FAIR-CT98-5064 Role of pectinases in determining the quality of flax fibres

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Objectives:

Most hemi (cellulose) degrading enzymes have a two-domain structure consisting of a catalytic domain and a cellulose-binding domain (CBD). The domains form well-defined units which are separated by a distinct linker region. When introduced in plants, bacterial CBD genes have been shown to alter growth properties, plant architecture or synthetic mechanisms within the plants. Transformed plants should acquire greater biomass, primarily through the acceleration of the process of cellulose synthesis. This proposal aimed to identify the role of cell wall modifying enzymes, more specific pectinases in determining the quality of cellulose flax fibres. This was to be accomplished by isolating endogenous flax genes encoding pectic enzymes which would be studied both in vitro and in vivo. The objectives were:

• To study the role of flax pectinases by in vitro production systems
• To define the role of specific pectic isoenzymes (such as pectin methyl esterases) during the development of flax plants.

Activities and Results:

During the first phase, in vitro studies focued on the role of flax-specific pectinase enzymes through isolation of full length cDNA sequences and protein production using a suitable eukaryotic expression system. The enzymes were to be analysed for functionality using a broad range of commercially-available substrates. During phase 2, isolated DNA sequences and in vitro produced pectinases were to be used to characterise the enzymes in situ during flax development. These studies were expected to elucidate the role of specific isoforms during fibre development both at a molecular and a biochemical level.

The expression of the CBD in E. coli using the pQE60 expression system (Qiagene) was not successful. Even as a 6xhis tagged protein, the product (mw 3,58 Kda) could not be detected. As short peptides (<5Kda) are sometimes unstable in E. coli, the CBD had to be expressed as a fusion protein. The sequence coding for the CBD was amplified using new primers and the product was then inserted in the pGEX-4T- I plasmid (Pharmacia) which allowed the production of a GST (gluthation S transferase) fusion protein. This GST, exhibiting a molecular weight of 25 Kda, could be easily expressed in E. coli. Cleavage of the protein of interest from the fusion protein could be achieved using a specific protease site. The recombinant plasmid has been introduced in E. coli M15 and the expression checked by SDS-PAGE. Once the expression conditions are optimised in E. coli, a large-scale production of the CBD will be possible. Purified peptides will first be purified by affinity chromatography, using the CBD property to bind cellulose. The purified peptide will then be tested for its binding capacities on different (hemi)cellulose substrates. CBD genes will be inserted first in a plant model (Arabidopsis thaliana) to study the cell wall modifications, and then in flax (Linum usitatissimum) to modify cellulosic fibres production. Recombinant protein including a CBD and a pectinase (such as pectate lyase) will be engineered, produced in E coli and then tested in vitro to modify fibre surface.

Keywords: cellulose, cellulose-binding domain, fusion protein, pectinase, flax

Contacts

Scientific Supervisor

• Trinette STOLLE-SMITS / ATO Dept Fibre and Paper Technology / NETHERLANDS