS 2001

the activity of these enzymes were collected separately and assayed for the activities degrading acharan sulfate and heparan sulfate. Fr-a, exhibiting acharan sulfate lyase activity, was dialyzed against 50 mm sodium phosphate
buffer, pH 7.0 for the further purification. The dialyzed 
enzyme preparation (330 mL) was applied to a HA Ultrogel 
column (2.5 10 cm) equilibrated with 50 mm sodium
phosphate buffer, pH 7.0. After being washed with 500 mL of the same buffer, the column was eluted with an 800-mL linear gradient, of 50±400 mm sodium phosphate buffer
(pH 7.0) at a flow rate of 120 mL´h21. The active fractions 
were pooled and dialyzed twice against 2 L of 50 mm 
sodium phosphate buffer, pH 7.0. The dialyzed enzyme 
(165 mL) was loaded onto a SP±Sephadex C-25 column 
(3 30 cm) equilibrated with 50 mm sodium phosphate
buffer, pH 7.0. After washing the unadsorbed proteins with 800 mL of the same buffer, an 800-mL linear KCl gradient (0±0.5 m) in 50 mm sodium phosphate buffer, pH 7.0 was performed to elute the acharan sulfate lyase at a flow rate of 75 mL´h21. Two active fractions (ASL1, fractions 91±99; ASL2, fractions 118±120) were investigated for homo-
geneity by SDS/PAGE.

Enzyme activity assays
The activities of GAG lyases, including acharan sulfate lyase, were measured according to the previously published procedure [14]. The activity was calculated from the
change of absorbance per min using an extinction coefficient of 3800 m21 for products [1 U ˆ 1 mmol of
4-deoxy-a-l-threo-hex-4-enopyranosyl uronic acid (DUA) containing product formed per min] [15]. The specific
activity was calculated by dividing the mmol product produced per min by mg protein in the cuvette. Protein concentration was measured by a Bradford assay using bovine serum albumin as a standard [16].

Characterization of acharan sulfate lyases
SDS/PAGE was performed for the determination of 
molecular mass according to Laemmli's procedure [17]. 
The gels were stained with Coomassie Brilliant Blue R-250 
solution and then further stained with silver. The pI value of 
heparinase was determined by isoelectric focusing (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-filtration 
using Sephacryl S-300 HR column (1.6 70 cm) cali-
brated with gel filtration low molecular mass calibration 
kit (from Sigma Co.) and high molecular calibration kit
(from Amersham Pharmacia Biotech). The pH optimum of 
acharan sulfate lyases were determined using 50 mm
sodium phosphate buffer (pH 6.0±8.5). Temperature depen-
dency of the enzyme was investigated by measuring enzyme activity at different temperatures (25±60 8C). To investigate the effect of divalent metal ions and KCl on the lyase activity, divalent metal ion (final concentration,
100 mm), chemical modifying agents (50 mm) and KCl (0±500 mm) were added into the reaction mixture. Kinetic constants of acharan sulfate lyases were determined by measuring the initial rates at various substrate concentra-
tions (200, 400, 600, 1000, 2000 and 3000 mg) under the standard reaction conditions. 
 

 

q FEBS 2001

The 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 mg of acharan sulfate, de-O-sulfated acharan 
sulfate and N-sulfoacharan sulfate were used in this assay. 
 Amino-acid composition analysis was performed on an 
Applied Biosystem model 420/130 Derivatizer/Amino-acid 
Analyzer using phenyl isothiocyanate precolumn derivati-
zation chemistry. Hydrolysis was perfomed by using 6 m 
hydrochloric acid/0.1% phenol at 155 8C for 1 h. 
 Internal amino-acid sequences of two purified acharan 
sulfate lyases were analyzed on a Applied Biosystem 
protein sequencer model 492.

RESULTS
Purification of acharan sulfate lyases
Bacteroides stercoris HJ-15, which degrades a variety of 
GAGs including heparin, heparan sulfate and chondroitin 
sulfates [13], constitutively produced acharan sulfate lyase 
activity. However, when induced with acharan sulfate or 
heparin, total acharan sulfate activity increased by about 
3.5-fold (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. Acharan 
sulfate lyase activity passed through these columns without 
binding to the matrices. The effluent was further purified to
































Fig. 1. Elution profile of CM±Sephadex
C-50 ion exchange chromatography (A)
and SP Sephadex column chromatography
(B). Solid circle, acharan sulfate lyase
activity; open triangle, heparin lyase activity;
solid line only, absorbance at 280 nm.
 

New bacteroidal heparinase (Eur. J. Biochem. 268) 2637

homogeneity by a series of CM±Sephadex C-50 column chromatography (Fig. 1A), hydroxyapatite Ultrogel chro-
matography and finally SP±Sephadex C-25 column chro-
matography (Fig. 1B). The specific activity and total
activity at each purification step are summarized in 
Table 1. The acharan sulfate lyase activity was divided 
into two parts by SP±Sephadex C-25 chromatography. The 
specific activity of the first eluted fraction (acharan sulfate 
lyase 1) had 50.5 U´mg protein21 with a yield of 15.5%, 
and that of the second eluted fraction (acharan sulfate lyase 
2) was 76.7 U´mg protein21 with a yield of 11.6%. Both 
acharan sulfate lyases were apparently homogeneous by 
SDS/PAGE and their molecular masses were identically 
estimated to be 83 000 Da, respectively (Fig. 2).


Characterization of two acharan sulfate lyases
The molecular masses of acharan sulfate lyase 1 and 2 under nondenaturing conditions were determined by gel filtration (Fig. 2). Both acharan sulfate lyase 1 and 2 were estimated to be about 83000 Da. It suggests that both
acharan sulfate lyase 1 and 2 are composed of one subunit. The optimal pHs of acharan sulfate lyases were determined to be 7.2±7.5 for both acharan sulfate and heparin. (Fig. 3) and the optimum temperature for the maximal activity was shown at 45 8C (Fig. 4).
Both acharan sulfate lyase 1 and 2 activities were slightly 
increased by addition of Mg21 or Mn21, whereas they were 
severely inhibited by Cu21 and Ni21 (Table 2). Particularly, 

oli