Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100

Abstract

We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving ['4C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three-to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 ,umol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two-to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 ,Lmol/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively.

Allelic Variation of Bile Salt Hydrolase Genes in Lactobacillus salivarius Does Not Determine Bile Resistance Levels

“…The enzymatic properties of the more active Bsh1 protein of JCM1046 were determined for comparison with those of other bacterial Bsh enzymes. The K m of L. salivarius Bsh1 JCM1046 for TCA (2.02 mM) is higher than those from L. johnsonii (0.76 mM [37]) and B. longum (1.12 mM [29]), suggesting that TCA is a better substrate for these enzymes. However, the C-terminal His tag present on the Bsh1 JCM1046 enzyme may affect the affinity for the substrate.…”

Hypocholesterolaemic Effects of Probiotics

“…The peptide-like bond between the bile acid and taurine or glycine is not cleaved by most proteolytic enzymes, but it is by bile salt hydrolase (BSH), an intracellular enzyme [99]. Deconjugation of bile salts in vitro, has been demonstrated by intestinal bacteria, such as Clostridium [100], Lactobacillus [101], Streptococcus [101], and Bifidobacterium [100].…”

Comparison of Acid and Bile Tolerances, Cholesterol Assimilation, and CLA Production in Probiotic Lactobacillus acidophilus Strains

“…Deconjugation of bile acids may play an important role in host serum cholesterol levels since deconjugated bile acids do not function as well as conjugated bile acids in the solublization and absorption of lipids. Bile salt hydrolase (BSH) catalyzes the hydrolysis of conjugated bile acids to produce free bile acids and amino acids (Lundeen and Savage, 1990 …”