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

Published online by Cambridge University Press:  01 May 1997

M. MÜLLER
Affiliation:
Centre for Agricultural Landscape and Land Use Research Müncheberg, Institute of Microbial Ecology and Soil Biology, Gutshof 7, D-14641 Paulinenaue, Germany
W. SEYFARTH
Affiliation:
Centre for Agricultural Landscape and Land Use Research Müncheberg, Institute of Microbial Ecology and Soil Biology, Gutshof 7, D-14641 Paulinenaue, Germany
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Abstract

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.

Type
Research Article
Copyright
Trustees of the New Phytologist 1997

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