J. Microbiol. Biotechnol. (2011), 21(8), 838–845 doi: 10.4014/jmb.1103.03028 First published online 2 June 2011

Molecular Characterization of Bile Salt Hydrolase from animalis subsp. lactis Bi30

Jarocki, Piotr*

Department of Biotechnology, Human and Food Commodities, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland Received: March 16, 2011 / Revised: May 10, 2011 / Accepted: May 11, 2011

The present work describes the identification, purification, intestinal immune system [6, 33], and decrease lactose and characterization of bile salt hydrolase (BSH) from intolerance [14], and can reduce the effects of diarrhea, Bifidobacterium animalis subsp. lactis. The enzyme was constipation, and inflammatory bowel disease [21]. Moreover, purified to electrophoretic homogeneity by hydrophobic some evidence suggests that bifidobacteria can prevent or chromatography, ion-exchange chromatography and delay the onset of colorectal cancer [20], and finally, by ultrafiltration. SDS-PAGE analysis of putative BSH and reducing serum cholesterol concentration, to decrease the gel filtration revealed that the analyzed protein is risk of cardiovascular disease [29]. presumably a tetramer composed of four monomers each In recent years, the possibility of using of about 35 kDa. The purified enzyme was analyzed by to lower the cholesterol level in hypercholesterolemic liquid chromatography coupled to LTQ FT ICR mass patients and to prevent hypercholesterolemia in healthy spectrometry and unambiguously identified as a bile salt people has been extensively analyzed [8, 32]. Abundant hydrolase from B. animalis. The isoelectric point of the studies have shown various mechanisms for the cholesterol studied protein was estimated to be around pH 4.9. The lowering action of lactic acid bacteria [23, 26, 29]. The most pH optimum of the purified BSH is between 4.7 to 6.5, promising route of this process is the occurrence of bile and the temperature optimum is around 50oC. The BSH salt hydrolase (BSH) activity in such intestinal commensals of B. animalis could deconjugate all tested bile salts, with such as Bifidobacterium. BSH is an enzyme that catalyzes clear preference for glycine-conjugated bile salts over the hydrolysis of glycine- and -conjugated bile salts taurine-conjugated forms. Genetic analysis of the bsh into amino acid residues and bile acids [16, 27]. Free bile showed high similarity to the previously sequenced bsh acids are less efficiently reabsorbed than conjugated bile gene from B. animalis and confirmed the usefulness of salts, which results in the excretion of larger amounts of bile salt hydrolase as a genetic marker for B. animalis deconjugated bile salts via feces. Therefore, bile salt identification. hydrolase activity could lead to the increased requirement Keywords: Bifidobacterium animalis, bile salt hydrolase, for cholesterol, which is a precursor for the synthesis of cholesterol, bile salts bile salts, and finally could bring about a lowering of the serum cholesterol level [2]. On the other hand, some researchers suggest that BSH activity may also cause detrimental effects on human health. It has been proposed Genus Bifidobacterium constitutes a group of Gram-positive, that bile salt hydrolase activity in enteric bacteria might nonmotile, nonsporulating, non-gas-producing, catalase- have a major role in the formation of gallstones [4], lipid negative, and anaerobic bacteria that are natural inhabitants malabsorption [27], and might even promote an activation of human and animal gastrointestinal tracts. Because of of carcinogens [3, 25]. These effects seem to be especially their different health-promoting properties, some of these important as some bifidobacteria strains such as Bifidobacterium characteristic Y- or V-shaped rods have been intensively animalis Bb12 and Bifidobacterium animalis DN-173 010 investigated for industrial application as food additives and are commonly used as food additives in the dairy industry. probiotic pharmaceuticals [1, 9, 28, 34]. Many studies have In this study, we present the characterization of the bile shown that members of Bifidobacterium may stimulate the salt hydrolase activity of Bifidobacterium animalis Bi30. Although the bsh promoter and operon-like structure *Corresponding author Phone: +48 81 462 33 51; Fax: +48 81 462 34 00; containing the bsh gene from Bifidobacterium animalis E-mail: [email protected] were previously described [19], the BSH enzyme from this 839 Piotr Jarocki species has not yet been characterized. In our work, BSH sodium-acetate buffer (pH 4.5) with 10 mM 2-mercaptoethanol and o was purified and selected biochemical characteristics were then incubated at 37 C for 1 h in 0.1 M sodium-phosphate buffer analyzed. Next, the whole bsh gene was amplified and (pH 6.0) containing 10 mM 2-mercaptoethanol and 10 mM sodium sequenced, and the obtained result was aligned with bsh salt of taurodeoxycholic acid. The BSH activity in the gel was genes present in GenBank. visualized by the formation of a white precipitate of deoxycholic acid at the position of the enzyme.

Purification and Molecular Mass Estimation of BSH MATERIALS AND METHODS The cell-free extract was loaded onto a Phenyl-Sepharose hydrophobic interaction chromatography (HIC) column (1×5 cm), which had been Microorganisms and Culture Conditions equilibrated in 50 mM sodium-phosphate buffer containing 0.8 M Bifidobacterium B. animalis B. strains used in this work, Bi30, sodium sulfate at a flow rate of 1 ml/min. The absorbed proteins catenulatum B. infantis B. longum KD14, ATCC 15697, and KN29, were eluted with a decreasing linear sodium sulfate gradient (0.8 to were obtained from the Food Science Institute of Animal Reproduction 0 M) and the collected fractions (2 ml) were assayed for BSH and Food Research of The Polish Academy of Sciences, Olsztyn, activity. The fractions containing more than 50% of the peak activity Poland. All strains were routinely anaerobically cultured in a modified o were pooled, desalted, and concentrated using the Vivaspin 20 Garche’s medium at 37 C [5]. To detect bile salt hydrolase activity, centrifugal concentrator (10,000 MWCO; Sartorius, AG, Goettingen, a solidified medium was supplemented with 0.5% (w/v) of the Germany). The concentrated BSH active fraction obtained from HIC sodium salt of taurodeoxycholic acid (TDCA). Plates were incubated was applied to a Q-Sepharose anion-exchange chromatography column o Escherichia coli anaerobically for 72 h at 37 C [7]. XL1 Blue (Stratagene (1×5 cm) that was equilibrated with 50 mM bis-Tris propane buffer Inc.) was used as a host, and pGEM-T Easy vector (Promega) was (pH 6.5). The elution was performed using a linear gradient of 1 M E. coli used for cloning of the PCR product and DNA sequencing. sodium chloride in 50 mM bis-Tris propane buffer (pH 6.5). The - o cells were routinely cultured in Luria Bertani (LB) broth at 37 C flow rate was 1 ml/min. Fractions (2 ml) were collected and assayed with vigorous shaking or on LB medium solidified with 1.5% agar, for BSH activity. The active fractions were then concentrated and µ and ampicillin (100 g/ml) was added, if necessary. used for further characterization. The protein purity was examined by SDS-PAGE according to Laemmli using a stacking gel containing Cell Extracts Preparation and BSH Assays 4% acrylamide and 12% resolving gel. The accurate subunit For cell-free extracts preparation, cells of overnight culture were molecular mass was determined by comparing its migration with the ×g o harvested by centrifugation at 10,000 for 10 min at 4 C. The cell molecular mass standards (Fermentas). The proteins were visualized pellet was washed twice in 0.1 M sodium-phosphate buffer (pH 7.0) by staining with Coomassie Brilliant Blue R-250. The native molecular and then resuspended into 20 ml of the same buffer containing mass of BSH was estimated by size-exclusion chromatography using 10 mM 2-mecaptoethanol (Sigma Aldrich). The cells were disrupted by a Sephacryl S-300 HR 1.5×100 cm column (Sigma Aldrich). The sonication for 3 min with constant cooling, followed by centrifugation enzyme was eluted with 0.1 M sodium-phosphate buffer (pH 7.0) ×g o - o at 20,000 for 10 min at 4 C. The supernatant was stored at 20 C. containing 0.15 M sodium chloride at the flow rate of 0.5 ml/min. BSH activity assay was performed by determining the amount of The molecular weight standard kit was obtained from Bio-Rad. All liberated amino acids from hydrolysis of the amide bond of chromatographic separations were performed using a BioLogic Duo conjugated bile salts as described previously [16, 30], with several Flow system (Bio-Rad). modifications. To 180 µl of 0.1 M sodium-phosphate buffer (pH 6.0) containing 10 mM 2-mercaptoethanol, 10 µl of 200 mM human bile Isoelectric Focusing and 2D Electrophoresis salt mix (12% taurocholic acid, 8% taurodeoxycholic acid, 12% In order to improve the isoelectric focusing, protein samples were taurochenodeoxycholic acid, 23% glycocholic acid, 16% glycodeoxycholic prepared with a 2D Clean-Up kit (Amersham Biosciences) and then acid, and 23% glycochenodeoxycholic acid) and 10 µl of the protein protein pellets were dissolved in rehydration buffer (Bio-Rad) sample in the appropriate dilution were added. The bile salts mixture containing 8 M urea, 2% (w/v) CHAPS, 50 mM DTT, 0.2% (v/v) was used to measure the overall activity of bile salt hydrolase. In Bio-Lyte 3/10 ampholyte, and 0.001% (w/v) Bromophenol Blue order to determine substrate specificity, reactions with individual (Bio-Rad). Proteins were subjected to active rehydration (12 h; 50 V; bile salts (final concentration, 10 mM) were prepared. The reaction o - o 20 C) on 7 cm, pH 3 10 linear IPG strips and then to isoelectric mixture was incubated at 37 C for 30 min. The enzymatic reaction focusing by using Protean IEF (Bio-Rad) for a total of 14 kVh at was terminated with 200 µl of 15% (w/v) TCA and then the 20oC under oil to prevent evaporation. After focusing, the ×g samples were centrifuged at 14,000 to remove the precipitate. IPG strips were equilibrated in 6 M urea, 2% (v/v) glycerol, and The amounts of amino acids released from the conjugated bile salts 0.375 M Tris-HCl (pH 8.8), reduced with 2% (w/v) DTT and were measured by the ninhydrin assay. One unit of BSH activity alkylated with 135 mM iodoacetamide. For the second dimension, was defined as the amount of enzyme that can liberate 1 µmol of the IEF strips were transferred to 10% polyacrylamide gels, sealed amino acid from a substrate per minute. with 0.5% (w/v) low melting point agarose containing Bromophenol Blue in 25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS, and Activity Staining on a Polyacrylamide Gel then the electrophoresis was carried out at 200 V for 40 min using The identification of BSH activity in cell-free extracts and in purified Mini Protean (Bio-Rad). protein sample was carried out in a nondenaturating 10% (w/v) To obtain the precise determination of the isoelectric point, wide acrylamide gel with Laemmli buffer system, ommiting SDS [10]. pH range strips (pH 3.0 to 10.0, 17 cm) were used. After isoelectric After electrophoretic separation, the gel was washed twice in 0.4 M focusing (IEF), the proteins were visualized with CBB R250. BILE SALT HYDROLASE FROM BIFIDOBACTERIUM ANIMALIS SUBSP. LACTIS 840

Mass Spectrometry bromide staining. The purified PCR product was ligated into pGEM-T In order to achieve the protein identification, the peptides mixture Easy vector according to the manufacturer’s procedure. The ligation was analyzed by liquid chromatography coupled to LTQ FT ICR mixture was transformed into E. coli XL1 Blue competent cells. mass spectrometry (Hybrid-2D-Linear Quadrupole Ion Trap - Fourier The purified recombinant plasmid was sequenced with the T7 and Transform Ion Cyclotron Resonance Mass Spectrometer, Thermo SP6 universal primers using the capillary sequencing system – 3730 Electron Corp.) in the Mass Spectrometry Laboratory, Institute of Genetic Analyzer (Applied Biosystems). The gene sequences obtained Biochemistry and Biophysics PAS, Warsaw, Poland. Prior to the in this study and those from the databases were analyzed by the analysis, gel slices were subjected to standard “in-gel digestion” BLAST and Clustal W. procedure, during which proteins were reduced with 100 mM DTT The total RNA was isolated with a Total RNA Minikit (A&A (for 30 min at 56oC), alkylated with iodoacetamide (45 min in darkroom Biotechnology, Poland) and then treated with DNase I (Fermentas). at room temperature), digested overnight with trypsin (sequencing The reverse transcription of the prepared RNA sample and PCR grade modified trypsin; Promega V5111), and then eluted from the amplification of cDNA were performed by using a GeneAmp RNA gel with 0.1% TFA and 2% ACN. The obtained peptide mixture was PCR Kit (Applied Biosystems) according to the manufacturer’s applied to a RP-18 precolumn (LC Packings) using water containing recommendations. The PCR amplification was done with internal 0.1% TFA as a mobile phase and next transferred to a nano-HPLC primers for the bsh gene (Bifbsh2F, position 46-68, and Bifbsh1R, RP-18 column (nanoACQUITY UPLC BEHC18; Waters 186003545) position 445-461) in a final volume of 20 µl (including 4 µl from using an acetonitrile gradient (0%-60% AcN in 30 min) in the the reverse transcription reaction) under standard conditions with a presence of 0.05% formic acid with the flowrate of 150 nl/min. The total of 35 cycles (94oC 1 min, 55oC annealing 1 min, 72oC 1 min). column outlet was directly coupled to an ion source of LTQ-FT-MS working in the regime of data-dependent MS to MS/MS switch. The Nucleotide Sequence Accession Numbers acquired raw data were analyzed by Mascot Distiller followed by Sequences generated in this work are available in the GenBank Mascot Search (Matrix Science, London, UK; locally installed http:// database under the accession numbers HQ845205 and HQ845206. proteom.pl/mascot) against the NCBI non-redundant database. The search parameters for precursor and product ions mass tolerance were respectively ± 40 ppm and ±0.8 Da, with allowance made for RESULTS one missed semiTrypsin, fixed modifications of cysteine through carbamidomethylation, and variable modification through lysine Detection of Bile Salt Hydrolase Activity carbamidomethylation and methionine oxidation. Four strains of Bifidobacterium were screened for their Partial Characterization of Bile Salt Hydrolase bile salt hydrolase activity using the solidified Garche’s The temperature dependence of the enzyme activity was measured by assaying the purified enzyme in 0.1 M sodium-phosphate buffer (pH 6.0) at various temperatures (26-70oC). The pH optimum of the investigated enzyme was determined at pH values ranging from 3.6 to 8 using 50 mM acetate buffer (pH 3.6-5.6) and 0.1 M phosphate buffer (pH 5.7-8.0). The sensitivity of the BSH to metals and inhibitors was examined using the following compounds: EDTA, iodoacetamide, PMSF (dissolved in 70% ethanol), periodic acid, HgCl2, CuCl2, CaCl2, MgCl2, MgSO4, and NaCl. All assays were performed under the standard conditions described above with TDCA as the substrate.

PCR Amplification, DNA Sequencing, and Reverse Transcription The chromosomal DNA of B. animalis Bi30 was prepared from overnight culture, according to the procedure described in a previous paper [13]. The species of the strain Bi30 was confirmed by amplification and sequencing of the 16S rRNA gene using universal primers 27F and 1525R [15]. In order to amplify the whole bsh gene, primers BSH-F (5'-AGTCCATATGTGCACAGCCGTTCGTTTTG-3') and BSH-R (5'-AGTCAAGCTTCACTTGCTCTGCAACTGG-3') [19] were used. The PCR was carried out in a total volume of 20 µl of reaction mixture containing 0.25 U of Taq DNA polymerase (Fermentas), 200 µM of each deoxynucleoside triphosphate, 0.5 µM of each primer, 50 ng of bacterial DNA, and PCR buffer (Fermentas). o The amplification was conducted with template denaturation at 94 C Bifidobacterium for 5 min, followed by 30 cycles (denaturation for 40 s at 94oC, Fig. 1. Detection of bile salt hydrolase in animalis subsp. lactis. annealing for 30 s at 62oC, and extension for 1 min at 72oC) and a o An agar plate assay was performed on solid medium supplemented with final extension for 10 min at 72 C. The PCR products were separated taurodeoxycholic acid. A. Control plate without bile salts. B. Plate on 1% agarose gel by electrophoresis and then visualized by ethidium containing 0.5% TDCA. 841 Piotr Jarocki

Bifidobacterium strains, B. animalis subsp. lactis definitely had the highest specific activity in comparison with B. catenulatum, B. infantis, and B. longum. A high BSH activity for B. animalis and B. longum was also obtained from whole cell suspensions (data not shown). Additionally, no activity of the enzyme or only a little bile salt hydrolase activity was detected in spent culture media. These results suggest that bile salt hydrolase in B. animalis is probably located in an intracellular fraction. The occurrence of bile salt hydrolase in the examined strains was also confirmed by activity staining in a nondenaturing gel (Fig. 3A). According Fig. 2. Comparison of bile salt hydrolase activity in four to a previous paper [11], for cell-free extracts of B. animalis, Bifidobacterium strains. B.cat, Bifidobacterium catenulatum KD14; B.inf, Bifidobacterium infantis B. longum, and B. catenulatum, the BSH activity was ATCC 15697; B.long, Bifidobacterium longum KN29; B.anim, Bifidobacterium presented by two segments with different electrophoretic animalis Bi30. mobilities. Another migration pattern was obtained for B. infantis, which was represented in the gel by a single BSH medium containing 0.5% TDCA. Two different forms of BSH activity band. Finally, the ability of the Bifidobacterium activity were obtained for the strains used in this work. strains used in this study to deconjugate bile salts was proved. The deconjugation activity of B. animalis subps. lactis was observed as characteristic white precipitates of deoxycholic Purification and Characterization of BSH from B. acid around bacterial colonies (Fig. 1). A similar effect of animalis subps. lactis occurrence of bile salt hydrolase activity was obtained for For the purification of bile salt hydrolase from B. animalis B. longum KN29, whereas B. infantis ATCC 15697 and B. subsp. lactis, the cell pellet was first disrupted by sonication catenulatum KD14 produced opaque white colonies without and then the cell-free extract containing BSH enzyme was precipitate halos. All tested strains produced similar loaded onto a HIC column. The BSH activity was found in colonies on plates without bile salts (data not shown). Each fractions eluted near the end of the sodium-sulfate gradient strain was also tested for BSH activity in a two-step (Fig. 4A). Subsequently, the fractions exhibiting bile salt procedure by determining the amount of amino acids hydrolase activity were pooled, concentrated, and passed over released from conjugated bile salts. In our experiment, all an anion-exchange chromatography column. The proteins examined Bifidobacterium strains showed a different degree with BSH activity were eluted at NaCl concentrations of BSH activity (Fig. 2). However, among the tested between 0.12 and 0.18 M. The elution profile from Q- Sepharose is shown in Fig. 4B. The purity and accurate molecular mass of the analyzed enzyme were examined by SDS-PAGE (Fig. 3B). The purified extract contained proteins with BSH activity migrating as the major band with molecular mass close to 35 kDa. The molecular mass of the native enzyme was estimated by gel filtration and was found to be between 120 and 140 kDa (Fig. 4C) These results suggest that putative BSH from B. animalis subsp. lactis is presumably a tetramer composed of four monomers each of about 35 kDa. For further identification of putative bile salt hydrolase, the purified proteins were subjected to two-dimensional gel electrophoresis (2DE) using 7 cm, linear 3-10 IPG strips (Fig. 5A), followed by MS and peptide mass fingerprinting. The major protein spot with an approximate isoelectric point between pH 4.0 and 5.0 Fig. 3. Electrophoretic examination of the tested bile salt hydrolases. and molecular mass near 35 kDa was excised from the A. Activity staining on a nondenaturing polyacrylamide gel. Lanes: 1, cell- 2DE gel. Next, after digestion with trypsin, the obtained free extract of B. catenulatum KD14; 2, cell-free extract of B. infantis ATCC 15697; 3, cell-free extract of B. longum KN29; 4, cell-free extract of peptide mixture was analyzed by nano-HPLC coupled to B. animalis Bi30; 5, purified BSH from B. animalis Bi30. B. SDS-PAGE an LTQ FT ICR mass spectrometer. The MS/MS spectra of cell extracts at each purification step. Lanes: 1, molecular weight were evaluated by using the Mascot against NCBI marker; 2, cell-free extract of B. animalis Bi30; 3, pooled fractions from databases. The obtained ion spectra represented 14 unique hydrophobic interaction chromatography (HIC); 4, pooled fractions from ion-exchange chromatography (IEC); 5, active fractions from HIC plus IEC. tryptic peptides with individual ion scores > 52 (overall Mascot score value of 5,024) and a maximum protein BILE SALT HYDROLASE FROM BIFIDOBACTERIUM ANIMALIS SUBSP. LACTIS 842

Fig. 5. Two-dimensional electrophoresis (A) and isoelectric point determination (B) of the purified BSH from B. animalis Putative BSH from B. animalis Bi30 is marked by an arrow.

isoelectric point of the purified BSH was performed using broad pH range strips (pH 3.0 to 10.0, 17 cm). Finally, the pI value of BSH from Bifidobacterium animalis subsp. lactis was estimated to be around pH 4.9 (Fig. 5B).

Partial Characterization of BSH Activity Among the six analyzed human bile salts, the purified enzyme exhibited the highest level of activity with glycocholic acid (defined as 100% activity). Furthermore, BSH from Bifidobacterium animalis subsp. lactis showed Fig. 4. Purification and molecular mass estimation of the BSH a clear preference for glycine-conjugated bile acids over from Bifidobacterium animalis subsp. lactis. the forms of bile salts with taurine instead of glycine, and A . Chromatography elution profiles of the BSH from B. Elution profile for only a little difference was observed with di- or BSH from Phenyl-Sepharose hydrophobic interaction chromatography. Solid lane: Protein (A 214 nm); dotted line: sodium sulfate gradient; trihydroxyconjugated bile salts. (Fig. 7). The studies on the dashed line: BSH activity. B. Elution profile for BSH from Q-Sepharose effect of pH on BSH activity showed a broad pH optimum anion-exchange chromatography column. Solid lane: Protein (A 214 nm); ranging from pH 4.7 to 6.5, with maximal catalytic activity dotted line: sodium chloride gradient; dashed line: BSH activity. C. Molecular weight estimation of BSH from B. animalis by gel filtration on at 5.1 (Fig. 8A). As shown in Fig. 7B, bile salt hydrolase Sephacryl S-300. had the highest activity at around 50°C, with 85% and 82% of maximum activity at 45oC and 55oC, respectively. A significant decrease in enzyme activity was observed at sequence coverage of 68%, indicating identity or an temperatures higher than 60oC. At 70oC, 90% of the extensive homology to database BSH proteins (Fig. 6). catalytic activity was lost. The effect of various ions and These data allowed for the unambiguous identification of the inhibitors on BSH activity are summarized in Table 1. purified protein as a bile salt hydrolase from Bifidobacterium Among the inhibitors tested, periodic acid, HgCl2, and animalis subsp. lactis. The precise determination of the CuCl2 caused strong inhibition of purified enzyme.

Fig. 6. Comparison of peptides identified by LC MS\MS with the amino acid sequence of BSH of B. animalis subsp. lactis KL61. The peptides identified by mass spectrometry analysis of the purified enzyme from B. animalis subsp. lactis Bi30 are underlined. 843 Piotr Jarocki

Table 1. Effects of various chemicals on BSH activity. Reagents Concentrations (mM) % Residual activity EDTA 30 75.7 Iodoacetamide 30 75.8 PMSF 0.5 90.9 Periodic acid 3 0.6 HgCl2 0.2 2.2 CuCl2 51.4 CaCl2 30 91.2 MgCl2 30 107.6 MgSO4 30 112.6 Fig. 7. Substrate specificity of purified BSH from Bifidobacterium NaCl 50 98.2 animalis Bi30. Six major bile salts are shown: glycocholic acid (GCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid sequenced. The obtained results (GenBank Accession No. (TCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic (TCDCA). HQ845205) unambiguously showed that strain Bi30 is a member of Bifidobacterium animalis subsp. lactis. To Interestingly, iodoacetamide, which is also an SH enzyme confirm that Bifidobacterium animalis subsp. lactis Bi30 inhibitor, reduced deconjugation activity only to approximately possesses the gene encoding BSH protein, PCR with 75%. A little inhibition was also observed with the metal primers BSH-F and BSH-R was performed. The specific enzyme inhibitor (EDTA) and serine enzyme inhibitor product was successfully amplified as an approximately (PMSF). Of those tested, Mg2+ was the only ion that 950 bp fragment (Fig. 9A). The PCR product was ligated stimulated catalytic activity of BSH from Bifidobacterium to TA cloning vector pGEM-T Easy, which was transformed animalis subsp. lactis. into Escherichia coli XL1 Blue competent cells. The recombinant clones were isolated and verified by restriction bsh Gene Sequence Analysis enzyme digestion and PCR with primers BSH-F and BSH- In order to precisely determine the species of the isolate used in this study, a partial gene encoding 16S rRNA was

Fig. 9. PCR amplification and confirmation of expression of the bsh gene from B. animalis. A. PCR amplification of bsh gene from B. animalis. Lanes: 1, Molecular weight marker (in base pairs); 2, PCR product obtained with genomic DNA of B. animalis Bi30 used as a template; 3, Amplification performed using recombinant plasmid pGEM-BSH-Bi30-2 as a template. B. Confirmation of bsh gene expression in Bifidobacterium animalis subsp. lactis. Lanes: 1, molecular weight marker (in base pairs); 2, RT-PCR detection of an Fig. 8. Effects of pH (A) and temperature (B) on the activity of internal fragment of bsh gene from B. animalis Bi30; 3, negative control purified bile salt hydrolase from B. animalis. (RT-PCR reaction without reverse transcriptase). BILE SALT HYDROLASE FROM BIFIDOBACTERIUM ANIMALIS SUBSP. LACTIS 844

R. The resulting plasmid, designated as pGEM-BSH-Bi30- close to 4.9. Kim et al. [16] showed that a pI value between 2, which harbored the insert with the size of approximately pH 4.0 and 5.0 is characteristic for BSHs originated from 950 bp (Fig. 9A), was then sequenced. The comparison of Bifidobacterium. the obtained sequence (GenBank Accession No. HQ845206) The biochemical study of the substrate specificity of the with the NCBI database sequences by BLAST analysis purified BSH has demonstrated that the analyzed enzyme revealed a very high similarity (99.8%) to the bsh gene of can hydrolyze both glycine and taurine conjugates of bile B. animalis, previously published by Kim and Lee [19]. acids. Our results also confirmed previous reports that Additionally, bsh from strain Bi30 was found to share a BSH enzymes exhibit an unquestionable preference for signficantly lower similarity (below 70 %) with bsh genes glycine-conjugated bile salts over taurine-conjugated forms from other species of the genus Bifidobacterium. On the [11, 16, 31]. Similar to other BSHs from and basis of the obtained bsh sequence, the theoretical molecular Bifidobacterium, the analyzed enzyme showed a broad mass and isoelectric point of the deduced protein were pH optimum over the range from pH 4.7 to 6.5. The calculated to be 35,022 Da and 4.70, respectively. These characteristic loss of activity below pH 4.0, related to results are in good agreement with the measured data the decrease of substrate solubility, was also observed. obtained in biochemical studies. The deduced amino acid Interestingly, we proved that the maximum activity of sequence of BSH from Bifidobacterium animalis subsp. BSH from B. animalis occurred at approximately 50oC. To lactis was also consistent with the results obtained from date, reported temperature optima for BSH activity in lactic the analysis of the purified enzyme by LC MS/MS. acid bacteria were between 30oC and 45oC [11, 12, 18, 24, Finally, the expression of the analyzed gene was confirmed 31]. The obtained data also showed that reagents known as by the RT-PCR detection of a 415 bp internal fragment of sulfhydryl-enzyme inhibitors significantly inhibited the bsh from Bifidobacterium animalis subsp. lactis (Fig. 9B). purified BSH. It confirmed that BSHs require a free thiol group for catalytic activity. The performed genetic studies proved that the primers DISCUSSION set (BSH-F and BSH-R) described previously by Kim and Lee [19] is very useful for specific amplification of bsh Many studies have shown that the ability to catalyze the genes from B. animalis. Additionally, the sequence analysis deconjugation of bile salts is a common characteristic of the amplified gene has revealed a very high level of among members of lactic acid bacteria [22, 30]. In this homology to a previously sequenced bsh gene from B. work, four strains from the genus Bifidobacterium were animalis [19] and significantly lower similarity with other screened for BSH activity. Three different experiments (a bsh genes from the genus Bifidobacterium. It confirmed plate test, an enzymatic assay, and a native PAGE) confirmed the utility of the bsh gene as a valuable molecular marker that all strains used in this study exhibited bile salt for the genetic identification of Bifidobacterium animalis hydrolase activity. In comparison with other tested strains, subsp. lactis among other bifidobacteria. Bifidobacterium animalis subsp. lactis definitely had the In conclusion, the given study describes the biochemical highest deconjugation activity towards the bile salts mixture. and genetic characterizations of a bile salt hydrolase from A two-step chromatographic procedure was performed Bifidobacterium animalis subsp. lactis. The presented for the purification of the BSH enzyme from B. animalis. results could be useful for a better understanding of the A single protein band (with molecular mass around 35 kDa) physiological role and the genetic diversity of BSH in the indicated the homogeneity of the BSH enzyme was obtained genus Bifidobacterium. In further studies, an overexpression after SDS-PAGE analysis of purified protein sample. and a structural analysis of BSH from Bifidobacterium Additionally, the BSH activity staining on native PAGE animalis subsp. lactis will be performed. The significance revealed the same activity patterns for the cell-free extract of BSH activity for the BSH-producing microorganisms as and the purified BSH. To confirm that the purified enzyme well as for the mammalian hosts will also be studied. is BSH, LC MS/MS analysis of protein sample excised from a 2DE gel was performed. The obtained results allowed for an unambiguous identification of the purified protein as a Acknowledgment homolog of bile salt hydrolase. The gel filtration and SDS-PAGE mobility data for the BSH suggested that the I am grateful to Agata Malinowska for help in an analyzed enzyme was a tetramer with an approximate interpretation of the MS/MS data. native molecular mass between 120 and 140 kDa. This is in good agreement with previous papers, stating that the REFERENCES native bile salt hydrolases in the members of the genus Bifidobacterium are typically tetramers [16, 17, 31]. The 1. Arunachalam, K. D. 1999. 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