Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1995-05-09
1999-08-17
Carlson, Karen Cochrane
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 697, 4353201, 435831, 536 232, 536 234, 536 237, C12P 1900, C12P 2104, C12N 100, C07H 2104
Patent
active
059392894
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns DNA compounds encompassing sequences coding for enzymes having mannuronan C-5-epimerase activity, a process for the preparation of such enzymes, the use of said genetic sequences in production of alginates having a definite G/M ratio and block structure, and the production of alginates having a definite G/M ratio by inactivating said genetic sequences.
Throughout this application, reference is made to publications from the scientific and patent literature. Publications so cited are hereby incorporated in their entirety by reference.
In this application the term gene is used to indicate a genetic sequence which encodes a protein, independent of whether the protein encoded by this genetic sequence is expressed or not in the natural host organism under those conditions.
Alginates are a family of polysaccharides, which are synthesized in brown algae as well as in bacteria, such as Azotobacter vinelandii and Azotobacter chroococcum. Alginates are also synthesized by some strains of Pseudomonas sp.
Chemically, alginates are unbranched binary copolymers of 1-4 linked .beta.-D-mannuronic acid, termed M, and its C-5 epimer .alpha.-L-guluronic acid, termed G.
Alginates derived from seaweeds and Azotobacter are generally true block copolymers where the monomers are arranged in homopolymeric stretches of M, termed M blocks, and homopolymeric stretches of G, termed G blocks, interspaced with regions containing both monomers, normally termed alternating blocks or MG blocks. The composition and sequential structure of alginates vary widely depending on the source. Alginates produced by Pseudomonas, however, do not have any G blocks.
Several functional properties such as the capacity to form gels and the binding of water depend on the M/G ratio and on the length of the various blocks. A relatively high content of G blocks, for instance gives good gelling properties, due to ionic cross linking of chains which takes place when Ca.sup.2+ -ions are added to an alginate solution. The composition and block structure also influence on the immunological properties of shown that alginates with a high content of mannuronic acid blocks are very potent nontoxic immunostimulants.
At present industrial production of alginates rely exclusively on algal sources. The range in composition is however limited as the highest content of guluronic acid to be found is 75% and the lowest 25%. Furthermore there are no suitable sources for alginate with a G content in the range of 42-54%. In the field of biotechnology or biomedicine, polymers with extreme compositions, such as a high G for immobilization of O., Biotechnol. Bioeng. 33, 79-86, (1989)! and a high M (90-100%) as are of major interest.
The key enzyme responsible for generation of the G blocks is called mannuronan C-5-epimerase. It was previously thought that only one enzyme having a certain amino acid sequence would exhibit this activity. It has now surprisingly been found that there exist at least five genes encoding enzymes having this activity. Some of these enzymes differ in molecular weight and amino acid sequence. The genes were found adjacent to each other in the bacterium Azotobacter vinelandii. It has also been found that the amino acid sequence of the enzyme affects the activity of the enzyme, not only in terms of potency but also in the type of alginate formed, for example, altering the content of guluronic acid and the single/block G content of the alginate. 297-308! the isolation of mannuronan C-5-epimerase from liquid cultures of Azotobacter vinelandii is reported. In the following, this epimerase will be termed mannuronan C-5-epimerase (2), and the DNA sequence encoding for it will correspondingly be denominated E2. (1982)!, the purification of mannuronan C-5-epimerase (2) by affinity chromatography on alginate sepharose is disclosed. In a separate paper (1985) 273-283! the characterization of this enzyme is disclosed. Further, the activity of the enzyme is described as an ability to epimerize both bacterial and algal alginate having a wide
REFERENCES:
Skjak-Braek et al. (1985) Carbohydrate Res. 139, 273-283.
Keil (1990) J. Gen. Microbiol. 136, 607-613.
Skjak-Braek, et al, Chem. Abstracts, vol. 102, No. 23, Abstract No. 200110x, p. 269 (Jun. 10, 1985).
Skjak-Braek, et al, Carbohydrate Research, vol. 103, pp. 133-136 (1982).
Haug, A. et al, Carbohydrate Research, vol. 17, pp. 297-308 (1971).
Patent Abstracts of Japan, vol. 13, No. 29, JP-A-63-233797 (Sep. 29, 1988).
Ertesv.ang.g Helga
Larsen Bj.o slashed.rn
Skj.ang.k-Braek Gudmund
Valla Svein
Carlson Karen Cochrane
Nobipol
Pronova Biopolymer a.s.
Slobodyansky Elizabeth
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