http://orcid.org/0000-0001-6906-5257
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ABSTRACT
Bile salt hydrolase (BSH), found commonly in intestinal species of Lactobacillus and Bifidobacterium, catalyzes the hydrolysis of glycine or taurine-conjugated bile acids into the amino acids and free bile acids. Deconjugated bile acids potentially play an important role in the reduction of blood cholesterol level and formation of some gastrointestinal diseases such as cholestasis, gallstone formation, and colon cancer. Although the crystal and three-dimensional structures of BSH enzyme are known, the working mechanism of catalytic activity of such an important BSH enzyme is not known very well. Previous in silico analysis of multiple BSH has identified that Arginine-16 (R16) and Aspartate-19 (D19) were catalytically important residues in the active site of BSH. To confirm the function of these amino acids, in this study, BSH enzyme from Lactobacillus plantarum B14 strain was cloned into Escherichia coli and strictly conserved polar R16 and D19 amino acids of BSH enzyme were substituted for hydrophobic Phenylalanine-16 (F16) and Leucine-19 (L19) amino acids, respectively, by polymerase chain reaction (PCR)-based site-directed mutagenesis. The effects of the mutations on catalytic activity and structure of the BSH enzymes were detected by ninhidrin assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. Research results showed that although F16 mutation led to loss of enzyme activity completely, L19 mutation led to abolishment of the synthesis of BSH enzyme. These results indicated that R16 and D19 amino acids located in loop I of the BSH enzyme might be critical for catalytic activity and assembly of the BSH enzyme respectively.