3). All sourdough Weissella strains revealed a dextransucrase at 180 kDa, which is similar to the dextransucrase visualized from the reference NRRL B-512F strain. A similar band pattern was obtained for both conditions of culture i.e. with sucrose or glucose as the carbon source (Fig. 3a and b, respectively). Conversely, no active band was detected with the reference strain NRRL B-512F when cultivated in glucose growth conditions (Fig. 3b); thus confirming the well-known MDV3100 molecular weight sucrose induction of dextransucrase. The most intense bands were observed for soluble fractions that previously exhibited higher enzyme activity in DNS assays. This confirms that W. cibaria and W. confusa 180 kDa dextransucrase

is mainly soluble and is produced either with sucrose or with glucose as the carbon source. Besides, an additional faint band was detected at 300 kDa for the W. confusa DSM 20196T strain and only in sucrose growth conditions (Fig. 3a, not visible in the supernatant), suggesting the presence of an additional sucrose-inducible dextransucrase. The sourdough strain K39, which produced the highest soluble enzyme activity, was selected to perform Bortezomib supplier protein sequencing in order to design specific primers. Indeed, a first

attempt to detect dextransucrase encoding genes from W. cibaria and W. confusa strains (Bounaix et al., 2009) was unsuccessful using degenerate primers DegFor-DegRev (Kralj et al., 2003), targeting conserved regions within the catalytic domains of LAB glucansucrases (Fig. 4), as also reported by several authors (Tieking et al., 2003; Di Cagno et al., 2006; van der Meulen

et al., 2007; Schwab et al., 2008). As shown in Fig. 4a, six peptides were generated during microsequencing of the K39 soluble 180 kDa dextransucrase, and the peptide sequences showed similarity with the glucansucrase GTFKg3 of Lactobacillus fermentum Kg3 (Kralj et al., 2004). A first set of degenerate primers bMAR1F-bMAR2R was designed (Fig. 4b and Table 1). PCR amplification yielded a 2500-bp amplicon from W. cibaria K39 DNA. Partial sequencing of this fragment allowed to design nondegenerate oligonucleotide primers dsrK39For through and dsrK39Rev (Fig. 4b and Table 1). Using these primers, a 1950-bp fragment was obtained from W. cibaria strains DNA, but no fragment was amplified from the two W. confusa strains DNA. Partial sequencing of the PCR products from strain K39 confirmed the similarity to dextransucrase (Fig. 5). The corresponding predicted amino acid sequence, named DSRK39, showed >98% identity to GTFKg3 of L. fermentum Kg3 (Kralj et al., 2004) and DSRWC from W. cibaria CMU (Kang et al., 2009) as well as 64.4% identity to DSR-S from L. mesenteroides NRRL B-512F, which is in agreement with the dextransucrase activity. Notably, the regions in the vicinity of the catalytic triad (D, E, D) are relatively conserved in these enzymes.