Bile salt deconjugation by Lactobacillus plantarum 80 and its implication for bacterial toxicity

The effects of bile salts on the survival of lactobacilli were investigated using glycocholic acid, cholic acid and deoxycholic acid as model compounds and the bile salt hydrolase active Lactobacillus plantarum 80 (BSH⁺) and its BSH negative mutant. The detrimental effects of cholic acid, i.e. growth inhibition and cytotoxicity at a concentration of 1 and 5 mmol l⁻¹, respectively, were considered to be due to the hydrophobic protonated form of the molecule, which brings about membrane damage. The conversion of glycocholic acid to cholic acid by the BSH active L. plantarum 80 caused a growth inhibition which was comparable with the inhibition observed in the broth supplemented with 1 mmol l⁻¹ cholic acid. Deoxycholic acid caused toxicity through membrane damage when the compound was in solution. Its toxicity disappeared in the culture broth as the molecule precipitated. In case of cholic acid, the toxicity could be removed by buffering the solution at pH 7·0. It was calculated that at this pH most of the cholic acid molecules were ionized. The results led to the formulation of an extended hypothesis about the ecological significance of bile salt transformations. Primary deconjugation is carried out to counteract intracellular acidification. Yet, the deconjugated molecule can be harmful at moderately acidic pH-values. In this case, the BSH⁺ strains could effectively profit from their activity in case they are associated with 7α-dehydroxylating bacteria which dehydroxylate the deconjugated bile salts. The dehydroxylated molecule has a low solubility and precipitates at moderately acidic pH.     

Relationship between bile salt hydrolase activity, changes in the internal pH and tolerance to bile acids in lactic acid bacteria

The effect of the conjugated bile acid (BA) on the microbial internal pH (pHin) values in lactic acid bacteria with and without ability to hydrolyze bile salts (BSH[+] and BSH[-] strains, respectively) was evaluated. BSH(+) strains showed a gradual increase in the pHin following the addition of conjugated BA; this behavior was more pronounced with GDCA than with TDCA may be due to the higher affinity of BSH for the glyco-conjugates acids. Conversely, the BSH(-) strains showed a decrease in internal pH probably as a consequence of weak acid accumulation. As expected, a decrease in the cytoplasmatic pH affected the cell survival in this last group of strains, while the BSH(+) strains were more resistant to the toxic effect of BA. PURPOSE OF WORK: To evaluate bile salt hydrolase activities, changes in the internal pH and cell survival to bile acids in lactic acid bacteria to establish the relationship between these parameters.     

Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.     

Bile Salt Hydrolase Activity and Resistance to Toxicity of Conjugated Bile Salts Are Unrelated Properties in Lactobacilli

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.     

Quantitative Detection of 15 Serum Bile Acid Metabolic Products by LC/MS/MS in the Diagnosis of Primary Biliary Cholangitis

To determine 15 bile acid metabolic products in human serum by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and value their diagnostic outcome in primary biliary cholangitis (PBC). Serum from 20 healthy controls and 26 patients with PBC were collected and went LC/MS/MS analysis of 15 bile acid metabolic products. The test results were analyzed by bile acid metabolomics, and the potential biomarkers were screened and their diagnostic performance was judged by statistical methods such as principal component and partial least squares discriminant analysis and area under curve (AUC). 8 differential metabolites can be screened out: Deoxycholic acid (DCA), Glycine deoxycholic acid (GDCA), Lithocholic acid (LCA), Glycine ursodeoxycholic acid (GUDCA), Taurolithocholic acid (TLCA), Tauroursodeoxycholic acid (TUDCA), Taurodeoxycholic acid (TDCA), Glycine chenodeoxycholic acid (GCDCA). The performance of the biomarkers was evaluated by the AUC, specificity and sensitivity. In conclusion, DCA, GDCA, LCA, GUDCA, TLCA, TUDCA, TDCA and GCDCA were identified as eight potential biomarkers to distinguish between healthy people and PBC patients by multivariate statistical analysis, which provided reliable experimental basis for clinical practice.     

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.     

Co-mutagenicity of glyco- and tauro-deoxycholic acids in the Ames test

Mutagenicity and co-mutagenicity of glyco- and tauro-deoxycholic acids (GDCA and TDCA), which are abundant in human bile, were examined by the Ames test. The two chemicals were not mutagenic for themselves to Salmonella typhimurium TA98 and TA100, with and without S9 mix. They enhanced, however, the mutagenic activities of the pro-mutagens, 2-aminoanthracene (2AA) and benzo[a]pyrene (BaP), for both TA98 and TA100 with S9 mix. They were more co-mutagenic for the pro-mutagens on TA98 than on TA100. On TA98, the mutagenic activities of 2AA with GDCA (5 μmol/plate) and with TDCA (5 μmol/plate) were 9.7-fold and 11.8-fold as high as that of the corresponding control (2AA only), respectively. BaP with GDCA (2.5 μmol/plate) and with TDCA (2.5 μmol/plate) showed 2.8-fold and 3.0-fold increases over the corresponding control level (BaP only), respectively. It is hence concluded that GDCA and TDCA may enhance the activity of some mutagens existing in bile.     

In-vitro assessment of probiotic potential of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WU-P19 isolated from a traditional fermented herb

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.     

High-performance liquid chromatographic mass spectrometric method for the determination of ursodeoxycholic acid and its glycine and taurine conjugates in human plasma

A novel sensitive high-performance liquid chromatography-electrospray mass spectrometry method has been developed for the determination of ursodeoxycholic acid (UDCA) and its glycine and taurine conjugates, glycoursodeoxycholic acid (GDCA) and tauroursodeoxycholic acid (TDCA). The procedure involved a solid phase extraction of UDCA, GDCA, TDCA and the internal standard, 23-nordeoxycholic acid from human plasma on a C18 Bond Elut cartridge. Chromatography was performed by isocratic reverse phase separation with methanol/25 mM ammonium acetate (40/60, v/v) containing 0.05% acetic acid on a C18 column with embedded polar functional group. Detection was achieved using an LC-MS/MS system. The standard curve was linear over a working range of 10-3000 ng/ml for all analytes and gave an average correlation coefficient of 0.9992 or better during validation. The absolute recovery for UDCA, GDCA, TDCA and the internal standard was 87.3, 83.7, 79.5 and 95.8%, respectively. This method is simple, sensitive and suitable for pharmacokinetics, bioequivalence or clinical studies.     

Effects of bile salts on bile formation in rabbits

The effects of different species of bile salts: deoxycholate, taurochenodeoxycholate, ursodeoxycholate, glycodeoxycholate, tauroursodeoxycholate, chenodeoxycholate and cholate (DCA, TCDC, UDCA, GDCA, TUDC, CDCA, CA) on bile secretion were examined in anesthetized rabbits using two different infusion routes. When bile salts were infused intravenously, all bile salts (except for TCDC) significantly increased the volume of bile and bile salt excretion, but their respective efficiency for bile formation was different. The concentration of bicarbonate ion in the bile significantly increased during the choleretic periods induced by DCA, UDCA, GDCA and CDCA but remained unchanged with the other bile salts (CA, TCDC, TUDC). In rabbits, where a bile salt solution was infused in the duodenum and then drained from the intestine through an incision in the distal part of duodenum, none of these bile salts affected bile secretion. The effects of intravenously administered bile salts on rabbit bile secretion are different in terms of their choleretic potency and bicarbonate excretion depending on the species of bile salts used. Furthermore, it was concluded that the intraduodenal infusion of UDCA, which was found to stimulate the pancreatic exocrine function, did not affect bile secretion.     

Combined use of Lactobacillus reuteri and soygerm powder as food supplement

AIMS: The survival of Lactobacillus reuteri when challenged with glycodeoxycholic acid (GDCA), deoxycholic acid (DCA) and soygerm powder was investigated. Moreover, the impact of Lact. reuteri on the bioavailability of isoflavones present in soygerm powder was examined. METHODS AND RESULTS: The strain experienced a die-off when adding 2 or 3 mmol l-1 bile salts, with more pronounced effects in the case of DCA. By means of a haemolysis test it was shown that toxicity could be due to membrane damage. When 4 g l-1 soygerm powder was added, the Lactobacillus strain survived the bile salt burden better (P < or = 0.05) and the membrane damage in the haemolysis test decreased (P < or = 0.05). The Lact. strain cleaved beta-glycosidic isoflavones during fermentation of milk supplemented with soygerm powder. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The interactions between the Lactobacillus strain and soygerm powder suggest that combining both in fermented milk can exhibit advantageous probiotic effects. The relevance of the combination of the strain and the soygerm powder should be studied under more relevant physiological conditions.     

Characterization of conjugated and unconjugated bile acid transport via human organic solute transporter α/β

Bile acids are biosynthesized from cholesterol in hepatocytes and usually localize in the enterohepatic circulation system. This system is regulated by several transporters that are expressed in the liver and intestine. Organic solute transporter (OST) α/β, which is known as a bidirectional transporter for some organic anions, contributes to the transport of bile acids; however, the transport properties of individual bile acids are not well understood. In this study, we investigated the transport properties of five bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], ursodeoxycholic acid [UDCA], and lithocholic acid [LCA]) together with their glycine and taurine conjugates mediated by OSTα/β. Of the unconjugated bile acids, CA, CDCA, DCA, and LCA were taken up by OSTαβ/MDCKII cells more rapidly than mock cells, but no significant increase in the uptake of UDCA was observed. On the contrary, all glycine- and taurine-conjugated bile acids showed a significant increase in the uptake by OSTαβ/MDCKII cells. Saturable OSTα/β-mediated transports of CDCA, DCA, glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurochenodeoxycholic acid (TCDCA), and taurolithocholic acid (TLCA) were observed. The apparent Michaelis constants of CDCA, DCA, GCDCA, GDCA, GLCA, TCDCA, and TLCA for OSTα/β were 23.0 ± 4.0, 14.9 ± 1.9, 864.2 ± 80.7, 586.4 ± 43.2, 12.8 ± 0.5, 723.7 ± 4.8, and 23.9 ± 0.3 μM, respectively. However, the transport of other bile acids was not saturable. Our results indicate that OSTα/β has a low affinity but a high capacity for transporting bile acids.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation

D.A. DIONYSIUS, P.A. GRIEVE AND A.C. VOS. 1992. Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H₂O₂. When low levels of thiocyanate (SCN^-) were used in the GO system, H₂O₂ was detected and lactoperoxidase (LP) was inactivated when SCN^- was depleted. With 10-fold higher SCN^-, LP remained active and H₂O₂ was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H₂O₂ in a system employing low SCN^-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H₂O₂ was not detected during the course of the experiment. At high SCN^- levels, relatively high concentrations of oxidation product were produced immediately, with H₂O₂ undetectable. The results suggest that the final product of the LP reaction depends on the method of H₂O₂ generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37°C.     

Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (H₂O₂) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol H₂O₂/min/10⁸ colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM H₂O₂, survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10⁵ CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01.     

降胆固醇乳酸菌的筛选和分析比较

目的比较7株乳酸菌各项生理特性,筛选高效、安全降解胆固醇的益生菌株,为后期开发应用奠定基础。方法对选定菌株依次进行体外降胆固醇、胆盐水解酶(BSH)活力、人工胃肠液及胆盐耐受性、细胞粘附性能的测定和比较,并分析抗生素耐药性和抑制病原菌活性,最后利用16S rRNA基因测序确定细菌种属。结果不同菌株各项生理指标存在差异。其中菌株ZL-2、JQI-7和PC-26的BSH酶活分别为0.010、0.015和0.030 U,可高效清除体外胆固醇,3株菌体外降解胆固醇率在58%以上;并且耐酸、耐胆盐,具备较好的HT--29细胞粘附性,不存在抗生素耐药性,可有效抑制大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌和阴沟肠杆菌生长。结论菌株ZL-2、JQI-7和PC-26满足降胆固醇益生菌株的生物学特性,可用于进一步研究和开发。     

Induction of the PhoE porin by NaCI as the basis for salt-induced acid sensitivity in Escherichia coli

Z. LAZIM, T.J. HUMPHREY AND R.J. ROWBURY. 1996. Organisms grown in low salt broth (LSB) are acid resistant but become sensitive on growth for 30-60 min with 300 mmol 1⁻¹ added NaCl. Salt-induced acid sensitivity only occurs in relA⁺ strains and sensitization is abolished by glucose, this catabolite repression effect being reversed by cAMP. The finding that sensitization did not occur in a phoE strain but did occur in a phoE⁺ derivative of it suggested that the response might result from PhoE induction, since PhoE acts as the major outer membrane (OM) proton pore under most conditions. In agreement with this, low-salt broth (LSB)-grown cells of a chromosomally lac⁻ strain carrying pJP102 ( phoE-lacZ ) produced low levels of β-galactosidase but growth with added NaCl led to rapid and appreciable induction. Also, a phoA mutant carrying a phoE-phoA fusion produced little alkaline phosphatase after growth in LSB but much more in LSB with added NaCl. Increased β-galactosidase synthesis (in phoE-lacZ strains) in the presence of NaCl was abolished by glucose, this effect being reversible by cAMP, and there was more NaCl-induced synthesis of this enzyme in relA⁺ strains.
Accordingly, it appears that addition of NaCl to LSB leads to acid sensitivity because it induces synthesis of the OM proton pore PhoE.     

Deconjugation of bile salts by Bacteroids and Clostridium

Deconjugation of bile salts by four strains of Bacteroides and four strains of Clostridium was studied by use of resting cells and cell-free culture supernatants. Bacteroids strains yielded active cells but showed relatively low bile salt hydrolase (BSH) activity in the culture supernatants while the reverse was the case for the spore-forming clostridial strains. BSH was formed constitutively and was oxygen insensitive. The optimum pH was between 4.5 and 5.0. Marked substrate specificity was found in two strains, one Clostridium and one Bacteroides, which showed restricted activity against taurine conjugates. Bacteroides in general attacked the taurine conjugates of dihydroxy bile acids more readily than the trihydroxy taurine conjugates. Deconjugated bile acid moieties were further modified by some resting cells, depending on the bacterial strain while no enzymatic activity other than that of BSH was found in the culture supernatants. Cells of B. fragilis 2536 performed 7 alpha-dehydrogenation when the pH of the medium allowed the reaction, and this oxidative process was markedly enhanced in the presence of an abundant supply of oxygen as a terminal electron acceptor. C. perfringens PB 6K produced the 3- keto product in addition to the 3 beta-hydroxy derivative of the liberated bile acids and the formation of the latter derivative seemed to take place without preliminary deconjugation.     

唾液乳杆菌BSH1及其突变体的底物特异性

为了解析胆盐水解酶催化中心中关键氨基酸位点与其底物特异性的关系,以大肠杆菌pET-20b(+)表达系统为分子改造平台,采用理性设计,结合氨基酸定点突变的方法,成功构建了唾液乳杆菌Lactobacillus salivarius胆盐水解酶BSH1的7种突变体。通过对比L.salivarius BSH1及其突变体对6种结合胆盐的底物特异性表明,7种突变体对不同的结合胆盐的水解活性有所改变。结果说明,Cys2和Thr264分别是BSH1催化TCA和GCA的关键残基,且对酶的催化活性的保持具有关键作用。其中,高保守性的氨基酸位点Cys2不是BSH1唯一的活性位点,而其他突变的氨基酸位点可能作为BSH1的结合位点参与了底物的结合,也可能影响了底物进入BSH1活性中心的通道或底物结合口袋的体积与形状,进而影响了BSH1对不同结合胆盐的水解活性。     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance

Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDeltapH) was involved in Lactobacillus bile salt resistance.