| Cardiovascular disease has become the primary cause of human death and treatment for cardiovascular disease is the current research focus.Reducing the total cholesterol level in serum is an important means to prevent cardiovascular disease.Bile salt hydrolase(BSH,EC 3.5.1.24)can hydrolyze and bind bile salts and play an important role in the cholesterol-lowering mechanism of lactic acid bacteria.Lactic acid bacteria-derived bile salt hydrolase could reduce cholesterol which could be used as a potentially safe and healthy treatment method for hyperlipidemia.Therefore,in this study,lactic acid bacteria with high-yielding bile salt hydrolase was screened from the bank ofhuman lactic acid bacteria.Then,the factors affecting the expression of bile salt hydrolase in wild bacteria were analyzed.Finally,the chemical properties and protein active sites of the bile salt hydrolase were explored using molecular biology and bioinformatics methods.This work was helpful to the application of bile salt hydrolase.The specific research content was as follows:(1)Screening of high-yield bile salt hydrolase strains and their probiotic properties.Using calcareous diameter as the screening index,30 strains of bile salt hydrolase positive bacteria were obtained from the human lactic acid bacteria which were isolated and stored in the laboratory,and the positive rate was 47.6%.Then,bile salt hydrolase activity and cholesterol degradation ability of 8 lactic acid bacteria with the largest calcareous diameter were tested.Finally,strain 58 with enzyme activity of 1.34 U/mg and cholesterol degradation rate of 57%was selected.By 16S rDNA sequencing,strain 58 wasidentified as Lactobacillus plantarum.The probiotic properties of Lactobacillus plantarum 58 were determined.The survival rate of strain 58 in artificial gastric juice,0.1%bile salt medium,0.3%bile salt medium was 43.73%,46.56%and 19.34%,respectively.After treating by 1%,2%,3%sodium chloride,the survival rate was seperately 85.1%,46.95%and 33.09%.Meanwhile,stressing by 0.1%bile salt,the hydrophobicity of the strain 58 increased from 57.09%to 63.68%and the self-aggregation ability decreased from 53.47%to 33.78%at 8 h.(2)The influence of fermentation conditions on the expression of bile hydrolase.The growth and production of bile salt hydrolase of strain 58 were measured.The results showed that enzyme activity kept increasing before the initial stage of the stable period.Moreover,different carbon and nitrogen sources not only affected the growth of the strain but also affected the enzyme activity of bile salt hydrolase.Strain 58 was showed the the highest enzyme activity of bile salt hydrolase When the single nitrogen source was soybean peptone and the single carbon source was glucose.Also,pH and temperature played improtant roles on the expression of bile salt hydrolase.And the optimal initial pH and temperature for cultivation was 6.0 and 37℃,respectively.Furthermore,enzyme activity of bile salt hydrolase could be increased by the addition of bile salt and sulfhydryl protective agent during the fermentation.(3)Study on the enzymatic properties of recombinant bile salt hydrolase.According to the reported bile salt hydrolase sequence,primers were designed by homologous sequence alignment.Then,the bsh fragment of strain 58 was amplifiedand the expression vector pBSH for bile salt hydrolase was constructed.The recombinant vector was transferred into Escherichia coli JM109 for recombinant expression.The enzymatic properties of the bile salt hydrolase of the recombinant bacteria were determined.the optimum reaction temperature and pH were 35℃ and 6.0,respectively.The enzyme performed better thermal stability at 30℃ for 1 h while the enzyme was almost inactivated at 60℃ for 1 h.Moreover,the bile salt hydrolase was activated by alcohol and inhibited by methanol,ethanol and metal ions.The hydrolysis preference of strain 58 to bile salt was:GCA>TCA>TDCA.(4)Analysis of the active site of bile salt hydrolase of strain 58.Through amino acid sequence comparison,six conserved sites,Cys2,Argl6,Asp19,Asn79,Asn170 and Arg223,were identified from the amino acid sequence ofbile salt hydrolase;BSH(PDB ID:5gs7),derived from Enterococcus faecalis,was used for online modeling.Then,bile salt hydrolase model TfBSH of strain 58 was constructed by POCASA to split the monomers.TfBSH spatial structure showed that the six conserved sites were distributed around the active center of Cys2.Furthermore,the bile salt hydrolase protein model derived from Clostridium perfringens(PDB ID:2RLC)was used as a template for protein overlay analysis.Ser136 was found to combine with the substrate GCA and Pro 134 and Ser136 were found also combining with GCA by molecular docking.Finally,6 mutant strains of R16A,N79A,P134S,S136A,S136P and S136D were constructed and the corresponding enzyme activity was measured.The result showed that there was scarce enzyme activity of mutant R16A.and the enzyme activity of mutant S136A and mutant S136P were decreased by 92.41%and 73.86%,respectively.These results were indicated that Arg16 and Ser136 were closely related to the bile salt hydrolase activity of strain 58. |
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