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Polysaccharide molecular engineering - The use of mannuronan C-5 epimerases for the upgrading of alginates and heteromannuronans (POLYENG)

   
Project

QLK3-1999-00034

Cell factory area

3.3.3

EU Contribution

1 176 487 Euro

Duration

36 months

Type

Research project

Starting date

01-01-2000

Keywords
A. vinelandi
C-5 epimerases
heteromannuronans
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ABSTRACT
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POLYENG aims to develop a set of highly efficient biomolecules, alginates and other uronic acid containing bio-polymers, with a wide variety of programmable properties designed for advanced drug delivery systems or as encapsulation matrix for cells. The technology is based on a unique set of recently-discovered polymer modifying C-5 epimerases with the capacity to introduce sites for co-operative ion binding into soluble polyuronates. Through enzymatic modification we will control the sequential structure of these polymers and thus tailor properties essential to their use as gel matrices in pharmaceutical drug delivery, in bio-reactors, and cell therapeutic systems. POLYENG will develop these enzymes from bacteria and algae into robust tools. The epimerisation process will be optimised and the structure and functional properties of the materials will be evaluated both on molecular bases and in applications.

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OBJECTIVES
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The project aims to produce high added value polysaccharides with new and improved gelling properties designed for advanced drug delivery systems. The processes will be based on enzyme modification of naturally occurring or chemically-modified uronans using a range of recently discovered mannuronan C-5 epimerases from bacteria and brown algae. Genetic methods together with biochemical and biophysical techniques will be used to unravel the mechanism of action of mannuronan C-5 epimerases and to improve and broaden their bio-technological potential. We will produce the recombinant epimerases from bacteria and algae and use them to modify alginates. The enzymes will also be applied to natural and artificial heterouronans to generate a novel family of polymers. The functional properties of tailor-made polymers will be evaluated in pharmaceuticals and in biotechnological and bio-medical applications.

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DESCRIPTION OF THE WORK
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The work will be divided into three main tasks:

  • Production and characterisation of the polymer modifying enzymes.
  • Production and investigation of the functional properties of the modified polysaccharides.
  • Exploitation of the new polymers in specific applications.

A combination of genetic methods, crystallography, NMR-spectroscopy and Atom Force Microscopy (AFM) will be used to unravel the mechanism of the action of mannuronan C-5 epimerases from A.vinelandii in order to improve and broaden their biotechnological potential for large scale bio-conversion of uronans. The enzymes will be further modulated through formation of hybrid enzymes, site-specific mutagenesis and construction of epimerases with new and improved functional properties by random enzyme evolution. The corresponding C-5 epimerases genes in brown algal will also be identified and over-expressed and characterised to obtain new, brown algal C-5 epimerases. The enzyme from bacteria AlgE 1-7 and eventually those from algae will be produced by fermentation of recombinant organisms. Substrate will be extracted from brown algae, prepared by fermentation, or by regioselective oxidation of, glucomannans and gallactomannans. Structural features of the modified polymers, will be investigated by 1H and 13C NMR spectroscopies, the chiroptical properties by means of circular dichroism measurements while the solution and gelling properties will be examined by means of static and dynamic laser light scattering experiments, and rheological methods. The performance of the modified polymers will be analysed in specific applications related to drug delivery systems for the gastric-intestinal tract and the use of alginate gel-entrapment materials for insulin-producing cells for cell therapy.

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DELIVERABLES
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  • Recombinant organisms with the 7 different epimerases from Azotobacter vinelandii.
  • Recombinant enzymes form A. vinelandii.
  • The crystal structure, reaction mechanism and mode of action of the epimerises.
  • Hybrid enzymes.
  • Gene and cDNA sequence of brown algal C-5 epimerases.
  • High molecular weight Mannuronan as substrates.
  • A range of tailor-made alginates with improved gelling properties.
  • A range of new heterouronans.
  • Drug formulations based on modified polymers in drug delivery systems.
  • Basal in vitro and in vivo evaluation of the new polymers as encapsulation material in cell therapy.

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CONSORTIUM
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COORDINATOR
  Dr. Gudmund Skjak-BrŠk
Department of Biotechnology
Norwegian University for Science and Technology
7491 Trondheim, Norway
Tel: +47 73593323
Fax: +47 73593340
Gudmund@chembio.ntnu.no

PARTNERS
  Dr. Bernard Kloareg
Centre d'Etudes d'OcÚanologie et de Biologie Marine
CNRS UPR 4601
29680 Roscoff, France
Tel: +33 298292330
Fax: +33 298292324
kloareg@sb-roscoff.fr

Dr. Stellan Sandler
Department of Medical Cell Biology
Uppsala University
PO Box 571, Husargatan 3,
751 23 Uppsala, Sweden
Tel: +46 184714430
Fax: +4618556401
Stellan.Sandler@medcellbiol.uu.se

Dr. Peter Dettmar
Reckitt & Colman
Dansom Lane
HU8 7DS Hull, United Kingdom
Tel: +44 1482582266
Fax: +44 1482582532
pdettmar@reccol.com

Dr. Vittorio Crescenzi
Department of Chemistry
University "La Sapienza"
00185 Rome, Italy
Tel: +39 0649913630
Fax: +39 064457112
crescenzi@axrma.uniroma1.it

Dr. Edvar Ons°yen
FMC Biopolymer
PO Box 494, Tomtegaten 36
3002 Drammen, Norway
Tel: +47 32203529
Fax: +47 32293510
Edvar_Onsoyen@fmc.com
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