Bile Salt Hydrolase Activity

The bile salt hydrolyzing activity of the purified protein was evaluated as described previously (Allain et al., 2018b; Kusada et al., 2021). The purified LpBSH (100 μg/100 μl) was mixed with eight different conjugated bile salts (each 0.24 mg/100 μl) and incubated at 37°C. Each bile salt solution treated with buffer (instead of LpBSH) was used as a negative control. The bile salt hydrolysis reaction was stopped by adding 200 μl of 15% trichloroacetic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and the proteins were precipitated by centrifugation at 10,000 × g for 15 min. The supernatant (80 μl) was then mixed with 680 μl of 0.3 M borate buffer with 1% SDS (pH 9.5) and 80 μl of 0.3% 2,4,6-trinitrobenzenesulfonic acid solution (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan). This mixture was incubated for 30 min at room temperature in the dark. The released glycine or taurine was measured at 416 nm using a SPARK 10M multimode microplate reader (TECAN, Männedorf, Switzerland). The tested conjugated bile salts were glycocholic acid (GCA, Sigma-Aldrich), glycochenodeoxycholic acid (GCDCA, Sigma-Aldrich), glycodeoxycholic acid (GDCA, Sigma-Aldrich), glycoursodeoxycholic acid (GUDCA, Tokyo Kasei Kogyo), taurocholic acid (TCA, Nacalai Tesque), taurochenodeoxycholic acid (TCDCA, Sigma-Aldrich), taurodeoxycholic acid (TDCA, Nacalai Tesque), and tauroursodeoxycholic acid (TUDCA, Sigma-Aldrich). Three independent experiments were performed (n = 24 each). Statistical analyses were performed using GraphPad Prism software (version 8.0; GraphPad Software, San Diego, CA, United States). The Student’s t-test was used, and a p-value less than 0.05 (P < 0.05) was considered statistically significant.