FAIR-CT97-5008 Protein engineering of a xylanase to improve its agricultural uses

Contacts




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Agriculture : FAIR Area 1.2 - Green Chemicals and Polymers Chain : FAIR Marie Curie Research Training Grants : Fine Chemicals

Objectives:

The overall objective of this research project was to use knowledge of the structure/function relationship of xylanases to:

• improve the thermostability of industrially important hemicellulases
• alter the catalytic residues in the active site of xylanases.

This should increase their resistance to organic solvents so that these enzymes can be used to synthesise molecules containing polysaccharides that will be of considerable use in the food industry.

Activities and Results:

The first year of the programme focussed on the modification of active site residues that have the potential to alter the catalytic activity of the model enzyme, xylanase A (XYLA) from Pseudomonas. Previous studies in the laboratory have shown that XYLA can accommodate seven xylose residues in its active site. The region of the enzyme that binds one xylose moiety is known as a sub site. Cleavage of the substrate occurs between subsites -I and +I, and the number assigned to the other subsites indicates how close it is to the site of bond cleavage. In the first year of the project site-directed mutagenesis was used to substitute several active residues with alanine. The results obtained can be summarised as follows:

Disruption of either subsite -2 or +1 by mutations E43A and F181A, respectively, reduced the activity of XYLA against xylooligosaccharides with a dp>7 50-100 fold. In contrast these mutants retained full catalytic activity against xylan.

The generation of xylanases whose end-products are xylooligosaccharides, rather than xylose and xylobiose could have important implications in the agriculture and food industries where oligosaccharides are playing an increasingly important role.

In addition to modify the reaction products produced by XYLA, the importance of Trp- 313 and Asp-248, which are located at the +1 subsite, has also been probed. These residues were substituted for alanine and the biochemical properties of the mutant enzymes were evaluated. Both W313A and D248 A were 100-fold less active against xylan-than native XYLA.

Conclusions:

Interestingly both the k_cat and K_m of these enzymes against substrates such as dinitrophenyl-beta-cellobioside, which have excellent leaving groups, were reduced 100 fold suggesting that the mutations were influencing the position and/or ionisation state of the catalytic nucleophile, Glu- 127, rather than the catalytic nucleophile.

Keywords: Xylanases, protein engineering

Contacts

Scientific Supervisor

• Harry GILBERT / University of Newcastle-upon-Tyne Dept of Biological & Nutritional Sciences / UNITED KINGDOM