Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii.

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg-1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL-1 and 647.93 U·mg-1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K-1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.

Isolation, Purification, and Characterization of Heparinase from Streptomyces variabilis MTCC 12266.

Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.

Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death. Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions. RESULTS: Quantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments. We characterized hepP and its protein product HepP. hepP was cloned from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14ΔhepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14. CONCLUSIONS: Changes within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing.

Polycation-induced benzoperylene probe excimer formation and the ratiometric detection of heparin and heparinase.

A benzoperylene probe excimer emission in an aqueous buffer solution is observed for the first time, and a novel ratiometric fluorescence method based on the probe excimer emission for the sensitive detection of heparin and heparinase is demonstrated. A negatively charged benzoperylene derivative, 6-(benzo[ghi]perylene-1,2-dicarboxylic imide-yl)hexanoic acid (BPDI), was employed. A polycation, poly(diallyldimethylammonium) chloride (poly-DDA), could induce aggregation of BPDI through noncovalent interactions. A decrease of BPDI monomer emission and a simultaneous increase of BPDI excimer emission were observed. Upon the addition of heparin, the strong binding between heparin and poly-DDA caused release of BPDI monomer molecules, and an excimer-monomer emission signal transition was detected. However, after the enzymatic hydrolysis of heparin by heparinase, heparin was hydrolyzed into small fragments, which weakened the competitive binding of heparin to poly-DDA. Poly-DDA induced aggregation of BPDI, and a monomer-excimer emission signal transition was detected. Our assay is simple, rapid, inexpensive, sensitive and selective, which could facilitate the heparin and heparinase related biochemical and biomedical research.

Expression of HpaI in Pichia pastoris and optimization of conditions for the heparinase I production.

Heparinase I has important applications in the fields of biomedicine and pharmaceuticals. The heparinase I gene (HpaI) from Flavobacterium heparinum was cloned and overexpressed in Pichia pastoris GS115, and the conditions for the heparinase I production were optimized by RSM. PCR analysis indicated that HpaI was integrated into the P. pastoris GS115 genome. The concentrations of key factors that affected the heparinase I activity were optimized, and were as follows: oleic acid, 0.07%, liquid volume in flask, 34.3 ml/L, and methanol, 0.96%. Under the optimal conditions, the activity of heparinase I was up to 323 U/L in shake flask. A maximal heparinase I activity of 398.5 U/L from the transformant 2 was achieved in a 5L fermentor. This study demonstrates the overproduction of heparinase I by recombinant P. pastoris.

Rational design of a tripartite fusion protein of heparinase I enables one-step affinity purification and real-time activity detection.

Enzymatic degradation of heparin has great potential as an ecological and specific way to produce low molecular weight heparin. However, the commercial use of heparinase I (HepA), one of the most important heparin lyases, has been hampered by low productivity and poor thermostability. Fusion with green fluorescent protein (GFP) or maltose-binding protein (MBP) has shown potential in facilitating the industrial use of HepA. Thus, tripartite fusion of GFP, MBP and HepA would be a promising approach. Therefore, in the present study, the tripartite fusion strategy was systematically studied, mainly focusing on the fusion order and the linker sequence, to obtain a fusion protein offering one-step purification and real-time detection of HepA activity by fluorescence as well as high HepA activity and thermostability. Our results show that fusion order is important for MBP binding affinity and HepA activity, while the linker sequences at domain junctions have significant effects on protein expression level, HepA activity and thermostability as well as GFP fluorescence. The best tripartite fusion was identified as MBP-(EAAAK)(3)-GFP-(GGGGS)(3)-HepA, which shows potential to facilitate the production of HepA and its application in industrial preparation of low molecular weight heparin.

Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli.

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP-HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP-HepI offered a 30.6% increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca2+ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP-HepI showed a specific activity of 56.3 IU/mg, 206% higher than that of the wild type obtained without Ca2+ addition, along with a 177% increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP-HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.

Expression of heparinase I of Bacteroides stercoris HJ-15 and its degradation tendency toward heparin-like glycosaminoglycans.

Recombinant heparinase I was cloned from Bacteroides stercoris HJ-15 (BSrhepI), overexpressed in Escherichia coli, and intensively characterized. The complete gene of BSrhepI was identified by Southern blotting, and was overexpressed as an inclusion body. The inclusion body was solubilized with 4 M guanidine-HCl, and the denatured BSrhepI was easily purified using Ni(2+)-affinity column chromatography. The purified but denatured enzyme was then successfully refolded by dialysis against 50 mM Tris-HCl (pH 7.0) containing 1mM DTT and CaCl(2). BSrhepI was most active in 50mM Tris-HCl buffer containing 300 mM NaCl, 10 mM CaCl(2), and 1 mM DTT (pH 7.0) at 37°C. This enzyme digested not only heparin, but also heparan sulfate. Through comparative HPLC-analysis of each degraded product of heparin and heparan sulfate by digestion with BSrhepI or flavobacterial heparinase I, we verified that BSrhepI has a broader spectrum of substrate specificities than other reported heparinases.

Cloning, expression and characterization of acharan sulfate-degrading heparin lyase II from Bacteroides stercoris HJ-15.

AIMS: This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15. METHODS AND RESULTS: The heparinase II gene from Bact. stercoris HJ-15 was identified by Southern blotting and the sequence was deposited in GenBank. The gene was cloned and overexpressed in Escherichia coli, and rHepII was purified using two simple ion-exchange column chromatography steps. Enzymatic properties and substrate specificities of rHepII were assessed and its kinetic constants were calculated. Heparin-like glycosaminoglycans (HLGAGs) were digested with rHepII under optimal reaction conditions, and the products were analysed by SAX-HPLC. CONCLUSIONS: The heparinase II gene is 2322-bp long and consists of 773 amino acids. rHepII is most active in 50 mmol l(-1) sodium phosphate buffer with 75 mmol l(-1) NaCl (pH 7.4) at 32 degrees C, and the activity is stable at 4 degrees C for 15 days on storage. Acharan sulfate is the best substrate for rHepII, followed by heparan sulfate and heparin. The major degradation products were verified as highly sulfated disaccharides through SAX-HPLC analysis. It means that rHepII prefers iduronic acid over glucuronic acid on the HLGAG structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides easy and certain means for obtaining large amounts of pure rHepII and also provides important information regarding the tendencies of this enzyme and its digested products. rHepII digests HLGAGs in a different manner than heparinases from Flavobacterium heparinum; therefore, we anticipate that rHepII will be a powerful tool for studies of GAGs and GAGs lyases.

Purification and characterization of a novel heparin degrading enzyme from Aspergillus flavus (MTCC-8654).

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/microg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 degrees C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (beta-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K (m) and V (max) as 2.2 x 10(-5 )M and 30.8 mM min(-1), respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.

Rapid purification and characterization of a novel heparin degrading enzyme from Acinetobacter calcoaceticus.

An intracellularly produced constitutive heparinase was isolated from the periplasmic space of Acinetobacter calcoaceticus by freeze fracturing and purified 51.2-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 41 IU/mug protein with a 120000Da molecular mass. The enzyme activity was maximum at 35 degrees C in the presence of 250mM NaCl at pH 7.5. The enzyme activity was inhibited in the presence of Ba(2+), Hg(2+), Cd(2+), IAA and DEPC, and enhanced by the presence of Cu(2+), Fe(2+) ions and reducing agents. Inhibition of enzyme activity by iodoacetic acid and enhancement of enzyme activity in the presence of reducing agents indicated that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The affinity of the enzyme for different glycosaminoglycans studied varied and showed high affinity for heparin with a K(m) value of 0.026mM. In situ gel digestion of the purified protein with trypsin did not show any homology with heparinase I. Depolymerization of heparin and fractionation of the oligosachharides yielded heparin disaccharides as main product. This suggests a catalytic similarity and structural dissimilarity of heparinase from Acinetobacter with heparinase I.

Purification and properties of recombinant GST-heparinase III and optimization of cultivation conditions.

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.

High yield, purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli.

Heparinase I from Flavobacterium heparinum, a source of diverse polysaccharidases, suffers from low yields, insufficient purity for structural studies and insolubility when expressed as a recombinant product in Escherichia coli that is devoid of glycosaminoglycan polysaccharidases. In this study, cDNA coding for the orthologue of F. heparinum heparinase I was constructed from genomic information from the mammalian gut symbiont Bacteroides thetaiotaomicron and expressed in E. coli as a fusion protein with GST at the N-terminus. This resulted in high yield (30 mg/g dry bacteria) of soluble product and facilitated one-step affinity purification to homogeneity. Purified heparinase I bearing the GST fusion exhibited a K(m) of 2.3 microM and V(max) of 42.7 micromol/min with a specific activity of 164 U/mg with heparin (average 12,000 Da) as substrate. The results indicate a 2-fold improvement in yield, specific activity and affinity for heparin as substrate over previous reports. The data suggest that the heparinase I from the gut symbiont exhibits a higher intrinsic affinity for heparin than that from F. heparinum. The purified GST fusion enzyme exhibited a requirement for Ca(2+) and a pH optimum between 6.7 and 7.3 that was similar to the enzyme freed of the N-terminal GST portion. Our study revealed that catalytic activity of heparinase I requires a reducing environment. The GST facilitated immobilization of heparinase I in solid phase either for clinical purposes or for structural studies in absence of interference by contaminating polysaccharidases.

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum.

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.

Preparation and structural determination of large oligosaccharides derived from acharan sulfate.

The structures of a series of large oligosaccharides derived from acharan sulfate were characterized. Acharan sulfate is an unusual glycosaminoglycan isolated from the giant African snail, Achatina fulica. Oligosaccharides from decasaccharide to hexadecasaccharide were enzymatically prepared using heparin lyase II and purified. Capillary electrophoresis and gel electrophoresis confirmed the purity of these oligosaccharides. Their structures, determined by ESI-MS and NMR, were consistent with the major repeating sequence in acharan sulfate, -->4)-alpha-d-GlcN(p)Ac-(1-->4)-alpha-l-IdoA(p)2S-(1-->, terminated by 4-linked alpha-d-GlcN(p)Ac residue at the reducing end and by 4,5-unsaturated pyranosyluronic acid 2-sulfate at the non-reducing end.

Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15.

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.

Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium.

A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively. The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources.

Purification and characterization of novel salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15.

Salt-active acharan sulfate lyase (no EC number) has been purified from Bacteroides stercoris HJ-15, which was isolated 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 micro mol x min-1 x mg-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 acharan sulfate lyase was 94 kDa by SDS/PAGE and gel filtration. The salt-active acharan sulfate lyase showed optimal activity at pH 7.2 and 40 degrees C. Salt-active acharan sulfate lyase activity was potently inhibited by Cu2+, Ni2+ and Zn2+. 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 purified heparin lyases. However, these findings suggest that the purified salt-active acharan sulfate lyase may belong to heparin lyase II.

Cloning, sequencing, and expression of the gene from bacillus circulans that codes for a heparinase that degrades both heparin and heparan sulfate.

The gene, designated hep, coding for a heparinase that degrades both heparin and heparan sulfate, was cloned from Bacillus circulans HpT298. Nucleotide sequence analysis showed that the open reading frame of the hep gene consists of 3,150 bp, encoding a precursor protein of 1,050 amino acids with a molecular mass of 116.5 kDa. A homology search found that the deduced amino acid sequence has partial similarity with enzymes belonging to the family of acidic polysaccharide lyases that degrade chondroitin sulfate and hyaluronic acid. Recombinant mature heparinase (111.2 kDa) was produced by the addition of IPTG from Escherichia coli harboring pETHEP with an open reading frame of the mature hep gene and was purified to homogeneity by SDS-polyacrylamide gel electrophoresis. Analyses of substrate specificity and degraded disaccharides indicated that the recombinant enzyme acts on both heparin and HS, as does heparinase purified from the wild-type strain.

Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans.

A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.