CN112695027A - Immobilized enzyme nanofiber membrane for realizing synchronization of enzymolysis reaction and product purification, and preparation and application thereof - Google Patents

Immobilized enzyme nanofiber membrane for realizing synchronization of enzymolysis reaction and product purification, and preparation and application thereof Download PDF

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CN112695027A
CN112695027A CN202011645220.1A CN202011645220A CN112695027A CN 112695027 A CN112695027 A CN 112695027A CN 202011645220 A CN202011645220 A CN 202011645220A CN 112695027 A CN112695027 A CN 112695027A
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enzyme
immobilized enzyme
nanofiber membrane
mofs
amino
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CN112695027B (en
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叶勇
叶传珍
唐小月
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South China University of Technology SCUT
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Abstract

The invention belongs to the technical field of immobilized enzymes, and discloses an immobilized enzyme nanofiber membrane for realizing synchronization of enzymolysis reaction and product purification, and preparation and application thereof. The method comprises the following steps: 1) reacting enzyme with MOFs material with amino to obtain NH of immobilized enzyme2-MOFs; the enzyme is protease and/or amylase; 2) will be fixedNH of chemolase2Preparing electrostatic spinning solution from MOFs and high molecular polymer; 3) and (3) 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, porosity, high strength, simplicity and convenience in operation, high efficiency and the like. When the nano fiber membrane is used in enzymolysis reaction, the enzymolysis product passes through the membrane to realize separation and purification of the product, so that the enzymolysis reaction and the product purification are carried out synchronously. The immobilized enzyme nanofiber membrane is used for preparing polypeptide and/or oligosaccharide.

Description

Immobilized enzyme nanofiber membrane for realizing synchronization of enzymolysis reaction and product purification, and preparation and application thereof
Technical Field
The invention belongs to the technical field of immobilized enzymes, and particularly relates to an immobilized enzyme nanofiber membrane for realizing synchronization of 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 using immobilized enzyme nano-cellulose.
Background
When the biological enzyme is used for catalyzing reaction, the free enzyme is easy to denature, inactivate and unstable, and if the free enzyme is fixed, the catalytic activity of the enzyme is kept, so that the stability of the enzyme is improved, the solid-liquid separation is easy, and the reuse of the enzyme is realized. There are various methods for immobilizing enzymes, such as adsorption, crosslinking, entrapping and carrier binding, but these methods all have the disadvantages of low enzyme binding strength or too low enzyme carrying amount, resulting in low enzyme recovery or low 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 immobilized enzyme is immobilized by electrostatic self-assembly, which causes insufficient binding strength of the enzyme and is not easy to separate and recover. Patent application CN108396023A discloses a grinding method for preparing magnetic MOF material and using it for enzyme immobilization, resulting in an immobilized enzyme material that can be recycled, but it still has the disadvantages 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 sufficiently contacted with the substrate, and the catalytic effect of the enzyme is reduced.
The current immobilized enzyme catalytic reaction still adopts a two-step method, namely, the immobilized enzyme is mixed with a substrate for reaction, and then the enzyme and a product are separated and purified, but the immobilized enzyme which is synchronously carried out in 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 invention aims to provide an immobilized enzyme nanofiber membrane for realizing synchronization of enzymolysis reaction and product purification and a preparation method thereof.
The invention also aims to provide application of the immobilized enzyme nanofiber membrane. The invention realizes the synchronous implementation of the enzymolysis reaction and the product purification by using the immobilized enzyme nano-cellulose. The invention uses immobilized enzyme nano-cellulose for rapidly preparing polypeptide or oligosaccharide.
The purpose of the invention is realized by the following technical scheme:
a preparation method of an immobilized enzyme nanofiber membrane comprises the following steps:
1) reacting enzyme with MOFs material with amino to obtain NH of immobilized enzyme2-MOFs; the enzyme is protease and/or amylase;
2) NH of immobilized enzyme2Preparing electrostatic spinning solution from MOFs and high molecular polymer;
3) and (3) 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 the step 1) is carried out in a phosphate buffer solution with the pH value of 5-8; the dosage of the phosphate buffer solution with the pH value of 5-8 is 1-1.5 times of the mass of the enzyme.
The reaction condition in the step 1) is that the reaction is carried out for 24-48 h at 25-40 ℃.
NH of the immobilized enzyme in step 2)2-the mass ratio of the MOFs to the high molecular weight polymer is (1-10): (10-15).
In the step 2), the high molecular polymer 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 the step 2) is to fix NH of the enzyme2-MOFs and high molecular weight polymer are mixed in an organic solvent, and high-pressure microjet is adopted for processing to obtain an electrostatic spinning solution;
in the step 2), the organic solvent is more than one of N, N-dimethylformamide, dimethyl sulfoxide and chloroform;
NH of immobilized enzyme2-MOFs and high molecular weight polymer in the electrostatic spinning solution at a mass concentration of 8% >. up to12％。
The conditions of the high-pressure micro-jet are as follows: the pressure is 5000-.
And 2) heating the electrostatic spinning solution before electrostatic spinning, and filtering. The heating treatment is carried out under the condition of keeping the temperature at 40-50 ℃ for 50-70 min.
The electrostatic spinning conditions 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 aluminum-based MOFs material with amino is obtained by reacting 2-amino terephthalic acid with an aluminum source.
The MOFs material with amino is prepared by the following method: carrying out solvent thermal reaction on 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 to 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, N-methylpyrrolidone 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 150-180 ℃ for 8-10 h.
The refluxing is carried out in an organic solvent; the organic solvent is more than one of N, N-dimethylformamide, N-methylpyrrolidone and dimethyl sulfoxide.
After the solvothermal reaction is finished, removing the solvent in the system, adding an organic solvent, and then refluxing.
The adding amount of the organic solvent is 3-5 times of the amount of the product after the solvent in the system is removed.
The drying condition is drying for 24-36 h at 30-60 ℃.
The immobilized enzyme nanofiber membrane is prepared by the method.
The immobilized enzyme nanofiber membrane is applied to the synchronization of the enzymolysis reaction and the purification of the enzymolysis product, namely, the immobilized enzyme nanofiber is used for preparing the enzymolysis product, so that the enzymolysis reaction and the purification of the enzymolysis product 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 and/or starch; the enzymolysis product is polypeptide and/or oligosaccharide.
The reaction condition is room temperature reaction for 6-24 h; 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 solution with the pH value of 5-8 is 1: (5-30).
After the immobilized fibrous membrane is subjected to enzymolysis reaction, the molecular weight of an obtained enzymolysis product is 500-3000 Da.
If the fiber membrane is prepared by electrostatic spinning, then the fiber membrane is mixed with enzyme for immobilization, the method can only obtain enzyme which is adsorbed and immobilized or enzyme which is coated by the membrane, the enzyme is easy to dissociate and unstable, and the enzyme and the substrate can not be in sufficient contact reaction; if the enzyme and the polymer are directly subjected to electrostatic spinning, the difficulties of easy inactivation of the enzyme and poor dispersibility, which result in poor effectiveness, exist. The method of the invention ensures that the enzyme is stably fixed without inactivation, can fully contact and react with the substrate, can react on one side of the membrane, and collects the reaction product on the other side, thereby realizing the synchronization of the enzymolysis reaction and the product purification.
The principle of the invention is as follows:
the invention utilizes NH2-dehydrating and condensing amino groups on the MOFs and carboxyl groups in the protease, and completing enzyme immobilization through the action of an amide bond; using high-pressure microjet technology to fix NH of enzyme2Preparing an electrostatic spinning precursor from the MOFs material and a high molecular polymer, and finally adjusting spinning conditions and spinning by using an electrostatic spinning technologyAnd (3) electrospinning the electrostatic spinning precursor to obtain the composite nanofiber membrane with 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, porosity, high strength, simplicity and convenience in operation, high efficiency, high recovery rate and the like.
(2) When the nano fiber membrane is used in enzymolysis reaction, the enzymolysis product passes through the membrane to realize separation and purification of the product, so that the enzymolysis reaction and the product purification are carried out synchronously.
(3) The method of the invention 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 micrograph 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 the embodiments of the present invention are not limited thereto.
Example 1
(1) Dissolving 1g of 2-aminoterephthalic acid and 1.5g of aluminum chloride 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 reacting for 24 hours at 150 ℃; filtering, adding dimethyl amide with the mass of 3 times of that of the filter residue, refluxing at 150 ℃ for 8h, filtering, and drying the filter residue at 30 ℃ for 36h to obtain the MOFs material (NH) with amino2-MOFs)。
(2) 10g of papain was dispersed in 10g of phosphate buffer pH8 with 10g of NH2mixing-MOFs materials, and reacting at 25 ℃ for 48 hours to obtain NH of immobilized enzyme2-MOFs。
(3) NH 10g of immobilized enzyme2-MOFs and 10g polyacrylonitrile (molecular weight 1000kDa) were dissolved in dimethylformamide to prepare 8% solution, and the electrospinning solution was prepared by using high pressure microjet with pressure of 5000PSI and cycle number of 8.
(4) The electrospinning solution was heated to 40 ℃ for 70min (heating was done to keep the solution stable), filtered, and the filtrate was electrospun at a flow rate of 1ml/h, a voltage of 12kv, and a take-up distance of 10 cm. Collecting the nanofiber to obtain the immobilized enzyme nanofiber membrane.
(5) Dispersing 10g of papain powder in 50g of phosphate buffer solution with the pH value of 8, placing the mixture on an immobilized papain nanofiber membrane for reaction at room temperature for 6 hours, collecting the solution permeating the membrane, and freeze-drying to obtain the purified papain. The pore diameter of the immobilized enzyme nanofiber membrane prepared by the embodiment is 1-2 nm, the enzyme loading amount is 25%, the enzyme activity recovery rate is 96%, and the 5-day enzyme activity recovery rate is 93%.
Example 2
(1) Dissolving 1g of 2-aminoterephthalic acid and 3g of aluminum chloride 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 reacting for 12 hours at 200 ℃; filtering, adding 5 times of dimethyl amide into the filter residue, refluxing at 180 deg.C for 8 hr, filtering, and drying the filter residue at 60 deg.C for 24 hr to obtain MOFs material (NH) with amino group2-MOFs)。
(2) 10g of alpha-amylase were dispersed in 15g of phosphate buffer pH5 with 30g of NH2mixing-MOFs materials, and reacting at 40 ℃ for 24h to obtain NH of immobilized enzyme2-MOFs。
(3) NH 10g of immobilized enzyme2-MOFs and 15g of polyurethane (molecular weight 1500kDa) were dissolved in dimethylformamide to prepare a 12% solution, which was prepared into electrospinning solution using high pressure microjets at 30000PSI with 3 cycles.
(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, wherein the receiving distance is 20 cm. Collecting the nanofiber to obtain the immobilized enzyme nanofiber membrane.
(5) Dispersing 10g of corn starch in 300g of phosphate buffer solution with the pH value of 5, placing the mixture on an immobilized alpha-amylase nanofiber membrane for reaction for 24 hours, collecting the solution permeating the membrane, and freeze-drying to obtain the purified corn oligosaccharide. The immobilized enzyme nanofiber membrane prepared by the embodiment has the aperture of 5-7 nm, the enzyme loading amount of 10% and the enzyme activity recovery rate of 92%.
Example 3
(1) Dissolving 1g of 2-aminoterephthalic acid and 2g of aluminum chloride in a mixed solvent of dimethylformamide and deionized water (volume ratio is 1: 1.5) to prepare a solution with mass concentration of 35%, and reacting for 16h at 180 ℃; filtering, adding dimethylformamide with the mass 4 times of that of the filter residue, refluxing at 160 ℃ for 9h, filtering, and drying the filter residue at 50 ℃ for 28h to obtain the MOFs material (NH) with amino2-MOFs). The invention selects the aluminum-based NH2MOFs have more ordered coordination sites for amino groups, which help to immobilize proteases orderly, and aluminum-based MOF structures are more tailorable than ZIF structures.
(2) 10g of alkaline protease are dispersed in 12g of phosphate buffer pH7 with 20g of NH2mixing-MOFs materials, and reacting at 30 ℃ for 36 hours to obtain NH of immobilized enzyme2-MOFs。
(3) NH 10g of immobilized enzyme2-MOFs and 12g polycaprolactone (molecular weight 1000kDa) were dissolved in dimethylformamide to prepare a 10% solution, which was prepared into electrospinning solution using high pressure microjet with pressure of 10000PSI and cycle number of 5.
(4) Heating the electrostatic spinning solution to 45 ℃ for 60min, filtering, and carrying out electrostatic spinning on the filtrate at the flow rate of 1.5ml/h and the voltage of 13kv and the receiving distance of 15 cm. Collecting the nanofiber to obtain the immobilized enzyme nanofiber membrane.
(5) Dispersing 10g of almond protein powder in 100g of phosphate buffer solution with the pH value of 7, placing on an immobilized alkaline protease nanofiber membrane for reaction for 12h, collecting the solution permeating the membrane, and freeze-drying to obtain the purified almond polypeptide. The immobilized enzyme nanofiber membrane prepared by the embodiment has the aperture of 3-5 nm, the enzyme loading amount of 15% and the enzyme activity recovery rate of 94%.
Comparative example 1 (changing the order of enzyme immobilization)
First NH2Electrospinning a solution of MOFs material and polymer, immersing the obtained fiber membrane in an enzyme solution for immobilization, each condition being the same as in example 2.
As a result: the enzyme loading amount is 4%, and the enzyme activity recovery rates of 0 day and 5 days are 90% and 30% respectively, which indicates that the effect of electrostatic spinning and then enzyme adsorption and immobilization is poor.
Comparative example 2 (amount of the enzyme to be used)
This comparative example differs from example 2 in that: the amount of enzyme used was 40 g.
As a result: the enzyme loading amount is 12%, and the enzyme activity recovery rates of 0 day and 5 days are 28% and 25% respectively, which shows that the enzyme loading amount is not changed greatly when the enzyme dosage is increased, but the enzyme activity recovery rate is low, because the enzyme dosage is large, the fixation is incomplete.
Comparative example 3 (NH of enlarged immobilized enzyme2-amount of MOFs)
This comparative example differs from example 2 in that: NH of immobilized enzyme2The amount of MOFs is 30 g.
As a result: the enzyme loading is 15 percent, the enzyme activity recovery rates in 0 day and 5 days are 75 percent and 60 percent respectively, which shows that the NH of the immobilized enzyme is increased2The use amount of MOFs, the change of enzyme loading amount, although increased, the recovery rate of enzyme activity is lower because of part of NH of immobilized enzyme2The MOFs cannot be immobilized by the fiber membrane.
Comparative example 4 (high speed homogeneous instead of high pressure micro jet)
This comparative example differs from example 2 in that: high-speed homogenization is adopted to replace high-pressure micro-jet, 20000rpm and 15 min.
As a result: the enzyme loading amount is 8 percent, and the enzyme activity recovery rates of 0 day and 5 days are 85 percent and 80 percent respectively, which shows that the enzyme loading amount and the enzyme activity recovery rate are reduced by high-speed homogenization, which causes NH of the immobilized enzyme2The effects are worsened by the accumulation in the operation continued after the MOFs.
And (3) performance testing:
(1) electron microscopy 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, adhering the nanofiber membrane on the sample table by using conductive adhesive, spraying gold, and observing in a Hitachi SU-8220 scanning electron microscope.
The experimental results are as follows: FIG. 1 is a scanning electron micrograph of the immobilized protease nanofiber membrane obtained in example 1. As can be seen from the figure, the immobilized papain nanofiber membrane prepared in example 1 has uniform meshes and is beneficial to the permeation of reaction products. The nanofiber has no attached particles, which shows that the enzyme and the high polymer material are well fused, and the combination is firm and cannot fall off.
The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A preparation method of an immobilized enzyme nanofiber membrane is characterized by comprising the following steps: the method comprises the following steps:
1) reacting enzyme with MOFs material with amino to obtain NH of immobilized enzyme2-MOFs; the enzyme is protease and/or amylase;
2) NH of immobilized enzyme2Preparing electrostatic spinning solution from MOFs and high molecular polymer;
3) and (3) carrying out electrostatic spinning on the electrostatic spinning solution to obtain the immobilized enzyme nanofiber membrane.
2. The method for preparing the immobilized enzyme nanofiber membrane according to claim 1, characterized in that: the mass ratio of the enzyme to the MOFs material with amino groups in the step 1) is 1: (1-3);
NH of the immobilized enzyme in step 2)2-the mass ratio of the MOFs to the high molecular weight polymer is (1-10): (10-15);
the specific step of the step 2) is to fix NH of the enzyme2-MOFs and high molecular weight polymer are mixed in an organic solvent, and high pressure micro-jet flow is adopted for processing to obtain the electrostatic spinning solution.
3. The method for preparing the immobilized enzyme nanofiber membrane as claimed in claim 2, wherein: the conditions of the high-pressure microjet in the specific preparation step of the step 2) are as follows: the pressure is 5000-;
in the specific preparation step of the step 2), the organic solvent is more than one of N, N-dimethylformamide, dimethyl sulfoxide and chloroform;
in the specific preparation step of step 2), NH of the immobilized enzyme2The mass concentration of the-MOFs and the high molecular polymer in the electrostatic spinning solution is 8-12%.
4. The method for preparing the immobilized enzyme nanofiber membrane according to claim 1, characterized in that:
the MOFs material with amino in the step 1) is an aluminum-based MOFs material with amino;
in the step 2), the high molecular polymer is one or more of polyacrylonitrile, polyurethane, polylactic acid and polycaprolactone;
the electrostatic spinning conditions 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 reaction in the step 1) is carried out in a phosphate buffer solution with the pH value of 5-8;
the reaction condition in the step 1) is that the reaction is carried out for 24-48 h at 25-40 ℃;
the molecular weight of the high molecular polymer in the step 2) is 1000-1500 kDa.
5. The method for preparing the immobilized enzyme nanofiber membrane according to claim 4, characterized in that: in the step 1), the aluminum-based MOFs material with amino is obtained by reacting 2-amino terephthalic acid with an aluminum source.
6. The method for preparing the immobilized enzyme nanofiber membrane according to claim 4, characterized in that: the MOFs material with amino is prepared by the following method: carrying out solvent thermal reaction on 2-amino terephthalic acid and an aluminum source in a mixed solvent, refluxing, filtering and drying to obtain the MOFs material with amino.
7. The method for preparing an immobilized enzyme nanofiber membrane according to claim 6, characterized in that: in the preparation of MOFs materials with amino groups, the mass ratio of the 2-amino terephthalic acid to the aluminum source is 1: (1.5-3);
in the preparation of the MOFs material with amino, 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, N-methylpyrrolidone and dimethyl sulfoxide;
in the preparation of the MOFs material with amino, the aluminum source is aluminum chloride; the mass concentration of the 2-amino terephthalic acid and the aluminum source in the mixed solvent is 30-40 percent;
in the preparation of the MOFs material with amino, the solvothermal reaction condition is that the reaction is carried out for 12-24 hours at 150-200 ℃; the reflux condition is 150-180 ℃ for 8-10 h;
the refluxing is carried out in an organic solvent; the organic solvent is more than one of N, N-dimethylformamide, N-methylpyrrolidone and dimethyl sulfoxide.
8. An immobilized enzyme nanofiber membrane obtained by the preparation method of any one of claims 1 to 7.
9. The use of the immobilized enzyme nanofiber membrane of claim 1, wherein: the immobilized enzyme nanofiber membrane is used for preparing an enzymolysis product, and the synchronous implementation of enzymolysis reaction and enzymolysis product purification is realized.
10. Use according to claim 9, characterized in that: 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.
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