The Preparation Of Functional Magnetic Nano-carrier Via Atrp And Application For Lipase Immobilization

Lipases (EC3.1.1.3) can catalyze the hydrolysis of ester at oil-water interface and the reverse synthesis of esters via esterification and transesterification in non-aqueous medium. Lipases have broad application prospectsin the biodiesel, fine chemical, pharmaceutical and food industries. However, the large-scale industrial applications of natural lipases have been limited due to bad operation and environmental stability, high costs of enzymes and non-reusability. In order to overcome these deficiencies of free lipase, the exploration of lipase immobilized method and the preparation of new immobilized carriers have drawed more and more attention. Among diverse supports for lipase immobilization, magnetic nanoparticles have attracted increasing attention because of their super-paramagnetic behavior, higher specific surface areas and low-cost. In the present study, lipase from Burkholderia cepacia was immobilized onto the surface of PGMA-b-PDMAEMA-grafted-Fe3O4magnetic nanoparticles through electrostatic adsorption and covalent coupling, and its properties and application in the resolution of (R,S)-phenylethanol were investigated. The specific contents and results were as follows:A block copolymer of dimethylaminoethyl methacrylate (DMAEMA) and glycidyl methacrylate (GMA) was grafted onto the surface of magnetic nanoparticles (Fe3O4) via ATRP to achieve bi-functionalized block copolymer PGMA-b-PDMAEMA-grafted-Fe3O4magnetic nanoparticles with both amino and epoxy groups. Two kinds of functional groups successfully grafted onto Fe3O4nanoparticles surface was proved by FT-IR. TGA results showed the grafting rate of DMAEMA and GMA on the surface of Fe3O4nanoparticles were1.54%wt and6.0%wt, respectively. The trans spinel crystal structure of magnetic nanocarrier was confirmed by XRD. SEM photograph showed the magnetic carriers were of uniform spherical shape with30±5nm.Lipase was immobilized onto bi-functionalized magnetic nanoparticles, the optimized conditions were:30mg PGMA-b-PDMAEMA-grafted-Fe3O4magnetic nanoparticles and25mL1.2mg/mL lipase in citrate buffer solution (50mmol/L, pH6.0) were placed in a shaking incubator at180rpm for12h at25℃, the immobilization efficiency and recovery of lipase were50%and43%, respectively, the specific activity of immobilized lipase was16U/mg. The optimum pH of free and immobilized lipase were pH7.0and pH9.0respectively, while the optimum temperature of the immobilized lipase ranged from37℃to 65℃, whereas that of the free lipase was60℃. Additionally, compared with free lipase, the immobilized lipase exhibited improved thermal stability, good tolerance to organic solvents with high log P, and higher pH stability at pH9.0. After six consecutive cycles, the residual activity of the immobilized lipase was still over55%of its initial activity.The chiral resolution of (R,S)-phenylethanol by immobilized lipase on bi-functionalized magnetic was studied in non-aqueous medium. The optimized conditions of chiral resolution reaction:0.05mol/L (R,S)-phenylethanol,1.0mol/L ethenylethanoate and30mg immobilized lipase were dispersed in5mL heptane solvent, and mixed in a shaking incubator at200rpm for48h at37℃, the conversion and enantioselectivity (ees) of immobilized lipase were42.4%and61.9%, respectively, the conversion and enantioselectivity (ees) of free lipase were36.2%and35.4%, respectively. After six consecutive cycles in non-aqueous medium, the conversion and enantioselectivity (ees) of immobilized lipase were still over32.6%and45.5%.