Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394
Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394
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DOI：
10.1007/s11033-013-2607-2
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发表时间：
2013-08-01
影响因子：
2.8
通讯作者：
Kondapalli, Narendrababu
中科院分区：
文献类型：
--
作者：
Kumar, Rajesh;Rajkumar, Hemalatha;Kondapalli, Narendrababu
胆盐水解酶(BSH)活性益生菌在体内降解胆汁酸氨基酸结合物，从而触发肝脏对胆固醇的消耗，合成新的胆汁，从而降低胆固醇。因此，本研究以胆盐水解力作为筛选发酵乳杆菌NCDO394的标准，并对其编码bsh的基因进行了鉴定和克隆。得到的BSH基因核苷酸序列包含一个978个核苷酸的开放阅读框(ORF)，编码325个氨基酸的预测蛋白质，理论等电点为6.39。此外，推测的BSH蛋白与发酵乳杆菌的BSHS有很高的相似性，与其他乳杆菌的Penclin V酰胺酶也有很大的相似性。发酵乳杆菌BSH蛋白中5个重要的催化氨基酸高度保守，而这些活性中心周围的4个氨基酸基序与其他BSH蛋白的氨基酸序列不一致。将发酵乳杆菌bsh基因亚克隆到pET-28b(+)载体上，用0.05 mM异丙基硫代半乳糖苷(IPTG)诱导表达。利用Ni+2-NTA柱层析对重组BSH(RBsh)进行了纯化，并对底物专一性、pH和温度进行了鉴定。RBsh水解人胆盐的六种主要成分，对甘氨酸结合的胆盐有轻微的选择性。RBsh的最适pH为6，其酶活在pH 5以下和pH 7以上时下降。该酶在65℃时仍能稳定发挥作用，在37℃时酶活性最高。
Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni+2-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 A degrees C while showed its maximum activity at 37 A degrees C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.