Abstract
BackgroundTannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum.ResultsThe genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917T, respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The Km values of the enzymes on each galloyl ester were comparable; however, the kcat/Km values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity.ConclusionTwo tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases.
Highlights
Tannase catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid
Tannase production has been identified in other bacterial species [1], including lactobacilli species of Lactobacillus plantarum, Lactobacillus paraplantarum, and Lactobacillus pentosus, which were isolated from fermented vegetables [6,7]
L. plantarum ATCC 14917T, L. paraplantarum NOS120 and L. pentosus 21A-3 were used as DNA donor strains for the gene cloning and expression of each of the tannases
Summary
Tannase (tannin acyl hydrolase, EC 3.1.1.20) catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found in fungal species and many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. We identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. Tannase (tannin acyl hydrolase, EC 3.1.1.20) catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins that occur widely in the plant kingdom and are considered to be a protective strategy against microbial attack [1]. Tannase production has been identified in other bacterial species [1], including lactobacilli species of Lactobacillus plantarum, Lactobacillus paraplantarum, and Lactobacillus pentosus, which were isolated from fermented vegetables [6,7]. Little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus
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