Purification and characterization of a recombinant β-D-xylosidase from Thermobifida fusca TM51.
The subject of our investigations was a recombinant β-D-xylosidase (TfBXyl43) from Thermobifida fusca TM51 which was expressed in E. coli BL21DE3 and was purified to apparent homogeneity. The SDS-PAGE investigations demonstrated that the molecular weight of the monomer unit is 62.5 kDa but the native-PAGE studies indicated that the mass of the enzyme is 240-250 kDa which proves the presence of a characteristic homo oligomer quaternary structure in solution phase. Optimal parameters of the enzyme activity were at pH 6.0 and 50 °C. The enzyme showed little stability under pH 4.5 and above 60 °C. The substrate specificity investigations indicated that the TfBXyl43 is an exo-glycosidase, hydrolyzing only xylobiose and -triose from the nonreducing end. Besides the enzyme shows very high specificity on the glycon part of the substrate, since it can only hydrolyze β-D-xylopyranoside derivatives. The importance of hydrophobic interactions in the binding of the substrates are supported that the enzyme can hydrolize about four times more efficiently the artificial p-nitrophenyl-β-D-xylopyranoside substrate compared to the natural one, xylobiose. Furthermore we could detect transxylosidase activity both in the case of xylobiose and p-nitrophenyl-β-D-xylopyranoside donors which is the first example among the inverting β-D-xylosidases from T. fusca.
Department of Genetics and Applied Microbiology, University of Debrecen, Debrecen, Hungary. fcsaba007@gmail.com
Csaba Attila
CA
Fekete
László
L
Kiss
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D004795:Enzyme Stability; D006027:Glycosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D006027:Glycosides; D011994:Recombinant Proteins; C008646:4-nitrophenyl beta-D-xyloside; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase