Furthermore, it has been speculated that their tannase activities AZD2014 purchase in the human alimentary tract have significant effects on pharmacological aspects of dietary tannins that are prevalent in beverages and teas [8]. Recently, we identified tanLpl (=lp_2956) within the genome of L. plantarum WCFS1 and found it to be similar to a known bacterial tannase gene in Staphylococcus lugdunensis[9]. Subsequently, tanLpl was expressed in E. coli[9, 10] to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. MX69 ic50 paraplantarum and L. pentosus. In this

study, we aim to identify tannase genes in other lactobacilli, such as L. paraplantarum and L. pentosus and to compare them with tanLpl in L. plantarum as well as to distinguish their structural and enzymological characteristics. Methods Bacterial strains, plasmids, and media Bacterial strains used in the study and their respective sources are listed in supplementary Additional file 1: Table S1. A total of 27 tannase-producing closely related Lactobacillus species isolates, consisting of 8 isolates of L. plantarum, 9 isolates of L. paraplantarum, 10 isolates of L. pentosus were used to study the identification of their tannase encoding genes and the determination of those sequences. The taxonomic identity of L. plantarum, L. paraplantarum, and L. pentosus

were determined by a 16S/23S rRNA gene spacer region targeted PCR assay [6]. Among them, L. plantarum ATCC 14917T, L. paraplantarum ARS-1620 NOS120 and L. pentosus 21A-3 were used as DNA donor strains for the gene cloning and expression of each of the tannases. Escherichia coli HST08 (TaKaRa Bio Inc., Shiga, Japan) strain was employed for recombinant plasmid construction. Bacillus others subtilis RIK 1285 (TaKaRa) was used as the host for gene expression and enzyme purification. The pGEM®-T Easy

cloning vector (Promega, Madison, USA) and pBE-S vector (TaKaRa) were utilized for the gene cloning for DNA sequencing and heterologous expression of tannase encoding genes in B. subtilis, respectively. The lactobacilli strains were cultured statically at 37°C in MRS broth (Difco, Detroit, USA) or on MRS supplemented with 1.5% agar before the experiment. Chemicals Methyl gallate (MG), epicatechin gallate (ECg) epigallocatechin gallate (EGCg) catechin gallate (Cg), and gallocatechin gallate (GCg), used as substrates for enzyme assay of tannases, were obtained from Wako Pure Chemical Industries., Ltd. (Osaka, Japan). In addition, (−)-epigallocatechin-3-O–(3-O–methyl) gallate (EGCg3″Me) was purchased from Nagara Science (Gifu, Japan). Structures of substrates are shown in Additional file 1: Figure S1. All substrates were dissolved in 50 mM phosphate buffer (pH 6.8) containing 1% ascorbic acid (Wako) at a final concentration of 0.5 mM or 1 mM. DNA manipulation Genomic DNA from the bacterial strains was prepared following the method of Marmur [11].