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.     

DECREASE IN DYNAMIC VISCOSITY AND AVERAGE MOLECULAR WEIGHT OF ALGINATE FROM LAMINARIA DIGITATA DURING ALKALINE EXTRACTION1

Alginates are natural polysaccharides that are extracted from brown seaweeds and widely used for their rheological properties. The central step in the extraction protocol used in the alginate industry is the alkaline extraction, which requires several hours. In this study, a significant decrease in alginate dynamic viscosity was observed after 2 h of alkaline treatment. Intrinsic viscosity and average molecular weight of alginates from alkaline extractions 1–4 h in duration were determined, indicating depolymerization of alginates: average molecular weight decreased significantly during the extraction, falling by a factor of 5 between 1 and 4 h of extraction. These results suggested that reducing extraction time could enable preserving the rheological properties of the extracted alginates.     

A Novel Alginate Lyase with High Activity on Acetylated Alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03

Summary To exploit alginate lyase which could degrade bacterial alginates, degenerate PCR and long range-inverse PCR (LR-IPCR) were used to isolate alginate lyase genes from soil bacteria. Gene algL, an alginate lyase-encoding gene from Pseudomonas sp. QD03 was cloned, and it was composed of a 1122 bp open reading frame (ORF) encoding 373 amino acid residues with the calculated molecular mass of 42.2 kDa. The deduced protein had a potential N-terminal signal peptide of 20 amino acid residues that was consistent with its proposed periplasmic location. Gene algL was expressed in pET24a (+)/E. coli BL21 (DE3) system. The recombinant AlgL was purified to electrophoretic homogeneity using affinity chromatography. The molecular weight of AlgL was estimated to be 42.8 kDa by SDS-PAGE. AlgL exhibited maximal activity at pH 7.5 and 37 °C. Na⁺, K⁺, Ca²⁺ and Ba²⁺ significantly enhanced the activity of AlgL. AlgL could degrade alginate and mannuronate blocks, but hardly degrade guluronate blocks. In particular, AlgL could degrade acetylated alginate of Pseudomonas aeruginosa FRD1 (approximately 0.54 mol of O-acetyl group per mol of alginate). It might be possible to use alginate lyase AlgL as an adjuvant therapeutic medicine for the treatment of disease associated with P. aeruginosa infection.     

Components in the inoculum determine the kinetics of Azotobacter vinelandii cultures and the molecular weight of its alginate

In cultures of Azotobacter vinelandii inoculated using washed cells (avoiding exhausted broth components) alginates of a higher molecular weight (1200 kDa) than those obtained in cultures conventionally inoculated (350 kDa), were produced. Also, when comparing conventionally inoculated cultures with those inoculated with washed-cells, the alginate lyase activity was delayed and the final polymer concentration decreased from 4.8 to 3.5 g l^–1. This suggests that components in the exhausted inoculum broth play important regulatory roles in alginate biosynthesis and needs to be taken into account when describing polymer biosynthesis.     

Seasonal studies on the alginate and its biochemical composition I: Sargassum polycystum (Fucales), Phaeophyceae

Investigations were made on the brown seaweed Sargassum polycystum C. Agardh collected from Rameswaram Coast, Tamil Nadu. The alginates extracted from ‘leaf’, ‘stem’ and entire thallus of S. polycystum were investigated for their viscosity and chemical 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) for six different seasons between August 1998 and November 1999. Significant seasonal variation (P< 0.05) was observed with high yield of alginate in February. The alginate extracted from the ‘leaf’ region showed a maximum yield whereas the ‘stem’ region exhibited maximum viscosity. The amount of G‐block was found to be more than M‐ and MG‐blocks in all the samples tested. The amount of G‐block was high in ‘stem’ followed by leaf and entire thallus. A positive correlation was recorded between viscosity and G‐block. Among the three alginates, the ratio of M/G was low in the ‘stem’ followed by ‘leaf’ and entire thallus.     

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.     

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.     

Resistance of biofilms containing alginate‐producing bacteria to disintegration by an alginate degrading enzyme (Algl)

Pure culture biofilms of Pseudomonas aeruginosa (strains 8830 and ATCC 700829) and mixed population biofilms composed of Pseudomonas aeruginosa (ATCC 700829), Pseudomonas fluorescens (ATCC 700830), and Klebsiellapneumoniae (ATCC 700831) were treated with an alginate‐degrading enzyme (AlgL). The enzyme effectively depolymerized the mannuronic acid rich (92%), partially O‐acetylated bacterial alginate produced by P. aeruginosa (8830), both in dilute solution and in a gel‐like, concentrated state. However, both biofilms were unaffected by the presence of the enzyme. These findings suggest either that bacterial alginates do not contribute significantly to the cohesiveness of biofilms or that the alginate is protected from enzymatic degradation in biofilms.     

The use of bacterial alginates to prepare biocontrol formulations

Summary Formulations which are economical and which can deliver a viable organism are critical to developing successful biocontrol products for plant pathogens. In the present study, alginates derived from commercial kelp and produced byAzotobacter vinelandii isolates ATCC 9104 and 12 837 were compared in their ability to form stable, biodegradable granular formulations of the biocontrol fungiTalaromyces flavus andGliocladium virens. Bacteria were grown in shake flask cultures (180 rpm) at 32°C for 104 h. The cultures were monitored for pH, dissolved oxygen, glucose concentration, dry cell weight, and alginate dry weight. Aqueous solutions of the bacterial alginates, as well as the kelp-derived alginate products, gelled readily in 0.25 M calcium chloride. Mannuronate (M) and guluronate (G) compositions of the alginate samples were determined by circular dichroism. M/G ratios for cultures of isolate 12837 averaged 0.98±0.18; for isolate 9104, 1.59±0.12; and for kelp, 1.54±0.39. The viability ofT. flavus in the kelp and bacterial alginate formulations were similar over 84 days. An exploratory experiment indicated good viability ofG. virens using the same bacterial alginates. This study demonstrated a practical use for bacterial alginate as a potentially less costly substitute for kelp alginate in the preparation of biocontrol agent formulations.     

Bacterial alginates: from biosynthesis to applications

Alginate is a polysaccharide belonging to the family of linear (unbranched), non-repeating copolymers, consisting of variable amounts of β-d-mannuronic acid and its C5-epimer α- l-guluronic acid linked via β-1,4-glycosidic bonds. Like DNA, alginate is a negatively charged polymer, imparting material properties ranging from viscous solutions to gel-like structures in the presence of divalent cations. Bacterial alginates are synthesized by only two bacterial genera, Pseudomonas and Azotobacter, and have been extensively studied over the last 40 years. While primarily synthesized in form of polymannuronic acid, alginate undergoes chemical modifications comprising acetylation and epimerization, which occurs during periplasmic transfer and before final export through the outer membrane. Alginate with its unique material properties and characteristics has been increasingly considered as biomaterial for medical applications. The genetic modification of alginate producing microorganisms could enable biotechnological production of new alginates with unique, tailor-made properties, suitable for medical and industrial applications.     

Alginate production by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> in a stirred draft fermenter

In this study alginate production by Pseudomonas mendocina in a laboratory-scale fermenter was investigated. In the experiments the effect of temperature (25–31°C) and agitation (500–620 rev min⁻¹) at a constant air flow of 10 v/v/h were evaluated in relation to the rate of glucose bioconversion to alginate using response surface methodology (RSM). The fermenter configuration was also adapted to a system with a screw mixer and draft tube, due to the change in rheological characteristics of the fermentation broth. The adjusted model indicates a temperature of 29.1°C and agitation of 553 rev min⁻¹ for optimum alginate synthesis. In this fermentation system a Y _p/s of 44.8% was achieved. The alginate synthesized by P. mendocina showed a partially acetylated pattern as previously reported for alginates obtained from other Pseudomonas spp and Azotobacter vinelandii.     

一株产褐藻胶裂解酶的需钠弧菌筛选及酶解产物分析

[背景]褐藻胶裂解酶种类丰富、降解机制多样,是高效环保降解褐藻胶、制备褐藻寡糖的工具酶,成为褐藻植物高值化开发利用的研究热点.[目的]从海泥中筛选获得褐藻胶裂解酶高效产酶菌株,确定菌株发酵产酶最优条件,鉴定和分析酶降解产物,进而解析该酶的降解特性.[方法]以褐藻胶为唯一碳源,从海带养殖场附近海泥中筛选菌株,通过形态学观...     

Determination of the diadic composition of alginate by means of circular dichroism: a fast and accurate improved method

Circular dichroism (CD) is presented as a reliable and sensitive method of determining the diadic frequency composition of alginate (F(GG), F(MM) and F(GM+MG)). The availability of samples, very largely or even completely conforming to the limiting structures of polymannuronate (MM)(n), polyguluronate (GG)(n) and polyalternating MG (MG)(n), respectively, allowed the limiting CD spectra for each alginate diad to be obtained. These showed very different CD behaviour, thus pointing out the crucial importance of the neighbouring residue in chiroptical properties. Using an iterative best-fit procedure, the diadic composition of commercial alginates could be obtained from their respective CD spectra by means of a linear combination of the spectra of the three limiting diads. The results were found in excellent agreement with the composition parameters obtained by 1H NMR spectroscopy.     

Extraction, isolation and cadmium binding of alginate from Sargassum spp.

Sargassum brown algal species have recently shown promise for use in flow-through column systems that rely on a passive ion-exchange mechanism for the remediation of toxic heavy metals such as Pd, Cd, and Zn from contaminated waters. To elucidate the metal binding mechanism and optimise this so-called biosorption process, detailed information on the biochemistry of the raw biomass and the alginate in particular is essential. This study focuses on the detailed characterisation (e.g., percentage of yield, block co-polymer structure) of the various fractions of material isolated from S. fluitans and S. oligocystum following a (i) standard neutral, (ii) alkaline (NaOH) and (iii) high-temperature alkaline alginate (80 °C; Na₂CO₃) extraction. Results indicate that the alginate yield was independent of the temperature or the extraction method employed (21.1 to 22.8% and 18.9 to 20.5% yields for S. fluitans and S. oligocystum, respectively). Furthermore, ¹H-nuclear magnetic resonance (NMR) analyses revealed that the alginates isolated by the three methods displayed nearly identical doublet -L-guluronic acid frequencies (F _GG; between 0.55 to 0.58 for both S. fluitans and S. oligocystum). Cadmium binding experiments (pH 4.5) further demonstrated that the three alginate extracts have similar metal binding capacities (uptake ranging from 1.59 to 1.81 mmol Cd/gram). The implementation of the high-temperature alkaline extraction procedure resulted in the isolation of a new acid-soluble fraction (ASF), capable of binding cadmium at pH 4.5, which cannot be isolated by the standard neutral extraction protocol. A preliminary characterisation of this ASF revealed the presence of minor quantities of proteins and sulphated polysaccharides, as well as traces of alginate and possibly other low-molecular weight uronic acid-containing polymers.     

Production of algal gels from the brown alga, <Emphasis Type="Italic">Laminaria japonica</Emphasis> Aresch., and their biotechnological applications

Alginic acid is localised in the cell walls and intercellular spaces of the brown alga, Laminaria japonica Aresch., and the salts of this compound occur mainly as calcium alginates. However, the alginates in this alga do not have the viscosity and the ability to create and stabilise structural products. Hence, the structure and properties of the alginates in Laminaria were changed by chemical modification to produce new products such as sodium alginates and other substances capable of forming gels. The rheological properties of the algal gel from Laminaria depended on the properties of the sodium alginate. Heating the algal product up to 90°C did not change its physical and chemical properties; the viscosity did not differ from that of the initial product. The viscosity and molecular weight of the sodium alginate isolated from the algal gel were stable from 20°C up to 95°C. However, about 30% of the viscosity was lost at 100°C. Recipes and various methods of preparing the gel products as fish sauces, jelly-like fish products, fruit jellies, drinks, cosmetic and pharmaceutical products are presented. The algal gel and the gel products did not lose their integrity with heat processing. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 4. Conversion of alginic acid to sodium alginate, drying and milling

The last three steps of the alginate production process were studied:conversion of alginic acid to sodium alginate, drying, and milling. Threemethods were used to follow the conversion reaction: measuring the pH (a) intheethanol-water liquid of the reaction mixture, (b) after dissolving a sample ofthe fiber taken from the reaction mixture, (c) after dissolving the driedsodiumalginate obtained from the reaction. To obtain a neutral dried sodium alginate,in the first method the pH should be adjusted to 9, and in the second the pHshould be adjusted to 8. The best method to control the reaction was todissolvea sample of the fiber and adjust the pH to 8. The best proportion to reach thecritical point, where pH just begins to rise, was 0.25 parts of sodiumcarbonateto 1 part of alginate in the initial dry algae. A pH above 7 may produce abreakdown of the molecule, reducing significantly the viscosity of the finalalginate. Four different temperatures were used to dry the alginate: 50, 60,70,and 80 °C. Drying time to reach 12% moisture ranged from 1.5h at 80 °C to 3 h at 50°C. The best drying temperature was 60 °C for2.5 h. The effect of drying temperature on alginate viscosity wasdependent on the alginate type. Low and medium viscosity alginates were notsignificantly affected, but alginate with high viscosity was reduced by 40 to54% using the temperature range of 60 to 80 °C. A fixed hammermill was used to reduce the particle size of the dried sodium alginate.Particlesize measurements showed that after a first milling the product contained 76%large particles (20–60 mesh) and 24% fine particles (80–120 mesh).After a third milling the product still contained 42.9% large particles. Nosignificant effect was found on alginate viscosity because of the millingsteps.     

Determination of alginate composition by a simple enzymatic assay

The action of alginate lyases may be easily followed in a UV-spectrophotometer, since each cut of the alginate chain will create an unsaturated unit at the non-reducing end with a strong absorbance at 230 nm. During prolonged incubation, this absorbance will approach an apparent endpoint level that reflects the initial substrate concentration. On this basis, a standardized assay has been developed. A combination of purified mannuronate lyase from Haliotis tuberculata and purified guluronate lyase from Klebsiella pneumoniae is applied to get quantitative concentration estimates that do not depend on alginate composition. The production of alginate in Azotobacter vinelandii is included as an example of application. Most important, by applying both enzymes alone and in combination, the block composition of the alginate may be estimated. Data for a series of widely different alginates have been compared with those obtained by NMR.     

Extraction and characterization of an alginate from the brown seaweed <Emphasis Type="Italic">Sargassum turbinarioides</Emphasis> Grunow

Matrix polysaccharide from the brown algae Sargassum turbinarioides collected in the coastal waters of Nosy Be (Madagascar) in the Indian Ocean was isolated and its structure was studied by ¹H-NMR spectroscopy, FT-IR, SEC-MALLS and HPAEC. An alginate with a molecular weight of 5.528 × 10⁵ g mol⁻¹ was identified as sole polysaccharide. Values of the M/G ratio, F _GG, F _MM and F _GM (or F _GM) blocks were measured at respectively 0.94, 0.39, 0.36 and 0.25 and compared with those of alginates from other Sargassum species. This sodium alginate appeared similar to some of the other Sargassum alginates with M/G < 1, high values of homopolymeric blocks (η < 1) and significant polyguluronic block content.     

In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Evaluation of steam explosion as pretreatment in agar extraction fromGracilaria dura (C. Agardh) J. Agardh (Gracilariaceae,Rhodophyta)

Steam explosion was investigated as a pretreatment step in the isolation of agar from the macroalgaGracilaria dura. As compared to conventional procedures, the yield of agar obtained using this method on alkali (Na₂CO₃) conditioned algal material was higher. Extractions performed first at 95 °C and then at 121 °C showed that the major fraction of the agar was extracted at 95 °C, independently of the pretreatment. The efficiency of sulphate hydrolysis during steam explosion ofG. dura previously conditioned in Na₂CO₃, was similar to that of a NaOH based alkali pretreatment. Except for a lower nitrogen content of the sample obtained after NaOH based alkali pretreatment and a higher 6-O-methyl--d-galactose content in the agar after steam explosion, the chemical composition of the agars showed no significant difference. Agars extracted after steam explosion had melting temperature, gel strength and apparent modulus of elasticity lower than those of corresponding native and alkali (NaOH) pretreated samples, but comparable to those of a commercial sample.Author for correspondence