CN110724682A - Method for preparing immobilized enzyme from zeolite imidazole ester framework compound - Google Patents
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Abstract
The invention discloses a method for preparing immobilized enzyme by using a zeolite imidazole ester framework compound. The method comprises the following steps: respectively preparing a 2-methylimidazole solution, a zinc acetate aqueous solution and a polyethylene glycol aqueous solution; centrifuging the zymogen liquid, removing supernatant, adding the polyethylene glycol aqueous solution, performing ultrasonic treatment to obtain a polyethylene glycol/carboxypeptidase A compound solution, performing ultrafiltration on the compound solution, and transferring the polyethylene glycol/carboxypeptidase A compound into a 2-methylimidazole aqueous solution by using a 2-methylimidazole aqueous solution to form a mixed solution; and adding a zinc acetate aqueous solution into the mixed solution, magnetically stirring, standing for aging, centrifugally collecting a solid product, washing with water, and drying to obtain the immobilized enzyme prepared from the zeolite imidazole ester framework compound. The method can greatly improve the stability and the loading rate of the enzyme, prolong the storage time of the enzyme, and the prepared compound of the zeolite imidazole ester framework compound loaded with the carboxypeptidase A has the advantages of simple recovery operation, economy, high efficiency and high reusability.
Description
Technical Field
The invention relates to an immobilized enzyme technology, which is applicable to the industrial fields of food safety and the like, in particular to a method for preparing immobilized enzyme from a zeolite imidazole ester framework compound.
Background
Examples of the enzyme immobilization include physical adsorption, coupling, entrapment, and crosslinking. The metal organic framework compound can be used as a carrier material for enzyme immobilization. The method for forming the complex enzyme by physical adsorption has a certain limitation because the pore size of most metal organic framework compounds is far smaller than the size of the enzyme, even if the pore size of the metal organic framework compounds is close to or larger than the size of the enzyme, if the combination between the metal organic framework compounds is relatively weak, the enzyme is likely to be washed away, and the loading rate of the existing adsorption method is about 19 percent. Coupling is through chemical cross-linking between carrier and enzyme, and enzyme and carrier are strong in binding force, but the structure of enzyme molecule is easily influenced. The entrapping method is to encapsulate an enzyme in a polymer gel or semi-permeable membrane microcapsule, but this method is liable to cause chemical polymerization, resulting in a decrease in enzyme activity.
Carboxypeptidase is a biodegradable enzyme that is effective in degrading OTA, and it is now well recognized that there is only a three-dimensional crystal structure of CPA and CPB, in which Carboxypeptidase A (CPA) is present in mammalian pancreas and has a relative molecular mass of 34.6kDa, and each enzyme molecule contains a Zn molecule2+As a prosthetic group, the enzyme protein is a single polypeptide chain with about 300 amino acid residues and a molecular size of 5 × 4.2 × 3.8. Related studies have demonstrated that carboxypeptidase A can degrade ochratoxin A to ochratoxin alpha and L-beta-phenylalanine. However, the free carboxypeptidase A has poor stability in the using process, such as thermal stability, pH tolerance range and tolerance to organic reagents, and the like, and the free carboxypeptidase A is difficult to separate and purify after degrading a substrate, is not favorable for recycling and is greatly limited in practical production application.
Disclosure of Invention
The invention aims to provide a method for preparing immobilized enzyme by using a zeolite imidazole ester framework compound.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
a method for preparing immobilized enzyme by zeolite imidazole ester framework compound comprises the following steps:
(1) adding 2-methylimidazole into distilled water or ultrapure water to prepare a 2-methylimidazole solution with the concentration of 10-210 g/L, adding zinc acetate into distilled water or ultrapure water to prepare a zinc acetate aqueous solution with the concentration of 65-95 g/L, and preparing a polyethylene glycol aqueous solution with the concentration of 10-20 g/L;
(2) centrifuging the zymogen liquid with the concentration of 24-28 g/L for 3-7 minutes, removing supernatant, adding the polyethylene glycol aqueous solution obtained in the step (1), and performing ultrasonic treatment for 10-20 minutes to obtain a polyethylene glycol/carboxypeptidase A compound solution;
(3) ultrafiltering the polyethylene glycol/carboxypeptidase A compound solution obtained in the step (2) for 20-40 minutes, and transferring the polyethylene glycol/carboxypeptidase A compound into a 2-methylimidazole water solution by using the 2-methylimidazole water solution prepared in the step (1) to form a mixed solution; and (2) immediately adding the zinc acetate aqueous solution obtained in the step (1) into the mixed solution, magnetically stirring for 3-10 minutes, standing and aging for 5-24 hours, centrifugally collecting a solid product, washing with water for 1-3 times, and drying to obtain the immobilized enzyme prepared from the zeolite imidazole ester framework compound.
Preferably, the enzyme stock solution in the step (2) is bovine pancreatic carboxypeptidase A enzyme stock solution (the immobilized enzyme obtained correspondingly is a zeolite imidazole ester framework compound loaded carboxypeptidase A).
Preferably, the concentration of the 2-methylimidazole aqueous solution in the step (1) is 13-198 g/L, and the concentration of the zinc acetate aqueous solution is 70-88 g/L.
Preferably, the mass ratio of the 2-methylimidazole to the zinc acetate to the enzyme stock solution to the polyethylene glycol is 106-4: 9-30: 1-2: 1.
Preferably, the drying in the step (3) is freeze drying for 40 to 60 hours at a temperature of between 30 ℃ below zero and 50 ℃ below zero, or natural drying for 20 to 30 hours at room temperature.
Preferably, the rotating speed of the centrifugation is 8000-12000 r/min.
The working principle of the invention is as follows: the amino acid has a structure similar to Ca2+And Zn2+Etc., thereby causing biomineralization. During the crystal growth process, the amido bond of carboxypeptidase A is connected with Zn2+And (3) interacting to regulate the embedding mode of the zeolite imidazole ester framework compound and the enzyme. Intermolecular hydrogen bonding and hydrophobic bonding interactions, in which the affinity of biological macromolecules (enzymes) for organic molecules (imidazoles) plays a crucial role. In addition, the synthesis conditions also affect the morphology of the porous backbone compound formed around the enzyme.
The invention has the beneficial effects that:
the immobilized enzyme prepared by the bionics mineralization method can ensure the stability of the enzyme to the greatest extent, prevent the leaching of the enzyme, ensure the reusability of the enzyme to the greatest extent and contribute to reducing the process cost. Another significant advantage is that there is no restriction on the size of the enzyme, even for enzymes having a size larger than the pore size of the metal-organic framework compound, the enzyme can be immobilized on the support material by this method.
After immobilization of the enzyme, the storage time of the enzyme increases. The residual activity after two months of storage of free carboxypeptidase A was 4%, and the residual activity after two months of storage of the zeolitic imidazolate framework compound-loaded carboxypeptidase A complex was 52.43%. The residual activity of the zeolitic imidazolate framework loaded carboxypeptidase A complex in 50% ethanol/phosphate buffer was 93.11%, whereas the free enzyme was only 12.42%. The activity of the compound of zeolite imidazole ester framework loaded with carboxypeptidase A can still be kept 97% after 7 times of recycling, while the residual activity of free carboxypeptidase A is only 62.47% after 7 times of recycling.
The method is simple to operate, economical and efficient, the immobilized enzyme is stable in property and high in reusability, and the loading rate of the zeolite imidazole ester framework compound loaded with carboxypeptidase A reaches 70%.
Drawings
A, C and E in FIG. 1 are electron micrographs of the zeolitic imidazolate framework compounds synthesized in comparative examples 1-3, respectively, and FIGS. B, D and F are carboxypeptidase A-loaded complexes of the zeolitic imidazolate framework compounds synthesized in examples 1-3;
FIG. 2A is a Fourier infrared spectrum diagram of the compound and the complex synthesized in comparative example 1 and example 1, FIG. 2B is an XRD diagram of the compound and the complex synthesized in comparative example 2 and example 2, and FIG. 2C is a TGA diagram of the complex synthesized in example 3;
a, B, C of FIG. 3 is a graph showing the formation of products from the decomposition of the substrate catalyzed by the free carboxypeptidase A and the zeolitic imidazolate framework compound-loaded carboxypeptidase A complex of examples 1-3, respectively;
FIG. 4 is a graph of the effect of days of storage on free carboxypeptidase A of control example 1 and carboxypeptidase A complex loading the zeolitic imidazolate framework compound of example 1; FIG. B is a graph of the effect of varying proportions of ethanol content on the free carboxypeptidase A of control example 1 and the carboxypeptidase A complex supported on the zeolitic imidazolate framework compound of example 1;
FIG. 5 is a graph of the effect of temperature on the free carboxypeptidase A of control example 2 and the carboxypeptidase A complex loaded with the zeolitic imidazolate framework compound of example 2;
FIG. 6 is a graph of the number of cycles of free carboxypeptidase A of example 3 and a complex of carboxypeptidase A loaded with a zeolitic imidazolate framework compound and control 3.
Detailed Description
The present invention is further illustrated with reference to specific examples, which do not specify specific conditions, which protocols are generally carried out under conventional conditions, or according to conditions recommended by the manufacturer of the kit. The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples.
The complexes described below all refer to the zeolitic imidazolate framework compound-supported carboxypeptidase A complex obtained in the examples, which is a zeolitic imidazolate framework compound.
Example 1 Synthesis of a Zeolite Imidazol ester framework Compound Supported carboxypeptidase A Complex
(1) Weighing 0.0657g of 2-methylimidazole, adding 5mL of ultrapure water into the 2-methylimidazole solution to prepare a 2-methylimidazole aqueous solution, adding 0.439g of zinc acetate into 5mL of ultrapure water to prepare a zinc acetate aqueous solution, and preparing 1mL of polyethylene glycol aqueous solution with the concentration of 15 mg/mL;
(2) taking 960 microliters (0.025g) of zymogen liquid by using a liquid transfer gun, placing the zymogen liquid into a 1.5mL centrifuge tube, centrifuging for 5 minutes, removing supernatant, adding the 1mL polyethylene glycol aqueous solution prepared in the step (1) into the centrifuge tube, and performing ultrasonic treatment for 15 minutes to form polyethylene glycol/carboxypeptidase A compound solution of polyethylene glycol-coated carboxypeptidase A;
(3) putting the polyethylene glycol/carboxypeptidase A compound solution into an ultrafiltration centrifugal tube for ultrafiltration for 30 minutes, pouring off filtrate in an ultrafiltration tube sleeve, and flushing a lining tube of the ultrafiltration centrifugal tube by using the 2-methylimidazole aqueous solution prepared in the step (1) until the polyethylene glycol/carboxypeptidase A compound in the lining tube is completely transferred into the 2-methylimidazole aqueous solution to form a mixed solution; immediately adding the zinc acetate aqueous solution in the step (1) into the mixed solution, placing the mixed solution into a 20ml small bottle for reaction, placing a magnetic rotor in the small bottle, uniformly stirring for 5 minutes, standing and aging for 5 hours, centrifuging at 10000r/min to collect a solid product and a supernatant for testing, centrifuging again after washing once to collect the solid product and the supernatant after washing once, and freeze-drying the product (-40 ℃, 48 hours).
Comparative example 1 Synthesis of Zeolite Imidazol ester framework Compound
0.0657g of 2-methylimidazole is weighed and added into 5ml of ultrapure water to prepare a solution, and then 0.439g of zinc acetate is added into 5ml of water to prepare an aqueous solution. Mixing the two prepared solutions, placing the two solutions into a 20ml small bottle for reaction, placing a magnetic rotor into the small bottle, uniformly stirring the mixture for 5 minutes, standing and aging the mixture for 5 hours, centrifuging the mixture at 10000r/min to collect a product, and freeze-drying the product (-40 ℃, 48 hours). To obtain the zeolite imidazole ester framework compound.
Example 2 Synthesis of Zeolite Imidazol ester framework Compound Supported carboxypeptidase A Complex
(1) 1.0526g of 2-methylimidazole is weighed and added into 8mL of ultrapure water to prepare 2-methylimidazole water solution, 0.1407g of zinc acetate is added into 2mL of ultrapure water to prepare zinc acetate water solution, and 1mL of polyethylene glycol water solution with the concentration of 15mg/mL is prepared;
(2) taking 960 microliters (0.025g) of zymogen liquid by using a liquid transfer gun, placing the zymogen liquid into a 1.5mL centrifuge tube, centrifuging (10000r/min) for 5 minutes, removing supernatant, adding 1mL of polyethylene glycol aqueous solution prepared in the step (1) into the centrifuge tube, and performing ultrasonic treatment for 15 minutes to form polyethylene glycol/carboxypeptidase A compound solution of polyethylene glycol-coated carboxypeptidase A;
(3) putting the polyethylene glycol/carboxypeptidase A compound solution into an ultrafiltration centrifugal tube for ultrafiltration for 30 minutes, pouring off filtrate in an ultrafiltration tube sleeve, and flushing a lining tube of the ultrafiltration centrifugal tube by using the 2-methylimidazole aqueous solution prepared in the step (1) until the polyethylene glycol/carboxypeptidase A compound in the lining tube is completely transferred into the 2-methylimidazole aqueous solution to form a mixed solution; and (2) immediately adding the zinc acetate aqueous solution in the step (1) into the mixed solution, placing the mixed solution into a 20ml small bottle for reaction, placing a magnetic rotor into the small bottle, uniformly stirring for 5 minutes, standing and aging for 24 hours, centrifuging at 10000r/min to collect a solid product and a supernatant for testing, centrifuging again after washing once to collect the solid product and the supernatant after washing once, and naturally drying the product at room temperature for 24 hours.
Comparative example 2 Synthesis of Zeolite Imidazol ester framework Compound
1.0526g of 2-methylimidazole is weighed and added into 8ml of ultrapure water to prepare a solution, and 0.1407g of zinc acetate is added into 2ml of water to prepare an aqueous solution. Mixing the prepared two solutions, placing the two solutions into a 20ml small bottle for reaction, placing a magnetic rotor into the small bottle, uniformly stirring the two solutions for 5 minutes, standing and aging the mixture for 24 hours, centrifuging the mixture at 10000r/min to collect a product, and naturally drying the product for 24 hours at room temperature. To obtain the zeolite imidazole ester framework compound.
Example 3 Synthesis of Zeolite Imidazol ester framework Compound Supported carboxypeptidase A Complex
(1) 1.5787g of 2-methylimidazole is weighed and added into 8mL of ultrapure water to prepare 2-methylimidazole water solution, 0.1407g of zinc acetate is added into 2mL of ultrapure water to prepare zinc acetate water solution, and 1mL of polyethylene glycol water solution with the concentration of 15mg/mL is prepared;
(2) taking 960 microliters (0.025g) of zymogen liquid by using a liquid transfer gun, placing the zymogen liquid into a 1.5mL centrifuge tube, centrifuging (10000r/min) for 5 minutes, removing supernatant, adding 1mL of polyethylene glycol aqueous solution prepared in the step (1) into the centrifuge tube, and performing ultrasonic treatment for 15 minutes to form polyethylene glycol/carboxypeptidase A compound solution of polyethylene glycol-coated carboxypeptidase A;
(3) putting the polyethylene glycol/carboxypeptidase A compound solution into an ultrafiltration centrifugal tube for ultrafiltration for 30 minutes, pouring off filtrate in an ultrafiltration tube sleeve, and flushing a lining tube of the ultrafiltration centrifugal tube by using the 2-methylimidazole aqueous solution prepared in the step (1) until the polyethylene glycol/carboxypeptidase A compound in the lining tube is completely transferred into the 2-methylimidazole aqueous solution to form a mixed solution; and (2) immediately adding the zinc acetate aqueous solution in the step (1) into the mixed solution, placing the mixed solution into a 20ml small bottle for reaction, placing a magnetic rotor into the small bottle, uniformly stirring for 5 minutes, standing and aging for 10 hours, centrifuging at 10000r/min to collect a solid product and a supernatant for testing, centrifuging again after washing once to collect the solid product and the supernatant after washing once, and naturally drying the product at room temperature for 24 hours.
Comparative example 3 Synthesis of zeolitic imidazolate framework Compound
Firstly, 1.5787g of 2-methylimidazole is weighed and added into 8ml of ultrapure water to prepare a solution, then 0.1407g of zinc acetate is added into 2ml of ultrapure water to prepare an aqueous solution, the prepared two solutions are mixed and placed into a 20ml small bottle to react, a magnetic rotor is placed into the small bottle to be uniformly stirred for 5 minutes, standing and aging are carried out for 10 hours, then a product is centrifugally collected at 10000r/min, and the product is naturally dried for 24 hours at room temperature. To obtain the zeolite imidazole ester framework compound.
Detection method
1. Electron microscopy characterization of zeolitic imidazolate framework compounds and zeolitic imidazolate framework compound-loaded carboxypeptidase A complex
2. Zeolitic imidazolate framework compounds loaded carboxypeptidase a complex and compound characterization: (1) fourier infrared spectroscopy (control 1, example 1), (2) XRD (control 2, example 2), (3) thermogravimetry (example 3)
3. Zeolite imidazate framework compound-supported carboxypeptidase A complex and activity identification of zeolite imidazate framework compound
(1) Degradation of hippuric acid-L-phenylalanine to hippuric acid and L-phenylalanine by the Complex (example 1)
EXAMPLE 1 Activity characterization of the synthesized Complex, carboxypeptidase A complex was loaded with free carboxypeptidase A and a zeolitic imidazolate compound, respectively, to catalytically decompose hippuric acid-L-phenylalanine at 25 deg.C for the same time to produce hippuric acid and L-phenylalanine. And detecting the generated substance by using a high performance liquid chromatograph. Detection conditions are as follows: a C18 column; an ultraviolet detector; mobile phase A: KH of 20mM2PO4The pH value is 2.45; mobile phase B: acetonitrile, gradient elution; the column temperature is 30 ℃, the flow rate is 1mL/min, the wavelength is 210nm, and the sample injection amount is 10 uL.
(2) Complex degradation of ochratoxin A to ochratoxin alpha and L-beta-phenylalanine (example 2)
Example 2 characterization of the activity of the synthesized complex, the stock solution was 1mg/mL methanol-solubilized OTA, diluted with water to a 20ppm solution of OTA toxin, and carboxypeptidase a degraded ochratoxin a to produce OT α and L- β -phenylalanine products. And loading the carboxypeptidase A complex on the zeolite imidazole ester framework compound for 24 hours, then sampling 100 microliters, adding 400 microliters of chromatographic pure methanol, fully mixing uniformly, and then collecting filtrate through an ultrafiltration membrane of 10kD to be tested. Detection conditions are as follows: fluorescence detector (lambda)ex＝333nm，λem460nm) mobile phase acetonitrile/water/acetic acid (volume ratio 99:99:2) flow rate 0.8 ml/min.
(3) Degradation of the complex to Z-Phe-Leu to Z-Phe and L-leucine (example 3)
Example 3 identification of the activity of the synthesized complex, the activity of the complex was determined by the cadmium-indetrione method. And the enzymatic stability was determined by this method.
Preparation of potassium phosphate buffer: collecting 0.1361g KH2PO410mL of distilled water was added as solution A, and 0.1742g of K was taken2HPO4Adding 10mL of distilled water to prepare solution B, mixing 4mL of solution A and 6mL of solution B, adding water to dilute to 100mL, and adjusting the pH value to 7.5 by using NaOH.
The reaction formula is as follows: Z-Phe-Leu + H2O→Z-Phe+Leu
The Z-Phe-Leu substrate was dissolved in 0.1mol/L, pH 7.5 potassium phosphate buffer to a final concentration of 8 mmol/L. 1.8mL of the substrate solution was taken, 0.2mL of the diluted enzyme solution was added, the mixture was reacted in a thermostat water bath at 35 ℃ for 10min, and 50. mu.L of the enzyme-substrate reaction solution was taken and diluted to 1mL with water. And adding 2mL of cadmium-ninhydrin reagent, mixing, uniformly mixing by using a vortex oscillator, heating in water bath at 84 ℃ for 5min, cooling to room temperature, and measuring the light absorption value at 507 nm.
4. Stability of complexes and Compounds
(1) Effect of storage time on Complex and free carboxypeptidase A (control 1, example 1)
The complex enzyme and the free enzyme with the same protein content are respectively stored in 0.1M phosphate buffer solution (pH 7.5) at 4 ℃ for 2 days, 7 days, 14 days, 30 days and 65 days, and then are respectively taken out and incubated for 10min to determine the residual activity. The ratio of the immobilized carboxypeptidase A activity to its initial activity is defined as the residual activity.
(2) Effect of different ratios of ethanol content on Complex and free carboxypeptidase A (control 1, example 1)
Preparing ethanol/PBS solution according to the proportion of 15%, 20%, 30%, 40% and 50%, dissolving the substrate Z-Phe-Leu with the prepared solution to prepare 8mM substrate solution, adjusting the pH value to 7.5, adding the complex enzyme and the free enzyme with the same protein content respectively, incubating for 10 minutes, and determining the residual activity.
(3) Effect of temperature on Complex and free carboxypeptidase A (control example 2, example 2)
0.066g of Z-Phe-Leu substrate is added into 20mL of potassium phosphate buffer solution with the pH value of 7.5, 10 parts of substrate solution with 1.8mL of each is respectively put into 5 centrifugal tubes with 10mL, the incubation temperatures are set to be 25 ℃, 35 ℃, 45 ℃, 55 ℃ and 65 ℃, respectively, free enzyme and complex enzyme are respectively added for reaction for 10min, and then sampling is carried out for color reaction to measure the content of the generated L-leucine.
(4) Comparison of the number of cycles of Complex and free carboxypeptidase A (control 3, example 3)
Putting the substrate solution into an ultrafiltration centrifugal tube, adding free enzyme, incubating for 10min at 35 ℃, centrifuging for 30min at the rotating speed of 5000r/min by using a centrifuge, taking out filtrate to be tested, adding the substrate solution again, incubating, ultrafiltering and sampling, and circulating for 7 times. Adding the complex enzyme into a substrate solution, incubating for 10min at 35 ℃, centrifuging to take the supernatant to be tested, taking the sample to be tested, and adding the substrate solution again to carry out the next circulation.
The result of the detection
1. Electron microscope results
As can be seen from FIG. 1, the shape of the zeolitic imidazolate framework is influenced by the synthesis conditions, the shapes of the zinc ions and the imidazole synthesized in different proportions are different, the compound in example 1 is sheet-shaped, and the surface is rough and has irregular holes; the material synthesized in example 2 was in the shape of a regular dodecahedron; the dodecahedral shape of the material synthesized in example 3 is less regular than the former and somewhat close to spherical.
2. Complex and compound characterization
(1) Fourier infrared spectroscopy
As shown in FIG. 2A, curves a, b and c are the infrared spectra of the zeolitic imidazolate framework compound of comparative example 1, the carboxypeptidase A complex supported on the zeolitic imidazolate framework compound of example 1 and the carboxypeptidase A, respectively. As shown in c, carboxypeptidase A was at 1649cm-1And 1541cm-1The position corresponds to C ═ O stretching vibration, and the characteristic peak of the protein is 1640--1Within the range, corresponding to the amide I band vibration; 693cm in the infrared spectrum of ZIF-8 as shown in a-1Bands at positions corresponding to out-of-plane blending of 2-methylimidazolium rings at 836.3cm-1To 1307.9cm-1The specific peak appearing in the range of (a) is due to in-plane bending of 2-methylimidazole. For example, b is the infrared spectrum of ZIF-8/CPA at 1660cm-1The specific peak of the amide I band at the position, which is due to N-H bending and C-N stretching of carboxypeptidase A, indicates that there is an interaction between the protein and the metal oxide, and that the carbonyl group of the protein is simultaneously bonded to Zn of ZIF-82+And (4) ion coordination. During the crystal growth process, the amido bond of carboxypeptidase A is connected with Zn2+Interact to regulate the intercalation mode of ZIF-8 with enzymes. Intermolecular hydrogen bonding and hydrophobic bonding interactions, in which the affinity of biological macromolecules (enzymes) for organic molecules (imidazoles) plays a crucial role. In addition, the morphology of the porous skeleton formed around the enzyme is also influenced by the synthesis conditions。
(2) As shown in FIG. 2B, curve e shows the compound of zeolitic imidazolate framework of example 2 loaded with carboxypeptidase A complex, and curve f shows the compound of zeolitic imidazolate framework of comparative example 2. The peak positions are not greatly changed from the map, which shows that the properties of the compound are not changed before and after loading carboxypeptidase A.
(3) As shown in FIG. 2C, the curve is a thermogravimetric curve of the compound of the zeolite imidazole ester framework compound loaded with carboxypeptidase A in example 3, the weight loss due to moisture evaporation in the first stage is 9.192% at a temperature in the range of 100 ℃, and the weight loss due to moisture evaporation in the second stage is 8.484% at a temperature in the range of 150-250 ℃, which is attributed to the decomposition of protein, thereby indicating that carboxypeptidase A is successfully loaded on the zeolite imidazole ester framework compound.
3. Complex and compound activity identification
(1) As shown in FIG. 3A, the retention time of hippuric acid standard is 7.2min, the retention time of L-phenylalanine standard is 3.3min, and the retention time of hippuric acid-L-phenylalanine is 17.5 min. The free carboxypeptidase A and the complex enzyme are respectively used for catalyzing and decomposing hippuric acid-L-phenylalanine for the same time, and product peaks appear at 3.3min and 7.2 min. Description example 1 zeolitic imidazolate framework-supported carboxypeptidase A complex is active.
(2) OT α residence time 1.68min and OTA residence time 4.5min as shown in fig. 3B. Thus, the zeolitic imidazolate framework compound-loaded carboxypeptidase A complex of example 2 is active.
(3) As shown in FIG. 3C, the color of the substrate solution is light yellow after the chromogenic reaction when the complex is not added to the tube a, which indicates that the substrate is not decomposed, and the color of the tube b is pink after the chromogenic reaction when the complex is added to the tube b and the substrate is degraded after 1 hour of degradation, which indicates that the L-leucine is generated after the complex is added to the tube b and the substrate is degraded. Thus, the zeolitic imidazolate framework compound-loaded carboxypeptidase A complex of example 3 is active.
4. Enzymatic properties of zeolite imidazole ester framework compound loaded carboxypeptidase A
(1) Effect of storage time on Complex and free carboxypeptidase A (control 1, example 1)
As shown in fig. 4A, the activity of free carboxypeptidase a decreased to 4% after two months of storage, whereas the zeolitic imidazolate framework compound-loaded carboxypeptidase a complex preserved half of the original activity, with a residual activity of 52.43%. After the complex enzyme and the free carboxypeptidase A are stored in the potassium phosphate buffer solution for 7 days, the activity of the complex enzyme is reduced to 89.75 percent, and the activity of the free carboxypeptidase A is reduced by 14.93 percent compared with the activity of the complex enzyme. After the compound enzyme is stored for 14 days, the activity of the compound enzyme is basically kept stable, but the activity of the free carboxypeptidase A is greatly reduced, and the residual activity is only 37.86 percent. Indicating that the complex has a protective shell which can effectively protect the activity of the carboxypeptidase A.
(2) Effect of different ratios of ethanol content on Complex and free carboxypeptidase A (control 1, example 1)
Grains are fermented to produce alcohol under the influence of various factors in the grain storage process. Different ratios of ethanol solutions as shown in fig. 4B had different effects on complex enzyme and free carboxypeptidase a. The free carboxypeptidase A has certain tolerance to organic solvents, the activity of the free carboxypeptidase A is kept at about 97.01% when the ethanol content is lower than 20%, and once the ethanol content is higher than 20%, the activity of the free carboxypeptidase A begins to be greatly reduced, so that the advantage of the compound of the zeolite imidazole ester framework loaded with the carboxypeptidase A is reflected. The activity of free carboxypeptidase A was reduced by 21.4% from the original initial activity when the ethanol content reached 30%. When the ethanol content is increased to 40%, the activity of the free carboxypeptidase A is reduced to 36.55%, and the activity of the complex enzyme is still maintained at 90.73%. ethanol/PBS solution in the range of 15% -50% has no influence on the activity of the complex enzyme basically, relative free carboxypeptidase A has poor tolerance to organic solvent, in 50% ethanol/PBS solution, the activity of the free carboxypeptidase A is only 12.42% and the residual activity of the zeolite imidazole ester framework compound loaded carboxypeptidase A complex is 93.11%.
(3) Effect of temperature on Complex and free carboxypeptidase A (control example 2, example 2)
As can be seen from FIG. 5, the optimum temperature of free carboxypeptidase A is 25 deg.C, and the activity of the enzyme is slightly decreased when the temperature is higher than 25 deg.C, the activity of free carboxypeptidase A remains substantially stable when the temperature is in the range of 35 deg.C to 45 deg.C. When the temperature is higher than 45 ℃, the activity of the free carboxypeptidase A begins to gradually decrease, and when the temperature is higher than 55 ℃, the activity of the free carboxypeptidase A shows a great reduction trend. However, when the temperature is in the range of 25 ℃ to 35 ℃, the activity of the immobilized enzyme is continuously increased, and the optimum temperature of the immobilized enzyme is reached at 35 ℃. When the temperature was further raised to 45 ℃, the activity of the immobilized enzyme was slightly decreased from the peak. When the temperature is gradually increased from 45 ℃ to 65 ℃, the activity of the immobilized enzyme is basically kept stable, and the temperature stability is improved by 20 ℃. Compared with the free carboxypeptidase A, the temperature tolerance range of the immobilized enzyme is expanded, and the immobilized enzyme can decompose the substrate to generate L-leucine under the condition of not being in the extreme temperature condition, and the activity of the L-leucine is higher than that of the free carboxypeptidase A, which shows that the deformation of the three-dimensional structure of the carboxypeptidase A can be reduced under the high-temperature condition due to the protection effect of the MOF shell. While heat transfer is impeded to delay the denaturing process. Therefore, the zeolite imidazole ester framework compound has a protective effect on carboxypeptidase A.
(4) Comparison of the number of cycles of Complex and free carboxypeptidase A (control 3, example 3)
As shown in fig. 6, the compound of the zeolite imidazolate framework compound-supported carboxypeptidase a can still maintain 97% of residual activity after 7 cycles of recycling, but the residual activity of the free carboxypeptidase a after 7 cycles of recycling is 62.47%, the free carboxypeptidase a needs to be performed in a 10kD ultrafiltration centrifuge tube during the recycling experiment, at least 30min is consumed for each ultrafiltration, the ultrafiltration centrifuge tube is expensive, the activity of the free carboxypeptidase a after each recycling is reduced, and the recovery is extremely inconvenient and not beneficial for large-scale application. However, after the compound of the zeolite imidazole ester framework compound loaded with the carboxypeptidase A is circulated for one time, the compound enzyme can be directly recovered only by high-speed centrifugation for 5min, and the operation is simple and convenient.
The above description is only for the purpose of illustrating the technical solutions of the present invention and not for the purpose of limiting the same, and other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A method for preparing immobilized enzyme by a zeolite imidazole ester framework compound is characterized by comprising the following steps:
(1) adding 2-methylimidazole into distilled water or ultrapure water to prepare a 2-methylimidazole solution with the concentration of 10-210 g/L, adding zinc acetate into distilled water or ultrapure water to prepare a zinc acetate aqueous solution with the concentration of 65-95 g/L, and preparing a polyethylene glycol aqueous solution with the concentration of 10-20 g/L;
(2) centrifuging the zymogen liquid with the concentration of 24-28 g/L for 3-7 minutes, removing supernatant, adding the polyethylene glycol aqueous solution obtained in the step (1), and performing ultrasonic treatment for 10-20 minutes to obtain a polyethylene glycol/carboxypeptidase A compound solution;
(3) ultrafiltering the polyethylene glycol/carboxypeptidase A compound solution obtained in the step (2) for 20-40 minutes, and transferring the polyethylene glycol/carboxypeptidase A compound into a 2-methylimidazole water solution by using the 2-methylimidazole water solution prepared in the step (1) to form a mixed solution; and (2) immediately adding the zinc acetate aqueous solution obtained in the step (1) into the mixed solution, magnetically stirring for 3-10 minutes, standing and aging for 5-24 hours, centrifugally collecting a solid product, washing with water for 1-3 times, and drying to obtain the immobilized enzyme prepared from the zeolite imidazole ester framework compound.
2. The method for preparing immobilized enzyme by using zeolite imidazole ester framework compound according to claim 1, wherein the enzyme stock solution in step (2) is bovine pancreatic carboxypeptidase A enzyme stock solution.
3. The method for preparing immobilized enzyme by using zeolite imidazolate framework compound as claimed in claim 1, wherein the concentration of the 2-methylimidazole aqueous solution in the step (1) is 13-198 g/L, and the concentration of the zinc acetate aqueous solution is 70-88 g/L.
4. The method for preparing immobilized enzyme by using zeolite imidazolate framework compound according to claim 1, wherein the mass ratio of the 2-methylimidazole, the zinc acetate, the enzyme stock solution and the polyethylene glycol is 106-4: 9-30: 1-2: 1.
5. The method for preparing immobilized enzyme by zeolite imidazole ester framework compound according to claim 1, wherein the drying in step (3) is freeze-drying at-30 to-50 ℃ for 40 to 60 hours or natural drying at room temperature for 20 to 30 hours.
6. The method for preparing immobilized enzyme by using zeolite imidazolate framework compound according to claim 1, wherein the rotation speed of the centrifugation in the step (3) is 8000-12000 r/min.
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| CN111704722A (en) * | 2020-05-27 | 2020-09-25 | 山东大学 | Preparation method and application of zeolite imidazole type metal organic framework material with polyethylene glycol as mineralizer |
| CN111704722B (en) * | 2020-05-27 | 2021-09-07 | 山东大学 | Preparation method and application of zeolite imidazole type metal organic framework material with polyethylene glycol as mineralizer |
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