scholarly journals The biosynthesis of alginic acid by Azotobacter vinelandii

1975 ◽  
Vol 152 (3) ◽  
pp. 617-622 ◽  
Author(s):  
D F Pindar ◽  
C Bucke
Keyword(s):  
Alginic Acid ◽  
Azotobacter Vinelandii ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Polymeric Substance

The sequence of reactions by which alginic acid is biosynthesized from sucrose in Azotobacter vinelandii was determined both by feeding radioactive individual enzymes involved. Results indicate that the first polymeric substance formed in the synthesis is polymannuronic acid and that mannuronic acid units are epimerized to guluronic acid at the polymer level. Guluronic acid does not appear to be formed at the monomer level, either free or in combination with GDP.

EXOCELLULAR ALGINIC ACID FROM AZOTOBACTER VINELANDII

1966 ◽  
Vol 44 (9) ◽  
pp. 993-998 ◽  
Author(s):  
P. A. J. Gorin ◽  
J. F. T. Spencer
Keyword(s):  
Sodium Alginate ◽  
Small Proportion ◽  
Alginic Acid ◽  
Azotobacter Vinelandii ◽  
Specific Rotation ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Exocellular Polysaccharide

Azotobactervinelandii produces a partly acetylated exocellular polysaccharide which consists mainly of D-mannuronic acid units and a small proportion of L-guluronic acid units. It resembles alginic acid since it contains 4-O-linked mannuronosyl residues and guluronosyl residues which are 4-O- and (or) 5-O-linked. The specific rotation of bacterial sodium alginate is close to that of its algal counterpart, thus suggesting that the glycosidic configurations are similar.

In situ studies on alginic acid synthesis and other aspects of the metabolism of Laminaria digitata

1972 ◽  
Vol 50 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Johan A. Hellebust ◽  
Arne Haug
Keyword(s):  
Amino Acids ◽  
Time Course ◽  
Alginic Acid ◽  
Acid Synthesis ◽  
Short Term ◽  
Fresh Weight Basis ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Rate Of Photosynthesis

Amino acids, particularly alanine and aspartate, become more strongly labeled than mannitol in short-term 14C-photoassimilation experiments. The amino acids are the most likely sources of carbon for alginic acid synthesis and respiration in the dark, in contrast to mannitol, which appears to be relatively unavailable. Temperature is very important in determining the rate of loss of recent photoassimilate in L. digitata. The rate of photosynthesis, on a fresh weight basis, is much higher for blades than for stipes.The time course for incorporation of photoassimilated carbon into alginate differs for the stipe and blade both in light and dark periods. Very little 14C enters alginate in blades in the dark, while alginate in stipes acquires considerable amounts of activity during dark periods. Alginate in both blade and stipe acquires 14C predominantly in mannuronic acid residues of their alginate during short-term photoassimilation periods, while guluronic acid residues become relatively more rapidly labeled during dark periods.

Photosynthesis, translocation, and alginic acid synthesis in Laminaria digitata and Laminaria hyperborea

1972 ◽  
Vol 50 (1) ◽  
pp. 169-176 ◽  
Author(s):  
Johan A. Hellebust ◽  
Arne Haug
Keyword(s):  
Time Course ◽  
Alginic Acid ◽  
Acid Synthesis ◽  
Laminaria Hyperborea ◽  
Short Term ◽  
Fresh Weight Basis ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Isolated Cortex ◽  
Photosynthetic Capacities

New and old tissues of L. digitata blades have very similar photosynthetic capacities on a fresh weight basis. Very little of the photoassimilate goes into alginic acid, or other macromolecular substances in old blade tissues. Less than 1% of the photoassimilated 14C in the old blade portion of a L. digitata blade was translocated to the new blade tissues in a 5-h experiment. In contrast, there is rapid transport of photoassimilate from bark cells to cells of the underlying tissues of L. digitata and L. hyperborea stipe sections. Isolated cortex and medulla tissues of L. digitata stipes have significant photosynthetic capacities, but are probably so strongly shaded by the darkly pigmented bark cells that little photosynthesis can normally occur in these tissues.A larger proportion of the photoassimilated carbon enters alginate in the cortex and medulla than in the bark of L. digitata and L. hyperborea stipes in short-term experiments. The time course for incorporation of photosynthate into alginate in continuous and pulse-labeling experiments indicates the presence of relatively large pools of alginate precursors. A large proportion of the total 14C incorporated into alginate in short-term experiments is found in the "M–M" (mannuronic acid) and "M–G" (alternating mannuronic and guluronic acid) block components.

scholarly journals The A Modules of the Azotobacter vinelandii Mannuronan-C-5-Epimerase AlgE1 Are Sufficient for both Epimerization and Binding of Ca2+

1999 ◽  
Vol 181 (10) ◽  
pp. 3033-3038 ◽  
Author(s):  
Helga Ertesvåg ◽  
Svein Valla
Keyword(s):  
Amino Acids ◽  
Reaction Rate ◽  
Azotobacter Vinelandii ◽  
Binding Studies ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Catalytic Sites ◽  
Direct Binding ◽  
Protein Modules ◽  
Secreted Enzymes

ABSTRACT The industrially important polysaccharide alginate is composed of the two sugar monomers β-d-mannuronic acid (M) and its epimer α-l-guluronic acid (G). In the bacteriumAzotobacter vinelandii, the G residues originate from a polymer-level reaction catalyzed by one periplasmic and at least five secreted mannuronan C-5-epimerases. The secreted enzymes are composed of repeats of two protein modules designated A (385 amino acids) and R (153 amino acids). The modular structure of one of the epimerases, AlgE1, is A1R1R2R3A2R4. This enzyme has two catalytic sites for epimerization, each site introducing a different G distribution pattern, and in this article we report the DNA-level construction of a variety of truncated forms of the enzyme. Analyses of the properties of the corresponding proteins showed that an A module alone is sufficient for epimerization and that A1 catalyzed the formation of contiguous stretches of G residues in the polymer, while A2 introduces single G residues. These differences are predicted to strongly affect the physical and immunological properties of the reaction product. The epimerization reaction is Ca2+ dependent, and direct binding studies showed that both the A and R modules bind this cation. The R modules appeared to reduce the Ca2+ concentration needed for full activity and also stimulated the reaction rate when positioned both N and C terminally.

scholarly journals Characterization of Three New Azotobacter vinelandii Alginate Lyases, One of Which Is Involved in Cyst Germination

2009 ◽  
Vol 191 (15) ◽  
pp. 4845-4853 ◽  
Author(s):  
Martin Gimmestad ◽  
Helga Ertesvåg ◽  
Tonje Marita Bjerkan Heggeset ◽  
Olav Aarstad ◽  
Britt Iren Glærum Svanem ◽  
...  
Keyword(s):  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Growth Conditions ◽  
Type I ◽  
Wild Type ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Polysaccharide Lyases ◽  
Alginate Lyases

ABSTRACT Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic acid and α-l-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2+. All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

scholarly journals Cloning and Expression of Three NewAzotobacter vinelandii Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases

1999 ◽  
Vol 181 (1) ◽  
pp. 68-77 ◽  
Author(s):  
Britt Iren Glærum Svanem ◽  
Gudmund Skjåk-Bræk ◽  
Helga Ertesvåg ◽  
Svein Valla
Keyword(s):  
Escherichia Coli ◽  
Southern Blot Analysis ◽  
Azotobacter Vinelandii ◽  
Cloning And Expression ◽  
Terminal Part ◽  
Hybrid Gene ◽  
Mannuronic Acid ◽  
New Genes ◽  
Guluronic Acid ◽  
Commercially Important

ABSTRACT The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii(algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca2+-dependent polymer-level epimerization of β-d-mannuronic acid to α-l-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6,algE7, and algY. All three genes were sequenced and expressed in Escherichia coli. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5′-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5′ part ofalgE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.

The Fine Structure of Alginic Acid Components

1968 ◽  
Vol 26 ◽  
pp. 82-83
Author(s):  
S. Inoue ◽  
Y. Tanaka ◽  
S.C. Skoryna
Keyword(s):  
Fine Structure ◽  
Sodium Alginate ◽  
Uronic Acid ◽  
Alginic Acid ◽  
Brown Seaweed ◽  
Uronic Acids ◽  
Laminaria Hyperborea ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Hydrolysis Of

Sodium alginate, a brown seaweed polysaccharide, is capable of preventing intestinal absorption of radioactive strontium and its radiotoxicological importance is now well established. Alginic acid was found to be composed of 1,4-linked poly-L-guluronic acid and poly-D-mannuronic acid chains with some glycosidic linkages between these uronic acids.Polyguluronic and polymannuronic acid were prepared by acid hydrolysis of alginic acid from Laminaria hyperborea followed by fractionation of the partially degraded sodium alginate at pH 2.8. These components consist almost exclusively of a single uronic acid (polyguluronic acid: man/gul = 0.27; polymannuronic acid: man/gul = 10.7). Sodium polyguluronate inhibits absorption of radiostrontium more effectively than sodium polymannuronate.

scholarly journals Biochemical analysis of the processive mechanism for epimerization of alginate by mannuronan C-5 epimerase AlgE4

2004 ◽  
Vol 381 (1) ◽  
pp. 155-164 ◽  
Author(s):  
Cristiana CAMPA ◽  
Synnøve HOLTAN ◽  
Nadra NILSEN ◽  
Tonje M. BJERKAN ◽  
Bjørn T. STOKKE ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Soil Bacteria ◽  
Biochemical Analysis ◽  
Azotobacter Vinelandii ◽  
Minimum Size ◽  
The Third ◽  
Mannuronic Acid ◽  
Enzyme Substrate ◽  
Guluronic Acid ◽  
Ionisation Mass Spectrometry

The enzymes mannuronan C-5 epimerases catalyse the in-chain epimerisation of β-D-mannuronic acid to α-L-guluronic acid in the last step of alginate biosynthesis. The recombinant C-5 epimerase AlgE4, encoded by the soil bacteria Azotobacter vinelandii and expressed in Escherichia coli, exhibits a non-random mode of action when acting on mannuronan and alginates of various monomeric compositions. The observed residue sequence has been suggested previously to be due to either a preferred attack or a processive mode of action. Based on methodologies involving specific degrading enzymes, NMR, electrospray ionisation mass spectrometry and capillary electrophoresis we show here that on average 10 residues are epimerised for each enzyme–substrate encounter. A subsite model for the enzyme is analysed by the same methodology using native and 13C-labelled mannuronan oligomers as substrate for the AlgE4 epimerase. A hexameric oligomer is the minimum size to accommodate activity. For hexa-, hepta- and octameric substrates the third M residue from the non-reducing end is epimerised first.

scholarly journals Biochemical Properties and Substrate Specificities of a Recombinantly Produced Azotobacter vinelandiiAlginate Lyase

1998 ◽  
Vol 180 (15) ◽  
pp. 3779-3784 ◽  
Author(s):  
Helga Ertesvåg ◽  
Frode Erlien ◽  
Gudmund Skjåk-Bræk ◽  
Bernd H. A. Rehm ◽  
Svein Valla
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Azotobacter Vinelandii ◽  
Divalent Cations ◽  
Alginate Lyase ◽  
Biochemical Properties ◽  
Mature Protein ◽  
Substrate Specificities ◽  
Mannuronic Acid ◽  
Guluronic Acid

ABSTRACT Alginate is a polysaccharide composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene,algL, was cloned, sequenced, and expressed inEscherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL fromPseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the Km was found to be 4.6 × 10−4 M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.

scholarly journals Construction and analyses of hybrid Azotobacter vinelandii mannuronan C-5 epimerases with new epimerization pattern characteristics

2004 ◽  
Vol 381 (3) ◽  
pp. 813-821 ◽  
Author(s):  
Tonje M. BJERKAN ◽  
Bjørn E. LILLEHOV ◽  
Wenche I. STRAND ◽  
Gudmund SKJÅK-BRÆK ◽  
Svein VALLA ◽  
...  
Keyword(s):  
Azotobacter Vinelandii ◽  
Point Mutations ◽  
Distribution Patterns ◽  
Time Resolved ◽  
Mannuronic Acid ◽  
Monomer Sequence ◽  
Guluronic Acid ◽  
The Many ◽  
The Individual ◽  
Hybrid Enzymes

The secreted mannuronan C-5 epimerases from Azotobacter vinelandii form a family of seven homologous modular type enzymes, which appear to have evolved through duplications and point mutations in the individual modules. The catalytic A modules of these enzymes are responsible for generating the characteristic sequence distribution patterns of G residues in the industrially important polymer alginate by epimerizing M (β-D-mannuronic acid) moieties to G (α-L-guluronic acid). Forty-six different hybrid enzymes were constructed by exchanging parts of the sequences encoding the A modules of AlgE2 (generates consecutive stretches of G residues) and AlgE4 (generates alternating structures). These hybrid enzymes introduce a variety of new monomer-sequence patterns into their substrates, and some regions important for the subsite specificity or processivity of the enzymes were identified. By using time-resolved NMR spectroscopy, it became clear that the rates for introducing alternating structures and consecutive stretches of G residues are different for each enzyme, and that it is the ratio between these rates that determines the overall epimerization pattern. These findings open up new possibilities in biotechnology and in studies of the many biological functions of alginates.

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