Preparation Of Novel Magnetic Flexible Carrier And Its Application In Immobilization Of Lipase

Lipase?EC 3.1.1.3?,also named triglyceride hydrolase,which can catalyze the hydrolysis of glycerol to glycerol and fatty acids and catalyze the hydrolysis of carboxylic acids to alcohol compounds and fatty acids.Lipase shows superior chemical,regional and stereo selectivities in the reaction of catalytic esterification and transesterification.As the superior catalytic properties and the diverse selectivity to substrates of lipase,it has became an important object in the reasearch of enzyme immobilization.There are many advantages of immobilized enzyme,such as improving the stability of enzyme,achieving continuous operation and reducing the cost of production.Flexible covalent binding was used in this study based on the previous studies.The magnetic nanoparticles were coated by catechol derivatives with the property of biominetic adhesion,and then the chemical modification polysaccharides were grafted on the surface of the carrier.At last,the prepared carrier was used to immobilize lipase.The main results as follows:1.Poly dopamine and dialdehyde starch?DAS?were assembled on the Fe₃O₄ magnetic nanoparticles with the covalent assembly,which prepared the novel magnetic flexible carrier?Fe₃O₄-PDA-DAS?.The carrier was characterized by EA,SEM,VSM,XRD,TGA,FT-IR and Zeta potential.The results indicated that the novle carrier has superior superparamagnetic property and thermal stability,and the crystal structure not changed.The diameter of the novel magnetic nanoparticle was 209.6 nm.2.The novel magnetic biominetic adhesion carrier?CMCC/DHBH-Fe₃O₄?was prepared starting from magnetic nanoparticle?Fe₃O₄?,the magnetic nanoparticle was attached and encapsulated by the reductive products of carboxymethyl chitosan Schiff base.The novel carrier was characterized by EA,SEM,VSM,XRD,TGA,FT-IR and Zeta potential.CMCC/DHBH-Fe₃O₄ had superior superparamagnetic property and thermal stability,and the crystal structure not changed.The diameter of the novel magnetic nanoparticle was 207.7 nm.3.The mganetic carrier?Fe₃O₄-PDA-GA?prepared with glutaraldehyde was used as control.Response surface experiments were utilized to optimize the immobilization conditions of lipase based on the single factor experiment.The optimum conditions of the immobilization of lipase as follows:pH 7.5,2.2 mg lipase/10 mg carrier and 43.7? when Fe₃O₄-PDA-DAS was used as immobilization carrier,pH 8.0,2.1 mg lipase/10 mg carrier and 40.5? when Fe₃O₄-PDA-GA was used as immobilization carrier,pH 7.5,2.2 mg lipase/10 mg carrier and 43.7?when CMCC/DHBH-Fe₃O₄ was used as immobilization carrier.The predicted enzyme activity was consistent with the actual activity under the optimum conditions.With the determination of enzyme characters and Gauss fitting,the novel magnetic nanoparticles were better to stabilize the conformation of target enzyme,and it also can decrease the damage to the structure of enzyme.