[Purification and characterization of a beta-D-xylosidase from Leifsonia shinshuensis DICP 16].
A beta-D-xylosidase from Leifsonia shinshuensis DICP 16 was purified to apparent homogeneity using a combination of ammonium sulfate precipitation, DE 52 anion-exchange, Q-Sepharose Fast Flow anion-exchange, Toyopearl Butyl 650C hydrophobic-interaction and Sephacryl S-300 HR gel-permeation chromatography. The purified xylosidase consisted of two same subunits and had the relative molecular weight of 180 kD as determined by SDS-PAGE and gel-permeation chromatography. The maximal beta-D-xylosidase activity occurred at 55 degrees C and pH 7.0. It was stable at 45 degrees C and retained its original activity for 60 min. The stability declined rapidly when the temperature rose above 55 degrees C. The xylosidase was stable in the pH range from 6.0 to 11.0 for 20 h. At pH 7.0 and 45 degrees C the Km for p-nitrophenyl-beta-D-xylopyranoside (pNPX) was 1.04 mmol/L and the Vmx was 0.095 mmol nitrophenol/min/mg xylosidase. The enzyme was inhibited strongly by Fe2+ and Cu2+. It exhibited low levels of activity against other artificial substrates, compared to its activity against pNPX. When different natural xylosides were used as the substrates, the xylosidase showed distinct hydrolysis ability. It could hydrolyze 20-C, beta-(1-->6)-xyloside of ginsenoside Rb3 (G-Rb3) into ginsenoside Rd, but did not hydrolyze the other beta-D-glucosidic bonds of G-Rb3. Additionally, the xylosidase could not hydrolyze C-7 xylosyl-bearing taxanes.
Luan
Hongwei
H
Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China.
Hu
Ying
Y
Liu
Xingbao
X
Hao
Dacheng
D
Yang
Ling
L
D000192:Actinomycetales / Q000145:classification / Q000201:enzymology*; D000595:Amino Acid Sequence; D003470:Culture Media; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D003470:Culture Media; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase