|M.Sc Student||Ayelet Baumzweig|
|Subject||Protein Engineering in Xylanase T-6:|
Thermostability and Activity Improving
|Department||Department of Biotechnology and Food Engineering||Supervisors||Professor Emeritus Cogan Uri|
|Full Professor Shoham Yuval|
Xylanases (EC 184.108.40.206) are hemicellulases that hydrolyze the b 1-4 bond in the xylan backbone yielding short xylooligomers. Although enzymes are the most efficient catalysts known with an acceleration rate of over 1017, their industrial exploitation is often limited due to their insufficient stability, in particular thermostability. The focus of this study was to probe amino acids that contribute to the thermostability of xylanase T-6. Specific amino acid replacements were selected based on two criteria: amino-acid homology to xylanases from extremophiles, and by using an computational methodology that, based on the known crystal structure of the protein, predicts amino acid replacements that may contribute to the stability of the molecule. Overall 13 mutants were tested, including 2 double mutants. The S236F, E206M, I64E, and the E206M/I64E replacemts, exhibited increase in melting temperature. However with all of these mutants, the relative activity at 55C decreased, compared to the native protein. Based on the crystal structure of the enzyme, it appears that the increase in the melting temperature of the mutants, S236F and E206M, can be atributed to the stabilization of external loops. The I64E replacement, allowed the generation of a salt bridge with K62 in the loop that connects the b2 strand and a2 helix. In conclusion, it is evident that single amino acid replacements can effect both stability and activity. In all of the stabilizing mutations, improved stability was associated with loss in activity, presumably due to increase rigidity of the protein. Both approaches to predict useful amino replacements were only partially successful.