Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01.
J Appl Microbiol
; 114(1): 121-33, 2013 Jan.
Article
em En
| MEDLINE
| ID: mdl-23035872
ABSTRACT
AIMS:
To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. METHODS ANDRESULTS:
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 (Ni(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.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Ácidos e Sais Biliares
/
Amidoidrolases
/
Lactobacillus
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article