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Bile salt hydrolase (BSH) is a gut-bacterial enzyme that influences human health by altering the host fat digestion and cellular energy generation. BSH is essential for deconjugation of the glycine or taurine-conjugated bile salts in the small intestine of humans. Therefore, BSH may be a key microbiome target for the designing of new measures to control some diseases in humans. BSHs, a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, exhibit higher variation in substrate specificity. The phenylalanine-129 (F129) and leucine-138 (L138) in loop III of BSH, thought to be responsible for substrate specificity, are partially conserved in this superfamily. In this study, the aromatic-hydrophobic F129 and aliphatic-hydrophobic L138 of C-terminally His-tagged BSH from Lactobacillus plantarum B14 (LbBSH) was substituted for aliphatic-hydrophobic isoleucine (I) and negatively charged polar glutamate (E) amino acid, respectively, by site-directed mutagenesis and characterized using an Escherichia coli BLR(DE3) expression system. Although both mutations resulted in an assembled and stable recombinant BSHs (rBSHs), they altered the catalytic activity and substrate specificity of rBSH. This is the first experimental finding which confirmed that F129 and L138 were critical amino acids for the catalytic activity and substrate specificity turnover of BSH. [ABSTRACT FROM AUTHOR]

Microbial bile salt hydrolases (BSHs), a member of cholylglycine hydrolase (CGH) family, catalyze the hydrolysis of glycine and taurine-linked bile salts in the small intestine of human. BSH is evolutionarily related to penicillin V acylase (PVA) which hydrolyses a penicillin V and is also a member of CGH family. Although, five of the six amino acids, C2, R16, D19, N170, N79 and R223, supposed to be responsible for catalytic activity of BSH enzyme, are strictly conserved in all CGH family members, N79 is partially conserved in this family. In this study, in order to analyze the correlation between N79 and catalytic activity or substrate specificity of BSH, the polar and acidic N79 was substituted for the aliphatic and hydrophobic V79 by PCR-based site directed mutagenesis and mutant recombinant BSH was expressed in E. coli BLR(DE3). While the effects of the mutation on catalytic activity and substrate specificity of BSH were detected by ninhydrin assay. The effect of this mutation on the stability of the BSH was observed by SDS-PAGE analysis. Although V79 mutation resulted in stable BSH, it reduced the catalytic activity and altered substrate specificity of BSH. The results suggested that N79 might be important for substrate binding and catalytic turnover of BSH. [ABSTRACT FROM AUTHOR]

Bile salt hydrolase (BSH) activity, hypo-cholesterolemic effect, and probiotic properties have been reported for Enterococcus strains isolated from animal and human gut and fermented foods but not for strains isolated from environmental niches, like aquatic and terrestrial plants, soil, and water. The present study is the first report on isolation of Enterococcus faecium from rhizospheric soils that harbor the bsh gene, remove cholesterol in vitro , and possess essential and desirable probiotic attributes. Fifteen samples were collected from different sites located in northern, southern, and central regions of India, of which five yielded pure colonies that were named LR2, LR3, ER5, LR13, and VB1. These were identified by 16S rRNA gene sequencing as E. faecium and evaluated for BSH activity, cholesterol-lowering potential in vitro , and probiotic properties. Our results indicated that all the strains were capable of surviving the harsh conditions of the gastrointestinal tract and did not harbor any of the virulence genes. Though all strains showed the presence of bsh and potential for cholesterol removal, E. faecium strain LR13 showed a remarkable cholesterol removal capability and vancomycin susceptibility and possessed most of the desirable and essential attributes of a probiotic. Hence, it seems to be a fairly promising probiotic candidate that needs to be further evaluated in in vivo studies, especially for its hypo-cholesterolemic potential. [ABSTRACT FROM AUTHOR]

Introduction: Bile salt hydrolase (bsh) is a bile salt- or acid- hydrolysable enzyme synthesized by different members of the autochthonous gastrointestinal microbiota to survive and colonize the intestinal lumen of the host. The ability of probiotic strains to detoxify bile salt through production of bile salt hydrolase has often been considered as essential criteria for probiotic strain selection. Objectives: The objective was to find out the bile salt tolerance and hydrolysing activities of Lysinibacillus sphaericus PKA17. The study also aimed to reveal the sequence-structure relationship of L. sphaericus and Bacillus spp. bsh protein and determine the catalytic interaction pattern of L. sphaericus bsh protein with its substrates and potent inhibitors. Methods: In silico phylogenetic analysis of bsh protein sequences of L. sphaericus and Bacillus sp. were carried out. The tertiary structure prediction and molecular docking of L. sphaericus bsh protein were performed against different substrates and inhibitors. Results: L. sphaericus PKA17 was able to tolerate and hydrolyse bile salt. L. sphaericus bsh proteins share close phylogenetic affinity and showed sequential divergence with Bacillus species despite their structural resemblance. Their active site was also conserved. Theaflavin, gossypetin and phenethyl caffeate were found as potent active site inhibitors. Conclusion: L. sphaericus PKA17 has the potency to be used as successful probiotic. In silico studies with L. sphaericus and Bacillus sp. bsh protein showed structural and functional conservancy. The inhibitors of this bsh protein like theaflavin, gossypetin and phenethyl caffeate must be avoided during fish feed preparation while applying L. sphaericus as probiotics. [ABSTRACT FROM AUTHOR]

Bile salt hydrolase (BSH), found commonly in intestinal species of Lactobacillus and Bifidobacterium, catalyzes the hydrolysis of glycine or taurine-conjugated bile acids into the amino acids and free bile acids. Deconjugated bile acids potentially play an important role in the reduction of blood cholesterol level and formation of some gastrointestinal diseases such as cholestasis, gallstone formation, and colon cancer. Although the crystal and three-dimensional structures of BSH enzyme are known, the working mechanism of catalytic activity of such an important BSH enzyme is not known very well. Previous in silico analysis of multiple BSH has identified that Arginine-16 (R16) and Aspartate-19 (D19) were catalytically important residues in the active site of BSH. To confirm the function of these amino acids, in this study, BSH enzyme from Lactobacillus plantarum B14 strain was cloned into Escherichia coli and strictly conserved polar R16 and D19 amino acids of BSH enzyme were substituted for hydrophobic Phenylalanine-16 (F16) and Leucine-19 (L19) amino acids, respectively, by polymerase chain reaction (PCR)-based site-directed mutagenesis. The effects of the mutations on catalytic activity and structure of the BSH enzymes were detected by ninhidrin assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. Research results showed that although F16 mutation led to loss of enzyme activity completely, L19 mutation led to abolishment of the synthesis of BSH enzyme. These results indicated that R16 and D19 amino acids located in loop I of the BSH enzyme might be critical for catalytic activity and assembly of the BSH enzyme respectively. [ABSTRACT FROM AUTHOR]

Lactobacillus gasseri is one of the most likely probiotic candidates among many Lactobacillus species. Although bile salt resistance has been defined as an important criterion for selection of probiotic candidates since it allows probiotic bacteria to survive in the gut, both its capability and its related enzyme, bile salt hydrolase (BSH), in L. gasseri is still largely unknown. Here, we report that the well-known probiotic bacterium L. gasseri JCM1131T possesses BSH activity and bile salt resistance capability. Indeed, this strain apparently showed BSH activity on the plate assay and highly tolerated the primary bile salts and even taurine-conjugated secondary bile salt. We further isolated a putative BSH enzyme (LagBSH) from strain JCM1131T and characterized the enzymatic function. The purified LagBSH protein exhibited quite high deconjugation activity for taurocholic acid and taurochenodeoxycholic acid. The lagBSH gene was constitutively expressed in strain JCM1131T, suggesting that LagBSH likely contributes to bile salt resistance of the strain and may be associated with survival capability of strain JCM1131T within the human intestine by bile detoxification. Thus, this study first demonstrated the bile salt resistance and its responsible enzyme (BSH) activity in strain JCM1131T, which further supports the importance of the typical lactic acid bacterium as probiotics.

Bile salt hydrolase (BSH) activity in probiotic strains is usually correlated with the ability to lower serum cholesterol levels in hypercholesterolemic patients. The objective of this study was the evaluation of BSH in five probiotic strains of lactic acid bacteria (LAB) and a probiotic yeast. The activity was assessed using a qualitative direct plate test and a quantitative high-performance thin- layer chromatography assay. The six strains differed in their BSH substrate preference and activity. Lactobacillus plantarum DGIA1, a potentially probiotic strain isolated from a double cream cheese from Chiapas, Mexico, showed excellent deconjugation activities in the four tested bile acids (69, 100, 81, and 92% for sodium glycocholate, glycodeoxycholate, taurocholate, and taurodeoxycholate, respectively). In the case of the commercial probiotic yeast Saccharomyces boulardii, the deconjugation activities were good against sodium glycodeoxycholate, taurocholate, and taurodeoxycholate (100, 57, and 63%, respectively). These last two results are part of the novelty of the work. A weak deconjugative activity (5%) was observed in the case of sodium glycocholate. This is the first time that the BSH activity has been detected in this yeast.

Bile salt hydrolase (BSH) and penicillin V acylase (PVA) are related enzymes that are classified as choloylglycine hydrolases (CGH). BSH enzymes have attracted significant interest for their ability to modulate the composition of the bile acid pool, alter bile acid signaling events mediated by the host bile acid receptors FXR and TGR5 and influence cholesterol homeostasis in the host, while PVA enzymes have been widely utilised in an industrial capacity in the production of semi-synthetic antibiotics. The similarities between BSH and PVA enzymes suggest common evolution of these enzymes and shared mechanisms for substrate binding and catalysis. Here, we compare BSH and PVA through analysis of the distribution, phylogeny and biochemistry of these microbial enzymes. The development of new annotation approaches based upon functional enzyme analyses and the potential implications of BSH enzymes for host health are discussed.

Lactobacillus johnsonii, as a microorganism widely presenting in the intestinal tract of mammals, dairy products, and poultry, possesses various probiotic characteristics. Currently, many research focused on the probiotic functions and industrial applications of L. johnsonii. In this study, 98 strains of L. johnsonii were subjected to comparative genomic analyses to explore its genetic diversity. The results indicated that based on ANI values, d-DDH values, phylogenetic analysis, and whole-genome comparison, the 98 strains of L. johnsonii were classified into two phylogenetic clusters. The carbohydrate utilization genes, bacteriocin operon, extracellular polysaccharide gene cluster, and bile salt hydrolase encoding genes in L. johnsonii shared common features and exhibited systematic differences based on phylogenetic clustering. The main reason for the high intra-species gene diversity of L. johnsonii might be differences in phages and other mobile genetic elements. This study provides new insights into the genomic features and functional genes of L. johnsonii. [ABSTRACT FROM AUTHOR]

The hypocholesterolaemic effects of milk fermented by Lactobacillus casei F0822 with bile salt hydrolase activity were investigated in hamsters fed a cholesterol-rich diet. Fermented milk (FM) exhibited dose-dependent hypocholesterolaemic activity. Sterilized and low-dose FM did not significantly affect the lipid profile of hamsters, whereas high-dose FM significantly reduced the levels of serum total cholesterol (TC) and non-high-density lipoprotein cholesterol (non-HDL-C), as well as hepatic TC and esterified cholesterol (EC). The daily faecal total bile acid excretion was significantly negatively correlated with the serum TC and non-HDL-C, as well as with the hepatic TC and EC. High-dose FM significantly enhanced daily faecal total bile acid excretion, which attributed to the hydrolysis of conjugated bile acids and the binding of free bile acids by the cells of strain F0822. These data suggest that the FM may serve as a functional food and has the potential to regulate cholesterol metabolism in humans.

The human gut microbiota impacts host metabolism and has been implicated in the pathophysiology of obesity and metabolic syndromes. However, defining the roles of specific microbial activities and metabolites on host phenotypes has proven challenging due to the complexity of the microbiome-host ecosystem. Here, we identify strains from the abundant gut bacterial phylum Bacteroidetes that display selective bile salt hydrolase (BSH) activity. Using isogenic strains of wild-type and BSH-deleted Bacteroides thetaiotaomicron, we selectively modulated the levels of the bile acid tauro-β-muricholic acid in monocolonized gnotobiotic mice. B. thetaiotaomicron BSH mutant-colonized mice displayed altered metabolism, including reduced weight gain and respiratory exchange ratios, as well as transcriptional changes in metabolic, circadian rhythm, and immune pathways in the gut and liver. Our results demonstrate that metabolites generated by a single microbial gene and enzymatic activity can profoundly alter host metabolism and gene expression at local and organism-level scales.

ABSTRACT Various Lactobacillus species have been reported to deconjugate bile acids in the gastrointestinal tract (GIT) through the action of bile salt hydrolase (BSH) proteins. This function contributes to altering the gut microbiota composition and bile metabolism and detoxification and to lowering cholesterol levels. Here, we investigated the Lactobacillus BSH repertoire across 170 sequenced species. We used hidden Markov models to distinguish between BSH and closely related penicillin-V acylase (PVA) proteins. Even though BSH and PVA proteins have very different target substrates, they share high sequence similarity and are often misannotated. We determined that 82/170 (48.24%) species encoded PVA proteins, 39/170 (22.94%) species encoded BSH proteins, and 8/170 (4.71%) species encoded both BSH and PVA proteins, while 57/170 (33.53%) species encoded neither. Mapping the occurrence of BSH-encoding species onto a phylogenetic tree revealed that BSH-encoding lactobacilli primarily adopt the vertebrate-adapted lifestyle but not the environmental or plant-associated subsets. Phylogenetic analysis of the BSH sequences revealed two distinct clades, several conserved motifs, and the presence of six previously reported active-site residues. These data will guide future mechanistic studies of BSH activity and contribute to the development and selection of BSH-encoding Lactobacillus strains with therapeutic potential. IMPORTANCE Bile acids play an integral role in shaping the gut microbiota and host physiology by regulating metabolic signaling, weight gain, and serum cholesterol and liver triglyceride levels. Given these important roles of bile acids, we investigated the presence of bile salt hydrolase (BSH) in Lactobacillus genomes representing 170 different species, determined strain- and species-specific patterns of occurrences, and expanded on the diversity of the BSH repertoire in this genus. While our data showed that 28% of Lactobacillus species encode BSH proteins, these species are associated mainly with vertebrate-adapted niches, demonstrating selective pressure on lactobacilli to evolve to adapt to specific environments. These new data will allow targeted selection of specific strains of lactobacilli and BSH proteins for future mechanistic studies to explore their therapeutic potential for treating metabolic disorders.

The gut microbiota continues to fascinate scientists in many realms when it is considered that humans contain 90% bacteria. Correlations between changes in composition and activity of the gut microbiota and common disorders such as cancer, hypertension, hypercholesterolemia, inflammatory bowel diseases, obesity, oral health, etc. have been proposed. What is the real role of probiotics, prebiotics and synbiotics in influencing a healthy microbiota? Both in vitro evidences and in vivo clinical data have supported some of these new health claims, while recent molecular advancement has provided strong indications to support and justify the hypotheses. However, probiotics validity and health claims have continuously been rejected on the basis of “biomarker deficiency”. To battle the increase in health care costs, a preventive approach to medicine with the development of probiotics and prebiotics or symbiotic products is being advanced. This review discusses the potential beneficial effects of probiotics in preventing and treating certain diseases as well as current and future perspectives of probiotic research.

Cardiovascular diseases (CVDs) have become one of the leading causes of global mortality. The raised serum cholesterol levels and its progressive accumulation in arterial walls results in development of atherosclerosis that leads to most of the heart attacks and strokes. Use of probiotics has been implicated for several health benefits including their cholesterol lowering potential and hence management of CVDs. The current review aims to describe the association of gut microbiota, probiotics and their potential mechanisms responsible for hypocholesterolemic effects. Probiotics execute hypocholesterolemic effects through several mechanisms such as bile salt hydrolase activity (BSH), deconjugation of bile salts, cholesterol assimilation, coprecipitation of cholesterol with deconjugated bile salts, removal of cholesterol with cellular surfaces through physical forces, incorporation of cholesterol in the cell membrane of the probiotics, intestinal conversion of cholesterol in coprostanol, and inhibition of Niemann-Pick C1 like 1 (NPC1L1) cholesterol transporter in the enterocytes. However, the health benefits including the hypocholesterolemic effects appear to be a strain specific phenomenon. Further studies are necessary for better understanding of the in-depth molecular mechanisms governing the hypocholesterolemic effects and to establish the probiotics as a potential non-pharmaceutical approach for management of CVDs. [ABSTRACT FROM AUTHOR]

It is generally considered that bile salt hydrolase (BSH) activity is hardly detected in nonintestinal lactic acid bacteria (LAB). The aim of this study was to investigate the distribution and intensity of BSH activity in LAB isolated from naturally fermented vegetables and milk. A total of 624 lactic acid bacterial strains classified into 6 genera and 50 species were isolated from 144 naturally fermented vegetable samples and 103 naturally fermented milk samples, and their BSH activity was screened by gas chromatography with electron capture detection. The BSH-positive strains were further analyzed quantitatively for their deconjugation ability against six human-conjugated bile salts by HPLC based on the disappearance of the conjugated bile salts from the reaction mixture. The results showed that 39% of the strains possessed BSH activity distributed in 24 lactic acid bacterial species. The strains of the fermented vegetable origin showed a 0.5-fold higher incidence of BSH-positive strains than those of the fermented milk origin, and the lactic acid bacilli exhibited 2.5-fold higher incidence of BSH-positive strains than the lactic acid cocci in general. The strains of the fermented vegetable origin generally had greater bile salt deconjugation ability than those of the fermented milk origin. More than 97% and 93% of the BSH-positive strains exhibited a greater substrate preference for glycoconjugated bile salts than tauroconjugated bile salts and for dihydroxy bile salts than trihydroxy bile salts, respectively. This study demonstrated that BSH activity was also present in nonintestinal LAB. [ABSTRACT FROM AUTHOR]

Summary: Emerging scientific evidence suggests that the supplementation of probiotics may help to reduce/manage blood cholesterol levels in humans. We evaluated the in vitro cholesterol‐lowering activity of the probiotic strain Bacillus coagulansMTCC 5856. This probiotic de‐conjugated bile salts and liberated deoxycholic acid, confirming its bile salt hydrolase activity. Furthermore, B. coagulansMTCC 5856 also significantly (P < 0.05) reduced cholesterol levels in culture media under growing (48.42%), resting (live but suspended in buffer, 36.47%) and even heat‐killed (dead, 8.5%) conditions. Bacillus coagulansMTCC 5856 significantly reduced the cholesterol levels in cholesterol‐rich foods, such as egg yolk (39.79%), chicken liver (45.44%) and butter (49.51%), when incubated for 24 h in conditions mimicking the in vivo environment. Bacillus coagulansMTCC 5856 also produced significant (P < 0.05) amounts of propionic acid and butyric acid while fermenting cholesterol‐rich foods. The multitudinous ways by which B. coagulansMTCC 5856 reduces cholesterol levels endorses its application in functional food formulations and as a dietary ingredient for the management of hypercholesterolemia, potentially reducing the incidence of coronary heart disease and other related disabilities. The probiotic bacteria Bacillus coagulans MTCC 5856 exhibits in vitro cholesterol‐lowering activity via different mechanisms, including bile salt de‐conjugation, cholesterol removal, cholesterol binding to resting and dead cells and the production of short‐chain fatty acids. Further, B. coagulans MTCC 5856 helped remove cholesterol from cholesterol‐rich foods (egg yolk, liver and butter) under a hostile GI environment, thereby suggesting its ability to function in vivo. The findings reported in this study provide clear evidence of the effectiveness of the probiotic strain B. coagulans MTCC 5856 in the nutritional treatment of diet‐induced hypercholesterolemia. [ABSTRACT FROM AUTHOR]

Bile acids (BA), the major components of bile, are biological detergents that facilitate the emulsification and solubilization of dietary lipids and also display potent antimicrobial activity, the bacterial membranes being their main targets. Considering the complicated nature of the stress produced by bile and BA, the microorganism tolerance requires different defence mechanisms including the presence of efflux pumps, bile salt hydrolase (BSH) enzyme, the intrinsic capacity of cells to maintain intracellular homeostasis and modifications in the architecture and composition of the cell membrane. Besides, the expression of proteins involved in carbohydrate and fatty acid metabolism, amino acid and nitrogenous base biosynthesis, and general stress response are commonly affected by the presence of bile. Among the microbial transformations, deconjugation of BA by BSH is the most important. Several studies indicate that BSH activity affects both the host physiology and the microbiota. In fact, it was strongly suggested that BSH could play an important role in the colonization and survival of bacteria in the gut. Also, recent work has shown that BSH and free BA participate in a variety of metabolic processes that include regulation of dietary lipid absorption, cholesterol metabolism, and energy and inflammation homeostasis. In this review we summarize recent advances in the understanding of the mechanisms involved in the tolerance of bacteria to bile, with special emphasis on the contributions of studies applying an omic approach. Besides, the physiological and ecological role of BSH enzyme and its relevance to human health as well as the function of bile acid as metabolic regulator are also discussed. [ABSTRACT FROM AUTHOR]

The worldwide trend of limiting the use of antibiotic growth promoters (AGPs) in animal production creates challenges for the animal feed industry, thus necessitating the development of effective non‐antibiotic alternatives to improve animal performance. Increasing evidence has shown that the growth‐promoting effect of AGPs is highly correlated with the reduced activity of bile salt hydrolase (BSH, EC 3.5.1.24), an intestinal bacteria‐producing enzyme that has a negative impact on host fat digestion and energy harvest. Therefore, BSH inhibitors may become novel, attractive alternatives to AGPs. Detailed knowledge of BSH substrate preferences and the wealth of structural data on BSHs provide a solid foundation for rationally tailored BSH inhibitor design. This review focuses on the relationship between structure and function of BSHs based on the crystal structure, kinetic data, molecular docking and comparative structural analyses. The molecular basis for BSH substrate recognition is also discussed. Finally, recent advances and future prospectives in the development of potent, safe, and cost‐effective BSH inhibitors are described. [ABSTRACT FROM AUTHOR]

A bile salt hydrolase (bsh) enzyme deconjugate taurine and glycine-linked bile salts. Because of the strong implications between deconjugation of tauro- or gluco-conjugated salts and its positive or negative health consequences, the characterization of the bsh enzymes is important. bshs from different lactobacilli species, even strains, exhibit higher variation in sequence, kinetic properties, and substrate specificity. In the present study four bsh related genes from Lactobacillus plantarum GD2 strain were cloned and expressed in Escherichia coli BLR (DE3) strain. Amino acid residues of recombinant bshs were analyzed and their deconjugation abilities were tested with six human conjugated bile salts. Results indicated that the genetic distance among four related bsh genes in Lb. plantarum is far from each other and bsh2-4 enzymes share significant sequence homology specifically with penicillin V acylase (PVA) family members. Biochemical and the in silico analysis suggest that the bsh1 enzyme is a member of the bsh family while bsh2-4 enzymes are members of the PVA family. [ABSTRACT FROM AUTHOR]

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera ( Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo . Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol. [ABSTRACT FROM AUTHOR]

Bile Salt Hydrolase (BSH), a member of Cholylglycine hydrolase family, catalyzes the de-conjugation of bile acids and is evolutionarily related to penicillin V acylase (PVA) that hydrolyses a different substrate such as penicillin V. We report the three-dimensional structure of a BSH enzyme from the Gram-positive bacteria Enterococcus faecalis ( Ef BSH) which has manifold higher hydrolase activity compared to other known BSHs and displays unique allosteric catalytic property. The structural analysis revealed reduced secondary structure content compared to other known BSH structures, particularly devoid of an anti-parallel β-sheet in the assembly loop and part of a β-strand is converted to increase the length of a substrate binding loop 2. The analysis of the substrate binding pocket showed reduced volume owing to altered loop conformations and increased hydrophobicity contributed by a higher ratio of hydrophobic to hydrophilic groups present. The aromatic residues F18, Y20 and F65 participate in substrate binding. Thus, their mutation affects enzyme activity. Docking and Molecular Dynamics simulation studies showed effective polar complementarity present for the three hydroxyl (–OH) groups of GCA substrate in the binding site contributing to higher substrate specificity and efficient catalysis. These are unique features characteristics of this BSH enzyme and thought to contribute to its higher activity and specificity towards bile salts as well as allosteric effects. Further, mechanism of autocatalytic processing of Cholylglycine Hydrolases by the excision of an N-terminal Pre-peptide was examined by inserting different N-terminal pre-peptides in Ef BSH sequence. The results suggest that two serine residues next to nucleophile cysteine are essential for autocalytic processing to remove precursor peptide. Since pre-peptide is absent in Ef BSH the mutation of these serines is tolerated. This suggests that an evolution-mediated subordination of the pre-peptide excision site resulted in loss of pre-peptide in Ef BSH and other related Cholylglycine hydrolases. [ABSTRACT FROM AUTHOR]

Bile salt hydrolase (BSH) active probiotic strains are being used in the treatment of hypercholesterolemia related diseases. Understanding the mechanism of action of the BSH of probiotics is crucial. Even though Lactobacillus gasseri is probably one of the most beneficial probiotics on the market, there is no information on the catalytic activity and substrate preferences of its BSH. This study aims to detect the substrate specificity of the BSH of Lb. gasseri ATCC 33323 strain, to determine the optimum temperature and pH of the BSH's activity by ninhydrin assay. To do so, the BSH gene from Lb. gasseri ATCC 33323 was cloned into Escherichia coli XL1-Blue strain, expressed and characterized in E. coli BLR(DE3). Results indicated that even though the recombinant BSH (rBSH) was able to hydrolyze the six major human bile salts, there was an obvious preference for the glycine-conjugated bile salts and the maximum activities of the rBSH were recorded. [ABSTRACT FROM AUTHOR]

[Display omitted] • Synthesis of the conjugate of cholic acid with a coumarin fluorescent reporter and characterization as a bile salt hydrolase substrate. • Investigation of 7-nitrobenzo[ c ][1,2,5]oxadiazole as a scavenger for taurine, formed by bile salt hydrolase. • Establishment of a pre-column derivatization-based HPLC method to monitor activity of bile salt hydrolases. The bile acid pool has a profound impact on human health and disease. The intestinal microbiota initiates the metabolism of conjugated bile acids through a critical first step catalyzed by bacterial bile salt hydrolase (BSH) and provides unique contributions to the diversity of bile acids. There has been great interest in surveying BSH activity. We compared two substrates with either 2-(7-amino-4-methyl-coumarinyl)acetic acid or 7-amino-4-methyl-coumarin as fluorescent reporters of BSH activity. The BSH-catalyzed conversion of the natural substrate taurocholic acid was followed through an HPLC-based assay by applying 7-nitrobenzo[ c ][1,2,5]oxadiazole as scavenger for taurine, released in the enzymatic reaction. Hence, a new opportunity to monitor the activity of bile salt hydrolases was introduced. [ABSTRACT FROM AUTHOR]

A correlation between the use of antibiotics in early life and the excessive weight gain in later childhood has been shown in several large cohort studies all over the world. One hypothesis explaining this association is the pervasive impact that antibiotics may have on the intestinal microbiota, and this has been supported by recent mouse studies. Studies have shown dramatic changes in the intestinal microbiota of adults in response to oral antibiotic treatments. However, little is known about the impact of antibiotics on the intestinal microbiota of children, although antibiotics account for the majority of the medication prescribed to children in Western countries.

Probiotic bacteria are beneficial to the health of poultry animals, thus are used as alternative candidates for antibiotics used as growth promoters (AGPs). However, they also reduce the body weight gain due to innate bile salt hydrolase (BSH) activity. Hence, the addition of a suitable BSH inhibitor along with the probiotic feed can decrease the BSH activity. In this study, a BSH gene (981 bp) encoding 326-amino acids was identified from the genome of Lactobacillus gasseri FR4 (LgBSH). The LgBSH-encoding gene was cloned and purified using an Escherichia coli BL21 (DE3) expression system, and its molecular weight (37 kDa) was confirmed by SDS–PAGE and a Western blot analysis. LgBSH exhibited greater hydrolysis toward glyco-conjugated bile salts compared to tauro-conjugated bile salts. LgBSH displayed optimal activity at 52°C at a pH of 5.5, and activity was further increased by several reducing agents (DTT), surfactants (Triton X-100 and Tween 80), and organic solvents (isopropanol, butanol, and acetone). Riboflavin and penicillin V, respectively, inhibited LgBSH activity by 98.31 and 97.84%. A homology model of LgBSH was predicted using EfBSH (4WL3) as a template. Molecular docking analysis revealed that the glycocholic acid had lowest binding energy of -8.46 kcal/mol; on the other hand, inhibitors, i.e., riboflavin and penicillin V, had relatively higher binding energies of -6.25 and -7.38 kcal/mol, respectively. Our results suggest that L. gasseri FR4 along with riboflavin might be a potential alternative to AGPs for poultry animals.

Bile salt hydrolase (BSH), a widely distributed function of the gut microbiota, has a profound impact on host lipid metabolism and energy harvest. Recent studies suggest that BSH inhibitors are promising alternatives to antibiotic growth promoters (AGP) for enhanced animal growth performance and food safety. Using a high-purity BSH from Lactobacillus salivarius strain, we have identified a panel of BSH inhibitors. However, it is still unknown if these inhibitors also effectively inhibit the function of the BSH enzymes from other bacterial species with different sequence and substrate spectrum. In this study, we performed bioinformatics analysis and determined the inhibitory effect of identified BSH inhibitors on a BSH from L. acidophilus. Although the L. acidophilus BSH is phylogenetically distant from the L. salivarius BSH, sequence analysis and structure modeling indicated the two BSH enzymes contain conserved, catalytically important amino residues and domain. His-tagged recombinant BSH from L. acidophilus was further purified and used to determine inhibitory effect of specific compounds. Previously identified BSH inhibitors also exhibited potent inhibitory effects on the L. acidophilus BSH. In conclusion, this study demonstrated that the BSH from L. salivarius is an ideal candidate for screening BSH inhibitors, the promising alternatives to AGP for enhanced feed efficiency, growth performance and profitability of food animals.

The effects of a Tween 80 and CaCl2 mixture on the hypocholesterolemic activity of bile salt hydrolase (BSH)-active Lactobacillus casei F0422 were investigated in rats fed a cholesterol-rich diet for 3 weeks. This mixture significantly enhanced the hypocholesterolemic activity of strain F0422 in rats. The group administered drinking water supplemented with both strain F0422 and the Tween 80 and CaCl2 mixture had significantly lower levels of total serum cholesterol, low-density lipoprotein cholesterol and total liver cholesterol than did the group administered drinking water supplemented only with strain F0422 or the Tween 80 and CaCl2 mixture. The hypocholesterolemic activity of the combined strain F0422 and Tween 80 and CaCl2 mixture was attributed to the increase in fecal bile acid excretion through enhancement of bile salt tolerance and BSH catalytic activity of strain F0422 by Tween 80 and up-regulation of the adhesion of strain F0422 to intestinal epithelial cells by CaCl2.

A new in vitro method was developed to determine the bile tolerance of potentially probiotic lactobacilli. The overnight culture of various lactobacilli strains was inoculated into sterile, half-strength MRS broth supplemented with and without 0.3% (wt/vol) oxgall, buffered with 0.1 M sodium phosphate buffer at a final pH of 7.3, and incubated at 37 °C for 12 h under anaerobic conditions. The bile tolerance ability of the lactobacilli strains was expressed as the percentage of the propagation generations of the bacterial cells in the presence of oxgall to those in the absence of oxgall. The bile tolerance ability of 11 strains of 8 Lactobacillus species, including 3 bile salt hydrolase (BSH)-negative strains and 8 BSH-positive strains, was analyzed using the newly developed method and two traditional methods. The results showed that bile tolerance ability of the strains was considerably different depending on the analysis method used. The newly developed method mimics the physiological environment of the human small intestine, and avoids changes in pH and bile salt composition during the incubation period, which are drawbacks of the traditional bile tolerance test methods. Therefore, the analysis method developed in this study is more suitable to screen or compare the bile tolerance ability of lactobacilli strains. [ABSTRACT FROM AUTHOR]

Samples of fermented herbs were used to isolate lactic acid bacteria (LAB). Of a total of 19 isolates, eight were resistant both to gastric acid and bile salts (glycocholic acid, GCA; taurocholic acid, TCA; glycodeoxycholic acid, GDCA; and taurodeoxycholic acid, TDCA). Most isolates exhibited a pH-dependent surface hydrophobicity: a pH of 4 conferred a greater hydrophobicity compared to a pH of 7. Based on the hydrophobicity characteristics, the LAB isolate WU-P19 from the traditional fermented herb Oroxylum indicum was selected for further study. WU-P19 was identified as Lactobacillus plantarum WU-P19. The presence of bile salts GCA and GDCA in the culture medium induced production of the relevant bile salt hydrolase. Relative to controls, the presence of the bile salts in the culture medium affected the carbon and nitrogen contents of the cells and their hydrophobicity. Cells grown in a medium free of bile salts were morphologically different to cells grown in the presence of GCA and GDCA. WU-P19 was resistant to several antibiotics. It produced β-galactosidase and inhibited growth of the tested pathogenic bacteria at various levels. In vitro, L. plantarum WU-P19 adapted well to conditions typical of the various zones of the human gastrointestinal tract. In view of the promising results, in vivo evaluations are planned for the isolate WU-P19. [ABSTRACT FROM AUTHOR]

Clostridium difficile infection (CDI) is one of the most common nosocomial infections. Dysbiosis of the gut microbiota due to consumption of antibiotics is a major contributor to CDI. Recently, fecal microbiota transplantation (FMT) has been applied to treat CDI. However, FMT has important limitations including uncontrolled exposure to pathogens and standardization issues. Therefore, it is necessary to evaluate alternative treatment methods, such as bacteriotherapy, as well as the mechanism through which beneficial bacteria inhibit the growth of C. difficile. Here, we report bile acid-mediated inhibition of C. difficile by Bacteroides strains which can produce bile salt hydrolase (BSH). Bacteroides strains are not commonly used to treat CDI; however, as they comprise a large proportion of the intestinal microbiota, they can contribute to bile acid-mediated inhibition of C. difficile. The inhibitory effect on C. difficile growth increased with increasing bile acid concentration in the presence of Bacteroides ovatus SNUG 40239. Furthermore, this inhibitory effect on C. difficile growth was significantly attenuated when bile acid availability was reduced by cholestyramine, a bile acid sequestrant. The findings of this study are important due to the discovery of a new bacterial strain that in the presence of available bile acids inhibits growth of C. difficile. These results will facilitate development of novel bacteriotherapy strategies to control CDI. [ABSTRACT FROM AUTHOR]

Background: Probiotics is used as a promising approach in the prevention and treatment of hypercholesterolemia. Modification of bile acid metabolism through the deconjugation of bile salts by microbial bile salt hydrolase (BSH) is considered to be the core mechanism of the hypocholesterolemic effects of probiotics. Nevertheless, BSH activity is reported to be detrimental to the human host due to the generation of toxic secondary bile acids. Thus, the influence of probiotic intake on bile acid metabolism needs to be elucidated. We analyzed the bile acid levels and microbiota in human fecal samples after probiotic supplementation to assess the influence of probiotic intake on fecal bile acid levels. Two patients hospitalized for schizophrenia and dyslipidemia, receiving an atypical antipsychotic drug, were enrolled in this study (Subjects A and B). Both subjects received Lactobacillus rhamnosus GG (LGG) for 4 weeks, and no probiotics for the following 4 weeks. Fecal samples were collected at baseline and after 4 and 8 weeks. Results: Conjugated bile acids may be modified by indigenous intestinal bacteria into unconjugated bile acids and secondary bile acids through deconjugation reactions by BSH activity and the subsequent 7a-dehydroxylation pathway, respectively. In the fecal microbiota from Subject A, the relative abundance of Bifidobacterium increased after LGG supplementation (30%-49%). Most Bifidobacterium and Lactobacillus strains that colonize mammalian intestines may report BSH activity, and in general bifidobacteria reveals a higher BSH activity than lactobacilli. The fecal unconjugated bile acid and secondary bile acid levels in Subject A increased after the LGG supplementation (0.36-1.79 and 1.82-16.19 µmol/g respectively). Although the LGG supplementation appears to promote bile acid deconjugation, most of the unconjugated bile acids in Subject A appear to have been modified into secondary bile acids. Alternatively, in Subject B there were no significant changes throughout the study. Conclusion: We observed a significant increase in the fecal secondary bile acid levels after probiotic administration in one of our cases. Further studies are needed to elucidate the factors affecting 7a-dehydroxylation of bile acids to confirm the safety of using probiotics. [ABSTRACT FROM AUTHOR]

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Bile salt hydrolase (BSH) enzyme, commonly found in probiotic bacteria of gut origin, catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts allowing for colonization of the bacteria in the gut and contributing to a decrease in levels of cholesterol. However an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid can have harmful side-effects. The aim of this study was to characterize the activity of BSH enzymes from Lactobacillus rhamnosus E9, a popular probiotic strain. The bsh gene was cloned, expressed, purified and characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activities and substrate specificities of the recombinant BSH (rBSH) enzyme were examined using six different bile acids. Nucleotide sequence analysis results indicated that the bsh of E9 contained an open reading frame (ORF) of 1014 and nucleotides encoding a 338-amino acid protein with a molecular weight of 37 kDa. Five catalytically important amino acids and the amino acid motifs located around the active site were highly conserved. The rBSH showed a slight preference towards glycine-conjugated to tauro-conjugated bile salts. This confirms that it is a safe strain for probiotics and its preference for glycine-conjugated bile salts should be further investigated. [ABSTRACT FROM AUTHOR]

• Metabolites of loganin were generated in the intestine and liver, respectively. • 11 unstable dialdehyde intermediates of loganin were formed. • Dialdehyde intermediates were mainly formed in the intestine. • Dialdehyde intermediates improved intestinal bile salt hydrolase activities. • Loganin and its metabolites underwent severe in-source fragmentation. Loganin is an iridoid with potent pharmacological effects. Loganin contains a hemiacetal structure and can convert to dialdehyde intermediates after deglycosylation. We hypothesized that the metabolites of loganin with hemiacetal can generate reactive dialdehyde intermediates. This study aims to characterize the metabolic profiling of loganin and especially for the unstable dialdehyde intermediates by using ultra-performance liquid chromatograph-quadrupole orbitrap mass spectrometry. In this study, a total of 26 stable metabolites were identified in loganin-treated rats. Loganin underwent different metabolism in the intestine and liver, which was confirmed mainly by the metabolites in the hepatic portal vein. In the intestine, the major metabolic pathways were ester hydrolysis and deglycosylation, followed by methylation and dehydrogenation. The hepatic metabolism pathways were hydrogenation, hydroxylation, glucuronidation, and sulfonation. The circulating metabolites with high abundance were mainly derived from intestinal metabolism. Importantly, 11 unstable dialdehyde intermediates of loganin were identified and described for the first time. The dialdehyde intermediates were identified by their dihydropyridine conjugates with amino acids. The dialdehyde intermediates were mainly produced in the intestine. The dialdehyde intermediates enable covalent modification of intestinal proteins. Loganin can up-regulate the activity of intestinal bile salt hydrolase (BSH), catalyzing bile acid metabolism. The level of protein adducts was positively associated with BSH activity, indicating dialdehyde intermediates played a key role in the up-regulation of BSH activities. In conclusion, this study not only demonstrates the characteristic metabolic fate of loganin but also facilitates the understanding of the pharmacologic effects of dialdehyde intermediates. [ABSTRACT FROM AUTHOR]