CN111304187A - Method for directly and rapidly preparing cross-linked enzyme aggregate from cell lysate - Google Patents
Method for directly and rapidly preparing cross-linked enzyme aggregate from cell lysate Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
Abstract
The invention relates to a method for directly and rapidly preparing a cross-linked enzyme aggregate from a cell lysate, which comprises the following steps: firstly, dissolving a diyne cross-linking agent in isopropanol, and then dispersing the diyne cross-linking agent in cell disruption liquid supernatant of aldehyde ketone reductase mutants doped with different amounts of unnatural amino acids; and (3) putting the container filled with the mixture into a microwave reactor provided with a cooling module for reaction, and preparing a cross-linked enzyme aggregate and realizing the purification of target enzyme protein by utilizing the characteristic reaction between the reaction groups of the diyne cross-linking agent and the side chains of the unnatural amino acids. The invention obtains the mutant enzyme doped with the unnatural amino acid at different sites by changing the number of the mutant sites, realizes accurate and controllable multipoint crosslinking, and can effectively protect the active center of the enzyme protein and the enzyme catalytic activity. The method does not need to use metal ions, organic solvents and the like, is safe, reliable, green and environment-friendly, greatly saves the time and cost consumed by purification, and is suitable for industrial production.
Description
Technical Field
The invention relates to a preparation method of an enzyme aggregate, in particular to a method for directly and rapidly preparing a cross-linked enzyme aggregate from a cell lysate.
Background
The catalytic enzyme is produced from readily available renewable resources and is biodegradable. The enzyme-mediated biological catalytic reaction has high catalytic efficiency, chemoselectivity, regioselectivity and stereoselectivity, and has wide application in fermentation, chemistry, food industry and environmental management. However, such practical applications suffer from practical drawbacks of free enzymes, such as low thermostability, optimal pH range, low tolerance to most organic solvents and many metal ions, etc. In addition, enzymes lead to unavoidable purification and isolation steps. The immobilized enzyme provides reproducibility and improves enzyme stability, for example, tolerance to organic solvents, pH tolerance and thermostability.
Most immobilized enzymes rely on a specific support. However, the additional immobilized enzyme support results in dilution of the activity of the immobilized enzyme due to the introduction of most of the non-catalytic species. In order to overcome these disadvantages, techniques for immobilizing enzymes in the absence of a carrier have been of great interest since the development, which will reduce the space-time yield, specific activity, etc. in enzymatic reactions. Most typically, cross-linked enzyme aggregates. Cross-linked enzyme aggregates were first described in the early 20 s and were produced by dissolving the free enzyme in a water-soluble solvent and cross-linking the physical aggregates. Cross-linked enzyme aggregates are generally stable in organic solvents and the activity of the enzyme is not altered, and in some cases enhanced, compared to the non-immobilized enzyme. The cross-linked enzyme aggregates show a high resistance to organic solvents, extremely high pH values and high temperatures.
The preparation of traditional enzyme aggregates relies primarily on the glutaraldehyde process, which is generally the cross-linking agent of our choice because it is inexpensive and readily available in large quantities. The glutaraldehyde method has been used for more than decades to crosslink proteins, and has the defects that the reaction (Schiff base reaction) between the glutaraldehyde method and side chain amino groups is randomly carried out, and the active center of the enzyme protein is buried or destroyed, so that the enzyme catalytic activity is greatly reduced, and the final specific activity after the enzyme protein is crosslinked is only 10-20% of the initial activity.
In addition, one of the most troublesome problems in immobilized enzymes is the need to use pure enzymes, and purification of the enzymes is a lengthy and complicated process. Generally, purified proteins are widely used for immobilization on solid supports, but protein purification usually uses more other auxiliary consumables and reagents, and has high cost, long period and serious enzyme activity loss.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for directly and rapidly preparing a cross-linked enzyme aggregate from a cell lysate, namely a method for forming the enzyme aggregate by doping unnatural amino acids into enzyme with the assistance of microwave. In order to achieve precise and controllable immobilization of the enzyme, unnatural amino acids are used for insertion into the enzyme protein for targeted covalent immobilization, while microwave-assisted irradiation is used to complete the preparation of the enzyme aggregates; by changing the number of mutant sites, mutant enzymes which incorporate unnatural amino acids at different sites are obtained, thereby realizing accurate and controllable multi-site immobilization.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for directly and rapidly preparing cross-linked enzyme aggregates from cell lysates comprises the following steps: firstly, dissolving a diacetylene cross-linking agent in isopropanol, and then dispersing the diacetylene cross-linking agent in a phosphate buffer solution (potassium phosphate buffer solution, KBS) of a cell disruption solution supernatant of an aldehyde-ketone reductase mutant doped with different amounts of unnatural amino acids; setting the reaction molar ratio of the azide to the alkynyl; and (3) putting the container filled with the mixture into a microwave reactor provided with a cooling module for reaction, separating the fixed enzyme, and preparing a cross-linked enzyme aggregate by utilizing the characteristic reaction between the diyne cross-linking agent and the side chain reaction groups of the unnatural amino acids, thereby realizing the purification of the target enzyme protein. Wherein continuous illumination is induced by controlling the temperature difference between the microwave reaction system and the cooling module.
Preferably, the diyne crosslinking agent is 5,6,11, 12-tetrahydrodibenzo [ a, e ] cyclooctene.
Preferably, the aldehyde-ketone reductase comprises a three-point mutant aldehyde-ketone reductase and a five-point mutant aldehyde-ketone reductase. The selection of the mutation site of the aldehyde ketone reductase needs to lead the mutation site to be far away from the active center and the active site of the protein and mutate into TAG codon, and the three-point and five-point target gene mutation is carried out on the basis of a single point.
Preferably, the unnatural amino acid incorporated by the aldoketoreductase is para-azido-L-phenylalanine.
Preferably, the concentration of the phosphate buffer (potassium phosphate buffer, KBS) is 0.05-0.15 mol.L-1The pH value is 6.0-8.0.
Preferably, Escherichia coli is used as a host for inducing the expression of aldehyde ketone reductase gene, the obtained cell sediment is harvested by centrifugation, the rotation number of the centrifugation is 7000-9000 rpm, the time is 4-8 min, and the sediment is washed by KBS; resuspending the pellet in KBS and lysing the cells by ultrasonic treatment, wherein the addition amount of the KBS is 1/5-1/3 of the volume of the original bacterial liquid, and the concentration of the KBS is 0.05-0.15 mol.L-1The pH is 6.0-8.0, the ultrasonic crushing adopts an ice bath, the power is 300-500W, the crushing time is 8-12 min, and the crushing is stopped for 7s every 10 s; and centrifuging the soluble and insoluble parts after cell disruption, wherein the revolution number of the centrifugation is 9000-12000 rpm, and the time is 15-25 min, so as to separate and obtain a supernatant of the cell disruption solution.
Preferably, the diyne crosslinking agent is dissolved in isopropanol to a concentration of 6 to 10 mmol.L-1And then suspending the diacetylene cross-linking agent in 0.5-2 mL of KBS containing the supernatant of the multipoint mutant cell disruption solution, wherein the reaction molar ratio of azide to alkynyl is 1: 0.5 to 1.5.
Preferably, the microwave temperature is 5-25 ℃, the microwave power is 5-40W, and the microwave time is 1-6 min.
Preferably, the immobilized enzyme is separated by centrifugation at 10000-14000 Xg for 2-8 min and KBS (0.01 mol. L)-1pH 7.0) and detected using Bradford method until no protein was detected in the supernatant; and taking out and centrifuging the reactant, draining the supernatant by using an injector, washing the reactant for 2-5 times by using water, and drying in a vacuum oven at 20-40 ℃.
Preferably, the rapid microwave-assisted method for preparing the enzyme aggregate specifically comprises the following steps:
(1) Dissolving the diyne crosslinking agent in isopropanol to a concentration of 8 mmol.L-1；
(2) Making Escherichia coli as host for inducing aldehyde ketone reductase gene expression, centrifuging to obtain cell precipitate, wherein the centrifugation rotation number is 8000rpm, the time is 5min, and washing the precipitate with KBS; the pellet was resuspended in KBS (0.1 mol. L) and the cells lysed by sonication-1pH 7.0), adding 1/4 which is equal to the volume of the original bacterial liquid, carrying out ultrasonic crushing by adopting an ice bath with the power of 400W, and crushing for 10min, wherein the crushing time is set to be 3s and 7s after the crushing time is set to be 10 s; centrifuging the soluble and insoluble parts after cell disruption at 10000rpm for 20min to obtain cell disruption solution supernatant;
(3) the diacetylene cross-linking agent was suspended in 1mL of KBS (0.1 mol. L.) from the supernatant of cell disruption of an aldone reductase mutant doped with para-azido-L-phenylalanine at three and five points-1pH 7.0); the reaction molar ratio of azide to alkynyl is 1: 1;
(4) placing the container containing the above mixture into a microwave reactor equipped with a cooling module, and irradiating at 10 deg.C and 10W for 4 min;
(5) separating the immobilized enzyme, centrifuging at 12000Xg for 5min, and mixing with KBS (0.01 mol. L)-1pH 7.0) and detected using Bradford method until no protein was detected in the supernatant;
(6) the reaction was removed and centrifuged, the supernatant was drained with a syringe, and the enzyme aggregate was washed 4 times with water and dried in a vacuum oven at 30 ℃.
The present study focused on the use of cyclooctyne as a novel cross-linker and unnatural amino acid modified enzyme with azide groups for copper-free click chemistry to form complete enzyme aggregates. Studies have shown that site-specific binding of unnatural amino acids can immobilize proteins in a controlled manner. Genetic code expansion uses orthogonal aminoacyl-tRNA synthetase (aaRS) -tRNA pairs to direct the incorporation of an unnatural amino acid into a protein in response to the introduction of an unassigned codon (typically an amber stop codon, UAG) at a desired site in the gene. For protein immobilization, the azide-alkyne cycloaddition reaction (CuAAC) catalyzed by cuprous salts and its strain-promoted alkyne-azide cycloaddition (SPAAC) are both click-chemistry efficient reactions. However, copper salts of monovalent copper are known to be cytotoxic, so an effective alternative method of choice is cross-linking by the SPAAC reaction. Based on the above theory, the present invention selects a diyne crosslinker (5,6,11, 12-tetrahydrodibenzo [ a, e ] cyclooctene) as the azide crosslinker.
For the immobilization method, the present invention selects a microwave-assisted method. Microwaves are electromagnetic waves with frequencies ranging from 0.3GHz to 300GHz, which have found application in many organic syntheses. Microwave radiation results in direct coupling of molecules in the reaction system. Microwave radiation produces better results than traditional heating, because it can couple molecules directly by selective absorption. Thus, conventional heating reactions, which take several hours to complete, can be successfully completed using microwaves in a very short time. With respect to the microwave mechanism, when microwaves come into contact with polar compounds, heat is generated, and the generated heat reacts with molecules, thereby causing ion conduction and dipole rotation. However, one of the most troublesome problems in immobilized enzymes is purification of the enzyme. Generally purified proteins are widely used for immobilization on solid supports, but purification of proteins is often laborious and expensive. However, the experimental scheme designed by the invention can well solve the problem. The invention uses diyne cross-linking immobilization to produce enzyme aggregates and uses them as cross-linking agents to achieve multi-point covalent attachment of unnatural amino acids. Under the irradiation of low-temperature microwaves, through a copper-free click reaction, enzyme aggregate precipitates which are very easy to separate and have high specific activity are generated, and the processes of purifying and immobilizing the two units are combined together to form a single operation.
The present invention uses aldehyde-ketone reductase, and uses p-azido-L-phenylalanine instead of three and five mutation sites of aldehyde-ketone reductase, followed by multipoint incorporation to obtain free enzyme of aldehyde-ketone reductase. Crosslinking a diacetylene crosslinking agent and multipoint aldehyde ketone reductase through cycloaddition reaction, and determining the optimal fixing condition of 10 ℃ by screening microwave radiation conditions, wherein the power is 10W, and the optimum fixing condition is radiated for 4min, so that the immobilized enzyme activity which is equal to more than 200% of free enzyme is obtained; the invention also checks the influence of the mutation site on the thermal stability of the immobilized aldehyde ketone reductase and the growth condition of the enzyme aggregate under a scanning electron microscope, and finds that the immobilization rate of five points reaches 90 percent, and the enzyme activity is equivalent to 210 percent of that of the corresponding five-point free enzyme; the three-point immobilization rate is only 60%, and the enzyme activity is only equivalent to 120% of that of three-point free enzyme. In addition, free aldoketoreductase enzymes (including wild-type, three-point and five-point free aldoketoreductase enzymes) remained only 10% to 20% of the original activity after heating at 60 ℃ for 16 hours. However, the five-point immobilized aldoketoreductase retained 70% of its original activity after 16 hours and was higher than the three-point immobilized aldoketoreductase.
The invention obtains the mutant enzyme doped with the unnatural amino acid at different sites by changing the number of the mutant sites, thereby realizing accurate and controllable multi-point fixation, preparing the cross-linked enzyme aggregate, realizing the purification of the target enzyme protein, and effectively protecting the active center of the enzyme protein and the enzyme catalytic activity; the method disclosed by the invention does not need to use metal ions, organic solvents, ammonium sulfate and resin for purification, is safe, reliable, green and environment-friendly, greatly saves the time and cost for purification, and is suitable for industrial production.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the present invention;
FIG. 2 is a schematic representation of the effect of microwave temperature on enzyme aggregate immobilization rate and specific enzyme activity according to the present invention;
FIG. 3 is a schematic representation of the effect of microwave power on enzyme aggregate immobilization rate and specific enzyme activity according to the present invention;
FIG. 4 is a schematic representation of the effect of microwave time on enzyme aggregate immobilization rate and specific enzyme activity according to the present invention;
FIG. 5 is a schematic scanning electron microscope of enzyme aggregates at different times according to the present invention;
FIG. 6 is a graphical representation of the thermal stability (A, 30 ℃; B, 40 ℃; C, 50 ℃; D, 60 ℃) of the aldoketoreductase enzyme aggregates of the present invention.
Detailed Description
The present invention is further described with reference to the following specific examples, which are not intended to be limiting, but are intended to be exemplary in nature and not to be limiting, and all equivalent modifications and equivalents of the known art that are within the spirit and scope of the present invention are intended to be protected by the present invention.
A method for directly and rapidly preparing cross-linked enzyme aggregates from cell lysate comprises the following steps: dissolving a diyne cross-linking agent in isopropanol, and suspending the diyne cross-linking agent in KBS of cell disruption supernatant of aldo-ketoreductase mutants doped with different amounts of unnatural amino acids; setting the reaction molar ratio of the azide to the alkynyl; placing the container filled with the mixture into a microwave reactor equipped with a cooling module, irradiating for a certain time at a certain temperature and power, and inducing continuous illumination by controlling the temperature difference between the microwave reaction system and the cooling module; the immobilized enzyme was isolated and washed with KBS until no protein was detected in the supernatant. The characteristic reaction between the reaction groups of the diacetylene cross-linking agent and the side chains of the unnatural amino acids is utilized to prepare the cross-linked enzyme aggregate and realize the purification of the target enzyme protein, and the process is shown in figure 1.
Preparation of supernatant of cell disruption solution
Escherichia coli was used as a host for inducing the expression of the aldone reductase gene, and the obtained cell pellet was harvested by centrifugation at 8000rpm for 5min using KBS (0.1 mol. L)-1pH 7.0) washing the precipitate; the pellet was resuspended in KBS (0.1 mol. L)-1pH 7.0) and lysis of the cells by sonication, wherein KBS (0.1 mol. L)-1pH 7.0), adding 1/4 which is equal to the volume of the original bacterial liquid, carrying out ultrasonic crushing by adopting an ice bath with the power of 400w and the crushing time of 10min, wherein the crushing time is 7s after 3s is set every 10 s; the soluble and insoluble fractions after cell disruption were also separated by centrifugation at 10000rpm for 20min to obtain a supernatant of the cell disruption solution.
Example 1
Influence of microwave temperature on aldehyde ketone reductase aggregate immobilization yield and specific enzyme activity
Preparation of enzyme aggregates under continuous microwave irradiation: dissolving the diyne crosslinking agent in isopropanol to a concentration of 8 mmol.L-1Then, the diacetylene cross-linking agent was suspended in 1mL of a five-spot supernatant of the cell disruption solution of the azido-L-phenylalanine-incorporating aldone reductase mutant (0.1 mol. L)-1pH 7.0); the reaction molar ratio of azide to alkynyl is 1: 1; placing the container containing the mixture into a microwave reactor equipped with a cooling module, and irradiating at different temperatures under power of 10W for 4 min; continuous illumination is induced by controlling the temperature difference between the microwave reaction system and the cooling module.
The results of the effect of microwave temperature on the immobilization rate and specific enzyme activity of the enzyme aggregate are shown in FIG. 2, and FIG. 2 shows the effect of microwave temperature on the immobilization rate and activity of the immobilized enzyme. The optimal temperature at which the aldehyde ketoreductase enzyme aggregate was observed to maintain activity was 10 ℃. At this temperature, the specific activity of the immobilized enzyme aggregate on the carrier was 2.06 U.mg-1The yield can reach 90%, which means that the enzyme activity of the aldehyde ketoreductase enzyme aggregate can reach more than 200% of the enzyme activity of the corresponding five-point free enzyme compared with the activity of the free enzyme per se, and the immobilized yield is good. Studies have shown that microwaves are effective for uniform heating of the substrate and are not harmful to the enzyme, as there is no destructive effect on the properties of the enzyme, such as stability and substrate specificity. However, at higher temperatures, a significant amount of aldehyde ketoreductase enzyme aggregate inactivation was observed, although the immobilization rate did not change much. Since higher temperatures can result in a significant loss of aggregation activity of the aldoketoreductase enzyme, optimization means that the temperature is reduced to the most suitable. As the temperature increases, the microwave radiation causes the frequency factor to increase, thereby increasing the collisions of molecules, thereby providing them with more energy. The more energy they have, the greater the tendency for the electrons of the reacting molecules to jump to a higher energy state. The movement of electrons increases the randomness of the system, inevitably increases the entropy of the system and reduces the activation energy, thereby accelerating the synthesis speed. However, at excessively high temperatures under the microwave system, the protein structure is thermally induced to be destroyed due to the enzymeAnd weak ionic and hydrogen bond destruction to denature, resulting in a reduced reaction rate.
Example 2
Influence of microwave power on aldehyde ketone reductase aggregate immobilization yield and specific enzyme activity
Preparation of enzyme aggregates under continuous microwave irradiation: dissolving the diyne crosslinking agent in isopropanol to a concentration of 8 mmol.L-1Then, the diacetylene cross-linking agent was suspended in 1mL of a five-spot supernatant of the cell disruption solution of the azido-L-phenylalanine-incorporating aldone reductase mutant (0.1 mol. L)-1pH 7.0); the reaction molar ratio of azide to alkynyl is 1: 1; placing the container containing the mixture into a microwave reactor equipped with a cooling module, and irradiating at 10 deg.C for 4min under different microwave power; continuous illumination is induced by controlling the temperature difference between the microwave reaction system and the cooling module.
The results of the effect of microwave power on the immobilization rate and specific enzyme activity of the enzyme aggregate are shown in FIG. 3, FIG. 3 shows the effect of microwave power on the immobilization rate and specific enzyme activity of the aldehyde ketoreductase enzyme aggregate, and the effect of microwave radiation power on the immobilization rate and specific enzyme activity was observed in a power range of 5 to 40W. The results show that 10W is the optimum power for microwave radiation. At this power level, the activity of the aldoketoreductase enzyme aggregate after 4 minutes of covalent immobilization by microwave radiation was 2.06 U.mg-1More than 200% of the corresponding five-point free enzyme. However, at higher powers, the change in immobilization rate slowly increased, but significant aldehyde ketoreductase enzyme aggregate inactivation was also observed. Due to the high microwave power, the activity of the aldoketoreductase enzyme aggregate is greatly lost, so the power will be optimized to obtain the maximum specific enzyme activity. At high microwave powers, above 50% of the microwave capacity, above 480W, the temperature becomes difficult to control. Therefore, in order to consume less energy and obtain a more uniform temperature distribution, it is necessary to select a suitable input power. High microwave power results in drastic conformational changes in the three-dimensional structure of the enzyme. Experiments performed at low microwave power below 50W showed adequate results at the best parameters.
Example 3
Preparation of enzyme aggregates under continuous microwave irradiation: dissolving the diyne crosslinking agent in isopropanol to a concentration of 8 mmol.L-1Then, the diacetylene cross-linking agent was suspended in 1mL of a five-spot supernatant of the cell disruption solution of the azido-L-phenylalanine-incorporating aldone reductase mutant (0.1 mol. L)-1pH 7.0); the reaction molar ratio of azide to alkynyl is 1: 1; the vessel containing the mixture was placed in a microwave reactor equipped with a cooling module and irradiated for 4min at a power of 10W for various microwave times. Continuous illumination is induced by controlling the temperature difference between the microwave reaction system and the cooling module.
The results of the effect of microwave time on the immobilization rate of enzyme aggregates and specific enzyme activity are shown in FIG. 4, and FIG. 4 shows the effect of microwave time on the immobilization rate of aldehyde-ketone reductase enzyme aggregates and specific enzyme activity. In order to avoid the adverse effect of strong microwave radiation, when the influence of microwave radiation time on the aldehyde ketoreductase enzyme aggregate is researched, the microwave radiation power of 10W and the temperature of 10 ℃ are selected as the main power and temperature. The results show that 4 minutes is sufficient to allow perfect cross-linking of the aldoketoreductase enzyme aggregate and the diyne cross-linker. The yield of immobilized enzyme hardly changed with the increase of irradiation time, but the activity of enzyme was slightly decreased, and the activity of aldehyde ketoreductase enzyme aggregate was 2.06 U.mg-1More than 200% of the corresponding five-point free enzyme. Molecular rearrangement during microwave treatment changes the intermolecular spacing between proteins and changes their quaternary and tertiary structures.
The present invention irradiates the aldoketoreductase with different enzyme aggregates at different times, and performs scanning electron microscope tests in microwave at 1 minute, 2 minutes, 4 minutes and 6 minutes, respectively, and the results are shown in fig. 5, from which fig. 5 it can be seen that the enzyme aggregates become gradually larger, the lower right angle scale of the picture is 10um, and the size is hardly changed at 4min and 6 min. The diameter was about 10um, which demonstrates that we obtained the most complete aldoketoreductase enzyme aggregate within 4 minutes.
Example 4
Effect of the number of immobilization sites on immobilization Rate
In order to better select the number of mutation sites of the aldehyde ketoreductase enzyme aggregate, the invention implements the preparation and characterization of the enzyme aggregate of aldehyde ketoreductase with different numbers of mutation sites.
Preparation of enzyme aggregate by aldehyde ketone reductase with three mutation sites
Dissolving the diyne crosslinking agent in isopropanol to a concentration of 8 mmol.L-1Then, the diyne cross-linking agent was suspended in 1mL of KBS (0.1 mol. L.) from the supernatant of cell disruption solution of the azido-L-phenylalanine-incorporating aldone reductase mutant-1pH 7.0). The reaction molar ratio of azide to alkynyl is 1: 1. the vessel containing this mixture was placed in a microwave reactor equipped with a cooling module and irradiated at 10 ℃ and 10W for 4 min. Continuous illumination is induced by controlling the temperature difference between the microwave reaction system and the cooling module. Separating the immobilized enzyme, centrifuging at 12000Xg for 5min, and mixing with KBS (0.01 mol. L)-1pH 7.0) and detected using Bradford method until no protein was detected in the supernatant. The reaction was taken out and centrifuged, the supernatant was drained with a syringe, and the enzyme aggregate was washed 4 times with water, and tested for enzyme activity, immobilization rate and thermal stability.
Preparation of enzyme aggregate by five mutation site aldehyde ketone reductase
Dissolving the diyne crosslinking agent in isopropanol to a concentration of 8 mmol.L-1Then, the diacetylene cross-linking agent was suspended in 1mL of a five-spot supernatant of the cell disruption solution of the azido-L-phenylalanine-incorporating aldone reductase mutant (0.1 mol. L)-1pH 7.0). The reaction molar ratio of azide to alkynyl is 1: 1. the vessel containing this mixture was placed in a microwave reactor equipped with a cooling module and irradiated at 10 ℃ and 10W for 4 min. Continuous illumination is induced by controlling the temperature difference between the microwave reaction system and the cooling module. Separating the immobilized enzyme, centrifuging at 12000Xg for 5min, and mixing with KBS (0.01 mol. L)-1pH 7.0) and detected using Bradford method until no protein was detected in the supernatantAnd (4) quality. The reaction was taken out and centrifuged, the supernatant was drained with a syringe, and the enzyme aggregate was washed 4 times with water, and tested for immobilization rate and thermal stability.
The thermal stability of the aldehyde ketone reductase enzyme aggregate is shown in fig. 6, and the aldehyde ketone reductase five-point enzyme aggregate and the aldehyde ketone reductase three-point enzyme aggregate are found to be highly thermally stable enzymes, so that the performance of the multi-point immobilized enzyme is superior to that of free enzymes (including wild type, three-point and five-point free aldehyde ketone reductase) at the temperature of 30-60 ℃. Wherein the thermal stability at 30 ℃ is greater than 40 ℃, 50 ℃ and 60 ℃. The result shows that the thermal stability of the five-point aldehyde ketone reductase enzyme aggregate is greatly improved compared with that of the three-point aldehyde ketone reductase enzyme aggregate along with the increase of sites when the five-point aldehyde ketone reductase enzyme aggregate is fixed under microwave radiation. After heat treatment at 60 ℃ for 16 hours, the residual activity of the immobilized enzyme under continuous microwave irradiation was 70% and higher than that of the three-point immobilized aldehyde-ketone reductase. The free aldehyde ketone reductase (including wild type, three-point and five-point free aldehyde ketone reductase) is only 10-20% of the original activity, and the five-point immobilization rate is found to be more than 90%, and the enzyme activity is equivalent to more than 200% of that of the corresponding five-point free enzyme; the three-point immobilization rate is only 60%, and the enzyme activity is only equivalent to more than 120% of that of three-point free enzyme.
The enzyme aggregate prepared by the method of the invention is irradiated for 4min under the optimum fixed condition of 10 ℃ and power of 10w by screening the microwave radiation condition. Thus, an immobilized enzyme activity equal to 210% of the corresponding five-point free enzyme was obtained. The invention also checks the influence of the mutant site and the thermal stability of the immobilized aldehyde ketone reductase and the growth condition of the enzyme aggregate under a scanning electron microscope, and finds that the immobilization rate of five points reaches more than 90%, the enzyme activity is equivalent to more than 200% of that of the corresponding five-point free enzyme, the immobilization rate of three points is only 60%, and the enzyme activity is equivalent to more than 120% of that of the corresponding three-point free enzyme. In addition, free aldoketoreductase enzymes (including wild-type, three-point and five-point free aldoketoreductase enzymes) remained only 10% to 20% of the original activity after heating at 60 ℃ for 16 hours. However, the five-point immobilized aldoketoreductase retained 70% of its original activity after 16 hours and was higher than the three-point immobilized aldoketoreductase.
The invention obtains the mutant enzyme doped with the unnatural amino acid at different sites by changing the number of the mutant sites, thereby realizing accurate and controllable multi-site fixation, preparing the cross-linked enzyme aggregate and realizing the purification of the target enzyme protein. The method is safe and reliable, has less environmental pollution, greatly saves the time and cost consumed by purification, and is suitable for industrial production.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention and are not to be construed as limiting the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the scope of the invention as defined by the appended claims. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A method for directly and rapidly preparing cross-linked enzyme aggregate from cell lysate is characterized by comprising the following steps: firstly, dissolving a diacetylene cross-linking agent in isopropanol, and then dispersing the diacetylene cross-linking agent in a phosphate buffer solution of a cell disruption solution supernatant of an aldehyde-ketone reductase mutant doped with different amounts of unnatural amino acids; and (3) putting the container filled with the mixture into a microwave reactor provided with a cooling module for reaction, and then separating the immobilized enzyme.
2. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: the diyne cross-linking agent is 5,6,11, 12-tetrahydrodibenzo [ a, e ] cyclooctene.
3. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: the aldehyde-ketone reductase comprises three-point mutant aldehyde-ketone reductase and five-point mutant aldehyde-ketone reductase.
4. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: the unnatural amino acid incorporated by the aldoketoreductase is para-azido-L-phenylalanine.
5. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: the concentration of the phosphate buffer solution is 0.01-0.15 mol.L-1The pH value is 6.0-8.0.
6. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: making escherichia coli become a host for inducing aldehyde ketone reductase gene expression, centrifuging to obtain cell sediment, wherein the centrifugation revolution is 7000-9000 rpm, the centrifugation time is 4-8 min, and washing the sediment by using potassium phosphate buffer solution; resuspending the precipitate in potassium phosphate buffer solution, and lysing cells by ultrasonic treatment, wherein the addition amount of the potassium phosphate buffer solution is 1/5-1/3 of the volume of the original bacterial liquid, and the concentration of the potassium phosphate buffer solution is 0.05-0.15 mol.L-1The pH is 6.0-8.0, the ultrasonic crushing adopts an ice bath, the power is 300-500W, the crushing time is 8-12 min, and the crushing is stopped for 7s every 10 s; and centrifuging the soluble and insoluble parts after cell disruption, wherein the revolution number of the centrifugation is 9000-12000 rpm, and the time is 15-25 min, so as to separate and obtain a supernatant of the cell disruption solution.
7. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: dissolving a diyne crosslinking agent in isopropanol to a concentration of 6-10 mmol.L-1And then suspending the diacetylene cross-linking agent in 0.5-2 mL potassium phosphate buffer solution containing the supernatant of the multipoint mutant cell disruption solution, wherein the reaction molar ratio of azide to alkynyl is 1: 0.5 to 1.5.
8. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: the microwave temperature is 5-25 ℃, the microwave power is 5-40W, and the microwave time is 1-6 min.
9. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, wherein: separating the immobilized enzyme by using a centrifugation method, wherein the centrifugation rotation number is 10000-14000 Xg, the time is 2-8 min, washing the immobilized enzyme by using a phosphate buffer solution, and detecting the immobilized enzyme by using a Bradford method until no protein is detected in a supernatant; and taking out and centrifuging the reactant, draining the supernatant by using an injector, washing the reactant for 2-5 times by using potassium phosphate buffer solution, and drying in a vacuum oven at 20-40 ℃.
10. The method for directly and rapidly preparing the cross-linked enzyme aggregate from the cell lysate according to claim 1, which is characterized by comprising the following steps:
(1) dissolving the diyne crosslinking agent in isopropanol to a concentration of 8 mmol.L-1；
(2) Making Escherichia coli as host for inducing aldehyde ketone reductase gene expression, centrifuging to obtain cell precipitate, wherein the centrifugation rotation number is 8000rpm, the time is 5min, and washing the precipitate with potassium phosphate buffer solution; resuspending the pellet in potassium phosphate buffer (1/4) in an amount of 0.1 mol. L) and lysing the cells by sonication-1The pH value is 7.0, the ultrasonic crushing adopts an ice bath, the power is 400w, the crushing time is 10min, and the crushing is stopped for 7s every 10s after 3 s; centrifuging the soluble and insoluble parts after cell disruption at 10000rpm for 20min to obtain cell disruption solution supernatant;
(3) suspending the diyne cross-linking agent in potassium phosphate buffer solution of supernatant of aldehyde ketone reductase mutant cell disruption solution doped with 1mL of p-azidophenylalanine at three points and five points, wherein the concentration of the potassium phosphate buffer solution is 0.1 mol.L-1pH 7.0; the reaction molar ratio of azide to alkynyl is 1: 1;
(4) placing the container containing the above mixture into a microwave reactor equipped with a cooling module, and irradiating at 10 deg.C and 10W for 4 min;
(5) the immobilized enzyme was separated by centrifugation at 12000Xg for 5min, and washed with potassium phosphate buffer at a concentration of 0.01 mol. multidot.L-1pH 7.0; and are
Detection was performed using Bradford method until no protein was detected in the supernatant;
(6) the reaction was removed and centrifuged, the supernatant was drained with a syringe, and the enzyme aggregate was washed 4 times with water and dried in a vacuum oven at 30 ℃.
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