CN107893063A - A kind of method by epoxy cross-linking embedded magnetic nano particle immobilized enzyme - Google Patents

Abstract

The invention discloses a method for immobilizing enzymes by embedding magnetic nanoparticles through epoxy cross-linking, comprising the following steps: 1) adding the enzymes into a phosphate buffer solution with a pH of 6-9 at a temperature of 0-4°C, and stirring at a speed of 10-60rpm/min until completely dissolved; 2) adding superparamagnetic ferrite nanoparticles into the mixed solution prepared in step 1) at a temperature of 0-4°C, and Stir at a speed of 10-60rpm/min until the mixture is uniform; 3) Add epichlorohydrin dropwise to the mixed solution prepared in step 2) at a temperature of 0-4°C, and mix at 10-60rpm/min Under the condition of stirring at high speed, the cross-linking reaction was carried out for 24-48 h, and the enzyme/magnetic nanoparticle composite microspheres were prepared; 4) The enzyme/magnetic nanoparticle composite microspheres prepared in step 3) were separated by magnetic substances, and washed with deionized water 2 to 4 times, and then dispersed in phosphate buffer at 0 to 4°C and pH 6 to 9 for storage; the purpose is to realize a simple method of making full use of the excellent properties of non-surface-modified magnetic nanoparticles without damaging the enzyme activity. effective immobilization.

Description

A method for immobilizing enzymes by embedding magnetic nanoparticles through epoxy crosslinking

technical field

The invention relates to the technical field of enzyme immobilization, in particular to a method for immobilizing enzymes by embedding magnetic nanoparticles through epoxy crosslinking.

Background technique

Hybrid microspheres with polymer as the shell and inorganic magnetic material as the core have been one of the research hotspots in the past two decades. In this method, by modifying the surface of the magnetic particles, the polymerization reaction occurs on the surface of the magnetic particles and a reactive functional active group (such as: -OH, -COOH, -CHO, -NH2, etc.) is grafted to make the microspheres The surface has good biocompatibility, which can be directly combined with biological enzymes, cells, antibodies, drugs, metal ions and organic substances, etc., and can also be combined after chemical modification to meet different needs; there are magnetic substances inside, Under the action of an external magnetic field, it can be effectively enriched, separated, recovered and reused. However, the shell of the polymer hybrid material will increase the volume of the magnetic particles, and at the same time, it will interfere with the magnetic properties of the magnetic particles as a solid phase carrier. In addition, larger volumes of shell-coated magnetic particles and their polymers can lead to non-specific adsorption of pollutants. However, such non-specific binding is rare in small-volume, uncoated particles. M. Koneracka, P. Kopcanský, M. Timko, C.N. Ramchand, A. deSequeira, M. Trevan, Direct binding procedure of proteins and enzymes to finemagnetic particles, J. Mol. Catal. B Enzym 18 (2002) 13-18.

Enzyme immobilization methods can be divided into two categories: physical methods and chemical methods. The adsorption method usually has disadvantages such as the weak adsorption force and the loss of the enzyme from the carrier. In the embedding method, only small molecules can be embedded through the polymer network, and it is not suitable for macromolecular substrates. Due to the complex operation process and strong reaction conditions in the covalent method, the activity of the immobilized enzyme is low. The cross-linking method uses a bifunctional or multifunctional cross-linking reagent to form a covalent bond between the enzyme molecule and the cross-linking agent to achieve immobilization of the enzyme molecule. The disadvantage is that the cross-linking agent can bind to the active center of the enzyme molecule. It will cause enzyme inactivation or specificity change to a certain extent. How to make full use of the excellent properties of non-surface-modified magnetic nanoparticles to immobilize enzymes simply and effectively without damaging the enzyme activity has not yet been reported.

Contents of the invention

The invention provides a method for immobilizing enzymes by embedding magnetic nanoparticles through epoxy cross-linking, the purpose of which is to realize the simple and effective immobilization of enzymes without damaging the enzyme activity by making full use of the excellent properties of non-surface-modified magnetic nanoparticles .

The present invention is realized through the following technical solutions:

A method for immobilizing enzymes by embedding magnetic nanoparticles through epoxy crosslinking, characterized in that: comprising the steps of:

1) At a temperature of 0-4°C, add the enzyme into a phosphate buffer solution with a pH of 6-9, and stir at a speed of 10-60 rpm/min until it is completely dissolved;

2) Add superparamagnetic ferrite nanoparticles into the mixed solution prepared in step 1) at a temperature of 0-4°C, and stir at a speed of 10-60 rpm/min until uniformly mixed;

3) Add epichlorohydrin dropwise to the mixed solution prepared in step 2) at a temperature of 0-4°C, and cross-link for 24-48 h under the condition of stirring at a speed of 10-60 rpm/min , to prepare enzyme/magnetic nanoparticle composite microspheres;

4) The enzyme/magnetic nanoparticle composite microspheres prepared in step 3) are separated by magnetic substances, washed 2 to 4 times with deionized water, and then dispersed in phosphate buffer at 0-4°C and pH 6-9 for storage.

The technical improvement scheme that the present invention further solves is:

The phosphate buffer in the steps 1) and 4) is a sodium phosphate buffer with a concentration of 0.005-0.1M, or a potassium phosphate buffer.

The technical improvement scheme that the present invention further solves is:

The ferrite nanoparticles in the step 2) are prepared by co-precipitation method, and are Fe ₂ O ₄ , MnFe ₂ O ₄ , CuFe ₂ O ₄ , NiFe ₂ O ₄ , ZnFe ₂ O with a particle size of 10-20 nm. _4. MgFe ₂ O ₄ nanoparticles, or a mixture of the above nanoparticles.

The technical improvement scheme that the present invention further solves is:

The concentration ratio of the ferrite nanoparticles to the enzyme in the mixed solution prepared in the step 2) is 1 mg/ml: (0.01-1) mg/ml.

The technical improvement scheme that the present invention further solves is:

The concentration of epichlorohydrin in the solution in step 3) is 0.5-1.5M.

The technical improvement scheme that the present invention further solves is:

The enzyme immobilization rate in the enzyme/magnetic nanoparticle composite microsphere prepared in the step 3) is greater than 90%.

The technical improvement scheme that the present invention further solves is:

The enzyme in step 1) is pectinase, or amylase, or pepsin.

Compared with the prior art, the present invention has the following obvious advantages:

1. When the present invention is not wrapped by a polymer shell, the size of the magnetic nanoparticles will not increase, so their binding ability and magnetic properties will not be affected, and rapid separation can be achieved under external magnetic field conditions.

2. In the present invention, compared with the nanoparticles containing the coating layer, the unmodified magnetic nanoparticles will not undergo excessive non-specific binding without increasing the size.

3. Compared with the particles with shells, the present invention has faster and more sensitive magnetic response without the interference of outer polymer substances; the small particles without shells have larger surface area and higher binding capacity.

4. The combination of the enzyme of the present invention and the particles will not lead to structural changes and loss of activity, because the degree of cross-linking can be adjusted by adjusting the concentration of epichlorohydrin during the reaction, so that excessive cross-linking will not occur and the activity of the enzyme will not be inhibited.

5. As shown in Figure 2, the epichlorohydrin of the present invention can be covalently bonded to -NH2, -SH and -OH groups on the enzyme, and this type of cross-linking is easy to embed nanoparticles in the cross-linked enzyme structure Among them, it in turn promotes the immobilization of enzymes on the nanoparticle carrier; this crosslinking facilitates the formation of an enzyme crosslinking structure with magnetic nanoparticles as the carrier; in addition, when preparing nanoparticles by co-precipitation, the -OH formed on the particle surface It can also promote the cross-linking reaction initiated by epichlorohydrin, therefore, the immobilization effect in the present invention is more excellent.

6. The present invention is environmentally friendly, economical, and has a simple preparation process.

Description of drawings

Fig. 1 is the activity change situation of immobilized enzyme and free enzyme under 4 ℃ of storage conditions;

Figure 2 is a schematic diagram of the cross-linking reaction between enzyme and epichlorohydrin.

Detailed ways

Below in conjunction with embodiment technical solution of the present invention is described further:

Embodiment one,

The preparation steps are as follows:

1) Add 5mg of pectinase into 0.01M sodium phosphate buffer solution at pH 8 with 20rpm/min mechanical stirring at 0°C until completely dissolved;

2) Add 10 mg of MnFe ₂ O ₄ nanoparticles with a superparamagnetic particle size of 10 to 20 nm prepared by the co-precipitation method into the mixed solution prepared in step 1) of mechanical stirring at 20 rpm/min at a temperature of 0°C, until evenly mixed;

3) Add epichlorohydrin dropwise to the mixed solution prepared in step 2) at a temperature of 0°C and mechanical stirring at 20 rpm/min to a concentration of 0.6M, and cross-link under this condition After reacting for 30 h, pectinase/magnetic nanoparticle composite microspheres with pectinase immobilization rate over 90% were obtained;

4) The pectinase/magnetic nanoparticle composite microspheres prepared in step 3) were separated by magnetic substances, washed 3 times with deionized water, and then dispersed in 0.01M sodium phosphate buffer at 0°C and pH 8 for storage.

Embodiment two,

The preparation steps are as follows:

1) Add 4mg of amylase into pH5, 0.5M potassium phosphate buffer solution with mechanical stirring at 30rpm/min at a temperature of 4°C until completely dissolved;

2) Add 20 mg of CuFe ₂ O ₄ nanoparticles with a superparamagnetic particle size of 10-20 nm prepared by co-precipitation method into the mixed solution prepared in step 1) of mechanical stirring at 30 rpm/min at a temperature of 4°C, until evenly mixed;

3) Add epichlorohydrin dropwise to the mixed solution prepared in step 2) at a temperature of 4°C and mechanical stirring at 30rpm/min to a concentration of 0.5M, and exchange under this condition After 24 hours of joint reaction, amylase/magnetic nanoparticle composite microspheres with an amylase immobilization rate of more than 90% were obtained;

4) The amylase/magnetic nanoparticle composite microspheres prepared in step 3) were separated by magnetic substances, washed 3 times with deionized water, and then dispersed in 0.05M potassium phosphate buffer at 4°C and pH 5 for storage.

Embodiment three,

The preparation steps are as follows:

1) Add 5mg of pepsin into 0.01M potassium phosphate buffer solution of pH 4 with mechanical stirring at 20rpm/min at a temperature of 4°C until completely dissolved.

2) Add 30 mg of Fe ₂ O ₄ nanoparticles with a superparamagnetic particle size of 10-20 nm prepared by co-precipitation method into the mixed solution prepared in step 1) of mechanical stirring at 40 rpm/min at a temperature of 4°C, until well mixed.

3) Add epichlorohydrin dropwise to the mixed solution prepared in step 2) at a temperature of 4°C and 40rpm/min mechanical stirring to a concentration of 1.0M, and cross-link under this condition After 36 hours, pepsin/magnetic nanoparticle composite microspheres with pepsin immobilization rate over 90% were obtained.

4) The pepsin/magnetic nanoparticle composite microspheres prepared in step 3) were separated by magnetic substances, washed 3 times with deionized water, and then dispersed in 0.01M potassium phosphate buffer at 4°C and pH 4 for storage.

As shown in Figure 1, through the analysis of the changes in enzyme activity units (U) of immobilized and free pectinase, amylase and pepsin when stored at 4°C, it was found that the enzyme activities in the three examples were stored after 30 days of low temperature storage Afterwards, all were significantly higher than free enzymes.

Through the analysis of the change of enzyme activity units (U) of immobilized and free pectinase, amylase and pepsin before and after immobilization, it was found that after the enzyme activity units in the three examples were immobilized, they had no difference with the free enzyme activity units. Significant difference. As shown in the table below: category Enzyme activity before fixation (U) Enzyme activity after fixation (U) pectinase 2.07±0.03a 2.05±0.05a Amylase 26.03±0.15a 26.11±0.11a Pepsin 12.11±0.09a 12.05±0.13a

The same lowercase letters in each row of data represent no significant difference (p<0.05)

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not used to limit the protection scope of the present invention. Equivalent replacements or replacements made on the basis of the above-mentioned technical solutions all belong to the protection of the present invention. Scope, the scope of protection of the present invention shall be determined by the claims.

Claims (7)

1. a method for embedding magnetic nanoparticles immobilized enzyme by epoxy cross-linking, is characterized in that: comprise the steps: 1) At a temperature of 0-4°C, add the enzyme into a phosphate buffer solution with a pH of 6-9, and stir at a speed of 10-60 rpm/min until it is completely dissolved; 2) Add superparamagnetic ferrite nanoparticles into the mixed solution prepared in step 1) at a temperature of 0-4°C, and stir at a speed of 10-60 rpm/min until uniformly mixed; 3) Add epichlorohydrin dropwise to the mixed solution prepared in step 2) at a temperature of 0-4°C, and cross-link for 24-48 h under the condition of stirring at a speed of 10-60 rpm/min , to prepare enzyme/magnetic nanoparticle composite microspheres; 4) The enzyme/magnetic nanoparticle composite microspheres prepared in step 3) are separated by magnetic substances, washed 2 to 4 times with deionized water, and then dispersed in phosphate buffer at 0-4°C and pH 6-9 for storage. 2. A method for immobilizing enzymes by embedding magnetic nanoparticles through epoxy cross-linking according to claim 1, characterized in that: the phosphate buffer solution in the steps 1) and 4) has a concentration of 0.005-0.1M sodium phosphate buffer, or potassium phosphate buffer. 3. A method for immobilizing enzymes by epoxy cross-linking and embedding magnetic nanoparticles according to claim 1 or 2, characterized in that: the ferrite nanoparticles in step 2) are prepared by co-precipitation method, It is Fe ₂ O ₄ , MnFe ₂ O ₄ , CuFe ₂ O ₄ , NiFe ₂ O ₄ , ZnFe ₂ O ₄ , MgFe ₂ O ₄ nanoparticles with a particle diameter of 10-20 nm, or a mixture of the above nanoparticles. 4. A method for immobilizing enzymes by epoxy crosslinking and embedding magnetic nanoparticles according to claim 1 or 2, characterized in that: the ferrite nanoparticles and enzymes in the mixed solution prepared in step 2) The concentration ratio is 1 mg/ml: (0.01~1) mg/ml. 5. A method for immobilizing enzymes by epoxy crosslinking and embedding magnetic nanoparticles according to claim 1 or 2, characterized in that: the concentration of epichlorohydrin in the solution in step 3) is 0.5 ~1.5M. 6. A method for immobilizing enzymes by epoxy crosslinking and embedding magnetic nanoparticles according to claim 1 or 2, characterized in that: the enzyme in the enzyme/magnetic nanoparticle composite microspheres prepared in step 3) The immobilization rate is greater than 90%. 7. A method for immobilizing enzymes by embedding magnetic nanoparticles through epoxy cross-linking according to claim 1 or 2, characterized in that: the enzyme in step 1) is pectinase, or amylase, or pepsin.