İnsan kaynaklı Lactobacillus plantarum kökenli safra tuzu hidrolaz (STH) genının klonlanması ve STH enziminin yönlendirilmiş mutagenez ile karakterizasyonu

The gastrointestinal microbiota plays an important role in human physiology and is liable for nutritional, physiological and immunological functions of the host. Microbial colonization of gastrointestinal system significiantly affect the genetic factors of colorectal cancer etiology. The microbial products of the toxic secondary bile acids such as lithocholic acid and deoxycholic acid caused colon cancer by influencing inflammation, DNA damage and apoptosis induction. The hydrolysis of glycine or taurine-conjugated bile acids is the gateway reaction for the occurrence of secondary bile acids. BSH enzymes (EC 3.5.1.24) from various sources rather differ in characteristics, substrate preference and specificity. To show relationship between BSH enzymes and toxic secondary metabolites, structure of the BSH must be better understood, but the structure and reaction mechanism of such an important BSH enzyme are not known very well. Site-directed mutagenesis is used to understand the structure and function of the enzyme due to the importance of the substitution of the codons coding amino acids supposed to be responsible for substrate specificity. In this study, Lactobacillus plantarum B14 BSH gene encoding 324-amino acids was cloned and the codons of the partially conserved amino acids, Phe-18, Tyr-24, Asn-79, Leu-138 and Asn-180 supposed to be responsible for substrate specificity, substituted by site directed mutagenesis. All mutant BSHs were purified using an E. coli BLDRE3-pET22b expression system. The stability of the mutant enzymes and molecular weight 37 kDa were confirmed by SDS–PAGE. The high BSH activity of Lb. plantarum B14 was determined qualitatively by Direct Plate Assay and quantitatively by Ninhydrin assay. The effects of the mutations on substrate specificity and catalytic activities of BSH were investigated by partially purified of mutant BSH enzymes with six different bile salts. The study showed that Lb. plantarum B14-BSH exhibited greater hydrolysis toward glyco-conjugated bile salts compared to tauro-conjugated bile salts and the BSH activities of mutant enzymes showed signifciantly decreasing alternations towards different bile salts.