CN112695027B - Immobilized enzyme nanofiber membrane for realizing synchronous enzymolysis reaction and product purification, and preparation and application thereof - Google Patents
Immobilized enzyme nanofiber membrane for realizing synchronous enzymolysis reaction and product purification, and preparation and application thereof Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of immobilized enzymes, and particularly relates to an immobilized enzyme nanofiber membrane for realizing synchronous enzymolysis reaction and product purification, and a preparation method and application thereof. The immobilized enzyme nanofiber membrane is used for preparing polypeptide or oligosaccharide. The invention can realize synchronous enzymolysis reaction and product purification by utilizing immobilized enzyme nanocellulose.
Background
When the biological enzyme catalyzes the reaction, the free enzyme is easy to denature and inactivate and unstable, for example, the free enzyme is immobilized, so that the catalytic activity of the enzyme is maintained, the stability of the enzyme is improved, the solid-liquid separation is easy, and the repeated use of the enzyme is realized. There are various methods for immobilizing enzymes, such as adsorption, crosslinking, embedding and carrier binding, but these methods have the disadvantage of not having high enzyme binding strength or too low enzyme loading, resulting in low enzyme recovery or enzyme activity. Patent CN103756990a discloses a papain preparation and an immobilization method, wherein cellulose nanocrystals are mixed with a papain solution and a polyacrylamide solution, which has the advantages of recoverability, simple operation, mild conditions and the like, but the enzyme is immobilized through electrostatic self-assembly, so that the bonding strength of the enzyme is insufficient, and the separation and recovery are not easy. Patent application CN108396023a discloses a grinding method for preparing a magnetic MOF material and for enzyme immobilization, which yields an immobilized enzyme material that can be recycled, but it still has the drawbacks of insufficient enzyme loading and low binding strength. Although the binding strength of the enzyme can be improved by the chemical crosslinking method, the porosity is reduced, so that the enzyme cannot be fully contacted with the substrate, and the catalytic effect of the enzyme is reduced.
The existing immobilized enzyme catalytic reaction still adopts a two-step method, namely, the immobilized enzyme and a substrate are mixed to react and then are separated and purified, and the immobilized enzyme which is synchronously carried out by the reaction and the product purification is not reported.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide an immobilized enzyme nanofiber membrane for realizing synchronous enzymolysis reaction and product purification and a preparation method thereof.
Another object of the present invention is to provide the use of the immobilized enzyme nanofiber membrane. The invention realizes synchronous enzymolysis reaction and product purification by utilizing immobilized enzyme nanocellulose. The invention uses immobilized enzyme nanocellulose to rapidly prepare polypeptide or oligosaccharide.
The aim of the invention is achieved by the following technical scheme:
the preparation method of the immobilized enzyme nanofiber membrane 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 mass ratio of the enzyme to the MOFs material with amino groups in the step 1) is 1: (1-3).
The reaction in step 1) is carried out in phosphate buffer with pH of 5-8; the dosage of the phosphate buffer solution with the pH value of 5 to 8 is 1 to 1.5 times of the mass of the enzyme.
The reaction conditions in the step 1) are 25-40 ℃ for 24-48 h.
NH of immobilized enzyme in step 2) 2 The mass ratio of MOFs to high molecular polymer is (1-10): (10-15).
The high molecular polymer in the step 2) is one or more of polyacrylonitrile, polyurethane, polylactic acid and polycaprolactone; the molecular weight of the high molecular polymer is 1000-1500 kDa.
The specific step of step 2) is to immobilize NH of the enzyme 2 Mixing MOFs and a high molecular polymer in an organic solvent, and treating by adopting high-pressure microjet to obtain an electrostatic spinning solution;
the organic solvent in the step 2) is more than one of N, N-dimethylformamide, dimethyl sulfoxide and chloroform;
NH of immobilized enzyme 2 The mass concentration of the MOFs and the high molecular polymer in the electrostatic spinning solution is 8-12%.
The conditions of the high pressure microjet were: the pressure is 5000-30000PSI, and the cycle times are 3-8.
And 2) heating the electrostatic spinning solution in the step 2) before electrostatic spinning, and filtering. The heating treatment condition is that the temperature is 40-50 ℃ and the heating treatment is kept for 50-70 min.
The conditions of the electrostatic spinning in the step 3) are as follows: the flow rate is 1-2 ml/h, the voltage is 12-15 kv, and the receiving distance is 10-20 cm.
The MOFs material with amino groups in the step 1) is an aluminum-based MOFs material with amino groups.
The amino-bearing aluminum-based MOFs material is obtained by reacting 2-amino terephthalic acid with an aluminum source.
The MOFs material with the amino group is prepared by the following steps: and carrying out solvothermal reaction on the 2-amino terephthalic acid and an aluminum source in a mixed solvent, refluxing, filtering and drying to obtain the MOFs material with amino. The aluminum source is aluminum chloride.
The mass ratio of the 2-amino terephthalic 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 the water is 1: (1-2).
The organic solvent is more than one of N, N-dimethylformamide, azomethyl pyrrolidone and dimethyl sulfoxide.
The mass concentration of the 2-amino terephthalic acid and the aluminum source in the mixed solvent is 30-40%.
The solvothermal reaction condition is 150-200 ℃ for 12-24 h.
The reflux condition is that the reflux is carried out for 8-10 hours at 150-180 ℃.
The refluxing is carried out in an organic solvent; the organic solvent is more than one of N, N-dimethylformamide, azomethyl pyrrolidone and dimethyl sulfoxide.
After the solvothermal reaction is completed, removing the solvent in the system, adding an organic solvent, and then refluxing.
The addition amount of the organic solvent is 3-5 times of the mass of the product after the solvent in the system is removed.
The drying condition is 30-60 ℃ for 24-36 h.
The immobilized enzyme nanofiber membrane is prepared by the method.
The application of the immobilized enzyme nanofiber membrane in the synchronous enzymolysis reaction and enzymolysis product purification is that immobilized enzyme nanocellulose is used for preparing an enzymolysis product, so that the synchronous enzymolysis reaction and enzymolysis product purification is realized;
dispersing an enzyme substrate in a phosphate buffer solution, placing the phosphate buffer solution on an immobilized enzyme nanofiber membrane for reaction, collecting a solution penetrating through the membrane, and freeze-drying to obtain a purified enzyme product; the enzyme substrate is protein and/or starch; the enzymolysis product is polypeptide and/or oligosaccharide.
The reaction condition is that the reaction is carried out for 6 to 24 hours at room temperature; the phosphate buffer solution is a phosphate buffer solution with the pH value of 5-8; the mass ratio of the enzyme substrate to the phosphate buffer with the pH value of 5-8 is 1: (5-30).
After the enzymolysis reaction, the molecular weight of the obtained enzymolysis product of the immobilized fiber membrane is 500-3000 Da.
If the fiber membrane is prepared by electrostatic spinning, then the fiber membrane is mixed with enzyme for fixation, the method can only obtain enzyme for adsorbing and fixing the enzyme or enzyme coated by the membrane, the former enzyme is easy to dissociate and unstable, and the latter enzyme and the substrate cannot sufficiently contact and react; if the enzyme and the polymer are directly subjected to electrostatic spinning, the enzyme is easy to deactivate and the dispersibility is poor, so that the effectiveness is poor. The method of the invention ensures that the enzyme is stably immobilized and not deactivated, can fully contact and react with the substrate, and can react on one side of the membrane and collect the reaction product on the other side, thereby realizing the synchronization of enzymolysis reaction and product purification.
The principle of the invention is as follows:
the invention utilizes NH 2 The amino groups on MOFs are dehydrated and condensed with carboxyl groups in protease, and enzyme immobilization is completed through the action of amide bonds; NH of immobilized enzyme by high-pressure micro-jet technology 2 Preparing an electrostatic spinning precursor by the MOFs material and a high polymer, and finally, adjusting spinning conditions and spinning parameters by an electrostatic spinning technology, and electrospinning the electrostatic spinning precursor to obtain the composite nanofiber membrane with the adjustable network aperture.
Compared with the prior art, the invention has the following advantages and effects:
(1) The nanofiber membrane has the advantages of stable immobilized enzyme, multiple holes, high strength, simple and efficient operation, high recovery rate and the like.
(2) 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.
(3) The method can realize the adjustable network aperture of the nanofiber membrane, thereby selectively realizing the separation and purification of products with different molecular weights according to the molecular size.
Drawings
FIG. 1 is a scanning electron microscope image of the immobilized protease nanofiber membrane obtained in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
Example 1
(1) 1g of 2-amino terephthalic acid and 1.5g of aluminum chloride are dissolved in a mixed solvent of dimethylformamide and deionized water (volume ratio is 1:1) to prepare a solution with the mass concentration of 30%, and the solution is reacted for 24 hours at 150 ℃; filtering, adding 3 times of dimethylamide into the residue, refluxing at 150deg.C for 8 hr, filtering, and drying the residue at 30deg.C for 36 hr to obtain MOFs material (NH) with amino group 2 -MOFs)。
(2) 10g of papain was dispersed in 10g of phosphate buffer pH8, with 10gNH 2 Mixing MOFs materials, and reacting at 25 ℃ for 48 hours to obtain NH of immobilized enzyme 2 -MOFs。
(3) NH of immobilized enzyme 10g 2 MOFs and 10g of polyacrylonitrile (molecular weight 1000 kDa) were dissolved in dimethylformamide to prepare an 8% solution, and the solution was prepared into an electrostatic spinning solution by high-pressure microfluidics with a pressure of 5000PSI and a cycle number of 8.
(4) The electrospinning solution was heated to 40℃for 70min (heating was performed to maintain the stability of the solution), filtered, and the filtrate was electrospun at a flow rate of 1ml/h at a voltage of 12kv for a receiving distance of 10cm. Collecting the nanofiber to obtain the immobilized enzyme nanofiber membrane.
(5) Dispersing 10g of papain powder in 50g of phosphate buffer with pH of 8, placing on an immobilized papain nanofiber membrane, reacting for 6 hours at room temperature, collecting a solution penetrating through the membrane, and freeze-drying to obtain the purified papain polypeptide. The immobilized enzyme nanofiber membrane prepared in the embodiment has the pore diameter of 1-2 nm, the enzyme loading amount of 25%, the enzyme activity recovery rate of 96% and the enzyme activity recovery rate of 93% in 5 days.
Example 2
(1) 1g of 2-amino terephthalic acid and 3g of aluminum chloride are dissolved in a mixed solvent of dimethylformamide and deionized water (volume ratio is 1:2) to prepare a solution with the mass concentration of 40%, and the solution is reacted for 12 hours at 200 ℃; filtering, adding 5 times of dimethylformamide into the residue, refluxing at 180deg.C for 8 hr, filtering, and drying the residue at 60deg.C for 24 hr to obtain MOFs material (NH) with amino group 2 -MOFs)。
(2) 10g of alpha-amylase was dispersed in 15g of phosphate buffer pH5, with 30gNH 2 Mixing MOFs materials, and reacting at 40 ℃ for 24 hours to obtain NH of immobilized enzyme 2 -MOFs。
(3) NH of immobilized enzyme 10g 2 MOFs and 15g of polyurethane (molecular weight 1500 kDa) were dissolved in dimethylformamide to prepare a 12% solution, and the solution was prepared as an electrostatic spinning solution using high pressure microfluidics at a pressure of 30000PSI and a number of cycles of 3.
(4) Heating the electrostatic spinning solution to 50 ℃ for 50min, filtering, and carrying out electrostatic spinning on the filtrate at the flow rate of 2ml/h and the voltage of 15kv for a receiving distance of 20cm. Collecting the nanofiber to obtain the immobilized enzyme nanofiber membrane.
(5) 10g of corn starch is dispersed in 300g of phosphate buffer solution with pH5, and is placed on an immobilized alpha-amylase nanofiber membrane for reaction for 24 hours, and the solution which penetrates through the membrane is collected and freeze-dried, so that the purified corn oligosaccharide can be obtained. The immobilized enzyme nanofiber membrane prepared in the embodiment has the pore diameter of 5-7 nm, the enzyme loading amount of 10% and the enzyme activity recovery rate of 92%.
Example 3
(1) 1g of 2-amino terephthalic acid and 2g of aluminum chloride are dissolved in a mixed solvent of dimethylformamide and deionized water (volume ratio is 1:1.5) to prepare a solution with the mass concentration of 35%, and the solution is reacted for 16 hours at 180 ℃; filtering, and adding 4 times of dimethyl into the filter residueRefluxing dimethylformamide at 160 ℃ for 9h, filtering, and drying filter residue at 50 ℃ for 28h to obtain MOFs material (NH) with amino groups 2 -MOFs). The invention selects NH of aluminum base 2 MOFs have more ordered amino coordination sites, which help to immobilize proteases in order, and aluminum-based MOF structures are better tailorable than ZIF structures.
(2) 10g of alkaline protease was dispersed in 12g of phosphate buffer pH7, and 20. 20gNH 2 Mixing MOFs materials, and reacting at 30 ℃ for 36h to obtain NH of immobilized enzyme 2 -MOFs。
(3) NH of immobilized enzyme 10g 2 MOFs and 12g of polycaprolactone (molecular weight 1000 kDa) were dissolved in dimethylformamide to prepare a 10% solution, and the solution was prepared into an electrostatic spinning solution by high-pressure microfluidics at a pressure of 10000PSI and a cycle number of 5.
(4) The electrostatic spinning solution was heated to 45℃for 60min, filtered, and the filtrate was electrospun at a flow rate of 1.5ml/h and a voltage of 13kv at a receiving distance of 15cm. Collecting the nanofiber to obtain the immobilized enzyme nanofiber membrane.
(5) Dispersing 10g of almond protein powder in 100g of phosphate buffer with pH7, placing the almond protein powder on an immobilized alkaline protease nanofiber membrane for reaction for 12 hours, collecting a solution penetrating through the membrane, and freeze-drying to obtain purified almond polypeptide. The immobilized enzyme nanofiber membrane prepared in the embodiment has the pore diameter of 3-5 nm, the enzyme loading amount of 15% and the enzyme activity recovery rate of 94%.
Comparative example 1 (changing the order in which enzymes are immobilized)
NH is first put into 2 The MOFs material was electrospun with a solution of polymer and the resulting fibrous membrane was immobilized by immersing in an enzyme solution under the same conditions as in example 2.
Results: enzyme loading is 4%, and enzyme activity recovery rates in 0 day and 5 days are 90% and 30%, respectively, which shows that the effects of electrostatic spinning and enzyme adsorption and fixation are poor.
Comparative example 2 (increasing the amount of enzyme)
This comparative example differs from example 2 in that: the amount of enzyme used was 40g.
Results: enzyme loading is 12%, enzyme activity recovery rates in 0 day and 5 days are 28% and 25%, respectively, which shows that the enzyme loading is increased, the change of enzyme loading is small, but the enzyme activity recovery rate is low, and the fixation is incomplete due to the large enzyme loading.
Comparative example 3 (increase of NH of immobilized enzyme) 2 -the amount of MOFs
This comparative example differs from example 2 in that: NH of immobilized enzyme 2 The amount of MOFs was 30g.
Results: enzyme loading is 15%, enzyme activity recovery rates are 75% and 60% in 0 day and 5 day respectively, which shows that NH of immobilized enzyme is increased 2 The amount of MOFs used, although the enzyme loading was increased, the recovery of enzyme activity was lower because of the NH of the partially immobilized enzyme 2 MOFs cannot be immobilized by fibrous membranes.
Comparative example 4 (high speed homogenization instead of high pressure microfluidics)
This comparative example differs from example 2 in that: high-speed homogenization was used instead of high-pressure microfluidics at 20000rpm for 15min.
Results: enzyme loading is 8%, enzyme activity recovery rates in 0 day and 5 day are 85% and 80% respectively, which shows that high-speed homogenization leads to NH of immobilized enzyme 2 Aggregation in continued operation after MOFs, the effect is poor.
Performance test:
(1) Electron microscopic observation of the immobilized papain nanofiber membrane prepared in example 1
The experimental method comprises the following steps: cutting the nanofiber membrane into a size suitable for the size of a sample table of an instrument, bonding the nanofiber membrane on the sample table by using conductive adhesive, spraying gold, and placing the nanofiber membrane in a Hitachi SU-8220 scanning electron microscope for observation.
Experimental results: FIG. 1 is a scanning electron microscope image of the immobilized protease nanofiber membrane obtained in example 1. From the figure, the immobilized papain nanofiber membrane prepared in example 1 has uniform meshes, and is beneficial to permeation of reaction products. The nanofiber has no adhesion particles, which indicates that the enzyme and the high polymer material are well fused, and the combination is firm and can not fall off.
The above examples of the present invention are only examples for clearly illustrating the present invention, and are not limiting of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (6)
1. The preparation method of the immobilized enzyme nanofiber membrane is characterized by comprising the following steps of: 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 or amylase; the protease is one of papain and alkaline protease; the amylase is alpha-amylase;
2) NH of immobilized enzyme 2 -preparing an electrostatic spinning solution by MOFs and a high molecular polymer;
3) Carrying out electrostatic spinning on the electrostatic spinning solution to obtain an immobilized enzyme nanofiber membrane;
the mass ratio of the enzyme to the MOFs material with amino groups in the step 1) is 1: (1-3); the MOFs material with the amino group is prepared by the following steps: carrying out solvothermal reaction on 2-amino terephthalic acid and an aluminum source in a mixed solvent, refluxing, filtering and drying to obtain MOFs material with amino groups; the aluminum source is aluminum chloride; the mass ratio of the 2-amino terephthalic acid to the aluminum source in the mixed solvent is 1: (1.5-3); the solvothermal reaction condition is 150-200 ℃ for 12-24 h; the reflux condition is that reflux is carried out for 8-10 hours at 150-180 ℃;
NH of immobilized enzyme in step 2) 2 The mass ratio of MOFs to high molecular polymer is (1-10): (10-15);
the specific step of step 2) is to immobilize NH of the enzyme 2 Mixing MOFs and a high molecular polymer in an organic solvent, and treating by adopting high-pressure microjet to obtain an electrostatic spinning solution; the high molecular polymer is one of polyacrylonitrile, polyurethane, polylactic acid and polycaprolactone; NH of immobilized enzyme 2 Electrostatic spinning of MOFs and high molecular weight polymersThe mass concentration of the silk solution is 8% -12%; the conditions of the high pressure microjet were: the pressure is 5000-30000PSI, and the cycle times are 3-8 times;
the conditions of the electrostatic spinning in the step 3) are as follows: the flow rate is 1-2 mL/h, the voltage is 12-15 kv, and the receiving distance is 10-20 cm.
2. The method for preparing the immobilized enzyme nanofiber membrane according to claim 1, wherein the method comprises the following steps:
in the specific preparation step of the step 2), the organic solvent is N, N-dimethylformamide.
3. The method for preparing the immobilized enzyme nanofiber membrane according to claim 1, wherein the method comprises the following steps:
the reaction in step 1) is carried out in phosphate buffer with pH of 5-8;
the reaction condition in the step 1) is 25-40 ℃ for 24-48 h;
the molecular weight of the high molecular polymer in the step 2) is 1000-1500 kDa.
4. The method for preparing an immobilized enzyme nanofiber membrane according to claim 3, wherein:
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 the water is 1: (1-2);
the organic solvent is N, N-dimethylformamide;
the refluxing is carried out in an organic solvent; the organic solvent is N, N-dimethylformamide.
5. An immobilized enzyme nanofiber membrane obtained by the production process according to any one of claims 1 to 4.
6. The use of an immobilized enzyme nanofiber membrane according to claim 5, wherein: the immobilized enzyme nanofiber membrane is used for preparing an enzymolysis product, so that the enzymolysis reaction and the enzymolysis product purification are synchronously carried out; dispersing an enzyme substrate in a phosphate buffer solution, placing the phosphate buffer solution on an immobilized enzyme nanofiber membrane for reaction, collecting a solution penetrating through the membrane, and freeze-drying to obtain a purified enzyme product;
the enzyme substrate is protein or starch; the enzymolysis product is polypeptide or oligosaccharide.
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