Molecular cloning and characterization of a bile salt hydrolase from Lactobacillus acidophilus PF01.
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.
Oh
Hae-Keun
HK
Bio-Resources Institute, EASY BIO System, Cheonan 330-822, Korea.
Lee
Ji Yoon
JY
Lim
Soo Jin
SJ
Kim
Min Jeong
MJ
Kim
Geun-Bae
GB
Kim
Jung-Hoan
JH
Hong
Soon-Kwang
SK
Kang
Dae-Kyung
DK
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
D011994:Recombinant Proteins; D000581:Amidohydrolases; C023154:choloylglycine hydrolase