Preparation And Characterization Of Biomimetic Affinity Polymer Nanoparticles For Bt Protein

Bt protein is a class of parasporal crystal protein with high toxicity and high specific insecticidal activity to Lepidoptera,Diptera and Coleoptera larvae,which generated in the sporulation of Bacillus thuringiensis,also known as ?-endotoxin or insecticidal crystal protein.Because of its broad insecticidal activity spectrum,strong specificity and its almost non-toxic and harmless effects on humans and higher animals,it is widely used in Bt insecticides and transgenic Bt crops.Bt protein can enter the soil and aquatic ecosystems by the plant residues of transgenic crops,roots or root exudates,pollen falling and other ways.The environmental and ecological risks caused by the residue and enrichment in the environment have been widespread concerned,and the existing detection methods have many shortcomings.Therefore,how to realize the specific recognition,extraction,separation and purification of Bt protein in the environment will be of great significance.Biomimetic affinity adsorbent materials have been widely used in the extraction,separation and purification of proteins.A very hot method for preparing biomimetic affinity ligands is provided based on the principle of multiple complementary interactions among biological macromolecules,using common hydrophobic,hydrophilic and charged functional monomers to synthesize polymer nanoparticles with different affinities to the target polypeptides,proteins and polysaccharides by adjusting the species and ratio of functional monomers.Amino acid as a unit of polypeptide and protein,if it can be introduced into the side chain of polymer,can increase the possibility of forming secondary structure and more highly ordered structure through non-covalent interactions such as hydrogen bonding,which makes the structure of polymer closer to biological macromolecules.Amino acid based polymers show great potential applications in biomimetic materials and medical materials because of their good biocompatibility,biodegradability and environmental friendliness.Thus,N-Acryloyl amino acid can serveas an ideal functional monomer for the synthesis of biomimetic affinity polymer nanoparticles(NPs).In this paper,several N-Acryloyl amino acid functional monomers with different hydrophobic properties were synthesized from amino acids.A series of polymer nanoparticles with different affinities were synthesized by adjusting the ratio of amino acid monomers to other hydrophilic and hydrophobic monomers.A polymer nanoparticle with the highest affinity for Bt Cry1 Ab protein was screened from this synthetic polymer nanoparticle library.A systematic optimization of the adsorption conditions for Bt Cry1 Ab protein on the selected polymer nanoparticle was carried out.The adsorption performance include isothermal adsorption,adsorption kinetics,specific adsorption,adsorption-desorption of Bt Cry1 Ab protein on the surface of polymer nanoparticle was evaluated comprehensively.Specific work includes the following four parts:1.Four kinds of N-acyloly-L-amino acid monomers including N-acyloly-L-Glycine(AGly),N-acyloly-L-Alanine(AAla),N-acyloly-L-Valine(AVal)and N-acyloly-L-Phenylalanine(APhe)were synthesized by acylation reaction of amino acid with acryloyl chloride.The structures of N-acyloly-L-amino acid monomers were characterized by 1H NMR and FT-IR.A series of polymer NPs(AGly-NPs,AAla-NPs,AVal-NPs,APhe-NPs)with amino acid side chain were prepared by precipitation polymerization by adjusting the ratio of four N-acyloly-L-amino acid monomers with N-isopropylacrylamide and N-tert-Butylacrylamide.Its concentration,yield,particle size and Zeta potential were characterized.2.APhe-NP2(APhe 50%,NIPAm 48%)which exhibited much higher affinity to Bt Cry1 Ab protein was screened out from the synthetic polymer nanoparticle library in 10 m M pH 6.88 phosphate buffer.The adsorption conditions such as pH,phosphate concentration and salt concentration were optimized,and the optimum adsorption conditions were 10 mM pH 6.0 phosphate buffer.3.The adsorption kinetics experiments showed that the adsorption rate of APhe-NP2 to Bt Cry1 Ab protein was very fast,and the adsorption equilibrium can be reached at 8min.The isothermal adsorption experiments showed that the maximum adsorption capacity of APhe-NP2 to Bt Cry1 Ab protein could reach to 330.8 mg/g.The results obtained from SDS-PAGE analytis showed that Bt Cry1 Ab protein could be effectively captured by APhe-NP2 when buffer pH was 6.0,and almost completely released from APhe-NP2 when buffer pH was 8.0 or 10.0,indicating that the polymer nanoparticle was able to catch and release Bt Cry1 Ab protein in a pH-responsive manner.4.The adsorption capacity of APhe-NP2 to the homologous proteins of Bt Cry1 Ab,such as Bt Cry1 Ac,non-homologous proteins such as Bt Cry1 F protein and other common proteins including BSA and HSA with similar molecular weight or close isoelectric point,pepsin and ovalbumin with different molecular weight and isoelectric point was compared.It was found that the adsorption capacity of APhe-NP2 to other proteins was smaller than that of Bt Cry1Ab/Ac protein.Therefore,APhe-NP2 has better specific adsorption ability to Bt Cry1Ab/Ac protein under the optimum adsorption conditions.This can establish a theoretical and practical foundation for the specific extraction,separation and purification of Bt protein from environmental samples and transgenic crops by APhe-NP2.