z-logo
Premium
Molecular cloning, characterization and comparison of bile salt hydrolases from L actobacillus johnsonii PF 01
Author(s) -
Chae J.P.,
Valeriano V.D.,
Kim G.B.,
Kang D.K.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12027
Subject(s) - biochemistry , hydrolase , biology , enzyme , molecular cloning , microbiology and biotechnology , gene , chemistry , peptide sequence
Aims To clone, characterize and compare the bile salt hydrolase ( BSH ) genes of L actobacillus johnsonii PF 01. Methods and Results The BSH genes were amplified by polymerase chain reaction ( PCR ) using specific oligonucleotide primers, and the products were inserted into the p ET 21b expression vector. Escherichia coli BLR ( DE 3) cells were transformed with p ET 21b 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 ( N i 2+ ‐ 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. Conclusions 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.

This content is not available in your region!

Continue researching here.

Having issues? You can contact us here