Modification Of The Bond Selectivity Of Glycyrrhizin And Selection Of DNA Aptamers For β-glucuronidase

Glycyrrhizin （GL） can be transformed into glycyrrhetic acid monoglucuronide （GAMG）which has higher bioavailability by β-glucuronidase. However, it also uses GAMG assubstrate to generate glycyrrhetinic acid （GA）. The yield of GAMG was very low because ofthe low GL bond selectivity of β-glucuronidase. In addition, the low utilization rate of thefree enzyme, the higher cost of the catalytic reaction. In this thesis β-glucuronidase expressedby E.coli （PGUS-E） was as the research object. PGUS-E was modified to improve bondselectivity for GL by directed evolution, and single strand DNA aptamer with high affinityand specificity binding to PGUS-E was obtained by a method of systematic evolution ofligands by exponential enrichment （SELEX）, which laid the foundation for the specificimmobilization of β-glucuronidase. The main results are as follows:Error-prone PCR was performed to construct a mutant library of β-glucuronidase, andthe optimum addition amount of Mn²⁺、Mg²⁺were0.05mmol/L and7mmol/L respectively.Then thin-layer chromatography and high performance liquid chromatography assay werecarried out to screen the mutant library, and a mutant was selected. The selected mutant wasnamed PGUS-E（M51）, bond selectivity of which for GL was improved by41%. Enzymaticproperties analysis showed that the optimum pH and the optimum temperature of the catalyticreaction were not change obviously compared with wild type, the activity of PGUS-E（M51）was decreased by16.86%, while the thermal stability was improved by8-10%on thecondition of55-65°C. Sequence analysis revealed that there were five mutations in thesequence of β-glucuronidase, three of them were located in the carbohydrate binding domain,in which the key sites of affecting substrate bond selectivity may be located.SELEX based on affinity chromatography was performed to screen aptamers specificbinding of PGUS-E,80nt-ssDNA library including40random sequences has beenconstructed. Ni-NTA Sepharose was as solid phase separating medium to immobilizePGUS-E. PCR amplification on the optimum condition was annealing temperature at64°Cand the number of cycles of amplification for15rounds. Generating ssDNA used the methodof lambda exonuclease digestion. After12rounds of screening, seven different aptamersequences were obtained. The first and secondary structure of aptamer showed that therewere some similarities of these7sequences and the special structure of stem loop andhairpins might be their mainly existence form. The results of aptamer12-1and12-9,immobilized on magnetic beads, capturing PGUS-E showed that aptamer can be as theligands to immobilize PGUS-E specifically, and12-9better than12-1.This research showed that the selected mutant by directed evolution could increase theyield of GAMG. Aptamer were used as the ligands to bind PGUS-E specifically screening by SELEX technology. These results will provide the β-glucuronidase of excellent propertiesand a new way of immobilization for continuous production of GAMG.