Lactobacillus gasseri safra tuzu hidrolaz emzimlerinin klonlanması, karakterizasyon ve karşılastırması

Over the past decade, bile salt hydrolase (BSH) active probiotic strains are being used in the treatment of hypercholesterolemia related diseases. The aptitude of this BSH enzyme to deconjugate bile salts into free bile acids and glycine or taurine moiety has been a major criteria in the selection of probiotics. Not only does this deconjugation provides bile tolerance to the bacteria for its continued existence in the GI tract, it also plays a critical part in the reduction of the blood cholesterol level of the human host. Therefore, knowing and understanding the mechanism of action of the BSH enzymes of the probiotic being used in those treatments is of utmost importance. Even though Lactobacillus gasseri is probably one of the most beneficial probiotics on the market, there is little information on the mechanism of action of the catalytic activity and substrate preferences of the BSH enzyme of this microorganism. In this study, the bsh gene from Lb. gasseri ATCC 33323 strain was cloned, expressed, characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activity of the wild-type recombinant BSH (wrBSH) enzyme was then examined in different temperatures ranging from 25°C to 65°C and various pHs from 2.5 to 8.0 along with six different bile acids by ninhydrin assay. The expression of bsh gene was detected by SDS-PAGE. The ninhydrin test results indicated that wrBSH enzyme hydrolyzed six major human bile salts with an obvious preference towards glycine-conjugated compared to tauro-conjugated bile salts. Maximun activities of wrBSH enzyme were also recorded at low pH and high temperatures. Our findings support the suggestion that BSH enzymes recognize their substrates predominantly through their amino acid moieties rather than their cholate moieties. However, more extensive studies like site directed mutagenesis are needed to fully understand the catalytic activity as well as the substrate specificities of the BSH enzymes and to set the standards for the selection of probiotic bacteria. KEYWORDS: Probiotics, Lactobacillus gasseri, Bile Salt Hydrolase (BSH), Catalytic activity and Substrate specificity