Journal of Applied Microbiology, Vol.121, No.1, 263-276, 2016
Functional characterization of a novel -fructofuranosidase from Bifidobacterium longum subsp infantis ATCC 15697 on structurally diverse fructans
AimIn this study, we describe the isolation of a gene encoding a novel -fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697, and the characterization of the enzyme, the second one found in this strain, significantly different in primary sequence to the already reported bifidobacterial -fructofuranosidases. Methods and ResultsThe gene, found through genome-mining was expressed in Escherichia coli C41(DE3). The recombinant enzyme (B.longum_l1) has a molecular weight of 75kDa, with optimal activity at 50 degrees C, pH 60-65, and a remarkable stability with a half-life of 755h at 50 degrees C. B.longum_l1 has a wide specificity for fructans, hydrolysing all substrates through an exo-type mechanism, including Oligofructose P95 (2-1 fructooligosaccharides (FOS), DP 2-8), Raftilose Synergy 1(2-1 FOS & inulin, DP 2-60), Raftiline HP (inulin, DP 2-60), bacterial inulin (3000kDa) and levan (83 & 3500kDa), Agave fructans (mixed fructans, DP 3-29) and levan-type FOS (2-6 FOS, DP 2-8), with the highest relative activity and turnover number found for levan-type FOS. The apparent affinity of the enzyme for levan-type FOS and Oligofructose P95 was found to be 92 and 46mmoll(-1) (K-m) with a specific activity of 908 and 725molmin(-1)mg(-1) of protein (k(2)), respectively, more than twice the activity for sucrose. ConclusionB.longum_l1 is a wide substrate specificity enzyme, which may contribute to the competitiveness and persistence of this strain in the colon. Significance and Impact of the StudyThe bifidobacterial -fructofuranosidase activity was evaluated with a wide variety of substrates including noncommercial fructans, such as levan-type and mixed agave fructans. Its activity on these substrates certainly strengthens their commercial prebiotic character and contributes to the understanding of bifidobacteria stimulation by structurally diverse fructans.