Efficient screening methods for glucosyltransferase genes in Lactobacillus strains

Limited information is available about homopolysaccharide synthesis in the genus Lactobacillus. Using efficient screening techniques, extracellular glucosyltransferase (GTF) enzyme activity, resulting in α-glucan synthesis from sucrose, was detected in various lactobacilli. PCR with degenerate primers based on homologous boxes of known glucosyltransferase (gtf) genes of lactic acid bacteria strains allowed cloning of fragments of 10 putative gtf genes from eight different glucan producing Lactobacillus strains (five Lactobacillus reuteri strains, one Lactobacillus fermentum strain, one Lactobacillus sake strain and one Lactobacillus parabuchneri strain). Sequence analysis revealed that these lactobacilli possess a large variation of (putative) gtf genes, similar to what has been observed for Leuconostoc and Streptococcus strains. Homologs of GTFA of Lb. reuteri 121 (synthesizing reuteran, a unique glucan with α-(1→4) and α-(1→6) glycosidic bonds) were found in three of the four other Lb. reuteri strains tested. The other Lactobacillus GTF fragments showed the highest similarity with GTF enzymes of Leuconostoc spp.

Exopolysaccharide production by Lactobacillus reuteri, involving sucrase type of enzymes

Exopolysaccharides (EPSs) find numerous applications in the food as well as in the non-food industries. They can be used as for instance viscosifying, thickening, gelling or water binding agents. Furthermore certain EPSs are known to exert health promoting effects such as cholesterol lowering, immunomodulating, antitumoral and prebiotic activities. Using a new method, a large collection of Lactobacillus strains was screened on the production of EPS. One of the positive strains, strain 121, produced two different soluble homopolysaccharides during growth on sucrose, a fructan and glucan. This strain was identified as Lactobacillus reuteri, a probiotic strain and an excellent colonizer of the gastro-intestinal tract of a broad variety of hosts, including humans. L. reuteri 121 was selected for further research. Structure analysis of the polysaccharides produced by L. reuteri 121 revealed that the fructan was a linear levan with beta(2-6) linked fructosyl units. This was the first example of fructan synthesis by lactobacilli. The glucan possessed a unique highly branched structure with alpha(1-4) and alpha(1-6) linkages with alpha(1-4,6) branching points. Both polymers were synthesized by sucrase type of enzymes (glucosyl- and fructosyltransferases). These enzymes only need sucrose as substrate; the energy released by the cleavage of the glycosidic bond in sucrose is subsequently used for the polysaccharide synthesis reaction. During growth of L. reuteri on sucrose or maltose, the sucrases responsible for the synthesis of the glucan and the levan appeared to be completely bound to the cell wall, whereas during growth on sucrose part of the enzymes was released into the culture medium. The EPS production was not a stable characteristic in continuous cultures. Different spontaneous mutants appeared, such as the EPS negative mutant strain K24, lacking both the glucansucrase (a glucosyltransferase) and the levansucrase (a fructosyltransferase). Mutant 35-5, lacking levansucrase, appeared after a pH shift-down. Using PCR techniques with degenerated primers based on known glucansucrase or fructosyltransferase amino acid sequences, chromosomal fragments containing a glucansucrase (gtfA) or a fructosyltransferase (ftfA) were amplified. Both fragments were sequenced and characterized at the amino acid level and phylogenetic trees of both types of sucrases were constructed. Both the gtfA and the ftfA were cloned separately in Escherichia coli. Cell free extracts of the E. coli strain harboring the ftfA gene produced an inulosucrase, which synthesized inulin and fructose-oligosaccharides from sucrose. The recombinant glucansucrase and the L. reuteri glucansucrase synthesized the same unique glucan. These were the first examples of the isolation, characterization and cloning of Lactobacillus glucansucrase and fructosyltransferase genes

Role of asparagine 1134 in glucosidic bond and transglycosylation specificity of reuteransucrase from Lactobacillus reuteri 121

Glucansucrases from lactic acid bacteria convert sucrose into various α-glucans that differ greatly with respect to the glucosidic bonds present (e.g. dextran, mutan, alternan and reuteran). This study aimed to identify the structural features of the reuteransucrase from Lactobacillus reuteri 121 (GTFA) that determine its reaction specificity. We here report a detailed mutational analysis of a conserved region immediately next to the catalytic Asp1133 (putative transition-state stabilizing) residue in GTFA. The data show that Asn1134 is the main determinant of glucosidic bond product specificity in this reuteransucrase. Furthermore, mutations at this position greatly influenced the hydrolysis/transglycosylation ratio. Changes in this amino acid expands the range of glucan and gluco-oligosaccharide products synthesized from sucrose by mutant GTFA enzymes.