Biochemical Characterization of the Highly Thermostable ?-Xylosidase from Caldicellulosiruptor saccharolyticus

Date of Award


Degree Name

M.S. in Chemical Engineering


Department of Chemical and Materials Engineering


Advisor: Donald Comfort


There has recently been an increasing focus on the conversion of lignocellulosic biomass to biofuel as an alternative to petroleum. The current bottleneck for the process is efficient hydrolysis of lignocellulose into simple sugar molecules for fermentation to ethanol. Xylan represents the major hemicellulose in plants and is identified as the second most abundant polysaccharide on earth. The complete degradation of xylan requires several enzymes working synergistically, including endoxylanases and ?-xylosidases. ?-xylosidases are capable of hydrolyzing xylo-oligosaccharides to xylose. Thermostable ?-xylosidases are more desirable in biofuel production due to their ability to withstand harsh process conditions. This research characterizes glycoside hydrolase enzymes from the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, which are predicted to possess the ability to degrade xylan into the fermentable sugar xylose. Thermostable ?-xylosidase encoded by Csac_2409 of GH39 from C. saccharolyticus was recombinantly expressed by GenScript and the protein purified to 75% purity. The protein was then characterized to determine the substrate preference, optimal temperature, pH value, thermal stability, and kinetic constants. Thermostable ?-xylosidase showed activity over wide range of pH and temperature with optimal pH of 6.5 and temperature of 80░C. The enzyme indicated high thermal stability at 70░C with half-life close to 3 hours. Michaelis-Menten kinetic parameters, KM, V, kcat, and kcat/Km were determined to be 0.918 mM, 0.251 mM/min, 13.6 s-1, and 14.75 s-1mM-1, respectively. Understanding the function and optimal conditions of the enzyme could help the advancement of the lignocellulosic ethanol process, which would ultimately lead to less fossil fuel usage and more environmentally friendly transportation fuels.


Chemical Engineering, Biofuel, Lignocellulose, Thermostable, Xylosidase

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