Synchronized purification and immobilization of his-tagged β-glucosidase via Fe3O4/PMG core/shell magnetic nanoparticles

• DOI: 10.1038/srep41741
• Publication Date: 2017-01-30T10:18:29Z
• Authors: Yang Zhou; Shaofei Yuan; Qian Liu; Dandan Yan; Yun Wang; Li Gao; Juan Han; Haifeng Shi
• Keywords: Pyrrolidines; Recombinant Fusion Proteins; beta-Glucosidase; Glycine; Temperature; 02 engineering and technology; Hydrogen-Ion Concentration; Enzymes, Immobilized; Ferric Compounds; Article; Enzyme Activation; Kinetics; Enzyme Stability; Spectroscopy, Fourier Transform Infrared; Magnetite Nanoparticles; 0210 nano-technology

AbstractIn this paper, an efficient and convenient Fe3O4/PMG/IDA-Ni2+ nanoparticles that applied to purify and immobilize his-tagged β-glucosidase was synthesized, in which, Fe3O4/PMG (poly (N, N’-methylenebisacrylamide-co-glycidyl methacrylate) core/shell microspheres were synthesized firstly using distillation-precipitation polymerization, then iminodiacetic acid (IDA) was used to open epoxy rings on the shell of microspheres to the combination of Ni2+. The gene of β-glucosidase that was from Coptotermes formosanus Shiraki was amplified, cloned into the expression vector pET28a with an N-terminal His-tag, and expressed in E.coli BL21. The nanoparticles showed the same purification efficiency as commercial nickel column which was a frequently used method in the field of purifying his-tagged proteins from crude cell lysates. The results indicated that Fe3O4/PMG/IDA-Ni2+ nanoparticles can be considered as an excellent purification material. β-glucosidase was immobilized on the surface of Fe3O4/PMG/IDA-Ni2+ to form Fe3O4/PMG/IDA-β-glucosidase by means of covalent bound with imidazolyl and Ni2+. The immobilized β-glucosidase exhibited excellent catalytic activity and stabilities compared with free β-glucosidase. In addition, immobilized β-glucosidase can be recycled for many times and retain more than 65% of the original activity. The materials display enormous potential in the aspect of purifying and immobilizing enzyme.