Polymer Degradation and Stability 59 (1998) 85-91

0 1998 Elsevier Science Limited. All rights reserved

Printed in Northern Ireland
PII: s0141-3910(97)00179-1 0141-3910/98/519.00
ELSEVIER

Biosynthesis and applications of alginates

Helga Ertesvig* & Svein Valla
Unigen Center for Molecular Biology, Norwegian University of Science and Technology, N-7005 Trondheim, Norway

(Accepted 18 June 1997)

Alginate is a family of linear polysaccharides composed of mannuronic acid (M)

and guluronic acid (G). The polymer is used as a gel-former and viscosifier in a
wide range of industrial applications. It is also used for encapsulation of cells and
enzymes. The viscosity of alginate is mainly dependent on the polymer length,
while the gel-forming and water-binding properties and the degree of immuno-
genicity are determined by the fraction and distribution of G-residues. Alginates
are currently manufactured by harvesting brown algae, but in nature the polymer
is also produced by some bacteria belonging to the genera Azotobacter and
Pseudomonas. The biosynthesis of alginate has been mostly studied in Pseudo-
monas aeruginosa, where many of the involved proteins and genes have also been
identified. In both algae and bacteria the polymer is first produced as mannur-
onan, which is then epimerized by the enzyme mannuronan C-5epimerase. A
gene encoding a periplasmic epimerase has been identified in the alginate gene
clusters of P. aeruginosa and Azotobacter vinelandii. The A. vinelandii genome
also encodes a family of at least five secreted epimerases, each of which intro-
duces different distributions of G in the alginate. These enzymes can therefore be
used to modify alginates in vitro to obtain polysaccharides with the desired con-
tent and distribution pattern of G. Such alginates may become useful in applica-
tions where reproducible and specific physical properties are required. 0 1998
Elsevier Science Limited. All rights reserved

1 STRUCTURE OF ALGINATE
Alginate is a linear copolymer composed of l-4
linked /3-D-mannuronic acid (M) and its C-5-epimer
a-L-guluronic acid (Fig. 1). These monomers can be
organized in blocks of consecutive G-residues (G-
blocks), consecutive M-residues (M-blocks), or
alternating M and G (MG-blocks). The relative
amount of each block-type varies between different
alginates. As shown in Fig. 1, there are distinct
structural differences between the block-types. MG-
blocks form the most flexible chains and are more
soluble at lower pH than the other two block-types.
M-blocks have been found to be strongly immunos-
timulating. G-blocks form stiff chains, and two G-
blocks of more than 6 residues each can be cross-
linked by divalent cations (e.g. Ca2+, Ba2+, Sr2+),
leading to gel formation. 2,3 At low pH, protonized
high-molecular-mass alginates can form soft acidic
gels. In these gels, it is mostly the homopolymeric
*To whom correspondence should be addressed.
73598705; e-mail: helgae@unigen.ntnu.no
blocks which form the junctions, but the stability of
the gel is mostly dependent on the content of G-
blocks.4 By analysing the polymer by NMR it is now
possible to determine the frequencies of each mono-
mer, each of the four possible dimers, and each of
the eight possible trimers.5,6 However, it is not pos-
sible to estimate the distribution of block-lengths for
any alginate containing more than one type of block.
2 TRADITIONAL INDUSTRIAL USES OF
ALGINATE
Alginates for commercial uses are extracted from
brown algae, and the annual production is about
30 000 tons.7 The composition of the poly-
saccharide depends on the species used and also
whether the blade and stipe are separated before
extraction (Table 1). For most applications the
polymer is sold for US$S-20/kg, but the highest
purity qualities are sold for up to US$40 OOO/kg.
Fax: 47 Alginates have various industrial uses as viscosi-
fiers, stabilizers and gel-forming, film-forming or
85
86 H. ErtesvGg, S. Valla

a) b)

Fig. 1. Structure of a) B-D-mannuronic acid, b) cw-guluronic acid and c) alginate.

Table 1. Composition aud sequence parameters for algal 3 ALGINATES AS IMMOBILIZATION

alginatek8
MATERIALS
Source FG FM FGG FMM FGM,MG

Ascophyllum nodosum 0.10 0.90 0.04 0.84 0.06 In recent years alginates have been used for
(fruiting bodies) encapsulation of cells and enzymes.* When
Ascophyllum nodosum 0.36 0.64 0.16 0.44 0.20 droplets of a mixture of cells or enzymes and
(old tissue)
Macrocystis pyrifera 0.39 0.61 0.16 0.38 0.23
Na-alginate are mixed with a solution containing a
Laminaria hyperborea 0.55 0.45 0.38 0.28 0.17 gel-forming ion, the gel-beads will form instan-
(leaf) taneously. The encapsulated cells are protected
Laminaria hyperborea 0.68 0.32 0.56 0.20 0.12 against mechanical stress, while nutrients and
(stipe)
Laminaria hyperborea 0.75 0.25 0.66 0.16 0.09 metabolites can diffuse through the semi-permeable
(outer cortex) capsule. Gels with a high amount of G-blocks
Durvillea antartica 0.29 0.71 0.15 0.57 0.14 exhibit high porosity, low shrinkage during gel-
formation, and low swelling after drying. With an
increasing amount of M the gels become softer and
have a smaller pore size. The beads can later be
water-binding agents. These applications range coated by a polycation such as poly-L-lysin. This
from textile printing and manufacturing of cer- will stabilize and strengthen the gel network and
amics to production of welding rods and reduce the permeability.lO~ll Some examples of
water-treatment.7 The polymer is soluble in cold cells encapsulated in alginate are shown in Table 2.
water and forms thermostable gels. These properties Insulin-producing cells encapsulated in alginate
are utilized in the food industry in products are now being tested as an artificial pancreas for
like custard creams and restructured food. The treatment of Type I diabetes.25,26 The pores in the
polymer is also used as a stabilizer and thickener in capsules are large enough to permit free diffusion
a variety of beverages, ice-creams, emulsions and of glucose and insulin, while at the same time pro-
sauces. tecting the cells from the immune system.27 Since
The pharmaceutical industry7 uses alginates as mannuronan stimulates the immune system, it is
wound dressings and dental impression materials. important to use well-defined and homogenous
The polysaccharide is also used as a tablet binder or alginates for implantation purposes. On the other
disintegrant, and by carefully choosing the optimal hand, this property of mannuronan may be utilized
alginate quality one can obtain controlled release of when a stimulation of the immune system is
the drug. advantageous.28
 Biosynthesis and applications of alginates 87

Table 2. Examples of cells immobilized in alginate

~____
Cells/enzymes Product/purpose Reference

Enzymes Urease Stabilization of enzyme 12

High-alkaline phosphatase Stabilization of enzyme 13
Bacteria Pseudomonas putida Removal of HzS 14
Erwinia rhapontici Isomaltulose 15
Blue green algae Anabena sp. Ammonia 16
Fungi Aspergillus niger Citric acid 17
Saccharomyces cerevisiae Ethanol 18
Algae Microalgae Cryopreservation 19
Botryococcus braunii Hydrocarbons 20
Plant cells Carrot Dry artificial seeds 21
Digitalis lanata Digitoxins 22
Mammalian cells rhCSF-1 or LM-10 CSF-1 23
Hybridoma Monoclonal antibodies 24
Islets of Langerhans Insulin 25

4 OCCURRENCE OF ALGINATES IN NATURE different,36probably reflecting the different needs

Alginate exists as a matrix polysaccharide in all
marine brown algae. It is the most abundant
polysaccharide in the organisms, comprising up to
40% of their dry weight. The habitats of the dif-
ferent species differ with regard to exposure to
periodic drying due to the tide and waves, and this
is reflected in different requirements for stiffness,
elasticity and water-binding capacity between
species. Since these properties are related to the
relative content of each block-type in the polymer,
this probably explains why different species need to
produce different alginates (Table 1).
Alginate is also used as a capsular poly-
saccharide for the bacteria Azotobacter vinelandii,29
Azotobacter chroococcum30 and some species
belonging to the genus Pseudomonas.31-33 The
polymers from Pseudomonas do not contain G-
blocks, while alginates from A. vinelundii contain
all three block types. 34 The main difference
between algal and bacterial alginate is that some of
the bacterial M-residues are 0-acetylated at the 2
and/or 3 position. 34 A. vinefundii is able to differ-
entiate into a cyst, which can be viewed as a resting
stage of the bacterium encapsulated in a Ca2+-
alginate ge1.35The composition of alginates found
in the capsule of the vegetative cell and in the outer
(exine) and inner (intine) layers of the cyst coat is
Table 3. Alginates from different stages in tbe life-cycle of
A. vinek&ii.36
Sample Distribution of diuronides
FMM FMG
Capsular material 0.26 0.32
Cyst exine 0.046 0.26
Cyst intine 0.39 0.21
for gel-strength and water-retaining capacities
(Table 3). Wild-type Pseudomonas aeruginosu does
not produce alginate, but mutations leading to
derepression of the a-factor AlgU give the corre-
sponding mutant the ability to produce the poly-
mer as a response to environmental signals.37
5 BIOSYNTHESIS OF ALGINATE
The biosynthesis of alginate was first studied by
Lin and Hassid,38 who in a cell-free system from
Fucus gurdnerii detected the enzyme activities
necessary for the synthesis of mannuronan. A simi-
lar pathway has since been found in A. vinelundii39
and P. ueruginosdlo (Fig. 2). Starting with D-fructose-
6-phosphate, the sugar nucleotide GDP-D-mannose
is made and further oxidized to GDP-D-mannuronic
acid. This compound is then polymerized to
mannuronan. The polymer can be further modified
by an acetyl transferase and by a mannuronan
C-5-epimerase, which epimerizes mannuronic acid
residues to guluronic acid.
The genes involved in the biosynthesis of algi-
nates were first identified in P. ueruginosa. Later,
several of the corresponding genes were identified
in A. vinelundii, where they are organized in the
same manner. 41 The two genes ulgA42-43 and
ulgD,44,45 encoding the bifunctional enzyme phos-
phomannoisomerase/D-mannose- 1-phosphate gua-
nylyl transferase and guanosine diphosphate
mannose dehydrogenase, respectively, are flanking
a gene cluster (Fig. 3) encoding all the known bio-
FGG
synthetic genes except aZgC.40,46The amino acid
0.11 sequence of the membrane protein A1g847,48shows
0.43 similarity to some processive glycosyl-transferases,49
0.091
and it is probable that A1g4447,48and AlgX (also
88 H. Ertesvdg, S. Valla

D-fructose-6-phosphate mutated in mucA or mucB.37 Mutations in mucD

1 L also seem to affect the alginate biosynthesis.60 In
P. aeruginosa it has been found that AlgU is also
D-mannose-6-phosphate
necessary for transcription of AlgR, AlgZ and
2 1 AlgB,6,62 transcription-factors which bind to the
D-mannose-l -phosphate
algD-promoter. AlgR and AlgZ are necessary for
the transcription of algD, while AlgB is necessary
3 \1 for high-level-production of alginate. Several other
GDP-D-mannose genes have also been shown to have an effect on
alginate production in P. aeruginosa.40~60~63-66
4 1 The genetics of alginate biosynthesis in A. vine-
GDP-D-mannuronic acid landii have not been studied to the same extent, but
as this bacterium produces alginate constitutively,
5 L
some differences must exist. It has been shown that
mannuronan (ManA),, the expression of algL and algA, which are in the
6 L
downstream part of the alginate biosynthesis clus-
ter, are dependent on AlgU in P. aeruginosa, but
mannuronan-OAc not in A. vinelandii.43 Recently it has been reported
7 L that some strains of Pseudomonas syringae produce
alginate in response to copper.67
alginate
(-MGMMMGMGGGGMGMGGGM-)

Fig. 2. The pathway of alginate biosynthesis in bacteria. 1: 6 EPIMERIZATION, THE KEY TO

Phosphomannoisomerase, 2: phosphomannomutase, 3: D-
mannose-l-phosphate guanylyl transferase, 4: guanosine
diphosphate mannose dehydrogenase, 5: glucosyl transferase;
n designates the number of uranic acid residues in the polymer
chain, 6: acetyl transferase, 7: mannuronan C-Sepimerase.
designated Alg60) participate in the inner-membrane
polymerization complex. 4o It has recently been
suggested that the periplasmic protein AlgK552
facilitates the transport of the alginate to the outer-
membrane pore formed by AlgE53 (AlgJ54 in A.
vinelandii). The epimerase (AlgG),4,55 lyase
(AlgL)43,56 and acetylase (AlgF)57 are all peri-
plasmic enzymes that modify alginate, but are
dispensable for alginate production. AlgI and
AlgJ58 have recently been shown to be necessary
for acetylation.
In both P. aeruginosa and A. vinelandii tran-
scription of algD is dependent on the a-factor
AlgU.37,59 afgU is the first gene in the algU-
mucABCD-operon,59,60 and its synthesis is auto-
regulated. MucA is an antisigma-factor for AlgU,
and mucoid strains of P. aeruginosa are often
ALGINATE STRUCTURE
The most unique feature of alginate biosynthesis is
that the synthesis of this heteropolymer involves
only one nucleotide-sugar (GDP-M), while the G-
residues are introduced by epimerization at the
polymer level. The first evidence of this mechanism
was published by Hellebust and Haug68 in 1969.
They studied the photoassimilation of r4C03- into
alginate by the algae Laminaria digitata, and
observed that in the presence of light the radio-
activity was mainly incorporated into M-blocks,
while the amount of label in MG- and G-blocks
increased during the night. A possible explanation
was found 2years later, when an enzyme-activity
which was able to epimerize mannuronic acid resi-
dues on the polymer chain to guluronic acid resi-
dues was demonstrated in the culture-medium of
A. vinelandii.69 Later mannuronan C-5-epimerases
have been found both in P. aeruginosa55 and in
several species of brown algae.70,71 By controlling
the action of the C-Sepimerases, the organism can

I Iwo* \ 1
0000 5000

~r118.~~%?z?~SsleA.

Pig. 3. The alginate biosynthesis gene cluster in P. ueruginosa. Filled arrows show genes where the complementary genes have been
found in the corresponding order in A. vinelandii.The functions of the genes are described in the text.
 Biosynthesis and applications of alginates 89

determine the content and distribution of G and thus Dllll

most of the properties of the alginate produced. AlgE4
The periplasmic AlgG from P. aeruginosa is
probably unable to make G-blocks, as that
block-type has never been found in alginates
from this species. A mutant with a defective algG AIgEl
has been shown to produce mannuronan.* Thus
the P. aeruginosu genome probably encodes only
one epimerase.
AlgE2
The secreted epimerase from A. vinelundii has
been shown to need calcium for activity,(j9 whereas
AlgG from A. vinelundii is independent of this
cation.41 A calcium-dependent epimerase has been AlgE3
purified and was shown to have the capacity to
incorporate G into both MG- and G-blocks.73
During the experiments aimed at cloning the gene
for the secreted epimerase from A. vinelundii, it was AlgE5
found that the bacterium encodes a family of at
least five calcium-dependent epimerases.74,75 Five Fig. 4. The modular structure of the AlgE-epimerases from A.
of these (AlgEl-5) have been cloned, sequenced vinelandii. A- and R-groups with the same hatchings belong
and expressed in Escherichiu coli. All the enzymes to the same subgroups defined by the construction of evolu-
tionary trees. The arrows indicate the locations of the putative
have been shown to be functional epimerases. The Ca*+-binding motifs. Open arrows indicate sequences that
sequences of ulgG and ulgEl-5 show little similar- deviate from the consensus sequence LXGGAGXDX. The
ity, but when the deduced sequences of the AlgE- symbol at the end of some of the R-modules indicates the
epimerases were compared they revealed a strong presence of short amino acid sequences (linkers). S refers to
the terminal 19 amino acids. These do not belong to the A- or
homology. All of them can be viewed as composed R-modules.
of one or two A-modules, being about 485 amino
acids long, and l-7 copies of a 153 amino acid-long Table 4. Epimerization of al&ate with a low (5%) content of G
module designated R (Fig. 4). The R-modules each with AIgE2 and AlgE4
contain 4-6 copies of a nine amino acid motif,
Enzyme Fo FM Distribution of diuronides
which has been identified in many secreted pro-
teins. In an alkaline phosphatase from P. uerugi- FGG FMM FMG,GM
nosu this motif has been shown to bind Ca*+ .76 AlgE274 0.40 0.60 0.34 0.54 0.06
The current hypothesis is that the A-modules are AlgE475 0.45 0.55 0.05 0.20 0.35
responsible for binding of alginate and for epimer-
ization, while the R-modules are involved in Ca* + -
binding and secretion. However, more experiments activity of the two enzymes and thus the structure of
are needed to confirm this hypothesis. the epimerized alginate. At low calcium levels the
The biological function of all these epimerases is relative amount of G-blocks will be higher, and the
not yet understood. However, comparisons of algi- polymer will thus form a stronger gel in spite of the
nate epimerized by AlgE2 and by AlgE4 gave valu- low calcium concentration. Still, this does not
able information. The experiments were performed explain why the genome encodes at least five AlgE-
by epimerizing alginate having a low content of G- epimerases. Since the bacterium produces different
residues (about 5%) with partially purified AlgE2 alginates at different stages of its life-cycle, it is
and AlgE4. The results (Table 4) show a clear dif- tempting to speculate that it regulates the degree of
ference: while AlgE2 mainly introduces G as G- epimerization by expressing different epimerases at
blocks, AlgE4 makes MG-blocks. In a paper by different times. It may also be possible that the other
Ofstad and Larsen77 it was shown that A. vinelundii epimerases make different distributions of MG- and
seemed to produce at least two epimerases, and that G-blocks or different block-lengths than AlgE2 and
the G-block-forming epimerase needed less calcium AlgE4. The function of the periplasmic epimerase
for activity than the MG-block-forming epimerase. (AlgG) in A. vinelundii is also not yet clear. Thus, a
Thus, given a mixture of AlgE2 and AlgE4 the lot of work remains before the epimerization pro-
Ca*+-concentration will determine the relative cess in this bacterium is fully understood.
90 H. Ertesvdg, S. Valla
7 FUTURE PROSPECTS
In most of the industrial uses of alginate, the
properties which can be obtained by extracting
alginate from different algae or parts of algae
are satisfactory. But when it comes to encapsu-
lation of cells, especially for implantation, the
requirements for gel strength and homogeneity are
not so easily met. Much can be done by carefully
chasing of alginates and fractionation. An alter-
native to fractionation would be to epimerize
algal alginates with an initially low content of
G and G-blocks with recombinant enzymes.
One might use AlgE2 to obtain G-blocks, or
AlgE4 to convert M-blocks to MG-blocks. By
using a mixture of the two it should be possible to
obtain an alginate with the desired properties with
respect to gel-strength, pore-size and immunogen-
icity. Such an epimerized alginate would also be
more homogenous than that usually obtained from
natural sources.
An alternative to the use of algal alginate is to
produce the polymer in bacteria. Earlier attempts
to do this have mostly been hampered by the
presence of an alginate lyase in both P. aeruginosa
and A. vinelandii. Even though mucoid strains of
P. aeruginosa usually cease to produce alginate
when cultured in the laboratory, stable alginate-
producing mutants have been isolated.78 And as a
gene for alginate lyase now has been identified in
both species, it should be possible to inactivate it
and thus construct strains that are more suitable
for industrial production of alginate.
It may also be possible to tailor the polymer in
vivo by expressing one or more of the recombi-
nant AlgE-epimerases in an alginate-producing
host. It would probably be an advantage if this
host produced alginates with a very low G-content.
The algG-mutant of P. aeruginosa meets this
requirement, but it may not be convenient to use
an opportunistic human pathogen for fermen-
tation. The problem with A. vinelundii is that it
has several epimerase genes, all of which prob-
ably have to be inactivated in order to produce
mannuronan.
When the structure-function relationships of the
different epimerases are more fully understood, it
may be possible to create new epimerases with
desired technical properties by site-specific muta-
genesis or by exchanging modules or parts of
modules between different epimerase genes. These
could then be utilized to epimerize alginate in vitro
or in vivo.
REFERENCES
1. Otterlei,M., Ostgaard,K., Skjbk-Braek, G., Smidsred, O.,
Soon-Shiong, P. and Espevik, T., .I. Immunother., 1991,
10, 286.
2. Stokke, B. T., Smidsrerd, O., Bruheim, P. and Skjlk-Braek,
G., Macromolecules, 1991, 24, 4645.
3. Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J. C.
and Thorn, D., FEBS Lett., 1973,32, 195.
4. Draget, K. I., Skjbk-Brrek, G., Christensen, B. E.,
GBsersd, 0. and Smidsrsd, O., Carbohydr. Polym., 1996,
29, 209.
5. Grasdalen, H., Larsen, B. and Smidsrnd, 0.. Carbohydr.
Res., 1981, 89, 179.
6. Grasdalen, H., Carbohydr. Res., 1983, 118, 255.
7. Onserien, E., Carbohydr. Europe, 1996, 14, 26.
8. Skjik-Braek, G. and Martinsen, A. In Seaweed Resources
in Europe: Uses and Potential, ed. M. D. Guiry and G.
Blunden, 1991, p. 219.
9. Martinsen, A., Skjlk-Brrek, G. and Smidsrerd, O., Bio-
technol. Bioengng, 1989, 33, 79.
10. Thu, B., Bruheim, P., Espevik, T., Smidsrsd, O., Soon-
Shiong, P. and Skjik-Braek, G., Biomaterials, 1996, 17,
1031.
11. Thu, B., Bruheim, P., Espevik, T., Smidsrerd, O., Soon-
Shiong, P. and Skjik-Brrek, G., Biomaterials, 1996, 17,
1069.
12. Elchin, Y. M., Biomaterials, 1995, 16, 1157.
13. Roig, M. G., Rashid, D. H. and Kennedy, J. F., Appl.
Biochem. Biotechnol., 1995, 55, 95.
14. Chung, Y. C., Huang, C. and Tseng, C. P., J. Environ. Sci.
Hlth Part A: Environ. Sci. Engng Toxic Hazar. Subst.
Control, 1996, 31, 1263.
15. Cheetham, P. S. J., Garretth, C. and Clark, J., Biotechnol.
Bioengng, 1985,27,471.
16. Musgrave, S. C., Kerby, N. W., Codd, G. A. and Stewart,
W. D. P., Biotechnol. Lett., 1982, 4, 647.
17. Eikmeier, H. and Rehm, H. J., Naturforsh. Sect. C.
Biosci., 1987, 42, 408.
18. Oda, G., Sameyiuna, H. and Yameda, T. In Proceedings
Biotechnology, London, 1983, p. 597.
19. Hirata, K., Phunchindawan, M., Tukamoto, J., Goda, S.
and Miyamoto, K., Cryo. Lett., 1996, 17, 321.
20. Bailliez, C., Largeau, C. and Casadevall, E., Appl. Micro-
biol. Biotechnol., 1985,23, 99.
21. Timbert, R., Barbotin, J. N. and Thomas, D.. Plant Sci.,
1996, 120,215.
22. Alfermann, A. W., Schuller, I. and Reinhardt, E., Planta
Medica, 1980, 40, 218.
23. Maysinger, D., Berezovskaya, 0. and Feodoroff, S., Exp.
Neurol., 1996, 141, 47.
24. Duff, R. G., TIBTECH, 1983,3, 167.
25. Soon-Shiong, P., Feldman, E., Nelson, R., Heintz, R.,
Yao, Q., Yao, Y., Zheng, T., Merideth, N., Skjik-Braek,
G., Espevik, T., Smidsrcad, 0. and Sandford, P., Proc.
Nat1 Acad. Sci. USA, 1993,90, 5843.
26. Soon-Shiong, P., Heintz, R. E., Merideth, N., Yao, Q. X.,
Yao, Z., Zheng, T., Murphy, M., Moloney, M. K.,
Schmehl, M., Harris, M., Mendez, R., Mendez, R. and
Sandford, P. A., Lancet, 1994, 343, 950.
27. Kulseng, B., Thu, B., Espevik, T. and SkjHk-Brrek, G.,
Cell. Transplant., 1997, 6, in press.
28. Skjlk-Brrek, G. and Espevik, T., Carbohyd. Europe, 1996,
14, 19.
29. Gorin, P. A. J. and Spencer, J. F. T., Can. J. Chem., 1966,
44,993.
30. Cote, G. L. and Krull, L. H., Carbohydr. Res., 1988, 181,
143.
 Biosynthesis and applications
31. Linker, A. and Jones, R. S., Nature, 1964, 204, 187.
32. Govan, J. R. W., Fyfe, J. A. M. and Jarman, T. R., J.
Gen. Microbial., 1981, 125, 217.
33. Fett, W. F., Cescutti, P. and Wijey, C., J. Appl. Bacterial.,
1996, 81, 181.
34. Skjak-Brmk, G., Grasdalen, H. and Larsen, B., Carbo-
hydr. Res., 1986, 154, 239.
35. Sadoff, H. L., Bacterial. Rev., 1975, 39, 516.
36. Page, W. J. and Sadoff, H. L., J. Bacterial., 1975, 122,
145.
37. Deretic, V., Schurr, M. J., Boucher, J. C. and Martin,
D. W., J. Bacterial., 1994, 176, 2773.
38. Lin, T. Y. and Hassid, W. Z., J. Biol. Chem., 1966, 241,
5284.
39. Pindar, D. E. and Bucke, C., Biochem. J., 1975, 152, 617.
40. May, T. B. and Chakrabarthy, A. M., Trends Microbial.,
1994, 2, 151.
41. Rehm, B. H., Ertesvag, H. and Valla, S., J. Bacterial.,
1996, 178, 5884.
42. Shinabarger, D., Berry, A., May, T. B., Rothmel, R.,
Fialho, A. and Chakrabarthy, A. M., J. Biol. Chem., 1991,
266, 2080.
43. Lloret, L., Barreto, R., Leon, R., Moreno, S., Martinez-
Salazar, J., Espin, G. and Soberon-Chavez, G., Mol.
Microbial., 1996, 21, 449.
44. Deretic, V., Gill, J. F. and Chakrabarty, A. M., Nucf.
Acids Res., 1987, 15,4567.
45. Campos, M.-E., Martinez-Salazar, J. M., Lloret, L.,
Moreno, S., Nuiiez, C., Espin, G. and Soberon-Chavez,
G., J. Bacterial., 1996, 178, 1793.
46. Zielinski, N. A., Chakrabarty, A. M. and Berry, A., J.
Biof. Chem., 1991,266,9754.
47. Maharaj, R., May, T. B., Wang, S.-K. and Chakrabarty,
A. M., Gene, 1993,136, 267.
48. Mejia-Ruiz, H., Moreno, S., Guzman, J., Najera, R. and
Soberon-Chavez, G., GenBank accession no. YO8819.
49. Saxena, I. M., Brown, R. M. Jr., Fevre, M., Geremia,
R. A. and Henrissat, B., J. Bacterial., 1995, 177, 1419.
50. Monday, S. R. and Schiller, N. L., J. Bacterial., 1996, 178,
625.
51. Aarons, S. J., Sutherland, I. W., Chakrabarty, A. M. and
Gallagher, M. P.. Microbiology, 1997, 143, 641.
52. Mejia-Ruiz, H., GenBank accession no. X98863.
53. Chu, L., May, T. B., Chakrabarty, A. M. and Misra,
T. K., Gene (1991) 1.
54. Rehm, B. H. A., Microbiology, 1996, 142, 873.
55. Franklin, M. J., Chitnis, C. E., Gacesa, P., Sonesson, A.,
White, D. C. and Ohman, D. E., J. Bacterial., 1994, 1821.
of alginates 91
56. Schiller, N. L., Monday, S. R., Boyd, C. M., Keen, N. T.
and Ohman, D. E., J. Bacterial., 1993, 175,478O.
57. Franklin, M. J. and Ohman, D. E., J. Bacterial., 1993,
175, 5057.
58. Franklin, M. J. and Ohman, D. E., J. Bacterial., 1996,
178, 2186.
59. Martinez-Salazar, J. M., Moreno, S., Najera, R., Boucher,
J. C., Espin, G., Sober&t-Chavez, G. and Deretic, V., J.
Bacterial., 1996, 178, 1800.
60. Boucher, J. C., Martinez-Salazar, J., Schurr, M. J., Mudd,
M. H., Yu, H. and Deretic, V., J. Bacterial., 1996, 178,
511.
61. Wozniak, D. J. and Ohman, D. E.. J. Batteriol., 1994,
176,6007.
62. Baynham, P. J. and Wozniak, D. J., Mol. Microbial.,
1996, 22, 97.
63. Delic-Attree, I., Touissant, B., Froger, A., Willison, J. C.
and Vignais, P. M., Microbiology, 1996, 142, 2785.
64. Hasset, D. J., Howell, M. L., Sokol, P. A., Vasil, M. L.
and Dean, G. E., J. Bacterial., 1997, 179, 1442.
65. Schlichtman, D., Kubo, M., Shankar, S. and Chakra-
barty, A. M., J. Bacterial., 1995, 177, 2469.
66. Yu, H., Mudd, M., Boucher, J. C., Schurr, M. J. and
Deretic, V., J. Bacterial., 1997, 179, 187.
67. Kidambi, S. P., Sundin, G. W., Palmer, D. A., Chakra-
barty, A. M. and Bender, C. L., Appl. Environ. Microbial.,
1995,61, 2172.
68. Hellebust, J. A. and Haug, A. In Proceedings of the 6th
International Seaweed Symposium, 1969, p. 463.
69. Larsen, B. and Haug, A., Carbohydr. Res., 1971, 20, 225.
70. Magdwick, J. C., Haug, A. and Larsen, B., Acta Chem.
&and., 1973, 27, 3592.
71. Ishikawa, M. and Nisizawa, K., Bull. Jpn. Sot. Sci. Fish.,
1981, 47, 889.
72. Chitnis, C. E. and Ohman, D. E., J. Bacterial., 1990, 172,
2894.
73. Skjbk-Brrek, G. and Larsen, B., Carbohydr. Res., 1985,
139, 273.
74. ErtesvPg, H., Doseth, B., Larsen, B., Skjak-Brick, G. and
Valla, S., J. Bacterial., 1994, 176, 2846.
75. Ertesvag, H., Hoidal, H. K., Hals, I. K., Rian, A., Doseth,
B. and Valla, S., Mol. Microbial., 1995, 16, 7 19.
76. Baumann, U., Wu, S., Flaherty, K. M. and McKay, D. B.,
EMBO J., 1993, 12, 3357.
77. Ofstad, R. and Larsen, B. In Proceedings of the 10th
International Seaweed Symposium, 1980, p. 485.
78. Darzins, A. and Chakrabarty, A. M., J. Bacterial., 1984,
159, 9.