Periplasmic Export of Bile Salt Hydrolase in Escherichia coli by the Twin-Arginine Signal Peptides

Abstract

Bile salt hydrolase (BSH, EC 3.5.1.24) is considered as an ideal way with lower cost and less side effects to release the risk of coronary heart disease caused by hypercholesterolemia. As bile salt hydrolase from Lactobacillus plantarum BBE7 could not be efficiently exported by PelB signal peptide of the general secretory (Sec) pathway, three twin-arginine signal peptides from twin-arginine translocation (Tat) pathway were synthesized, fused with bsh gene, inserted into expression vectors pET-20b(+) and pET-22b(+), and transformed into four different Escherichia coli hosts, respectively. Among the 24 recombinant bacteria obtained, E. coli BL21 (DE3) pLysS (pET-20b(+)-dmsA-bsh) showed the highest BSH activity in periplasmic fraction, which was further increased to 1.21 ± 0.03 U/mL by orthogonal experimental design. And, signal peptide dimethyl sulfoxide reductase subunit DmsA (DMSA) had the best activity of exported BSH. More importantly, the presence of BSH in the periplasm had proven to be caused by the export rather than cell leakage. For the first time, we report the periplasmic expression of BSH by signal peptides from the Tat pathway. This will lay a solid foundation for the purification and biochemical characterization of BSH from the supernatant, and strategies adopted here could be used for the periplasmic expression of other proteins in E. coli.

Taxonomic profiling and populational patterns of bacterial bile salt hydrolase (BSH) genes based on worldwide human gut microbiome

“…The BSH specific activity was determined by measuring the release of amino acids (glycine and taurine) from conjugated bile salts. The amounts of amino acids liberated by BSH were determined by ninhydrin assay [84]. Protein concentrations were determined using a Bradford Protein Assay kit (Beyotime, Nanjing, China).…”

Taxonomic Profiling and Populational Patterns of Bacterial Bile Salt Hydrolase (BSH) Genes on Worldwide Human Gut Microbiome

“…BSH specific activity was determined by measuring the release of amino acid from conjugated bile salts. The amounts of amino acids liberated by BSHs are determined by the ninhydrin assay (Dong et al, 2015). Protein concentrations were determined with the Bradford Protein Assay kit (Beyotime, Nanjing, China), and bovine serum albumin was used as standard.…”