CN113234716A - Method for treating immobilized enzyme by using strengthening liquid and application thereof - Google Patents
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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 the immobilized enzyme by using the strengthening liquid comprises the following steps: (1) adsorbing free enzyme onto 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 treated by the strengthening solution. 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-thaw resistance and the like of the enzyme are improved.
Description
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 acyltransferases, amidases, transaminases, ketoreductases, oxidases, monooxygenases, hydrolases and the like are used in reactions involving antibiotics, herbicides, pharmaceutical intermediates and new-generation therapeutics.
The enzyme preparation is generally added into the enzyme catalysis reaction in the form of enzyme powder, the free enzyme is difficult to separate and reuse once being added into the system, and the cost is high due to the fact that the price of the enzyme preparation is generally high and the cost is high due to single use. The enzyme immobilization is one of effective methods for solving the difficulty, and the enzyme immobilization limits the biological enzyme to a carrier material, so that the biological enzyme is insoluble in a reaction medium, and has a series of advantages of high storage stability, easy separation and recovery, repeated use, continuous and controllable operation, simple and convenient process and the like while keeping the characteristics of high efficiency, specificity and mild enzyme catalytic reaction, and therefore, the enzyme immobilization is increasingly valued by researchers.
The prior immobilized enzyme technology is mainly used most commonly by four methods, namely a covalent bonding method, an adsorption method, a crosslinking method and an embedding method, but the suitable immobilization methods for different enzymes are different, and the four immobilization methods have advantages and disadvantages respectively. Wherein, the adsorption method is the earliest appearing immobilization method, the adsorption method can be divided into two methods, namely ion exchange adsorption and physical adsorption, the method has mild conditions, and the conformation of the enzyme can not be changed to a great extent basically, so that the catalysis of the enzyme can not be greatly influenced; however, the enzyme and the carrier have weak binding force, so that under some special conditions, such as high salt concentration, high temperature and the like, the enzyme is easy to fall off from the carrier and pollute catalytic reaction products, a serious enzyme leakage problem is faced, the recycling performance is poor, and the improvement of the recycling performance of the immobilized enzyme becomes a problem to be solved urgently.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention aims at providing a method for treating immobilized enzyme by using strengthening liquid, which aims at prolonging the service life and improving the recycling times of the immobilized enzyme by further treating the immobilized enzyme by using a biomimetic mineralization post-treatment technology.
It is still another object of the present invention to provide an application of the above method for treating an immobilized enzyme with a strengthening liquid.
The purpose of the invention is realized by the following technical scheme:
a method for treating immobilized enzyme by using strengthening liquid comprises the following steps:
(1) adsorbing free enzyme onto 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 treated by the strengthening solution.
The free enzyme in step (1) includes, but is not limited to, at least one of nucleic acid synthase, nucleic acid ligase, telomerase, sugar isomerase, racemase, glutamine synthetase, aldolase, phosphatase, methyltransferase, phosphorylase, dehydrogenase, catalase, acyltransferase, amidase, transaminase, ketoreductase, oxidase, monooxygenase, penicillin acylase, and hydrolase;
the hydrolytic enzyme includes but is not limited to at least one of lipase, amylase, protease, cellulase, pectinase and lactase.
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 carrier in the step (1) preferably comprises but is not limited to at least one of macroporous resin, porous silica particles and metal-organic framework material; the solid phase porous carrier is more preferably at least one of solid phase porous aminated resin and porous metal organic framework carrier.
The porous metal organic framework carrier is prepared by the following preparation method:
1) FeCl is added3·6H2Dissolving O and terephthalic acid in N, N-dimethylformamide, performing ultrasonic treatment to clarify, performing high-temperature sealing reaction, cooling to room temperature, centrifuging, collecting precipitate, cleaning, and vacuum drying to obtain metal organic framework NH2-MIL-101(Fe)；
2) Reacting NH2Dispersing MIL-101(Fe) in phosphoric acid solution, performing ultrasonic treatment, slowly stirring, reacting in water bath, cooling, centrifuging, collecting solid, washing, and vacuum drying to obtain porous metal organic framework carrier。
In step 1), the FeCl3·6H2Preferably, the molar ratio of O to terephthalic acid is 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 at 120 ℃ for 30 h.
In the step 1), the washing is preferably carried out by sequentially using DMF, ultrapure water and absolute ethyl alcohol; the number of cleaning is preferably 2-3.
In the step 1), the vacuum drying condition is preferably 60-100 ℃ for 22-26 h; more preferably 80 ℃ for 24 h.
In step 2), the NH2-MIL-101(Fe) and phosphoric acid solution are preferably calculated in a mass (mg) to volume (mL) ratio of 450-550: 45-55; more preferably in 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 30 mM.
In the step 2), the reaction condition under the water bath condition is preferably 30-50 ℃ for 0.5-3.5 h under the water bath condition; more preferably, the reaction is carried out for 1h under the condition of water bath at 40 ℃.
In step 2), the washing reagent is preferably deionized water and absolute ethyl alcohol.
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 solution 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 methyltrimethoxysilane, methyldiethoxysilane, ethyltriethoxysilane, methyltriethoxysilane, Tetraethylorthosilicate (TEOS), Tetramethoxysilane (TMOS), bis (trimethylsilyl) acetamide (BSA), 3-Aminopropyltriethoxysilane (APTES), 3-mercaptopropyltrimethoxysilane (MPTMS), Trimethylchlorosilane (TMCS), chlorotriethoxysilane (TECS), octyltrimethoxysilane, cyclohexylmethyldimethoxysilane, trimethoxysilane, triethoxysilane, benzyltriethoxysilane, vinyltrimethoxysilane, isobutyltriethoxysilane, tetraisopropyltitanate, methyl titanate, tetraethyltitanate, tetrabutyltitanate, tetrapropyltitanate, oligomers capable of forming insoluble calcium salts, calcium carbonate, Calcium phosphate oligomers, and at least one of metal organic frameworks ZIF-8, ZIF-67 that are water phase preparable; more preferably at least one of Tetraethylorthosilicate (TEOS), Tetramethoxysilane (TMOS), and 3-Aminopropyltriethoxysilane (APTES).
The buffer solution with the pH value of 3-10 is preferably at least one of a phosphate buffer solution, an acetic acid-sodium acetate buffer solution, a HEPES buffer solution, a Tris-HCl buffer solution and a carbonate buffer solution; more preferably at least one of an acetic acid-sodium acetate buffer solution of pH 3.0 to 3.9 and 50 to 100mM and a PBS buffer solution of pH 8.5 and 100 mM.
In the strengthening solution, a small molecular mineralization reagent and a buffer solution with the pH value of 3-10 are preferably mixed according to the molar concentration ratio of 10-1000: 100.
The ratio of the strengthening liquid to the immobilized enzyme in the step (2) is preferably 100-1000: 1-2 by volume.
The incubation condition in the step (2) is preferably that the co-incubation is carried out for 0.15-24 h under the shaking condition of 4-37 ℃; the oscillation is at least one of vortex oscillation and inversion oscillation.
The buffer solution in the step (2) is preferably at least one of acetic acid-sodium acetate buffer solution, glycine-sodium hydroxide buffer solution, HEPES buffer solution and Tris-HCl buffer solution.
The pH value of the buffer solution in the step (2) is preferably 4.5-8.0, and the molar concentration is 20-100 mM.
The method for treating the 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-thaw stability, organic solvent stability, recycling capability and the like, and the free enzyme is introduced into a pore channel structure of a porous solid phase carrier by adopting a pore adsorption method to realize the immobilization of the enzyme; further, the immobilized enzyme is further immobilized by adopting a biomimetic mineralization method through the strengthening liquid, so that the service life of the immobilized enzyme is prolonged, and the recycling times of the immobilized enzyme are increased.
(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-thaw resistance and the like of the enzyme are improved.
Drawings
FIG. 1 shows a metal organic framework NH2Transmission electron microscopy results of MIL-101(Fe) and porous metal organic framework support; wherein the diagram (a) is a metal organic framework NH2Transmission electron microscopy result plot of MIL-101 (Fe); FIG. b is a transmission electron microscope result image of the porous metal-organic framework support.
FIG. 2 is a graph showing the results of the recycling capacity of immobilized enzymes treated by four groups of strengthening liquids, 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 capabilities of the immobilized enzyme treated with the enhancing solution without adding the small molecule mineralization reagent obtained in comparative example 1, the immobilized enzyme treated with the enhancing solution without performing the co-incubation process obtained in comparative example 2, the immobilized enzyme treated with the enhancing solution without washing to remove the small molecule mineralization reagent obtained in comparative example 3, and the immobilized enzyme treated with the enhancing solution without using the porous solid phase carrier obtained in comparative example 4.
FIG. 4 is a graph showing the thermal stability results of GOx @ MOF-TMOS and free glucose oxidase.
FIG. 5 is a graph of organic solvent tolerance results for 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 the embodiments of the present invention are not limited thereto.
The enzyme activity determination method comprises the following steps:
1. glucose oxidase Activity assay
1.1BCA protein assay Standard Curve is drawn:
8 tubes were numbered, 1000. mu.g/mL 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 the BCA kit (Pierce)TMBCA protein quantitative analysis kit, Seimerfeishi science and technology company) prepares working solution C required by color reaction according to the volume ratio of 50:1, 25 mul of prepared standard solution is added into 200 mul of working solution C, the working solution C is fully and uniformly mixed and reacts for 2h at 25 ℃, then an enzyme-labeling instrument is used for measuring the absorbance value at 562nm, the x axis is used as the absorbance value, the y axis is used as the protein concentration, a protein standard curve is drawn, the linear regression equation of the protein standard curve is that y is 0.6886x-0.0015, R is2＝0.9996。
1.2BCA kit method for detecting protein content:
adding 200 mul of working solution C into 25 mul of samples needing to be detected for protein content, fully and uniformly mixing, reacting for 2h at 25 ℃, measuring the light absorption value at 562nm by using an enzyme-labeling instrument, and converting the concentration of the protein in the samples according to a standard curve for subsequent calculation.
1.3ABTS+Drawing a standard curve:
numbering 8 tubes, diluting 1000 μ M ABTS standard solution with deionized water to 1mL of 200 μ M, 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M and 0 μ M respectively, adding 100 μ L, 10 μ M hydrogen peroxide and 15 μ L, 0.25mg/mL horseradish peroxidase, reacting for 10min, measuring absorbance at 420nm with microplate reader, and taking x-axis as absorbance value and y-axis as ABTS standard solution+Concentration, plotting ABTS+Standard curve with linear regression equation of y-25 x-4.1, R2＝0.999。
1.4 glucose oxidase Activity measurement, 96-well plate method
The principle is as follows:
96-well plate reaction solution system (total volume 205. mu.L): 100. mu.L of 0.4mM ABTS solution was mixed with 100. mu.L of 6.0mM β -D (+) glucose substrate solution as a substrate solution, 3. mu.L of 0.8mg/mL horseradish peroxidase solution and 2. mu.L of free enzyme or immobilized enzyme solution were added to 200. mu.L of the substrate solution, and A was continuously monitored at 25 ℃400nmThe 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 (mL) of the enzyme added to the reaction system.
1Unit is defined as the cascade catalyzing the formation of 1. mu. mol of ABTS per minute of beta-D-glucose at pH 3.0, T.gtoreq.25 deg.C+The amount of glucose oxidase required (where HRP activity is excessive, assuming 100% activity in the cascade, i.e.H produced2O2Can be immediately combined with HRP to complete subsequent color reaction).
2. Lipase Activity assay
2.1 plotting p-nitrophenol standard curve:
0.02, 0.04, 0.06, 0.08, 0.12, 0.16mL of a p-nitrophenol mother liquor (2mM) was diluted to 220. mu.L with 50mM HEPES buffer solution having pH7.0, and the absorbance at 410nm was measured. Taking the concentration y (corresponding concentration is respectively 0.01, 0.02, 0.03, 0.04, 0.06 and 0.08, unit: mM) of p-nitrophenol as an ordinate and the light absorption value x as an abscissa, drawing a standard curve, wherein the linear regression equation is that y is 0.0675x-0.0069, R is2＝0.996。
2.2 Lipase Activity assay, 96-well plate method
The principle is as follows: the p-nitrophenol ester is a substrate which is most widely applied in the determination of the hydrolysis activity of the lipase, the p-nitrophenol produced by hydrolyzing the p-nitrobenzoic palmitate ester by the lipase is yellow, and the light absorption value is obtained at 410 nm.
96-well plate reaction solution system (total volume 440. mu.L): mu.L of 8.89mM p-nitrophenylpalmitate solution and 162. mu.L of 50mM HEPES buffer solution at pH7.0 were mixed to prepare a substrate solution, 40. mu.L of a free enzyme or immobilized enzyme solution was added to the substrate solution, the substrate solution was reacted at 37 ℃ for 5 minutes, 220. mu.L of absolute ethanol was added thereto to terminate the reaction, and 220. mu.L of the mixture was taken and the absorbance at 410nm was measured. Enzyme activity was calculated according to the following formula.
Wherein: 0.440 represents total system volume (mL);
0.040 represents the volume (mL) of enzyme added to the reaction system.
1Unit is defined as the amount of lipase required for the cascade to catalyze the hydrolysis of p-nitrophenol ester to 1. mu. mol p-nitrophenol per minute at pH7.0, T.37 ℃.
Example 1: preparation of porous metal organic framework carrier
A porous metal organic framework support comprising the steps of:
(1) FeCl is added3·6H2O (675mg, 2.45mmol) and terephthalic acid (H)2BDC) (206mg, 1.24mmol) is dissolved in 30mL of N, N-Dimethylformamide (DMF), ultrasonic treatment is carried out for 2min until the mixture is clear, then the mixture is placed in a reaction kettle and sealed, the mixture is fully reacted in an oven at 120 ℃ for 30h, the mixture is cooled to room temperature, the precipitate is centrifugally collected, the precipitate is sequentially washed with DMF, ultrapure water and absolute ethyl alcohol for 2-3 times, and the mixture is dried under vacuum at 80 ℃ for 24h, so that the metal organic framework NH is obtained2-MIL-101(Fe)；
(2) 500mg of the above NH2Redispersing MIL-101(Fe) in 50mL of 30mM phosphoric acid solution, performing ultrasonic treatment for 10min, slowly stirring, heating and reacting for 1h under the condition of 40 ℃ water bath, cooling to room temperature, centrifuging and collecting solid parts, and respectively washing with sufficient deionized water and absolute ethyl alcohol to obtain 2-And (3) drying for 12 hours in vacuum to ensure that no organic solvent remains, and drying to obtain the porous metal organic framework carrier.
Example 2
A method for treating immobilized enzyme by using strengthening liquid comprises the following steps:
(1) adsorbing 0.2mg of free glucose oxidase (purchased from Shanghai Yingxin laboratory facilities, Ltd., cat # TX 202042; the same below) onto 1mg of a solid-phase porous aminated resin (purchased from Xian blue, New science and technology materials, Ltd., cat # LXTE-705; the same below) to obtain an immobilized enzyme;
(2) a100 mM acetic acid-sodium acetate buffer solution containing 10mM tetraethyl orthosilicate (TEOS) and having a pH of 3.9 is used as a strengthening solution, the strengthening solution is added into an immobilized enzyme according to a volume ratio of 1000:1 (strengthening solution: immobilized enzyme) and incubated for 24 hours under reverse oscillation at 4 ℃, and then the immobilized enzyme (GOx @ LXTE-705-TEOS) treated by the strengthening solution is obtained by centrifugal washing with an acetic acid-sodium acetate buffer solution with a pH of 5.0 and a concentration of 50 mM.
Example 3
A method for treating immobilized enzyme by using strengthening liquid comprises the following steps:
(1) adsorbing 0.2mg of free glucose oxidase onto 1mg of the porous metal organic framework carrier prepared in example 1 to obtain an immobilized enzyme;
(2) a100 mM acetic acid-sodium acetate buffer solution containing 10mM Tetramethoxysilane (TMOS) and having a pH of 3.0 was used as a strengthening solution, the strengthening solution was added to an immobilized enzyme at a volume ratio of 100:1 (strengthening solution: immobilized enzyme) and incubated at 37 ℃ for 10min under reverse shaking, and then the immobilized enzyme (GOx MOF @ TMOS) treated with the strengthening solution was obtained by centrifugation and washing with a 50mM acetic acid-sodium acetate buffer solution having a pH of 5.5.
Example 4
A method for treating immobilized enzyme by using strengthening liquid comprises the following steps:
(1) adsorbing 0.3mg of free lipase (purchased from Shanghai Aladdin Biotechnology Ltd., product number: L298994) onto 1mg of solid-phase porous aminated resin (purchased from Xian Langxing scientific and New Material Ltd., product number: LXTE-705) to obtain an immobilized enzyme;
(2) a100 mM PBS buffer solution containing 500mM 3-Aminopropyltriethoxysilane (APTES) and having a pH of 8.5 was used as a strengthening solution, the strengthening solution was added to the immobilized enzyme at a volume ratio of 500:2 (strengthening solution: immobilized enzyme) and incubated at 4 ℃ for 15 hours under vortex oscillation, and then centrifuged and washed with a 100mM glycine-sodium hydroxide buffer solution having a pH of 8.0 to obtain a strengthening solution-treated immobilized enzyme (PLL @ LXTE-705-APTES).
Example 5
A method for treating immobilized enzyme by using strengthening liquid comprises the following steps:
(1) adsorbing 0.3mg of free lipase onto 1mg of the porous metal organic framework support prepared in example 1 to obtain an immobilized enzyme;
(2) a100 mM acetic acid-sodium acetate buffer solution containing 1000mM 3-Aminopropyltriethoxysilane (APTES) and having a pH of 3.0 was used as an enhancing solution, the enhancing solution was added to an immobilized enzyme at a volume ratio of 1000:1 (enhancing solution: immobilized enzyme) and incubated at 25 ℃ for 10min with inversion, and then washed by centrifugation using a Tris-HCl buffer solution having a pH of 4.5 and a concentration of 20mM to obtain an enhancing solution-treated immobilized enzyme (PLL @ MOF-APTES).
Comparative example 1:
a method for treating immobilized enzyme by using strengthening liquid without adding small molecule mineralizing reagent comprises the following steps:
(1) adsorbing 0.3mg of free lipase onto 1mg of solid-phase porous aminated resin (purchased from New science and technology materials Co., Ltd., product number: LXTE-705) to obtain an immobilized enzyme;
(2) 100mM acetic acid-sodium acetate buffer solution with pH of 3.9 is added into immobilized enzyme according to the volume ratio of 1000:1 (buffer solution: immobilized enzyme) for co-incubation for 24h under reversed oscillation at 4 ℃, and then the immobilized enzyme treated by the strengthening solution without adding small molecule mineralization reagent is obtained by centrifugal washing with acetic acid-sodium acetate buffer solution with pH of 5.0 and concentration of 50 mM.
Comparative example 2:
a method for treating immobilized enzyme with strengthening liquid without co-incubation process comprises the following steps:
(1) adsorbing 0.3mg of free lipase onto 1mg of solid-phase porous aminated resin (purchased from New science and technology materials Co., Ltd., product number: LXTE-705) to obtain an immobilized enzyme;
(2) a100 mM acetic acid-sodium acetate buffer solution containing 10mM tetraethyl orthosilicate (TEOS) and having a pH of 3.9 was used as a strengthening solution, an immobilized enzyme was added to the strengthening solution at a volume ratio of 1000:1 (strengthening solution: immobilized enzyme), mixing was performed at 4 ℃ with vortex oscillation, and after uniform mixing, centrifugal washing was immediately performed using an acetic acid-sodium acetate buffer solution having a pH of 5.0 and a concentration of 50mM to obtain an immobilized enzyme treated with the strengthening solution without a co-incubation process.
Comparative example 3:
a method for treating immobilized enzyme by using strengthening liquid without washing to remove small molecule mineralizing reagent comprises the following steps:
(1) adsorbing 0.3mg of free glucose oxidase onto 1mg of solid phase porous aminated resin (purchased from New science and technology materials Co., Ltd., product number: LXTE-705) to obtain immobilized enzyme;
(2) taking 100mM acetic acid-sodium acetate buffer solution with pH of 3.9 and containing 10mM tetraethyl orthosilicate (TEOS) as strengthening solution, adding the strengthening solution into immobilized enzyme according to the volume ratio of 1000:1 (strengthening solution: immobilized enzyme) for co-incubation for 24h under reverse oscillation at 4 ℃, and directly recovering the immobilized enzyme treated by the strengthening solution without washing and removing the small molecular mineralization reagent without washing the buffer solution.
Comparative example 4:
a method for treating an immobilized enzyme with a strengthening liquid without using a porous solid phase carrier, comprising the steps of:
(1) 0.2mg of free glucose oxidase was adsorbed to 1mg of NH prepared in example 12-MIL-101(Fe) to obtain an immobilized enzyme;
(2) taking 100mM acetic acid-sodium acetate buffer solution containing 10mM tetraethyl orthosilicate (TEOS) and having a pH value of 3.9 as strengthening solution, adding the strengthening solution into the immobilized enzyme according to the volume ratio of 1000:1 (strengthening solution: immobilized enzyme) for co-incubation for 24h under reverse oscillation at 4 ℃, and then centrifugally washing by using 50mM acetic acid-sodium acetate buffer solution with a pH value of 5.0 to obtain the immobilized enzyme treated by the strengthening solution without using a porous solid phase carrier.
And (3) performance testing:
NH prepared as described in example 12-Transmission Electron Microscopy (TEM) analysis of MIL-101(Fe) and porous metal organic framework support, washing with appropriate amount of deionized water for 2-3 times, formulating to appropriate concentration with anhydrous ethanol, dropping the sample on copper mesh, drying thoroughly, observing its morphology with transmission electron microscope (Talos L120C) and taking pictures, the results are shown in fig. 1.
The results show that NH was synthesized2MIL-101(Fe) is regular octahedron, the surface is smooth, and no mesopores exist on the nano particles; NH after etching treatment2MIL-101(Fe) porous metal organic framework support, the surface is rough, and obvious mesoporous structure can be observed.
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 were tested for recycling ability of immobilized enzyme after treatment with four strengthening liquids: the immobilized enzyme treated with 8mL or 10mg/mL of the enhancing solution was placed in a 10mL EP tube, and the precipitate was collected by centrifugation, resuspended in a reaction solution (obtained by mixing 100. mu.L or 0.4mM ABTS solution with 100. mu.L or 6.0mM beta-D (+) glucose substrate solution as a substrate solution, and adding 3. mu.L of 0.8mg/mL horseradish peroxidase solution to 200. mu.L of the substrate solution), allowed to stand at room temperature for 1 hour, centrifuged, the supernatant was discarded, washed 2 times with a 50mM acetic acid buffer solution having a pH of 5.0, and then resuspended in a 50mM acetic acid buffer solution having a pH of 5.0, and the enzyme activity of the resuspended immobilized enzyme was measured. The above steps are repeated for 10 times, and the relative activity is calculated by taking the 0 th enzyme activity as 100%, and the result is shown in fig. 2.
The immobilized enzymes treated by four groups of strengthening liquid, namely GOx @ LXTE-705-TEOS, GOx @ MOF-TMOS, PLL @ LXTE-705-APTES and PLL @ MOF-APTES, have relative activity of more than 60% after being recycled for 10 times, which shows that the immobilized enzymes obtained after being treated by the strengthening liquid have good recycling capability.
The ability of the immobilized enzyme treated with the enhancing solution without the addition of the small molecule mineralization reagent obtained in comparative example 1, the immobilized enzyme treated with the enhancing solution without the co-incubation process obtained in comparative example 2, the immobilized enzyme treated with the enhancing solution without washing to remove the small molecule mineralization reagent obtained in comparative example 3, and the immobilized enzyme treated with the enhancing solution without the use of the porous solid phase carrier obtained in comparative example 4 was tested: the immobilized enzyme treated by 8mL and 10mg/mL of the strengthening solution is put in a 10mL EP tube, the precipitate is collected by centrifugation, the reaction solution (the reaction solution is obtained by mixing 100 mu L and 0.4mMABTS solution with 100 mu L and 6.0mM beta-D (+) glucose substrate solution as substrate solution, and 3 mu L of 0.8mg/mL horseradish peroxidase solution is added into 200 mu L substrate solution) is used for resuspending, the reaction is kept still for 1h at room temperature, the supernatant is discarded after centrifugation, the reaction solution is washed for 2 times by acetic acid buffer solution with pH of 5.0 and concentration of 50mM, then the acetic acid buffer solution with pH of 5.0 and concentration of 50mM is used for resuspension, and the enzyme activity of the immobilized enzyme after resuspension is measured. 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 control examples after 10 cycles of use is less than 60%, wherein 49% of the relative activity of the immobilized enzyme (control example 1) which is not added with the small-molecule mineralization reagent and treated by the strengthening liquid is reserved after 10 cycles of use, and compared with more than 60% of the relative activity of the immobilized enzyme after 10 cycles of use, which is obtained in example 2, of the immobilized enzyme treated by the strengthening liquid, the small-molecule mineralization reagent can strengthen the cycle use capability of the immobilized enzyme. The immobilized enzyme treated by the strengthening liquid (comparative example 2) without the co-incubation process has 43% of relative activity after 10 times of use, and compared with the immobilized enzyme treated by the strengthening liquid obtained in example 2, which has more than 60% of relative activity after 10 times of use, the activity of the small molecule mineralization reagent needs a certain time, and the co-incubation treatment can completely strengthen the reaction of the strengthening liquid. The immobilized enzyme treated by the strengthening liquid (comparative example 3) without washing and removing the small molecule mineralization reagent has 55% of relative activity after 10 times of use, and compared with the immobilized enzyme treated by the strengthening liquid obtained in example 2 and having more than 60% of relative activity after 10 times of use, the immobilized enzyme shows that the small molecule mineralization reagent has certain influence on enzymatic reaction. The immobilized enzyme (comparative example 4) which is not used with the porous solid phase carrier and is treated by the strengthening liquid has the relative activity of only 8 percent after being recycled for 10 times, which shows that the porous solid phase carrier has great influence on the immobilized enzyme, and the enzyme which is not used with the porous solid phase carrier is easy to leak from the carrier to cause the loss of the enzyme activity.
Testing the thermal stability of the immobilized glucose oxidase treated by the free glucose oxidase and the strengthening solution obtained in example 3:
taking 8mL of immobilized glucose oxidase treated by 10mg/mL of strengthening solution into a 10mL EP tube, centrifugally collecting precipitates, washing for 2 times by using an acetic acid buffer solution with pH of 5.0 and concentration of 50mM, then re-suspending by using the acetic acid buffer solution with pH of 5.0 and concentration of 50mM, placing in a water bath kettle at 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 placing in the water bath kettle as 100%.
Taking 8mL of free glucose oxidase with pH of 5.0 and concentration of 10mg/mL prepared by 50mM acetic acid buffer solution, placing the free glucose oxidase in a 10mL EP tube, placing the tube in a 70 ℃ water bath, sampling 100 mu L every 15min, measuring the enzyme activity of the free glucose oxidase, and calculating the relative activity by taking the enzyme activity before placing the tube in the water bath as 100%. The results are shown in FIG. 4.
Under the high-temperature treatment of 70 ℃, only 70% of relative activity is left after 15min of the free glucose oxidase, and 98% of relative activity is remained after 15min of the immobilized glucose oxidase treated by the strengthening solution, so that the activity is basically not lost; after 75min, only 29.4% of relative activity of the free glucose oxidase remained, while 64% of relative activity of the immobilized glucose oxidase after the strengthening solution treatment remained. This shows that the thermal stability of the immobilized glucose oxidase treated by the strengthening solution is greatly improved compared with that of the free glucose oxidase.
Testing the organic solvent resistance of the immobilized glucose oxidase treated by the free glucose oxidase and the strengthening solution obtained in example 3:
taking 8mL and 10mg/mL immobilized enzyme treated by the strengthening solution into a 10mL EP tube, centrifuging, collecting precipitates, resuspending the precipitates by acetone, standing the precipitates for 24 hours at room temperature, centrifuging, discarding the supernatant, washing the precipitates for 2 times by using an acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, then resuspending the precipitates by using 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 acetone as 100 percent. Preparing 10mg/mL free glucose oxidase solution by using 8mL of acetone, standing the solution in a 10mL EP tube at room temperature for 24 hours, measuring the enzyme activity of the free enzyme, and calculating the relative activity by taking the enzyme activity before mixing the acetone as 100%.
Taking 8mL and 10mg/mL immobilized enzyme treated by the strengthening solution into a 10mL EP tube, centrifuging, collecting precipitates, resuspending the precipitates by DMF, standing the precipitates for 24 hours at room temperature, centrifuging, discarding the supernatant, washing the precipitates for 2 times by an acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, then resuspending the precipitates by 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 DMF as 100 percent. Preparing 10mg/mL free glucose oxidase solution by 8mL DMF, standing for 24h at room temperature in a 10mL EP tube, measuring the enzyme activity of the free enzyme, and calculating the relative activity by taking the enzyme activity before mixing the DMF as 100%.
Taking 8mL and 10mg/mL immobilized enzyme treated by the strengthening solution into a 10mL EP tube, centrifuging, collecting precipitates, resuspending by isopropanol, standing for 24h at room temperature, centrifuging, discarding a supernatant, washing for 2 times by using an acetic acid buffer solution with the pH of 5.0 and the concentration of 50mM, then resuspending 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 resuspension, and calculating the relative activity by taking the enzyme activity before mixing with the isopropanol as 100%. Preparing 10mg/mL free glucose oxidase solution by using 8mL of isopropanol, standing the solution in a 10mL EP tube at room temperature for 24 hours, measuring the enzyme activity of the free enzyme, and calculating the relative activity by taking the enzyme activity before mixing the isopropanol as 100%. The results are shown in FIG. 5.
As can be seen from fig. 5, when the immobilized glucose oxidase and the free glucose oxidase after the treatment with the enhancing solution were treated with different organic solvents, the immobilized glucose oxidase after the treatment with the enhancing solution exhibited more excellent resistance to the organic solvents.
Repeated freeze-thaw stability testing of free glucose oxidase and immobilized glucose oxidase treated with the strengthening solution obtained in example 3:
and (2) putting the immobilized enzyme treated by 8mL and 10mg/mL of strengthening solution into a 10mL EP tube, centrifugally collecting precipitates, washing for 2 times by using an acetic acid buffer solution with pH of 5.0 and concentration of 50mM, then re-suspending by using the acetic acid buffer solution with pH of 5.0 and concentration of 50mM, freezing for 30min in a refrigerator at-80 ℃, taking out, incubating for 10min at 25 ℃ for re-melting, repeating the freezing and thawing operation, sampling 100 mu L of immobilized enzyme solution after each freezing and thawing, and calculating the relative activity by taking the enzyme activity of the 0 th time as 100%.
Taking 8mL of free glucose oxidase of 10mg/mL prepared by using acetic acid buffer solution of which the pH value is 5.0 and the concentration is 50mM, placing the free glucose oxidase in a 10mL EP tube, freezing the free glucose oxidase in a refrigerator at the temperature of minus 80 ℃ for 30min, taking out the free glucose oxidase, incubating the cooled free glucose oxidase at the temperature of 25 ℃ for 10min, thawing the free glucose oxidase again, repeating the freezing and thawing operation, sampling 100 mu L of the free glucose oxidase after each freezing and thawing operation to determine the enzyme activity of the free glucose oxidase, and calculating the relative activity by taking the enzyme activity of the 0 th time as 100%. The results are shown in FIG. 6.
73% of the relative activity of the free glucose oxidase is remained after 10 times of repeated freeze thawing, and 87% of the relative activity of the immobilized glucose oxidase after the strengthening solution treatment is still remained after 10 times of repeated freeze thawing, which indicates that the immobilized glucose oxidase after the strengthening solution treatment is greatly improved in repeated freeze thawing stability compared with the free glucose oxidase.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for treating immobilized enzyme by using strengthening liquid is characterized by comprising the following steps:
(1) adsorbing free enzyme onto 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 treated by the strengthening solution.
2. The method of claim 1,
the strengthening solution 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 methyltrimethoxysilane, methyldiethoxysilane, ethyltriethoxysilane, methyltriethoxysilane, tetraethyl orthosilicate, tetramethoxysilane, bis (trimethylsilyl) acetamide, 3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, trimethylchlorosilane, chlorotriethoxysilane, octyltrimethoxysilane, cyclohexylmethyldimethoxysilane, trimethoxysilane, triethoxysilane, benzyltriethoxysilane, vinyltrimethoxysilane, isobutyltriethoxysilane, tetraisopropyltitanate, methyl titanate, tetraethyl titanate, tetrabutyl titanate, tetrapropyl titanate which can form titanium dioxide, calcium carbonate oligomer which can form insoluble calcium salt, calcium phosphate oligomer, and metal organic framework ZIF-8, which can be prepared in aqueous phase, At least one of ZIF-67;
the buffer solution with the pH value of 3-10 is one of phosphate buffer solution, acetic acid-sodium acetate buffer solution, HEPES buffer solution, Tris-HCl buffer solution and carbonate buffer solution.
3. The method of claim 2,
the small molecule mineralizing reagent is at least one of tetraethyl orthosilicate, tetramethoxysilane and 3-aminopropyltriethoxysilane;
the buffer solution with the pH of 3-10 is at least one of an acetic acid-sodium acetate buffer solution with the pH of 3.0-3.9 and 50-100 mM and a phosphate buffer solution with the pH of 8.5 and 100 mM;
in the strengthening solution, a small molecular mineralization reagent and a buffer solution with the pH value of 3-10 are mixed according to the molar concentration ratio of 10-1000: 100.
4. The method according to claim 1, wherein the incubation in step (2) is performed under shaking conditions at 4-37 ℃ for 0.15-24 h;
and (3) calculating the reinforcing liquid and the immobilized enzyme in the step (2) according to the volume ratio of 100-1000: 1-2.
5. The method of claim 1, wherein the solid phase porous carrier in step (1) comprises at least one of but not limited to macroporous resin, porous silica particles, and metal-organic framework material;
the free enzyme in step (1) includes, but is not limited to, at least one of nucleic acid synthase, nucleic acid ligase, telomerase, sugar isomerase, racemase, glutamine synthetase, aldolase, phosphatase, methyltransferase, phosphorylase, dehydrogenase, catalase, acyltransferase, amidase, transaminase, ketoreductase, oxidase, monooxygenase, penicillin acylase, and hydrolase.
6. The method of claim 5,
the solid phase porous carrier is at least one of solid phase porous aminated resin and a porous metal organic framework carrier;
the free enzyme is at least one of free glucose oxidase, free lipase and free penicillin acylase.
7. The method of claim 6,
the porous metal organic framework carrier is prepared by the following preparation method:
1) FeCl is added3·6H2Dissolving O and terephthalic acid in N, N-dimethylformamide, performing ultrasonic treatment to clarify, performing high-temperature sealing reaction, cooling to room temperature, centrifuging, collecting precipitate, cleaning, and vacuum drying to obtain metal organic framework NH2-MIL-101(Fe)；
2) Reacting NH2Dispersing MIL-101(Fe) in a phosphoric acid solution, performing ultrasonic treatment, slowly stirring, reacting in a water bath, cooling, centrifuging, collecting a solid, washing, and performing vacuum drying to obtain the porous metal organic framework carrier.
8. The method of claim 7,
in step 1), the FeCl3·6H2Calculating the molar ratio of O to terephthalic acid (TPA) to 2-3: 1-2;
in the step 1), the high-temperature sealing reaction is carried out for 25-32 h at 100-140 ℃;
in the step 1), the cleaning is sequentially carried out by using DMF, ultrapure water and absolute ethyl alcohol;
in the step 1), the vacuum drying condition is vacuum drying for 22-26 h at the temperature of 60-100 ℃;
in step 2), the NH2-MIL-101(Fe) and phosphoric acid solution in a 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 0.5-3.5 hours under the water bath condition of 30-50 ℃.
9. The method of claim 1,
the buffer solution 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;
the pH value of the buffer solution in the step (2) is 4.5-8.0, and the molar concentration is 20-100 mM.
10. Use of the method for treating an immobilized enzyme with a strengthening liquid according to any one of claims 1 to 9 for enzyme immobilization.
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