Effects of Structural Changes in Bile Salt Hydrolase Enzyme on Biocatalytic Efficiency and Activation Energy at Working pH and Temperature Conditions

Abstract

Microbial bile salt hydrolases (BSHs) catalyse the hydrolysis of glycine and taurine-linked bile salts in the small intestine of humans. Achieving the effects of structural changes in BSH molecules on biocatalytic efficiency ( k cat / K m ) and activation energy ( E a ) is necessary to determine biocatalytic performances of the enzymes. Amino acids responsible for biocatalytic activity or substrate specificity in BSH molecules were modified to determine the effects of structural changes on k cat / K m values and E a values of the bioconversion reactions. Purified wild type positive control enzyme (pCON2) and mutant recombinant target enzymes (F18L and Y24L) reacted with six conjugated pure bile salt substrates at working temperature and pH conditions. The results of the hydrolysis conversion analysis conducted at various pH conditions were used to estimate k cat / K m , and the assays conducted at various temperature conditions were used to approximate E a of the biocatalytic reactions. The quantified k cat / K m value was found remarkably highest with mutant recombinant enzymes (Y24L), while the efficiency value with wild type (pCON2) was determined as lowest, indicating that the structural modifications in BSH molecules showed higher values. The alterations with the mutant-type enzymes F18L and Y24L resulted in decreasing k cat / K m and increasing E a estimations of the hydrolysis conversion reactions.