• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.838-845
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• 2011

The present work describes the identification, purification, and characterization of bile salt hydrolase (BSH) from Bifidobacterium animalis subsp. lactis. The enzyme was purified to electrophoretic homogeneity by hydrophobic chromatography, ion-exchange chromatography and ultrafiltration. SDS-PAGE analysis of putative BSH and gel filtration revealed that the analyzed protein is presumably a tetramer composed of four monomers each of about 35 kDa. The purified enzyme was analyzed by liquid chromatography coupled to LTQ FT ICR mass spectrometry and unambiguously identified as a bile salt hydrolase from B. animalis. The isoelectric point of the studied protein was estimated to be around pH 4.9. The pH optimum of the purified BSH is between 4.7 to 6.5, and the temperature optimum is around 50oC. The BSH of B. animalis could deconjugate all tested bile salts, with clear preference for glycine-conjugated bile salts over taurine-conjugated forms. Genetic analysis of the bsh showed high similarity to the previously sequenced bsh gene from B. animalis and confirmed the usefulness of bile salt hydrolase as a genetic marker for B. animalis identification.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.10
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• pp.1505-1512
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• 2005

Bile salt deconjugation is the most biologically significant reaction among the bacterial alterations of bile acids in the gastrointestinal tract of human and animal. The responsible enzyme, bile salt hydrolase (BSH), catalyzes the hydrolysis of glycineand/or taurine-conjugated bile salts into amino acid residues and deconjugated bile acids. Herein we review current knowledge on the distribution of BSH activity among various microorganisms with respect to their biochemical and molecular characteristics. The proposed physiological impact of BSH activity on the host animal as well as on the BSH-producing bacterial cells is discussed. BSH activity of the probiotic strains is examined on the basis of BSH hypothesis, which was proposed to explain cholesterol-lowering effects of probiotics. Finally, the potential applications of BSH research are briefly discussed.

• Journal of Dairy Science and Biotechnology
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• v.29 no.1
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• pp.49-54
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• 2011

Bile salt hydrolase (BSH, EC 3.5.1.24) activity, which cleaves amide bond between carboxyl group (bile acid) and amino group (glycine or taurine), is commonly detected in gut-associated species of human and animal. During the screening of BSH active strains from the fecal samples of elderly human volunteers, strain CU30-2 was isolated on the basis of the highly active BSH producing activity. A bsh gene of the isolate was cloned into the pET22b expression vector and overexpressed in Escherichia coli BL21 (DE3) Gold by induction with 1mM IPTG. The overexpressed BSH enzyme with 6x His-tag was purified with apparent homogeneity using a $Ni^+$-NTA agarose column and characterized. The BSH enzyme of E. faecalis CU30-2 exhibited approximately 50 times higher activity against glycol-conjugated bile salts than tauro-conjugated bile salts having the highest activity against glycocholic acid. Considering the prevalence of E. faecalis strains in the human GI tract and glycol-conjugates dominated bile acid composition of human bile, further study is needed to investigate the impact of the BSH activity exerted by E. faecalis strains to the host as well as to the BSH producing strains.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2100-2105
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• 2015

Probiotic bacteria must have not only tolerance against bile salt but also no genes for antibiotic resistance. Leuconostoc citreum is a dominant lactic acid bacterium in various fermented foods, but it is not regarded as a probiotic because it lacks bile salt resistance. Therefore, we aimed to construct a bile salt-resistant L. citreum strain by transforming it with a bile salt hydrolase gene (bsh). We obtained the 1,001 bp bsh gene from the chromosomal DNA of Lactobacillus plantarum and subcloned it into the pCB4170 vector under a constitutive P710 promoter. The resulting vector, pCB4170BSH was transformed into L. citreum CB2567 by electroporation, and bile salt-resistant transformants were selected. Upon incubation with glycodeoxycholic acid sodium salt (GDCA), the L. citreum transformants grew and formed colonies, successfully transcribed the bsh gene, and expressed the BSH enzyme. The recombinant strain grew in up to 0.3% (w/v) GDCA, conditions unsuitable for the host strain. In in vitro digestion conditions of 10 mM bile salt, the transformant was over 67.6% viable, whereas only 0.8% of the host strain survived.

• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1047-1052
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• 2006

A bile salt hydrolase (BSH) was purified from Lactobacillus plantarum CK 102 and its enzymatic properties were characterized. This enzyme was successfully purified using ion-exchange chromatography with Q-Excellose and hydrophobic interaction chromatography with Butyl-Excellose. The purified enzyme showed a single protein band of 37 kDa by SDS-polyacrylamide gel electrophoresis, which was similar to the molecular weight of known BSHs. The amino acid sequence of GLGLPGDLSSMSR, determined by MALDI-TOF, was identical to that of BSH of L. plantarum WCFS1. Although this BSH hydrolyzed all of the six major human bile salts, glycine-conjugated bile acid was the best substrate, based on its specificity and $K_{m}$ value. Among the various substrates, the purified enzyme maximally hydrolyzed glycocholate with apparent $K_{m}$ and $V_{max}$ values of 0.5 mM and 94 nmol/min/mg, respectively. The optimal pH of the enzyme ranged from 5.8 to 6.3. This enzyme was strongly inhibited by thiol enzyme inhibitors such as iodoacetate and periodic acid.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.222-226
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• 2010

This study was carried out to isolate and characterize the Lactobacillus plantarum with bile salt deconjugation activity that was isolated from Kimchi. Some isolates were selected and identified as L. plantarum by 16S rRNA gene sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole cell protein patterns. They were assayed to determine their capacities to express bile salt hydrolase (BSH) activity. Among the identified strains, L. plantarum CIB 001 showed the highest level of BSH activity. Then, resistance to gastric acidity and bile condition were analyzed for further characterization. This strain was able to maintain viability for 1h at pH 2.0 and to survive in a MRS (deMan, Rogosa, and Sharpe) broth with 1.0% of bile acids. L. plantarum CIB 001 would potentially be useful in the food industry as probiotics.

• Preventive Nutrition and Food Science
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• v.11 no.2
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• pp.140-145
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• 2006

Seventy eight bacterial strains were isolated from several jeotgals using MRS and M 17 agar media. The probiotic properties such as tolerance of extreme growth condition, production of antimicrobial compound, production of hydrogen peroxide, and enzymatic activity of bile salt hydrolase were investigated. DHK 4, 10, 21 and 74 strains showed_a strong tolerance property against extreme conditions such as low pH and 0.5% oxgall-supplemented medium. DHK 10 and 47 strains produced hydrogen peroxide on TMB agar plate. DHK 8 and 10 strains produced antimicrobial compounds onto MRS agar against E. facalis. DHK 4, 6, 21, 29, 33, 63 and 87 strains had high activities of bile salt hydrolase. Especially, DHK 10 displayed a strong probiotic candidate; the abilities to produce the antimicrobial compound, hydrogen peroxide, and bile salt hydrolase. All these strains are assumed to be useful probiotic candidates. Among 78, twenty seven strains which have probiotic properties were tentatively identified by 16S rRNA sequencing. Among them, 7 Lactobacillus spp., 6 Leuconosotoc spp., 2 Weisella spp., 1 Pediococcus sp., 1 Staphylococcus sp., 1 Enterococcus sp. and 2 Streptococcus spp. were tentatively identified.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.449-456
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• 2008

Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a $Ni^{2+}$-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately $40^{\circ}C$. The enzyme maintained approximately 70% of its maximum activity even at $60^{\circ}C$, whereas its activity rapidly decreased at below $37^{\circ}C$. The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.

• Microbiology and Biotechnology Letters
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• v.27 no.5
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• pp.385-390
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• 1999

To investigate bile salt hydrolase activities of the bacterial strains isolated from fermented milk products, 21 strains of Lactobacillus were tested for their abilities to produced cholic acid from taurocholic and glycocholic acids. The production of cholic acid was measured by HPLC analysis during the growth in broth media for 24hrs. All strains of Lactobacillus acidophilus and L. plantarum deconjugated both taurocholate and glycocholate, whereas none strains of L. delbrueckii subsp. bulgaricus, L. casei subsp. casei, L. casei subsp. rhamnosus, L, reuteri did. L. acidophilus stains isolated from yogurts had the higher decojugation activities on glycocholate than taurocholate, however, L. acidophilus 1009 isolated from the human intestine showed the similar deconjugation activities on both taurocholate and glycocholate.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.231-240
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• 2011

The objective of this study was to evaluate the acid and bile tolerance, bile salt hydrolase (BSH) activity, and cholesterol assimilation ability of lactic acid bacteria isolated from mustard leaf kimchi. MLK11, MLK22, MLK27, MLK41, and MLK67 were relatively acid- and bile-tolerant strains, with more than $10^5$ CFU/ml after incubation in simulated gastric juice and intestinal fluid, while MLK53 was the most sensitive strain to acid and bile. Strains MLK22 and MLK67 deconjugated the highest level of sodium glycocholate with more than 3.5 mM of cholic acid released, while deconjugation was lowest by strains MLK13 and MLK41 which released only 1.35 mM and 1.16 mM, respectively. Specially, strains MLK22 and MLK67 showed higher deconjugation of sodium glycocholate compared to sodium taurocholate and conjugated bile mixture. Although strains MLK22 and MLK67 exhibited maximal BSH activity at the stationary phase, MLK22 had somewhat higher total BSH activity compared to MLK67 towards both sodium glycocholate and sodium taurocholate. Meanwhile, cholesterol removal varied among tested strains (p<0.05) and ranged from 5.22 to 39.16 ${\mu}g$/ml. Especially, MLK67 strain assimilated the highest level of cholesterol in media supplemented with 0.3% oxgall, cholic acid, and taurocholic acid (p<0.05). According to physiological and biological characteristics, pattern of carbohydrate fermentation, and 16S rDNA sequence, strain MLK67 that may be considered as probiotic strain due to acid and bile tolerance and cholesterol-lowering effects was identified as Pediococcus pentosaceus MLK67.