Lactobacillus plantarum B14'den elde edilen rekombinant mutant safra tuzu hidroz (STH) enzimlerinin fonksiyonel ve yapısal analizi

Bile salt hydrolase (BSH), synthesized by some intestinal bacteria, catalyzes the hydrolysis of taurine or glycine-linked bile acid into the amino acid and deconjugated bile acid in human intestine. 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. Because of the link between BSH and human health, the structural and biochemical features of BSHs necessitate an intense study to better understand their role in regulating bacterial and host metabolism. BSHs exhibit higher variations in their catalytic activities and substrate specificities. However, six amino acids, supposed to be responsible from catalytic activity, are totaly conserved in all members of the BSH family. In order to analyze the correlation between two of these strictly conserved amino acids and catalytic activity of BSH, the asparagine-170 (N170) and arginine-223 (R223) amino acids were substituted for the valine-170 (V170) and phenylalanine-223 (F223) amino acids respectively by PCR-based site directed mutagenesis. The mutant recombinant BSHs (rBSHs) were expressed in E. coli BLR(DE3) strain. While the effect of the mutations on catalytic activity was detected by direct plate and ninhidrin assays, the effect of the mutations on formation of the BSHs was observed by SDS-PAGE analysis. It was found that V170 and F223 mutations resulted in prevention of BSH formation. This is the first experimental work showing that the N170 and R223 could be critical amino acids for folding or stability of the BSH, but not for the catalytic activity of this enzyme as predicted by in silico analysis of BSH related data in literature.