Polyamine-assisted natural polyphenol rapid and stable modification magnetic nano immobilized enzyme carrier and application thereof
阅读说明:本技术 聚胺辅助天然多酚快速、稳定修饰磁性纳米固定化酶载体及应用 (Polyamine-assisted natural polyphenol rapid and stable modification magnetic nano immobilized enzyme carrier and application thereof ) 是由 陈超 汤文 王平 庄家丰 马同昊 于 2019-09-09 设计创作,主要内容包括:本发明提供了一种聚胺辅助实现天然多酚快速且稳定修饰的磁性纳米固定化酶载体,包括磁性纳米颗粒,表面经天然多酚和聚胺类修饰。本发明提供了相应的制备方法、固定化酶及制备方法、应用。本发明提供了一种利用聚胺加速天然多酚聚合且稳定沉淀的仿生黏附策略制备天然多酚/聚胺二元体系修饰的磁性纳米颗粒固定化酶的方法,为固定化酶技术提供了新方法。本发明首次合成了天然多酚/聚胺二元体系修饰的磁性四氧化三铁纳米颗粒,用于酶的固定化,具有超顺磁性、良好的生物相容性以及高的酶负载量,为固定化酶提供了新的酶固定化载体。本发明将固定化的脂肪酶在交变磁场中催化废弃食用油生产生物柴油,具有较高的催化效率,表现出巨大的工业应用前景。(The invention provides a magnetic nano immobilized enzyme carrier for realizing quick and stable modification of natural polyphenol by polyamine, which comprises magnetic nano particles, wherein the surfaces of the magnetic nano particles are modified by natural polyphenol and polyamine. The invention provides a corresponding preparation method, immobilized enzyme, a preparation method and application. The invention provides a method for preparing a magnetic nanoparticle immobilized enzyme modified by a natural polyphenol/polyamine binary system by utilizing a bionic adhesion strategy of polyamine for accelerating polymerization of natural polyphenol and stabilizing precipitation, and provides a new method for an immobilized enzyme technology. The magnetic ferroferric oxide nano-particles modified by a natural polyphenol/polyamine binary system are synthesized for the first time, are used for enzyme immobilization, have superparamagnetism, good biocompatibility and high enzyme loading capacity, and provide a new enzyme immobilization carrier for immobilized enzymes. The invention catalyzes the waste edible oil to produce the biodiesel by the immobilized lipase in the alternating magnetic field, has higher catalytic efficiency and shows huge industrial application prospect.)
1. A magnetic nano immobilized enzyme carrier for realizing quick and stable modification of natural polyphenol by polyamine is characterized by comprising magnetic nano particles, wherein the surfaces of the magnetic nano particles are modified by the natural polyphenol and polyamine.
2. the magnetic nano-immobilized enzyme carrier for realizing rapid and stable modification of natural polyphenol by the assistance of polyamine according to claim 1, wherein the polyamine is one or more of tetraethylenepentamine, polyetherimide, triethylenetetramine or diethylenetriamine.
3. the magnetic nano-immobilized enzyme carrier for realizing rapid and stable modification of natural polyphenol by the aid of polyamine of claim 1, wherein the natural polyphenol is one or more of tannic acid, epigallocatechin gallate, epicatechin gallate or epigallocatechin.
4. The magnetic nano-immobilized enzyme carrier for realizing rapid and stable modification of natural polyphenol by the aid of polyamine according to claim 1, wherein the magnetic nano-particles are ferroferric oxide magnetic nano-particles.
5. A method for preparing magnetic nano-immobilized enzyme carrier assisted by polyamine to realize rapid and stable modification of natural polyphenol as claimed in claim 1, which comprises the steps of:
(1) Preparing magnetic nanoparticles;
(2) under the alkalescent condition, the surface of the magnetic nano-particles is functionally modified by adopting a natural polyphenol/polyamine binary system.
6. The method for preparing a magnetic nano-immobilized enzyme carrier assisted by polyamine to realize rapid and stable modification of natural polyphenol according to claim 5, wherein in the step (2), the mass ratio of tannic acid/polyamine capable of forming a stable polymer layer is 8: 1-3: 1.
7. The method for preparing a magnetic nano-immobilized enzyme carrier assisted by polyamine to realize rapid and stable modification of natural polyphenol according to claim 5, wherein in the step (2), the co-precipitation time of the tannin/polyamine binary system capable of forming a stable polymer layer is 4-10 h.
8. An immobilized enzyme immobilized on the modified surface of the magnetic immobilized nanoenzyme carrier of claim 1, wherein the enzyme is a water-soluble enzyme.
9. Use of the immobilized enzyme of claim 8 in the catalytic production of biodiesel under an alternating magnetic field.
Technical Field
The invention relates to an enzyme immobilization technology, in particular to preparation and application of a magnetic nano enzyme immobilization carrier for realizing rapid and stable modification of natural polyphenol by polyamine assistance, and belongs to the fields of high polymer materials and biological catalysis science.
Background
In recent years, biodiesel has been receiving more and more attention as a biodegradable, environmentally friendly and renewable biofuel. It can be produced by using waste oil, animal fat and vegetable oil through transesterification reaction under the action of a biological catalyst or a chemical catalyst. At present, the supply of the biodiesel mainly comes from alkali or acid catalytic conversion, but the quality and the yield of the biodiesel are seriously reduced due to the potential disadvantages of environmental pollution, high energy demand, non-ideal reaction and the like. Compared with chemical catalysis, enzymatic catalysis (such as lipase) is increasingly regarded by people with the advantages of environmental friendliness, mild operating conditions, low energy consumption and the like. However, free lipases have problems in industrial applications due to their difficulty in recycling, their susceptibility to inactivation in organic solvents, and their high operating costs.
In order to overcome the above bottleneck problems, researchers have been looking for efficient enzyme immobilization carriers suitable for lipases in recent years to improve enzyme stability, significantly reduce costs, and achieve enzyme recycling. At present, various nano materials such as mesoporous materials, magnetic materials, polymer materials and the like are successfully designed and synthesized as enzyme immobilization carriers.
In various nano materials, the magnetic nano particles have wide application prospect in the field of enzyme immobilization due to the advantages of high specific surface area, low mass transfer resistance, good biocompatibility, easy separation and recovery and the like. However, direct immobilization of enzymes on the surface of magnetic nanoparticles can seriously affect the catalytic performance and loading capacity of immobilized enzymes, mainly due to their inherent chemical inertness and lack of active sites for enzyme immobilization. In addition, steric hindrance and high mass transfer resistance exist between the surface of the magnetic nanoparticle and the immobilized enzyme. Therefore, the surface modification of the magnetic nanoparticles by using the natural polymer which has a multi-active functional group, is biodegradable and has good biocompatibility can significantly improve the immobilization efficiency of the enzyme.
To date, green chemistry provides an environmentally friendly strategy for the preparation of functional nanostructures. Among them, the use of natural polyphenols to modify magnetic nanoparticles to enhance their interaction with enzymes or to provide additional enzyme covalent immobilization functional groups for carriers has received increasing attention. Among them, tannic acid is a natural polyphenol compound extracted from plants, and has been widely used in functionalized nanoparticles. Specifically, tannic acid can be oxidized and polymerized to form a poly (tannic acid) layer on the surface of the magnetic nanoparticles, which has been proved to be a general platform for enzyme immobilization, and the enzyme immobilization is realized through Michael reaction or Schiff base reaction. However, the surface modification of pure tannic acid has some disadvantages in enzyme immobilization, such as long time for forming poly (tannic acid) layer and poor mechanical stability of the formed tannic acid polymer layer. Polydopamine can adhere rapidly and tightly to various substrate materials by both covalent and non-covalent interactions, as compared to pure tannic acid. In addition, it has been shown that amino and phenolic groups are two key components in forming covalent bonds in the polydopamine layer. Inspired by this, the present study can be confident to conclude that molecules containing phenolic and amino groups may have adhesive properties, capable of rapid oxidative polymerization to form stable polymer layers similar to polydopamine. Considering that tannic acid contains abundant phenolic groups and polyamines and also abundant amino groups, the combination of both may have a chemical structure similar to dopamine, so that the tannic acid/polyamine binary system may be oxidatively polymerized to form a stable polymer layer for enzyme immobilization.
aiming at the problems that the modification of a substrate material by a tannic acid layer consumes long time, the mechanical stability of the substrate material is poor, and the practical application of the tannic acid layer in the field of immobilized enzymes is seriously hindered, the patent provides a strategy for rapidly and stably modifying tannic acid on the surface of magnetic nanoparticles for enzyme immobilization by using polyamine, and successfully synthesizes a tannic acid/polyamine binary system modified ferroferric oxide nanoparticle compound (Fe3O 4-pTAPA). The compound carrier has the advantages of good mechanical stability, biocompatibility, dispersibility, magnetic responsiveness, covalent connection with enzyme and high enzyme loading capacity, thereby providing an immobilized enzyme carrier with excellent performance for the enzyme and having huge industrial application potential in the field of enzyme catalysis.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides an enzyme-loaded magnetic nano immobilized enzyme carrier with good biocompatibility, high stability and high loading capacity, an immobilized enzyme, a corresponding preparation method and application, and can solve the technical problems that the formation of a tannic acid layer takes long time, the mechanical stability on a substrate material is poor, and the practical application of the tannic acid layer in the field of immobilized enzymes is seriously hindered.
The invention provides a magnetic nano-immobilized enzyme carrier which is assisted by polyamine to realize quick and stable modification of natural polyphenol, comprising magnetic nano-particles, wherein the surfaces of the magnetic nano-particles are modified by natural polyphenol and polyamine. The immobilized enzyme carrier has good biocompatibility, high stability and high loading capacity.
Preferably, the polyamine is one or more of Tetraethylenepentamine (TEPA), Polyetherimide (PEI), triethylenetetramine (TETA) or Diethylenetriamine (DETA).
Preferably, the natural polyphenol is one or more of Tannic Acid (TA), epigallocatechin gallate (EGCG), epicatechin gallate (ECG) and Epigallocatechin (EGC).
Preferably, the magnetic nanoparticles are ferroferric oxide magnetic nanoparticles.
The second purpose of the invention is to provide a preparation method of the magnetic nano immobilized enzyme carrier which is assisted by polyamine to realize quick and stable modification of natural polyphenol, comprising the following steps:
(1) Preparing magnetic nanoparticles; preferably, the Fe3O4 magnetic nanoparticles (Fe3O4NPs) are prepared using a solvothermal method;
(2) Under the alkalescent condition, the surface of the magnetic nano-particles is functionally modified by adopting a natural polyphenol/polyamine binary system.
In the preparation method and the product obtained by the preparation method, polyamine-assisted tannic acid rapidly modifies the surface of the magnetic nanoparticles.
Preferably, in the step (2), the mass ratio of the tannic acid to the polyamine is 8: 1-3: 1.
Preferably, in the step (2), the coprecipitation time of the tannin/polyamine binary system is 4 to 10 hours.
The third purpose of the invention is to provide an immobilized enzyme, which is obtained by immobilizing any water-soluble enzyme on the modified surface of the magnetic nano immobilized enzyme carrier.
The fourth purpose of the invention is to provide a preparation method of the immobilized enzyme, which comprises the following steps: and adding the immobilized enzyme carrier into an enzyme solution to perform immobilized enzyme.
Wherein, the enzyme can be any water-soluble enzyme; the initial enzyme adding amount is 50-350mg/g carrier; the enzyme immobilization time is 1-9 h.
A fifth object of the present invention is to provide an application of immobilized enzyme for food, biodiesel production, sewage treatment, chiral drug production, etc., particularly for producing biodiesel under an alternating magnetic field, wherein the alternating magnetic field is generated by an alternating magnetic field generator, and the alternating magnetic field generator mainly consists of a control panel, an iron coil, a glass reactor and a transformer.
The invention has the advantages that:
(1) The invention provides a method for preparing a magnetic nanoparticle immobilized enzyme modified by a natural polyphenol/polyamine binary system by utilizing a bionic adhesion strategy of polyamine for accelerating polymerization of natural polyphenol and stabilizing precipitation, and provides a new method for an immobilized enzyme technology.
(2) The magnetic ferroferric oxide nano-particles modified by a natural polyphenol/polyamine binary system are synthesized for the first time, are used for enzyme immobilization, have superparamagnetism, good biocompatibility and high enzyme loading capacity, and provide a new enzyme immobilization carrier for immobilized enzymes.
(3) The invention greatly improves the stability and the reusability of the enzyme by utilizing a novel immobilization method.
(4) The invention catalyzes the waste edible oil to produce the biodiesel by the immobilized lipase under the alternating magnetic field, has higher catalytic efficiency and shows huge industrial application prospect.
Drawings
FIG. 1: a basic principle diagram of preparation and application of magnetic ferroferric oxide (Fe3O4) nano particles modified by a tannic acid/polyamine binary system.
FIGS. 2A to 2D: and (3) a morphology and structure characterization diagram of the prepared functionalized magnetic Fe3O4 nano-particles. Wherein, FIG. 2A is a transmission electron micrograph of Fe3O4 before modification, FIG. 2B is a transmission electron micrograph of Fe3O4 after modification (Fe3O4-pTAPA), FIG. 2C is an infrared spectrum chromatogram of the prepared magnetic Fe3O4, Fe3O4-pTAPA and Fe3O4-pTAPA-CALB nanoparticles, and FIG. 2D is a hysteresis curve chart of the prepared magnetic Fe3O4, Fe3O4-pTAPA and Fe3O4-pTAPA-CALB nanoparticles.
FIGS. 3A to 3D: the prepared functionalized magnetic Fe3O4 nanoparticles are examined and mapped to mechanical stability and biocompatibility. FIG. 3A is a thermogravimetric analysis graph of the prepared magnetic Fe3O4, Fe3O4-pTA and Fe3O4-pTAPA nanoparticles; FIG. 3B shows the polymer residual rates of Fe3O4-pTA and Fe3O4-pTAPA magnetic nanoparticles at different times; FIG. 3C shows the cell viability of L-02 cells incubated with various concentrations of Fe3O4-pTAPA nanoparticles; FIG. 3D is a light microscope photograph of L-02 cells cultured with and without Fe3O4-pTAPA nanoparticles.
FIGS. 4A to 4B: and (4) optimizing the immobilized enzyme process. FIG. 4A is a graph of the effect of TA/TEPA binary system codeposition time on enzyme loading and relative activity; FIG. 4B is a graph of the effect of added CALB concentration on enzyme loading and relative activity.
FIGS. 5A to 5D: the enzymatic properties of the immobilized enzyme. FIGS. 5A-5B are graphs of the effect of pH and temperature on the hydrolytic activity of free and immobilized enzymes; FIG. 5C is the thermostability of free enzyme and immobilized enzyme at 40 ℃; fig. 5D is a graph of the tolerance of free and immobilized enzymes in the presence of 30% and 60% methanol.
Fig. 6A to 6D: producing the biodiesel by using the immobilized enzyme. FIG. 6A is a schematic representation of biodiesel production from waste edible oil catalyzed by free enzyme and immobilized enzyme; FIG. 6B is a photograph of an AC magnetic field generator homemade in a laboratory; FIG. 6C is a graph of the effect of different applied alternating magnetic field frequencies on immobilized enzyme catalyzed biodiesel production at maximum magnetic field strength; FIG. 6D shows the reusability of immobilized enzyme for biodiesel production under an alternating magnetic field.
Detailed Description
for a better understanding of the present invention, reference will now be made to the following examples. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
As shown in fig. 1: according to the invention, magnetic response type ferroferric oxide Fe3O4 is used as a substrate material, three galloyl groups of Tannic Acid (TA) firstly react with FeIII on the surface of a ferroferric oxide (Fe3O4) nanoparticle to form a stable octahedral complex. Meanwhile, the phenol group in TA is oxidized into a quinoid structure under an alkaline condition, and then further reacted with an amino group in polyamine through michael addition reaction/schiff base reaction to form a carbon-nitrogen bond (C-N bond) at the ortho position of the benzene ring. The resulting poly (tannic acid/polyamine) layer adheres tightly to the magnetic Fe3O4 surface by covalent and non-covalent interactions. Finally, the immobilization is achieved by covalent bonds between the enzyme and the support described above, without using any chemical cross-linking agent.