One-step Purification And Immobilization Of β-glucuronidase Based On Aptamer And Its Application

With the rapid development of global industrialization, energy and environment crisis has become a problem confronting the whole of mankind. In the process of industrial production, this problem is particularly prominent. Therefore, it is extremely urgent to find a reasonable way to solve these issues. The production process which dominated by biocatalysis brings new opportunity for this. Using biological catalysis instead of traditional chemical catalysis appear to hold more promise, on account of its good selectivity, specificity, efficiency and green with enzyme catalyst. But the drawbacks of free enzyme limit its industrial applications while the enzyme immobilization is a good way to overcome these disadvantages. Traditional enzyme immobilization methods have many disadvantages and in order to adapt to the large-scale promotion of biocatalysis and better meet the demand of industrial, developing a new type of enzyme immobilization technology seems more and more urgent.Aptamers are oligonucleic acid that exhibit high affinity and specificity to their corresponding target and wildly used in molecular capture, biological imaging, biological detection, clinical diagnosis, drug delivery and so on. The affinity and sepecificity of aptamer make a broad development potential in the aspects of enzyme immobilization. This thesis is based on the purpose of developing new enzyme immobilization method and the application platform for biological catalytic conversion of glycyrrhizin. Aptamers for β-glucuronidases(PGUS-E) were obtained by our modified SELEX method and then covalently connected to the graphene oxide and magnetic bead for one step purification and immobilization PGUS-E. The immobilized PGUS-E was further used as catalyst for conversion of glycyrrhizin. The main research contents and results are as follows:(1) The SELEX and characterization of aptamers for β-glucuronidasesThe β-glucuronidases that purified by nickel ion affinity chromatography, ion exchange and molecular sieve was immobilized on Ni-NTA magnetic beads as targets for the aptamer selection. The ss DNA library which contain 40 nt random sequence goes into the next cycle after incubation, isolation, amplification and preparation ss DNA. In order to improve the specificity, counter selection was performed after every three cycle of screening. The amount of beads, the incubation time of target-coated beads with ss DNA, and the wash times were chosen as control variables to accelerate the screening efficiency. In view of the requirement of the catalytic reaction temperature, the incubation temperature increased to 40 ℃ and another 6 rounds of selection were completed. 16 different ss DNA sequences were obtained after cloning and sequencing and their affinity was determined by surface plasmon resonance technology. The results showed that the dissociation constants between aptamers and PGUS-E reached nanomole.(2) Covalent Binding Aptamers on supporter and its characterizationGraphene oxide was choosed as supporter due to its large specific surface area and good water-solubility. It was modified by chemicals to improve the content of carboxyl groups and then the amino modified aptamer covalently linked to its surface. The results were characterized by UV-visible absorption spectra, fluorescence microscope, FTIR and so on. Also, the same works were did with carboxyl functionalized silica. It showed that the capacity of graphene oxide for Apt5 and Apt9 were 29.08 μg/mg and 35.03 μg/mg while for magnetic beads this value were 10.685 μg/mg and 13.565 μg/mg.(3) The interactions between aptamer and PGUS-EThe influence of ionic strength and p H value on the interactions between aptamer and PGUS-E was studied. The ionic strength was controlled by changing concentration of Na Cl. It found that the interactions were destroyed with 0.3 M Na Cl and the beads could be regenerated with 1 M Na Cl. In all cases investigated, the effect of p H was not sensitive as ionic strength and only a little PGUS-E escaped from the constraints of aptamer at p H of 4 and 7. Adsorption behaviors of aptamer to PGUS-E were fitted and this process could be described and predicted by Tenkin isothermal adsorption model, pseudo-second-order kinetics model, Elovich and Webber-Morris model.(4) The applications of aptamers for β-glucuronidasesThe aptamers were used for one step purification and immobilization β-glucuronidases to catalysis glycyrrhizin. The maximum PGUS-E capturing capacity of aptamer modified magnetic beads was found to be nearly 32 μg/mg. The immobilized PGUS-E on aptamer-based magnetic beads showed good reusability, and the conversion of glycyrrhizin still remained 74.1% for Apt5 and 56% for Apt5 after 7 cycles. In addition, the aptamer-modified beads support can be easily regenerated, and the conversion rate of glycyrrhizin was still 62.7% for Apt5 and 58% for Apt9 after the 7th cycle of regeneration. A trace PGUS-E detection method with low detection limit, simple opration, high sensitivity and high speed acquisition was established based on aptamer. The PGUS-E purified by aptamer using ours method has a higher purity and enzyme activity than that by the conventional nickel ion affinity chromatography. Meanwhile, it could used for purification of low abundance protein PGUS.In conclusion, this technology based on aptamer can realize one step purification and immobilization PGUS-E from cell lysates. It has strong stability, simple operation, recuperability and reusability. What is more, it can be used as a new type of enzyme immobilization.