|M.Sc Student||Yael Grimpel|
|Subject||Clonning, Expression and Biochemical Characterization of|
Acetylxylan Estrases from Geobacillus
|Department||Department of Biotechnology and Food Engineering||Supervisor||Full Professor Shoham Yuval|
|Full Thesis text - in Hebrew|
The aim of this study was to clone, express and characterize two acetylxylan esterases from Geobacillus stearothermophilus T-6, a thermophilic bacterium that possesses an extensive xylanolytic system. Acetylxylan esterases (E.C. 188.8.131.52) catalyze the hydrolysis of ester bonds between acetic acid and xylan and are part of the enzymatic system allowing the utilization of hemicellulose.
The two putative acetylxylan esterases genes, axe1 (615-bp) and axe2 (660-bp), exhibit homology to carbohydrate esterases from family CE4 and to the lipolytic enzymes G-D-S-L family, respectively. Both genes appear to lack a leader sequence and their gene product is likely to be intracellular. The genes were cloned via PCR and over-expressed in Escherichia coli. Under standard growth conditions Axe1 formed inclusion bodies. When growth was carried out in the presence of sorbitol and betaine at 18 oC 40% of the protein was soluble. Axe1 was purified from the soluble cell extract by gel chromatography, yielding ~60 mg of protein per liter culture. Based on gel filtration Axe1 is a dimer in solution. The expression of Axe2 in E. coli provided a soluble product and following heat treatment and gel filtration, about 180 mg of protein per 1 liter culture with an overall yield of 29% was obtained. Based on gel chromatography Axe2 is a hexamer in solution.
The activity of the enzymes was checked towards several substrates including, chemically acetylated xylan, xylobiose peracetate and para nitrophenyl derivatives of C2-C18 carbon chain. Axe1 hydrolyzed half of the available acetyl residues from chemically acetylated xylan, but showed no activity towards xylobiose peracetate and the chromophore substrates. Axe2 gave kcat/Km values of 0.24 sec-1mM-1 towards xylobiose peracetate, 0.45 sec-1mM-1 towards 4-nitrophenyl acetate, 0.04 sec-1mM-1 towards 2-nitrophenyl acetate and 0.15 sec-1mM-1 towards 4-nitrophenyl propionate. Axe2 was inactive on acetylxylan or long carbon chain chromophore substrates. These results indicate that the role of Axe1 is to hydrolyze ester bonds between acetic acid and large xylooligosaccarides, whereas Axe2 hydrolyzes acetyl residues from one or two xylose units.