CN111675825B - Preparation method of microporous membrane attached with trypsin and application of microporous membrane in proteolysis - Google Patents
Preparation method of microporous membrane attached with trypsin and application of microporous membrane in proteolysis Download PDFInfo
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
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- C12Y—ENZYMES
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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Abstract
The invention discloses a preparation method of a microporous membrane attached with trypsin, which takes polymethyl methacrylate as a template agent and amphiphilic polymer polyethylene glycol block-polyethylene glycol as a stabilizer to successfully prepare self-organizing microporous membrane PP under the optimal condition. The obtained microporous membrane is used as a host material, and trypsin is loaded in pores on the surface of the membrane to form the microporous membrane attached with the trypsin. The invention realizes a simple and easy-to-operate enzyme immobilization approach, the prepared porous membrane with the trypsin has uniform structure, good stability and good trypsin activity, can efficiently hydrolyze protein and can be recycled, compared with free enzyme, the porous membrane with the trypsin has the advantages of convenient recovery, repeated use, low cost and the like, and has important application value in the fields of food processing, new product development, food preservation and the like.
Description
Technical Field
The invention belongs to the field of enzyme immobilization and food proteolysis, and particularly relates to a trypsin immobilization method based on a microporous membrane material and application of a microporous membrane of immobilized enzyme in proteolysis.
Background
The enzyme is an indispensable role in the food field as a biocatalyst. The catalytic performance of the enzyme is widely applied to the aspects of new product development and food preservation, and the food components can be analyzed and detected by utilizing the sensitivity and specificity of the enzyme catalysis. The traditional free enzyme has high catalytic efficiency, mild reaction conditions and selectivity and specificity. However, in the actual production process, there are problems such as poor stability, difficulty in recovery, difficulty in controlling side reactions, and the like, and the application of the enzyme is extremely limited.
The enzyme immobilization technique is a technique in which an enzyme is physically or chemically confined on a certain carrier or in a certain space, but a specific catalytic reaction thereof can be carried out, and the enzyme can be recovered and reused. In recent years, research on novel efficient enzyme immobilization methods and development of tools have become a hot issue for researchers to study. For example, a sensor is prepared by using a well-known enzyme linked immunosorbent assay (Elisa) and is applied to the fields of food quality supervision, medical drug delivery, environmental pollution monitoring and the like. Compared with free enzyme, the immobilized enzyme has the unique advantages of high catalytic efficiency, easy reaction control, good storage stability, high temperature and solvent sensitivity and the like, thereby arousing great interest of people.
The ideal conditions for enzyme immobilization are order and high efficiency of the loaded enzyme. At present, various solid porous inorganic or organic materials, such as ordered mesoporous materials, have been successfully developed into high-load enzyme immobilized solid carriers. There are three main immobilization methods: encapsulation, carrier binding and crosslinking. Immobilization strategies for enzymes have been widely adopted, such as top town technology, photolithography, colloidal crystals, microphase separation self-assembly methods, and the like. However, in the existing technologies, enzymes are generally fixed in carriers and only suitable for water-soluble small molecular substrates, and large molecular substrates are often blocked by the carriers and are not easy to contact with the enzymes, so that catalytic activity is difficult to exert, and most of the enzymes are high in investment cost when being used for the first time.
The trypsin is a proteolytic enzyme extracted from cattle, sheep or pig pancreas, has great potential in improving the functional characteristics of food protein such as solubility, foamability, emulsibility, gelation and the like in food processing, and can also be used for determining the in-vitro digestibility of the food protein and determining the protein sequence of the food in food science research. However, during the enzymatic reaction, the state of the enzyme and environmental conditions such as temperature, pH, etc. have a great influence on these functions. Trypsin immobilization may result in enzymes with good thermal stability, pH stability and specificity. Some technical methods such as magnetic iron oxide nanoparticle immobilized trypsin have been reported so far, but such methods generally reduce the activity of the enzyme; although covalent bonds can be tightly bound to proteins when chemical coupling is used, the attachment of multiple covalent bonds may reduce the activity of the enzyme; in addition, the coating method and the crosslinking polymerization method are not only cumbersome to operate and poor in reusability, but also may encounter mass transfer and diffusion resistance of the substrate or product. Therefore, it is necessary to develop a means for immobilizing trypsin that is environmentally friendly, reusable, and inexpensive.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a simple method for fixing trypsin based on a microporous membrane material, and the prepared microporous membrane with the trypsin can be used for hydrolyzing various food proteins and can be repeatedly used.
The invention is realized by the following technical scheme:
a method for preparing a microporous membrane with trypsin attached thereto, comprising the steps of:
s1, preparation of microporous membrane PP: adding polymethyl methacrylate (PMMA) and polyethylene glycol-polypropylene glycol-polyethylene glycol (P123) into dichloromethane, and performing ultrasonic treatment to completely dissolve the PMMA and the polyethylene glycol-polypropylene glycol-polyethylene glycol to obtain a microporous membrane solution; directly dripping the microporous membrane solution on a substrate, placing the substrate at the temperature of 20-30 ℃ and the humidity of 60-90% for 6-10 hours, and obtaining a substrate attached with microporous membrane PP, wherein the film formed on the substrate is the microporous membrane PP; the substrate is a glass slide, a mica sheet and a silicon wafer;
wherein in the microporous membrane solution, the concentration of PMMA is 4-8 mg/mL, and the concentration of P123 is 0.001-0.003 mg/mL;
s2, patterned microporous membrane capture of trypsin: diluting trypsin (Try) to 0.4-0.6 mg/mL by using PBS buffer solution with pH7.4 to obtain Try solution; soaking the substrate attached with the microporous membrane PP in the step S1 in the Try solution, and then washing with deionized water to wash away unadsorbed trypsin; drying the microporous membrane on the substrate in the air, and forming a film on the substrate after drying in the air, wherein the microporous membrane PPT attached with trypsin can be torn off from the substrate.
Preferably, the concentration of PMMA in the step S1 is 6mg/mL, and the concentration of P123 is 0.001 mg/mL; the substrate is a glass slide; the ultrasonic wave is specifically 40KHz in frequency and 30 seconds in time; the humidity is 85 percent, and the temperature is 25 ℃; the volume of the microporous membrane solution dropped on the substrate was 10. mu.L.
Preferably, the trypsin solution in step S2 is 0.6 mg/mL; the soaking time is 15-30 min; the drying time is 12-24 h.
Alternatively, the method for preparing the trypsin-attached microporous membrane comprises the following steps:
s1, preparation of microporous membrane PP: adding PMMA and P123 into dichloromethane, and performing 40KHz ultrasonic treatment for 30S to obtain a microporous membrane solution; in the microporous membrane solution, the concentration of PMMA is 6mg/mL, and the concentration of P123 is 0.001 mg/mL;
directly dripping 10 mu L of the microporous membrane solution on a glass slide, placing the glass slide in a humidity box with the temperature of 25 ℃ and the humidity of 85% for 4 hours, volatilizing an organic solvent under the condition of high humidity, attaching water vapor, and forming a film on the glass slide into microporous membrane PP to obtain the glass slide attached with the microporous membrane PP;
s2, patterned microporous membrane capture of trypsin: diluting trypsin (Try) with PBS buffer solution (pH7.4) to 0.6mg/mL to obtain Try solution; wherein the enzyme activity of the trypsin is 186U/g;
soaking the slide attached with the microporous membrane PP in the step S1 in 10ml of the Try solution for 30min, and then washing with deionized water to wash away unadsorbed trypsin; and (3) drying the microporous membrane on the glass slide in the air for 24h, wherein the membrane formed on the dried glass slide is microporous membrane PPT attached with trypsin, and the microporous membrane PPT attached with trypsin can be torn off from the glass slide.
The invention also provides application of the microporous membrane PPT attached with the trypsin, the microporous membrane PPT attached with the trypsin has the activity of the trypsin, can replace the trypsin, can be used for hydrolyzing protein and the like, the microporous membrane PPT attached with the trypsin is used for hydrolyzing the protein, and the microporous membrane PPT attached with the trypsin is added during protein hydrolysis; preferably, the protein is casein.
The microporous membrane (PPT) attached with the trypsin is used for hydrolyzing casein and can be recycled;
the invention has the beneficial effects that:
(1) according to the invention, the preparation of the microporous membrane is improved by adding P123, the prepared microporous membrane has uniform aperture, and the immobilization of trypsin is realized;
(2) the appearance of the microporous membrane PP is characterized by an optical microscope, a scanning electron microscope and an atomic force microscope, so that the prepared microporous membrane has a good structure, and the pores are uniform and compact in size;
(3) the thermogravimetric curve shows that the prepared microporous membrane PP has good thermal stability before 180 ℃;
(4) the trypsin activity in the microporous membrane attached with trypsin is kept well and is more than 86% of the initial trypsin activity.
(5) The microporous membrane with the trypsin has the activity of the trypsin, can hydrolyze various proteins such as casein and the like, and has the characteristics of being recyclable and reusable.
(6) The defects of complex operation, poor reusability, non-environment-friendly materials and the like in the prior art are overcome, a simple, convenient and green method for enzyme immobilization based on a microporous membrane is provided, a new way is developed for enzyme immobilization, and potential application prospects are hopefully provided in the aspects of catalytic application in food industry, food biological detection and the like.
In conclusion, the invention takes the nontoxic water drops as the template, and has green initiative and low consumption. With the evaporation of the low boiling point polymer organic solution, the water vapor rapidly cools on the cooling surface and controllably condenses on the membrane surface, subsequently forming an array structure. Compared with the traditional method, the ordered microporous membrane loaded trypsin prepared by the green, simple and low-cost method is expected to be used for hydrolyzing various food proteins, can be repeatedly utilized, has low cost, and has important application prospects in the fields of food processing, new product development, food preservation and the like.
Drawings
FIG. 1 is an optical microscope photograph of a microporous film PP obtained in example 1 of the present invention; the microporous membrane PP shows a porous structure at 20 times magnification, with many more regular cavities.
FIG. 2 is an optical microscope photograph of a pure PMMA film prepared by comparative example 1 of the present invention without adding a surfactant; as can be seen from the figure, when the surfactant P123 is not added, the porous morphology of the PMMA film is poor, the PMMA film is provided with small cavities, and large gaps are formed among the cavities; the membrane is not favorable for capturing enzyme in subsequent experiments due to low enzyme loading rate.
FIG. 3 is an optical microscope photograph of a film PM obtained in comparative example 2 of the present invention; it can be seen from the figure that no distinct morphological structure is visible under the microscope under low humidity conditions.
FIG. 4 is a scanning electron micrograph of the microporous film PP obtained in example 1 of the present invention, at 2000 times magnification; it can be seen from the figure that the microporous film PP can observe a wide range of porous structures and the pores are closely arranged.
FIG. 5 is a 2D image of an AFM of the PP microporous film obtained in example 1 of the present invention, which shows that the film has a good porous morphology and pores with a size of 2 μm to 3 μm, and the obtained result is consistent with the SEM result.
FIG. 6 is a 3D atomic force microscope image of the PP microporous film obtained in example 1; when the film structure was observed from a stereoscopic angle, the image showed a film thickness of about 3 μm.
FIG. 7 is a graph showing a thermal weight loss test of the microporous film PP obtained in example 1 of the present invention; it can be seen from the figure that the thermal stability of the porous film before 180 ℃ is good.
FIG. 8 is a contact angle test chart of PP obtained from the microporous film of example 1; it can be seen from the figure that the porous membrane prepared by us has an average contact angle of 80.1 ° (< 90 °), and is hydrophilic.
FIG. 9 is a SDS-PAGE gel electrophoresis chart of casein hydrolyzability and reproducibility tests of trypsin-attached microporous membrane (PPT) prepared in example 2 of the present invention; in FIG. 9, the lanes are: lane 1 is Marker, lane 2 is casein, lane 3 is microporous membrane (PPT) with trypsin attached to hydrolyze casein once, and lane 4 is microporous membrane (PPT) with trypsin attached to hydrolyze casein twice, demonstrating that trypsin attached to microporous membrane can hydrolyze casein and can be repeatedly used.
Detailed Description
The invention is described in more detail by the following examples, which are not intended to limit the invention.
A method for preparing a microporous membrane attached with trypsin comprises the following steps:
s1, preparing a microporous membrane. Adding polymethyl methacrylate (PMMA) and polyethylene glycol-polypropylene glycol-polyethylene glycol (P123) into dichloromethane, and performing ultrasonic treatment to completely dissolve the materials to obtain microporous membrane solution; the microporous membrane solution was dropped directly onto a glass slide, which was placed in a humidity cabinet. The slide glass is placed in a humidity chamber for 6-10 hours to obtain a microporous membrane (PP). Wherein the concentration of PMMA in the microporous membrane solution is 4-8 mg/mL, and the concentration of P123 is 0.001-0.003 mg/mL.
S2, patterned microporous membrane capture of trypsin: trypsin (Try) was diluted to 0.4-0.6 mg/mL with PBS buffer (pH 7.4). And (3) soaking the microporous membrane prepared in the step (S1) in the prepared Try solution for 15-30 min, and washing with deionized water for 3 times to wash away unadsorbed trypsin. Drying the microporous membrane in air, and airing to obtain the microporous membrane (PPT) attached with trypsin.
Preferably, the concentration of PMMA in step S1 is 6mg/mL, and the concentration of P123 is 0.001 mg/mL.
Preferably, the ultrasound in step S1 has a frequency of 40KHz and a time of 30 seconds.
Preferably, the humidity in step S1 is 85% and the temperature is 25 ℃.
Preferably, the microporous membrane solution of step S1 is 10 μ L.
Preferably, the trypsin solution of step S2 is 0.6 mg/mL.
The invention also provides application of the microporous membrane (PPT) attached with the trypsin, and the microporous membrane (PPT) attached with the trypsin is used for hydrolyzing casein and can be recycled; is used for hydrolyzing bovine serum albumin and can be recycled; the soybean protein hydrolysate is used for hydrolyzing soybean protein isolate and can be recycled; can be used for hydrolyzing gelatin and recycling.
Immobilization method for immobilizing trypsin on self-organized porous surface
1. Preparation of the film
(1) And (3) preparing a microporous membrane (PP).
PMMA (6mg/mL) and P123(0.001mg/mL) were added to methylene chloride, and the mixture was sonicated for 30s, with the humidity set at 85% and the temperature set at 25 ℃. The mixed solution (10. mu.L) was dropped directly onto a slide glass, which was placed in a humidity chamber. Under high humidity conditions, the organic solvent evaporates and water vapor adheres. The slide glass was placed in a humidity chamber for 4 hours to obtain a microporous membrane (PP).
(2) Trypsin was captured on a patterned microporous membrane (PP).
Trypsin (Try) was diluted to 0.4-0.6 mg/mL with PBS buffer (pH 7.4). And (3) soaking the microporous membrane prepared in the step (S1) in the prepared Try solution for 15-30 min, and washing with deionized water for 3 times to wash away unadsorbed trypsin. Drying the microporous membrane in air, and airing to obtain the microporous membrane (PPT) attached with trypsin.
2. Study of topography
Based on evaporation of volatile solvent and steam condensation, microporous membrane (PP) is synthesized by adopting a breathing mode method to form a regular microporous array. The breathing pattern method can prepare micro-scale and nano-scale ordered porous membranes, which are used as carriers of bioactive substances, and comprises the steps of firstly completely dissolving PMMA (6mg/mL) and P123(0.001mg/mL) in dichloromethane to form a polymer organic solution, then compounding the polymer organic solution with an organic solvent, and then covering a clean glass substrate in a high-humidity environment with the polymer solution. After 6 hours, it was dried in air to give a milky white film. The morphological structure of the microporous membrane PP is observed by an optical microscope, a scanning electron microscope and an atomic force microscope. The result shows that the PP microporous membrane has a better porous structure, the PM membrane prepared under the low humidity condition cannot see an obvious morphological structure, and the PMMA membrane prepared without adding the surfactant has uneven and sparse pores.
3. Stability study
In order to make the prepared membrane material hopefully applied to the fields of food processing and biological detection, the thermal stability of the membrane material is tested. The microporous film PP was placed in a platinum crucible and its thermal stability was measured by thermogravimetric analysis (TGA). And (3) characterizing the microporous membrane by using a thermogravimetric analyzer, and finding that the membrane has good thermal stability.
4. Study of surface wettability
The research on the surface wettability of the solid material has important application value in the fields of food processing, environmental protection and the like, such as new oil-water separation materials, liquid food container coatings and the like. For this purpose, we used a DSA-25 contact angle measuring instrument to measure the surface wettability of the PP material of the microporous film. Under water-air conditions, PP has an average contact angle of 80.1 ° (< 90 °), indicating its hydrophilicity.
5. Investigation of enzyme Activity
The material was used with a microporous membrane (PPT) with trypsin attached and its activity was measured. A1 mmol/L solution of the N-benzoyl-L-arginine ethyl ester hydrochloride substrate was prepared at 25 ℃. And a 1mmol/L hydrochloric acid solution is prepared. To the cuvette, 2.8ml of the substrate solution and 0.2ml of hydrochloric acid solution were added. Zero at 253nm in a spectrophotometer as a blank control. To another cuvette, 2.8ml of the same substrate solution and 0.2ml of trypsin solution were added as a control and the timing was started. Read every half minute for a total of 3 minutes. According to the experimental results, the trypsin on the load membrane still has high activity.
6. Study of the hydrolysis and recyclability of the enzyme
The protease PPT loaded on the membrane was tested by SDS-PAGE gel electrophoresis, and the enzyme hydrolysis was verified by hydrolyzing the immobilized protease. A2 mg/mL casein solution was prepared for the experiment. Wherein the molecular weight represents the hydrolytic capacity of the enzyme. The molecular weight of the protein including the protein body, the hydrolysis of the protein by trypsin fixed on the membrane and the secondary hydrolysis is obtained by observation. 10% of concentrated gel and 5% of separation gel are selected for electrophoresis, and the loading amount is 8 mu L. The gel was stained with Coomassie brilliant blue, destained with ethanol and photographed with a gel imager. The observation result shows that the activity of the immobilized enzyme is not changed, and the enzyme can not only hydrolyze casein but also carry out secondary utilization according to the molecular weight, and the good repeatability is verified.
Example 1
A method for preparing a microporous membrane attached with trypsin comprises the following steps:
s1, preparation of microporous membrane PP: adding PMMA and P123 into dichloromethane, and performing 40KHz ultrasonic treatment for 30S to obtain a microporous membrane solution; in the microporous membrane solution, the concentration of PMMA is 6mg/mL, and the concentration of P123 is 0.001 mg/mL;
directly dripping 10 mu L of the microporous membrane solution on a glass slide, placing the glass slide in a humidity box with the temperature of 25 ℃ and the humidity of 85% for 4 hours, volatilizing an organic solvent under the condition of high humidity, attaching water vapor, and forming a film on the glass slide into microporous membrane PP to obtain the glass slide attached with the microporous membrane PP;
s2, patterned microporous membrane capture of trypsin: diluting trypsin (Try) with PBS buffer solution (pH7.4) to 0.6mg/mL to obtain Try solution; wherein the enzyme activity of the trypsin is 186U/g;
soaking the slide attached with the microporous membrane PP in the step S1 in 10ml of the Try solution for 30min, and then washing with deionized water to wash away unadsorbed trypsin; and (3) drying the microporous membrane on the glass slide in the air for 24h, wherein the membrane formed on the dried glass slide is microporous membrane PPT attached with trypsin, and the microporous membrane PPT attached with trypsin can be torn off from the glass slide.
This embodiment may further include pretreatment steps such as preparation of a buffer solution and washing of a beaker.
The microporous film PP obtained in step S1 of this example was observed by an optical microscope, as shown in fig. 1. It can be preliminarily observed that the porous membrane is in a dense and uniform porous shape in a large range.
The microporous film PP obtained in step S1 of this example is observed by scanning electron microscope, as shown in fig. 4. When the magnification was 2000 times each, a wide range of porous structures were observed in PP with a tight pore arrangement.
The microporous film PP obtained in step S1 of this example was observed by an atomic force microscope, as shown in fig. 5 and 6. The good appearance of the prepared film and the uniform and compact holes can be further observed from the perspective. The thickness of the film is about 3 μm, and the size of the pores is 2 to 3 μm.
The microporous film PP obtained in step S1 of this example was placed in a platinum crucible and its thermal stability was measured by a thermogravimetric analyzer (TGA). As shown in FIG. 7, the weight of PP remained relatively stable until 180 deg.C, indicating that-PP had good thermal stability at 180 deg.C.
The surface wettability of the microporous film PP obtained in step S1 of this example was measured by a contact angle measuring apparatus. As shown in FIG. 8, the average contact angle of PP under water-air conditions was 80.1 ° (< 90 °), indicating that PP is hydrophilic.
Taking the microporous membrane PP obtained in the step S1 of the embodiment, tearing the microporous membrane PP from the glass slide for standby; taking the microporous membrane PPT attached with trypsin prepared in the step S2 of the embodiment, and tearing the microporous membrane PPT from a glass slide for later use; the following enzyme activity test and test of the properties of hydrolyzed proteins were conducted.
Activity test of immobilized trypsin:
the trypsin activity of the trypsin-attached microporous membrane PPT prepared in this example was measured by the following method:
a1 mmol/L solution of the N-benzoyl-L-arginine ethyl ester hydrochloride substrate was prepared at 25 ℃. And a 1mmol/L hydrochloric acid solution is prepared. To the cuvette were added 2.8ml of the substrate solution and 0.2g of the microporous membrane PP prepared in step S1 of example 1. Zero at 253nm in a spectrophotometer as a blank control. In another cuvette, 2.8ml of the same substrate solution and 0.2g of trypsin-attached microporous membrane PPT were added as a control group and the timekeeping was started. Read every half minute for a total of 3 minutes. According to the experimental result, the trypsin on the loading membrane still has high activity, which is 160U/g.
And (3) testing the performance of the hydrolyzed protein:
casein was hydrolyzed using the microporous membrane PPT with trypsin prepared in step S2 of this example, and the degree of hydrolysis was evaluated by SDS-PAGE gel electrophoresis, which was specifically performed as follows:
preparing a casein solution with the concentration of 2 mg/mL; 20mg of the microporous membrane PPT with the trypsin (namely the microporous membrane PPT with the trypsin is torn off from a glass slide) is placed in 10ml of the protein solution, the protein solution is hydrolyzed for 5min at 25 ℃, the microporous membrane PPT with the trypsin is taken out, the hydrolyzed protein solution is subjected to SDS-PAGE electrophoresis, 10% of concentrated gel and 5% of separation gel are selected for electrophoresis, and the loading amount is 8 mu L. The electrophoresis gel was stained with Coomassie brilliant blue, decolorized with ethanol, and photographed with a gel imager. The results are shown in fig. 9, which demonstrates that the immobilized protease has good proteolytic ability;
the casein solution was again hydrolyzed using the hydrolyzed (i.e., once used) trypsin-attached microporous membrane PPT, and the degree of hydrolysis was evaluated by SDS-PAGE gel electrophoresis, as shown in fig. 9, demonstrating that the immobilized protease after use still has a good proteolytic ability.
After the first hydrolysis, the activity of the immobilized trypsin is not changed, and the molecular weight shows that the microporous membrane PPT attached with the trypsin can hydrolyze various proteins, can be reutilized, and verifies the good repeatability of the microporous membrane PPT.
Comparative example 1
To demonstrate that the addition of P123 enables the PP membrane described in example 1 of the present invention to have a cavity structure that traps enzymes, we repeated the procedure of example 1 without adding the surfactant P123.
S1, preparation of film (PMMA): adding a PMMA solution into dichloromethane, and performing ultrasonic treatment at 40KHz for 30S to obtain a microporous membrane solution; in the microporous membrane solution, the concentration of PMMA is 6mg/mL
S2, directly dripping 10 mu L of the microporous membrane solution on a glass slide, placing the glass slide in a humidity box with the humidity set to 85% and the temperature set to 25 ℃ for 6 hours, volatilizing the organic solvent under the high humidity condition, and attaching water vapor to obtain the PMMA membrane.
When the PMMA film prepared in the present comparative example was observed using an optical microscope, as shown in fig. 2, the pure PMMA film without the surfactant P123 added had small cavities with large gaps between the cavities. The porous membrane is not favorable for capturing enzyme in subsequent experiments due to low enzyme loading rate.
Comparative example 2
And (4) repeating the step S1 in the embodiment 1 under the condition of 20-30% relative humidity of air.
S1, preparation of microporous membrane PM: adding PMMA and P123 into dichloromethane, and performing 40KHz ultrasonic treatment for 30S to obtain a microporous membrane solution; in the microporous membrane solution, the concentration of PMMA is 6mg/mL, and the concentration of P123 is 0.001 mg/mL;
directly dripping 10 mu L of the microporous membrane solution on a glass slide, placing the glass slide in a humidity box with the temperature of 25 ℃ and the humidity of 20-30% for 4 hours, volatilizing an organic solvent under the high humidity condition, attaching water vapor, and forming a microporous membrane as a film on the glass slide to obtain the glass slide attached with the microporous membrane PM; when the film prepared in this comparative example was observed with an optical microscope, as shown in fig. 3, it was found that no distinct morphological structure was observed under the microscope at a humidity of 20% to 30%.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (10)
1. A method for preparing a microporous membrane with trypsin attached thereto, comprising the steps of:
s1, preparation of microporous membrane PP: adding polymethyl methacrylate and polyethylene glycol-polypropylene glycol-polyethylene glycol into dichloromethane, and dissolving by ultrasonic to obtain microporous membrane solution; dripping the microporous membrane solution on a substrate, placing the substrate at the temperature of 20-30 ℃ and the humidity of 60-90% for 6-10 hours, and obtaining a substrate attached with microporous membrane PP, wherein the film formed on the substrate is the microporous membrane PP;
wherein, in the microporous membrane solution, the concentration of the polymethyl methacrylate is 4-8 mg/mL, and the concentration of the polyethylene glycol-polypropylene glycol-polyethylene glycol is 0.001-0.003 mg/mL; the substrate is a glass slide, a mica sheet or a silicon wafer;
s2, patterned microporous membrane capture trypsin: diluting trypsin to 0.4-0.6 mg/mL by using PBS (phosphate buffer solution) with pH7.4 to obtain a Try solution; soaking the substrate attached with the microporous membrane PP in the step S1 in the Try solution, and washing away unadsorbed trypsin by using deionized water; and drying the microporous membrane on the substrate in the air, and forming a film on the substrate after air drying, wherein the microporous membrane is the microporous membrane PPT attached with trypsin.
2. The method of claim 1, wherein the concentration of the polymethylmethacrylate is 6mg/mL and the concentration of the polyethylene glycol-polypropylene glycol-polyethylene glycol is 0.001mg/mL in the microporous membrane solution of step S1.
3. The method of claim 1, wherein the ultrasonic frequency of step S1 is 40KHz for 30 seconds.
4. The method of claim 1, wherein the humidity of step S1 is 85% and the temperature is 25 ℃.
5. The method of claim 1, wherein the volume of the microporous membrane solution dropped on the substrate in step S1 is 10 μ L.
6. The method for preparing a microporous membrane with trypsin according to claim 1, wherein the soaking time in step S2 is 15-30 min.
7. The method for preparing a microporous membrane with trypsin according to claim 1, wherein the drying time in step S2 is 12-24 hours.
8. The method of producing the microporous membrane with trypsin attached according to claim 1, comprising the steps of:
s1, preparation of microporous membrane PP: adding polymethyl methacrylate and polyethylene glycol-polypropylene glycol-polyethylene glycol into dichloromethane, and performing 40KHz ultrasonic treatment for 30S to obtain microporous membrane solution; in the microporous membrane solution, the concentration of polymethyl methacrylate is 6mg/mL, and the concentration of polyethylene glycol-polypropylene glycol-polyethylene glycol is 0.001 mg/mL;
dripping 10 mu L of the microporous membrane solution on a glass slide, placing the glass slide in a humidity box with the temperature of 25 ℃ and the humidity of 85% for 4 hours, and obtaining a glass slide attached with microporous membrane PP, wherein the film formed on the glass slide is the microporous membrane PP;
s2, patterned microporous membrane capture of trypsin: diluting trypsin to 0.6mg/mL by using PBS buffer solution with pH7.4 to obtain Try solution;
soaking the slide attached with the microporous membrane PP in the step S1 in 10ml of the Try solution for 30min, and then washing away unadsorbed trypsin by deionized water; and (3) drying the microporous membrane on the glass slide in the air for 24h, wherein the film formed on the dried glass slide is microporous membrane PPT attached with trypsin.
9. Use of microporous membrane PPT with attached trypsin prepared according to any one of claims 1 to 8, wherein said microporous membrane PPT with attached trypsin is active for the hydrolysis of proteins and wherein said microporous membrane PPT with attached trypsin is added during the proteolysis.
10. Use of the microporous membrane PPT with trypsin according to claim 9, wherein said protein is casein.
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