The average dN/dS ratios for three lactobacilli tannase was 0.1373 suggesting that these genes are under neutral (dN/dS = 1) or purifying selection (dN/dS < 1). The levels of sequence identity to other known bacterial tannases,
such as TanA from S. lugdunensis and two putative tannase-coding genes from the whole genome sequence of S. gallolyticus UCN34 (GenBank accession no. YP_003430356 and YP_003431024) were less than 30% (Additional file 1: Figure S2). However, alignment analysis GSK3326595 cell line revealed that these enzymes contained a highly conserved Gly-X-Ser-X-Gly motif (e.g. the 161th to 165th positions of TanLpl sequence), typical of the catalytic triad with a nucleophilic serine found in serine hydrolases [18] (Additional file 1: Figure S2). Although the enzymes were supposed to be secreted, SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/)
analysis failed to suggest any https://www.selleckchem.com/products/netarsudil-ar-13324.html plausible signal peptide sequence. We sequenced the tannase-coding genes from 24 additional isolates of L. plantarum, L. paraplantarum, and L. pentosus (Additional file 1: Table S1). Their amino acid sequences composed the clades subdividing the species ranged from 99.3%-100% for L. plantarum, 95.5%-100% for L. paraplantarum, and 93.8%-100% for L. pentosus (Figure 1). The comparative analysis revealed that the lactobacilli tannase genes had a restricted diversity, forming a distinct phylogenetic cluster among the known tannases (Additional file 1: Figure S3). TanLpl, TanLpa, and TanLpe are representing a novel subfamily as they showed low amino acid
see more sequence similarity less than 60% with any other reported tannases in DDBJ/EMBL/GenBank databases. Figure Atazanavir 1 Neighbor-joining phylogenetic consensus tree based on amino acid sequences of TanLpl, TanLpa, and TanLpe. The deduced amino acid sequences of TanLpl, TanLpa, and TanLpe were aligned by the ClustalW method using the MEGA5 software package [12]. Phylogenetic trees were constructed using the neighbor-joining method [13] with MEGA5. The percentage of similarity between nucleotide sequences was calculated using BioEdit software [14]. The analysis was based on 469 residues for TanLpl and TanLpa sequences, and 470 residues for TanLpe sequences. The tannase genes of the L. plantarum WCFS1 (GenBank accession no. YP_004890536) and L. pentosus IG1 (GenBank accession no. CCC17686) were used to align with the corresponding genes obtained in this study. The stability of the groupings was estimated by bootstrap analysis with 1,000 replications. The information of used strains and DDBJ accession numbers are listed in Additional file 1: Table S1. Expression and purification of recombinant tannase It should be noted that we did not obtain any clone that secreted a measurable amount of recombinant tannase protein in the spent medium. Therefore, we obtained the purified recombinant enzymes from bacterial cells of the clones of transformed B.