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The key enzyme in the fructose-6-phosphate shunt in bifidobacteria, Fructose-6-phosphate phosphoketolase (F6PPK; E.C. 18.104.22.168.), was purified to electrophoretic homogeneity for the first time from Bifidobacterium longum (BB536).A three-step procedure comprising acetone fractionation followed by fast protein liquid chromatography (FPLC) resulted in a 30-fold purification. The purified enzyme had a molecular mass of 300 ± 5 kDa as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 93 ± 1 kDa and 59 ± 0.5 kDa, as determined by SDS-PAGE.The deduced N-terminal amino acid sequences of the two subunits revealed no significant similarity between them and other proteins when compared to the data bases of EMBL and SWISS-PROT, indicating that this could be the first report on N-terminal amino acid sequence of F6PPK.The data from this study will be used to design oligonucleotide probe specific for bifidobacteria and to study the gene encoded F6PPK.