Modification Of β-Glucuronidase Substrate Specificity And Immobilization By DNA Aptamers

Glycyrrhizin(GL) is an important class of glycosides, whicn has many medicinal activities sucn as anti-tumor, anti-viral, anti-inflammatory, anti-hypertensive, anti-hyperlipidemic and the promotion of Adrenocorticotropic hormone and so on, meanwhile, its has been widely used as food additives and fine chemicals in food and cosmetic industry. Compared with GL, its derivatives glycyrrhetic acid 3-O-mono-β-D-glucuronide(GAMG) has better bioavailability, biological safety, medicinal therapeutic value, and also higher sweetness, and solubility, which facilitates GAMG to become a promising substitute for GL.PGUS-E(recombinant β-glucuronidase expressed in E coli) derived from fungal, can enzymatically hydrolyze GL into GAMG, but a large quantity of byproduct-glycyrrhizic acid is also produced in the meantime due to the low substrate specificity of PGUS-E for GL. In order to improve the substrate specificity of PGUS-E, this thesis focus on a series molecular modification of enzyme PGUS-E including the construction of expression system for high-throughput screening of PGUS-E with high substrate specificity, molecular modification of PGUS-E by irrational and semi-rational design, and the study of mechanism for PGUS-E substrate specificity. Meanwhile, this thesis also explores the screening of the specific nucleic acid aptamers and immobilization of PGUS-E. The main results are as follows:E.coli DH5 a was as a host for PGUS-E expression, in order to eliminate the interference of inherent β- glucuronidase(gusA) of E.coli DH5 a, the gene uidA of E.coli DH5 a was knocked out by Red homologouse Recombination using 50 nt homology arms. PCR validation and enzyme characterization of gusA show that the gene uidA was successfully knocked out. A Multi-function plasmid was constructed for screening the mutant with high substrate specificity towards GAMG. The vector contained two main functional regions: the expression of β-glucuronidase under different strength constitutive promoters, and the bacteriophage lyase SRRZ under the temperature-induced PL promoter. Then the expression efficiency of PGUS-E with promoters of different strength was investigated, and the effect of the expression of bacteriophage lyase SRRZ on lysis efficiency of the host under different temperature was optimized.Then, an in vitro directed evolution selection strategy was introduced to improve the substrate specificity of the PGUS-E by Error-PCR, and different concentrations of MnCl2 were tested to the mutation rate at 1-2/kb. Three transformants(C9, F72 and M51) with improved substrate specificity were selected from ~10,000 colonies through a one-pot method, where the substrate selectivity was increased by 25%, 37% and 41%, respectively. The three-dimensional structure model of PGUS-E was constructed using the Swiss-Model; Structure analysis showed that the mutation sites were located in loops or a-helixes that were far away from the activity site, but the changes at α helix structure of TIM barrel domain had a great influence on the improvement of the substrate specificity.Semi-rational modification of PGUS-E was performed to further improve its substrate specificity.The hot sites among the entrance to the substrate channel were identified by homology alignment and docking GL, GAMG and glucuronic acid with PGUS-E crystal structure with Autodock/Vina software and analyzing the complex structure. For all the selected hot-sites, site-directed saturation mutagenesis was carried out. The key sites of Ala365 and Arg563 were obtained by screening ~6,000 colonies, and the substrate specificity was improved to 65% and 77%, respectively. After combining the mutations Ala365 and Arg563 site, the substrate specificity of mutant A365 H / R563 E, A365 T / R563 E reached 96% and 95%, respectively. The structural change in the mutation site was analyzed,the interaction between GAMG sugar moiety structure and the side chain of Thr365/His365 by van der Waals and π-π force,which weakened the affinity of GAMG and active catalytic sites,and the carboxyl chain of R563 E are probably confined to the groove and are in contant with the residue Asp162 through hydrogen bond or van der Waals interactions, which may will contribute to the tightly closed conformation of substrate binding pocket of triterpenoid with PGUS-E,and the mechanism for the improvement substrate specificity was elucidated from the molecular level.The SELEX process based on magnetic beads was established to screen aptamers specific showing high affinity to PGUS-E. A DNA library containing 440 random sequences was designed and synthesized, and magnetic beads were used to fix PGUS-E as a solid phase separation medium. The annealing temperature of ddDNA libraries, the number of amplification cycles and the preparation protocol of ssDNA were optimized. After 18 rounds of screening, 11 different Aptamer sequences were obtained. The main secondary structure was neck ring and hairpin. The interaction between Apt5 and PGUS-E was measured on BIAcore. The affinity was tested to capture the target protein PGUS-E and the results showed that aptamers Apt5 and PGUS-E has a high affinity of 200 nM, which reached the antibody-antigen levels. There was no loss in its activity after being recycled 7 times and the activity of PGUS-E always kept over 72%, and the regeneration of Apt5 reached to 91%, demonstrating a high operational stability. These results will provide a new way of one-step purification and immobilization f of PGUS-E.