Escolar Documentos
Profissional Documentos
Cultura Documentos
1 STRUCTURE OF ALGINATE blocks which form the junctions, but the stability of
the gel is mostly dependent on the content of G-
Alginate is a linear copolymer composed of l-4 blocks.4 By analysing the polymer by NMR it is now
linked /3-D-mannuronic acid (M) and its C-5-epimer possible to determine the frequencies of each mono-
a-L-guluronic acid (Fig. 1). These monomers can be mer, each of the four possible dimers, and each of
organized in blocks of consecutive G-residues (G- the eight possible trimers.5,6 However, it is not pos-
blocks), consecutive M-residues (M-blocks), or sible to estimate the distribution of block-lengths for
alternating M and G (MG-blocks). The relative any alginate containing more than one type of block.
amount of each block-type varies between different
alginates. As shown in Fig. 1, there are distinct
structural differences between the block-types. MG- 2 TRADITIONAL INDUSTRIAL USES OF
blocks form the most flexible chains and are more ALGINATE
soluble at lower pH than the other two block-types.
M-blocks have been found to be strongly immunos- Alginates for commercial uses are extracted from
timulating. G-blocks form stiff chains, and two G- brown algae, and the annual production is about
blocks of more than 6 residues each can be cross- 30 000 tons.7 The composition of the poly-
linked by divalent cations (e.g. Ca2+, Ba2+, Sr2+), saccharide depends on the species used and also
leading to gel formation. 2,3 At low pH, protonized whether the blade and stipe are separated before
high-molecular-mass alginates can form soft acidic extraction (Table 1). For most applications the
gels. In these gels, it is mostly the homopolymeric polymer is sold for US$S-20/kg, but the highest
purity qualities are sold for up to US$40 OOO/kg.
*To whom correspondence should be addressed. Fax: 47 Alginates have various industrial uses as viscosi-
73598705; e-mail: helgae@unigen.ntnu.no fiers, stabilizers and gel-forming, film-forming or
85
86 H. ErtesvGg, S. Valla
a) b)
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
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
In most of the industrial uses of alginate, the 1. Otterlei,M., Ostgaard,K., Skjbk-Braek, G., Smidsred, O.,
properties which can be obtained by extracting Soon-Shiong, P. and Espevik, T., .I. Immunother., 1991,
10, 286.
alginate from different algae or parts of algae 2. Stokke, B. T., Smidsrerd, O., Bruheim, P. and Skjlk-Braek,
are satisfactory. But when it comes to encapsu- G., Macromolecules, 1991, 24, 4645.
lation of cells, especially for implantation, the 3. Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J. C.
requirements for gel strength and homogeneity are and Thorn, D., FEBS Lett., 1973,32, 195.
4. Draget, K. I., Skjbk-Brrek, G., Christensen, B. E.,
not so easily met. Much can be done by carefully GBsersd, 0. and Smidsrsd, O., Carbohydr. Polym., 1996,
chasing of alginates and fractionation. An alter- 29, 209.
native to fractionation would be to epimerize 5. Grasdalen, H., Larsen, B. and Smidsrnd, 0.. Carbohydr.
Res., 1981, 89, 179.
algal alginates with an initially low content of 6. Grasdalen, H., Carbohydr. Res., 1983, 118, 255.
G and G-blocks with recombinant enzymes. 7. Onserien, E., Carbohydr. Europe, 1996, 14, 26.
One might use AlgE2 to obtain G-blocks, or 8. Skjik-Braek, G. and Martinsen, A. In Seaweed Resources
in Europe: Uses and Potential, ed. M. D. Guiry and G.
AlgE4 to convert M-blocks to MG-blocks. By Blunden, 1991, p. 219.
using a mixture of the two it should be possible to 9. Martinsen, A., Skjlk-Brrek, G. and Smidsrerd, O., Bio-
obtain an alginate with the desired properties with technol. Bioengng, 1989, 33, 79.
10. Thu, B., Bruheim, P., Espevik, T., Smidsrsd, O., Soon-
respect to gel-strength, pore-size and immunogen- Shiong, P. and Skjik-Braek, G., Biomaterials, 1996, 17,
icity. Such an epimerized alginate would also be 1031.
more homogenous than that usually obtained from 11. Thu, B., Bruheim, P., Espevik, T., Smidsrerd, O., Soon-
natural sources. Shiong, P. and Skjik-Brrek, G., Biomaterials, 1996, 17,
1069.
An alternative to the use of algal alginate is to 12. Elchin, Y. M., Biomaterials, 1995, 16, 1157.
produce the polymer in bacteria. Earlier attempts 13. Roig, M. G., Rashid, D. H. and Kennedy, J. F., Appl.
to do this have mostly been hampered by the Biochem. Biotechnol., 1995, 55, 95.
14. Chung, Y. C., Huang, C. and Tseng, C. P., J. Environ. Sci.
presence of an alginate lyase in both P. aeruginosa Hlth Part A: Environ. Sci. Engng Toxic Hazar. Subst.
and A. vinelandii. Even though mucoid strains of Control, 1996, 31, 1263.
P. aeruginosa usually cease to produce alginate 15. Cheetham, P. S. J., Garretth, C. and Clark, J., Biotechnol.
Bioengng, 1985,27,471.
when cultured in the laboratory, stable alginate- 16. Musgrave, S. C., Kerby, N. W., Codd, G. A. and Stewart,
producing mutants have been isolated.78 And as a W. D. P., Biotechnol. Lett., 1982, 4, 647.
gene for alginate lyase now has been identified in 17. Eikmeier, H. and Rehm, H. J., Naturforsh. Sect. C.
Biosci., 1987, 42, 408.
both species, it should be possible to inactivate it
18. Oda, G., Sameyiuna, H. and Yameda, T. In Proceedings
and thus construct strains that are more suitable Biotechnology, London, 1983, p. 597.
for industrial production of alginate. 19. Hirata, K., Phunchindawan, M., Tukamoto, J., Goda, S.
It may also be possible to tailor the polymer in and Miyamoto, K., Cryo. Lett., 1996, 17, 321.
20. Bailliez, C., Largeau, C. and Casadevall, E., Appl. Micro-
vivo by expressing one or more of the recombi- biol. Biotechnol., 1985,23, 99.
nant AlgE-epimerases in an alginate-producing 21. Timbert, R., Barbotin, J. N. and Thomas, D.. Plant Sci.,
host. It would probably be an advantage if this 1996, 120,215.
22. Alfermann, A. W., Schuller, I. and Reinhardt, E., Planta
host produced alginates with a very low G-content. Medica, 1980, 40, 218.
The algG-mutant of P. aeruginosa meets this 23. Maysinger, D., Berezovskaya, 0. and Feodoroff, S., Exp.
requirement, but it may not be convenient to use Neurol., 1996, 141, 47.
24. Duff, R. G., TIBTECH, 1983,3, 167.
an opportunistic human pathogen for fermen-
25. Soon-Shiong, P., Feldman, E., Nelson, R., Heintz, R.,
tation. The problem with A. vinelundii is that it Yao, Q., Yao, Y., Zheng, T., Merideth, N., Skjik-Braek,
has several epimerase genes, all of which prob- G., Espevik, T., Smidsrcad, 0. and Sandford, P., Proc.
ably have to be inactivated in order to produce Nat1 Acad. Sci. USA, 1993,90, 5843.
26. Soon-Shiong, P., Heintz, R. E., Merideth, N., Yao, Q. X.,
mannuronan. Yao, Z., Zheng, T., Murphy, M., Moloney, M. K.,
When the structure-function relationships of the Schmehl, M., Harris, M., Mendez, R., Mendez, R. and
different epimerases are more fully understood, it Sandford, P. A., Lancet, 1994, 343, 950.
27. Kulseng, B., Thu, B., Espevik, T. and SkjHk-Brrek, G.,
may be possible to create new epimerases with Cell. Transplant., 1997, 6, in press.
desired technical properties by site-specific muta- 28. Skjlk-Brrek, G. and Espevik, T., Carbohyd. Europe, 1996,
genesis or by exchanging modules or parts of 14, 19.
29. Gorin, P. A. J. and Spencer, J. F. T., Can. J. Chem., 1966,
modules between different epimerase genes. These
44,993.
could then be utilized to epimerize alginate in vitro 30. Cote, G. L. and Krull, L. H., Carbohydr. Res., 1988, 181,
or in vivo. 143.
Biosynthesis and applications of alginates 91
31. Linker, A. and Jones, R. S., Nature, 1964, 204, 187. 56. Schiller, N. L., Monday, S. R., Boyd, C. M., Keen, N. T.
32. Govan, J. R. W., Fyfe, J. A. M. and Jarman, T. R., J. and Ohman, D. E., J. Bacterial., 1993, 175,478O.
Gen. Microbial., 1981, 125, 217. 57. Franklin, M. J. and Ohman, D. E., J. Bacterial., 1993,
33. Fett, W. F., Cescutti, P. and Wijey, C., J. Appl. Bacterial., 175, 5057.
1996, 81, 181. 58. Franklin, M. J. and Ohman, D. E., J. Bacterial., 1996,
34. Skjak-Brmk, G., Grasdalen, H. and Larsen, B., Carbo- 178, 2186.
hydr. Res., 1986, 154, 239. 59. Martinez-Salazar, J. M., Moreno, S., Najera, R., Boucher,
35. Sadoff, H. L., Bacterial. Rev., 1975, 39, 516. J. C., Espin, G., Sober&t-Chavez, G. and Deretic, V., J.
36. Page, W. J. and Sadoff, H. L., J. Bacterial., 1975, 122, Bacterial., 1996, 178, 1800.
145. 60. Boucher, J. C., Martinez-Salazar, J., Schurr, M. J., Mudd,
37. Deretic, V., Schurr, M. J., Boucher, J. C. and Martin, M. H., Yu, H. and Deretic, V., J. Bacterial., 1996, 178,
D. W., J. Bacterial., 1994, 176, 2773. 511.
38. Lin, T. Y. and Hassid, W. Z., J. Biol. Chem., 1966, 241, 61. Wozniak, D. J. and Ohman, D. E.. J. Batteriol., 1994,
5284. 176,6007.
39. Pindar, D. E. and Bucke, C., Biochem. J., 1975, 152, 617. 62. Baynham, P. J. and Wozniak, D. J., Mol. Microbial.,
40. May, T. B. and Chakrabarthy, A. M., Trends Microbial., 1996, 22, 97.
1994, 2, 151. 63. Delic-Attree, I., Touissant, B., Froger, A., Willison, J. C.
41. Rehm, B. H., Ertesvag, H. and Valla, S., J. Bacterial., and Vignais, P. M., Microbiology, 1996, 142, 2785.
1996, 178, 5884. 64. Hasset, D. J., Howell, M. L., Sokol, P. A., Vasil, M. L.
42. Shinabarger, D., Berry, A., May, T. B., Rothmel, R., and Dean, G. E., J. Bacterial., 1997, 179, 1442.
Fialho, A. and Chakrabarthy, A. M., J. Biol. Chem., 1991, 65. Schlichtman, D., Kubo, M., Shankar, S. and Chakra-
266, 2080. barty, A. M., J. Bacterial., 1995, 177, 2469.
43. Lloret, L., Barreto, R., Leon, R., Moreno, S., Martinez- 66. Yu, H., Mudd, M., Boucher, J. C., Schurr, M. J. and
Salazar, J., Espin, G. and Soberon-Chavez, G., Mol. Deretic, V., J. Bacterial., 1997, 179, 187.
Microbial., 1996, 21, 449. 67. Kidambi, S. P., Sundin, G. W., Palmer, D. A., Chakra-
44. Deretic, V., Gill, J. F. and Chakrabarty, A. M., Nucf. barty, A. M. and Bender, C. L., Appl. Environ. Microbial.,
Acids Res., 1987, 15,4567. 1995,61, 2172.
45. Campos, M.-E., Martinez-Salazar, J. M., Lloret, L., 68. Hellebust, J. A. and Haug, A. In Proceedings of the 6th
Moreno, S., Nuiiez, C., Espin, G. and Soberon-Chavez, International Seaweed Symposium, 1969, p. 463.
G., J. Bacterial., 1996, 178, 1793. 69. Larsen, B. and Haug, A., Carbohydr. Res., 1971, 20, 225.
46. Zielinski, N. A., Chakrabarty, A. M. and Berry, A., J. 70. Magdwick, J. C., Haug, A. and Larsen, B., Acta Chem.
Biof. Chem., 1991,266,9754. &and., 1973, 27, 3592.
47. Maharaj, R., May, T. B., Wang, S.-K. and Chakrabarty, 71. Ishikawa, M. and Nisizawa, K., Bull. Jpn. Sot. Sci. Fish.,
A. M., Gene, 1993,136, 267. 1981, 47, 889.
48. Mejia-Ruiz, H., Moreno, S., Guzman, J., Najera, R. and 72. Chitnis, C. E. and Ohman, D. E., J. Bacterial., 1990, 172,
Soberon-Chavez, G., GenBank accession no. YO8819. 2894.
49. Saxena, I. M., Brown, R. M. Jr., Fevre, M., Geremia, 73. Skjbk-Brrek, G. and Larsen, B., Carbohydr. Res., 1985,
R. A. and Henrissat, B., J. Bacterial., 1995, 177, 1419. 139, 273.
50. Monday, S. R. and Schiller, N. L., J. Bacterial., 1996, 178, 74. ErtesvPg, H., Doseth, B., Larsen, B., Skjak-Brick, G. and
625. Valla, S., J. Bacterial., 1994, 176, 2846.
51. Aarons, S. J., Sutherland, I. W., Chakrabarty, A. M. and 75. Ertesvag, H., Hoidal, H. K., Hals, I. K., Rian, A., Doseth,
Gallagher, M. P.. Microbiology, 1997, 143, 641. B. and Valla, S., Mol. Microbial., 1995, 16, 7 19.
52. Mejia-Ruiz, H., GenBank accession no. X98863. 76. Baumann, U., Wu, S., Flaherty, K. M. and McKay, D. B.,
53. Chu, L., May, T. B., Chakrabarty, A. M. and Misra, EMBO J., 1993, 12, 3357.
T. K., Gene (1991) 1. 77. Ofstad, R. and Larsen, B. In Proceedings of the 10th
54. Rehm, B. H. A., Microbiology, 1996, 142, 873. International Seaweed Symposium, 1980, p. 485.
55. Franklin, M. J., Chitnis, C. E., Gacesa, P., Sonesson, A., 78. Darzins, A. and Chakrabarty, A. M., J. Bacterial., 1984,
White, D. C. and Ohman, D. E., J. Bacterial., 1994, 1821. 159, 9.