plantarum, that has 99% amino acid identity to TanLpl. They identified Ser163, His451, and Asp419 as a catalytic triad with a nucleophilic serine within the pentapeptide sequence motif selleckchem Gly161-X-Ser163-X-Gly165 of the crystal structure. Alignment analysis indicated that all the three lactobacilli tannases, TanLpl,

TanLpa, and TanLpe contained the conserved Gly-X-Ser-X-Gly motif in their amino acid sequences as the catalytic triad (Additional file 1: Figure S1). In addition, we found that amino acid residues of Asp421, Lys343, and Glu357, considered to play a key role in binding of the enzyme to them corresponding galloyl site of buy XAV-939 the substrate [19], were also conserved. We sequenced a total of 28 possible lactobacilli tannase genes, forming selleck a distinct phylogenetic clade among the tannase genes reported in databases. No other bacterial tannases in databases showed higher than 60% amino acid sequence similarity with TanLpl, TanLpa, or TanLpe, suggesting that the three lactobacilli tannases form a novel independent lineage of tannase superfamily. Although an increasing number of genome sequencing reports are revealing that bacteria possess various tannase genes, only few of them have been cloned and expressed in heterologous hosts [20]. We thus undertook the gene expression and protein purification of TanLpl, TanLpa, and TanLpe in B. subtilis. However, the recombinant tannases were not readily secreted into the culture medium, but were

trapped within the cell walls. In agreement with our previous report [9], much the optimum temperature and pH for activities of TanLpl were 40°C and 8.5, respectively. On the other hand, Rodríguez et al. [21] reported that cell-free extracts

of the type strain L. plantarum CECT 748T (=ATCC 14917T) had optimal tannase activity at pH 5.0 and at 30°C. According to the available genome information of L. plantarum ATCC 14917T, this strain is known to have at least two unique tannase genes in its genome, i.e., tanLpl and another gene (GenBank accession no. ZP_07077992). It might be possible that Rodríguez et al. [21] worked with the second one. The optimum temperature and pH of TanLpa were similar to those of TanLpl, whereas TanLpe was weaker at temperatures higher than 40°C. The number of proline residues was reported to contribute to the enzyme thermo-stability [22]. The difference might be due to the lower proline content of TanLpe (21 proline residues), compared with TanLpl (23 proline residues) and Tanlpa (25 proline residues). Most of lactobacilli species are acid tolerant reflecting the fact that they produce various organic acids during fermentation, and thought until recently, to be generally not considered alkali sensitive. Nevertheless, Sawatari et al. [23] reported that some lactobacilli strains including L. plantarum and L. pentosus originating from plant materials showed growth at pH up to 8.9 and alkali tolerance of the glycolytic enzymes of the strains. Moreover, in turned out that L.