title: Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394.
abstract: Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni+2-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 °C while showed its maximum activity at 37 °C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.
authors.affiliation: Department of Microbiology & Immunology, National Institute of Nutrition, Hyderabad, 500007, India. rajesh.mbu@gmail.com
authors.forename: Rajesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Rajkumar
authors.forename: Manoj
authors.initials: M
authors.lastname: Kumar
authors.forename: Sudarshan Reddy
authors.initials: SR
authors.lastname: Varikuti
authors.forename: Ramakrishna
authors.initials: R
authors.lastname: Athimamula
authors.forename: Mohd
authors.initials: M
authors.lastname: Shujauddin
authors.forename: Ramesh
authors.initials: R
authors.lastname: Ramagoni
authors.forename: Narendrababu
authors.initials: N
authors.lastname: Kondapalli
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside; D052197:Limosilactobacillus fermentum / Q000201:enzymology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D019936:Probiotics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D007544:Isopropyl Thiogalactoside; D000581:Amidohydrolases; C023154:choloylglycine hydrolase