CN112695027B - An immobilized enzyme nanofiber membrane that realizes synchronization of enzymatic hydrolysis reaction and product purification and its preparation and application - Google Patents

Abstract

The invention belongs to the technical field of immobilized enzymes, and discloses an immobilized enzyme nanofiber membrane for realizing synchronous enzymolysis and product purification, and preparation and application thereof. The method comprises the following steps: 1) Reacting enzyme with MOFs material with amino group to obtain NH of immobilized enzyme ₂ -MOFs; the enzyme is protease and/or amylase; 2) NH of immobilized enzyme ₂ -preparing an electrostatic spinning solution by MOFs and a high molecular polymer; 3) And carrying out electrostatic spinning on the electrostatic spinning solution to obtain the immobilized enzyme nanofiber membrane. The nanofiber membrane has the advantages of stable immobilized enzyme, high enzyme activity recovery rate, multiple holes, high strength, simplicity and high efficiency in operation and the like. In the enzymolysis reaction of the nanofiber membrane, the separation and purification of the enzymolysis product are realized through the membrane, so that the enzymolysis reaction and the product purification are synchronously carried out. The immobilized enzyme nanofiber membrane is used for preparing polypeptide and/or oligosaccharide.

Description

An immobilized enzyme nanofiber membrane that realizes synchronization of enzymatic hydrolysis reaction and product purification and Preparation and application

Technical field

The invention belongs to the technical field of immobilized enzymes, and specifically relates to an immobilized enzyme nanofiber membrane that realizes synchronization of enzymatic hydrolysis reaction and product purification and its preparation method and application. The immobilized enzyme nanofiber membrane is used to prepare polypeptides or oligosaccharides. The present invention utilizes immobilized enzyme nanocellulose to realize simultaneous enzymatic hydrolysis reaction and product purification.

Background technique

When biological enzymes catalyze reactions, free enzymes are prone to denaturation, inactivation, and instability. If they are immobilized to retain the catalytic activity of the enzyme, their stability will be improved, and solid-liquid separation will be easier to achieve, thereby realizing the enzyme's catalytic activity. reuse. There are many methods for immobilizing enzymes, such as adsorption, cross-linking, encapsulation and carrier binding. However, these methods all have the disadvantages of low enzyme binding strength or low enzyme loading, resulting in low enzyme recovery or Enzyme activity is low. Patent CN103756990A discloses a papain preparation and immobilization method. Cellulose nanocrystals are mixed with papain solution and polyacrylamide solution. It has the advantages of recyclability, easy operation, and mild conditions. However, it uses electrostatic self-assembly. Immobilized enzymes result in insufficient binding strength and difficulty in separation and recovery. Patent application CN108396023A discloses a grinding method to prepare magnetic MOF materials and use them for immobilizing enzymes. An immobilized enzyme material that can be recycled is obtained. However, it still has the defects of insufficient enzyme loading and low binding strength. Although the binding strength of the enzyme can be improved through chemical cross-linking, the void ratio is reduced, preventing the enzyme from fully contacting the substrate, and reducing the catalytic effect of the enzyme.

Current immobilized enzyme-catalyzed reactions still use a two-step method, that is, the immobilized enzyme and the substrate are mixed and reacted, and then the enzyme and product are separated and purified. However, there are no reports on immobilized enzymes in which the reaction and product purification are performed simultaneously.

Contents of the invention

In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide an immobilized enzyme nanofiber membrane that can synchronize enzymatic hydrolysis reaction and product purification and a preparation method thereof.

Another object of the present invention is to provide the application of the above-mentioned immobilized enzyme nanofiber membrane. The present invention utilizes immobilized enzyme nanocellulose to realize simultaneous enzymatic hydrolysis reaction and product purification. In the present invention, immobilized enzyme nanocellulose is used to rapidly prepare polypeptides or oligosaccharides.

The object of the present invention is achieved through the following technical solutions:

A method for preparing an immobilized enzyme nanofiber membrane, including the following steps:

1) React the enzyme with MOFs materials containing amino groups to obtain NH ₂ -MOFs with immobilized enzyme; the enzyme is protease and/or amylase;

2) Prepare an electrospinning solution by mixing NH ₂ -MOFs with immobilized enzyme and high molecular polymer;

3) Electrospinning the electrospinning solution to obtain an immobilized enzyme nanofiber membrane.

The mass ratio of the enzyme described in step 1) to the MOFs material with amino groups is 1: (1-3).

The reaction described in step 1) is carried out in a phosphate buffer with a pH of 5 to 8; the amount of the phosphate buffer of a pH of 5 to 8 is 1 to 1.5 times the mass of the enzyme.

The conditions for the reaction described in step 1) are 25-40°C for 24-48 hours.

In step 2), the mass ratio of the NH ₂ -MOFs immobilized enzyme to the polymer is (1-10): (10-15).

The high molecular polymer in step 2) is one or more of polyacrylonitrile, polyurethane, polylactic acid, and polycaprolactone; the molecular weight of the high molecular polymer is 1000 to 1500 kDa.

The specific steps of step 2) are to mix the NH ₂ -MOFs with immobilized enzyme and high molecular polymer in an organic solvent, and process it with high-pressure microjet to obtain an electrospinning solution;

The organic solvent described in step 2) is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and chloroform;

The mass concentration of immobilized enzyme NH ₂ -MOFs and high molecular polymer in the electrospinning solution is 8% to 12%.

The conditions for high-pressure microjet flow are: the pressure is 5000-30000 PSI, and the number of cycles is 3 to 8 times.

The electrospinning solution described in step 2) is heated and filtered before electrospinning. The heat treatment conditions are 40-50°C for 50-70 minutes.

The conditions for electrospinning in step 3) are: flow rate 1~2ml/h, voltage 12~15kv, and receiving distance 10~20cm.

The MOFs material with amino groups described in step 1) is an aluminum-based MOFs material with amino groups.

Aluminum-based MOFs materials with amino groups are obtained by reacting 2-aminoterephthalic acid with an aluminum source.

The MOFs material with an amino group is specifically prepared by the following method: performing a solvothermal reaction between 2-aminoterephthalic acid and an aluminum source in a mixed solvent, refluxing, filtering, and drying to obtain the MOFs material with an amino group. The aluminum source is aluminum chloride.

The mass ratio of the 2-aminoterephthalic acid to the aluminum source is 1: (1.5-3).

The mixed solvent is an organic solvent and water; the volume ratio of the organic solvent to water is 1: (1-2).

The organic solvent is at least one of N,N-dimethylformamide, nitrogen methylpyrrolidone and dimethyl sulfoxide.

The mass concentration of 2-aminoterephthalic acid and aluminum source in the mixed solvent is 30% to 40%.

The conditions of the solvothermal reaction are 150-200°C for 12-24 hours.

The reflux conditions are refluxing at 150-180°C for 8-10 hours.

The reflux is performed in an organic solvent; the organic solvent is at least one of N,N-dimethylformamide, nitrogen methylpyrrolidone, and dimethyl sulfoxide.

After the solvothermal reaction is completed, the solvent in the system is removed, an organic solvent is added, and then refluxed.

The added amount of the organic solvent is 3 to 5 times the mass of the product after removing the solvent in the system.

The drying conditions are 30-60°C for 24-36 hours.

The immobilized enzyme nanofiber membrane is prepared by the above method.

The application of the immobilized enzyme nanofiber membrane in the simultaneous enzymatic hydrolysis reaction and enzymatic hydrolysis product purification is to use the immobilized enzyme nanofiber membrane to prepare the enzymatic hydrolysis product, so as to realize the simultaneous enzymatic hydrolysis reaction and enzymatic hydrolysis product purification;

Specifically, the enzyme substrate is dispersed in a phosphate buffer, placed on an immobilized enzyme nanofiber membrane for reaction, the solution that passes through the membrane is collected, and freeze-dried to obtain a purified enzyme product; the enzyme substrate is protein and/or Starch; enzymatic hydrolysis products are polypeptides and/or oligosaccharides.

The conditions of the reaction are room temperature reaction for 6 to 24 hours; the phosphate buffer is a phosphate buffer of pH 5 to 8; the mass ratio of the enzyme substrate to the phosphate buffer of pH 5 to 8 is 1: (5 to 30).

After the enzymatic hydrolysis reaction of the immobilized fiber membrane of the present invention, the molecular weight of the enzymatic hydrolysis product obtained is 500-3000 Da.

If the fiber membrane is first prepared by electrospinning, and then the fiber membrane is mixed with the enzyme for immobilization, this method can only obtain the adsorbed and immobilized enzyme or the membrane-coated enzyme. The former enzyme is prone to dissociation and instability, and the latter enzyme and the substrate The substances cannot fully contact and react; if the enzyme and polymer are directly electrospun, there are difficulties in that the enzyme is easily inactivated and has poor dispersion, resulting in poor effectiveness. The method of the present invention ensures that the enzyme is stably immobilized without being inactivated, and can fully contact and react with the substrate. It can react on one side of the membrane and collect the reaction product on the other side to achieve synchronization of enzymatic hydrolysis reaction and product purification.

Principle of the invention:

The present invention uses the amino group on NH ₂ -MOFs and the carboxyl group in protease to carry out dehydration condensation, and completes the immobilization of the enzyme through the action of amide bonds; it uses high-pressure micro-jet technology to combine the NH ₂ -MOFs material of the immobilized enzyme with The high molecular polymer is prepared into an electrospinning precursor. Finally, the spinning conditions and parameters are adjusted through electrospinning technology, and the electrospinning precursor is electrospun to obtain a composite nanofiber membrane with adjustable network pore size.

Compared with the existing technology, the present invention has the following advantages and effects:

(1) The nanofiber membrane of the present invention has the advantages of stable immobilized enzyme, porousness, high strength, easy and efficient operation, and high recovery rate.

(2) During the enzymatic hydrolysis reaction of the nanofiber membrane of the present invention, the enzymatic hydrolysis product passes through the membrane to realize the separation and purification of the product. Therefore, the enzymatic hydrolysis reaction and product purification are performed simultaneously.

(3) The method of the present invention can be used to realize that the network pore size of the nanofiber membrane can be adjusted, so that the separation and purification of products with different molecular weights can be selectively achieved according to the molecular size.

Description of the drawings

Figure 1 is a scanning electron microscope image of the immobilized protease nanofiber membrane obtained in Example 1.

Detailed ways

The present invention will be described in further detail below with reference to examples, but the implementation of the present invention is not limited thereto.

Example 1

(1) Dissolve 1g 2-aminoterephthalic acid and 1.5g aluminum chloride in a mixed solvent of dimethylformamide and deionized water (volume ratio 1:1) to prepare a solution with a mass concentration of 30%. React at 150°C for 24 hours; filter, add 3 times its mass of dimethylamide to the filter residue, reflux at 150°C for 8 hours, filter, and dry the filter residue at 30°C for 36 hours to obtain MOFs materials with amino groups (NH ₂ -MOFs).

(2) Disperse 10g of papain in 10g of pH 8 phosphate buffer, mix with 10g of NH ₂ -MOFs material, and react at 25°C for 48 hours to obtain NH ₂ -MOFs of immobilized enzyme.

(3) 10g of enzyme-immobilized NH ₂ -MOFs and 10g of polyacrylonitrile (molecular weight: 1000kDa) were dissolved in dimethylamide to form an 8% solution. Use a high-pressure microorganism with a pressure of 5000PSI and a cycle number of 8 times. The jet is prepared into an electrospinning solution.

(4) Heat the electrospinning solution to 40°C for 70 minutes (heating is to maintain the stability of the solution), filter, and electrospinning the filtrate with a flow rate of 1ml/h, a voltage of 12kv, and a receiving distance of 10cm. The nanofibers are collected to obtain the immobilized enzyme nanofiber membrane.

(5) Disperse 10g of papaya protein powder in 50g of pH 8 phosphate buffer, place it on the immobilized papain nanofiber membrane and react at room temperature for 6 hours. The solution that permeates the membrane is collected and freeze-dried to obtain purified papaya polypeptide. The immobilized enzyme nanofiber membrane prepared in this example has a pore size of 1 to 2 nm, an enzyme loading amount of 25%, an enzyme activity recovery rate of 96%, and a 5-day enzyme activity recovery rate of 93%.

Example 2

(1) Dissolve 1g 2-aminoterephthalic acid and 3g aluminum chloride in a mixed solvent of dimethylformamide and deionized water (volume ratio 1:2) to prepare a solution with a mass concentration of 40%, 200 React at ℃ for 12 hours; filter, add 5 times its mass of dimethylamide to the filter residue, reflux at 180°C for 8 hours, filter, and dry the filter residue at 60°C for 24 hours to obtain MOFs materials with amino groups (NH ₂ -MOFs).

(2) Disperse 10g of α-amylase in 15g of pH 5 phosphate buffer, mix with 30g of NH ₂ -MOFs material, and react at 40°C for 24 hours to obtain NH ₂ -MOFs of immobilized enzyme.

(3) 10g of enzyme-immobilized NH ₂ -MOFs and 15g of polyurethane (molecular weight: 1500kDa) were dissolved in dimethylformamide to form a 12% solution, using a high-pressure microjet with a pressure of 30,000 PSI and a cycle count of 3 times. Prepare an electrospinning liquid solution.

(4) Heat the electrospinning solution to 50°C for 50 minutes, filter, and perform electrospinning on the filtrate with a flow rate of 2 ml/h, a voltage of 15 kv, and a receiving distance of 20 cm. The nanofibers are collected to obtain the immobilized enzyme nanofiber membrane.

(5) Disperse 10g of corn starch in 300g of pH 5 phosphate buffer, place it on the immobilized α-amylase nanofiber membrane and react for 24 hours. Collect the solution that permeates the membrane and freeze-dry it to obtain purified corn oligosaccharides. . The immobilized enzyme nanofiber membrane prepared in this example has a pore size of 5 to 7 nm, an enzyme loading amount of 10%, and an enzyme activity recovery rate of 92%.

Example 3

(1) Dissolve 1g 2-aminoterephthalic acid and 2g aluminum chloride in a mixed solvent of dimethylformamide and deionized water (volume ratio 1:1.5) to prepare a solution with a mass concentration of 35%, 180 React at ℃ for 16 hours; filter, add 4 times its mass of dimethylformamide to the filter residue, reflux at 160°C for 9 hours, filter, and dry the filter residue at 50°C for 28 hours to obtain MOFs materials with amino groups (NH ₂ -MOFs). The aluminum-based NH ₂ -MOFs selected in the present invention have more ordered amino coordination sites, which are helpful for orderly immobilization of proteases, and the aluminum-based MOF structure is more tailorable than the ZIF structure.

(2) Disperse 10g of alkaline protease in 12g of pH 7 phosphate buffer, mix with 20g of NH ₂ -MOFs material, and react at 30°C for 36 hours to obtain NH ₂ -MOFs of immobilized enzyme.

(3) 10g of enzyme-immobilized NH ₂ -MOFs and 12g of polycaprolactone (molecular weight: 1000kDa) were dissolved in dimethylformamide to form a 10% solution. The pressure was 10000PSI and the number of cycles was 5. High-voltage microjet is used to prepare electrospinning liquid solution.

(4) Heat the electrospinning solution to 45°C for 60 minutes, filter, and perform electrospinning on the filtrate with a flow rate of 1.5ml/h, a voltage of 13kv, and a receiving distance of 15cm. The nanofibers are collected to obtain the immobilized enzyme nanofiber membrane.

(5) Disperse 10g of almond protein powder in 100g of pH 7 phosphate buffer, place it on the immobilized alkaline protease nanofiber membrane and react for 12 hours. Collect the solution that permeates the membrane and freeze-dry it to obtain purified almond polypeptide. The immobilized enzyme nanofiber membrane prepared in this example has a pore size of 3 to 5 nm, an enzyme loading amount of 15%, and an enzyme activity recovery rate of 94%.

Comparative Example 1 (change the order of enzyme immobilization)

First, the solution of NH ₂ -MOFs material and polymer is electrospun, and the obtained fiber membrane is soaked in the enzyme solution and fixed. The conditions are the same as in Example 2.

Results: The loading amount of enzyme was 4%, and the recovery rates of enzyme activity at 0 and 5 days were 90% and 30% respectively, indicating that the effect of electrospinning first and then adsorbing and immobilizing the enzyme was poor.

Comparative Example 2 (increase the dosage of enzyme)

The difference between this comparative example and Example 2 is that the dosage of enzyme is 40g.

Results: The loading amount of enzyme was 12%, and the recovery rate of enzyme activity on 0 days and 5 days was 28% and 25% respectively, indicating that increasing the amount of enzyme would not change much the amount of enzyme loading, but the recovery rate of enzyme activity was low because of the large amount of enzyme. Resulting in incomplete fixation.

Comparative Example 3 (increasing the amount of NH ₂ -MOFs for immobilized enzyme)

The difference between this comparative example and Example 2 is that the amount of NH ₂ -MOFs for immobilized enzyme is 30g.

Results: The enzyme loading amount was 15%, and the enzyme activity recovery rates on 0 days and 5 days were 75% and 60% respectively, indicating that increasing the amount of NH ₂ -MOFs for immobilized enzymes will increase the enzyme loading amount. The recovery rate of enzyme activity is low because some of the NH ₂ -MOFs immobilized enzyme cannot be immobilized by the fiber membrane.

Comparative Example 4 (high-speed homogenization instead of high-pressure microjet)

The difference between this comparative example and Example 2 is that high-speed homogenization is used instead of high-pressure microjet, 20000rpm, 15min.

Results: The enzyme loading amount was 8%, and the enzyme activity recovery rates on 0 days and 5 days were 85% and 80% respectively, indicating that both the enzyme loading amount and the enzyme activity recovery rate were reduced during high-speed homogenization. This is caused by high-speed homogenization. The NH ₂ -MOFs that immobilize enzymes aggregate during continued operation, and the effect becomes worse.

Performance Testing:

(1) Electron microscope observation of the immobilized papain nanofiber membrane prepared in Example 1

Experimental method: Cut the nanofiber membrane into a size suitable for the size of the instrument sample stage, then bond it to the sample stage with conductive glue, spray gold, and place it in a Hitachi SU-8220 scanning electron microscope for observation.

Experimental results: Figure 1 is a scanning electron microscope image of the immobilized protease nanofiber membrane obtained in Example 1. It can be seen from the figure that the immobilized papain nanofiber membrane prepared in Example 1 has a uniform mesh, which is conducive to the penetration of the reaction product. The nanofiber has no attached particles, indicating that the enzyme and polymer material are well integrated and firmly bonded without falling off.

The above-described embodiments of the present invention are only examples to clearly illustrate the present invention, but are not intended to limit the implementation of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (6)

1. A method for preparing an immobilized enzyme nanofiber membrane, which is characterized in that it includes the following steps: 1) React the enzyme with the MOFs material containing amino groups to obtain NH ₂ -MOFs with immobilized enzyme; the enzyme is protease or amylase; the protease is one of papain and alkaline protease; the starch The enzyme is α-amylase; 2) Prepare an electrospinning solution by mixing NH ₂ -MOFs with immobilized enzyme and high molecular polymer; 3) Electrospinning the electrospinning solution to obtain an immobilized enzyme nanofiber membrane; The mass ratio of the enzyme and the amino-containing MOFs material in step 1) is 1: (1-3); the amino-containing MOFs material is specifically prepared by the following method: combining 2-aminoterephthalic acid and The aluminum source is subjected to a solvothermal reaction in a mixed solvent, refluxed, filtered, and dried to obtain MOFs materials with amino groups; the aluminum source is aluminum chloride; the mass of 2-aminoterephthalic acid and the aluminum source in the mixed solvent The ratio is 1: (1.5~3); the solvothermal reaction conditions are 150~200°C for 12~24h; the reflux conditions are 150~180°C reflux for 8~10h; In step 2), the mass ratio of NH ₂ -MOFs and polymer for immobilized enzyme is (1～10): (10～15); The specific steps of step 2) are to mix the NH ₂ -MOFs with immobilized enzyme and a high-molecular polymer in an organic solvent, and use high-pressure microjet for treatment to obtain an electrospinning solution; the high-molecular polymer is polyacrylonitrile. , one of polyurethane, polylactic acid, and polycaprolactone; the mass concentration of NH ₂ -MOFs and polymers for immobilized enzymes in the electrospinning solution is 8% to 12%; the conditions for high-pressure microjet are: The pressure is 5000-30000 PSI, and the number of cycles is 3 to 8 times; The conditions for electrospinning described in step 3) are: flow rate 1-2mL/h, voltage 12-15kv, and receiving distance 10-20cm. 2. The preparation method of immobilized enzyme nanofiber membrane according to claim 1, characterized in that: In the specific preparation step of step 2), the organic solvent is N,N-dimethylformamide. 3. The preparation method of immobilized enzyme nanofiber membrane according to claim 1, characterized in that: The reaction described in step 1) is carried out in a phosphate buffer with a pH of 5 to 8; The reaction conditions described in step 1) are 25-40°C for 24-48 hours; The molecular weight of the high molecular polymer described in step 2) is 1000-1500kDa. 4. The preparation method of immobilized enzyme nanofiber membrane according to claim 3, characterized in that: In the preparation of MOFs materials with amino groups, the mixed solvent is an organic solvent and water; the volume ratio of the organic solvent to water is 1: (1~2); The organic solvent is N,N-dimethylformamide; The reflux is performed in an organic solvent; the organic solvent is N,N-dimethylformamide. 5. An immobilized enzyme nanofiber membrane obtained by the preparation method according to any one of claims 1 to 4. 6. The application of the immobilized enzyme nanofiber membrane according to claim 5, characterized in that: the immobilized enzyme nanofiber membrane is used to prepare enzymatic hydrolysis products to achieve simultaneous enzymatic hydrolysis reaction and enzymatic hydrolysis product purification; specifically: Disperse the enzyme substrate in phosphate buffer, place it on the immobilized enzyme nanofiber membrane for reaction, collect the solution that penetrates the membrane, and freeze-dry to obtain the purified enzyme product; The enzyme substrate is protein or starch; the enzymatic hydrolysis product is polypeptide or oligosaccharide.