Recombinant α-NAcetylgalactosaminidase from Marine Bacterium-Modifying A Erythrocyte Antigens.

A plasmid based on pET-40b was constructed to synthesize recombinant α-N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426T (α-AlNaGal) in Escherichia coli cells. The yield of α-Al- NaGal attains 10 mg/ml with activity of 49.7 ± 1.3 U at 16°C, concentration of inductor 2 mM, and cultivation for 12 h. Techniques such as anion exchange, metal affinity and gel filtration chromatography to purify α-AlNaGal were applied. α-AlNaGal is a homodimer with a molecular weight of 164 kDa. This enzyme is stable at up to 50°C with a temperature range optimum activity of 20-37°C. Furthermore, its activity is independent of the presence of metal ions in the incubation medium. 1H NMR spectroscopy revealed that α-AlNaGal catalyzes the hydrolysis of the O-glycosidic bond with retention of anomeric stereochemistry and possesses a mechanism of action identical to that of other glycoside hydrolases of the 109 family. α-AlNaGal reduces the serological activity of A erythrocytes at pH 7.3. This property of α-AlNaGal can potentially be used for enzymatic conversion of A and AB erythrocytes to blood group O erythrocytes.

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus.

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked ß-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked ß-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.

Structure of the O-specific polysaccharide of the marine bacterium Arenibacter palladensis KMM 3961(T) containing 2-acetamido-2-deoxy-l-galacturonic acid.

The O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Arenibacter palladensis type strain KMM 3961(T) and studied by chemical methods and (1)H and (13)C NMR spectroscopy including 2D COSY, TOCSY, (1)H,(13)C HSQC, and HMBC experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units containing two mannose residues (Man), one 2-acetamido-2-deoxy-D-galactose residue (D-GalNAc), and one 2-acetamido-2-deoxy-L-galacturonic acid residue (L-GalNAcA) and having the following structure: →2)-α-D-Manp-(1→6)-α-D-Manp-(1→4)-α-L-GalpNAcA-(1→3)-ß-D-GalpNAc-(1→.

Structure of an acidic O-specific polysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form).

An acidic O-specific polysaccharide containing L-rhamnose, 2-acetamido-2-deoxy-D-galactose, 2,6-dideoxy-2-(N-acetyl-L-threonine)amino-D-galactose, and 2-acetamido-2-deoxy-D-mannuronic acid was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form) followed by gel-permeation chromatography. The polysaccharide was subjected to Smith degradation to give a modified polysaccharide with trisaccharide repeating unit containing L-threonine. The initial and modified polysaccharides were studied by sugar analysis and 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, ROESY, and HSQC experiments, and the structure of the branched tetrasaccharide repeating unit of the polysaccharide was established.

[Polysaccharide and lipid composition of the brown seaweed Laminaria gurjanovae].

Polysaccharide and lipid composition of the Pacific brown seaweed Laminaria gurjanovae is determined. Alginic acid is shown to be the main polysaccharide of its biomass (about 28%); it consists of mannuronic and guluronic acid residues at a ratio of 3 : 1. The yield of water-soluble polymannuronic acid is low and does not exceed 1.1% of dry biomass. High laminaran content (about 22%) is found, whereas the yield of fucoidan is no more than 3.6%. Laminaran consists of two fractions, soluble and insoluble in cold water, their ratio is 2.5 : 1. Practically, insoluble laminaran is a linear 1,3-beta-D-glucan, and the soluble fraction was shown to be 1,3;1,6- 3-D-glucan. The oligosaccharide products of desulfation or partial acidic hydrolysis of fucoidan were studied by MALDI TOF MS; they were found to be fuco- and galactooligosaccharides. The fucoidan is suggested to be a highly sulfated partially acetylated galactofucan (Fuc/Gal is -1 : 1). The main lipid components of the dried L. gurjanovae are neutral lipids and glyceroglycolipids, whereas phospholipids are found in minor amounts. The main fatty acid components of lipids are 14 : 0, 16 : 0, 16 : 1 n-7, 18 : 1 n-7, and 18 : 2 n-6 acids.

Enzymatic transformation of biologically active 1,3;1,6-beta-D-glucan. Structure and activity of resulting fragments.

The fragmentation of the biologically active 1,3;1,6-beta-D-glucan Antivir by endo-1,3-beta-D-glucanase LIV from crystalline styles of the marine mollusk Spisula sachalinensis was carried out. It was found that low molecular mass oligomers possessing a stabilizing effect on membranes and anti-viral activity against tobacco mosaic virus appeared in the process of enzymatic hydrolysis of Antivir. Biological activity of 1,3;1,6-beta-D-glucooligo- and polysaccharides was found to be associated with molecular mass (polymerization degree (n) not less than 14) and with presence of intralinked beta-1,6-connected monosaccharide residues. Probably, decrease in molecular mass is compensated by increase in number of intralinked beta-1,6-connected monosaccharide residues.

[An unusual lipid A from a marine bacterium Chryseobacterium scophtalmum CIP 104199T].

The hydrolysis of defatted cells of the marine bacterium Chryseobacterium scophtalmum CIP 104199T with 10% acetic acid (3 h, 100 degrees C) led to an unusual lipid A (LA) (yield 0.6%), obtained for the first time. Using chemical analysis, FAB MS, and NMR spectroscopy, it was shown to be D-glucosamine 1-phosphate acylated with (R)-3-hydroxy-15-methylhexadecanoic and (R)-3-hydroxy-13-methyltetradecanoic acids at the C2 and C3 atoms, respectively. It is similar to the monosaccharide biosynthetic precursor of lipopolysaccharide (LPS), so-called lipid X (LX). Unlike LX, LA can be isolated by the treatment of bacteria with organic solvents only after the preliminary acidic hydrolysis of the cells, which suggests that LA might be strongly, probably chemically, linked to other components of the outer membrane. However, LPS cannot be such a component, because extraction with phenol-water or phenol-chloroform-petroleum ether mixtures in high yields (5.34% and 0.5%, respectively) leads to preparations that do not contain 3-deoxy-D-manno-oct-2-ulopyranosonic acid, 3-hydroxyalkanoic acids, or LA.

[The structure of uncommon lipid A from the marine bacterium Marinomonas communis ATCC 27118T].

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118T with 1% AcOH. Using chemical analysis and ID and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be beta-(1',6)-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-[(R)-3-hydroxydecanoyloxy)]decanoic acid, and (R)-3-hydroxydecanoic acid at the C2, C2' and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M. communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.

Comparative study of carrageenans from reproductive and sterile forms of Tichocarpus crinitus (Gmel.) Rupr (Rhodophyta, Tichocarpaceae).

A comparative study of the structure and properties of the sulfated polysaccharides (carrageenans) isolated from the vegetative and reproductive forms of the red alga Tichocarpus crinitus was performed. The polysaccharides were separated into the gelling (KCl-insoluble) and non-gelling (KCl-soluble) fractions by precipitation with 4% KCl. The total content of polysaccharides extracted from the reproductive form of the alga was 1.8-fold more than that extracted from the vegetative form, and in the first case, the gelling polysaccharides mostly accumulated. The gelling polysaccharides from the vegetative form have the highest molecular weight (354 kD). According to the results of FT-IR and 13C-NMR spectroscopy, the gelling polysaccharide fractions from both forms are kappa/beta carrageenans. The differences concern the content of the kappa- and beta-disaccharide units and the presence of a small content of the sulfated disaccharide segments (precursors of the kappa-carrageenans) in the polysaccharide from the reproductive form of the alga. The non-gelling polysaccharide fractions from both forms of the plant are mixtures of sulfated galactans with a low content of 3,6-anhydrogalactose.

[Polysaccharides of diatoms occurring in Lake Baikal].

Polysaccharide composition of neutral, acid- and alkali-soluble fractions of the diatoms Stephanodiscus meyerii Genkal et Popovsk and Aulacoseira baicalensis (K. Meyer) Simonsen of Lake Baikal has been studied. Neutral polysaccharides were represented by chrysolaminarans (1-->3;1-->6-beta-D-glucans). The chrysolaminaran from S. meyerii consists of the high- and low-molecular-weight fractions (40 and 2-5 kDa, respectively) and contains a large number of beta-1-->6-bound glucose residues. The chrysolaminaran from A. baicalensis is a low-molecular-weight 1-->3:1-->6-beta-D-glucan containing a small number of beta-1-->6 bonds, with mannitol being attached to the reducing unit of its chain. Acid- and alkali-soluble polysaccharide fractions are practically absent in S. meyerii. The alkali-soluble fraction from A. baicalensis is a low-molecular-weight (2-kDa) glycoprotein, the carbohydrate moiety of which is represented by a heteropolysaccharide.

[Elucidation of structure of lipid A from the marine Gram-negative bacterium Pseudoalteromonas haloplanktis ATCC 14393T]. / Ustanovlenie struktury lipida A iz morskoi gramotritsatel'noi bakterii Pseudoalteromonas haloplanktis ATSS 14393T.

The chemical structure of lipid A from the marine gamma-proteobacterium Pseudoalteromonas haloplanktis ATCC 14393T, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be beta-1,6-glucosaminobiose 1,4'-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3' and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2', respectively.

Structure of lipid A from the marine gamma-proteobacterium Marinomonas vaga ATCC 27119T lipopolysaccharide.

The chemical structure of a novel lipid A obtained as a major component on hydrolysis of LPS from the marine gamma-proteobacterium Marinomonas vaga ATCC 27119T with 1% AcOH was determined. Using chemical analysis and NMR data, it was shown to be beta-1,6-glucosaminobiose 1-phosphate acylated with R-3-hydroxydecanoic acid (at position 3), and R-3-dodecanoyloxydecanoic (or R-3-decanoyloxydecanoic) acid and R-3-(R-3-hydroxydecanoyl)oxydecanoic acids (at the 2- or 2;-positions). The absence of a fatty acid at the 3;-position and a phosphoryl group at the 4;-position, and also the presence of R-3-acyloxyalkanoic acid with R-3-hydroxyalkanoic acid as the secondary acid are unique features distinguishing the M. vaga lipid A from other ones.

Intracellular alginolytic enzymes of the marine bacterium Pseudoalteromonas citrea KMM 3297.

The marine bacterium Pseudoalteromonas citrea KMM 3297 is an associate of the holothurian Apostichopus japonicus. When grown in a medium containing glucose, the strain produces two intracellular alginolytic enzymes, AlI and AlII. Fucoidan from the brown alga Fucus evanescens induces synthesis of one more alginolytic enzyme, AlIII. These enzymes were separated using anion-exchange chromatography. The alginate lyase AlI completely retains its activity at 35 degrees C, AlII and AlIII being stable at 45 degrees C. The alginate lyases exhibit maximal activities in the range of pH 7-8. The molecular weights of AlI, AlII, and AlIII determined by gel filtration are 25, 79, and 61 kD, respectively. All the investigated enzymes are endo-type alginate lyases. They catalyze degradation of polyguluronate (poly-G) and polymannuronate (poly-M) yielding oligosaccharides of the polymerization degree of 5 > or = n > or = 3 with the unsaturated bond between the C4 and C5 atoms of the non-reducing terminus. A mixture of these three enzymes exhibits synergism while acting on the polymeric substrate. The Km values of the alginate lyase AlI for poly-G and poly-M are 24 and 34 micro g/ml, respectively. Alginate lyase AlIII exhibits less affinity to poly-M (Km = 130.0 microg/ml) than to poly-G (Km = 40.0 microg/ml). NaCl (0.2 M), MgCl2 and MgSO4 (0.01 M) activate all three enzymes more than twofold. The presence of several alginolytic enzymes of different specificity provides efficient destruction of alginic acids of brown algae by the strain P. citrea KMM 3297.

Structure of O-specific polysaccharide from Pseudoalteromonas nigrifaciens strain KMM 161.

On mild acid degradation of the lipopolysaccharide of the marine microorganism Pseudoalteromonas nigrifaciens KMM 161 an O-specific polysaccharide containing D-galactose, 2-acetamido-2-deoxy-D-glucose, 3,6-dideoxy-3-(4-hydroxybutyramido)-D-galactose, and 2-acetamido-2-deoxy-L-guluronic acid residues was obtained. From the results of Smith degradation, O-deacetylation of the polysaccharide, and NMR spectroscopy the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established [see reaction]. It should be noted that the same structure occurs in the antigenic polysaccharide of Pseudoalteromonas nigrifaciens KMM 158 described earlier as Alteromonas macleodii 2MM6.

[Degradation of fucoidan by the marine proteobacterium Pseudoalteromonas citrea]. / Osobennosti degradatsii fukoidana morskimi proteobakteriiami Pseudoalteromonas citrea.

It was found that Pseudoalteromonas citrea strains KMM 3296 and KMM 3298 isolated from the brown algae Fucus evanescens and Chorda filum, respectively, and strain 3297 isolated from the sea cucumber Apostichopus japonicus are able to degrade fucoidans. The fucoidanases of these strains efficiently degraded the fucoidan of brown algae at pH 6.5-7.0 and remained active at 40-50 degrees C. The endo-type hydrolysis of fucoidan resulted in the formation of sulfated alpha-L-fucooligosaccharides. The other nine strains of P. citrea studied (including the type strain of this species), which were isolated from other habitats, were not able to degrade fucoidan.

O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770.

The O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770 was obtained by mild acid degradation of the corresponding lipopolysaccharide and found to contain D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine residues in equimolar ratio. Based on methylation analysis, periodate oxidation, and 13C-NMR spectroscopy data of native and modified polysaccharides, the following structure of the trisaccharide repeating unit of the O-specific polysaccharide was established: [structure: see text]

[The structure of sulfated cauloside C]. / Struktura sul'fatirovannogo kaulozida C.

The structure of the sulfated analogue of cauloside C, a biologically active triterpenoid glycoside, was elucidated to be 3-O-[beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl]-he deragenin 23,4',4",6"-tetrasulfate pentasodium salt by the comparison of its 13C NMR spectrum with that of cauloside C potassium salt.

New diterpenic altrosides of the fungus Acremonium striatisporum isolated from a sea cucumber.

Two new diterpenic glycosides, virescenosides M (1) and N (2), have been isolated from a marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Their structures were determined on the basis of MS and NMR data as beta-D-altropyranosido-19-7-oxo-isopimara-8,15-diene-2alp ha, 3beta-diol (1) and beta-D-altropyranosido-19-isopimara-7, 15-diene-2alpha,3beta,6beta-triol (2). Three other altrosides (3-5), identified as virescenosides A, B, and C from the terrestrial strain Acremonium luzulae, were also isolated. The cytotoxic activity of the virescenosides was examined.

Structure of sulfated O-specific polysaccharide of the marine bacterium Pseudoalteromonas marinoglutinosa KMM 232.

A sulfated O-specific polysaccharide containing D-mannose, L-rhamnose, and the sulfate group was obtained by mild acid hydrolysis of lipopolysaccharide (S-form) of the marine bacterium Pseudoalteromonas marinoglutinosa KMM 232. Based on analysis of methylation and 13C-NMR spectroscopy of native and desulfated polysaccharides, the following structure of disaccharide repeat unit in the O-specific polysaccharide has been established: [scheme]. This is the first report of a sulfated O-specific polysaccharide isolated from gram-negative bacteria.

[Structure of teichoic acids from marine microorganisms Bacillus subtilis and Bacillus licheniformis]. / Struktura teikhoevykh kislot iz morskikh mikroorganizmov Bacillus subtilis i B. licheniformis.

Teichoic acids from the cell walls of marine bacilli Bacillus subtilis CMM (Collection of Marine Microorganisms) 234 (R-1) and B. licheniformis CMM 454 (1-1G-2) were isolated and characterized. These teichoic acids were found to have identical structures and are composed of the glucose, ribitol, and phosphoric acid residues. On the basis of 13C NMR and 31P NMR spectra of the teichoic acids and the products of their dephosphorylation, we established the following structure for the biopolymer: poly[-->5)-4-O-beta-D-glucopyranosylribitol-(1-phospho].