CN110540986A - Polyamine-assisted natural polyphenol rapid and stable modification magnetic nano immobilized enzyme carrier and application thereof - Google Patents

Abstract

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.

Description

Polyamine-assisted natural polyphenol rapid and stable modification magnetic nano immobilized enzyme carrier and application thereof

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.

Example 1:

Preparation of magnetic nano enzyme immobilized carrier for realizing rapid and stable modification of tannic acid under assistance of polyamine and preparation of corresponding immobilized enzyme

(1) Preparing magnetic ferroferric oxide nanoparticles (Fe3O4NPs) by a solvothermal method:

Polyacrylic acid (PAA,150mg), sodium acetate (NaAc,6.0g), and ferric chloride hexahydrate (FeCl 3.6H2O, 1.62g) were dissolved in a mixed solution composed of diethylene glycol and ethylene glycol, respectively. After vigorous mechanical stirring, the mixture was heated hermetically at 200 ℃ for 12 hours. After the reaction is finished, separating and collecting the magnetic particles by using a magnet, and respectively cleaning the magnetic particles for 3 times by using deionized water and absolute ethyl alcohol to obtain black magnetic particles, and drying the black magnetic particles for 24 hours in vacuum.

(2) preparation of Tannic Acid (TA)/polyamine (TEPA) binary system functionalized magnetic nanoparticles (Fe3O4-pTAPA NPs):

Adding the Fe3O4 nanoparticles prepared in step (1) into Tris-HCl (pH 8.5) buffer solution containing 2mg/mL TA, and adding TEPA into the mixed solution for reaction for a certain period of time. And finally, washing the synthesized nano material for multiple times by using deionized water and ethanol, and drying for 24 hours in vacuum. Obtaining the TA/TEPA binary system functionalized magnetic nanoparticles (Fe3O4-pTAPA NPs).

As a control experiment, nanoparticles synthesized without the addition of TEPA were named Fe3O4-pTA NPs.

(3) Preparation of magnetic nanoparticles functionalized by epigallocatechin gallate (EGCG)/polyamine (DETA) binary system (Fe3O4-pEGDE NPs):

Adding the Fe3O4 nanoparticles prepared in the step (1) into Tris-HCl (pH 8.5) buffer solution containing 3mg/mL of EGCG, and then adding DETA into the mixed solution according to a certain mass ratio for reaction for a period of time. And finally, washing the synthesized nano material for multiple times by using deionized water and ethanol, and drying for 24 hours in vacuum. EGCG/DETA binary system functionalized magnetic nanoparticles (Fe3O4-pEGDE NPs) are obtained.

Wherein, the step (3) can be used as an alternative of the step (2).

(4) Immobilized lipase

This example selects the lipase CALB as a model enzyme, and immobilizes the enzyme on Fe3O4-pTAPA nanoparticles by a simple covalent binding method.

Specifically, a certain amount of Fe3O4-pTAPA nanoparticles were dispersed in a buffer solution containing lipase CALB by ultrasound, and reacted for 6h with shaking at 180 rpm. Subsequently, the immobilized lipase CALB (Fe3O4-pTAPA-CALB) was separated with a magnet and washed several times with a phosphate buffer to remove the unligated lipase CALB. The lipase stock finally obtained was stored at 4 ℃ until use.

(5) Characterization of

in this example, the synthesized material was characterized by a transmission electron microscope, an infrared spectrum, and a vibration sample magnetometer, and the results are shown in fig. 2A to 2D. As shown in fig. 2A, transmission electron microscopy showed that the average diameter of the synthesized spherical Fe3O4 nanoparticles of the present invention was about 100 nm; as shown in fig. 2B, Fe3O4-pTAPA obtained after TA/TEPA binary system modification showed a distinct irregular layer around Fe3O4 nanoparticles, indicating that TA/TEPA binary system was successfully modified on Fe3O4 nanoparticles; as shown in the IR spectrum of FIG. 2C, compared with the unmodified Fe3O4, several new absorption peaks appeared after modification by the TA/TEPA binary system, the new absorption peaks at 1222 and 1437cm-1 can be attributed to the C-N stretch band and amide II of TEPA, and a wider absorption band near 1614cm-1, which may be related to the aromatic ring of TA, further proving that the Fe3O4 nanoparticles are successfully modified by the TA/TEPA binary system; after CALB immobilization, a new absorption peak appeared at 1200cm-1 for Fe3O4-pTAPA-CALB, which may be related to the stretching of Schiff base reaction to form C-N bond during immobilization, indicating that CALB was successfully covalently immobilized on Fe3O4-pTAPA nanoparticles. As shown in FIG. 2D, the saturation magnetic strengths of the Fe3O4, Fe3O4-pTAPA and Fe3O4-pTAPA-CALB nanoparticles were 62.8, 59.7 and 44.0emu/g, respectively. Due to the modification of the TA/TEPA binary system and the further fixation of the lipase CALB, the magnetic strength of the material is gradually reduced. This result also clearly demonstrates the formation of a pTAPA layer oxidatively polymerized by the TA/TEPA binary system and the immobilization of the lipase CALB. Although the saturation magnetization of the Fe3O4-pTAPA-CALB nanoparticles was smaller than that of the Fe3O4 nanoparticles, it is evident from the inset in fig. 2D that the magnet still rapidly separated the immobilized lipase CALB from the aqueous solution within 20 seconds.

In conclusion, the above results indicate that the Fe3O4-pTAPA nanoparticles were successfully synthesized and the immobilized lipase CALB was successfully constructed. In addition, the immobilized enzyme can be rapidly recovered from the reaction liquid through a magnet, and the catalyst can be recycled.

Example 2:

Examination of mechanical stability and biocompatibility of functionalized magnetic Fe3O4 nanoparticles

Mechanical stability investigation: 0.5mg/mL of Fe3O4-pTA or Fe3O4-pTAPA nanoparticles were dispersed in phosphate buffer (20mM, pH 7.0) and stirred for 7 days. Samples were taken from the above solution daily for thermogravimetric analysis (TGA). The calculation formula of the polymer residual rate is as follows:

Polymer residue (%). Wi/W0X 100

Wherein W0 is the thermal weight loss of Fe3O4-pTAPA or Fe3O4-pTA nano-particles in 0 day, and Wi is the thermal weight loss of Fe3O4-pTAPA or Fe3O4-pTA nano-particles in a certain time.

Examination of biocompatibility: l-02 cells were seeded in 96-well plates and cultured overnight at 37 ℃. Then, the original culture medium is replaced by fresh culture medium of Fe3O4-pTAPA nano-particles with different concentrations for 24h, and finally the cytotoxicity of the Fe3O4-pTAPA nano-particles is determined by an MTT method.

As can be seen from FIG. 3A, the weight loss of Fe3O4-pTA was 25.1 wt%, while the weight loss of Fe3O4-pTAPA nanoparticles increased to 51.2 wt%. This phenomenon can be explained by that, under weakly basic conditions, Tannic Acid (TA) providing a phenolic group and polyamine (TEPA) providing an amino group can rapidly form a covalent bond, and the formed pTAPA layer is tightly adhered to the surface of Fe3O4 nanoparticles by covalent and non-covalent interactions. Thus, this result indicates that the presence of polyamine (TEPA) can accelerate the polymerization of TA on Fe3O4 nanoparticles.

As shown in fig. 3B, the polymer residue of Fe3O4-pTAPA nanoparticles remained above 80.9% after stirring for 7 days, while the polymer residue of Fe3O 4-ptaa nanoparticles decreased to 53.2%, indicating that polyamine (TEPA) can improve the mechanical stability of the tannic acid polymer layer. This result may be due to the oxidation of the phenolic group in TA to a quinoid structure, which is susceptible to michael addition reactions and schiff base reactions with the amino groups in the polyamine, resulting in a dense polymer layer coating the surface of the support material.

FIG. 3C shows that the cell viability of L-02 cells is still higher than 85% at concentrations of Fe3O4-pTAPA nanoparticles as high as 200. mu.g/mL, indicating good biocompatibility and low cytotoxicity of Fe3O4-pTAPA nanoparticles.

morphological images of L-02 cells treated with or without the addition of Fe3O4-pTAPA nanoparticles were observed with an optical microscope as shown in FIG. 3D. Compared with the control group of L-02 cells, the L-02 cells treated by the Fe3O4-pTAPA nanoparticles have no obvious damage to the cell morphology, and the synthetic nanoparticles have good biocompatibility.

The results show that the polyamine can accelerate the oxidative polymerization of tannic acid on the surface of the magnetic ferroferric oxide nanoparticles, and the synthesized Fe3O4-pTAPA nanoparticles have good mechanical stability and biocompatibility.

Example 3: optimization of lipase immobilization Process

In the enzyme immobilization process, the influence of the co-deposition time (4-10) of a TA/TEPA binary system and the addition amount (50-350mg/g carrier) of the initial lipase CALB on the loading capacity and the enzyme activity of the enzyme is examined. The enzyme activity is determined according to a p-NPP method, the highest enzyme activity is 100 percent, and the relative enzyme activity and the enzyme carrying capacity under different immobilization process conditions are obtained.

As shown in fig. 4A and 4B, the optimum immobilized enzyme conditions were: the TA/TEPA binary coprecipitation time is 7h and the addition amount of immobilized lipase is 250mg/g carrier.

Example 4: enzymatic Properties of immobilized Lipase

(1) Optimum pH and temperature

The hydrolysis activity of the lipase is measured by adopting a p-NPP method, the influence of different pH (4-10) and different temperature (20-80 ℃) on the catalytic activity of free and immobilized lipase CALB is researched, and the relative enzyme activities are compared.

(2) Temperature stability

The enzyme activity was measured by p-NPP method using appropriate samples with gentle stirring at 40 ℃ in phosphate buffer (10mM, pH 7.0) at regular intervals, and then the relative activities of free and immobilized CALB were calculated.

(3) Methanol tolerance

Respectively culturing the free enzyme and the immobilized lipase CALB in methanol solutions (v/v, 30 percent and 60 percent) with different concentrations for a certain time, taking a proper amount of samples, measuring the enzyme activities of the free and immobilized CALB by a p-NPP method, and comparing the methanol tolerance.

As shown in fig. 5A to 5D, compared with free enzyme, the immobilized enzyme has significantly improved pH, temperature, thermal stability and methanol tolerance, which is mainly because the rigidity of the immobilized enzyme is enhanced by the multi-point covalent immobilization of the enzyme on the carrier, thereby effectively avoiding drastic change of conformation of the immobilized enzyme in different micro environments.

Example 5: production of biodiesel by immobilized lipase in alternating magnetic field

The catalytic reaction was carried out in a 10mL stoppered shake flask, and the reaction system was as follows: 2.0g of waste edible oil, absolute methanol in a molar ratio of oil to methanol of 1:3, a molecular sieve (40 wt% oil) and a certain amount of free lipase CALB or immobilized lipase CALB. During the reaction, the addition of methanol was carried out in three times. The reaction was carried out at 40 ℃ with a stirring speed of 200rpm and a reaction time of 96h at the maximum. At the same time, samples were taken from the reaction system at regular intervals and quantitatively analyzed by gas chromatography. In addition, when the influence of the magnetic field of the alternating magnetic field on the immobilized enzyme is studied, the addition amount of the immobilized enzyme is reduced to half of the original amount, the reaction is carried out for 84h, and other conditions are not changed.

As shown in fig. 6A, the catalytic performance of the immobilized lipase CALB was significantly higher than that of the free lipase CALB. This phenomenon may be due to the presence of an excess of methanol in the reaction system, which has poor solubility in the waste cooking oil, which may result in inactivation of the lipase CALB, whereas the methanol tolerance of the immobilized lipase CALB is significantly improved, thus improving the production efficiency of biodiesel.

interestingly, we have previously reported that enzymes and cofactors immobilized on superparamagnetic nanoparticles can significantly increase the catalytic reaction rate under alternating magnetic fields. Fig. 6B shows a photograph of an ac magnetic field generator made by our laboratory, which consists of a control panel, an iron coil, a glass reactor and a transformer. Wherein, there are 5 grades of magnetic field frequency (25-500 Hz) and 4 grades of magnetic field intensity (lowest, low, high and highest) on the control panel.

As can be seen from FIG. 6C, the catalytic efficiency of the immobilized lipase CALB was higher under the action of the alternating magnetic field than that without the alternating magnetic field, and the reaction rate increased with the increase of the frequency of the magnetic field. The result shows that the Fe3O4-pTAPA-CALB nano-particles are like a micro stirrer under the alternating magnetic field, so that the collision frequency of the immobilized lipase CALB and the substrate can be obviously improved, and the production efficiency of the biodiesel is improved. Therefore, these interesting results indicate that the alternating magnetic field can promote the catalytic production of biodiesel from Fe3O4-pTAPA-CALB, and the biodiesel production efficiency is further improved with the increase of the frequency and intensity of the alternating magnetic field.

As shown in FIG. 6D, Fe3O4-pTAPA-CALB had good reusability.

In conclusion, a series of morphological and structural representations prove that the method skillfully solves the problems of long time consumption and poor mechanical stability of the formation of the tannic acid layer by introducing polyamine, and successfully synthesizes the magnetic nanoparticles modified by the tannic acid/polyamine binary system. The magnetic nano-particles synthesized by the method have the advantages of good mechanical stability, biocompatibility, dispersibility, magnetic responsiveness, covalent connection with enzyme and high enzyme loading capacity. The immobilized lipase prepared by the method can be used for producing biodiesel by efficiently utilizing waste edible oil under an alternating magnetic field, and has good reusability. In addition, the magnetic carrier synthesized by the present invention is suitable for immobilization of any water-soluble enzyme. The preparation method is simple and environment-friendly, and is suitable for large-scale production

In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Claims (9)

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.