Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon
2013; Elsevier BV; Volume: 54; Issue: 11 Linguagem: Inglês
10.1194/jlr.m039834
ISSN1539-7262
AutoresJayoung Lee, Hisashi Arai, Yusuke Nakamura, Satoru Fukiya, Masaru Wada, Atsushi Yokota,
Tópico(s)Digestive system and related health
ResumoBile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90–100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7β-hydroxysteroid dehydrogenase (7β-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7β-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon. Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90–100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7β-hydroxysteroid dehydrogenase (7β-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7β-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon. The dynamic balance of bile acid composition in the colon is influenced by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes (1Ridlon J.M. Kang D.J. Hylemon P.B. Bile salt biotransformations by human intestinal bacteria.J. Lipid Res. 2006; 47: 241-259Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 2Begley M. Gahan C.G.M. Hill C. The interaction between bacteria and bile.FEMS Microbiol. Rev. 2005; 29: 625-651Crossref PubMed Scopus (1140) Google Scholar, 3Monte M.J. Marin J.J.G. Antelo A. Vazquez-Tato J. Bile acids: chemistry, physiology, and pathophysiology.World J. Gastroenterol. 2009; 15: 804-816Crossref PubMed Scopus (372) Google Scholar). However, our knowledge is still far from being able to predict changes in the dynamic balance because we have a limited catalog of bile acid-converting bacteria and little information about the enzymes involved in bile acid conversion. Primary bile acids are synthesized from cholesterol in the liver and secreted into the duodenum as the main component of bile (2Begley M. Gahan C.G.M. Hill C. The interaction between bacteria and bile.FEMS Microbiol. Rev. 2005; 29: 625-651Crossref PubMed Scopus (1140) Google Scholar, 3Monte M.J. Marin J.J.G. Antelo A. Vazquez-Tato J. Bile acids: chemistry, physiology, and pathophysiology.World J. Gastroenterol. 2009; 15: 804-816Crossref PubMed Scopus (372) Google Scholar, 4Hofmann A.F. Bile acids: the good, the bad, and the ugly.News Physiol. Sci. 1999; 14: 24-29PubMed Google Scholar). While bile acids contribute to the emulsification, digestion, and absorption of dietary lipids, some secondary bile acids formed from the primary bile acids by bacterial biotransformation (1Ridlon J.M. Kang D.J. Hylemon P.B. Bile salt biotransformations by human intestinal bacteria.J. Lipid Res. 2006; 47: 241-259Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 2Begley M. 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Gastroenterol. 2009; 15: 804-816Crossref PubMed Scopus (372) Google Scholar), bile acid may be responsible for the alteration in the gut microbiota composition in response to a high-fat diet. Thus, bile acid is likely involved in development of metabolic syndrome by altering the gut microbiota composition during high-fat diet intake. Gaining an understanding of the regulatory mechanism(s) of the bile acid pool through characterization of the bacteria involved in bile acid conversion is important for host health. Accordingly, we focused on UDCA, which is a beneficial secondary bile acid, although it represents less than 2% of total biliary and fecal bile acids in humans (1Ridlon J.M. Kang D.J. Hylemon P.B. Bile salt biotransformations by human intestinal bacteria.J. Lipid Res. 2006; 47: 241-259Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 12Bachrach W.H. Hofmann A.F. Ursodeoxycholic acid in the treatment of cholesterol cholelithiasis. Part I.Dig. Dis. Sci. 1982; 27: 737-761Crossref PubMed Scopus (229) Google Scholar). UDCA is produced from chenodeoxycholic acid (CDCA; 3α, 7α-dihydroxy-5β-cholan-24-oic acid) by successive reactions catalyzed by 7α- and 7β-hydroxysteroid dehydrogenases (HSDH) of intestinal bacteria (24Fedorowski T. Salen G. Tint G.S. Mosbach E. Transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal bacteria.Gastroenterology. 1979; 77: 1068-1073Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 25Hirano S. Masuda N. Oda H. In vitro transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal flora, with particular reference to the mutual conversion between the two bile acids.J. Lipid Res. 1981; 22: 735-743Abstract Full Text PDF PubMed Google Scholar, 26Lepercq P. Gérard P. Béguet F. Raibaud P. Grill J.P. Relano P. Cayuela C. Juste C. Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces.FEMS Microbiol. Lett. 2004; 235: 65-72Crossref PubMed Google Scholar), with 7-oxo-lithocholic acid (7-oxo-LCA; 3α-hydroxy-7oxo-5β-cholan-24-oic acid) as an intermediate (27Fromm H. Sarva R.P. Bazzoli F. Formation of ursodeoxycholic acid from chenodeoxycholic acid in the human colon: studies of the role of 7-ketolithocholic acid as an intermediate.J. Lipid Res. 1983; 24: 841-853Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). In the present study, we screened and identified a novel UDCA-producing bacterium, Ruminococcus gnavus N53, from human feces. As R. gnavus has been identified as a common core species of the human intestinal microbiota (28Qin J. Li R. Raes J. Arumugam M. Burgdorf K.S. Manichanh C. Nielsen T. Pons N. Levenez F. Yamada T. et al.A human gut microbial gene catalogue established by metagenomic sequencing.Nature. 2010; 464: 59-65Crossref PubMed Scopus (7267) Google Scholar), it is important to investigate UDCA production by this bacterium to understand formation of the UDCA pool in the human colon. Thus, we investigated the bile acid conversion reaction mediated by this bacterium and conducted a functional characterization of recombinant 7β-HSDH. UDCA-producing R. gnavus N53 was isolated from human feces and characterized in our laboratory. R. gnavus ATCC 29149T, a reference strain, and Collinsella aerofaciens ATCC 25986T, another UDCA-producing bacterium (29Hirano S. Masuda N. Epimerization of the 7-hydroxy group of bile acids by the combination of two kinds of microorganisms with 7α- and 7β-hydroxysteroid dehydrogenase activity, respectively.J. Lipid Res. 1981; 22: 1060-1068Abstract Full Text PDF PubMed Google Scholar, 30Hirano S. Masuda N. Characterization of NADP-dependent 7β-hydroxysteroid dehydrogenases from Peptostreptococcus productusEubacterium aerofaciens.Appl. Environ. Microbiol. 1982; 43: 1057-1063Crossref PubMed Google Scholar), were obtained from the Japan Collection of Microorganisms (JCM; Tsukuba, Ibaraki, Japan). The strains were grown at 37°C in Gifu anaerobic medium broth (GAM broth; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) under anaerobic conditions (80% N2, 10% CO2, and 10% H2) in an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI). When necessary, 15 g/l agar was added to solidify the medium. The media were kept in the anaerobic chamber at least for 24 h after autoclaving and before use. Luria-Bertani (LB) medium (plate/broth) and M9 liquid medium containing ampicillin (100 μg/ml) were used to culture Escherichia coli JM109 transformants. Strains were cultured aerobically at 37°C unless otherwise specified. UDCA was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Sodium UDCA and 7-oxo-LCA were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Sodium 7-oxo-LCA was prepared by neutralizing free 7-oxo-LCA with NaOH using a method described previously (31Fung B.M. Thomas Jr, L. The motion of aromatic molecules in bile acid micelles.Chem. Phys. Lipids. 1979; 25: 141-148Crossref Scopus (10) Google Scholar). Sodium 7-oxo-LCA was used as the substrate for measuring enzyme activity because it dissolves easily in water, while free 7-oxo-LCA does not. Bile acids in culture broths or enzyme reaction mixtures were characterized either by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), or gas chromatography-mass spectrometry (GC-MS), as described previously (32Fukiya S. Arata M. Kawashima H. Yoshida D. Kaneko M. Minamida K. Watanabe J. Ogura Y. Uchida K. Itoh K. et al.Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces.FEMS Microbiol. Lett. 2009; 293: 263-270Crossref PubMed Scopus (64) Google Scholar). Methods for the extraction of bile acids and sample preparation were as described by Fukiya et al. (32Fukiya S. Arata M. Kawashima H. Yoshida D. Kaneko M. Minamida K. Watanabe J. Ogura Y. Uchida K. Itoh K. et al.Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces.FEMS Microbiol. Lett. 2009; 293: 263-270Crossref PubMed Scopus (64) Google Scholar). UDCA-producing bacteria were screened using methods described previously (32Fukiya S. Arata M. Kawashima H. Yoshida D. Kaneko M. Minamida K. Watanabe J. Ogura Y. Uchida K. Itoh K. et al.Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces.FEMS Microbiol. Lett. 2009; 293: 263-270Crossref PubMed Scopus (64) Google Scholar). Fecal samples were provided by a healthy Japanese adult male. Samples were homogenized, diluted, and plated on 1/4 GAM agar medium. Colonies on the plates were picked and cultured in 200 μl GAM broth containing either 0.1 mM 7-oxo-LCA or UDCA (both as free acids) in 96-well microtiter plates for 48 h. Detection of bile acid conversion in the culture broths was carried out by TLC. Culture samples showing corresponding spots with either 7-oxo-LCA or UDCA by TLC were further analyzed by GC-MS. Identification of the candidate strain by 16S rDNA sequencing and its biochemical characterization using the API 20A and Rapid ID 32A kits (bioMérieux SA, Marcy-l'Etoile, France) were performed as described previously (32Fukiya S. Arata M. Kawashima H. Yoshida D. Kaneko M. Minamida K. Watanabe J. Ogura Y. Uchida K. Itoh K. et al.Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces.FEMS Microbiol. Lett. 2009; 293: 263-270Crossref PubMed Scopus (64) Google Scholar). R. gnavus N53 and its type strain ATCC 29149T, as well as C. aerofaciens ATCC 25986T, another UDCA-producing bacterium (29Hirano S. Masuda N. Epimerization of the 7-hydroxy group of bile acids by the combination of two kinds of microorganisms with 7α- and 7β-hydroxysteroid dehydrogenase activity, respectively.J. Lipid Res. 1981; 22: 1060-1068Abstract Full Text PDF PubMed Google Scholar, 30Hirano S. Masuda N. Characterization of NADP-dependent 7β-hydroxysteroid dehydrogenases from Peptostreptococcus productusEubacterium aerofaciens.Appl. Environ. Microbiol. 1982; 43: 1057-1063Crossref PubMed Google Scholar), were precultured in 5 ml GAM broth in screw-capped vials at 37°C in an anaerobic chamber until stationary phase. The cultures were then inoculated into 50 ml GAM broth in 200 ml screw-cap bottles supplemented with 1 mM 7-oxo-LCA at an initial optical density at 660 nm (OD660) of 0.01 and cultured under the same conditions. During culture, 5 ml of the culture broth was withdrawn periodically. Growth was measured at OD660 using a photometer (MiniPhoto518R; TAITEC Corp., Koshigaya, Saitama, Japan), and bile acid concentration was determined by HPLC. Cells were harvested by centrifugation at 7,000 g for 10 min. Pellets (0.2 g wet cells) were resuspended in 1 ml extraction buffer containing 0.1 M Tris, 3 mM EDTA, 1 mM dithiothreitol (DTT; Wako Pure Chemical Industries Ltd., Osaka, Japan), and 10% (v/v) glycerol (adjusted to pH 8.0 with HCl). Cell suspensions were disrupted under cooling conditions by sonication with 1 min pulses, followed by 1 min breaks repeatedly for 15 min using a Bioruptor UCD-200T (COSMO BIO Co. Ltd., Tokyo, Japan). Cell debris was removed by centrifugation (46,000 g, 4°C) for 40 min, and the supernatant was used as the crude enzyme solution. The assay mixture for the UDCA-forming reaction (forward reaction) contained, in a total volume of 1 ml, 10 mM Tris-HCl buffer (pH 6.0), 250 μM NADPH, 1 mM sodium 7-oxo-LCA, and 10 μl enzyme solution, while that for the 7-oxo-LCA-forming reaction (reverse reaction) contained 10 mM glycine-NaOH buffer (pH 10.0), 1 mM NADP+, 2 mM sodium UDCA, and 10 μl enzyme solution. The reactions were carried out at 37°C with addition of the substrate. Reaction mixtures without the substrate served as controls. The 7β-HSDH activity was determined by the change in NADP(H) concentration by monitoring absorbance at 340 nm with a spectrophotometer (DU800; Beckman Coulter Inc., Brea, CA). Enzyme activity was calculated using a molar extinction coefficient of 6.22 mM−1×cm−1 for NADPH. The protein concentration was determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) with BSA as the standard. Enzyme activity was expressed as micromoles of NADPH oxidized or NADP+ reduced per minute per milligram of protein. Total genomic DNA was extracted from both R. gnavus N53 and ATCC 29149T using ISOPLANT II (Nippon Gene Co. Ltd., Tokyo, Japan) and used as the template for PCR amplification. The gene putatively encoding 7β-HSDH (hypothetical protein RUMGNA_02585; accession number, ZP_02041813; GenBank database, ) was amplified from the genomic DNA of R. gnavus strains by PCR using synthetic primers: forward (5′-ttaaGCATGCATGACATTGAGAGAAAAATA-3′) and reverse (5′-taatCTGCAGTTATTCTTGATAGAAAGATC-3′). The underlined bases denote the SphI and PstI restriction sites, respectively. The PCR conditions were as follows: initial 2 min denaturation at 94°C followed by 30 cycles of amplification at 98°C for 10 s, 50°C for 30 s, and 68°C for 1 min, and then elongation at 68°C for 7 min. The PCR product from N53 was further purified using the MinElute PCR Purification Kit (QIAGEN GmbH, Hilden, Germany). The PCR product and vector pQE30 (ampicillin resistance; QIAGEN) containing an N-terminal six-His tag were double-digested with SphI and PstI, and then purified using the MinElute Reaction Cleanup Kit (QIAGEN). The digested pQE30 was dephosphorylated and ligated with the PCR product using Ligation high Ver.2 (Toyobo Co. Ltd., Osaka, Japan). The plasmids were transformed into E. coli JM109 and were purified from colonies on LB plates containing ampicillin. The insert DNA was sequenced using standard primers. The resulting plasmid was named pQE30-7β-HSDH and was also used for the expression of 7β-HSDH for enzyme purification. E. coli JM109 containing pQE30-7β-HSDH was precultured overnight in 5 ml LB broth containing ampicillin. The preculture was inoculated into 50 ml M9 medium containing ampicillin at an initial OD660 of 0.01. 7β-HSDH gene expression was induced by adding isopropyl β-d-thiogalactoside at a final concentration of 0.1 mM to exponentially growing cells at an OD660 of 0.4, at which time the culture temperature was shifted from 37°C to 25°C. After 22 h of culture at 25°C, the cells were collected by centrifugation at 7,000 g for 10 min at 4°C. Cell-free extract for 7β-HSDH purification was prepared using the method described above. The cell-free extract, after membrane filtration (Minisart, 0.20 µm; Sartorius Stedim Biotech GmbH, Goettingen, Germany), was applied to a TALON Single Step Column (Clontech Laboratories Inc., Mountain View, CA) equilibrated with equilibration/wash buffer (50 mM sodium phosphate buffer at pH 8.0, 300 mM NaCl). Nonabsorbed materials were removed by washing the column with two column bed volumes of equilibration/wash buffer. Weakly bound proteins were removed by washing with two column bed volumes of wash-2 buffer (50 mM sodium phosphate buffer at pH 8.0, 300 mM NaCl, 7.5 mM imidazole). His-tagged 7β-HSDH protein was eluted with 1 ml elution buffer (50 mM sodium phosphate buffer at pH 8.0, 300 mM NaCl, 150 mM imidazole). The eluate was dialyzed in a dialysis tube (TOR-3K; Nippon Genetics Co. Ltd., Tokyo, Japan) with a molecular weight cutoff of 3.5 kDa against 1 l extraction buffer containing 0.1 M Tris, 3 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol (adjusted to pH 8.0 with HCl). The solution was then concentrated by Microcon (YM-10; Millipore, Billerica, MA), resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by staining with Coomassie Brilliant Blue R-250 (Wako Pure Chemical Industries) followed by destaining in methanol:acetic acid:water (40:7:53). Partial N-terminal amino acid sequencing was performed. The purified enzyme was subjected to SDS-PAGE and electrically transferred to a polyvinylidene difluoride membrane (Bio-Rad) using transfer buffer containing 10 mM CAPS and 10% methanol (adjusted to pH 11.0 with NaOH). Transferred protein was stained with 0.25% Coomassie Brilliant Blue R-250. The band was excised and sequenced by the Instrumental Analysis Division, Equipment Management Center, Creative Research Institution at Hokkaido University. As a result of screening for UDCA-producing bacteria from human feces, 1,482 colonies were isolated; these were evaluated for their ability to convert 7-oxo-LCA to UDCA by TLC. Consequently, we isolated strain N53, which gave a spot corresponding to UDCA by TLC after culture in GAM broth containing 7-oxo-LCA (Fig. 2). The bile acids extracted from the culture medium were characterized by GC-MS; their retention times (RT) corresponded to 7-oxo-LCA (RT, 12.2 min) and UDCA (RT, 12.6 min), respectively. The m/z values for the fragment ions corresponding to the conversion products (213, 255, 355, 370, and 460) were identical to authentic UDCA. 16S rDNA sequencing showed that the strain N53 was closely related to R. gnavus ATCC 29149T (99.85% sequence homology). 7β-HSDH activity has been reported for several intestinal bacteria, such as Collinsella aerofaciens 25986T (29Hirano S. Masuda N. Epimerization of the 7-hydroxy group of bile acids by the combination of two kinds of microorganisms with 7α- and 7β-hydroxysteroid dehydrogenase activity, respectively.J. Lipid Res. 1981; 22: 1060-1068Abstract Full Text PDF PubMed Google Scholar, 30Hirano S. Masuda N. Characterization of NADP-dependent 7β-hydroxysteroid dehydrogenases from Peptostreptococcus productusEubacterium aerofaciens.Appl. Environ. Microbiol. 1982; 43: 1057-1063Crossref PubMed Google Scholar), Ruminococcus productus b-52 (29Hirano S. Masuda N. Epimerization of the 7-hydroxy group of bile acids by the combination of two kinds of microorganisms with 7α- and 7β-hydroxysteroid dehydrogenase activity, respectively.J. Lipid Res. 1981; 22: 1060-1068Abstract Full Text PDF PubMed Google Scholar, 30Hirano S. Masuda N. Characterization of NADP-dependent 7β-hydroxysteroid dehydrogenases from Peptostreptococcus productusEubacterium aerofaciens.Appl. Environ. Microbiol. 1982; 43: 1057-1063Crossref PubMed Google Scholar, 33Masuda N. Oda H. Tanaka H. Purification and characterization of NADP-dependent 7β-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52.Biochim. Biophys. Acta. 1983; 755: 65-69Crossref PubMed Scopus (13) Google Scholar), Ruminococcus sp. PO1-3 (34Akao T. Akao T. Kobashi K. Purification and characterization of 7β-hydroxysteroid dehydrogenase from Ruminococcus sp. of human intestine.J. Biochem. 1987; 102: 613-619Crossref PubMed Scopus (11) Google Scholar), and Clostridium baratii (26Lepercq P. Gérard P. Béguet F. Raibaud P. Grill J.P. Relano P. Cayuela C. Juste C. Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces.FEMS Microbiol. Lett. 2004; 235: 65-72Crossref PubMed Google Scholar), but not for R. gnavus. Biochemical tests of strain N53 and the R. gnavus type strain ATCC 29149T with API 20 and Rapid API ID 32A showed a few differences in sugar assimilation (i.e., saccharose; supplementary Table I) and enzyme activity (α-fucosidase, leucine arylamidase, pyroglutamic acid arylamidase, and alanine arylamidase; supplementary Table II) between the two strains. From these phylogenetic and biochemical data, strain N53 was identified as R. gnavus. Thus, we identified the R. gnavus N53 isolate as a novel UDCA producer. To identify the conversion reaction catalyzed by 7β-HSDH, R. gnavus N53 was cultured in GAM broth supplemented with 7-oxo-LCA (Fig. 3A). The type strain ATCC 29149T (Fig. 3C) and the known UDCA-producing bacterium C. aerofaciens ATCC 25986T (Fig. 3B) were also cultured for comparison. The three bacterial strains nearly reached stationary phase 6 h after inoculation. All of these strains were found to convert 7-oxo-LCA to UDCA, and their conversion rates peaked (>90%) during stationary phase, suggesting that the reductive conversion reaction was growth-dependent. In contrast, when R. gnavus N53 was cultured in the presence of UDCA, 7-oxo-LCA was not detected (data not shown), indicating that the oxidative reaction with whole cells was unable to proceed. Measurement of the 7β-HSDH activity of the cells cultured for 12 h revealed that R. gnavus N53 showed the highest specific activity
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