title: Molecular cloning and characterization of a bile salt hydrolase from Lactobacillus acidophilus PF01.
abstract: 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 Ni2+-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 40oC. The enzyme maintained approximately 70% of its maximum activity even at 60 degrees , whereas its activity rapidly decreased at below 37 degrees . The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.
authors.affiliation: Bio-Resources Institute, EASY BIO System, Cheonan 330-822, Korea.
authors.forename: Hae-Keun
authors.initials: HK
authors.lastname: Oh
authors.forename: Ji Yoon
authors.initials: JY
authors.lastname: Lee
authors.forename: Soo Jin
authors.initials: SJ
authors.lastname: Lim
authors.forename: Min Jeong
authors.initials: MJ
authors.lastname: Kim
authors.forename: Geun-Bae
authors.initials: GB
authors.lastname: Kim
authors.forename: Jung-Hoan
authors.initials: JH
authors.lastname: Kim
authors.forename: Soon-Kwang
authors.initials: SK
authors.lastname: Hong
authors.forename: Dae-Kyung
authors.initials: DK
authors.lastname: Kang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D015870:Gene Expression; D007779:Lactobacillus acidophilus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine / Q000382:microbiology
chemical_list: D011994:Recombinant Proteins; D000581:Amidohydrolases; C023154:choloylglycine hydrolase