Purification and substrate specificity of an extracellular fructanhydrolase from Lactobacillus paracasei ssp. paracasei P 4134

A novel extracellular fructanhydrolase was isolated from the culture filtrate of Lactobacillus paracasei ssp. paracasei P 4134 grown on a mineral medium supplemented with fructan extracted from Timothy ( Phleum pratense L.) as the only carbon source. The enzyme was purified by a combination of ammonium sulphate precipitation, affinity chromatography, preparative isoelectric focusing and anion‐exchange chromatography. As a result of these procedures, the specific enzyme activity increased 93‐fold, with a final yield of 28·4%. The substrate‐specific activities against different fructan types were determined by incubating the enzyme fractions with fructan extracted from Timothy (predominantly β‐2,6 fructosyl‐fructose linkages), inulin from Dahlia tubers (mostly β‐2,1 fructosyl‐fructose linkages) and sucrose. The purified enzyme catalysed the hydrolysis of β‐2,6‐linked fructan more rapidly than the β‐2,1 linkages of inulin. Additionally, the enzyme showed low ability to hydrolyse sucrose. Fructose was the main product of the degradation of Timothy fructan and inulin, indicating a high exohydrolytic activity of the enzyme. It is proposed that the fructan‐degrading enzyme from L. paracasei ssp. paracasei P 4134 is a β‐ D ‐fructan‐fructohydrolase (EC 3.2.1.80). The enzyme preparation showed a single protein band in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis with a mobility corresponding to molecular weight of c . 42 kDa. It was concluded that only one molecular weight of fructan‐degrading enzyme exists in L. paracasei ssp. paracasei P 4134.