| Bile salt hydrolase(EC 3.5.1.24, BSH), the N-terminal nucleophilic(Ntn) enzyme deconjugating conjugated bile salt into free bile acid and glycine/taurine, widely existed in intestinal microflora. Expression of BSH is one of ways to increase bile salt tolerance for numerous microorganism in the intestinal tract. However, the exact structure-function relationship of BSH is not clear. Thus, there is not enough theoretical guidance for the application of BSH into the biological agent. In this dissertation, the correlation in the structure and property of BSH as well as bile salt tolerance of cells was deeply analyzed using Lactobacillus salivarius LMG14476 BSH as the object and Lactococcus lactis NZ9000 as the indicator. Moreover, the molecular mechanism of BSH was preliminarily discussed. The main results of this dissertation were described as follows: 1. The effects of BSH expression on the bile salt tolerance of Lactococcus lactisTwo bile salt hydrolases(BSH1 and BSH2) from L. salivatius LMG14476 were expressed in L. lactis NZ9000, respectively. Through comparing the growth perfomance of recombinant strains stressed by porcine bile salt, it was found that the growth performances of BSH1 and BSH2 expressing strains were, respectively, enhanced 2.8- and 2.4-fold than the parental strain after bile salt stressing 20 h, suggesting the bile salt tolerance of the strain could be obviously increased through expression of BSH. In addition, the growth performance of BSH1 expressed strains was 1.3-fold higher than that of BSH2 after 15 h, indicating the biological function of BSH1 was better than BSH2. Based on the observation of cellular morphology as well as the qualitative and quantitative analysis on intracellular bile acid, it was found that intracellular bile acid of BSH2 expressed strain was 1.5-fold higher than the strain expressing BSH1, indicating BSH1 producing fewer bile acid contribute to enhance bile salt tolerance of cells. 2. Quantitative characterization of BSH catalytic propertiesAnalysis on the enzymatic properties showed that the optimum pH range of BSH1 and BSH2 were 5.5-7.0, and the optimum temperature were amongst 20-50 oC. The enzymatic reaction kinetic curves of BSHs to six conjugated bile salts displayed sigmoidal type. The values of the Hill coefficient were greater than 1.0, indicating these two BSHs were the allosteric enzymes with positive cooperativity. The kcat and kcat/S0.5 values of BSH1 were both less than BSH2, demonstrating the catalytic efficiency and substrate affinity of BSH1 were both lower than BSH2. In addition, the features of the kinetic sigmoidal curves for both BSH1 and BSH2 were disappeared in the presence of DTT using GCDCA as the substrate. The decrease in kcat and increase in kcat/S0.5 indicated DTT weakened the allosteric feature, reduced the catalytic efficiency, and increased substrate affinity of BSH. 3. Analysis on the structure-function relationship of BSHThe energy map of BSH catalytic reaction showed two distinct thermodynamic events from exothermic to endothermic process detected by the isothermal titration microcalorimetric(ITC) technique, verifying the allosteric properties of BSH. Moreover, the calorimetric curves of each titration with the conjugated bile salt into BSH showed an initial endothermic peak in the hydrolytic process, indicating BSH firstly bond with bile acid group of bile salt molecule through hydrophobic interaction. It was also found that three bile acid groups combined with the substrate pocket at the surface of BSH1 through the hydrophobic interaction. The UV-vis absorption spectroscopy showed that the bile acid groups induced the characteristic absorption peak of BSH1 took place red shift from 192 nm to 201, indicating the combination of bile acid groups resulted in the extension of the side chains and relaxation of protein skeleton for BSH1, facilitating the exposure of the active site Cys2 buried in hydrophobic pocket.It was predicted that the binding sites of bile acid groups were Tyr24, Ala58, Phe65, Phe130 and Leu134 in BSH1 through the amino acid sequence alignment with C. perfringens 13 BSH which crystal structure has been reported. The amino acid residues Tyr24, Phe65, Phe130, Leu134 were replaced by alanine, respectively, and Ala58 was replaced by glutamine. The mutant Y24 A could not hydrolyze GCA and GDCA. The mutants A58 Q and F65 A could not hydrolyze GCA. The substrate specificity of the mutant F130 and L134 A were changed to TCA>GDCA>GCDCA=TDCA>GCA>TCDCA and TCDCA>TCA>GDCA=GCDCA> GCA>TDCA, respectivrly. These results indicated that the substrate binding capacity of BSH could be improved or reduced through weakening the hydrophobicity of the amino acid located at the substrate binding pocket of BSH surface, resulting in increased substrate selectivity. 4. Molecular modification of BSH strengthening bile salt tolerance of cellsThe mutant library of BSH was constructed using the site-saturation mutagenesis and DNA shuffling. According to the biomass of the strain under bile salt stress, ten recombinant strains exhibited 6-15% increases in biomass were selected from 10,000 transformants. After bile salt stressing 15 h, the growth performance of the strain NZ-bsh1 v with the highest biomass increased 1.3-fold compared to the strain expressing BSH1, and the amount of the bile acid in NZ-bsh1 v was one fifth of that in BSH1 expressed strain. These results further demonstrated BSH producing fewer bile acid exhibited better biological performance.The BSH mutant in NZ-bsh1 v was overexpressed in E. coli BL21(DE3) and purified. The analysis on the enzymatic properties and electrophoresis showed that the subunit size of this BSH mutant was 6 kDa, which is the smallest functional unit of BSH reported so far. The BSH mutant only presented hydrolytic activity on trihydroxy bile salts(TCA and GCA), and its specific activities on TCA and GCA were 37.51±1.40 U·mg-1 and 69.74±1.69 U·mg-1, respectively. These results indicatd the substrate specificity of BSH mutant was increased, and the hydrolytic activity was reduced. The fluorescence spectrum showed that the maxmum emission wave-length of BSH mutant intrinsic fluorescence was 339 nm while that of BSH1 was 329 nm, suggesting the maxmum emission wave-length of the enzyme took place red shift. The fluorescence intensity of BSH mutant was also reduced. These data indicated that the conformation of the BSH mutant was more compact, the protein folding and surface hydrophobicity was decreased, and the thermal stability was increased compared with BSH1. 5. The effect of BSH on the bile salt response progress of cellsThe effects of BSH on the physiological activity of cells under bile salt stress were investigated. It was found that on the basis of the general bile salt response as the parental strain, the strain expressing BSH strengthen its bile salt resistance through enhancing the surface hydrophobicity, decreasing membrane fluidity, regulating the expression abundance of membrane protein, relieving the decline in intracellular pH, strengthening the FoF1-ATPase activity, increasing the ATP consumption, relieving the decline in the level of the carbon and nitrogen metabolism, enhancing the level of ammonia produced by ADI pathway.. In addition, compared to the wild BSH expressed strain, the reduction of vertical diffusion was slower in the strain expressing BSH mutant, and then a seris of the relevant response progresss were regulated in order to further enhance bile salt resistance. |
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