|M.Sc Student||Dolkinshtein Margarita|
|Subject||The Partial Genome Sequence of Geobacillus|
Stearothermophilus and Characterization of its
Acetylxylan Degrading Enzymes
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
The increase of the world’s energy demand and the depletion of crude oil, has given rise to the worldwide interest in alternative sources of energy. An important renewable resource is an ethanol produced from biomass. Cellulose and hemicellulose constitute the cell wall, forming 23-53% and 20-35% of the biomass, respectfully. Hemicellulose is a highly branched chain of carbohydrate polymers, mostly xylan, arabinan and galactan, substituted by varied side chains, with an important role in reducing the accessibility of enzymes to the main chain. Hemicellulases are a diverse group of enzymes - glycoside hydrolases and carbohydrate esterases - that degrade hemicellulose.
Geobacillus stearothermophilus T6 is a soil bacterium possessing an impressive set of hemicellulases, as well as transport and regulatory elements, allowing it to degrade xylan and arabinan.
In the current project, the partial genome sequence of G. stearothermophilus T6 was solved, with a total of 258 DNA fragments with the maximal length of 147 Kb. The main reason for the large number of contigs is proposed to be the abundance of transposable genetic elements.
The new arabinogalactan utilization cluster was discovered, and the possibility of horizontal gene transfer for the new arabinogalactan, as well, as for xylan/arabinan utilization clusters was discussed.
Two acetylxylan esterases from G. stearothermophilus T6, Axe1 and Axe2, were cloned in the recombinant E.coli culture. The enzymatic activity was tested on acetylxylan, xylobiose peracetate and the synthetic substrate 4-nitro phenyl acetate. Axe2 showed wide activity, acting on all the substrates, while Axe1 was not active on xylobiose peracetate, and had three orders of magnitude less Kcat/Km for 4-nitro phenyl acetate, than Axe2. Axe1 was assigned to the carbohydrate esterases family 4 according to sequence similarity. Axe2 does not show significant similarity to any of the known carbohydrate esterases family. Here we demonstrate that Axe2 is an active acetylxylan esterase, and hence there should be defined a new family of carbohydrate esterases for it.
The results hint that the two enzymes play different roles in the cell, being the synergists of different types of the hemicellulases degrading the main sugar backbone.