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

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.


Introduction
BSH enzyme, a member of cholylglycine hydrolase (CGH) family, is produced by the intestinal tract microbiota bacteria, such as Lactobacillus, Clostridium, Bacteroides, and Bifidobacterium, and catalyses the hydrolysis bioreaction of glycine or taurine-conjugated bile acids to produce taurine or glycinamino acids and bile salts. 1,2 Through bile salt deconjugation, BSH enzymes function as specific signalling and regulating molecules in multiple physiological processes, and contribute to the treatment effect of human diseases such as hypercholesterolemia, colon cancer, diabetes, inflammation, antimetabolic functionality, anti-immune homeostasis, giardial activity, and cardiovascular diseases. 3,4 However, various studies have stated that extra deconjugation of tauro-conjugated bile salts and secondary bile acid production during the hydrolysis can cause formation of deoxicholic and lithocholic acids in the gastrointestinal tract, which may result in undefined toxic effects resulting in colon cancer. 5 Determination of the function of BSH amino acids that are responsible for the substrate specificity or biocatalytic activity can supply information on the role of formation of secondary toxic metabolites. 6 Therefore, the molecular structure of BSH was altered by modifying the amino acids that are hypothetically responsible for the specificity or the activity following site-directed mutagenesis procedures.
BSHs differ in subunit size and composition, parameters of pH and temperature optimum, kinetic properties, substrate specificity, gene organisation and regulation. To understand the characteristic features of BSH, the bsh gene is cloned into the expression vector, and production of the active pure BSH protein is required. Future structure analysis of BSH allows to discover key residues of the active site and substrate-binding pocket, and provide information on the substrate selectivity of BSHs. Two BSHs (BSH1 and BSH2) from Lb. salivarius were expressed and purified in a heterologous manner, and BSH1 showed a wide optimum pH range of 5.5 to 7.0, while a narrower pH range of 5.5 to 6.0 was determined for BSH2. The kinetic curves of BSH1 and BSH2 were found to be similar to the hyperbolic forms of Michaelis-Menten kinetics in the presence of dithiothreitol (DTT). The results of the obtained Michaelis-Menten kinetics constant (K m ) were found to be significant at pH (6.0) and temperature (37 °C). 1 The modifications in the amino acid residues can affect the biocatalytic performances, and consequently k cat /K m and E a of the enzymes in the substrate hydrolysis reactions. The evaluation of the biocatalytic efficiency can be calculated at working pH and constant temperature, and E a can be estimated at working temperature and constant pH.
BSH and the other enzymes contain biocatalytically active group residues as binding sites or active sites that are directly dependent on physiological conditions such as pH and temperature. The dependency dominates the activity of enzymes and affects the amino acid chains of biocatalytic

Effects of Structural Changes in Bile Salt
Hydrolase Enzyme on Biocatalytic Efficiency and Activation Energy at Working pH and Temperature Conditions side residues. The other active groups of the enzyme sites coordinate formation of the enzyme-substrate complex and product. Except for ideal conditions, the extremely lower and higher pH and temperature values reduce the biocatalytic activity and damage the molecular structures of the enzymes. BSH enzymes have specific working pH and temperature conditions, and if the pH and temperature gradients are out of the ideal limits, then apparent activity loss and denaturation occur.
Amino acid residues in the active site chains of enzymes can be charged positively or negatively or protonated or deprotonated by increasing or decreasing the pH values, adjusting the activity of the enzymes. Low pH gives protonated negative charge value to the enzyme solution and to the binding sites, and high pH positively charges the sites. The negative and positive charges affect the strength of the bond between the amino acids in the sites of the enzymes and substrate that indicate the biocatalytic activity. 7 The Michaelis-Menten equation, used to model the reaction rate V (1/time), is based on the reaction constant (K m ) of the substrates (S) and the maximum reaction rate (V max ). The Michaelis-Menten equation was linearised to obtain K m and V max constants using the Eadie-Hofstee linearisation approach, one of the most used linearisation methods. 8 According to the Eadie-Hofstee approximation equation, the linear equation of the Michaelis-Menten function was obtained by plotting the graph according to V vs V/S for estimation of V m from the intersection of y axis, and V m /K m from the intersection of x axis. 9,10 The model of an enzyme-catalysed reaction mechanism consists of the binding of the total amount of enzyme (e T ) to substrate, and then the catalysis of the reaction and release of the product. The binding of the substrate to the enzyme is determined by the affinity constant K A = k 1 /k −1 and the Michaelis-Menten reaction constants (K m ), which are K S = (k 2 + k −1 )/k 1 for the substrate, and K P = (k 2 + k −1 )/ k −2 for the product. The maximum velocity (V m ) for the forward enzyme reaction equals to the multiplication of the biocatalytic rate constant (k 2 ) and the total amount of enzyme (e T ). Overall kinetic constants can be arranged in various combinations, and Michaelis-Menten velocity of enzyme kinetics was approached for the substrate concentration. 11 The BSH enzyme hydrolysis is strictly temperature-dependent and actual temperature conditions are needed for ideal biocatalytic activity. The temperature had a noticeable direct effect on the kinetic energy of the molecular structure. Therefore, there is occurrence possibility of clever inactivation at low temperatures and severe denaturation at high temperatures in enzyme molecules. The temperature impacts the value of kinetic constants, which affect the E a that is the function of the kinetic constants and reaction temperature, defined as the critical energy of the conversion reactions. 12 Enzymes decrease the E a of the specific reactions, resulting in the increase in the reaction rates by specifically binding more strongly at the transition state than at the reactant state. 13 The Arrhenius equation was used to determine the E a of enzymatic reactions, that can also be written in linear equation form, which was applied for plotting lnk vs 1/T. The value of −E a /R was cal-culated from the slope, and lnA from the intercept of the plot. The k cat and reaction temperature values were used in Arrhenius equation for determination of the E a to obtain maximum conversion of the substrates and high product formation in enzyme-catalysed reactions. 14 The equations of Michaelis-Menten (V) and the maximum velocity (V m ), and the Arrhenius equation (k) and their linearised forms are given in Table 1.
The molecular structure of the amino acid residues of BSH enzymes was engineered according to the amino acid modification applications. 2 Ninhydrin reagent test was conducted for detection of the presence of amino acids in the sample, having the reaction taking place between the amino group and the ninhydrin reagent. 21 This research aimed to investigate the effects of the structural changes in the BSH molecules on k cat /K m and E a at ideal pH and temperature ranges.

Materials and methods
The assays were performed according to the standard experimental procedures using six primary and secondary bile acids (glycocholic acid (GCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA)) as the substrates. The bile acid standards and other chemicals were obtained from Sigma-Aldrich. The BSH enzymes were obtained through transferring the amplified bile salt hydrolase (BSH) gene from Lactobacillus plantarum to Escherichia coli. Partially purified wild type enzyme (pCON2) and mutant recombinant target enzymes (F18L and Y24L) were used in the duplicated hydrolysis conversion assays carried out at a) pH values of 3.0, 4.0, 5.0, and 7.0 at a constant temperature of 37 °C, and at b) 25, 37, 45, 55, and 65 °C at a constant pH value of 6.0. The standard Ninhydrin assay method was applied to detect the activity of wild-type recombinant and mutant BSH enzymes. 21 The conversion data of the substrates observed at different pH values at constant temperature were used to determine the biocatalytic efficiency, and the data collected at differ-ent temperature and constant pH conditions were applied to assess E a , consistent with previously accomplished studies.

Results
The data plots of Eadie-Hofstee have given the most satisfactory linear lines among the three equations. Effects of structural changes on k cat /K m and E a of human-sourced purified wild type and mutant BSHs were investigated at ideal pH and temperature ranges. The molecular structure of recombinant BSHs were altered by modifying the amino acids evidently responsible for biocatalytic activity following the site-directed mutation procedures.
The six bile salts as pure substrates were applied for the hydrolysis reactions with the purified enzymes at ideal pH and temperature conditions. The biocatalytic activities and the reaction mechanisms of the BSH enzymes were evaluated by estimating the overall Michaelis-Menten kinetic constants for the converted substrate concentrations. The reaction kinetic constants, the rate of binding of substrates to the enzymes (K m ), and the maximum reaction rate (V m ) values were determined using the hydrolysis conversions data for the experimented pH and temperature values. The bioprocess of an enzyme-catalysed reaction mechanism consists of the binding of the total amount of enzyme (e T ) to substrate, and then the catalysis of the reaction and the release of the product. The biocatalytic rate constant (k cat ), ratio of maximum velocity (V m ), and bioreaction constant (K m ) were determined to approximate the biocatalytic efficiency values. The kinetic constants were used to model the reaction rates (V) for the determination of biocatalytic efficiency, and E a for the enzymes and substrates. The biocatalytic efficiency and E a values of purified wild type positive control enzyme (pCON2) were compared to the purified mutant recombinant enzymes (F18L and Y24L) at the investigated pH and temperature values.

Effect of structural changes on biocatalytic efficiency
In this part of the research, the purified BSH enzymes were applied to hydrolyse the six pure conjugated bile salts as substrates at varied pH and constant temperature conditions to approximate the biocatalytic efficiency values of the BSHs. The hydrolysis conversion data of the six substrates measured at the experimented pH values were applied to determine K m and V m , which were used to estimate V and k cat /K m of the BSHs.
The hydrolysis conversions of the six pure bile salt substrates were tested with purified wild and mutant enzymes (F18L and Y24L) at different pH and fixed temperature conditions as presented in Fig. 1. The reaction rate (V) was predicted upon quantifying approximation of K m and V m , and the graphs of the reaction rate (V) vs hydrolysed amount of the six substrates and experimented pH values are shown in Fig. 2.
The biocatalytic rate constant determinations were achieved using V m estimations and e T values, which were determined by activity of the purified mutant recombinant enzymes. The ratio of K m over k cat was used to attain k cat / K m assessments of the BSHs bioprocess. The biocatalytic efficiency graphs for the six bile salt substrates hydrolysed by the BSH enzymes are presented in Fig. 3.

Effect of structural changes on E a
Effect of structural alterations in the molecular structures of the BSH enzymes on E a was evaluated in this section. The amino acids responsible for biocatalytic activity of BSHs molecules were modified to determine the effects of structural changes on E a at ideal temperature gradients. The six pure conjugated bile salt substrates were reacted with purified BSH enzymes at different temperatures and fixed pH conditions. The hydrolysis conversions data were used to determine K m and V m values, which were used to estimate the reaction rates (V).
The hydrolysis conversions of the six bile salt substrates tested with purified wild and mutant enzymes (F18L and Y24L) at different pH and fixed temperature conditions are presented in Fig. 4.  and recombinant enzymes. The estimated k cat and experimented temperature values were used to evaluate the E a as illustrated in Fig. 6.

Discussion
Modifications in molecular structure of the recombinant BSHs are presumed to affect the biocatalytic performances of BSHs due to the biocatalytically active group residues of the enzymes that control the hydrolysis reactions at ideal surrounding pH and temperature conditions.
The purified wild type pCON2 was used as the positive control enzyme, and the purified mutant recombinant enzymes (F18L and Y24L) were used as the target enzymes in hydrolysis of the six pure conjugated bile salts (GCA, GDCA, GCDCA, TCA, TDCA and TCDCA) to estimate the biocatalytic efficiency and E a at different pH and temperature conditions.
The biocatalytic performances were investigated by determination of biocatalytic efficiency estimated from hydrolysis reactions at ideal pH and constant temperature, and E a approximated from hydrolysis reactions at ideal temperatures and constant pH, in accordance with the studies accomplished using natural and mutant enzymes.  The BSHs were applied to the six substrates for hydrolysis conversions to assess the reaction rate (V) and biocatalytic efficiency constants of BSHs at ideal pH values of 3.0, 4.0, 5.0, 7.0 and constant 37 °C temperature conditions. The hydrolysis conversions of the six substrates at experimented pH ranges are given in Fig. 1, which indicates overall high conversions in glyco-conjugated bile salt substrates with wild type control enzyme (pCON2) at 4.0, 5.0, 7.0 pH points that were found ideal pH ranges for the whole conversion reactions. The lowest conversions were observed with the tauro-conjugated bile salt substrates, and mutant recombinant enzyme (F18L) performed low biocatalytic activity when compared to the other conversions. The reaction rate of hydrolysis conversions at experimented pH ranges is presented in Fig. 2, which indicates overall high reaction rate in glyco-conjugated bile salt substrates with wild type enzyme (pCON2) and the mutant recombinant enzyme (Y24L). Low reaction rate was recorded with mutant recombinant enzyme (F18L), which performed lower conversion reactions when compared to the other enzymes, indicating low enzyme-substrate interactions and low force of the bonds between the amino acids in active sites of the enzymes and binding sites of the substrates. Fig. 3 shows significantly highest biocatalytic efficiency value for mutant recombinant enzymes (Y24L). Dramatically low reaction rate values were recorded at pH 3.0 due to the low pH, which reduced the biocatalytic activity of the enzymes. The pH parameter reflected the binding progress of the enzymes to the substrates; thus, the low pH reduced binding k cat /K m values, which resulted in lower yield in enzyme reactions. 16 The biocatalytic efficiency value of mutant recombinant enzymes (Y24L) was found significantly highest, and the value of wild type (pCON2) was determined as lowest, suggesting high biocatalytic performance of the mutant recombinant enzymes over the wild type (pCON2) enzyme. The modifications in molecular structure of BSHs showed higher values. The biocatalytic efficiency of hydrolysis reactions of the glyco-conjugated bile salts was found slightly higher at ideal pH 5.5 and 41 °C temperature conditions. 12 The data of hydrolysis conversions of the six conjugated GCA, GDCA, GCDCA, TCA, TDCA, and TCDCA bile salt substrates were reacted with wild type enzyme positive control (pCON2), and mutant recombinant target enzymes (F18L and Y24L) at 25, 37, 45, 55, 65 (°C) temperatures and constant pH 6.0 conditions were used to quantify the reaction rate and E a values. The ideal temperature range was observed at 25 and 37 °C temperatures, as shown in Fig. 4, in which the highest hydrolysis conversions were recorded with reaction of wild type enzyme (pCON2) and the mutant recombinant enzyme (Y24L) with the glyco-conjugated substrates. The highest conversion occurred at 37 °C representing the tested highest conversion. Evident low conversions were observed with the tauro-conjugated bile salt substrates, and mutant recombinant enzyme (F18L) performed with low biocatalytic activity compared to the other enzymes.
The BSHs were exerted to the six substrates for hydrolysis conversions to determine the reaction rate and biocatalytic efficiency coefficients of BSHs at experimented temperature conditions. The reaction rates of the conversions of the glycol-conjugated substrates with the reaction of wild type enzyme (pCON2) and the mutant recombinant enzymes (Y24L) were estimated as highest at ideal temperature points 25 and 37 °C, as shown in Fig. 5. Remarkably low reaction rates were detected at 45, 55, and 65 °C temperature conditions.
The E a values for the biocatalytic conversion reactions were approximated according to the studies completed previ-ously using the kinetic constants and experimented temperature values. The estimated E a results are presented in Fig. 6, which reveals a high E a value in mutant-type enzymes (F18L and Y24L) reactions, and lower E a was recorded for the conversion reactions performed by wild type enzyme (pCON2). The alterations in molecular structure of BSHs led to a significant increase in E a of the hydrolysis reactions.

Conclusion
Six conjugated pure bile salt substrates were used for the hydrolysis conversion analyses with purified wild type positive control enzyme (pCON2) and purified mutant recombinant enzymes (F18L and Y24L). The wild type (pCON2) enzyme demonstrated a low value, and the mutant (Y24L) enzyme showed a high k cat /K m value, demonstrating that structural modifications in BSH molecules result in higher k cat /K m values. The estimated E a was found low with wild type enzyme pCON2, and the modified mutant-type enzymes (F18L and Y24L) resulted in higher E a values, indicating that the modifications had increased the E a of the enzyme reactions. This study shows that the modification of the amino acids responsible for the activity in the BSH enzyme molecular structure, lowered k cat /K m and increased E a values of hydrolysis conversion reactions.