Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Cloning and Characterization of a Bile Salt Hydrolase from Lactobacillus acidophilus PF01

  • Oh, Hae-Keun (Bio-Resources Institute, EASY BIO System) ;
  • Lee, Ji-Yoon (Division of Agricultural Biotechnology, Seoul National University) ;
  • Lim, Soo-Jin (Department of Animal Resources Science, Dankook University) ;
  • Kim, Min-Jeong (Department of Animal Resources Science, Dankook University) ;
  • Kim, Geun-Bae (Department of Animal Science and Technology, Chungang University) ;
  • Kim, Jung-Hoan (School of Natural Food Sciences, Eulji University) ;
  • Hong, Soon-Kwang (Division of Bioscience and Bioinformatics, Myongji University) ;
  • Kang, Dae-Kyung (Department of Animal Resources Science, Dankook University)
  • Published : 2008.03.31
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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 $Ni^{2+}$-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 $40^{\circ}C$. The enzyme maintained approximately 70% of its maximum activity even at $60^{\circ}C$, whereas its activity rapidly decreased at below $37^{\circ}C$. The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.

Keywords

References

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