Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products.
Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.
Anwar
Munir A
MA
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Kralj
Slavko
S
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Piqué
Anna Villar
AV
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Leemhuis
Hans
H
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
van der Maarel
Marc J E C
MJEC
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Dijkhuizen
Lubbert
L
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005630:Fructans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007444:Inulin / Q000378:metabolism*; D007700:Kinetics; D007778:Lactobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019936:Probiotics / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005630:Fructans; D007444:Inulin; C072599:levan; D006026:Glycoside Hydrolases; C021512:levanase