CN113234716B - Method for treating immobilized enzyme by using strengthening liquid and application thereof - Google Patents

Method for treating immobilized enzyme by using strengthening liquid and application thereof Download PDF

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CN113234716B
CN113234716B CN202110591445.1A CN202110591445A CN113234716B CN 113234716 B CN113234716 B CN 113234716B CN 202110591445 A CN202110591445 A CN 202110591445A CN 113234716 B CN113234716 B CN 113234716B
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CN113234716A (en
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朱伟
周壮
雷琪
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South China University of Technology SCUT
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Abstract

The invention discloses a method for treating immobilized enzyme by using strengthening liquid and application thereof, belonging to the technical field of enzyme engineering. The method for treating immobilized enzyme by using the strengthening liquid comprises the following steps: (1) Adsorbing the free enzyme to a solid phase porous carrier to obtain immobilized enzyme; (2) Adding the strengthening solution into the immobilized enzyme for co-incubation, and then washing by using a buffer solution to obtain the immobilized enzyme after the strengthening solution treatment. The biomimetic mineralization process provides a rigid shielding environment for enzyme molecules, so that the influence of external adverse environment on enzyme activity is effectively reduced, and the characteristics of recycling stability, thermal stability, organic solvent tolerance, repeated freeze thawing resistance and the like of the enzyme molecules are improved.

Description

Method for treating immobilized enzyme by using strengthening liquid and application thereof
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for treating immobilized enzyme by using strengthening liquid and application thereof.
Background
The use of microbial cells or isolated or engineered enzymes has led to significant advances in biocatalysis and to a shift in the manner of manufacture. Many classes of enzymes such as acylases, amidases, transaminases, ketoreductases, oxidases, monooxygenases, hydrolases and the like are used in the production of reactions involving antibiotics, herbicides, pharmaceutical intermediates and new era therapeutics.
Enzyme preparations are generally added to enzyme catalyzed reactions in the form of enzyme powders, which are difficult to separate and reuse once added to the system, and single use results in higher costs due to the generally higher price of enzyme preparations. Enzyme immobilization is one of the effective methods for solving the difficulty, and limits biological enzymes to a carrier material, so that the biological enzymes are insoluble in a reaction medium, and the method has the advantages of high storage stability, easiness in separation and recovery, repeated use, continuous and controllable operation, simple and convenient process and the like while maintaining the efficient, specific and mild enzyme catalytic reaction characteristics, so that the immobilization of the enzymes is increasingly valued by researchers.
The current immobilized enzyme technology mainly adopts four most common methods, namely a covalent bonding method, an adsorption method, a crosslinking method and an embedding method, but the immobilization methods suitable for different enzymes are different, and the four immobilization methods have advantages and disadvantages. The adsorption method is the earliest immobilization method, and can be divided into two types, namely ion exchange adsorption and physical adsorption, and the method has mild conditions and basically does not change the conformation of the enzyme to a great extent, so that the catalysis of the enzyme is not greatly influenced; however, the enzyme and the carrier have weak binding force, so that under some special conditions, such as higher salt concentration, high temperature and the like, the enzyme is easy to fall off from the carrier and pollute catalytic reaction products, serious enzyme leakage problems are faced, the recycling performance is poor, and the problem to be solved is urgent to improve the recycling property of the immobilized enzyme.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the primary aim of the invention is to provide a method for treating immobilized enzyme by using strengthening liquid, wherein the immobilized enzyme is further treated by using biomimetic mineralization post-treatment technology, and the aim is to improve the service life and the recycling times of the immobilized enzyme.
It is still another object of the present invention to provide the use of the method for treating an immobilized enzyme with a strengthening solution as described above.
The invention aims at realizing the following technical scheme:
a method for treating an immobilized enzyme with a strengthening solution, comprising the steps of:
(1) Adsorbing the free enzyme to a solid phase porous carrier to obtain immobilized enzyme;
(2) Adding the strengthening solution into the immobilized enzyme for co-incubation, and then washing by using a buffer solution to obtain the immobilized enzyme after the strengthening solution treatment.
The free enzymes described in step (1) include, but are not limited to, at least one of nucleic acid synthases, nucleic acid ligases, telomerases, sugar isomerases, racemases, glutamine synthases, aldolases, phosphatases, methyltransferases, phosphatases, dehydrogenases, catalases, acyltransferases, amidases, transaminases, ketoreductases, oxidases, monooxygenases, penicillin acylases, and hydrolases;
the hydrolytic enzymes include, but are not limited to, at least one of lipases, amylases, proteases, cellulases, pectinases, and lactases.
The free enzyme is further preferably at least one of free glucose oxidase, free lipase and free penicillin acylase; more preferably at least one of free glucose oxidase and free lipase.
The solid phase porous support described in step (1) preferably includes, but is not limited to, at least one of macroporous resins, porous silica particles, and metal-organic framework materials; the solid phase porous carrier is more preferably at least one of a solid phase porous amination resin and a porous metal organic framework carrier.
The porous metal organic framework carrier is prepared by the following preparation method:
1) FeCl is added 3 ·6H 2 Dissolving O and terephthalic acid in N, N-dimethylformamide, performing ultrasonic to clarify, performing high-temperature sealing reaction, cooling to room temperature, centrifugally collecting precipitate, cleaning, and vacuum drying to obtain a metal organic framework NH 2 -MIL-101(Fe)；
2) NH is added to 2 MIL-101 (Fe) is dispersed in phosphoric acid solution, ultrasonic treatment, slow stirring, reaction under the water bath condition, cooling, centrifugal collection of solid, washing and vacuum drying are carried out, thus obtaining the porous metal organic framework carrier.
In step 1), the FeCl 3 ·6H 2 O and terephthalic acid are preferably calculated according to a molar ratio of 2-3:1-2; more preferably in a molar ratio of 2.45:1.24.
In the step 1), the conditions of the high-temperature sealing reaction are preferably 100-140 ℃ for 25-32 h; more preferably 120℃for 30 hours.
In the step 1), the cleaning is preferably sequentially performed by DMF, ultrapure water and absolute ethanol; the number of times of the washing is preferably 2 to 3.
In the step 1), the vacuum drying condition is preferably 60-100 ℃ vacuum drying for 22-26 hours; more preferably at 80 c for 24 hours.
In step 2), the NH 2 The ratio of MIL-101 (Fe) to phosphoric acid is preferably 450-550:45-55; more preferably calculated as a mass (mg) to volume (mL) ratio of 10:1.
In the step 2), the concentration of the phosphoric acid solution is preferably 20-60 mM; more preferably 30mM.
In the step 2), the reaction condition under the water bath condition is preferably 30-50 ℃ water bath condition for 0.5-3.5 h; more preferably, the reaction is carried out for 1 hour in a water bath at 40 ℃.
In step 2), the washing reagent is preferably deionized water and absolute ethanol.
The mass ratio of the free enzyme to the solid phase porous carrier in the step (1) is preferably 0.2-0.3:1.
The strengthening liquid in the step (2) consists of a micromolecular mineralization reagent and a buffer solution with the pH value of 3-10;
the small molecule mineralizing agent is preferably at least one of methyltrimethoxysilane, methyldiethoxysilane, ethyltriethoxysilane, methyltriethoxysilane, tetraethylorthosilicate (TEOS), tetramethoxysilane (TMOS), bis (trimethylsilyl) acetamide (BSA), 3-aminopropyl triethoxysilane (APTES), 3-mercaptopropyl trimethoxysilane (MPTMS), trimethylchlorosilane (TMCS), chlorotriethoxysilane (TECS), octyltrimethoxysilane, cyclohexylmethyldimethoxysilane, trimethoxysilane, triethoxysilane, benzyltriethoxysilane, vinyltrimethoxysilane, isobutyltriethoxysilane, tetraisopropyl titanate, methyl titanate, tetraethyltitanate, tetrabutyltitanate, tetrapropyltitanate, calcium carbonate oligomer capable of forming insoluble calcium salts, calcium phosphate oligomer capable of being prepared in aqueous phase, and metal organic frameworks ZIF-8, ZIF-67 capable of being prepared in aqueous phase; more preferably at least one of tetraethyl orthosilicate (TEOS), tetramethoxysilane (TMOS) and 3-aminopropyl triethoxysilane (APTES).
The buffer solution with the pH value of 3-10 is preferably at least one of phosphate buffer solution, acetic acid-sodium acetate buffer solution, HEPES buffer solution, tris-HCl buffer solution and carbonate buffer solution; more preferably at least one of acetic acid-sodium acetate buffer having a pH of 3.0 to 3.9 and 50 to 100mM and PBS buffer having a pH of 8.5 and 100mM.
In the strengthening solution, the micromolecular mineralization reagent and the buffer solution with the pH value of 3-10 are preferably mixed according to the molar concentration ratio of 10-1000:100.
The volume ratio of the strengthening liquid to the immobilized enzyme in the step (2) is preferably 100-1000:1-2.
The incubation condition in the step (2) is preferably that the co-incubation is carried out for 0.15 to 24 hours under the shaking condition at the temperature of 4 to 37 ℃; the oscillation is at least one of vortex oscillation and inversion oscillation.
The buffer in step (2) is preferably at least one of acetic acid-sodium acetate buffer, glycine-sodium hydroxide buffer, HEPES buffer and Tris-HCl buffer.
The pH of the buffer solution in the step (2) is preferably 4.5 to 8.0 and the molar concentration is 20 to 100mM.
The method for treating immobilized enzyme by using the strengthening liquid is applied to enzyme immobilization.
The preparation method of the invention has the following advantages and beneficial effects:
(1) The invention aims to overcome the defects of free enzyme in the aspects of thermal stability, freeze thawing stability, organic solvent stability, recycling capability and the like, and adopts a pore adsorption method to introduce the free enzyme into a pore channel structure of a porous solid phase carrier so as to realize the immobilization of the enzyme; further, the immobilized enzyme is further immobilized by adopting the strengthening liquid through a biomimetic mineralization method, so that the service life and the recycling times of the immobilized enzyme are prolonged.
(2) The biomimetic mineralization process provides a rigid shielding environment for enzyme molecules, so that the influence of external adverse environment on enzyme activity is effectively reduced, and the characteristics of recycling stability, thermal stability, organic solvent tolerance, repeated freeze thawing resistance and the like of the enzyme molecules are improved.
Drawings
FIG. 1 is a metal organic framework NH 2 Transmission electron microscopy results for MILs-101 (Fe) and porous metal-organic framework supports; wherein figure (a) is a metal organic framework NH 2 -a transmission electron microscope result map of MILs-101 (Fe); and (b) is a transmission electron microscope result graph of the porous metal organic framework carrier.
FIG. 2 is a graph showing the results of the recycling ability of the immobilized enzyme after treatment with four sets of strengthening solutions, GOx@LXTE-705-TEOS, GOx@MOF-TMOS, PLL@LXTE-705-APTES, and PLL@MOF-APTES.
FIG. 3 is a graph showing the results of the recycling ability of the immobilized enzyme treated with the strengthening solution without adding the small molecule mineralizer obtained in comparative example 1, the immobilized enzyme treated with the strengthening solution without performing the co-incubation process obtained in comparative example 2, the immobilized enzyme treated with the strengthening solution without washing to remove the small molecule mineralizer obtained in comparative example 3, and the immobilized enzyme treated with the strengthening solution without using the porous solid support obtained in comparative example 4.
FIG. 4 is a graph showing the results of the thermostability of GOx@MOF-TMOS and free glucose oxidase.
FIG. 5 is a graph showing the results of organic solvent tolerance of GOx@MOF-TMOS and free glucose oxidase.
FIG. 6 is a graph showing the results of repeated freeze-thaw stability of GOx@MOF-TMOS and free glucose oxidase.
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.
The enzyme activity determination method comprises the following steps:
1. glucose oxidase Activity assay
1.1 drawing a standard curve for BCA protein content measurement:
8 tubes were numbered and 1000. Mu.g/mL of BSA standard solution was diluted with deionized water to 1000. Mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL, 62.5. Mu.g/mL, 31.25. Mu.g/mL, 15.625. Mu.g/mL and 0. Mu.g/mL, respectively, and BCA kit (Pierce TM BCA protein quantitative analysis kit, the solution A and the solution B in the Siemens technology company) are prepared into working solution C required by the color reaction according to the proportion of 50:1 by volume, 25 mu L of prepared standard solution is added into 200 mu L of working solution C, the mixture is fully and uniformly mixed and then reacted for 2 hours at 25 ℃, then an enzyme-labeled instrument is used for measuring the absorbance at 562nm, the x axis is used as absorbance value, the y axis is used as protein concentration, a protein standard curve is drawn, and the linear regression equation is y= 0.6886x-0.0015, R 2 ＝0.9996。
1.2BCA kit assay for protein content:
and adding 200 mu L of working solution C into 25 mu L of sample to be detected for protein content, fully and uniformly mixing, reacting for 2 hours at 25 ℃, measuring the absorbance at 562nm by using an enzyme-labeling instrument, and converting the concentration of protein in the sample according to a standard curve for subsequent calculation.
1.3ABTS + Drawing a standard curve:
numbering 8 tubes, diluting 1000. Mu.M ABTS standard solution with deionized water to 1mL of 200. Mu.M, 100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M and 0. Mu.M, adding 100. Mu.L, 10. Mu.M hydrogen peroxide and 15. Mu.L, 0.25mg/mL horseradish peroxidase, reacting for 10min, measuring the absorbance at 420nm with a microplate reader, and taking x-axis as absorbance and y-axis as ABTS + Concentration, map ABTS + Standard curve with linear regression equation y=25x—4.1, r 2 ＝0.999。
1.4 measurement of glucose oxidase Activity, 96 well plate method
Principle of:
96-well plate reaction solution system (total volume 205 μl): 100 mu L of 0.4mM ABTS solution and 100 mu L of 6.0mM beta-D (+) glucose substrate solution are mixed as substrate solution, 3 mu L of 0.8mg/mL horseradish peroxidase solution is added into 200 mu L of substrate solution, 2 mu L of free enzyme or immobilized enzyme solution is added, and A is continuously monitored at 25 DEG C 400nm The enzyme activity was calculated according to the following formula.
Wherein 0.303 represents the total volume of the system (mL);
0.010 represents the volume of enzyme (mL) added to the reaction system.
1Unit determinationMeaning that the cascade catalyzes the production of 1. Mu. Mol of ABTS per minute of beta-D-glucose at pH3.0, T=25℃ + The amount of glucose oxidase required (where HRP activity is excessive, its activity in the cascade was considered to be 100%, i.e., H produced) 2 O 2 Can immediately bind to HRP to complete subsequent color reaction).
2. Lipase Activity assay
2.1 drawing of p-nitrophenol standard curve:
0.02, 0.04, 0.06, 0.08, 0.12 and 0.16mL of p-nitrophenol mother liquor (2 mM) was diluted to 220. Mu.L with HEPES buffer at pH7.0 and 50mM concentration, and absorbance at 410nm was measured. The concentration y of p-nitrophenol (corresponding concentrations are respectively 0.01, 0.02, 0.03, 0.04, 0.06 and 0.08, unit: mM) is taken as an ordinate, the absorbance value x is taken as an abscissa, a standard curve is drawn, the linear regression equation is y=0.0675x-0.0069, R 2 ＝0.996。
2.2 Lipase Activity assay, 96 well plate method
Principle of: the p-nitrophenol ester is a substrate which is most widely used in the measurement of lipase hydrolysis activity, and the lipase hydrolyzes p-nitrophenol palm eleostearate to generate yellow p-nitrophenol, and has a light absorption value at 410 nm.
96-well plate reaction solution system (total volume 440 μl): 18. Mu.L of p-nitrophenyl palm acid ester solution (8.89 mM) was mixed with 162. Mu.L of HEPES buffer solution (pH 7.0) at 50mM concentration as a substrate solution, 40. Mu.L of free enzyme or immobilized enzyme solution was added, the reaction was terminated by rapidly adding 220. Mu.L of absolute ethyl alcohol after reacting at 37℃for 5 minutes, and the absorbance at 410nm was measured by taking 220. Mu.L of the mixed solution. Enzyme activity calculation was performed according to the following formula.
Wherein: 0.440 represents the total volume of the system (mL);
0.040 represents the volume of enzyme added to the reaction system (mL).
1Unit is defined as the amount of lipase required to catalyze the hydrolysis of p-nitrophenol ester to 1. Mu. Mol of p-nitrophenol per minute at pH7.0, T=37℃.
Example 1: preparation of porous metal organic framework supports
A porous metal-organic framework support comprising the steps of:
(1) FeCl is added 3 ·6H 2 O (6755 mg,2.45 mmol) and terephthalic acid (H) 2 BDC) (206 mg,1.24 mmol) is dissolved in 30mL of N, N-Dimethylformamide (DMF), ultrasonic treatment is carried out for 2min until clarification, then the mixture is placed in a reaction kettle for sealing, then the mixture is fully reacted in a baking oven at 120 ℃ for 30h, cooled to room temperature, centrifugally collected and precipitated part is washed for 2 to 3 times by DMF, ultrapure water and absolute ethyl alcohol in sequence, and dried for 24h under vacuum at 80 ℃ to obtain the metal organic framework NH 2 -MIL-101(Fe)；
(2) 500mg of the NH as described above 2 And (3) re-dispersing MIL-101 (Fe) in 50mL of 30mM phosphoric acid solution, carrying out ultrasonic treatment for 10min, slowly stirring, heating and reacting for 1h under the water bath condition of 40 ℃, cooling to room temperature, centrifugally collecting solid parts, respectively washing with enough deionized water and absolute ethyl alcohol for 2-3 times, drying under vacuum for 12h, ensuring no organic solvent residue, and drying to obtain the porous metal-organic framework carrier.
Example 2
A method for treating an immobilized enzyme with a strengthening solution, comprising the steps of:
(1) 0.2mg of free glucose oxidase (purchased from Shanghai Yingxin laboratory equipment Co., ltd., product No. TX202042; hereinafter the same) was adsorbed onto 1mg of a solid phase porous amination resin (purchased from Sian blue and Xiao technology New Material Co., ltd., product No. LXTE-705; hereinafter the same) to obtain an immobilized enzyme;
(2) 100mM acetic acid-sodium acetate buffer with pH of 3.9 containing 10mM tetraethyl orthosilicate (TEOS) is used as strengthening solution, the strengthening solution is added into the immobilized enzyme according to the volume ratio of 1000:1 (strengthening solution: immobilized enzyme) to perform co-incubation for 24 hours under the reverse oscillation of 4 ℃, and then the immobilized enzyme (GOx@LXTE-705-TEOS) treated by the strengthening solution is obtained by centrifugal washing with acetic acid-sodium acetate buffer with pH of 5.0 and concentration of 50 mM.
Example 3
A method for treating an immobilized enzyme with a strengthening solution, comprising the steps of:
(1) Adsorbing 0.2mg of free glucose oxidase to 1mg of the porous metal-organic framework carrier prepared in example 1 to obtain immobilized enzyme;
(2) 100mM acetic acid-sodium acetate buffer with pH of 3.0 and containing 10mM Tetramethoxysilane (TMOS) is used as strengthening liquid, the strengthening liquid is added into the immobilized enzyme according to the volume ratio of 100:1 (strengthening liquid: immobilized enzyme) to perform co-incubation for 10min under reverse oscillation at 37 ℃, and then the immobilized enzyme (immobilized glucose oxidase after strengthening liquid treatment, GOx@MOF-TMOS) after strengthening liquid treatment is obtained by centrifugal washing with acetic acid-sodium acetate buffer with pH of 5.5 and concentration of 50 mM.
Example 4
A method for treating an immobilized enzyme with a strengthening solution, comprising the steps of:
(1) 0.3mg of free lipase (available from Shanghai Ala Biotechnology Co., ltd., product number: L298994) was adsorbed onto 1mg of a solid phase porous amination resin (available from Sian blue Xiao technology New Material Co., ltd., product number: LXTE-705) to obtain an immobilized enzyme;
(2) 100mM PBS buffer solution with pH of 8.5 and containing 500mM 3-aminopropyl triethoxysilane (APTES) is used as strengthening solution, the strengthening solution is added into immobilized enzyme according to the volume ratio of 500:2 (strengthening solution: immobilized enzyme), and the mixture is incubated for 15 hours under vortex oscillation at 4 ℃, and then glycine-sodium hydroxide buffer solution with pH of 8.0 and concentration of 100mM is used for centrifugal washing, so as to obtain immobilized enzyme (PLL@LXTE-705-APTES) after the strengthening solution treatment.
Example 5
A method for treating an immobilized enzyme with a strengthening solution, comprising the steps of:
(1) Adsorbing 0.3mg of free lipase onto 1mg of the porous metal-organic framework carrier prepared in example 1 to obtain immobilized enzyme;
(2) 100mM acetic acid-sodium acetate buffer with pH of 3.0 and 1000mM 3-aminopropyl triethoxysilane (APTES) is used as strengthening solution, the strengthening solution is added into immobilized enzyme according to the volume ratio of 1000:1 (strengthening solution: immobilized enzyme) to perform co-incubation for 10min under reversed oscillation at 25 ℃, and then Tris-HCl buffer with pH of 4.5 and concentration of 20mM is used for centrifugal washing, so as to obtain immobilized enzyme (PLL@MOF-APTES) after strengthening solution treatment.
Comparative example 1:
a method for treating immobilized enzyme by using strengthening liquid without adding small molecule mineralizing agent, comprising the following steps:
(1) Adsorbing 0.3mg of free lipase onto 1mg of solid phase porous amination resin (purchased from SiAn blue and Xiao technology New Material Co., ltd., product number: LXTE-705) to obtain immobilized enzyme;
(2) Adding 100mM acetic acid-sodium acetate buffer with pH of 3.9 into immobilized enzyme according to the volume ratio of 1000:1 (buffer: immobilized enzyme), incubating for 24h under reversed oscillation at 4 ℃, and centrifugally washing by using acetic acid-sodium acetate buffer with pH of 5.0 and concentration of 50mM to obtain immobilized enzyme treated by strengthening solution without adding small molecule mineralizing agent.
Comparative example 2:
a method for treating immobilized enzymes with a strengthening solution without co-incubation, comprising the steps of:
(1) Adsorbing 0.3mg of free lipase onto 1mg of solid phase porous amination resin (purchased from SiAn blue and Xiao technology New Material Co., ltd., product number: LXTE-705) to obtain immobilized enzyme;
(2) 100mM acetic acid-sodium acetate buffer with pH of 3.9 containing 10mM tetraethyl orthosilicate (TEOS) is used as strengthening liquid, the strengthening liquid is added into immobilized enzyme according to the volume ratio of 1000:1 (strengthening liquid: immobilized enzyme), the mixture is mixed under vortex oscillation at 4 ℃, and the mixture is immediately centrifugally washed by using acetic acid-sodium acetate buffer with pH of 5.0 and concentration of 50mM after uniform mixing, thus obtaining the immobilized enzyme which is not treated by the strengthening liquid in the co-incubation process.
Comparative example 3:
a method for treating immobilized enzyme with a strengthening solution without washing to remove small molecule mineralizing agent, comprising the steps of:
(1) Adsorbing 0.3mg of free glucose oxidase onto 1mg of solid phase porous amination resin (purchased from SiAn blue and Xiao technology New Material Co., ltd., product number: LXTE-705) to obtain immobilized enzyme;
(2) 100mM acetic acid-sodium acetate buffer solution with pH of 3.9 and containing 10mM tetraethyl orthosilicate (TEOS) is used as strengthening solution, the strengthening solution is added into the immobilized enzyme according to the volume ratio of 1000:1 (strengthening solution: immobilized enzyme), and the mixture is incubated for 24 hours under the reverse oscillation of 4 ℃, the washing operation of the buffer solution is not carried out, and the immobilized enzyme which is not washed to remove the micromolecule mineralizer and is treated by the strengthening solution is obtained through direct recovery.
Comparative example 4:
a method for treating an immobilized enzyme with a strengthening solution without using a porous solid support, comprising the steps of:
(1) 0.2mg of free glucose oxidase was adsorbed to 1mg of NH prepared in example 1 2 -MIL-101 (Fe) to obtain an immobilized enzyme;
(2) 100mM acetic acid-sodium acetate buffer solution with pH of 3.9 and containing 10mM tetraethyl orthosilicate (TEOS) is used as strengthening solution, the strengthening solution is added into the immobilized enzyme according to the volume ratio of 1000:1 (strengthening solution: immobilized enzyme) to perform co-incubation for 24 hours under the reverse oscillation of 4 ℃, and then the immobilized enzyme treated by the strengthening solution without using a porous solid phase carrier is obtained by centrifugal washing with the acetic acid-sodium acetate buffer solution with pH of 5.0 and concentration of 50 mM.
Performance test:
NH produced by the method described in example 1 2 Analysis of MIL-101 (Fe) and a Transmission Electron Microscope (TEM) of the porous metal-organic frame carrier, washing 2-3 times with a proper amount of deionized water, preparing into a proper concentration with absolute ethanol, dropping the sample on a copper mesh, sufficiently drying, observing the morphology with a transmission electron microscope (Talos L120C) and photographing, and the result is shown in FIG. 1.
The results showed that the synthesized NH 2 MIL-101 (Fe) is a regular octahedron with smooth surface and no mesoporous on the nano particles; NH after etching treatment 2 MIL-101 (Fe) porous metallo-organicThe surface of the frame carrier is rough, and obvious mesoporous structure can be observed.
Test of the ability to recycle immobilized enzyme after treatment with four strengthening solutions, GOx@LXTE-705-TEOS obtained in example 2, GOx@MOF-TMOS obtained in example 3, PLL@LXTE-705-APTES obtained in example 4, and PLL@MOF-APTES obtained in example 5: taking 8mL of immobilized enzyme treated by 10mg/mL of strengthening solution, centrifuging and collecting precipitate, using a reaction solution (the reaction solution is obtained by mixing 100 mu L of 0.4mM ABTS solution with 100 mu L of 6.0mM beta-D (+) glucose substrate solution to obtain a substrate solution, adding 3 mu L of 0.8mg/mL of horseradish peroxidase solution into 200 mu L of the substrate solution to obtain a substrate solution), standing at room temperature for reaction for 1h, centrifuging, discarding supernatant, washing 2 times by using an acetic acid buffer solution with pH of 5.0 and concentration of 50mM, and then re-suspending by using an acetic acid buffer solution with pH of 5.0 and concentration of 50mM to measure the enzyme activity of the immobilized enzyme after re-suspending. The above steps were repeated 10 times, and the relative activity was calculated with the 0 th enzyme activity as 100%, and the results are shown in FIG. 2.
The immobilized enzymes treated by four groups of strengthening solutions, namely GOx@LXTE-705-TEOS, GOx@MOF-TMOS, PLL@LXTE-705-APTES and PLL@MOF-APTES, all maintain relative activity of more than 60% after 10 times of cyclic use, which indicates that the immobilized enzymes obtained after the strengthening solution treatment have good cyclic use capability.
The immobilized enzyme treated with a strengthening solution without adding a small molecule mineralization reagent obtained in comparative example 1, the immobilized enzyme treated with a strengthening solution without performing a co-incubation process obtained in comparative example 2, the immobilized enzyme treated with a strengthening solution without washing to remove a small molecule mineralization reagent obtained in comparative example 3, and the four immobilized enzymes of immobilized enzyme treated with a strengthening solution without using a porous solid phase carrier obtained in comparative example 4 were tested for their ability to be recycled: taking 8mL of immobilized enzyme treated by 10mg/mL of strengthening solution, centrifuging and collecting precipitate, using a reaction solution (the reaction solution is obtained by mixing 100 mu L of 0.4mM of MABTS solution with 100 mu L of 6.0mM of beta-D (+) glucose substrate solution as a substrate solution, adding 3 mu L of 0.8mg/mL of horseradish peroxidase solution into 200 mu L of the substrate solution, standing at room temperature for reaction for 1h, centrifuging, discarding supernatant, washing 2 times by using an acetic acid buffer solution with pH of 5.0 and concentration of 50mM, and then re-suspending by using an acetic acid buffer solution with pH of 5.0 and concentration of 50mM, and measuring the enzyme activity of the immobilized enzyme after re-suspending). The above steps were repeated 10 times, and the relative activity was calculated with the 0 th enzyme activity as 100%, and the results are shown in FIG. 3.
The relative activity of the four groups of comparison examples is less than 60% after 10 cycles of use, wherein the immobilized enzyme treated by the strengthening liquid (comparison example 1) without adding the small molecule mineralization reagent has 49% relative activity after 10 cycles of use, and compared with the immobilized enzyme treated by the strengthening liquid obtained in example 2, which has more than 60% relative activity after 10 cycles of use, the immobilized enzyme can strengthen the recycling capability of the immobilized enzyme. The immobilized enzyme treated by the strengthening solution (comparative example 2) which is not subjected to the co-incubation process has 43% of relative activity after 10 times of use, and compared with the immobilized enzyme treated by the strengthening solution obtained in example 2 which has more than 60% of relative activity after 10 times of use, the immobilized enzyme shows that the effect of the small molecule mineralizing agent needs a certain time, and the co-incubation treatment can fully play a role in strengthening the reaction of the strengthening solution. The immobilized enzyme treated with the strengthening solution (comparative example 3) which is not washed to remove the small molecule mineralization reagent has 55% of relative activity after 10 uses, and compared with the immobilized enzyme treated with the strengthening solution obtained in example 2 which has more than 60% of relative activity after 10 uses, the immobilized enzyme shows that the small molecule mineralization reagent has a certain influence on enzymatic reaction. The relative activity of the immobilized enzyme treated by the strengthening liquid (comparative example 4) without using the porous solid phase carrier is only 8% after 10 times of recycling, which proves that the porous solid phase carrier has great influence on the immobilized enzyme, and the enzyme without using the porous solid phase carrier is easy to leak from the carrier to cause the loss of the enzyme activity.
Thermal stability ability test of immobilized glucose oxidase after treatment with free glucose oxidase and the strengthening solution obtained in example 3:
taking 8mL of immobilized glucose oxidase treated by 10mg/mL of strengthening solution, centrifuging in a 10mL EP tube, collecting precipitate, washing 2 times by using acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, re-suspending by using the acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, placing in a water bath kettle with the temperature of 70 ℃, sampling 100 mu L every 15min, measuring the enzyme activity of the immobilized enzyme, and calculating the relative activity by taking the enzyme activity before being placed in the water bath kettle as 100%.
8mL of free glucose oxidase with pH of 5.0 and concentration of 10mg/mL prepared by 50mM acetic buffer solution is taken in a 10mL EP tube, placed in a water bath kettle at 70 ℃, 100 mu L is sampled every 15min, and the enzyme activity of the free enzyme is measured, and the relative activity is calculated by taking the enzyme activity of the free enzyme as 100% before being placed in the water bath kettle. The results are shown in FIG. 4.
The relative activity of the free glucose oxidase is only 70% after 15min at the high temperature of 70 ℃, and the immobilized glucose oxidase after the strengthening liquid treatment has 98% relative activity after 15min and basically has no activity loss; after 75min, only 29.4% of the relative activity of free glucose oxidase remained, while the immobilized glucose oxidase after the strengthening solution treatment remained 64% of the relative activity. This demonstrates that the immobilized glucose oxidase after the strengthening solution treatment has greatly improved thermal stability compared with free glucose oxidase.
Organic solvent tolerance test of immobilized glucose oxidase after treatment with free glucose oxidase and the strengthening solution obtained in example 3:
taking 8mL of immobilized enzyme treated by 10mg/mL of strengthening solution, centrifuging, collecting precipitate, re-suspending by using acetone, standing for 24 hours at room temperature, centrifuging, discarding supernatant, washing for 2 times by using acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, re-suspending by using acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, measuring the enzyme activity of the re-suspended immobilized enzyme, and calculating relative activity by taking the enzyme activity before mixing acetone as 100%. A10 mg/mL solution of free glucose oxidase was prepared with 8mL of acetone, and the mixture was allowed to stand at room temperature for 24 hours in a 10mL EP tube, and the enzyme activity of the free glucose oxidase was measured, and the relative activity was calculated by taking the enzyme activity before mixing with acetone as 100%.
Taking 8mL of immobilized enzyme treated by 10mg/mL of strengthening solution, placing the immobilized enzyme in a 10mL EP tube, centrifuging, collecting precipitate, re-suspending the immobilized enzyme by DMF, standing the immobilized enzyme at room temperature for 24 hours, centrifuging, discarding supernatant, washing the immobilized enzyme for 2 times by using an acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, re-suspending the immobilized enzyme by using an acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, measuring the enzyme activity of the immobilized enzyme after re-suspending, and calculating the relative activity by taking the enzyme activity before mixing DMF as 100%. A10 mg/mL solution of free glucose oxidase was prepared with 8mL of DMF in a 10mL EP tube, and the mixture was allowed to stand at room temperature for 24 hours to determine the enzyme activity of the free enzyme, and the relative activity was calculated by taking the enzyme activity before DMF mixing as 100%.
Taking 8mL of immobilized enzyme treated by 10mg/mL of strengthening solution, placing the immobilized enzyme in a 10mL EP tube, centrifuging, collecting precipitate, resuspending the immobilized enzyme with isopropanol, standing the immobilized enzyme at room temperature for 24 hours, centrifuging, discarding supernatant, washing the immobilized enzyme for 2 times by using an acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, resuspending the immobilized enzyme with the acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, measuring the enzyme activity of the immobilized enzyme after resuspension, and calculating the relative activity by taking the enzyme activity before mixing the isopropanol as 100%. A10 mg/mL solution of free glucose oxidase was prepared with 8mL of isopropanol in a 10mL EP tube, and the mixture was allowed to stand at room temperature for 24 hours to determine the enzyme activity of the free enzyme, and the relative activity was calculated by taking the enzyme activity before mixing with isopropanol as 100%. The results are shown in FIG. 5.
As can be seen from fig. 5, the immobilized glucose oxidase and the free glucose oxidase after the strengthening solution treatment were treated with different organic solvents, respectively, and the immobilized glucose oxidase after the strengthening solution treatment exhibited more excellent resistance to the organic solvents.
Repeated freeze thawing stability performance test of free glucose oxidase and immobilized glucose oxidase after the treatment with the strengthening liquid was obtained in example 3:
taking 8mL of immobilized enzyme treated by 10mg/mL of strengthening solution, placing the immobilized enzyme treated by the strengthening solution in a 10mL EP tube, centrifugally collecting precipitate, washing the precipitate for 2 times by using an acetic acid buffer solution with the pH value of 5.0 and the concentration of 50mM, re-suspending the precipitate by using the acetic acid buffer solution with the pH value of 5.0 and the concentration of 50mM, freezing the precipitate in a refrigerator with the temperature of-80 ℃ for 30min, taking out the solution, incubating the solution for 10min at the temperature of 25 ℃ for remelting, repeating the freeze thawing operation, sampling 100 mu L of the immobilized enzyme solution after every freeze thawing, and calculating the relative activity by taking the enzyme activity of the 0 th time as 100%.
8mL of free glucose oxidase prepared with acetic acid buffer solution with pH of 5.0 and concentration of 50mM was taken out in a 10mL EP tube, the tube was placed in a refrigerator at-80 ℃ for 30min, and after taking out, the tube was incubated at 25 ℃ for 10min for remelting, the above freeze thawing operation was repeated, and 100. Mu.L of sample was taken after every freeze thawing to determine the enzyme activity of the free enzyme, and the relative activity was calculated with the enzyme activity at time 0 being 100%. The results are shown in FIG. 6.
The relative activity of the free glucose oxidase after repeated freezing and thawing for 10 times is 73%, and the relative activity of the immobilized glucose oxidase after the strengthening liquid treatment after repeated freezing and thawing for 10 times is 87%, which indicates that the immobilized glucose oxidase after the strengthening liquid treatment is greatly improved in repeated freezing and thawing stability compared with the free glucose oxidase.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (7)

1. A method for treating an immobilized enzyme with a strengthening solution, comprising the steps of:
(1) Adsorbing the free enzyme to a solid phase porous carrier to obtain immobilized enzyme;
(2) Adding the strengthening solution into the immobilized enzyme for co-incubation, and then washing by using a buffer solution B to obtain the immobilized enzyme treated by the strengthening solution;
the solid phase porous carrier in the step (1) is solid phase porous amination resin;
the strengthening liquid in the step (2) consists of a micromolecular mineralization reagent and a buffer solution A with the pH value of 3-10;
the micromolecular mineralizing agent is methyltrimethoxysilane, methyldiethoxysilane, ethyltriethoxysilane, methyltriethoxysilane, tetraethyl orthosilicate, tetramethoxysilane, bis (trimethylsilyl) acetamide, 3-aminopropyl triethoxysilane, 3-mercaptopropyl trimethoxysilane, trimethylchlorosilane, chlorotriethoxysilane, octyl trimethoxysilane, cyclohexyl methyldimethoxysilane, trimethoxysilane, triethoxysilane, benzyl triethoxysilane, vinyl trimethoxysilane and isobutyl triethoxysilane which can form silicon dioxide;
the buffer solution A with the pH value of 3-10 is at least one of acetic acid-sodium acetate buffer solution with the pH value of 3.0-3.9 and 50-100 mM and phosphate buffer solution with the pH value of 8.5 and 100 mM;
in the strengthening liquid in the step (2), small molecule mineralizing agent and buffer solution A with pH of 3-10 are mixed according to the molar concentration ratio of 10-1000:100;
the volume ratio of the strengthening liquid to the immobilized enzyme in the step (2) is 100-1000:1-2;
the incubation condition in the step (2) is that co-incubation is carried out for 0.15-24 h under the oscillation condition of 4-37 ℃;
the buffer solution B in the step (2) is at least one of acetic acid-sodium acetate buffer solution, glycine-sodium hydroxide buffer solution, HEPES buffer solution and Tris-HCl buffer solution.
2. A method for treating an immobilized enzyme with a strengthening solution, comprising the steps of:
(1) Adsorbing the free enzyme to a solid phase porous carrier to obtain immobilized enzyme;
(2) Adding the strengthening solution into the immobilized enzyme for co-incubation, and then washing by using a buffer solution B to obtain the immobilized enzyme treated by the strengthening solution;
the solid phase porous carrier in the step (1) is a porous metal organic framework carrier;
the porous metal organic framework carrier is prepared by the following preparation method:
1) FeCl is added 3 ·6H 2 Dissolving O and terephthalic acid in N, N-dimethylformamide, performing ultrasonic to clarify, performing high-temperature sealing reaction, cooling to room temperature, centrifugally collecting precipitate, cleaning, and vacuum drying to obtain a metal organic framework NH 2 -MIL-101(Fe)；
2) NH is added to 2 MIL-101 (Fe) dispersed in phosphorusIn acid solution, carrying out ultrasonic treatment, slowly stirring, reacting under the water bath condition, cooling, centrifugally collecting solids, washing and vacuum drying to obtain the porous metal organic framework carrier;
the strengthening liquid in the step (2) consists of a micromolecular mineralization reagent and a buffer solution A with the pH value of 3-10;
the micromolecular mineralizing agent is methyltrimethoxysilane, methyldiethoxysilane, ethyltriethoxysilane, methyltriethoxysilane, tetraethyl orthosilicate, tetramethoxysilane, bis (trimethylsilyl) acetamide, 3-aminopropyl triethoxysilane, 3-mercaptopropyl trimethoxysilane, trimethylchlorosilane, chlorotriethoxysilane, octyl trimethoxysilane, cyclohexyl methyldimethoxysilane, trimethoxysilane, triethoxysilane, benzyl triethoxysilane, vinyl trimethoxysilane and isobutyl triethoxysilane which can form silicon dioxide;
the buffer solution A with the pH value of 3-10 is at least one of acetic acid-sodium acetate buffer solution with the pH value of 3.0-3.9 and 50-100 mM and phosphate buffer solution with the pH value of 8.5 and 100 mM;
in the strengthening liquid in the step (2), small molecule mineralizing agent and buffer solution A with pH of 3-10 are mixed according to the molar concentration ratio of 10-1000:100;
the volume ratio of the strengthening liquid to the immobilized enzyme in the step (2) is 100-1000:1-2;
the incubation condition in the step (2) is that co-incubation is carried out for 0.15-24 h under the oscillation condition of 4-37 ℃;
the buffer solution B in the step (2) is at least one of acetic acid-sodium acetate buffer solution, glycine-sodium hydroxide buffer solution, HEPES buffer solution and Tris-HCl buffer solution.
3. A method according to claim 1 or 2, characterized in that,
the free enzyme in step (1) comprises at least one of a nucleic acid synthase, a nucleic acid ligase, a telomerase, a sugar isomerase, a racemase, a glutamine synthase, an aldolase, a phosphatase, a transmethylase, a phosphorylase, a dehydrogenase, a catalase, an acyltransferase, an amidase, a transaminase, a ketoreductase, an oxidase, a monooxygenase, a penicillin acylase, and a hydrolase.
4. The method of claim 3, wherein the step of,
the free enzyme is at least one of free glucose oxidase, free lipase and free penicillin acylase.
5. The method of claim 2, wherein the step of determining the position of the substrate comprises,
in step 1), the FeCl 3 ·6H 2 O and terephthalic acid are calculated according to a molar ratio of 2-3:1-2;
in the step 1), the conditions of the high-temperature sealing reaction are 100-140 ℃ for 25-32 h;
in the step 1), the cleaning is sequentially performed by DMF, ultrapure water and absolute ethyl alcohol;
in the step 1), the vacuum drying condition is that the vacuum drying is carried out for 22-26 hours at the temperature of 60-100 ℃;
in step 2), the NH 2 MIL-101 (Fe) and phosphoric acid solution are calculated according to the mass mg to volume mL ratio of 450-550:45-55;
in the step 2), the concentration of the phosphoric acid solution is 20-60 mM;
in the step 2), the reaction condition under the water bath condition is that the reaction is carried out for 0.5 to 3.5 hours under the water bath condition of 30 to 50 ℃.
6. A method according to claim 1 or 2, characterized in that,
the pH of the buffer solution B in the step (2) is 4.5-8.0, and the molar concentration is 20-100 mM.
7. Use of the method for immobilizing an enzyme by treating an immobilized enzyme with a strengthening solution according to any one of claims 1 to 6.
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