title: Synchronized purification and immobilization of his-tagged β-glucosidase via Fe3O4/PMG core/shell magnetic nanoparticles.
abstract: In 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.
authors.affiliation: Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Shaofei
authors.initials: S
authors.lastname: Yuan
authors.affiliation: School of Chemistry and Chemical Engineering, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Yan
authors.affiliation: School of Chemistry and Chemical Engineering, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.affiliation: School of Food and Biological Engineering, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Han
authors.affiliation: Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Shi
mesh_terms: D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005290:Ferric Compounds / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D058185:Magnetite Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D011759:Pyrrolidines / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D005290:Ferric Compounds; D058185:Magnetite Nanoparticles; C442886:N-(2-pyrrolidine-methyl)-N-(thymine-1-acetyl)-glycine; D011759:Pyrrolidines; D011993:Recombinant Fusion Proteins; C000499:ferric oxide; D001617:beta-Glucosidase; D005998:Glycine