Both authors contributed equally to this work.
Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01
Article first published online: 29 OCT 2012
© 2012 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 114, Issue 1, pages 121–133, January 2013
How to Cite
Chae, J.P., Valeriano, V.D., Kim, G.-B. and Kang, D.-K. (2013), Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01. Journal of Applied Microbiology, 114: 121–133. doi: 10.1111/jam.12027
- Issue published online: 12 DEC 2012
- Article first published online: 29 OCT 2012
- Accepted manuscript online: 4 OCT 2012 10:49AM EST
- Manuscript Accepted: 27 SEP 2012
- Manuscript Revised: 25 SEP 2012
- Manuscript Received: 17 JUN 2012
- Next-Generation BioGreen 21 Program. Grant Number: PJ00812701
- bile salt hydrolase;
- glyco-conjugated bile salts;
- Lactobacillus johnsonii ;
- substrate specificity;
- tauro-conjugated bile salts
To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.
Methods and Results
The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l−1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni2+-NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.
BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation.
Significance and Impact of the Study
This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.