Production of exopolysaccharides from a thermophilic microorganism isolated from a marine hot spring in flegrean areas

A thermophilic strain isolated from sea sand at Maronti, near Sant' Angelo (Ischia), is described. The organism grows well at an optimal temperature of 60 °C at pH 7.0. The thermophilic bacterium, named strain 4004, produces an exocellular polysaccharide (EPS) in yields of 90 mg/l. The EPS fraction was produced with all substrates tested, although a higher yield was obtained with sucrose or trehalose as sole carbon source. During growth, the EPS content was proportional to the biomass. Three fractions (EPS1, EPS2, EPS3) were obtained after purification. Quantitative monosaccharide analysis of the EPSs revealed the presence of mannose:glucose:galactose in a relative ratio of 0.5:1.0:0.3 in EPS1, mannose:glucose:galactose in a relative ratio of 1.0:0.3:trace in EPS2, and galactose:mannose:glucosamine:arabinose in a relative ratio of 1.0:0.8:0.4:0.2 in EPS3. The average molecular mass of EPS3 was determined to be 1×10⁶ Da. From comparison of the chemical shift values in ¹H and ¹³C spectra, we conclude that EPS3 presents a pentasaccharide repeating unit. Electronic Publication     

Formation of α-1,2- and α-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-α-d-mannose, p-nitrophenyl-α-d-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-α-d-mannose or with mannose were α-linked. The dissaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-α-1,2-mannose and mannose-α-1,3-mannose. Synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-α-1,3-mannose was severely inhibited by Triton X-100.     

Synthesis of alpha 1----6-mannooligosaccharides in Mycobacterium smegmatis. Function of beta-mannosylphosphoryldecaprenol as the mannosyl donor

Incubation of a membrane fraction from Mycobacterium smegmatis cells with GDP-mannose and free mannose at pH 7 in presence of Mg2+ ions resulted in the formation of a series of alpha 1----6-linked mannooligosaccharides with up to 12 mannoses. The membrane fraction also catalyzed incorporation of mannose from GDP-mannose into a lipid-soluble product with the properties of a mannosyl phospholipid. A similar product was formed by the incubation of the membrane protein with decaprenol phosphate and GDP-mannose, and it was characterized as beta-mannosylphosphoryldecaprenol. A pulse-chase experiment suggested that the mannosyl phospholipid was an intermediate in alpha 1----6-linked mannooligosaccharide synthesis, and the isolated beta-mannosylphosphoryldecaprenol was shown to function as a direct mannosyl donor on incubation with mannose, methyl alpha-D-mannoside, or alpha 1----6-linked mannooligosaccharides as acceptors. The Km values for mannose, methylmannoside, and alpha 1----6-linked mannobiose were 30-90 mM, whereas for alpha 1----6-linked mannotriose, mannotetraose, and mannopentaose the Km dropped to 2 mM. A weak enzymic activity was detected at pH 6 in the presence of both Mg2+ and Mn2+ ions that catalyzed addition of mannose in alpha 1----2 linkage to the longer alpha 1----6-mannooligosaccharides in a reaction that was specific for GDP-mannose as the donor. The membrane preparation also contained an endo-alpha 1----6-mannanase activity that degraded products longer than mannotriose by cleavage of trisaccharide units from the nonreducing end of the alpha 1----6-mannooligosaccharides.     

Studies on the structure of the oligosaccharide chains of alpha 1-acid glycoprotein associated with rough membranes from rat liver

The high mannose form of rat alpha 1-acid glycoprotein was isolated from rough membranes of rat liver using methods described previously. The high mannose glycopeptides were prepared by Pronase digestion, and oligosaccharides were isolated following digestion with endohexosaminidase-H. The structure of the carbohydrate chains of the high mannose glycopeptide and the oligosaccharides was examined by 300 MHz nuclear magnetic resonance spectroscopy. The glycopeptide contained a mixture of about equal amounts of AsnGlcNAc2Man9 and AsnGlcNAc2Man8. Analysis of the oligosaccharide fraction showed that it consisted of about equal amounts of GlcNAc Man9 and GlcNAc Man8; the GlcNAc Man8 fraction contained 85% of the "A" isomer (which was missing the terminal mannose from the middle antenna). The results suggested that mannose processing of alpha 1-acid glycoprotein in rough membranes of rat liver in vivo occurred only as far as the Man8 structure and that the "A" isomer was the main isomer formed.     

Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation

Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis

Structural analysis of the phosphomannan isolated from yeast-form cells of a pathogenic yeast, Candida albicans J-1012 strain, was conducted. Treatment of this phosphomannan (Fr. J) with 10 mM HCl at 100 degrees C for 60 min gave a mixture of beta-1,2-linked manno-oligosaccharides, from tetraose to biose plus mannose, and an acid-stable mannan moiety (Fr. J-a), which was then acetolyzed by means of an acetolysis medium, 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4, at 40 degrees C for 36 h in order to avoid cleavage of the beta-1,2 linkage. The resultant manno-oligosaccharide mixture was fractionated on a column of Bio-Gel P-2 to yield insufficiently resolved manno-oligosaccharide fractions higher than pentaose and lower manno-oligosaccharides ranging from tetraose to biose plus mannose. The higher manno-oligosaccharide fraction was then digested with the Arthrobacter GJM-1 alpha-mannosidase in order to cleave the enzyme-susceptible alpha-1,2 and alpha-1,3 linkages, leaving manno-oligosaccharides containing the beta-1,2 linkage at their nonreducing terminal sites, Manp beta 1----2Manp alpha 1----2Manp alpha 1----2Manp alpha 1----2Man, Manp beta 1----2Manp beta 1----2Manp alpha 1----2Manp alpha 1---- 2Manp alpha 1----2Man, and Manp beta 1----2Manp beta 1----2Manp beta 1----2Manp alpha 1---- 2Manp alpha 1----2Manp alpha 1----2Man. However, the result of acetolysis of Fr. J-a by means of a 10:10:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 13 h was significantly different from that obtained by the mild acetolysis method; i.e., the amount of mannose was apparently larger than that formed by the mild acetolysis method. In summary, a chemical structure for Fr. J as a highly branched mannan containing 14 different branching moieties was proposed.     

Polysaccharides from Extremophilic Microorganisms

Several marine thermophilic strains were analyzed for exopolysaccharide production. The screening process revealed that a significant number of thermophilic microorganisms were able to produce biopolymers, and some of them also revealed interesting chemical compositions. We have identified four new polysaccharides from thermophilic marine bacteria, with complex primary structures and with different repetitive units: a galacto-mannane type from strain number 4004 and mannane type for the other strains. The thermophilic Bacillus thermantarcticus produces two exocellular polysaccharides (EPS 1, EPS 2) that give the colonies a typical mucous character. The exopolysaccharide fraction was produced with all substrates assayed, although a higher yield 400 mg liter(-1) was obtained with mannose as carbon and energy source. NMR spectra confirmed that EPS 1 was a heteropolysaccharide of which the repeating unit was constituted by four different alpha-D-mannoses and three different beta-D-glucoses. It seems to be close to some xantan polymers. EPS 2 was a mannan. Four different alpha-D-mannoses were found as the repeating unit. Production and chemical studies of biopolymers produced by halophilic archaea, Haloarcula species were also reported.     

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.     

Isolation and characterization of two proteoheparan sulfate species of calf arterial tissue

When calf aortic tissue, preincubated under organ culture conditions in the presence of [35S]sulfate, was submitted to a sequential collagenase and elastase digestion and guanidinium chloride extraction, the bulk of proteoheparan sulfate was obtained in the elastase fraction. Ion-exchange chromatography on DEAE-cellulose of the elastase digest under dissociative conditions yielded a proteoglycan fraction that contained heparan sulfate as the sole glycosaminoglycan. The proteoheparan sulfate fraction was resolved into a high-molecular-mass (P-HS 1) and a low-molecular-mass (P-HS 2) fraction by gel filtration on Sephacryl S-400. P-HS 1 has a Mr of 175,000 and possesses four heparan sulfate side-chains (Mr 32,000) covalently bound to the protein core via a galactose- and xylose-containing polysaccharide-protein binding region. The protein core (Mr 38,000), which was obtained after deglycosylation of PG-HS 1 with trifluormethane sulfonic acid, contained in addition a few N-glycosidically linked oligosaccharide units representing a complex type with terminal neuraminic acid residues. P-HS 2 is a single-chain peptidoheparan sulfate of Mr of 38,000 containing one heparan sulfate chain (Mr 32,000) linked to a polypeptide (Mr 6000). The ratio of specific radioactivities of P-HS 1 and P-HS 2 was 1:0.66.     

Production and characterization of exopolysaccharides excreted by thermophilic bacteria from shallow,marine hydrothermal vents of Flegrean Ares (Italy)

Thermophilic microorganisms (4001-4014), described as aerobic or facultatively anaerobic, endospore forming with growth optima temperatures in the range of 60 to 80 degrees C, have been isolated from hot marine springs around Ischia and from hydrothermal vents in the gulf of Naples. Mucous colonies are been selected for the recovery of new strains producing exopolysaccharides (EPS). To induce the biosynthesis of new exopolysaccharides, different sugars were tested as carbon sources in the media. The production of EPS in the strain 4009 reached 60 mg/l using trehalose as carbon source, increasing the yield of about 1000 fold. The 4001-EPS was a mannan with a molecular weight of 380.000 D and with a complex primary structure. In fact, the analysis of the permethylated polysaccharide in GC-MS, showed the presence of mannose, glucose, galactose, mannosamine in the relative ratio of 1:0.1:tr :tr, respectively. Nuclear magnetic resonance spectrum of the exopolysaccharide confirmed the presence of a repetitive unity formed by seven monosaccharides, six with alpha gluco/galacto configuration and one residue with beta conformation.     

Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans

Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3–37%), nucleic acids (9–50%), and carbohydrates (3–21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.     

一株高温产粘菌株的筛选及其产胞外聚合物分析

从油井采出水中分离到一株高温产胞外聚合物的细菌MS-1,经16S rDNA基因序列分析属于芽孢杆菌属(Bacillus sp.).该菌能在60℃生长并产生胞外聚合物,其中胞外多糖含量为48.3%～54.5%.主要由甘露糖、葡萄糖、半乳糖组成,摩尔比分别是2.04:1.00:0.89.胞外聚合物中蛋白含量为37.2%～42.4%,主要由甲硫氨酸、亮氨酸、天门冬氨酸、丙氨酸、组氨酸和丝氨酸组成.利用透射电镜和环境扫描电镜对胞外聚合物的形成进行了观察.该菌的分离和研究为高温油藏的微生物调剖和驱油奠定了生物学基础.     

The effect of cultivation conditions on the physicochemical properties of the exopolysaccharide ethapolan

The physicochemical properties of the complex exopolysaccharide ethapolan (EPS) produced by Acinetobacter sp. 12S during growth on media with various C/N ratios and different concentrations of mineral components and phosphate buffer were studied. Irrespective of the cultivation conditions, the concentrations of carbohydrates (38–44%) and pyruvic acid (3.2–3.7%) in the total EPS, as well as in the acylated (AP) and nonacylated (NAP) polysaccharides obtained from them, were practically the same. The EPS, AP, and NAP were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3: 2: 1: 1. The polysaccharides contained different concentrations of mineral salts (6–28%), uronic acid (3.7–22.0%), and fatty acids (5.8–15.4%); they also differed in the ratio of acetylated and nonacetylated polysaccharides. Due to the differences in the chemical composition and molecular mass (500 kDa–1.5 MDa), the viscosities of the EPS solutions (in the presence of 0.1 M KCl, in the H⁺-form, and in Cu²⁺-glycine system) were different as well. The mechanisms responsible for changes in the physicochemical properties of the total EPS, AP, and NAP synthesized on various media are discussed.     

Isolation, ultrastructure and chemical composition of the outermost layer ("exo-layer") of the Epidermophyton floccosum cell wall.

The outer-most layer ("exo-layer") of the wall was isolated from cell walls of Epidermophyton floccosum. The pure cell walls, obtained by disruption in a Ribi cell fractionator, sonication and centrifugation, were digested with snail enzyme for 12 h. Thereafter, the exo-layer preparation was obtained as the fraction resistant to the snail enzyme. Electron microscopy showed that the exo-layer is a thin, stranded network structure 10-20 nm thick. Chemical analysis of the exo-layer showed that the main components are protein (63 percent), mannose (10 percent) and glucosamine (17 percent). Sodium dodecyl sulfate polyacrylamide gel electrophoresis has revealed that the main band is a glycoprotein containing mannose.     

Structural Properties of the Heparan Sulfate Proteoglycans of Brain

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.     

Oligomannoside biosynthesis in Mycobacterium smegmatis

A cell-free particulate enzyme system of Mycobacterium smegmatis ATCC 607 was shown to catalyze the incorporation of labeled mannose from GDP-[¹⁴C]mannose into endogenous acceptors to form a series of labeled neutral oligomannosides. These oligomannosides were devoid of amino sugar. The major oligomannoside product was characterized to be a trimannoside, O-α-d-mannopyranosyl-(1 → 2)-O-α-d-mannopyranosyl(1 → 2)-d-mannose and represented 46% of the total labeled oligomannoside product. The higher oligomannosides were shown to have either/or both α(1 → 2) and α(1 → 6) glycosidic linkages. A series of unlabeled endogenous oligosaccharides was isolated from the 105,000g supernatant fractions of the cell-free extracts of M. smegmatis and found to be chromatographically similar to the labeled oligomannosides synthesized by the cell-free system. The nature of the endogenous acceptor was not determined.     

Purification,structural features,antioxidant and moisture-preserving activities of an exopolysaccharide from Lachnum YM262

A water-soluble exopolysaccharide, designated as LEP-2a, was isolated from Lachnum YM262 and purified by DEAE-Cellulose 52 and Sepharose CL-6B chromatographic columns. LEP-2a was a homogeneous polysaccharide, with a molecular weight of 1.52 × 10⁵ Da. It was composed of mannose and galactose in a molar ratio of 20.6:1.0. Its structural features were investigated and elucidated by methylation analysis, periodate oxidation and Smith degradation, FT-IR and NMR spectroscopy. Based on obtained data, the backbone of LEP-2a consisted of 1,2-linked-α-d-mannose, 1,3-linked-α-d-mannose, 1,2,6-linked-α-d-mannose and 1,3-linked-β-d-galactose and the side chains were attached to the backbone at O-6 position of 1,2,6-linked-α-d-mannose. In vitro antioxidant activity assay proved that LEP-2a possessed significant scavenging activities on superoxide, hydroxyl and DPPH radical. Furthermore, LEP-2a had strong in vitro moisture-absorption and -retention capacities as compared to chitosan and glycerol. These results suggested that LEP-2a might have a good potential to be applied as a multifunctional cosmetic additive in cosmetics.     

The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum.

Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.     

Production and characterization of the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis> grown on olive mill-waste waters

Paenibacillus jamilae, a strain isolated from compost prepared with olive-mill wastewaters, produced an extracellular polysaccharide (EPS) when it was grown in a culture containing olive-mill waste waters (OMWW) as sole carbon and energy sources. Maximal EPS production in 100 mL batch-culture experiments (5.1 g L⁻¹) was reached with a concentration of 80% of OMWW as fermentation substrate (v/v). Although an inhibitory effect was observed on growth and EPS production when OMWW concentration was increased, an appreciable amount of EPS (2.7 g L⁻¹) was produced with undiluted OMWW. Sepharose CL-2B chromatography showed that the EPS presented two fractions, EPS I (>2000 kDa) and EPS II (500 kDa). Both fractions were characterized by GC-MS as two different acidic heteropolysaccharides containing glucose, galactose and mannose as the major components. The performed study made evident the possibility of using OMWW as substrate for the production of EPS by P. jamilae with a satisfactory yield.