E.Coli de Lactobacillus plantarum dan izole edilen safra tuzu hidrolaz enziminde Arg16phe and Asp19leu mutasyonlarının oluşturulması ve karakterizasyonu

Probiotics are thought to have an important role in the lowering of the high cholesterol level that is shown as the main reason for cardiovascular diseases. Drugs used for treatment are expensive and have a lot of side effects so promoting the importance of the probiotics. There are a lot of studies proving that probiotics especially, intestinal bacteria such as Lactobacillus and Bifidobacterium, are lowering the amount of cholesterol with their Bile salt hydrolase (BSH) enzymes, but we do not know much about this enzyme. To characterize BSH enzyme one of the methods is; site direct mutagenesis and the other method is crystallography. However, no mutagenesis study on BSH enzyme was found in literature except Cys2 amino acid. In this study, fully conserved amino acids, Arg-16 and Asp-19 of L. plantarum species, were substituted for phenylalanine and leucine amino acids respectively in pCON1 construct using appropriate primers. Afterwards, mutant bsh genes were transferred to pET22b expression vector. The recombinant mutant proteins were purified using a Ni2+ chelated column and then were analyzed in respect to their activity and stability by direct plate assay, ninhidrin assay and polyacrylamide gel electrophoresis, respectively.While Direct plate assay and Ninhidrin assay results indicated that all of the mutations resulted in inactive BSH enzymes, polyacrylamide gel electrophorese results showed that Arg-16 mutant BSH protein was folded, but Asp-19 mutant protein was not. Our results revealed that while Arg-16 amino acid is responsible for the catalytic activity of BSH enzyme, Asp-19 amino acid is required for the folding of the BSH enzyme and its stability. Therefore, this work emphasized that further site directed mutagenesis studies, especially on the amino acids supposed to be responsible for catalytic activity and bile salt substrate specificity of the BSH enzyme, are required.