Biofilm formation by exopolysaccharide mutants of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces an insoluble glucan in place of alternan and dextran. To test the effect of exopolysaccharide production on biofilm formation, these strains were cultured in a biofilm reactor. All strains grew well as biofilms, with comparable cell densities, including strain NRRL B-21414, which produces neither alternan nor dextran in planktonic cultures. However, the exopolysaccharide phenotype clearly affected the appearance of the biofilms and the sloughed-off biofilm material produced by these biofilms. For all strains, soluble glucansucrases and soluble polysaccharides produced by biofilm cultures appeared to be similar to those produced by planktonic cultures. Biofilms from all strains also contained insoluble polysaccharides. Strain R1510 biofilms contained an insoluble polysaccharide similar to that produced by planktonic cultures. For most other strains, the insoluble biofilm polysaccharides resembled a mixture of alternan and dextran. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Some structural features of an insoluble α-D-glucan from a mutant strain of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces two soluble extracellular α-D-glucans from sucrose: alternan and dextran. An unusual mutant strain derived from NRRL B-1355 has recently been isolated which produces practically no soluble polysaccharide, but significant amounts of an insoluble D-glucan. Methylation analysis shows it contains linear (1→3) and (1→6) linkages as well as (1→2) and (1→3) branch linkages. The insoluble glucan was partially digestible by endodextranase, giving rise to a series of oligosaccharides, a high-molecular weight soluble fraction and an insoluble residue. Treatment of the soluble dextranase-limit fraction with an α(1→2) debranching enzyme led to further dextranase susceptibility. Methylation, FTIR and NMR analyses of the dextranase-treated fractions indicate a non-uniform structure with domains bearing similarities to L. mesenteroides strain NRRL B-1299 dextran and to insoluble streptococcal D-glucans. Received 05 November 1998/ Accepted in revised form 31 March 1999     

Modification of alternan by dextranase

Alternan is a unique glucan with a backbone structure of alternating α-(1 → 6) and α-(1 → 3) linkages. Previously, we isolated strains of Penicillium sp. that modify native, high molecular weight alternan in a novel bioconversion process to a lower molecular weight form with solution viscosity properties similar to those of commercial gum arabic. The mechanism of this modification was unknown. Here, we report that these Penicillium sp. strains secrete dextranase during germination on alternan. Furthermore, alternan is modified in vitro by commercial dextranases, and dextranase-modified alternan appears to be identical to bioconversion-modified alternan. This is surprising, since alternan has long been considered to be resistant to dextranase. Results suggest that native alternan may have localized regions of consecutive α-(1 → 6) linkages that serve as substrates for dextranase. Dextranase treatment of native alternan, particularly with GRAS enzymes, may have practical advantages for the production of modified alternan as a gum arabic substitute. U.S. Department of Agriculture—Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Cloning,expression, and characterization of an insoluble glucan-producing glucansucrase from <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K _M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.     

Alternansucrase mutants of Leuconostoc mesenteroides strain NRRL B-21138

Alternan is a unique α-D-glucan of potential commercial interest, produced by rare strains of Leuconostoc mesenteroides. Natural isolates that produce alternan, such as NRRL B-1355, also produce dextran as a troublesome contaminant. We previously isolated mutants of strain NRRL B-1355 that are deficient in dextran production, including the highly stable strain NRRL B-21138. In the current work, we mutagenized strain NRRL B-21138 and screened survivors for further alterations in production of alternansucrase, the enzyme that catalyzes the synthesis of alternan from sucrose. Second generation mutants included highly stable strain NRRL B-21297, which produced four-fold elevated levels of alternansucrase without an increase in the proportion of dextransucrase activity. Such alternansucrase overproducing strains will facilitate studies of this enzyme, and may become valuable for the enzymatic production of alternan. Another highly stable mutant strain, NRRL B-21414, grew slowly on sucrose with negligible production of glucan or extracellular glucansucrase activity. This strain may prove useful as an expression host for glucansucrase genes. Received 30 July 1996/ Accepted in revised form 15 December 1996     

Isolation and Culture of a Marine Bacterium Degrading the Sulfated Fucans from Marine Brown Algae

Fucoidans are matrix polysaccharides from marine brown algae, consisting of an α-l-fucose backbone substituted by sulfate-ester groups and masked with ramifications containing other monosaccharide residues. In spite of their interest as biologically active compounds in a number of homologous and heterologous systems, no convenient sources with fucanase activity are available yet for the degradation of the fucalean algae. We here report on the isolation, characterization, and culture conditions of a bacterial strain capable of degrading various brown algal fucoidans. This bacterium, a member of the family Flavobacteriaceae, was shown to secrete fucoidan endo-hydrolase activity. An extracellular enzyme preparation was used to degrade the fucoidan from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of α-1→3-l-fucopyranose-2-sulfate-α-1→4-l-fucopyranose-2,3-disulfate, with the 3-linked residues at the nonreducing end.     

Purification of alternanase by affinity chromatography

The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating α(1→6), α(1→3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield. Electronic Publication     

A mutant strain of Leuconostoc mesenteroides B-1355 producing a glucosyltransferase synthesizing α(1→2) glucosidic linkages

A mutant strain (R1510) of Leuconostoc mesenteroides B-1355 was isolated which synthesized primarily an insoluble polysaccharide and little soluble polysaccharide when grown in sucrose-containing medium. Glucose or sucrose cultures of this strain produced a single intense band of GTF-1 activity of 240 kDa on SDS gels, and a number of faint, smaller bands. Oligosaccharides synthesized by strain R1510 from methyl-α-D-glucoside and sucrose included a trisaccharide whose structure contained an α(1→2) glucosidic linkage. This type of linkage has not been seen before in any products from strain B-1355 or its mutant derivatives. The structure of the purified trisaccharide was confirmed by ¹³C-nuclear magnetic resonance. The insoluble polysaccharide also contained α(1→2) branch linkages, as determined by methylation analysis, showing that synthesis of the linkages was not peculiar to methyl-α-D-glucoside. GTF-1, which had been excised with a razor blade from an SDS gel of a culture of the parent strain B-1355, produced the same trisaccharides as strain R1510, showing that GTF-1 from the wild-type strain was the same as GTF-1 from strain R1510. Mutant strains resembling strain R1510, but producing a single intense band of alternansucrase (200 kDa) instead of GTF-1 were also isolated, suggesting that mutations may be generated which diminished the activities for any two of the three GTFs of strain B1355 relative to the third. Strain R1554 produced a soluble form of alternansucrase, while strain R1588 produced a cell-associated form. The mechanism(s) by which specific GTFs become associated with the cells of L. mesenteroides was not explored. Received 12 May 1998/ Accepted in revised form 16 July 1998     

Isolation and Characterization of Microorganisms with Alternan Hydrolytic Activity

Alternan is an unusual α-D-glucan containing alternating (1 → 3), (1 → 6) linkages that exhibits remarkable resistance to enzymatic hydrolysis. The commercial potential of the polysaccharide may be enhanced by the ability to economically modify the native form into fractions of varying molecular weight. By employing isolation procedures with covalently dyed alternan as the substrate, several bacterial isolates that produced endohydrolytic activity were obtained in pure culture. The activity was confirmed by decreases in viscosity and by direct examination of the hydrolysis products with thin layer chromatography. Analysis of the hydrolysis products established that all isolates produced enzymes with identical alternan depolymerizing activity, producing a cyclic tetrasaccharide as a major product. All alternanase activity was shown to be extracellularly located. A single strain exhibited constitutive production of alternanase, while all other isolates required the presence of alternan in the growth media for enzyme production. All isolates were phenotypically similar, produced heat-resistant spores, and were tentatively identified as members of the genus Bacillus.     

Characterization of the <Emphasis Type="Italic">Pragia fontium</Emphasis> Lipopolysaccharides

Lipopolysaccharides (LPSs) of two strains Pragia fontium 97U116 and 27480 were isolated and characterized; they were close to those of other representatives of the family Enterobacteriaceae in fatty acid composition and contained, respectively, 3-hydroxytetradecanoic acid as the predominant component (45.8 and 45.1%), tetradecanoic (23.5 and 28.9%), hexadecanoic (12.6 and 7.9%), hexadecenoic (12.6 and 7.9%), and dodecanoic (4.9 and 4.2%) fatty acids. The O-specific polysaccharides consisted of linear penta- and tetrasaccharide repeating units: →2)-α-D-Galf-(1→3)-α-L-Rhap2Ac-(1→4)-α-D-GlcpNAc-(1→2)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→ →4)-β-D-ManpNAc3NAcA-(1→2)-α-L-Rhap-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→ The LPSs of P. fontium 97U116 and 27480 were serologically active and belonged to different serogroups; they were less toxic than those of strain E. coli O55:B5, but more pyrogenic than the Pyrogenal preparation.     

Structural peculiarities of the O-specific polysaccharides of <Emphasis Type="Italic">Azospirillum</Emphasis> bacteria of serogroup III

Lipopolysaccharides and O-specific polysaccharides were isolated from the outer membrane of bacterial cells of three strains belonging to two Azospirillum species, and their structures were established by monosaccharide analysis including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy. It was shown that while having the identical composition, the O-polysaccharides have different branched tetrasaccharide repeating units. Two neutral polysaccharides were found in the lipopolysaccharide of A. brasilense 54, and the structure for the predominant O-polysaccharide was determined. The structural data, together with results of serological studies, enabled assignment of strains examined to a novel serogroup, III. The chemical basis for the serological relatedness among the azospirilla of this serogroup is presumably the presence of a common →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→oligosaccharide motif in their O-polysaccharides.     

The genusCandida Berkhout

Glucose and mannose were demonstrated by chromatography in polysaccharides isolated by alkaline and acid extraction from cell walls. A mannan composed ofd-mannopyranose units bound by α(1–2) and α(1–6) bonds was isolated from the polysaccharides of the strainsCandida guilliermondii (Cast.) Langeron et Guerra andCandida guilliermondii var.membranaefaciens. Mannans isolated from these two strains have the same chemical structure and immunochemical properties. The polysaccharide isolated from cell walls of the strainCandida guilliermondii var.nitratophila has a different chemical structure and different immunochemical properties.     

Pullulan production by tropical isolates of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Tropical isolates of Aureobasidium pullulans previously isolated from distinct habitats in Thailand were characterized for their capacities to produce the valuable polysaccharide, pullulan. A. pullulans strain NRM2, the so-called “color variant” strain, was the best producer, yielding 25.1 g pullulan l⁻¹ after 7 days in sucrose medium with peptone as the nitrogen source. Pullulan from strain NRM2 was less pigmented than those from the other strains and was remarkably pure after a simple ethanol precipitation. The molecular weight of pullulan from all cultures dramatically decreased after 3 days growth, as analyzed by high performance size exclusion chromatography. Alpha-amylase with apparent activity against pullulan was expressed constitutively in sucrose-grown cultures and induced in starch-grown cultures. When the alpha-amylase inhibitor acarbose was added to the culture medium, pullulan of slightly higher molecular weight was obtained from late cultures, supporting the notion that alpha-amylase plays a role in the reduction of the molecular weight of pullulan during the production phase.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac ⁺ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac ⁺) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.     

α-(4-O-Methyl)-d-glucuronidase activity produced by the rumen anaerobic fungusPiromonas communis: A study of selected properties

The rumen anaerobic fungusPiromonas communis, unlike the rumen anaerobic fungiNeocallimastix frontalis andNeocallimastix patriciarum, produced extracellular α-(4-O-methyl)-d-glucuronidase when grown in cultures containing filter-paper, barley straw, birchwood xylan or birchwood sawdust as carbon source. The highest concentration of enzyme was produced in cultures containing birchwood sawdust. The aldobiouronic acidO-α-(4-O-methyl-d-glucopyran-osyluronic acid)-(1 → 2)-d-xylopyranose (MeGlcAXyl) was the best substrate of those tested: the aldotriouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid (1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl₂) and the aldotetraouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid)-(1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl₃) were also attacked but the rate fell as the degree of polymerisation increased. When the same substituted xylooligosaccharides were reduced to the corresponding alditols the enzyme activity disappeared. Similarly,p-nitrophenyl-α-d-glucuronide was not a substrate. Remarkably, the relative rates of attack shown by the α-(4-O-methyl)-d-glucuronidase on the aldouronic acids and on xylans extracted from birchwood, oat spelts and oat straw differed according to the carbon source used to produce the enzyme. The α-(4-O-methyl)-d-glucuronidase had a pH optimum of 5.5 and a temperature optimum of 50°C. On gel filtration the enzyme was shown to be associated with proteins covering the range 100–300 kDa, but a major peak of activity in the column effluent appeared to have a molecular mass of 103 kDa.     

A broader role for AmyR in <Emphasis Type="Italic">Aspergillus niger</Emphasis>: regulation of the utilisation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose or <Emphasis Type="SmallCaps">d</Emphasis>-galactose containing oligo- and polysaccharides

AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed.     

Di-<Emphasis Type="Italic">tert</Emphasis>-butylsilylene-directed α-selective synthesis of <Emphasis Type="Italic">p</Emphasis>-nitrophenyl T-antigen analogues

Seven analogues of p-nitrophenyl T-antigen [Galβ(1→3)GalNAcα(1→O)PNP] have been synthesized as potential substrates for elucidation of the substrate specificity of endo-α-N-acetylgalactosaminidase. These compounds, which are commercially unavailable, include: GlcNAcβ(1→3){GlcNAcβ(1→6)}GalNAcα(1→O)PNP [core 4 type], GalNAcα(1→3)GalNAcα(1→O)PNP [core 5 type], GlcNAcβ(1→6)GalNAcα(1→O)PNP [core 6 type], GalNAcα(1→6)GalNAcα(1→O)PNP [core 7 type], Galα(1→3)GalNAcα(1→O)PNP [core 8 type], Glcβ(1→3)GalNAcα(1→O)PNP and GalNAcβ(1→3)GalNAcα(1→O)PNP. The assembly of these synthetic probes was accomplished efficiently, based on di-tert-butylsilylene(DTBS)-directed α-galactosylation as a key reaction.     

Biosynthetic origin and longevity in vivo of α-d-mannopyranosyl-(1 → 4)-α-d-glucuronopyranosyl-(1 → 2)-myo-inositol, an unusual extracellular oligosaccharide produced by cultured rose cells

A non-reducing trisaccharide, α-D-mannopyranosyl-(1 → 4)-α-D-glucuronopyranosyl-(1 → 2)-myo-inositol (MGI) accumulated in the spent medium of cell-suspension cultures of `Paul's Scarlet' rose (Rosa sp.) predominantly during the period of rapid cell growth. This trisaccharide was also produced by cultures of sycamore (Acer pseudoplatanus L.) but not by those of the graminaceous monocots maize (Zea mays L.) and tall fescue grass (Festuca arundinacea Schreb.). When added to cultured Rosa cells, [¹⁴C]MGI was neither taken up by the cells nor bound to the cell surface and was not metabolised extracellularly. When D-[6-¹⁴C]glucuronic acid was fed to cultured Rosa cells, extracellular [¹⁴C]MGI started to appear only after a 5-h lag period, compared with a 0.5-h lag period for labelling of extracelluar polysaccharides. Furthermore, [¹⁴C]MGI continued to accumulate in the medium for at least 20 h after the accumulation of ¹⁴C-polymers had ceased. These observations indicate that extracellular MGI was produced from a slowly turning-over pool of a pre-formed intermediate. Structural considerations indicate that the intermediate could be a glucuronomannan or a phytoglycolipid (glycophosphosphingolipid). No Rosa polysaccharides could be found that generated MGI in the presence of living Rosa cells. We therefore favour phytoglycolipids as the probable biosynthetic origin of MGI. Received: 29 April 1999 / Accepted: 13 June 1999     

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase

Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an α-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083^T. It was found to act with retention of configuration (α→α), releasing α-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As α-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of α-galactosides using the transgalactosylation activity of Bi. adolescentisα-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 °C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp and tetrasaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate. Received: 15 March 1999 / Received revision: 8 June 1999 / Accepted: 11 June 1999     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.