Study On Preparation Of Regenerated Magnetic Carriers For Immobilization Glucoamylase

Immobilized enzymes as a versatile tool that allows repeated or continuous use of enzymes, easy recovery of substrates and products, easy purification, and in some cases, improved the performance of biological catalysts. However, in addition to the methods of adsorption, the carriers can not be recycled, and are discarded with the inactivation of the enzyme, which causes environmental pollution, waste of resources and production costs. Although the carriers can be regenerated via adsorption immobilization of enzymes, the combination between carriers with the enzyme is not solid. The loss of enzyme is more serious, which also limits its application. Therefore, the design and preparation of the carrier material can be recycled to become, one of the focuses of this research. Specifically, this paper carried out the following aspects of research and exploration:1. Enzymes Immobilized on Superparamagnetic Fe3O4@Clays NanocompositesWe present a simple method to produce superparamagnetic Fe3O4@Clays nanocomposites consisting of magnetic iron oxide nanoparticles orderly self-assembled on some restricted positions of nanoclays. The as-prepared Fe3O4@Clays have highly ordered structure, large surface area, and high magnetic sensitivity, as verified by transmission electron microscopy (TEM), IR spectroscopy, X-ray diffraction (XRD), nitrogen adsorption-desorption isotherms, and a vibrating sample magnetometer (VSM). Subsequently, the as-prepared Fe3O4@Clays treated with (γ-aminopropyl)triethoxysilane are used as immobilization material. The conjugation of glucoamylase, hereby chosen as model enzyme, onto the amino-functionalized magnetic nanoparticles by using glutaraldehyde as a coupling reagent is further demonstrated and assessed based on its activity, kinetics, and thermal stability as well as reusability. Inspired by the structural character of enzyme (containing functional residues that are ideal reaction sites for the immobilization of enzymes once more), two novel regenerated strategies of supports are successfully developed to regenerate the carriers at the end of the life of the immobilized glucoamylase. The quality of glucoamylase immobilized on the regenerated supports is also defined by determining of the enzyme activity, kinetics, thermal stability, and reusability. The results indicate that the strategies for the regeneration of supports are viable. The applicability of the regenerated strategies of supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase. The regenerated strategies also offer an attractive and flexible alternative to regenerate other traditional supports at the end of the life of the immobilized enzyme.2. Glycidyl Methacrylate Grafted on FeSBA-15 by ATRP Technique:Modified with Metal-ligand Complex as a Regenerated Support for Enzyme ImmobilizationMagnetic SBA-15 (FeSBA-15) was prepared via wet impregnation, calcination and reduction, and poly(glycidylmethacrylate) (PGMA) was grafted on the surface of FeSBA-15 using surface-initiated atom transfer radical polymerization (SI-ATRP) for a prescribed time. The epoxy groups of the PGMA were reacted with Cu(II) metal-ligand complex (i.e., imidazole or iminodiacetic acid) to form metal-chelate brush. The as-prepared supports have highly ordered structure, and characterized by transmission electron microscopy (TEM), IR spectroscopy, X-ray Photoelectron Spectroscopy (XPS) and a vibrating sample magnetometer (VSM). Subsequently, the functionalized FeSBA-15 as a regenerated support was used for enzyme immobilization. Glucoamylase was immobilized as a model enzyme on the regenerated supports through" metal-ion affinity interactions. The quality of glucoamylase immobilized on the regenerated supports is defined by determining of the enzyme activity, thermal stability, and reusability. The results indicate that the metal-chelate brushes offer an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.3. Reversible immobilization of glucoamylase onto magnetic carbon nanotubes functionalized with dendrimerMagnetic carbon nanotubes (MCNTs) with necklace-like nanostructures was prepared via solvothermal method, and hyperbranched poly(amidoamine) (PAMAM) was grafted on the surface of MCNTs on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of EDA, which could achieve generational growth under such uniform stepwise reactions. The termina1-NH2 groups from the dendritic PAMAM were reacted with differently functionalized groups to form functionalized MCNTs. Subsequently, enzyme was immobilized on the functionalized MCNTs through adsorption, covalent bond and metal-ion affinity interactions. The immobilization of glucoamylase, hereby chosen as model enzyme, onto the differently functionalized MCNTs is further demonstrated and assessed based on its activity, thermal stability as well as reusability. Besides ease in recovery by magnetic separation, the immobilized glucoamylase on functionalized MCNTs offers superior stability and reusability, without compromising the substrate specificity of free glucoamylase. Furthermore, the results indicate that the metal-chelate dendrimer offer an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.4. Reversible immobilization of glucoamylase onto magnetic chitosan nanocarrierA uniform particle size, monodisperse superparamagnetic Fe3O4 nanoparticles was prepared via solvothermal method. Then, chitosan was covalently linked to nanoparticles using a simple method, forming a shell-like pH-sensitive Fe3O4/CS magnetic nanocarrier. The as-prepared supports have highly ordered structure, and characterized by transmission electron microscopy (TEM), IR spectroscopy, and a vibrating sample magnetometer (VSM). The results show that the preparation of pH-sensitive carrier Fe3O4/CS is successful, and its magnetic strength is relatively high. Subsequently, the pH-sensitive Fe3O4/CS magnetic nanocarrier as a regenerated support was used for enzyme immobilization. The quality of glucoamylase immobilized on the regenerated supports is defined by determining of the enzyme activity, thermal stability, and reusability. The results indicate that the pH-sensitive offer an efficient route to immobilize enzymes.