CN110669753A - Multi-point immobilization method based on unnatural amino acid modified enzyme - Google Patents

Abstract

The invention relates to a multipoint immobilization method based on an unnatural amino acid modified enzyme, wherein an unnatural amino acid is used for modifying aldehyde ketone reductase, cyclooctyne modified amino activated epoxy resin polymer is used for immobilizing the enzyme, and the enzyme is used as a solidified carrier to realize multipoint covalent connection with the unnatural amino acid modified aldehyde ketone reductase, and one-step purification and immobilization of the enzyme are completed through alkyne-azide cycloaddition reaction. The enzyme activity of the non-natural amino acid modified immobilized aldehyde-ketone reductase prepared by the method is greatly improved compared with that of free enzyme, and the traditional complicated purification method of the aldehyde-ketone reductase is omitted by copper-free click reaction, wherein the five-point immobilization can realize the purification efficiency of 60%. 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.

Description

Multi-point immobilization method based on unnatural amino acid modified enzyme

Technical Field

The invention relates to an enzyme immobilization method, in particular to a multi-point immobilization method based on an unnatural amino acid modified enzyme.

Background

The catalytic enzyme is produced from readily available renewable resources and is biodegradable, essentially harmless and non-toxic. The use of enzymes has increased dramatically over the last decades, mainly in the fields of research methodology, pharmaceutical research, food modification, laundry and paper industry in the cosmetics industry. However, such practical applications are hampered by the vulnerability of the enzymes (e.g., low thermostability, narrow optimal pH range, low tolerance to most organic solvents and many metal ions, etc.). In addition, enzymes lead to unavoidable purification and isolation steps. Immobilized enzymes provide a simple and elegant solution to these challenges. Methods for immobilizing enzymes can be divided into three categories: binding to a solid support (carrier), entrapment and cross-linking. Binding to the preformed support may be physical (e.g. hydrophobic), ionic or covalent. These differ in the type, character, form and type of interaction formed, the carrier material used. Traditionally, all of these techniques have failed to control the direction in which the enzyme attaches or adsorbs to the surface, thereby minimizing unwanted enzyme-surface interactions, maximizing enzyme stability, and maximizing active site accessibility. Among the various immobilization strategies, the attachment of enzymes to solid surfaces by physical adsorption or covalent bonding establishes one of the best practical techniques. The formation of the covalent bond of the enzyme and the carrier is firm and irreversible, and the operation stability is higher.

Therefore, it is important to find an efficient covalent site-specific enzyme immobilization method. Site-directed mutagenesis allows the exchange of specific amino acids at any position in the protein sequence, thereby increasing control over the direction of ligation, as it is not limited to only N-terminal or C-terminal modifications. Furthermore, multiple site-directed mutations can be introduced into a single enzyme variant, not only allowing its targeted modification to a support, but also modulating the enzyme's interaction with the support and its catalytic behavior. Previous studies demonstrated that covalent site-specific immobilization techniques have great limitations at potential fixed positions, such as the N-terminal or C-terminal regions, and rely primarily on modification of functional groups in the side chains of 20 canonical amino acids. Traditional site-specific modification of canonical amino acids does not meet our need for new covalent immobilization, so we chose unnatural amino acids as important tools for immobilization.

Modification should occur at specific sites of the enzyme to maximize control over the orientation of the enzyme and maintain its maximum activity. 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 pairs (aaRS) -trnas 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. The orthogonal synthetase does not recognize endogenous trnas, particularly by aminoacylating an orthogonally homologous tRNA with an unnatural amino acid provided to (or synthesized by) the cell (which is not a potent substrate for the endogenous synthetase).

Also for protein fixation, cu (i) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted variants thereof (SPAAC) are becoming increasingly popular. Copper (I) salts are known to be cytotoxic and so studies have found that an effective alternative is in the form of a strain-promoted response. To improve the biocompatibility of azide-alkyne cycloadditions, we attempted to activate alkynes by methods other than metal catalysis (i.e., by ring strain). Research on strained alkenes and alkynes has continued until the 1960 s during which scientists have claimed that the least stable cycloalkyne cyclooctyne, when combined with the phenylazide, reacts "explosively". Based on the above theory, we selected cyclooctyne (BCN, Bicyclo [6.1.0] nonyne) as the azide crosslinker and modified it on a synthetic polymer support to accomplish the biological crosslinking of proteins. In place of inorganic materials, synthetic polymers including Polystyrene (PS), Polymethylmethacrylate (PMMA), Polycarbonate (PC) and poly (L-lactide) are another popular immobilized enzyme support in bioassays. The greatest advantage of synthetic polymers is that the support material, the monomer from which the polymer chain is built, can be chosen according to the requirements of the enzyme and the process in which the immobilized product is used. However, one of the most troublesome problems in immobilization 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.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a one-step purification and multi-point immobilization method based on an unnatural amino acid modified enzyme. The method introduces unnatural amino acid and is characterized in that the unnatural amino acid is used for modifying aldehyde ketone reductase, cyclooctyne modified amino activated epoxy resin polymer is used for immobilizing enzyme, the enzyme is used as a solid carrier to realize multi-point covalent connection with the aldehyde ketone reductase modified by the unnatural amino acid, and one-step purification and immobilization of the enzyme are completed through alkyne-azide cycloaddition reaction. The enzyme activity of the non-natural amino acid modified immobilized aldehyde-ketone reductase prepared by the method is greatly improved compared with that of free enzyme, and the traditional complicated purification method of the aldehyde-ketone reductase is omitted by copper-free click reaction, wherein the five-point immobilization can realize the purification efficiency of 60%. 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.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for multi-point immobilization based on an unnatural amino acid modified enzyme, comprising the steps of:

(1) synthesis and expression of unnatural amino acid-modified aldoketoreductase: modifying aldehyde ketone reductase by using unnatural amino acid to azido-L-phenylalanine to obtain aldehyde ketone reductase with different numbers of mutation sites;

(2) carrier activation (preparation of cyclooctyne modified amino epoxy resin): the epoxy resin is modified into an epoxy resin carrier with a cyclooctyne functional group through primary amino modification and secondary cyclooctyne modification;

(3) one-step purification and multi-point immobilization of the enzyme: the epoxy resin with cyclooctyne functional groups is used for immobilizing the aldehyde ketone reductase with different numbers of mutation sites, and one-step purification and immobilization of the enzyme are completed through alkyne-azide cycloaddition reaction.

Preferably, the aldehyde-ketone reductases include single point mutant aldehyde-ketone reductases, three point mutant aldehyde-ketone reductases, and five point mutant aldehyde-ketone reductases.

The invention takes single-point mutation aldehyde ketone reductase, three-point mutation aldehyde ketone reductase and five-point mutation aldehyde ketone reductase as research materials, epoxy resin is modified into epoxy resin with cyclooctyne functional groups through primary amino modification and secondary cyclooctyne modification, and three types of mutation enzymes and the modified epoxy resin are immobilized with enzyme, so that the enzyme with higher enzyme activity after one-step purification and immobilization is obtained, and the immobilization efficiency is greatly improved along with the increase of sites.

Preferably, the step (1) is specifically: the method comprises the steps of simulating the tertiary structure of a target aldehyde ketone reductase through SWISS-MODEL, carrying out site-directed mutagenesis on a target gene of the aldehyde ketone reductase to obtain a TAG codon through screening an active site, co-transforming the target gene of the aldehyde ketone reductase and an orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair pEVOL-pAzF into C321. delta A strain (MG1655), adding antibiotics and an inducer, and inducing to obtain the required aldehyde ketone reductase (target protein). In this technique, it is first necessary to introduce exogenous aaRS-tRNA pairs into the body, which do not interfere with the endogenous aaRS-tRNA pairs, and which specifically recognize and "bind" an unnatural amino acid to the corresponding cognate tRNA by aminoacylation. These aminoacyl-trnas carrying the unnatural amino acid then enter the ribosome and introduce the unnatural amino acid into the growing peptide chain by recognizing a specific nonsense codon in the mRNA.

The selection of the mutation site of the aldehyde ketone reductase in the step (1) is to ensure that the mutation site is far away from the active center of the protein and mutates into a TAG codon, and the multi-point target gene mutation is carried out on the basis of a single point.

Preferably, the amino modification (epoxy resin amination) process in the step (2) is as follows: and adding the epoxy resin polymer into the reaction aqueous solution for amino activation, adjusting the pH value of the solution to be alkaline, centrifuging, washing and drying after reaction to obtain an amino-activated epoxy resin carrier intermediate.

The amino modified epoxy resin is modified and fixed under alkaline conditions by using amino acid which is close to isoelectric point under alkaline conditions.

Preferably, the cyclooctynyl modification (esterification of carboxyl group of amino epoxy resin) in the step (2) is performed by: adding cyclooctyne (BCN), an amino activated epoxy resin carrier intermediate, Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) into Dichloromethane (DCM), and reacting to obtain the cyclooctyne functionalized epoxy resin.

Preferably, the step (3) is specifically: adding cyclooctyne functionalized resin carrier and harvested aldehyde ketone reductase (crude enzyme supernatant of cell lysate of cultured strain) with different numbers of mutation sites into phosphate buffer solution, carrying out immobilization by shaking incubation operation, washing the immobilized enzyme with sodium chloride solution and phosphate buffer solution, and completing accurate multi-point immobilization of target protein.

Preferably, the specific process of cyclooctynyl modification is as follows: adding an epoxy resin polymer into the reaction aqueous solution, adding 2eq of lysine, and adjusting the pH value of the solution to 9-11 by adding NaOH; reacting at room temperature overnight, 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 40-60 ℃.

Preferably, the cyclooctynyl modification process specifically comprises the following steps: cyclooctyne, an amino-activated epoxy resin carrier intermediate, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a molar ratio of 1: 1: 1.2: 0.5 addition to dichloromethane and reaction overnight in the dark at room temperature; after the reaction, the solution is washed 3-6 times with water, and the cyclooctyne functionalized resin is dried in a vacuum drying oven at 40-60 ℃.

Preferably, the method for multi-point immobilization based on an unnatural amino acid-modifying enzyme specifically comprises the following steps:

(1) synthesis and expression of unnatural amino acid-modified aldoketoreductase: the aldoketoreductase mutant cells were inoculated into LB medium containing 50. mu.g/mL ampicillin, 34. mu.g/mL chloramphenicol and 100. mu.g/mL kanamycin and cultured at 34 ℃ in a shaking incubator; when OD600 reached 0.5, inducer L- (+) -arabinose was added to a final concentration of 0.2% (w/v); at an OD600 of 0.6, protein expression was induced by adding 30ng/mL anhydrotetracycline and p-azido-L-phenylalanine as inducers to a final concentration of 1 mmol/L;

(2) activating a carrier: adding 1g of epoxy resin polymer and 15mg of lysine into 3mL of reaction aqueous solution, adjusting the pH value of the solution to 10 by adding 1mol/L NaOH, carrying out the reaction at room temperature overnight, taking out and centrifuging the reaction product, draining the supernatant by using an injector, washing the reaction product for 3 times by using water, and drying the reaction product in a vacuum oven at 50 ℃; adding 100. mu. moL of cyclooctyne, 100. mu. moL of amino-activated epoxy resin carrier intermediate, 120. mu. moL of dicyclohexylcarbodiimide and 50. mu. moL of 4-dimethylaminopyridine to 2mL of dichloromethane, and reacting overnight at room temperature in the dark; after the reaction, the solution was washed 4 times with water to remove excess DCC and DMAP, and the cyclooctyne functionalized resin was dried in a vacuum oven at 50 ℃;

(3) one-step purification and multi-point immobilization of the enzyme: 0.5g of cyclooctyne functionalized resin carrier and 3mL of single-point, three-point, five-point mutant crude enzyme supernatant of cell lysate of the harvested cultured strain were added to 1mL of phosphate buffer and incubated at 20 ℃ with shaking and run at 160rpm for 18 hours, and then the immobilized enzyme was washed twice with 1mol/L NaCl solution and 0.02mol/L phosphate buffer.

The enzyme activity of the non-natural amino acid modified immobilized aldehyde-ketone reductase prepared by the method is greatly improved compared with that of free enzyme, and the traditional complicated purification method of the aldehyde-ketone reductase is omitted by copper-free click reaction, wherein under the condition of 24h, the purification efficiency of 60 percent can be realized by five-point immobilization. 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.

Drawings

FIG. 1 is a schematic view of the immobilization process of the present invention;

FIG. 2 is the effect of immobilization time on the amount of immobilized enzyme;

FIG. 3 is the relative strength of single point mutase immobilization;

FIG. 4 is the relative strengths of three-point mutant enzyme immobilization;

FIG. 5 is the relative strength of five point mutant enzyme immobilization.

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 only in light of the teachings of the present invention and are not intended to be limiting.

According to the invention, an unnatural amino acid is used for modifying the azido-L-phenylalanine aldehyde ketone reductase, meanwhile, a cyclooctyne modified amino activated epoxy resin polymer is used for immobilizing the enzyme, the amino activated epoxy resin polymer is used as a solidified carrier to realize multi-point covalent connection with the unnatural amino acid modified aldehyde ketone reductase, and one-step purification and immobilization of the enzyme are completed through alkyne-azide cycloaddition reaction; the process is shown in figure 1.

Synthesis and expression of unnatural amino acid-modified aldoketoreductase

Simulating the tertiary structure of the target aldehyde ketone reductase by SWISS-MODEL, carrying out site-directed mutagenesis on a target gene into a TAG codon by screening an active site, cotransforming the target gene and an orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair pEVOL-pAzF into C321. delta. A strain (MG1655), adding an antibiotic and an inducer, and inducing to obtain the required aldehyde ketone reductase (target protein). The method specifically comprises the following steps: the aldoketoreductase mutant cells were inoculated into LB medium containing 50. mu.g/mL ampicillin, 34. mu.g/mL chloramphenicol and 100. mu.g/mL kanamycin and cultured at 34 ℃ in a shaking incubator; when OD600 reached 0.5, inducer L- (+) -arabinose was added to a final concentration of 0.2% (w/v); at an OD600 of 0.6, protein expression was induced by addition of 30ng/mL anhydrotetracycline and p-azido-L-phenylalanine as inducers to a final concentration of 1 mmol/L.

The invention uses single-point mutation aldehyde ketone reductase and three-point and five-point mutation aldehyde ketone reductase as research materials.

Modification of epoxy resin carriers

In order to select more preferable epoxy resin amination, the present invention screens precursor amino acids for epoxy resin amination, and performs epoxy resin amination using lysine, glutamic acid, and glycine, respectively.

Epoxy resin aminated by using BCN modified lysine

To an aqueous reaction solution (3mL) was added an epoxy resin polymer (120. mu. moL epoxy number, 1g), 2eq lysine (240. mu. moL, 15mg) was added, and the pH of the solution was adjusted to 10 by the addition of 1moL/L NaOH. The reaction was allowed to proceed at room temperature overnight, the reaction was removed and centrifuged, the supernatant was drained with a syringe, and the reaction was washed 3 times with water and dried in a vacuum oven at 50 ℃. Bicyclo [6.1.0] nonyne (bcn) (100 μmoL), an amino-activated epoxy resin-supported intermediate (100 μmoL), DCC (120 μmoL) and DMAP (50 μmoL) were added to 2mL of DCM and reacted at room temperature overnight in the dark. After the reaction, the solution was washed 4 times with aqueous solution to remove excess DCC and DMAP. The BCN functionalized resin was dried in a vacuum oven at 50 ℃.

Epoxy resin aminated by modifying glutamic acid with BCN

To an aqueous reaction solution (3mL) was added an epoxy resin polymer (120. mu. moL epoxy number, 1g), 2eq glutamic acid (240. mu. moL, 35mg) was added, and the pH of the solution was adjusted to 10 by the addition of 1moL/L NaOH. The reaction was allowed to proceed at room temperature overnight, the reaction was removed and centrifuged, the supernatant was drained with a syringe, and the reaction was washed 3 times with water and dried in a vacuum oven at 50 ℃. Bicyclo [6.1.0] nonyne (bcn) (100 μmoL), an amino-activated epoxy resin-supported intermediate (100 μmoL), DCC (120 μmoL) and DMAP (50 μmoL) were added to 2mL of DCM and reacted at room temperature overnight in the dark. After the reaction, the solution was washed 4 times with aqueous solution to remove excess DCC and DMAP. The BCN functionalized resin was dried in a vacuum oven at 50 ℃.

Glycine aminated epoxy resin modified by BCN

To an aqueous reaction solution (3mL) was added an epoxy resin polymer (120. mu. moL epoxy number, 1g), 2eq glycine (240. mu. moL, 18mg) was added, and the pH of the solution was adjusted to 10 by the addition of 1moL/L NaOH. The reaction was allowed to proceed at room temperature overnight, the reaction was removed and centrifuged, the supernatant was drained with a syringe, and the reaction was washed 3 times with water and dried in a vacuum oven at 50 ℃. BCN (100. mu. moL), amino-activated epoxy resin support intermediate (100. mu. moL), DCC (120. mu. moL) and DMAP (50. mu. moL) were added to 2mL DCM and reacted at room temperature overnight at a temperature in the dark. After the reaction, the solution was washed 4 times with aqueous solution to remove excess DCC and DMAP. The BCN functionalized resin was dried in a vacuum oven at 50 ℃.

By modification of different aminated resins with cyclooctyne, we have found that lysine immobilization is superior to glutamic acid and glycine. The isoelectric points of the three amino acids lysine, glycine and glutamic acid are respectively 9.74, 5.97 and 3.22. Lysine is more difficult to protonate under basic conditions than glycine and glutamic acid, which means that amino acid groups are easily exposed to the carrier and easily react with epoxy groups, which can be opened by amino groups under basic conditions to complete the modification of the group.

Enzyme immobilization time for screening

In order to better select the immobilization time, the invention screens the immobilization time of single-point mutation aldehyde ketone reductase on the cyclooctyne modified amino epoxy resin.

Immobilization of single-point mutant enzyme by using BCN modified amino acid aminated epoxy resin

BCN-functionalized resin support (0.5g) and 3mL of crude enzyme supernatant of harvested cell lysate of the cultured strain were added to 1mL of phosphate buffer (0.02M, pH 7.0) and run at 160rpm for 24 hours with shaking incubation at 20 ℃. And (3) carrying out an enzyme concentration test of the single-point mutant aldehyde ketone reductase, and then detecting the enzyme concentration of the supernatant in the immobilized enzyme every 4 hours to calculate the amount of the immobilized enzyme at different times. Then, the immobilized enzyme was washed twice with 1mol/L NaCl solution and 0.02mol/L phosphate buffer solution (pH 7.0).

The results of the effect of the immobilization time of the lysine-modified epoxy resin, the glutamic acid-modified epoxy resin, and the glycine-modified epoxy resin on the amount of immobilized enzyme are shown in fig. 2, and it can be seen from fig. 2 that the amounts of enzyme immobilized by the three types of immobilized carriers gradually increase with the increase of time, wherein the amount of immobilized enzyme reaches the maximum after 24 hours, and among the three types of carriers, the immobilization effect of the epoxy resin aminated by lysine modified with cyclooctyne is the best. Through experiments, the lysine has better fixation efficiency compared with the glutamic acid and glycine modified epoxy resin, and meanwhile, the amount of immobilized enzyme is gradually increased along with the increase of time and reaches the maximum after 24 hours.

Effect of the number of immobilization sites on immobilization Rate

In order to better select the number of the sites of the immobilized enzyme, the invention implements immobilization of aldehyde ketone reductase with different numbers of mutation sites.

Single mutation site aldehyde ketone reductase is subjected to one-step purification and immobilization

BCN-functionalized resin three amino acid modified epoxy resin and carrier (0.5g) and 3mL of the harvested single point mutant strain cell lysate crude enzyme supernatant were added to 1mL phosphate buffer (0.02mol/L, pH 7.0) and run at 160rpm for 24 hours at 20 ℃ with shaking incubation. And (3) carrying out an enzyme concentration test of the single-point mutant aldehyde ketone reductase, and then detecting the enzyme concentration of the supernatant in the immobilized enzyme every 4 hours to calculate the amount of the immobilized enzyme at different times. Then, the immobilized enzyme was washed twice with 1mol/L NaCl solution and 0.02mol/L phosphate buffer solution (pH 7.0).

The protein supernatant before immobilization, the protein supernatant after immobilization, and the non-target protein washed out at the first stage were subjected to SDS-PAGE, the whole immobilization process (including the crude enzyme supernatant before immobilization, the immobilized supernatant, and the supernatants of two 1mol/L NaCl-washed carriers) was analyzed by SDS-PAGE, and the relative intensity of the supernatants during immobilization was calculated using ImageJ software to obtain the actual target enzyme immobilization rate. The effect of the immobilization time on the amount of immobilized enzyme was shown in FIG. 3, and the purification immobilization rate of the lysine-modified epoxy resin was calculated to be 30%.

Three mutation site aldehyde ketone reductase is subjected to one-step purification and immobilization

BCN functionalized lysine modified epoxy resin and carrier (0.5g) and 3mL of the harvested crude enzyme supernatant of the three point mutant strain cell lysate were added to 1mL of phosphate buffer (0.02mol/L, pH 7.0) and incubated at 20 ℃ with shaking and run at 160rpm for 24 hours. And (3) carrying out an enzyme concentration test of the single-point mutant aldehyde ketone reductase, and then detecting the enzyme concentration of the supernatant in the immobilized enzyme every 4 hours to calculate the amount of the immobilized enzyme at different times. Then, the immobilized enzyme was washed twice with 1mol/L NaCl solution and 0.02mol/L phosphate buffer solution (pH 7.0).

The protein supernatant before immobilization, the protein supernatant after immobilization, and the non-target protein washed out at the first stage were subjected to SDS-PAGE, the whole immobilization process (including the crude enzyme supernatant before immobilization, the immobilized supernatant, and the supernatants of two 1mol/L NaCl-washed carriers) was analyzed by SDS-PAGE, and the relative intensity of the supernatants during immobilization was calculated using ImageJ software to obtain the actual target enzyme immobilization rate. The effect of the immobilization time on the amount of immobilized enzyme was shown in FIG. 4, and the purification immobilization rate of the lysine-modified epoxy resin was calculated to be 50%.

Five mutation site aldehyde ketone reductase is subjected to one-step purification and immobilization

BCN functionalized lysine-modified epoxy resin and carrier (0.5g) and 3mL of the crude enzyme supernatant of the harvested five-point mutated strain cell lysate were added to 1mL of phosphate buffer (0.02mol/L, pH 7.0) and incubated at 20 ℃ with shaking and run at 160rpm for 24 hours. And (3) carrying out an enzyme concentration test of the single-point mutant aldehyde ketone reductase, and then detecting the enzyme concentration of the supernatant in the immobilized enzyme every 4 hours to calculate the amount of the immobilized enzyme at different times. Then, the immobilized enzyme was washed twice with 1mol/L NaCl solution and 0.02mol/L phosphate buffer solution (pH 7.0).

The protein supernatant before immobilization, the protein supernatant after immobilization, and the non-target protein washed out at the first stage were subjected to SDS-PAGE, the whole immobilization process (including the crude enzyme supernatant before immobilization, the immobilized supernatant, and the supernatants of two 1mol/L NaCl-washed carriers) was analyzed by SDS-PAGE, and the relative intensity of the supernatants during immobilization was calculated using ImageJ software to obtain the actual target enzyme immobilization rate. The effect of the immobilization time on the amount of immobilized enzyme was shown in FIG. 5 when the lysine-modified epoxy resin was immobilized on the crude enzyme, and the purification immobilization rate of the lysine-modified epoxy resin was calculated to be 60%.

The method comprises the steps of selecting lysine modified epoxy resin to carry out single-point, three-point and five-point mutation of aldehyde ketone reductase for immobilization, carrying out 24-hour immobilization, washing resin immobilized enzyme twice by using 1mol/L NaCl to wash enzyme adsorbed on the epoxy resin so as to load the whole carrier on target mutant enzyme, carrying out SDS-PAGE on protein supernatant before immobilization, protein supernatant after immobilization and non-target protein washed out at the first stage, and analyzing the strip strength by software Image so as to obtain the expected immobilization yield. The single-point immobilization rate can reach 30%, the three-point effect can reach 50%, and the five-point pure enzyme immobilization rate can reach 60%.

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 (9)

1. A method for multi-point immobilization based on an unnatural amino acid-modified enzyme, comprising the steps of:

(1) synthesis and expression of unnatural amino acid-modified aldoketoreductase: modifying aldehyde ketone reductase by using unnatural amino acid to azido-L-phenylalanine to obtain aldehyde ketone reductase with different numbers of mutation sites;

(2) activating a carrier: the epoxy resin is modified into an epoxy resin carrier with a cyclooctyne functional group through primary amino modification and secondary cyclooctyne modification;

(3) one-step purification and multi-point immobilization of the enzyme: the epoxy resin with cyclooctyne functional groups is used for immobilizing the aldehyde ketone reductase with different numbers of mutation sites, and one-step purification and immobilization of the enzyme are completed through alkyne-azide cycloaddition reaction.

2. The method of claim 1, wherein the non-natural amino acid modifying enzyme-based multi-point immobilization comprises: the aldehyde ketone reductase comprises single-point mutation aldehyde ketone reductase, three-point mutation aldehyde ketone reductase and five-point mutation aldehyde ketone reductase.

3. The method for the multiple-point immobilization based on an unnatural amino acid-modifying enzyme according to claim 1 or 2, wherein the step (1) is specifically: through SWISS-MODEL to simulate the tertiary structure of the target aldehyde ketone reductase, through screening active sites, the target gene of the aldehyde ketone reductase is subjected to site-directed mutation to be TAG codon, the target gene of the aldehyde ketone reductase and an orthogonal aminoacyl-tRNA synthetase/tRNA pair pEVOL-pAzF are co-transformed to C321. delta. Astrain, and antibiotics and an inducer are added for induction to obtain the required aldehyde ketone reductase.

4. The method for multi-point immobilization based on an unnatural amino acid modifying enzyme according to claim 1 or 2, wherein the amino modification in step (2) is: and adding the epoxy resin polymer into the reaction aqueous solution for amino activation, adjusting the pH value of the solution to be alkaline, centrifuging, washing and drying after reaction to obtain an amino-activated epoxy resin carrier intermediate.

5. The method of claim 4, wherein the cyclooctynyl modification process in step (2) is: adding cyclooctyne, an amino activated epoxy resin carrier intermediate, dicyclohexylcarbodiimide and 4-dimethylaminopyridine into dichloromethane, and reacting to obtain the cyclooctyne functionalized epoxy resin.

6. The method for the multiple-point immobilization based on an unnatural amino acid-modifying enzyme according to claim 1 or 2, wherein the step (3) is specifically: the cyclooctyne functionalized resin carrier and the harvested aldehyde ketone reductase of different numbers of mutation sites are added into a phosphate buffer solution, and immobilized in a shaking incubation run, and the immobilized enzyme is washed with a sodium chloride solution and the phosphate buffer solution.

7. The method of claim 4, wherein the cyclooctynyl group modification comprises the following steps: adding an epoxy resin polymer into the reaction aqueous solution, adding 2eq of lysine, and adjusting the pH value of the solution to 9-11 by adding NaOH; reacting at room temperature overnight, 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 40-60 ℃.

8. The method of claim 5, wherein the cyclooctynyl modification is performed by: cyclooctyne, an amino-activated epoxy resin carrier intermediate, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a molar ratio of 1: 1: 1.2: 0.5 addition to dichloromethane and reaction overnight in the dark at room temperature; after the reaction, the solution is washed 3-6 times with water, and the cyclooctyne functionalized resin is dried in a vacuum drying oven at 40-60 ℃.

9. The method of claim 1, comprising the steps of:

(1) synthesis and expression of unnatural amino acid-modified aldoketoreductase: the aldoketoreductase mutant cells were inoculated into LB medium containing 50. mu.g/mL ampicillin, 34. mu.g/mL chloramphenicol and 100. mu.g/mL kanamycin and cultured at 34 ℃ in a shaking incubator; when OD600 reached 0.5, inducer L- (+) -arabinose was added to a final concentration of 0.2% (w/v); at an OD600 of 0.6, protein expression was induced by adding 30ng/mL anhydrotetracycline and p-azido-L-phenylalanine as inducers to a final concentration of 1 mmol/L;

(2) activating a carrier: adding 1g of epoxy resin polymer and 15mg of lysine into 3mL of reaction aqueous solution, adjusting the pH value of the solution to 10 by adding 1mol/L NaOH, carrying out the reaction at room temperature overnight, taking out and centrifuging the reaction product, draining the supernatant by using an injector, washing the reaction product for 3 times by using water, and drying the reaction product in a vacuum oven at 50 ℃; adding 100. mu. moL of cyclooctyne, 100. mu. moL of amino-activated epoxy resin carrier intermediate, 120. mu. moL of dicyclohexylcarbodiimide and 50. mu. moL of 4-dimethylaminopyridine to 2mL of dichloromethane, and reacting overnight at room temperature in the dark; after the reaction, the solution was washed 4 times with water and the cyclooctyne functionalized resin was dried in a vacuum oven at 50 ℃;

(3) one-step purification and multi-point immobilization of the enzyme: 0.5g of cyclooctyne functionalized resin carrier and 3mL of single-point, three-point, five-point mutant crude enzyme supernatant of cell lysate of the harvested cultured strain were added to 1mL of phosphate buffer and incubated at 20 ℃ with shaking and run at 160rpm for 18 hours, and then the immobilized enzyme was washed twice with 1mol/L NaCl solution and 0.02mol/L phosphate buffer.

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