Gene Screening,Cloning,Expression,Function Research And Molecular Modification Of 7α-HSDH

Bear bile is the drainage gallbladder of Selenarctos thibetanus Cuvier Asiatic Black Bear or Ursus arctos Linnaeus Brown Bear belonging to the family of bear animal.It is a traditional rare medicine.Ursodeoxycholic acid（UDCA）is the main medicinal component of bear bile,which combines with taurine to form tauroursodeoxycholic acid（TUDCA）.TUDCA is widely used in the treatment of various hepatobiliary diseases such as primary biliary cirrhosis.At present,TUDCA is mainly obtained through bear bile drainage or chemical synthesis.However,these two methods have the disadvantage of being ecologically and environmentally unfriendly.With the integration of biotechnology and engineering technology,it will become possible to apply biocatalytic technology to large-scale preparation of TUDCA.In our previous research,7α-hydroxysteroid dehydrogenase（7α-HSDH）and7β-hydroxysteroid dehydrogenase（7β-HSDH）are two key enzymes that drive the efficient biosynthesis of TUDCA from taurochenodeoxycholic acid（TCDCA）in vitro.Therefore,7α-HSDH is one of the key enzymes for the efficient biotransformation of TCDCA to TUDCA.The thermal stability and catalytic activity of 7α-HSDH are key factors in determining whether it can adapt to industrial production.In order to obtain 7α-HSDH with good thermal stability and high activity,a new 7α-HSDH（named St-2-2）was obtained in the black bear intestinal microbial metagenomic database.The paper systematically studies the enzymatic properties of St-2-2.Further based on bioinformatics analysis,the carboxyl terminus was molecularly modified to obtain variant type St-2-2 with good thermal stability and high activity.The reason for the improvement of the thermal stability of mutant I255Q was discussed.This study expands the natural enzyme bank of the 7α-HSDH and provides a new resource for the large-scale preparation of TUDCA using biocatalytic technology.The main research contents and results are as follows:（1）Based on the laboratory early bear gut microgenomics database,we obtained a new 7α-HSDH（named St-2-2）.1)Homology and phylogenetic tree analysis indicated that St-2-2 belongs to the SDRs family.2)St-2-2 has a subunit of 28.3 kDa and a natural size of 113.2 kDa,which is a tetramer,as determined by size exclusion chromatography and mass spectrometry.3)The catalytic activity of St-2-2 indicates that the catalytic activity of St-2-2 on TCDCA,GCDCA,TCA and GCA is significantly better than the five 7α-HSDHs found in our previous laboratories.Among them,the catalytic activity of St-2-2 on TCDCA was significantly higher than that of S1-a-1（p<0.01）,S1-a-2（p<0.05）,H1-a-1（p<0.001）,H1-a-2（p<0.001）and Y1-a-1（p<0.001）.The catalytic activity of St-2-2 was 1.7 times,1.2 times,10.3 times,15.8 times and 13.7 times that of these 7a-HSDHs,respectively.From the perspective of biocatalytic activity,St-2-2 has significant advantages in biosynthesis of TUDCA.（2）The enzymatic properties of St-2-2 were systematically studied using TCDCA as a substrate.1)The St-2-2 pH optimum study showed that St-2-2 is a basophilic enzyme with an optimum pH of 10.0.2)The best temperature study for St-2-2 indicates that St-2-2 is a mesophilic enzyme with an optimum temperature of 35^oC.3)The thermal stability study of St-2-2 showed that St-2-2 retained 65.4%of the enzyme activity after treatment at 4^oC for 80 h.The thermal stability of St-2-2 in the 37^oC environment was significantly better than the five 7α-HSDHs found in our previous laboratory.St-2-2retained 65.5%of enzyme activity after treatment at 37^oC for 30 hours,while S1-a-1and Y1-a-1 lost activity under the same conditions.The residual enzyme activities of S1-a-2,H1-a-1 and H1-a-2 under the same conditions were 51.2%,38.9%and 40.1%,respectively.4)The effect of metal ions on St-2-2 enzyme activity showed that Na⁺,Mg²⁺had an activation effect on St-2-2 enzyme activity at an appropriate concentration.When the Na⁺concentration reached 5 mM,the enzyme activity of St-2-2 increased by14.3%,and when Mg²⁺reached 2.5 mM,the enzyme activity of St-2-2 increased by30.5%;When the Cu²⁺and Mn²⁺concentrations reached 5 mM and 50 mM,respectively,the enzyme activity of St-2-2 was significantly inhibited（p<0.001）,and the corresponding enzyme activities were reduced to 8.8%and 34.2%,respectively.（3）Based on bioinformatics analysis,the carboxyl terminus of St-2-2 was molecularly modified to obtain six variant type St-2-2.1)The results of enzyme activity studies showed that all six mutants had enzyme activity.Among them,mutant I255Q significantly increased the catalytic activity of TCDCA and GCDCA（p<0.001）,and its enzyme activity was 2.7 times and 2.2 times that of St-2-2,respectively;In contrast,I255Q significantly reduced the catalytic activity of GCA and TCA（p<0.001）,and its enzyme activity decreased to 15.7%and 33.1%,respectively.Compared with the activity of wild-type St-2-2,the mutant K262R/K261Q/A259L had no significant difference in the catalytic activity of TCDCA and GCDCA,but the catalytic activity of GCA and TCA was significantly improved（p<0.001）,its enzyme activity was 2.0 times and 1.8 times that of St-2-2,respectively.2)The thermostability and Tm values of the variant type St-2-2 showed that the thermal stability and Tm of the mutants K262R,A259L,I255Q,K262R/K261Q/A259L were increased compared to the wild type St-2-2.Among them,the thermal stability and Tm value of I255Q were significantly improved compared to wild type St-2-2.The wild-type St-2-2 retained 76.2%of the enzyme activity after treatment at 50^oC for 30 min,while the mutant I255Q retained 96.9%of the enzyme activity under the same conditions,and the corresponding Tm was also increased by 7.8^oC.Similarly,the mutant K262R/K261Q/A259L retained 86.1%of the enzyme activity under the same conditions,and its corresponding Tm value increased by 1.7^oC.Mutants with improved activity and stability laid the foundation for large-scale preparation of TUDCA using biocatalytic technology.（4）We used SWISS-MODEL to model the homologous three-dimensional structure of wild-type St-2-2 and mutant I255Q,and used PyMOL to perform intermolecular force analysis on their 255 positions.The analysis showed that the Gln-255 position of the mutant I255Q formed a hydrogen bond with the Ser-253 compared to the Ile-255position of the wild type St-2-2.Hydrogen bonding is the main intermolecular forces for stabilizing the structure of the protein.Therefore,such a change increases the stability of the protein molecular chain,thereby increasing the thermal stability of the mutant I255Q.We determined their secondary structure by circular dichroism.The results showed that the I255Q relative to the secondary structure of St-2-2,theα-helix content of the mutant I255Q was reduced by 15.5%,while the content ofβ-sheet was increased by 12.4%.In the relationship between the structure and function of proteins,theα-helix content is reduced,and the increase inβ-sheet helps to stabilize the structure of the protein.The above results indicate that the formation of the hydrogen bond between the Gln-255 position and the Ser-253 of the mutant I255Q and the change in the secondary structure content after the mutation are responsible for the increase in the thermal stability of the mutant I255Q.