Preparation Of Magnetic Phenolic Biomimetic Adhesive Composite Materials For Lipase Immobilization

Lipase(triacylglycerol ester hydrolases, EC 3.1.1.3) is a kind of important biocatalyst with various biological activities, such as esterification, interesterification and hydrolysis reactions, and plays an important role in stereo-recognition of chiral substrates. However, the stability of free enzymes is pretty terrible, and their poor activity, hard to reuse and high cost are still needed to be resolved. Thus, the technology of enzyme immobilization was developed to overcome these disadvantage and inconveniences, which could improve the stability of enzyme, reduce the pollution of product and perform enzyme reuse and achieve the goal of successive production and cost reduction. Inspired by the biomimetic adhesive properties of catechol derivatives, we put forward to the strategies of constructing magnetic-phenolic systems. When applied for enzyme immobilization, these systems exhibited high efficiency and easy recyclability. The main contents of this dissertation were summarized as follows:(1) Mussel-inspired surface modification of magnetic@graphite nanosheets composite for efficient candida rugosa lipase immobilizationBy the facile adhesion way, the novel composite complex by polydopamine(PDA) and magnetic graphite nanosheets(Fe3O4@GNSs) has been successfully synthesized. The resulting composite was characterized by means of the scanning electron microscopy(SEM), transmission electron microscope(TEM), Fourier transform infrared spectra, Raman spectrums, vibrating sample magnetometer(VSM) etc.. Meanwhile, the PDA functionalized Fe3O4@GNSs was applied for candida rugosa lipase(CRL) immobilization covalently without any toxic coupling agent. Combing the superior chemical stability, high loading capacity of Fe3O4@GNSs and the well biocompatibility, functional characteristics of PDA, the Fe3O4@GNSs-PDA composite displayed several advantages, including the high enzyme capacity, enzyme activity and stability and resuability.(2) Facile synthesis of oxidic PEG modified magnetic polydopamine nanospheres for candida rugosa lipase immobilizationA versatile method for the design of polydopamine coated magnetic material with a brush-like structure used for CRL immobilization was reported in this work. Firstly, polydopamine(PDA) coated on the surface of Fe3O4 nanospheres(Fe3O4 NPs) with a controllable thickness via dip-coating process, and CRL can be immobilized on it directly via covalent bonding. Subsequently, PDA functionalized Fe3O4 NPs were modified with oxidic polyethylene glycol(PEG) to obtain the aldehyde groups and the brush-like structure of the magnetic supports were formed. After characterized with various methods, it was verified that the prepared magnetic NPs possessed good monodispersity and displayed high saturation magnetization(55.35 emu g-1) after modification. Meanwhile, the CRL was immobilized on it covalently and the resulted immobilized enzyme exhited higher enzyme activities(such as activity, stability and reusability) than magnetic PDA NPs immobilized CRL. Significantly, the versatility of polydopamine-inspired chemistry combined with the unique biological nature and tunability with dialdehyde PEG could evoke the efficiency of the CRL, and effectively protect enzyme from denaturation due to the rigidity of support.(3) Preparation of core-shell magnetic polydopamine/alginate biocomposite for candida rugosa lipase immobilizationA flexible, biocompatible and bioadhesive enzyme immobilizing material, which was synthesized based on the covalent assembly of biomimetic polymer and oxidized polysaccharide on magnetic nanoparticles(NPs), has been developed in this feasibility study. In this work, the bio-inspired polymer, polydopamine(PDA), was used to modify the well-monodispersed Fe3O4 NPs(mPDA NPs) with a controllable thickness via a dip-coating process, then the alginate di-aldehyde(ADA) was covalently assembled on the mPDA NPs and employed as a naturally occurring linking agent for CRL immobilization. The resulting support material was characterized by means of the transmission electron microscope(TEM), Fourier transform infrared spectra, X-ray diffraction(XRD), thermogravimetry(TG) analyser, and vibrating sample magnetometer(VSM). It was verified that the prepared mPDA NPs possessed distinct core-shell structure with uniform size and high saturation magnetization(57.62 emu g-1). For furhter application, the mPDA NPs was utilized in CRL immobilizing procedures and demonstrated can facilitate improving the enzyme activities.(4) Formulation of robust hybrid magnetic polydopamine microcapsules through hard-template mediated method for efficient enzyme immobilizationA mild and facile method for the construction of robust organic-inorganic hybrid magnetic microcapsules was developed by a hard-template mediated method coordinated with polydopamine(PDA) and Fe3O4 nanoparticles onto CaCO3 microparticle template. More specifically, vaterite type CaCO3 microparticle was prepared via the co-precipitaiton method, and CRL was encapsulated into the CaCO3 microparticle simultaneously. Based on the porous property(pore size 20-60 nm) and the ξ potential is positively charged under pH=7 of CaCO3 microparticles, negatively charged Fe3O4 nanoparticles(particle size< 20 nm) were adsorbed on the surface or into the lumen of porous CaCO3 microparticles through physical absorption and electrostatic interaction. Then the magnetic sacri?cial templates were coated with PDA through self-polymerization of dopamine to obtain the magnetic PDA-CaCO3 microparticles, which were followed by the template removal using EDTA to construct organic-inorganic hybrid magnetic microcapsules. Characterizations confirmed the microcapsules possess a robust hollow structure so that enzyme inside exists in a free state. The Fe3O4 nanoparticles acted critical factors in microcapsules for both recyclable component and tough scaffold to sustain the microcapsules away from collapse and fold. The encapsulated CRL was demonstrated to have several advantages, including increased encapsulation efficiency, enzyme activity and long-term storage stability.(5) Construction of enzyme immobilization system through metal-polyphenol assisted Fe3O4/chitosan hybrid microcapsulesMetal-polyphenol film consolidated Fe3O4/chitosan hybrid microcapsules were developed based on Fe3O4/chitosan aggregation and tannic acid-FeIII coating. Specifically, citrate modified Fe3O4 nanoparticles were adsorbed onto chitosan-citrate microaggregates via ion exchange. After continuous stirring, the citrate moieties in the internal part of chitosan-citrate microaggregates were released to the bulk solutions and caused the disassembly of chitosan-citrate microaggregates, thus generating the capsule lumen. To further obtain an intact and robust structure, metal-polyphenol(tannic acid-FeIII) film was utilized to form adhesive coatings on the surface of Fe3O4/ chitosan microcapsules. CRL was encapsulated into chitosan-citrate microaggregates during it was parpared. The microcapsule preparation process was mild and high efficient, it can be completed within 40 min. After studying the properties of immobilized CRL such as activity, kinetic behaviors, stability and reusability, it was proved that TA-FeIII consolidated Fe3O4/CS microcapsules exhibited more excellent properties than Fe3O4/CS microcapsules only.