Seasonal studies on alginate and its composition II: Turbinaria conoides (J.Ag.) Kütz. (Fucales, Phaeophyceae)

The alginates extracted from ‘leaf’, ‘stem’ and entire thallus of Turbinaria conoides (J.Ag.) Kütz. were investigated for their viscosity and biochemical constituents namely, β-D-mannuronic acid (M-block}), α-L-guluronic acid (G-Block) and alternating sequences of β–D-mannuronic acid and α-L-guluronic acid (MG-block). Substantial seasonal variation was recorded with high yield of alginate during premonsoon. The yield of alginate was maximum in ‘leaf’ region. In contrast to this, viscosity and G-block were maximum in the ‘stem’ region. A significant positive correlation was observed between viscosity and G-block}. The ratio of guluronic: mannuronic acid was also assessed. Low levels of M/G ratio were recorded in the ‘stem’ region followed by ‘leaf’ and entire thallus.     

The invasive brown seaweed Sargassum muticum as new resource for alginate in Morocco: Spectroscopic and rheological characterization

The Japanese brown seaweed Sargassum muticum, recently invaded several shorelines worldwide including the Atlantic coast of Morocco with large well‐established populations. Within the framework of a sustainable strategy to control this invasive seaweed, we report on extraction yield, spectroscopic characterization and rheological properties of alginate, a commercially valuable colloid, from harvested biomass of S. muticum. Extraction yield was about 25.6% on dry weight basis. Infrared spectroscopy analysis shows that the obtained Fourier transform infrared spectra of the extracted biopolymer exhibit strong similarities with that of the commercial alginate. Furthermore, Proton nuclear magnetic resonance spectroscopy revealed that S. muticum alginate has almost equal amounts of β‐D‐mannuronic acid (M; 49%) and α‐L‐guluronic acid (G; 51%) with an M/G ratio of 1.04 and a high content of heteropolymeric MG GM diads suggesting a sequence distribution of an alternated polymer type. Rheological measurements were performed at different sodium alginate concentrations, temperatures and shear rates. The hydrocolloid exhibited pseudoplastic behavior and showed shear thinning, particularly at high solution concentration and low temperature which is consistent with the rheological behavior reported for commercial alginates. Considering the abundance of S. muticum in the Northwestern Atlantic coast of Morocco and the quality of the extracted hydrogel, this invasive species could be considered as a potential source of alginates.     

Effect of molecular weight on the antioxidant property of low molecular weight alginate from Laminaria japonica

In this study, three alginate fractions with different molecular weights and ratios of mannuronic acid (M) to guluronic acid (G) were prepared by enzymatic hydrolysis and ultrafiltration to assess the antioxidant property of alginates from Laminaria japonica with molecular weight below 10 kDa. The antioxidant properties of different molecular weight alginates were evaluated by determining the scavenging abilities on superoxide, hydroxyl, and hypochlorous acid and inhibitory effect on Fe²⁺-induced lipid peroxidation in yolk homogenate. The results showed that low molecular weight alginates exhibited high scavenging capacities on superoxide, hydroxyl, and hypochlorous acid radicals and good inhibition of Fe²⁺-induced lipid peroxidation in yolk. By comparison, alginate A1 with molecular weight below 1 kDa and M/G of 1.84 had better scavenging activity on superoxide, hydroxyl, and hypochlorous acid radicals in vitro than A2 (1–6 kDa), A3 (6–10 kDa), ascorbic acid, and carnosine. With similar M/G ratio, A2 exhibited better antioxidant activity on superoxide and hypochlorous acid radicals than A3. However, fraction A3 with molecular weight of 6–10 kDa exhibited higher inhibitory ability on lipid peroxidation in yolk in vitro than A1 and A2. The results indicated that molecular weight played a more important role than M/G ratio on alginate to determine the antioxidant ability. By comparison, low molecular weight alginates composed of guluronic acid and mannuronic acid exhibited better antioxidant ability on oxygen free radicals than sulfated polysaccharides from L. japonica in our previous study and represent a good source of marine polysaccharide with potential application as natural antioxidant.     

Characterization of the alginates from algae harvested at the Egyptian Red Sea coast

The alginates from five species of brown algae from the Egyptian Red Sea coast, namely: Cystoseira trinode, Cystoseira myrica, Sargassum dentifolium, Sargassum asperifolium, and Sargassum latifolium, were isolated and their compositions and structures studied by 1H NMR spectroscopy. All the alginates studied contain more guluronic acid (G) than mannuronic acid (M) and have a homopolymeric block-type structure (eta<1). The intrinsic viscosity of the alginate samples range from 8.6 to 15.2 and the gel strength ranges from 10.97 to 15.51. The constitutional G- and M-blocks of alginates from two different species (C. trinode and S. latifolium) were separated after partial acid hydrolysis. The 1H NMR spectral data of the blocks GG and MM obtained by chemical fractionation were compared with those of polymeric alginates. The monomeric uronic acids were separated by complete acid hydrolysis of S. asperifolium alginate and the G and M monomers were characterized by 1H, 13C NMR spectroscopy as well as by paper electrophoresis.     

Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4

Alginate epimerases are large multidomain proteins capable of epimerising C5 on β‐D ‐mannuronic acid (M) turning it into α‐L ‐guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution‐state NMR are hampered by their size—the smallest epimerase, AlgE4, consisting of one A‐ and one R‐module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally ²H, ¹⁵N labeled AlgE4 isotopomeres (A‐[²H, ¹⁵N]‐R and [²H, ¹⁵N]‐A‐R) by protein trans‐splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild‐type AlgE4.     

Galactose-substituted alginate: preliminary characterization and study of gelling properties

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.     

Uronic acid sequence in alginate from different sources

Alginate may be considered as a block co-polymer of D-mannuronic and L-guluronic acids, and consists of three types of blocks: homopolymeric blocks of mannuronic acid (MM) and of guluronic acid (GG), and blocks with an alternating sequence (MG). The block composition of alginates has been characterized by a simple chemical method involving partial hydrolysis with acid, followed by fractional precipitation of the acid-resistant part of the alginate. Alginates from eleven different species of brown algae have been examined and, for five species, alginates from different tissues have been compared. The results indicate that young tissue is rich in MM blocks, and that the difference between the alginates from different species is mainly due to the alginates from the older parts of the plants. Extracellular alginates from two types of bacteria have been examined.     

Catalytic properties and specificity of a recombinant, overexpressed D-mannuronate lyase

Lysis of alginates and of their saturated and unsaturated fragments was monitored by 1H NMR spectroscopy. AlxM(B) alginate lyase performs beta-elimination on the mannuronic acid (M) residues. It does not cleave the guluronic acid (G) sequences, nor the M-G or the G-M diads. In consequence, it is a true mannuronate lyase. The end product of the reaction is O-(4-deoxy-alpha-L-ery-thro-hex-4-enopyranosyl-uronic acid)-(1->(4)-O-(beta-D-mannopyranosyluronic acid)-(1->4)-O-beta-D-mannpyranuronic acid. Viscosity measurements made during degradation of a polymannuronate alginate showed that AlxM(B) behaves as an endo-enzyme. HPLC analysis of the degradation products of oligomannuronates and oligoalginates suggested that the beta-elimination requires the interaction of the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers with different M/G ratio. Kinetic measurements showed that the specificity constant Vm/Km increased with the number of mannuronic acid residues. AlxM(B) may be reversibly inhibited by heteropolymeric blocks in a competitive manner.     

Variations in the alginate content and composition of Durvillaea antarctica and D. willana from southern New Zealand

The brown seaweeds Durvillaea antarctica andD. willana are dominant components of the lowerlittoral and upper sublittoral of exposed rocky shoresin southern New Zealand. Tissue samples of bothspecies, harvested from a site on the south-east coastof South Island over a period of 2 years, wereanalysed for alginate content and composition.Individuals of both species were further separatedinto different blade (lamina and palm) and stipe(cortex and medulla) fractions to assess variationwithin the thallus. On average the alginate contentand frequency of mannuronic acid (F_m) was higherin D. antarctica than in D. willana. Blades contained more alginate than stipes, laminaeand stipes were rich in mannuronic acid whereasholdfasts were rich in guluronic acid. Variations incomposition are considered to reflect the functionaldifferences of the tissue, giving flexibility to bladeand stipe and rigidity to the holdfast. Despitefluctuations in content and composition betweencollection times no seasonal trends in eithercomponent were apparent.     

Isolation of Marine Bacteria Capable of Producing Specific Lyases for Alginate Degradation

Alginate lyases (EC 4.2.2.3) were isolated from cultures of several marine bacterial isolates. The lyases were induced by native alginate and had activity toward both the mannuronic acid and the guluronic acid blocks of the alginate polymer. The guluronic acid-specific lyase was recovered from the medium, whereas the mannuronic acid-specific lyase was retained with the bacteria.     

Direct quantification of M/G ratio from C CP-MAS NMR spectra of alginate powders by multivariate curve resolution

Multivariate curve resolution (MCR) was applied to ¹³C cross-polarisation (CP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of non-depolymerised alginate powders obtained from brown seaweed plus a pure mannuronate sample isolated from Pseudomonas fluorescens for estimation of the mannuronic acid/guluronic acid ratio (M/G ratio). An excellent MCR model with a correlation coefficient of r² = 0.99 was established between the estimated M/G ratios and the M/G ratios obtained from the traditional ¹H solution state NMR method. The new method allows for successful determination of the M/G ratio independent of the calcium content (at least up to 2.4%, which was the upper limit in this study) with a root mean square error of prediction of 0.05. It is thus concluded that ¹³C CP-MAS NMR in combination with multivariate curve resolution is a reliable, convenient (no sample preparation is required) and relatively rapid method for M/G ratio determinations of alginates and it may serve as a good alternative to the chemical techniques traditionally used.     

Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae

This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-l-erythro-5-hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.     

Consequences of the association of calcium with alginate during batch culture of Azotobacter vinelandii

Summary Azotobacter vinelandii strain E was grown in batch culture in the presence of radioactive Ca⁴⁵. The partitioning of Ca⁴⁵ in solution and associated with extracellular polysaccharide (alginate) during the growth was studied. The amount of alginate produced was estimated and its composition was determined by ¹H-n.m.r. Alginate produced early in the growth cycle was characterised by high guluronic acid block mole fraction (0.45) whilst later, alginate with a preponderance of mannuronic acid blocks was observed. Mixed block synthesis occurred throughout the incubation. The level of free Ca²⁺ fell rapidly (from 97% to 22%) during the first 33 h of incubation.     

Alginate,mannitol, phenolic compounds and biological activities of two range-extending brown algae, <Emphasis Type="Italic">Sargassum mangarevense</Emphasis> and <Emphasis Type="Italic">Turbinaria ornata</Emphasis> (Phaeophyta: Fucales), from Tahiti (French Polynesia)

This study deals with two range-extending brown algae from Tahitian coral reefs, Sargassum mangarevense and Turbinaria ornata; their alginate properties, mannitol and phenolic contents, antioxidant and antimicrobial activities were determined. Turbinaria ornata showed the richest alginate content with the highest extraction yield (19.2 ± 1.3% dw). Their alginates also exhibited the highest viscosity (50 ± 18 mPa.s), but the M:G ratios (mannuronic acid to glucuronic acid) of alginates (1.25–1.42) were similar in both species. Alginate yield displayed spatial variations, but no significant seasonal changes. The highest mannitol content was found in S. mangarevense (12.2 ± 2.1% dw) during the austral winter. With respect to other tropical Fucales, both algae exhibited also a high phenolic content (2.45–2.85% dw) with significant spatio-temporal variations. Furthermore, high antioxidant activity and activity against Staphylococcus aureus were also detected in extracts. According to these preliminary results, these two range-extending algae are of key interest in numerous industrial areas.     

Preparation of stable alginate gel beads in electrolyte solutions using Ba2+ and Sr2+

Summary Alginate solutions with two different M/G (mannuronic acid/guluronic acid) ratios were added dropwise to SrCl₂ and BaCl₂ solutions. The low M/G ratios (0.27) Sr^– and Ba-alginate gel beads were more chemically and physically stable in electrolyte solutions than conventional Ca^– alginate gel beads. These gel beads with immobilized yeast cells had normal ethanol productivities.author to whom all correspondence should be addressed.     

Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.     

Mechanical properties of C-5 epimerized alginates

There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.     

Preliminary studies on the chemical characterization and antihyperlipidemic activity of polysaccharide from the brown alga Sargassum fusiforme

A polysaccharide (SFP) extracted from the brown alga Sargassum fusiforme (Harv.) Setch. was purified by chromatography on DEAE-Sephadex A50 and Sephadex G-100. Studies using paper chromatography (PC), electrophoresis and infrared spectroscopy (IR) indicated that SFP was a kind of alginate with a molecular weight of 16 000 and a molar ratio of mannuronic acid (M) to guluronic acid (G) of 2.75. Pharmacological experiments showed that SFP could markedly decrease the content of total cholesterol (TC), triglyceride (TG) and low density lipoprotein-cholesterol (LDL-C) in the serum of experimental hyperlipidemic rats, and significantly increase the level of high density lipoprotein-cholesterol (HDL-C).

     

Extraction and physicochemical characterization of Sargassum vulgare alginate from Brazil

Alginate fractions from Sargassum vulgare brown seaweed were characterized by (1)H NMR and fluorescence spectroscopy and by rheological measurements. The alginate extraction conditions were investigated. In order to carry out the structural and physicochemical characterization, samples extracted for 1 and 5h at 60 degrees C were further purified by re-precipitation with ethanol and denoted as SVLV (S. vulgare low viscosity) and SVHV (S. vulgare high viscosity), respectively. The M/G ratio values for SVLV and SVHV were 1.56 and 1.27, respectively, higher than the ratio for most Sargassum spp. alginates (0.19-0.82). The homopolymeric blocks F(GG) and F(MM) of these fractions characterized by (1)H NMR spectroscopy were 0.43 and 0.55 for SVHV and 0.36 and 0.58 for SVLV samples, respectively, these values typically being within 0.28-0.77 and 0.07-0.41, respectively. Therefore, the alginate samples from S. vulgare are much richer in mannuronic block structures than those from other Sargassum species. Values of M(w) for alginate samples were also calculated using intrinsic viscosity data. The M(w) value for SVLV (1.94 x 10(5)g/mol) was lower than that for SVHV (3.3 x 10(5)g/mol). Newtonian behavior was observed for a solution concentration as high as 0.7% for SVLV, while for SVHV the solutions behaved as a Newtonian fluid up to 0.5%. The optimal conditions for obtaining the alginates from S. vulgare were 60 degrees C and 5h extraction. Under these conditions, a more viscous alginate in higher yield was extracted from the seaweed biomass.