CN117965520A

CN117965520A - Method for preparing TA-APTES deposited coating enzyme film based on glycerol regulation

Info

Publication number: CN117965520A
Application number: CN202410068843.9A
Authority: CN
Inventors: 金垚; 张博; 杨增叶
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2024-01-17
Filing date: 2024-01-17
Publication date: 2024-05-03

Abstract

The invention discloses a method for preparing a Ta-aPTES deposition coating enzyme film based on glycerol regulation, which comprises the following steps: preparing a modified membrane by the base membrane grafted coating, and grafting biological enzyme to the modified membrane; in the preparation of the modified membrane by the base membrane grafted coating, the pretreated base membrane is soaked in a mixed solution of TA and APTES containing glycerol, and the mixed solution is oscillated at room temperature to form a uniform coating layer on the surface of the base membrane; wherein the glycerol accounts for 10-20% of the TA solution by mass percent; the TA is tannic acid; the APTES is 3-aminopropyl triethoxysilane. The method solves the problem that the conventional Ta-aPTES coating seriously damages the filtration performance of the modified membrane, and can improve the enzyme-carried efficiency and the enzyme activity of the modified membrane while improving the filtration performance of the membrane.

Description

Method for preparing TA-APTES deposited coating enzyme film based on glycerol regulation

Technical Field

The invention relates to a method for preparing a TA-APTES deposited coating enzyme film, in particular to a method for preparing a TA-APTES deposited coating enzyme film based on glycerol regulation.

Background

The enzymatic reaction is a green clean and high-efficiency reaction mode, and a high-sensitivity and high-selectivity biosensor, a green clean bioreactor and the like can be developed by utilizing biological enzymes. In order to overcome the shortcomings of instability and difficult recovery of enzymes, the utilization efficiency is improved, and enzyme immobilization is a potential mode. Common immobilization methods include adsorption, entrapment, cross-linking, covalent bonding, and the like; immobilized enzyme support platforms are of a wide variety, such as MOFs (Metal Organic Framework metal organic frameworks), co-deposits of dopamine compounds, silica, anodized alumina, titania, chitosan, metal nanoparticles, graphene oxide, and the like.

An enzyme membrane is a material that uses the membrane as an immobilization platform for enzymes. In recent years, a class of coatings of phenol-amine binary system inspired by mussel bionics is developed, and the coatings which are applied to preparation (Robust Coatings via Catechol-Amine Codeposition:Mechanism,Kinetics,and Application,ACS Appl.Mater.Interfaces.10(2018)5902-5908). of an enzyme film and are based on polydopamine are not only suitable for modifying the surfaces of various matrixes, but also have extremely high stability. However, polydopamine coatings have the disadvantages of high cost, neurotoxicity, cytotoxicity, and the like. With the development of polyphenol chemistry, tannic acid and APTES (3-aminopropyl triethoxy silane) can be polymerized to obtain the system, and the coating technology has the advantages of environmental protection, low cost, high enzyme-loaded efficiency and the like, and has wide research and application prospects.

At present, a great deal of research work expects to graft the coating on the surface of a filtration separation membrane to prepare a biocatalysis separation membrane coupling biocatalysis (screening drugs, decomposing pollutants or being used for membrane self-cleaning) and separation and purification, and the coating is applied to scenes (Tannic acid-aminopropyltriethoxysilane co-deposition modified polymer membrane forα-glucosidase immobilization,Journal of Chromatography A.1683(2022)463550;Nanostructured Polyphenol-Mediated Coating:a Versatile Platform for Enzyme Immobilization and Micropollutant Removal,Ind.Eng.Chem.Res.59(2020)2708-2717). such as drug purification, wastewater treatment and the like but the permeability of the filtration membrane is seriously attenuated. Therefore, both improvements in such coatings and development of high quality enzyme films are necessary.

Two membrane coating construction modes of the existing phenol-amine binary system are constructed, one is constructed for a Dopamine (DA) coating, and the other is constructed for a TA/APTES coating. For TA/APTES coating construction: a2 g/L TA solution was prepared in Tris-HCl buffer (pH 8.5) and 10g/LAPTES dissolved in absolute ethanol. The solution volume ratio of TA to APTES was 8:1. The manner of immobilizing the enzyme is further divided into Incubation mode (i.e., the manner of immersing and incubating) and Fouling-inducedmode (i.e., the manner of filtering and immobilizing). For TA/APTES coating, the enzyme solution was incubated with the modified membrane at Incubationmode for 2.5-4 h at 100 rpm.

For TA/APTES coatings, the disadvantage is mainly represented by a severe decay of the permeation capacity, especially for nanofiltration-grade separation membranes, which is almost completely lost. With respect to the two ways Incubation mode of immobilizing enzymes, the disadvantage is mainly represented by the low enzyme loading efficiency.

Disclosure of Invention

The invention aims to provide a method for preparing a TA-APTES deposition coating enzyme film based on glycerol regulation, which solves the problem that the conventional TA-APTES coating seriously damages the filtration performance of a modified film, and can improve the enzyme-carried efficiency and the enzyme activity of the modified film while improving the filtration performance of the film.

In order to achieve the above object, the present invention provides a method for preparing a TA-APTES deposited coating enzyme film based on glycerol regulation, comprising: preparing a modified membrane by the base membrane grafted coating, and grafting biological enzyme to the modified membrane; in the preparation of the modified film by the base film grafted coating, the pretreated base film is soaked in a mixed solution of TA and APTES containing 10-20% of glycerol by mass, and the mixed solution is oscillated at room temperature to form a uniform coating layer on the surface of the base film; wherein, TA is tannic acid; the APTES is 3-aminopropyl triethoxysilane.

Preferably, the base membrane is a PVDF ultrafiltration membrane.

Preferably, the PVDF ultrafiltration membrane has a molecular weight cut-off of 100 kDa.

Preferably, the shaking is performed at 100rpm for 18 hours.

Preferably, the pretreatment submerges the base film in water.

Preferably, the mixed solution of TA and APTES containing 10-20% of glycerol by mass percent is prepared as follows: and (3) preparing a solution A: adding TA into Tris-HCl buffer solution, adding 10-20% of glycerol by mass percent, and uniformly dispersing to obtain solution A; and (3) preparing a solution B: preparing an ethanol solution of APTES to obtain a solution B; and mixing the solution A and the solution B to obtain a mixed solution of TA and APTES containing glycerin.

Preferably, the Tris-HCl buffer has a ph=8.5.

Preferably, the volume ratio of the solution A to the solution B is 8:1; the concentration of the solution B is 10g/L; the ratio of the mass of TA to the volume of Tris-HCl buffer is 1g:500mL.

Preferably, the modified membrane grafted biological enzyme, the preparation comprises: the modified membrane was washed and dried, the membrane was fixed in an ultrafiltration cup, 50mL of 0.6g/L enzyme solution was added to the ultrafiltration cup, and the membrane was incubated at 120rpm for 4 hours. The immobilization of the enzyme is through covalent bond bonding formed by Michael addition and Schiff base reaction between amino groups on the enzyme and quinone groups on the coating.

Preferably, the enzyme solution is candida rugosa lipase which is selected by enzyme.

The method for preparing the TA-APTES deposited coating enzyme film based on glycerol regulation solves the problem that the conventional TA-APTES coating seriously damages the filtration performance of the modified film, and has the following advantages:

According to the invention, through an improved technology of glycerol regulation coating deposition, an original coating structure mode is optimized through a simple and green method, so that the enzyme activity on the membrane is increased to 2.5 times, and the pure water flux attenuation is reduced from 54% to 23%. Compared with the original coating, the method provided by the invention realizes the improvement of enzyme-carried efficiency and immobilized enzyme activity and the improvement of permeability.

The method adjusts the coating structure in a simple and green mode, and the prepared enzyme membrane gives consideration to and improves the selectivity-permeability, has higher catalytic activity and permeability capacity, thereby improving the production efficiency and being more suitable for the industrialized demand.

The method not only solves the problem of low enzyme loading efficiency of incubation mode to a certain extent, but also improves accessibility of enzyme and substrate. This technique is very advantageous for incubation mode enzyme immobilization which is itself suitable for long-term production.

Drawings

FIG. 1 is a diagram comparing the preparation process of the method of the present invention with that of the conventional method.

FIG. 2 shows SEM test results of experimental example 1 of the present invention; (a) is an SEM image of the original commercial film (Pristine); (b) SEM images of TA/APTES deposited films with glycerol addition amounts (wt%) of 0%, 20% and 40%, respectively.

FIG. 3 shows the FT-IR test result of experimental example 1 of the invention.

FIG. 4 shows the AFM test result I of experimental example 1 of the present invention; (a) AFM images of the original commercial film (Pristine); (b) AFM image of TA/APTES deposited film with 0% glycerol addition (wt%).

FIG. 5 shows a second AFM test result of experimental example 1 of the present invention; (c) AFM images of TA/APTES deposited films with 20% and 40% glycerol addition (wt%) were obtained, respectively.

Fig. 6 shows the contact angle test result of experimental example 1 of the present invention.

FIG. 7 shows the CLSM test results of Experimental example 1 of the present invention; (a) And (c) are CLSM test results of TA/APTES deposited films with glycerol addition amounts (wt%) of 0%, 20% and 40%, respectively.

FIG. 8 shows the results of the pure water permeation flux test of experimental example 2 of the present invention.

FIG. 9 shows the results of enzyme loading and enzyme activity test in experimental example 3 of the present invention.

Note that: pristine in the figure is the original commercial membrane, namely PVDF ultrafiltration membrane; TA represents 0GLY film, namely TA/APTES deposited film with 0 glycerol addition amount of non-immobilized enzyme; 40GLY represents TA/APTES deposited film with 20% glycerol addition of non-immobilized enzyme; 40GLY-E represents an enzyme membrane obtained by immobilizing 40GLY with an enzyme; 20GLY represents TA/APTES deposited film with 20% glycerol addition of non-immobilized enzyme; 10GLY represents a TA/APTES deposited film with a glycerol addition amount of 10% for the non-immobilized enzyme.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The materials used in the following examples and experimental examples:

Candida rugosa lipase CRL (Type VII, calibrated activity > >700U mg ^-1); tannic Acid (TA), APTES (3-aminopropyl triethoxysilane), glycerol (GLY), p-nitrophenyl palmitate (p-NPP) and p-nitrophenol (p-NP) were all purchased without further purification; PVDF ultrafiltration membrane with a molecular weight cut-off (MWCO) of 100 kDa.

Example 1

A TA-APTES deposited coating enzyme film is prepared by adopting glycerol regulation and control, and specifically comprises the following steps:

(1) Pretreatment of PVDF ultrafiltration membrane: immersing a dried PVDF ultrafiltration membrane in deionized water overnight for standby;

(2) And (3) preparing a solution A: weighing 0.2g of TA, adding into 100mL of Tris-HCl buffer solution (pH 8.5), adding 10% (w/w) of glycerol into the solution, and uniformly dispersing to obtain solution A;

(3) And (3) preparing a solution B: weighing APTES solution, and preparing 10g/L of APTES ethanol solution to obtain solution B;

(4) A, B liquid is uniformly mixed, and the volume ratio of TA to APTES solution is 8:1, immersing a pretreated PVDF ultrafiltration membrane in a A, B mixed solution, placing the mixed solution in a water bath shaking table at 25 ℃ and 100rpm, and oscillating for 18 hours to obtain a modified TA/APTES deposited membrane;

(5) The modified TA/APTES deposited film was dried after pure water washing, the film was fixed in an ultrafiltration cup, 50mL of 0.6g/L CRL-PBS enzyme solution (prepared by adding CRL to 50mM PBS) was added to the ultrafiltration cup, and the resultant was incubated at room temperature for 4 hours at 120rpm, and the enzyme film obtained by washing with PBS after incubation was designated as 10GLY-E.

Example 2

A TA-APTES deposited coated enzymatic film was prepared in substantially the same manner as in example 1, except that:

In step (2), 20% (w/w) of glycerin was added.

The enzyme membrane prepared in this example 2 was designated 20GLY-E.

Comparative example 1

in step (2), no glycerol is added.

The enzyme membrane prepared in this comparative example 1 was designated 0GLY-E.

Comparative example 2

in step (2), 30% (w/w) of glycerol was added.

The enzyme membrane prepared in this comparative example 2 was designated as 30GLY-E.

Comparative example 3

In step (2), 40% (w/w) of glycerin was added.

The enzyme membrane prepared in this comparative example 3 was designated 40GLY-E.

Experimental example 1 film surface characterization

1. Scanning electron microscope

The surface morphology of the modified film was observed by a field emission scanning electron microscope (FESEM, S4800, hitachi, japan).

As shown in fig. 2, SEM test results of experimental example 1 of the present invention show the surface morphology of four films, wherein (a) is SEM image of original commercial film (Pristine), and (b) to (d) are SEM images of TA/APTES deposited films with glycerol addition (wt%) of 0%, 20%, 40%, respectively. Compared with the original film (base film), the different glycerol addition amounts have different effects on the morphology of the deposited coating on the film surface. The TA-APTES deposition layer without glycerol is in a nanosphere deposition mode; the TA-APTES deposition layer of an appropriate amount of glycerin (20%) was in a platelet deposition mode, while the excess glycerin (40%) resulted in a deposition layer that was difficult to deposit on the base film surface.

2. Infrared spectrum

The crosslinking of the base film and the polycondensation reaction between TA and APTES were analyzed by Fourier transform attenuated total reflection infrared spectroscopy (ATR-FTIR, thermo Scientific, nicolet iS 50) to investigate what the glycerol might play. FT-IR spectra of the original PVDF ultrafiltration membrane, TA/APTES deposited membrane and modified membranes of different glycerol content are shown in FIG. 3. The spectral characteristic peaks of PVDF appear mainly at 1174 and 1404cm ^-1, respectively attributed to-CF ₂ flexural vibration and-CH ₂ flexural vibration. After modification with TA/APTES, the profile showed a stretching vibration at 1705cm ^-1 corresponding to c=o, which helped the oxidation of the phenolic hydroxyl groups of TA to quinone groups. The band at 1534cm ^-1 is assigned to the N-H bend (amide II).

3. Atomic force microscope

The film surface roughness was characterized using an atomic force microscope (AFM, bruker axs).

As shown in fig. 4 to 5, the AFM test results of experimental example 1 of the present invention show the roughness of four films. Compared with the original film, the roughness of the TA-APTES deposited film without glycerol is greatly improved, and the roughness of the film surface is gradually reduced along with the increase of glycerol. It can be seen that the deposited coating after glycerol adjustment is more uniform and flat.

4. Contact angle of water

The hydrophilicity of the membrane surface was studied by water contact angle measured by SL 200KB (Kino, USA).

As shown in FIG. 6 (WCA: water contact angle; pristine: PVDF ultrafiltration membrane; TA: 0GLY;20GLY: enzyme membrane prepared in example 2 of the present invention; 40GLY: enzyme membrane prepared in comparative example 3 of the present invention), the contact angle test results of experimental example 1 of the present invention are shown for the hydrophilicity degree of the four membranes. Compared with the original film, the hydrophilicity of the TA-APTES deposited film is improved to different degrees no matter whether glycerin is added or not. The 20% glycerol content showed similar hydrophilicity to the non-glycerol deposited film, indicating that the glycerol did not penetrate directly into the base film and that the hydrophilic functional groups on the glycerol were not grafted to the film surface. The deposited film with 40% of glycerol has stronger hydrophilicity, mainly because excessive glycerol is remained in the mesopores on the surface of the film and is difficult to remove.

5. Distribution of enzymes on membrane surface

After staining the enzyme protein with isothiocyanato luciferin (FTIC), the distribution of the enzyme on the membrane surface was observed using a fluorescence microscope, and the results are shown in fig. 7, wherein (a) is a glycerol-free TA/APTES deposited membrane, (b) is a TA/APTES deposited membrane with a glycerol addition of 20%, and (c) is a TA/APTES deposited membrane with a glycerol addition of 40%. The results showed that the fluorescent intensity of the enzyme protein detected on the enzyme membrane was highest when the glycerol addition amount was 20%, indicating that the membrane had the best effect on enzyme immobilization.

Experimental example 2 test of pure water permeation flux

The test of pure water permeation flux adopts dead-end ultrafiltration equipment, and is specifically as follows:

Filtration was performed on a laboratory scale dead-end filtration unit with an effective filtration area of the membrane module of 41.8cm ². After pre-pressing the membranes (inventive examples 1-2, comparative examples 1-3 or the raw membrane) at 25℃for 30min at 2bar, the pure water flux was measured at 1 bar. The pure water permeation flux J is calculated from the following formula:

Wherein V is the permeation volume in units of L over a time interval Deltat; a is the effective membrane area in m ².

As shown in FIG. 8 (Glycerol membrane is an enzyme membrane prepared in the examples of the present invention and comparative examples), the results of the pure water permeation flux test in experimental example 2 of the present invention are shown. Compared with the original film, the pure water permeation flux of the glycerol-free TA-APTES deposited film is obviously reduced, and the nanosphere deposited coating brings serious flux loss to the original film, and the loss is nearly 54%. While the introduction of glycerol improves this, the flux loss gradually decreases with increasing glycerol, and is minimal, only 23%, when glycerol is added in an amount of 20%. This result is determined by both the difference in hydrophilicity and the difference in surface structure.

Experimental example 3 testing of enzyme load and enzyme activity

1. Enzyme loading

The protein concentration of the residual enzyme solution (i.e., the residual enzyme solution after incubation immobilization of the crude enzyme solution in examples and comparative examples of the present invention) was measured by the modified Bradford method, corrected with 0 to 16. Mu.g/mL of Bovine Serum Albumin (BSA) solution. The samples were diluted as required to within the protein calibration curve. Enzyme solution and Bradford reagent at 1: mixing at a volume ratio of 1. After 5min incubation, absorbance was measured at 595 nm. The enzyme load mass is calculated from the following formula:

Enzyme loading mass = c _i×v_i-c_r×v_r-c_w×v_w

Wherein c is the soluble protein concentration; v is the volume of the solution at the corresponding concentration; subscripts i, r, and w represent the initial, recovery, and wash solutions, respectively.

The enzyme load is calculated from the following formula:

2. Enzyme activity

The lipase enzyme activity was determined by p-nitrophenol (p-NP) method, and was specifically as follows:

0.1g p-NPP (26.49 mM) was dissolved in 10mL isooctane (substrate solution). To the enzyme membranes prepared in the examples and comparative examples of the present invention, 0.04mL of the substrate solution and 0.4mLPBS solution (50 mM) were added, and the mixture was shaken at 45℃for 5 minutes. Finally, 0.55mL of 95% ethanol was added to inactivate the enzyme, 0.4mL of the supernatant was diluted 10-fold with PBS, and the absorbance was measured at 405 nm.

The enzyme activity is calculated by the following formula:

Wherein, C _E is the concentration of p-NP generated after the reaction, V _E is the volume of CRL solution, and t _E is the enzymatic reaction time.

The specific and relative enzyme activities were calculated from the following formulas:

as shown in FIG. 9, the enzyme loading and enzyme activity test results of experimental example 3 of the present invention are shown. The introduction of an appropriate amount of glycerol did increase the enzyme load to some extent, and at a glycerol addition of 10%, the enzyme load was maximum, which was consistent with the above. The enzyme activity measured on a TA-APTES deposited film of 10% glycerol was approximately 2.5 times that of a TA-APTES deposited film of no glycerol. As the amount of glycerol continues to increase, the enzyme loading begins to decrease, presumably due to a substantial decrease in membrane surface roughness, a decrease in residence time of free enzyme on the membrane surface, and insufficient reaction.

While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims

1. A method for preparing a TA-APTES deposited coating enzyme film based on glycerol regulation, comprising: preparing a modified membrane by the base membrane grafted coating, and grafting biological enzyme to the modified membrane;

in the preparation of the modified membrane by the base membrane grafted coating, the pretreated base membrane is soaked in a mixed solution of TA and APTES containing glycerol, and the mixed solution is oscillated at room temperature to form a uniform coating layer on the surface of the base membrane;

Wherein the glycerol accounts for 10-20% of the TA solution by mass percent;

The TA is tannic acid; the APTES is 3-aminopropyl triethoxysilane.

2. The method of claim 1, wherein the TA solution is Tris-HCl buffer of TA.

3. The method according to claim 1, wherein the glycerol-containing mixture of TA and APTES is prepared by:

and (3) preparing a solution A: adding TA into Tris-HCl buffer solution, adding 10-20% of glycerol by mass percent, and uniformly dispersing to obtain solution A;

and (3) preparing a solution B: preparing an alcohol solution of APTES to obtain a solution B;

and mixing the solution A and the solution B to obtain a mixed solution of TA and APTES containing glycerin.

4. A method according to claim 2 or 3, characterized in that the Tris-HCl buffer has a pH = 8.5.

5. A method according to claim 3, wherein the solution volume ratio of solution a to solution B is 8:1; the concentration of the solution B is 10g/L; the ratio of the mass of TA to the volume of Tris-HCl buffer is 1g:500mL.

6. The method according to claim 1, wherein the base membrane is a PVDF ultrafiltration membrane;

the molecular weight cut-off of the PVDF ultrafiltration membrane is 100k Da.

7. The method of claim 1, wherein the oscillating is at 100rpm for 18 hours.

8. The method of claim 1, wherein the pre-treatment submerges the base film in water.

9. The method of claim 1, wherein the modified membrane grafted bio-enzyme is prepared comprising:

The modified membrane was washed and dried, the membrane was fixed in an ultrafiltration cup, 50mL of 0.6g/L enzyme solution was added to the ultrafiltration cup, and the membrane was incubated at 120rpm for 4 hours.

10. The method of claim 9, wherein the enzyme solution is candida rugosa lipase.