Purification and properties of thermostable beta-xylosidase from immature stalks of Saccharum officinarum L. (sugar cane).
Thermostable beta-xylosidase was purified from immature sugar cane stalks to an electrophoretically homogeneous form by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose and P-cellulose columns, heat treatment (70 degrees C, 20 min) and gel filtration on a Sephadex G-100 column. The purification was about 165-fold in specific activity with a high recovery of 43%. The apparent molecular weight of the enzyme, as determined by gel filtration, was 62,000. In SDS-polyacrylamide gel electrophoresis, the purified enzyme was homogeneous and consisted of only one polypeptide, having a molecular weight of approximately 62,000. The optimum temperature and pH were found to be 75 degrees C and 4.85, respectively. The enzyme was thermostable and especially stable in the presence of D-xylose. The enzyme retained full activity after incubation at 70 degrees C for 60 min in the presence of 0.1% D-xylose and when heated at 75 degrees C in the presence of 1% D-xylose, the enzyme was stable up to 30 min. Among the various sugars tested, D-xylose was found to be most effective stabilizer. The Km and Vmax values were 2.05 mM and 20.4 mumol/mg/min, respectively. The substrate specificity of purified sugar cane beta-xylosidase was investigated with 16 substrates. It was not able to hydrolyze any p-nitrophenyl glycopyranosides, larch wood xylan, or sugar cane except for p- and o-nitrophenyl-beta-D-xylopyranosides. The enzyme hydrolyzed p-nitrophenyl-beta-D-xylopyranoside more rapidly than o-nitrophenyl-beta-D-xylopyranoside. The hydrolysis of p-nitrophenyl-beta-D-xylopyranoside was markedly inhibited by AgNO3, HgCl2, and D-xylose. Competitive inhibition was shown to occur with both HgCl2 and D-xylose. AgNO3 was found to be a non-competitive inhibitor. The enzyme lost 20% of its activity by photo-oxidation in the presence of methylen blue for 8 h. By polyacrylamide disc gel electrophoresis, the enzyme was found to contain carbohydrate. The enzyme was then hydrolyzed and the carbohydrate content found to be 13.5%, the constituent sugars being arabinose and galactose.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D006026:Glycoside Hydrolases / Q000032:analysis*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010777:Photochemistry; D010940:Plant Proteins / Q000032:analysis; D010944:Plants / Q000201:enzymology*; D014995:Xylosidases / Q000032:analysis*
D000596:Amino Acids; D002241:Carbohydrates; D010940:Plant Proteins; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase