Computational protocol: Effect of Bile Salt Hydrolase Inhibitors on a Bile Salt Hydrolase from Lactobacillus acidophilus

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[…] Bioinformatics analysis of BSH. A BSH gene from L. acidophilus PF01 has been identified and characterized by Hae-Keun et al. []; the nucleotide sequence of this BSH gene was deposited in the GenBank database (Accession No. EF536029). The BSH gene from the L. acidophilus PF01 strain [] were compared to those identified from L. salivarius NRRL B-30514 [] and from other diverse bacteria using the BLASTP program from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). To reveal the phylogenetic relationship, multiple sequence alignment of BSH sequences from different bacterial species and penicillin V acylase from Bacillus sphaericus (BPVA) was performed using the ClustalW program in MEGA 6.0 []. The dendrogram was constructed by neighbor-joining methods. To identify the conserved amino acid motifs potentially involved in BSH activity, multiple sequence alignment of BSH enzymes was performed using the ClustalW2 program.The modeling of BSH was performed in the Molecular Operating Environment (MOE), version 2008.10 (Chemical Computing Group, Montreal, QC). The BSH from Clostridium perfringens [] was chosen as template. Total 10 models were generated in the MOE homology module, using the AMBER99 force field. The one with highest packing score was chosen to superimpose with the C. perfringens BSH [], using the substitution matrix blosum62.Purification of recombinant BSH (rBSH). A pET-21b (+) vector-derived recombinant plasmid encoding recombinant L. acidophilus BSH [] was kindly provided by Dr. Dae-Kyung Kang (Dankook University, Korea). This recombinant plasmid bears a histidine-tagged rBSH gene with a full-length of BSH gene cloned from Lactobacillus acidophilus PF01, a commensal strain isolated from swine intestine []. In this study, this recombinant plasmid was introduced into the E. coli BL21 (DE3) host strain via transformation and desired transformants were selected on Luria-Bertani (LB) agar plates supplemented with ampicilin (100 μg/mL) following overnight incubation at 37 °C. The recombinant plasmid in one transformant, designated as JL1139, was extracted and subsequently sequenced; no mutations in the coding sequence of the BSH gene were detected. Expression and purification of the His-tagged rBSH from JL1139 were performed using the procedure described in previous publications [,,]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12% (wt/vol) polyacrylamid separating gel was performed to monitor production and purification of the rBSH. The purified rBSH was finally dialyzed against PBS buffer containing 10% of glycerol (pH 7.0) and stored in −80 °C freezer prior to use. Protein concentration was measured by BCA protein assay kit (Pierce).Effect of identified BSH inhibitors on the activity of BSH. The following three groups of compounds that have been identified as inhibitors for the L. salivarius BSH [,] were used in standard BSH assay in this study: (1) the approved feed additives used in food animals including CuCl2, CuSO4, ZnCl2, ZnSO4, NaHIO3, KIO3 and NaIO4; (2) the novel BSH inhibitors identified using high-throughput screening, which include caffeic acid phenethyl ester, riboflavin, epicatechin monogallate, gossypetin, menadione, and purpurogallin []; (3) the antibiotics that can inhibit BSH activity including oxytetracycline, demeclocycline hydrochloride, methacycline hydrochloride, doxycycline hydrochloride, roxarsone, and lincomycin [].A modified two-step standard BSH assay [] was performed to determine the inhibitory effect of the selected BSH inhibitors on the activity of the rBSH from L. acidophilus. Breifly, 10 µL of specific inhibitor,10 µL of rBSH (1.20 µg/µL), 168 µL of reaction buffer (0.1 M sodium-phosphate, pH 6.0) and 2 μL of 1 M DTT were mixed gently and incubated at 37 °C for 30 min. Then 10 μL of glycocholic acid (100 mM) was added in the 190 μL of reaction mix and the final reaction mix (total volume of 200 µL) was incubated at 37 °C for another 30 min. A 50-μL aliquot of the reaction mixture was then immediately mixed with 50 μL of 15% (w/v) trichloroacetic acid for stopping the reaction, followed by centrifugation for 5 min at 12,000 × g at room temperature to remove the precipitate. The supernatant was used in the second step, in which 50 μL of supernatant was thoroughly mixed with 950 μL of ninhydrin reagent mix (0.25 mL of 1% [wt/vol] ninhydrin in 0.5 M Sodium-citrate buffer, pH 5.5; 0.6 mL of glycerol; and 0.1 mL of 0.5 M sodium-citrate buffer, pH 5.5). A positive control (with BSH enzyme, without BSH inhibitor) and a negative control without BSH added were set up in each independent experiment. All assays were performed in triplicate. Percentage inhibition was calculated by dividing the inhibited activity (mean activity of control—mean residual activity of presence of a compound) relative to the mean activity of control and then multiplied by 100. […]

Pipeline specifications

Software tools BLASTP, Clustal W, MEGA
Application Phylogenetics
Organisms Lactobacillus acidophilus, Lactobacillus salivarius