Department of Animal Resources Science, Dankook University, Cheonan, Korea.
J Appl Microbiol. 2013 Jan;114(1):121-33. doi: 10.1111/jam.12027. Epub 2012 Oct 29.
To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.
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.
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.
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.
克隆、鉴定并比较约翰逊乳杆菌 PF01 的胆盐水解酶（BSH）基因。
采用聚合酶链反应（PCR），使用特异性寡核苷酸引物扩增 BSH 基因，将产物插入 pET21b 表达载体。将含有 BSH 基因的 pET21b 载体转化至大肠杆菌 BLR（DE3）细胞中，并用 0.1mmol l(-1)异丙基硫代-β-D-半乳糖苷诱导表达。采用镍-亚氨基二乙酸（Ni(2+) -NTA）琼脂糖柱纯化过表达的 BSH 酶，并对其活性进行鉴定。BSH A 最适作用于 pH5.0 和 55°C 时的牛磺胆酸盐，BSH C 最适作用于 pH5.0 和 70°C 时的甘胆酸盐。这些酶对特定胆酰基无优先活性。
BSH 酶在其底物特异性和特性方面存在差异，从而拓宽了其活性。尽管它们假定的底物结合位点缺乏保守性，但通过基序保守性，这些位点仍然具有功能。
这是首次从单一菌株中分离 BSH 酶并显示其对甘胆酸盐或牛磺胆酸盐具有水解酶活性的报道。未来的结构同源性研究和与底物特异性相关的定点突变研究可能阐明 BSH 酶的特异性。
