Purification and characterization of fructose 6 phosphate phosphoketolase from Bifidobactetium asteroides
K.G. Fandi, H.M. Ghazali*, A.M. Yazid and A.R. Raha
of Food Science and Biotechnology, Universiti Putra Malaysia, UPM 43400 Serdang, Selangor Darul Ehsan, Malaysia
Received 30 September 2000 / Accepted 18 November 2000
Abstract.
Fructose 6 phosphate phosphoketolase (F6PPK; EC 4.1.2.22), the key enzyme of the fructose 6 phosphate shunt in bifidobacteria, was purified and characterized from Bifidobacterium asteroides (ATTC 25909). The enzyme was purified to homogeneity using acetone fractionation at 40-70% saturation, Mono Q anion exchange and Superose 12 gel filtration. A single band was obtained on nondenaturing PAGE, and shown to be that of F6PPK following elution from preparative polyacrylamide gel. SDS PAGE revealed two distinct bands corresponding to proteins with molecular masses of 59 ± I and 53 ± 0.5 kDa. The intact enzyme had a relative molecular mass of 110 ± 5 kDa as estimated by gel filtration chromatography (Sephadex G 200), suggesting that the F6PPK of B. asteroides is probably a dimer composed of two subunits, a1ß1 The NH2 terminal amino acid of the a chain was found to be methionine, and no significant similarity was found to the deduced amino acid sequence. The enzyme was stable at pH 4.5 8.0 having the optimum activity at pH 6.0. The enzyme activity was stable below 42oC and the optimum temperature was 30oC. The apparent Km value of the enzyme for fructose 6 phosphate was 14.1 mM. Purified enzyme had no apparent requirement for thiamine pyrophospliate as cofactors. The enzyme activity was inactivated by Hg2+ and recovered after addition of dithiothretol, indicating that sulfhydryl group was involved in the enzyme activity
Keywords: Bifidobacteria, Fructose 6 phosphate phosphoketolase, Bifidobacterium asteriodes, NH2 terminal amino acid sequence
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