Oh Hae-Keun, Lee Ji Yoon, Lim Soo Jin, Kim Min Jeong, Kim Geun-Bae, Kim Jung-Hoan, Hong Soon-Kwang, Kang Dae-Kyung
Bio-Resources Institute, EASY BIO System, Cheonan 330-822, Korea.
J Microbiol Biotechnol. 2008 Mar;18(3):449-56.
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.
对从仔猪粪便中分离出的嗜酸乳杆菌PF01进行了胆盐水解酶（BSH）活性的表型筛选。从嗜酸乳杆菌PF01的基因组文库中鉴定并克隆了一个编码BSH的基因。通过核苷酸序列分析对bsh基因及其周边区域进行了表征，发现其包含一个951个核苷酸的单一开放阅读框（ORF），编码一个316个氨基酸的蛋白质。潜在的bsh启动子区域位于起始密码子的上游。从完整ORF推导的蛋白质与其他BSH具有高度相似性，并且位于活性位点周围的四个氨基酸基序FGRNXD、AGLNF、VLTNXP和GXGXGXXGXPGD高度保守。将bsh基因克隆到pET21b表达载体中，并通过用0.1mM异丙基硫代半乳糖苷诱导在大肠杆菌BLR(DE3)中表达。使用Ni2+-NTA琼脂糖柱对BSH酶进行了纯化，使其具有明显的均一性，并对其进行了表征。嗜酸乳杆菌PF01过表达的重组BSH酶对牛磺结合型胆盐具有水解活性，但对糖结合型胆盐没有水解活性。它对牛磺胆酸表现出最高活性。最大BSH活性出现在约40℃。即使在60℃时，该酶仍保持其最大活性的约70%，而在低于37℃时其活性迅速下降。最适pH为6，在pH 5以下和pH 7以上时，BSH活性迅速失活。
