Lactobacillus plantarum GD2'den safra tuzu hidrolaz genlerinin (BSH) moleküler klonlanması ve karakterizasyonu

Bile Salts Hydrolase (BSH) enzymes catalyse the hydrolysis of taurine and glycine-linked bile salts into amino acid residues. The precise function of BSHs is still unknown, but some hypothesis have been proposed about playing role in nutrition, cholesterol lowering and colorectal cancer etc. BSHs are frequently synthesized by intestinal commensal lactobacilli. BSHs from different lactobacilli strains exhibit higher variation in sequence, pH optimum, kinetic properties and substrate specificity. In this study, bsh1 gene from L. plantarum GD2 was cloned into pET22b expression vector and overexpressed. At first; the gene was amplified by PCR using by species specific oligonucleotide primers and then cloned into pJET1.0 or pBluescript II SK+ cloning vector and pET22b expression vector. After clones were transformed to XL1-Blue and BLR(DE3) Escherichia coli host strains respectively, the bsh1 gene was expressed in E.coli BLR(DE3) host by induction of 0.1 mmol 1-1 of isopropylthio galactopyranoside. The overexpressed recombinant protein was purified using B-PER 6xHis Fusion Protein Purification Kit and deconjugation ability of the enzyme was tested with six conjugated bile salts by ninhydrin protein assay. Four genes, bsh1, bsh2, bsh3 and bsh4 were identified in the genome sequence of L. plantarum GD2 and sequenced. Comparison of the deduced amino acid sequences of Bsh1 with previously known sequences revealed high homology with BSH enzymes of several microorganisms. Biochemical characterization of the purified Bsh1 revealed some distinct characteristics not observed in other species of Lactobacillus. The purified Bsh1 enzyme showed preferential activity against glyco-conjugated bile acids and show highest activity against glycodeoxycholic acids. In this study, the primary structure of Bsh1 enzyme was characterized and analysed to further studies. It is thought that site-directed mutagenesis on conserved amino acids of BSH enzyme and structural analysis will be required for the future studies to investigate key residues of the active site, substrate binding pocket and substrate selectivity of BSH enzyme.