scholarly journals Bile Salt Hydrolase of Bifidobacterium longum—Biochemical and Genetic Characterization

2000 ◽  
Vol 66 (6) ◽  
pp. 2502-2512 ◽  
Author(s):  
Hiroshi Tanaka ◽  
Honoo Hashiba ◽  
Jan Kok ◽  
Igor Mierau
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Bile Salt ◽  
Bile Salts ◽  
Bifidobacterium Longum ◽  
Bacillus Sphaericus ◽  
Site Directed Mutagenesis ◽  
Bile Salt Hydrolase ◽  
Ph Optimum ◽  
Human Bile

ABSTRACT A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized. Furthermore, we describe for the first time cloning and analysis of the gene encoding BSH (bsh) in a member of the genusBifidobacterium. The enzyme has a native molecular weight of 125,000 to 130,000 and a subunit molecular weight of 35,024, as determined from the deduced amino acid sequence, indicating that the enzyme is a tetramer. The pH optimum of B. longum BSH is between 5 and 7, and the temperature optimum is 40°C. The enzyme is strongly inhibited by thiol enzyme inhibitors, indicating that a Cys residue is likely to be involved in the catalytic reaction. The BSH ofB. longum can hydrolyze all six major human bile salts and at least two animal bile salts. A slight preference for glycine-conjugated bile acids was detected based on both the specificity and the Km values. The nucleotide sequence of bsh was determined and used for homology studies, transcript analysis, and construction and analysis of various mutants. The levels of homology with BSH of other bacteria and with penicillin V acylase (PVA) of Bacillus sphaericus were high. On the basis of the similarity of BSH and PVA, whose crystal structure has been elucidated, BSH can be classified as an N-terminal nucleophile hydrolase with Cys as the N-terminal amino acid. This classification was confirmed by the fact that a Cys1Ala exchange by site-directed mutagenesis resulted in an inactive protein. Reverse transcription-PCR experiments revealed that bsh is part of an operon containing at least two genes, bsh andglnE (GlnE is glutamine synthetase adenylyltransferase). Two UV-induced BSH-negative mutants and one spontaneous BSH-negative mutant were isolated from B. longum SBT2928 cultures and characterized. These mutants had point mutations that inactivatedbsh by premature termination, frameshift, or amino acid exchange.

scholarly journals Structural and Functional Analysis of a Conjugated Bile Salt Hydrolase from Bifidobacterium longum Reveals an Evolutionary Relationship with Penicillin V Acylase

2006 ◽  
Vol 281 (43) ◽  
pp. 32516-32525 ◽  
Author(s):  
R. Suresh Kumar ◽  
James A. Brannigan ◽  
Asmita A. Prabhune ◽  
Archana V. Pundle ◽  
Guy G. Dodson ◽  
...  
Keyword(s):  
Bile Salt ◽  
Tertiary Structure ◽  
Bifidobacterium Longum ◽  
Active Form ◽  
Three Dimensional ◽  
Evolutionary Relationship ◽  
Bacillus Sphaericus ◽  
Site Directed Mutagenesis ◽  
Bile Salt Hydrolase ◽  
Penicillin V Acylase

Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine- or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic αββα tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.

scholarly journals Cloning and Characterization of the Bile Salt Hydrolase Genes (bsh) from Bifidobacterium bifidum Strains

2004 ◽  
Vol 70 (9) ◽  
pp. 5603-5612 ◽  
Author(s):  
Geun-Bae Kim ◽  
Carol M. Miyamoto ◽  
Edward A. Meighen ◽  
Byong H. Lee
Keyword(s):  
Bile Salt ◽  
Active Sites ◽  
Biochemical Characterization ◽  
Bifidobacterium Longum ◽  
Pcr Amplification ◽  
Bacillus Sphaericus ◽  
Bifidobacterium Bifidum ◽  
Bile Salt Hydrolase ◽  
Cloned Gene

ABSTRACT Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.

Diversity of bile salt hydrolase activities in different lactobacilli toward human bile salts

2009 ◽  
Vol 60 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Jinkang Jiang ◽  
Xiaomin Hang ◽  
Min Zhang ◽  
Xianglong Liu ◽  
Daotang Li ◽  
...  
Keyword(s):  
Bile Salt ◽  
Bile Salts ◽  
Bile Salt Hydrolase ◽  
Human Bile

Studies on the Action Mechanism for Cholesterol-Lowering of Lactobacillus which Yields Bile Salt Hydrolase from Kefir Grains

2013 ◽  
Vol 781-784 ◽  
pp. 1336-1340
Author(s):  
Hui Liu ◽  
Yuan Hong Xie ◽  
Tao Han ◽  
Hong Xing Zhang
Keyword(s):  
Bile Salt ◽  
High Throughput Screening ◽  
Bile Salts ◽  
Lactobacillus Casei ◽  
Streptococcus Thermophilus ◽  
High Yield ◽  
Bile Salt Hydrolase ◽  
Action Mechanism ◽  
Cholesterol Lowering ◽  
Conjugated Bile Salts

Cholesterol-lowering strains were obtained by high throughput screening technology and ortho-phthalaldehyde method. We used oxford cup method to screen again to obtain strains of high yield bile salt hydrolase and illuminate action mechanism ofLactobacillusreducing cholesterol. Screened six strains had the ability of high yield bile salt hydrolase and good ferment ability. The results of identifying bacteria species: strain KTxKL1J1 wereLactobacillus casei, strain Tx wasStreptococcus thermophilus, strain KS4P1 wereLactococcus lactis subsp.lactis, where the last two bacteria were strain of high yield bile salt hydrolase to be few known in literature. This work showed that dissociation bile salts and cholesterol conjuncted sediments by bile salt hydrolase decomposing conjugated bile salts.

scholarly journals Characterization of a Thermoacidophilic l-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum

2005 ◽  
Vol 71 (12) ◽  
pp. 7888-7896 ◽  
Author(s):  
Sang-Jae Lee ◽  
Dong-Woo Lee ◽  
Eun-Ah Choe ◽  
Young-Ho Hong ◽  
Seong-Bo Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Bacillus Halodurans ◽  
Wild Type ◽  
Ph Optimum ◽  
Calculated Molecular Mass ◽  
Alicyclobacillus Acidocaldarius ◽  
Arabinose Isomerase

ABSTRACT The araA gene encoding l-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for l-arabinose and d-galactose were 48.0 mM (V max, 35.5 U/mg) and 129 mM (V max, 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (k cat/Km ) of each mutant at different pHs was significantly affected by an increase or decrease in V max. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.

scholarly journals Functional Analysis of Four Bile Salt Hydrolase and Penicillin Acylase Family Members in Lactobacillus plantarum WCFS1

2008 ◽  
Vol 74 (15) ◽  
pp. 4719-4726 ◽  
Author(s):  
Jolanda M. Lambert ◽  
Roger S. Bongers ◽  
Willem M. de Vos ◽  
Michiel Kleerebezem
Keyword(s):  
Bile Salt ◽  
Lactobacillus Plantarum ◽  
Bile Salts ◽  
Natural Habitat ◽  
Penicillin Acylase ◽  
Exponential Growth Phase ◽  
Bile Salt Hydrolase ◽  
Intestinal Microbes ◽  
Bsh Activity

ABSTRACT Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat.

Purification and characterization of a post-proline dipeptidyl aminopeptidase fromStreptococcus cremorisAM2

1990 ◽  
Vol 57 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Mary Booth ◽  
Ide Ni Fhaoláin ◽  
P. Vincent Jennings ◽  
Gerard O'Cuinn
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Cheese Ripening ◽  
Streptococcus Cremoris ◽  
Scissile Bond ◽  
Dipeptidyl Aminopeptidase

SummaryThe present study describes the purification of a post-proline dipeptidyl aminopeptidase from the cytoplasm ofStreptococcus cremorisAM2. On the basis of its elution from a calibrated Sephadex G200 column, the enzyme had a molecular weight of 117000 and exhibited a broad pH optimum activity between 6·0 and 9·0. The activity was most comprehensively inhibited by phenylmethylsulphonylfluoride and more modestly inhibited by 1,10-phenanthroline and 8-hydroxyquinoline but not by EDTA. A range of peptides containing either proline or alanine as the penultimate amino acid residue could act as substrates. The presence of proline on the carboxy side of the scissile bond prevented hydrolysis. However the enzyme could release Pro-Pro from Pro-Pro-Gly-Phe-Ser-Pro. The significance of this substrate specificity is considered in the context of removal of either single proline residues or prolylproline sequences from oligopeptides during cheese ripening.

scholarly journals The safety and potential probiotic properties analysis of Streptococcus alactolyticus strain FGM isolated from the chicken cecum

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Jingyan Zhang ◽  
Hong Zhang ◽  
Lei Wang ◽  
Kang Zhang ◽  
Zhengying Qiu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bile Salt ◽  
Hemolytic Activity ◽  
Gc Content ◽  
Amino Acid Sequences ◽  
Feed Additive ◽  
Bile Salt Hydrolase ◽  
Probiotic Properties ◽  
In Silico Studies

Abstract Purpose Streptococcus alactolyticus strain FGM is used to ferment Astragalus membranaceus to develop a novel feed additive for animals in China. This study aimed at characterizing the safety and potential probiotic features of the strain FGM in vitro. Methods The genome of S. alactolyticus strain FGM was sequenced and used for genomic in silico studies. It was evaluated for morphology, antibiotic susceptibility, hemolytic activity, acid tolerance, bile salt tolerance, adherence ability to Caco-2, and inhibitory pathogens activity. Result The GC content of the strain FGM was 40.38% and composed of 29 contigs. The annotation of coding genes revealed important characteristics of the germs, especially 151 genes annotated to biological adhesion. The strain FGM forecasted 43 amino acid sequences to be VF, but did not have a hemolytic gene, and neither did it show hemolytic activity in phenotypic analysis. Although 30 amino acid sequences were predicted to aid in resisting some antibiotics, the strain FGM just showed the resistance to trimoxazole and oxytetracycline, and intermediate resistance to kanamycin. FGM cells were showed the tolerance to pH 2 broth within 4 h, and 0.15~0.30% bile salt medium with the latter being attributed to the presence of bile-salt hydrolase. The strain FGM was shown to have the ability to adhere to Caco-2 cells and the adherence rate of 1.0 × 109 CFU/mL bacterial suspensions was 37.51%. Compared with Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus casei, the strain FGM showed a high capability to inhibit the diffusion of Escherichia coli O78 and reduce its adhesion on Caco-2 cells. Conclusion The results demonstrated the presence of probiotic potential and absence of adverse effects for the Streptococcus alactolyticus strain FGM in vitro, thus contributing to develop a safety and effective fermentation feed for animals.

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