Hydrolysis of Oligofructoses by the Recombinant β-Fructofuranosidase from Bifidobacterium lactis
Summary
The ability of the β- fructofuranosidase (EC 3.2.1.26) from Bifidobacterium lactis DSM 10140T to cleave a variety of fructooligosaccharides was characterised. We identified its gene on a cloned chromosomal DNA fragment by sequence similarity (69% identity) to the putative CscA protein encoded in the Bifidobacterium longum genome. The deduced amino acid sequence of 532 residues (59.4 kDa) appeared to be identical to the β- fructofuranosidase from the same strain recently described by Ehrmann et al. (Curr. Microbiol. 2003, 46, 391–397). However, the characterisation of the heterologously expressed enzyme showed several discrepancies to the referred study. First, the B. lactis β-fructofuranosidase gene was found to have 41% identity with CscA from E. coli in contrast to the 16% reported, therefore it was assigned to as CscA protein instead of BfrA. Second, we observed only low activity of the enzyme towards sucrose (6%) instead of the 100% previously reported. Instead, we measured highest activity (100%) of the enzyme with the oligofructose Raftilose® as a substrate compared with the inulin of low degree of polymerisation Raftiline® LS (29%) and the highly polymerised Raftiline® HP (10%). Altogether, the enzyme showed high affinity to terminal β(2–1) glycosyl linkages between fructose moieties. The Km values obtained for Raftilose®, Raftiline® LS and sucrose were 0.12, 7.08 and 8.37 mM, respectively, and Vmax values for the conversion to fructose were calculated to be 5, 21 and 17 mol/min per mg of protein, respectively. Growth of B. lactis was supported by fructans of low degree of polymerisation (Raftilose® and Raftiline® LS), whereas we observed no growth with highly polymerised inulin (Raftiline® HP).
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