Purification and characterization of an endoinulinase from Xanthomonas oryzae No.5

The extracellular endoinulinase from Xanthomonas oryzae No. 5 which converts inulin into inulooligosaccharides was purified from the culture broth by ammonium sulphate precipitation. followed by column chromatography on Phenyl-Sepharose and DEAE-Sephacel. The enzyme was purified 29-fold with a yield of 5.5%,, from the starting culture broth. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. and its molecular weight was estimated to be 139 kDa. The specific activity of the purified enzyme was 1372 U/mg. The enzyme activity was highest at pH 7.5 and 50 degreesC, and stable over a pH range of 6.0-9.0 and up to 45 degreesC. The reaction rates declined at inulin concentrations over 125 g/l indicating occurrence of substrate inhibition. The Michaelis constant (K-m) and maximum reaction velocity (V-max) of the endoinulinase were 16.7 g/l and 12.1 g/l-h, respectively. There was no significant difference in the product distribution from the reaction between crude and purified enzyme, where major product was penta-inulooligosaccharides. (C) 2002 Elsevier Science Ltd. All rights reserved.