Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.     

Alginate as an antiglycating agent for human serum albumin

Hyperglycemia and the accumulation of advanced glycation endproducts (AGEs) in tissues and serum have important roles in diabetic complications. Therefore, the identification of anti-glycation compounds is attracting considerable interest. In this study, the interaction of human serum albumin (HSA) with fructose, in the absence and presence of alginate, was studied by circular dichroism, absorbance and fluorescence techniques. The characterization study of AGEs was performed using autofluorescence, fibrillar formation, the increase in absorbance and the quantification of free lysine side chains. The results indicate that alginate inhibits the fructation of HSA as observed by a reduction in the formation of fluorescent AGEs and fibrils. Furthermore, alginate reduces the amount of modified lysine side chains, signified by the lack of increase in absorbance, and increases the helicity of this protein.     

Characterization of insoluble calcium alginates by solid-state NMR

Three series of 9 insoluble calcium alginate powders with different average calcium contents (1.5, 3.5 and 8%, w/w) are investigated by means of ¹³C solid-state NMR spectroscopy. The effect of the increased calcium content on the determination of the mannuronate (M) to guluronate (G) ratio from spectral deconvolution of the ¹³C CP/MAS spectra is discussed, and the variations observed are commented in function of possible structural modifications related to the interaction with the divalent cations. The possibility of using solid-state NMR spectroscopy for the quantification of the calcium content in unknown alginate samples is explored performing principal component analysis (PCA) of the spectra. The results obtained show that a clear separation of alginates with slightly different calcium content is possible. The proposed method relies on the sole use of the chemical shifts of the signals corresponding to pyranose carbons, suggesting that PCA of solid-state NMR data holds promises as a rapid and undestructive method for screening the calcium content of alginate-based materials with biomedical uses.     

Chemometric prediction of alginate monomer composition: A comparative spectroscopic study using IR, Raman, NIR and NMR

The potential of using infrared (IR), Raman and near infrared (NIR) spectroscopy combined with chemometrics for reliable and rapid determination of the ratio of mannuronic and guluronic acid (M/G ratio) in commercial sodium alginate powders has been investigated. The reference method for quantification of the M/G ratio was solution-state ¹H nuclear magnetic resonance (NMR) spectroscopy. For a set of 100 commercial alginate powders with a M/G ratio range of 0.5–2.1 quantitative calibrations using partial least squares regression (PLSR) were developed and compared for the three spectroscopic methods. All three spectroscopic methods yielded models with prediction errors (RMSEP) of 0.08 and correlation coefficients between 0.96 and 0.97. However, the model based on extended inverted signal corrected (EISC) Raman spectra stood out by only using one PLS component for the prediction. The results are comparable to that of the experimental error of the reference method estimated to be between 0.01 and 0.08.     

Alginate lyase from Pseudomonas aeruginosa CF1/M1 prefers the hexameric oligomannuronate as substrate

In order to investigate the catalytic properties of alginate lyase from Pseudomonas aeruginosa CF1/M1, a clinical isolate, regarding the capability to perform β-elimination on oligomannuronates of defined length (2–9), the alginate lyase was purified from periplasmic extracts. A purification method for unsaturated and saturated oligomannuronates applying anionic exchange chromatography on a FPLC apparatus was established. The alginate lyase showed the highest activity, when hexamers were provided as substrate. This indicated that the alginate lyase best accommodates a chain of six alginate residues in the active center. As a minimum chain length, the pentameric oligomannuronate was still accepted as substrate. Mannuronate oligomers shorter than the pentamer were not accepted as substrate for alginate lyase. Furthermore, oligomer pattern analysis of polymannuronate which was subjected to β-elimination by alginate lyase revealed that the trimer is the most abundant oligomer. These data indicated that β-elimination and cleavage occurred at mannuronic acid residue no. 3 of the accommodated hexameric alginate chain.     

An evaluation of tritium and fluorescence labelling combined with multi-detector SEC for the detection of carbonyl groups in polysaccharides

The carbonyl content of a pectic polysaccharide from Sphagnum papillosum (sphagnan) and periodate oxidised alginates was investigated using three different carbonyl labelling strategies combined with size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and on-line fluorescence or off-line tritium detection. The labelling strategies were tritium incorporation via NaB³H₄ reduction, and fluorescent labelling with carbazole carbonyl oxyamine (CCOA), or 2-aminobenzamide (2-AB), respectively. Carbonyl quantification was based on labelled pullulan, dextran and alginate standards possessing only the reducing end carbonyl group. As a result the carbonyl distribution in the polysaccharides could be determined. In sphagnan it was found that the carbonyl content increased with increasing molecular weight, whereas in periodate oxidised alginate the carbonyl content was as expected independent of the molecular weight. The methods proved useful for carbonyl detection in water soluble polysaccharides in general. The tritium incorporation method was preferred for alkali stable polysaccharides, while the CCOA method was most suitable for acid stable polysaccharides with low carbonyl content. The 2-AB method is applicable for all polysaccharides tested with varying carbonyl content; however, it lacks the ability to detect ketone functionalities.     

Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and secretion form a multiprotein complex spanning the inner and outer membranes. In the present study, we developed a method by which the porin AlgE was detected by immunogold labeling and transmission electron microscopy. Localization of the AlgE protein was found to depend on the presence of other proteins in the multiprotein complex. No correlation was found between the number of alginate factories and the alginate production level, nor were the numbers of these factories affected in an algC mutant that is unable to produce the precursor needed for alginate biosynthesis. Precursor availability and growth phase thus seem to be the main determinants for the alginate production rate in our strain. Clustering analysis demonstrated that the alginate multiprotein complexes were not distributed randomly over the entire outer cell membrane surface.     

Improved delivery of biocontrolPseudomonas and their antifungal metabolites using alginate polymers

Alginate polymer was evaluated as a carrier for seed inoculation with a genetically modified strainPseudomonas fluorescens F113LacZY, which protects sugar-beet againstPythium-mediated damping-off. F113LacZY survived in alginate beads at 5 log₁₀ CFU/ bead or higher counts for 8 weeks of storage, regardless of the conditions of incubation. In plant inoculation experiments, colonisation of the growing area of the root by F113LacZY, derived from alginate beads placed in the soil next to the seed or from an alginate coating around the seeds, was improved compared with application of just free cells of the strain. F113LacZY trapped in alginate beads was an effective producer of antifungal phloroglucinols as indicated by direct HPLC quantification of phloroglucinols and in vitro inhibition of both the indicator bacteriumBacillus subtilis A1 and the pathogenic fungusPythium ultimum. Alginate polymer represents a promising carrier for the delivery of biocontrol inoculants for root colonisation and production of antifungal metabolites.     

Quantification of biofilm exopolysaccharides using an in situ assay with periodic acid–Schiff reagent

A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid–Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins).     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola I. Indications for a Polysaccharide Resembling Alginic Acid in Seven P. syringae Pathovars

Extracellular polysaccharides (EPS) produced by Pseudomonas syringae pv. phaseolicola are obviously composed of two main components: the long known levan consisting of fructose, and a mannuronan consisting mainly of mannuronic acid (manA), thus resembling alginic acid (alginate). The identification of manA was established by TLC utilizing different developing systems, and by cellulose acetate electrophoresis in different buffers. References were authentic uronic acids and hydrolyzed authentic alginate. A rough quantification of the “alginate” present in crude EPS was achieved with a selective colour reaction which largely excluded compounds other than uronic acids. Levan was only synthesized with sucrose as primary carbon source. When grown on several other sugars and related compounds “alginate” was the predominant component of the EPS. Additionally, rhamnose, fucose, glucose and amino sugars were found in some instances in hydrolysates of crude EPS, suggesting the release of lipopolysaccharides (LPS) from the bacterial cell walls during culture. Growth on carbon sources not related to sugars resulted in these “LPS” as the main constituent of EPS. After cultivation with sucrose, the “alginate” was restricted to the “slime” fraction of the EPS. In the “capsular” fraction, levan was predominating. A screening program revealed the capacity to synthesize the “alginate” in six additional P. syringae pathovars: pisi, lachrymans, aptata, tomato, syringae, and glycinea. All of the strains tested so far produced levan from sucrose, however, the “alginate” was formed not by all of them. There was a tendency that fresh isolates produced more “alginate” than strains subcultured for an extended time in vitro. This was also true for the total amount of EPS.     

Electrostatic encapsulation and growth of plant cell cultures in alginate.

The growth of callus tissue from African Violets, encapsulated in alginate using electrostatics, was investigated as well as the mechanism of alginate droplet formation. Alginate microbeads as small as 500 (+/-50) microns in diameter could be produced by electrostatic extrusion directly from a plastic syringe (1900 micron extrusion orifice), in the absence of a needle. Video analysis of the mechanism of electrostatic alginate droplet formation from the syringe showed the development of a Taylor cone-like droplet which extended to form a thin strand that then broke up into droplets. Autoclaving of the alginate/medium solution significantly reduced its viscosity, giving smaller beads. Calculated microbead diameters agreed well with experimental values. Callus tissue from leaf explants was successfully immobilized and cultured using electrostatic extrusion. Tissue immobilized using 4% alginate in medium and cultured on agar grew best, producing a complete plantlet within four months. The long-term aim is to develop an effective method for large production of artificial seeds.     

A comparison of the poly(hexamethylenebiguanidinium chloride) assay and a neutral equivalent method for the determination of alginates in industrial liquors extracted from brown seaweed

The poly(hexamethylenebiguanidinium chloride) assay and a neutral equivalent method have been used to estimate the sodium alginate content of industrial liquors extracted from brown seaweed. With the liquor samples examined, agreement to within 5% was achieved with the two methods under defined alginate concentration conditions. Statistical evaluation of the data using a paired comparison t-test has shown that a difference of only 0·10±0·05 can be expected with 95% confidence, between the two methods in the analysis of liquor samples with sodium alginate contents in the range 2·00–3·83 mg ml⁻¹. In the majority of cases the PHMBH⁺ Cl⁻ method gave a slightly higher estimate that the neutral equivalent method. Consideration of the practical aspects of the two alternative methods allows this difference to be explained. The simplicity of the PHMBH⁺ Cl⁻ assay renders it suitable for the rapid screening of large numbers of alginate samples.     

Hydrophobic modification of sodium alginate and its application in drug controlled release

Sodium alginate was hydrophobically modified by coupling of polybutyl methacrylate onto the alginate. The polybutyl methacrylate was previously prepared through polymerization of butyl methacrylate in the presence of 2-amino-ethanethiol as a chain transfer agent. The structure of the product was characterized by Fourier-transformed infrared spectrometry, nuclear magnetic resonance (¹HNMR) and thermogravimetry. The result of fluorescence analysis showed that the hydrophobicity of the modified alginate was obviously increased. The modified alginate conjugate was used for immobilization of bovine serum albumin in the presence of calcium chloride. In addition, the release behavior of the drug-loaded alginate in deionized water and Tris–HCl buffer solution (pH 7.2) was investigated. It was found that the modified sodium alginate possessed prolonged release behavior compared to unmodified sodium alginate, and it had potential application in controlled release as a drug carrier.     

Direct determination of alginate content in brown algae by near infra-red (NIR) spectroscopy

The methods used to quantify total alginate in brown algal tissue are time-consuming and may also be misleading, so faster and simpler methods for measuring alginate content would be beneficial in a variety of applications. This study reports on the use of near infra-red (NIR) analysis to monitor the alginate content of Laminaria hyperborea stipe during biodegradation. NIR reflectance spectra were recorded for 78 different freeze-dried samples of its stipe. The samples were collected during several biological degradation experiments and the total alginate content varied from 2.2 to 40.8% Na-alginate (w/w), determined by established methods based on ion exchange. Data analysis was performed using multivariate calibration methods in order to relate the spectral data to the alginate content. PLS2 analysis revealed some dependence on material type, probably reflecting differences in polyphenol content. In the end, a PLS1 model with 9 components was selected. The calculated model was validated both with internal data and with an external test set. Internal full cross validation explained 96.6% of the variance in alginate content. The external validation showed that the PLS1 model was able to predict the alginate concentration with a root mean square prediction accuracy of 2.1%. This revised version was published online in June 2006 with corrections to the Cover Date.     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

Induced circular dichroism of the interaction between pinacyanol and algal alginates

The interaction between pinacyanol chloride and sodium alginate or guluronate-rich alginate is found to effect profound changes in the visible absorbance and circular dichroism spectra. Two different types of aggregates are observed depending on the relative dye/alginate concentrations. With a dye/alginate ratio at 1:1, a complex is deduced based on an analysis of Job’s method and conductometric titrations. Another complex forms at 1:10 dye/alginate ratio and only in the presence of alginate or guluronate-rich alginate. The two aggregates are in dynamic equilibrium according to the presence of isosbestic points in the visible spectra. The effects of pH and divalent cations on the spectra are studied. The 1:10 complex is damaged by addition of hydrochloric acid and divalent cations; however, at low concentration of these agents the spectra indicate conversion of the complex into the 1:1 aggregate. Models for the two complexes are proposed taking into account the preference of guluronate binding sites for chelating ions.     

Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes

Aims:  Isolation of novel alginate degrading bacteria for the disposal of seaweed waste in composting process.
Methods and Results:  Decomposition of alginate polymers was checked by the 3,5-dinitrosalicylic acid (DNS) method for reducing sugar, and absorbance at 235 nm for unsaturated sugar. A bacterium A7 was isolated from wakame compost and confirmed to belong to the genus Gracilibacillus by partial 16S rDNA analysis. The optimum condition for the growth of A7 in a medium containing 5 g l⁻¹ of sodium alginate is as follows: pH, 8·5–9·5; NaCl, 0·5 mol l⁻¹; temperature, 30°C and polypeptone as nutrient content, 2–5 g l⁻¹. In a laboratory-scale composting experiment, the alginate content in wakame compost decreased to 14·3% after 72 h of composting from an initial value of 36%, indicating the effectiveness of alginate decomposition of A7 in wakame composting.
Conclusions:  The bacterium A7 was found to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process.
Significance and Impact of the Study:  Development of new methods for the disposal of marine wastes and production of functional products.     

Isolation of water-soluble alginate from brown algae

Summary Water-soluble alginate was obtained from an aqueous extract of Kjellmaniella crassifolia by precipitation with HCl, calcium acetate or 20% ethanol in the presence of 0.05 M MgCl₂ Of these precipitation procedures, MgCl₂-ethanol gave the purest alginate preparation as judged by electrophoresis. The thin-layer and gas-liquid chromatography of its acid hydrolysate, and the IR spectra analysis of the whole alginate, suggested that the water-soluble alginate is similar to ordinary water-insoluble and alkali-soluble alginate such as Kelco alginate.However, the alginate obtained in the present work contained a great excess of mannuronic acid residues, giving an M:G ratio of about 13. Its molecular weight distribution was rather broad as with Kelco alginate, but the molecular weight of its major component was estimated to be 500 000 amu, whereas that Kelco alginate measured on the same column under the same condition was 1 700 000 amu. This suggests that water-soluble alginate was far smaller in average molecular size than Kelco alginate.     

Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2

A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40?°C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate.     

In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa

An enzymatic in vitro alginate polymerization assay was developed by using 14C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing Pseudomonas aeruginosa FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the alg8 gene. Surprisingly, this resulted in significant alginate overproduction of the complemented P. aeruginosa Deltaalg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. (1)H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7 to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane. A structural model of Alg8 based on the structure of SpsA from Bacillus subtilis was developed.