Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08.
The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D: -xylopyranose (4NPX) and p-nitrophenyl-alpha-L: -arabinofuranose (4NPA), and it was found that the ratio k (cat)/K (m) 4NPA/k (cat)/K (m) 4NPX was approximately 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k (cat)/K (m) muA/k (cat)/K (m) muX was approximately 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K (i) of 6.8 +/- 0.62 mM and xylose K (i) of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t (1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.
Wagschal
Kurt
K
Western Regional Research Center, USDA Agricultural Research Service, Albany, CA 94710, USA. kurt.wagschal@ars.usda.gov
Heng
Chamroeun
C
Lee
Charles C
CC
Robertson
George H
GH
Orts
William J
WJ
Wong
Dominic W S
DW
D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism; D001406:Bacillaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D009005:Monosaccharides / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D009005:Monosaccharides; D009844:Oligosaccharides; D014990:Xylans; C431714:arabinofuranose; C085118:arabinoxylan; D001089:Arabinose; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase