CN117904097A - Self-assembled protein scaffold co-immobilized enzyme and application thereof - Google Patents
Self-assembled protein scaffold co-immobilized enzyme and application thereof Download PDFInfo
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- CN117904097A CN117904097A CN202410071694.1A CN202410071694A CN117904097A CN 117904097 A CN117904097 A CN 117904097A CN 202410071694 A CN202410071694 A CN 202410071694A CN 117904097 A CN117904097 A CN 117904097A
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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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
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/098—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer formed in the presence of the enzymes or microbial cells
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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
- 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)
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses a self-assembled protein scaffold co-immobilized enzyme and application thereof, wherein the self-assembled protein scaffold co-immobilized enzyme is obtained by adopting EutM-SpyCatcher/SpyTag system to immobilize R-omega-aminotransferase and alcohol dehydrogenase; an immobilized multienzyme catalytic system is constructed in vitro, so that the cascade reaction of ATTA and ADH double enzymes is realized, a high-efficiency coenzyme regeneration system is generated, the NADPH regeneration efficiency is improved, and sufficient NADPH is provided for biocatalysis reaction, so that the cascade action and the catalytic efficiency of enzymes are enhanced; meanwhile, the protein scaffold also provides a good environment for amination reaction, and improves the thermal stability of enzyme.
Description
Technical Field
The invention relates to a self-assembled protein scaffold, which co-immobilizes R-omega-aminotransferase from Aspergillus terreus and alcohol dehydrogenase and applies the same to asymmetric selective amination of 1- (4-methoxyphenyl) acetone to prepare (R) -1- (4-methoxyphenyl) propan-2-amine.
Background
(R) -1- (4-methoxyphenyl) propan-2-amine ((R) -1- (4-methoxyphenyl) propan-2-amine, 4-MPPA), CAS number: 58993-79-6, the molecular formula is: c 10H15 NO, molecular weight: 165.23, density is: 0.99g/mL, boiling point: is insoluble in water at 90-95 deg.C, and is easily dissolved in organic solvent such as ethyl acetate. The (R) -1- (4-methoxyphenyl) propan-2-amine is a key chiral intermediate for synthesizing various medicines such as formoterol which is a powerful medicine for treating asthma and chronic obstructive pulmonary disease, so how to economically, environmentally-friendly and efficiently prepare the (R) -1- (4-methoxyphenyl) propan-2-amine is one of research hotspots in the related fields at home and abroad.
Biocatalysis has found wide application as a substitute for chemical catalysis in sustainable chemistry, where the synthesis of key intermediates for chiral drugs is prominent in the pharmaceutical industry. With the development of biosynthesis and metabolic engineering, researchers construct artificial multi-enzyme complex systems in microorganisms as well as in vitro based on engineering design and gene editing. To achieve the desired catalytic effect, numerous strategies for multi-enzyme cascade immobilization have been developed, including carrier-mediated co-immobilization, enzyme scaffolds, protein fusion, and the like.
The multienzyme co-immobilization technology has a plurality of advantages, so that enzyme molecules have good stability and recycling property, the performance of synergetic catalysis among the multienzymes is fully exerted, and the catalysis efficiency is improved. By genetically engineering bacterial microchamber proteins EutM from Salmonella enterica, they can spontaneously assemble into crystal arrays in vivo and in vitro to form a stable protein scaffold. The SpyCatcher gene is fused to the C-end of EutM, the short peptide SpyTag gene is fused to the R-ATTA of the target protein and the N-end of ADH, the target protein is immobilized by spontaneously forming a protein scaffold by self-assembled protein EutM and spontaneously forming an isopeptide bond by the SpyCatcher/SpyTag system, and a protein-based scaffold co-immobilized biocatalysis system is created. Compared with the free enzyme mixture catalytic cascade reaction, the protein scaffold co-immobilized multienzyme catalytic system provides a modularized and easy-to-use immobilized system, provides a stable microenvironment for amination reaction, and improves the thermal stability of enzymes; and the protein scaffold is utilized to arrange the multiple enzymes in a short distance, so as to trigger a substrate channel effect, realize the efficient utilization and cascade catalysis of cofactors, and further improve the catalysis efficiency.
Disclosure of Invention
The invention aims to provide a method for synthesizing (R) -1- (4-methoxyphenyl) propan-2-amine ((R) -4-MPPA) by double enzyme cascade of self-assembled protein bracket co-immobilized R-omega-aminotransferase (R-omega-ATTA) and Alcohol Dehydrogenase (ADH), which utilizes the self-assembled protein bracket immobilized aminotransferase and alcohol dehydrogenase to form an efficient multienzyme cascade reaction system for coenzyme regeneration circulation, improves the catalytic efficiency and stability of multienzyme, reduces the cost and provides reference for the subsequent co-immobilized double enzyme cascade catalytic process.
The technical scheme adopted by the invention is as follows:
In a first aspect, the invention provides a self-assembled protein scaffold co-immobilized enzyme, wherein the self-assembled protein scaffold co-immobilized enzyme is obtained by immobilizing R-omega-aminotransferase and alcohol dehydrogenase by adopting a EutM-SpyCatcher/SpyTag system; the amino acid sequence of the R-omega-aminotransferase is shown in SEQ ID NO:14, the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO: shown at 18.
The double-enzyme system obtained by EutM-SpyCatcher/SpyTag system can effectively improve the thermal stability of the enzyme and further improve the enzymatic reaction temperature, the conversion rate and the ee value.
Further, the self-assembled protein scaffold co-immobilized enzyme is constructed by the following method:
mixing recombinant protein SpyTag-R-omega-aminotransferase (SpyTag-ATTA), recombinant protein SpyTag-alcohol dehydrogenase (SpyTag-ADH) and recombinant protein EutM-SpyCatcher in water, incubating for 1-12h (preferably 2 h) at 0-37 ℃ (preferably 4 ℃), ultrafiltering, and freeze-drying the obtained retentate to obtain the self-assembled protein scaffold co-immobilized enzyme.
The recombinant protein SpyTag-R-omega-aminotransferase is obtained by connecting a short peptide SpyTag with R-omega-aminotransferase; the recombinant protein SpyTag-alcohol dehydrogenase is obtained by connecting a short peptide SpyTag with R-omega-aminotransferase, and the recombinant protein EutM-SpyCatcher is obtained by connecting a SpyCatcher domain with EutM protein (connected to the C end of EutM).
The incubation process is gently swayed every half hour to promote assembly. Preferably, each recombinant protein is prepared as an aqueous solution and then mixed, and the concentration of each aqueous solution is 2 mu M.
In the embodiment of the invention, the amino acid sequence of the recombinant protein SpyTag-R-omega-aminotransferase is shown in SEQ ID NO: shown at 16; the amino acid sequence of the recombinant protein SpyTag-ethanol dehydrogenase is shown as SEQ ID NO: shown at 20; the amino acid sequence of the recombinant protein EutM-SpyCatcher is shown in SEQ ID NO: shown at 6.
Preferably, the molar ratio of the recombinant protein SpyTag-R- ω -transaminase, recombinant protein SpyTag-alcohol dehydrogenase to recombinant protein EutM-SpyCatcher is 1:0.5-2:1-7 (preferably 1:1:3).
Further, the procedure of freeze drying is: pre-freezing in a refrigerator at-80deg.C for 8 hr, and vacuum drying at-65deg.C for 24 hr.
The catalytic system is a co-immobilized enzyme catalytic system formed by fusing a SpyCatcher domain to the C-terminal of EutM to form a EutM-SpyCatcher domain scaffold building block, wherein the SpyCatcher domain on the scaffold forms a covalent isopeptide bond with a target protein R-omega-aminotransferase (R-omega-ATTA) and an Alcohol Dehydrogenase (ADH) fused with the SpyTag domain.
Preferably, the pure enzyme of the recombinant protein EutM-SpyCatcher is prepared as follows: (1) Introducing recombinant plasmid pET22b-EutM-SpyCatcher into competent escherichia coli BL21 (DE 3), inoculating single colony into LB medium containing 50-100 mug/mL (preferably 50 mug/mL) ampicillin, and culturing at 37 ℃ and 150-200rpm (preferably 180 rpm) for overnight to obtain seed solution; (2) Transferring seed solution into fresh LB medium containing 50-100 μg/mL (preferably 50 μg/mL) ampicillin according to 1% -5% (preferably 3%) inoculation amount, fermenting and culturing at 37 ℃ and 150-200rpm (preferably 180 rpm) until the optical density (OD 600) at 600nm is 0.6-1.0 (preferably 0.8), adding isopropyl-beta-thiogalactoside (IPTG) with the final concentration of 0.1-1.0mM (preferably 0.5M), and continuously inducing and culturing in a constant temperature shaking table at 16-30 ℃ (preferably 18 ℃) for 8-24h (preferably 16 h); centrifuging the fermentation broth at 4deg.C and 8000rpm for 10min, washing the precipitate with 0.9% (v/w) physiological saline for 1-4 times (preferably 3 times), and collecting wet cells; (3) Resuspending the wet cells harvested in the step (2) in a pH 7.0 and 100mM sodium phosphate buffer solution at 50g/L, and performing ultrasonic disruption for 15min under the conditions of power of 360W, work for 3s and intermittent time of 7 s; collecting the crushed liquid, centrifuging at a speed of 8000rpm and a temperature of 4 ℃ for 10min, and taking supernatant to obtain crude enzyme liquid; (4) The crude enzyme solution filtered by a 0.45 mu m filter membrane is mixed with a chromatographic medium of a reduction-resistant nickel column (BeyoGold TM His-tag) according to a volume ratio of 8:1 mixing (preferably placing in ice cubes, slowly shaking in a shaking table at 40rpm for 1 h), loading onto a reduction-resistant nickel column (BeyoGold TM His-tag), washing the column with non-denaturing washing liquid for 5 times to remove impurity proteins, and eluting 1-2 column volumes each time; eluting with non-denaturing eluent for 6-10 times (preferably 8 times), eluting 1-2 column volumes each time, collecting eluent containing target protein, dialyzing with 0.01M, pH 8.0.0 sodium phosphate buffer for 24 hr, replacing the dialysate for three times, and lyophilizing the retentate (pre-freezing in-80deg.C refrigerator for 8 hr, and vacuum drying at-65deg.C for 24 hr) to obtain pure enzyme; the non-denaturing washing is pH8.0, 50mM sodium dihydrogen phosphate buffer containing 300mM NaCl and 2mM imidazole; the non-denaturing eluate is pH8.0, 50mM sodium dihydrogen phosphate buffer containing 300mM NaCl and 50mM imidazole.
In the examples of the present invention, the recombinant protein SpyTag-R- ω -transaminase, the recombinant protein SpyTag-alcohol dehydrogenase, and the recombinant protein EutM-SpyCatcher were prepared by the following procedures: constructing recombinant expression plasmid, transferring the recombinant expression plasmid into host cell, inducing expression, purifying and separating. The above operation is a routine procedure in the art, and it is preferable that the recombinant expression plasmid vector of the recombinant protein SpyTag-R-omega-aminotransferase and the recombinant protein SpyTag-alcohol dehydrogenase is pET28a, and the recombinant expression plasmid vector of the recombinant protein EutM-SpyCatcher is pET22b; the host cells of the recombinant proteins are all E.coli BL21 (DE 3).
The recombinant plasmid pET22b-EutM-SpyCatcher is constructed by the following method: (1) The EutM gene is constructed between Xba I and BamH I restriction enzyme sites of pET-22b plasmid to obtain plasmid pET22b-EutM; the nucleotide sequence of EutM gene is shown as SEQ ID NO:1 is shown in the specification;
The plasmid pET22b-EutM is subjected to double digestion by using restriction enzymes BamH I and Xho I to obtain a linearization vector;
(2) Inserting the SpyCatcher gene between BamH I and Xho I sites of the pET-28a (+) plasmid to obtain a plasmid pET-28a-SpyCatcher; the nucleotide sequence of the SpyCatcher gene is shown in SEQ ID NO:3 is shown in the figure;
Taking the plasmid pET-28a-SpyCatcher as a template, and carrying out PCR amplification by using the following primer pairs to obtain a target gene fragment:
forward primer CATCACCACAGCCAGGATCCGGTAGTGGTGGTAGTGGTG
Reverse primer TGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTC;
(3) And (3) seamlessly connecting the linearization vector in the step (1) and the target gene fragment in the step (2) to obtain the recombinant plasmid pET22b-EutM-SpyCatcher.
Furthermore, the pure enzymes of the recombinant proteins SpyTag-ATTA and SpyTag-ADH adopt the preparation method of the EutM-SpyCatcher pure enzyme, and the difference is that: ① The recombinant plasmids in the step (1) are pET28a-SpyTag-ATTA and pET28a-SpyTag-ADH respectively; ② The temperature of the induction culture in the step (2) is 16-30 ℃ (preferably 23 ℃).
Furthermore, the construction method of the recombinant plasmid pET28a-SpyTag-ATTA is the same as that of pET22b-EutM-SpyCatcher, and the difference is that: in the step (1), SEQ ID NO:9, constructing a SpyTag gene between Nde I and BamH I restriction sites of a pET28a (+) plasmid to obtain a plasmid pET28a-SpyTag; then, restriction enzymes BamH I and Xho I are used for enzyme digestion to obtain a linearization vector; the step (2) is as follows: replacement of the SpyCatcher gene with SEQ ID NO:13 to obtain plasmid pET-28a-ATTA; taking the plasmid pET-28a-ATTA as a template, and carrying out PCR amplification by using the following primer pairs to obtain a target gene fragment:
forward primer TGGTGTAGACGGATTCGCGAGCATGGATAAAGTTTTCG
Reverse primer TGGTGGTGGTGGTGCTCGAGTTAGTTACGTTCGTTGTAGTCAATT.
Furthermore, the construction method of the recombinant plasmid pET28a-ADH is the same as that of pET28a-SpyTag-ATTA, and the difference is that: the step (1) is used; the step (2) is as follows: replacement of the ATTA aminotransferase gene with the amino acid sequence as set forth in SEQ ID NO:17 to obtain a plasmid pET228a-ADH; taking the plasmid pET-28a-ADH as a template, and carrying out PCR amplification by using the following primer pairs to obtain a target gene fragment:
forward primer TGGTGTAGACGGATTCATGAGCATTCCGGAAACCCAGA;
reverse primer TGGTGGTGGTGGTGCTCGAGTTTGCTGGTATCAACAACATAACGA.
In a second aspect, the invention also provides an application of the self-assembled protein scaffold immobilized enzyme in the synthesis of (R) -1- (4-methoxyphenyl) propan-2-amine ((R) -4-MPPA) by asymmetric reduction of 1- (4-methoxyphenyl) acetone.
Specifically, the application is as follows: the self-assembled protein scaffold immobilized enzyme is used as a catalyst, 1- (4-methoxyphenyl) acetone is used as a substrate, dimethyl sulfoxide (DMSO) is used as a cosolvent, isopropylamine hydrochloride is used as an ammonia donor, pyridoxal phosphate (PLP) is used as a coenzyme, NAD + is used as a cofactor, ethanol is used as an auxiliary substrate, a buffer solution with pH of 6-10 is used as a reaction medium to form a reaction system, the reaction system is mixed and stirred in a shaking table at 25-45 ℃ (preferably 32 ℃) and 150-200rpm (preferably 180 rpm) for 12-36h (preferably 24 h), and the obtained reaction solution is separated and purified to obtain (R) -1- (4-methoxyphenyl) propan-2-amine.
Preferably, in the reaction system, the final concentration of the catalyst is 0.5-5g/L (preferably 1 g/L), the final concentration of the 1- (4-methoxyphenyl) acetone is 25-125mM (preferably 125 mM), the final concentration of the isopropylamine hydrochloride is 25-125mM (preferably 100 mM), the final concentration of the pyridoxal phosphate is 0.5-1.5mM (preferably 1 mM), the final concentration of the NAD + is 0.5-1.5mM (preferably 1 mM), the final concentration of the cosubstrate is 25-125mM (preferably 50 mM), and the volume of the cosolvent is 10% -30% (preferably 20%) of the volume of the reaction system.
Preferably, the buffer is a pH 7.0, 100mM sodium phosphate buffer.
Preferably, the separation is purified as: and centrifuging the reaction liquid, collecting supernatant, adding ethyl acetate with the same volume as the supernatant for extraction, collecting an organic phase, drying by anhydrous sodium sulfate, and volatilizing the solvent at normal temperature to obtain (R) -1- (4-methoxyphenyl) propan-2-amine.
Compared with the prior art, the invention has the beneficial effects that:
The method adopts EutM brackets for immobilization of R-aminotransferase (R-ATTA) and Alcohol Dehydrogenase (ADH) for the first time, and has the advantages of simplifying reaction process, being convenient to operate, catalyzing efficiently and the like. An immobilized multienzyme catalytic system is constructed in vitro, so that the cascade reaction of the double enzymes ATTA and ADH is realized, a high-efficiency coenzyme regeneration system is generated, the regeneration efficiency of NADH is improved, and sufficient NADH is provided for the biocatalysis reaction, so that the cascade action and the catalysis efficiency of the enzymes are enhanced; meanwhile, the protein scaffold also provides a good environment for amination reaction, and improves the thermal stability of enzyme. The method of the invention improves the catalytic yield of 84.7 percent (free double enzyme) to 97.7 percent (co-immobilized double enzyme) when the substrate concentration is 125mM, and obviously improves the amination efficiency of the substrate 1- (4-methoxyphenyl) acetone, thereby establishing a process for preparing R-4-MPPA by efficiently aminating 1- (4-methoxyphenyl) acetone.
Drawings
FIG. 1 is an SDS-PAGE protein electrophoresis of EutM-SPYCATCHER (A) and EutM (B) after purification in example 1; lane M represents the standard molecule Maker.
FIG. 2 is an SDS-PAGE protein electrophoresis of purified SpyTag-GFP (A) and GFP (B) of example 2; lane M represents the standard molecule Maker.
FIG. 3 is an SDS-PAGE protein electrophoresis (A) and a fluorescence (B) of the assembly of example 3EutM-SpyCatcher and SpyTag-GFP; lane M represents the standard molecule Maker.
FIG. 4 is an SDS-PAGE protein electrophoresis of SpyTag-ATTA purified in example 4; lane M represents the standard molecule Maker.
FIG. 5 is an SDS-PAGE protein electrophoresis of the purified SpyTag-ADH of example 5; lane M represents the standard molecule Maker.
FIG. 6 is a TEM characterization of the self-assembled protein scaffold co-immobilized enzyme of example 7.
FIG. 7 is a schematic illustration of the mechanism of reaction of 1- (4-methoxyphenyl) propanone catalyzed by self-assembled protein immobilized bis-enzyme to form (R) -1- (4-methoxyphenyl) propan-2-amine.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
The BeyoGold TM His-tag Purification Resin protein purification column is reduction-resistant chelate type and is purchased from Shanghai Biyun Tian biotechnology Co.
LB liquid medium composition: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 7.0 pH. LB solid medium composition: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 18g/L of agar, wherein the solvent is water and the pH value is 7.0.
Example 1: eutM expression and preparation of SpyCatcher fusion proteins
1. Construction of E.coli DH5 alpha/pET 22b-EutM
EutM self-assembled protein gene (nucleotide sequence is shown as SEQ ID NO:1, and is synthesized by Beijing engine family biological company) is inserted between Xba I and BamH I restriction sites of pET-22b (+) plasmid, and is transferred into DH5 alpha competence of escherichia coli to construct engineering bacteria E.coli DH5 alpha/pET 22b-EutM, and the amino acid sequence is shown as SEQ ID NO: 2.
2. Construction of recombinant plasmid pET-28 a-SpyCatcher:
Inserting SpyCatcher protein gene (nucleotide sequence shown as SEQ ID NO:3, entrusted to synthesis of Beijing engine family biological company) between BamH I and Xho I restriction sites of pET-28a (+) plasmid, transferring into DH5 alpha competence of escherichia coli, and constructing engineering bacterium E.coli DH5 alpha/pET-28 a-SpyCatcher, wherein the amino acid sequence is shown as SEQ ID NO: 4.
1. Construction of recombinant plasmid pET-22 b-EutM-SpyCatcher:
(1) Extracting plasmid pET22b-EutM of engineering bacteria E.coli DH5 alpha/pET 22b-EutM constructed in the step 1; (2) Extracting plasmid pET-28a-SpyCatcher of engineering bacteria E.coli DH5 alpha/pET-28 a-SpyCatcher constructed in the step 2 as a PCR amplification template, and amplifying the SpyCatcher gene; forward primer F CATCACCACAGCCAGGATCCGGTAGTGGTGGTAGTGGTG; reverse primer R TGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTC; carrying out agarose gel electrophoresis on the PCR product, and recovering and purifying the gel; (3) Plasmid pET22b-EutM was digested with restriction enzymes BamH I and Xho I at 37℃for 3h; performing agarose gel electrophoresis on the enzyme-digested product, and recovering and purifying the carrier fragment; (4) Uniformly mixing a large fragment of the linearized pET22b-EutM vector after enzyme digestion and the SpyCatcher gene fragment after glue recovery according to a molar ratio of 1:3, adding the mixture into a seamless cloning system (the total volume of the system is 10 mu L), and carrying out enzyme-linked reaction on ice for 15min; (5) The enzyme-linked product is transformed into E.coli DH5 alpha competent cells, activated for 60min, coated on LB solid medium containing 100 mug/mL ampicillin, placed at 37 ℃ for half an hour, and then inverted and cultured overnight at 37 ℃; (6) And (3) selecting single bacterial colony on the plate to perform colony PCR to screen positive clones, and obtaining plasmid pET22b-EutM-SpyCatcher after sequencing and verifying correctness, wherein the nucleotide sequence of the plasmid pET22b-EutM-SpyCatcher is shown as SEQ ID NO:5, the amino acid sequence is shown as SEQ ID NO: shown at 6.
SEQ ID NO：5
ATGGAAGCGCTGGGTATGATTGAAACACGTGGTCTGGTTGCACTGATTGAAGCGAGTGATACAATGGTTAAAGCGGCGCGTGTTAAACTGGTTGGTGTGAAACAGATTGGTGGTGGTCTGTGTACGGCTATGGTTCGTGGTGATGTTGCAGCTTGTAAAGCAGCGACCGATGCGGGTGCAGCAGCAGCACAGCGTATTGGTGAACTGGTTAGTGTGCATGTTATTCCGCGTCCGCATGGTGATCTGGAAGAAGTTTTTCCGATTAGTTTTAAAGGCGATAGCAATATTGGATCCATGGGTGCTATGGTTGATACGCTGAGTGGTCTGAGTAGTGAACAGGGTCAGAGCGGTGATATGACAATTGAAGAAGATAGTGCAACGCATATTAAATTTAGTAAACGCGATGAAGATGGTAAAGAACTGGCAGGTGCAACAATGGAACTGCGTGATAGTAGCGGTAAAACCATTAGTACCTGGATTAGCGATGGTCAGGTGAAAGATTTTTATCTGTATCCAGGTAAATATACCTTTGTGGAAACTGCAGCACCGGATGGTTATGAAGTTGCAACCGCAATTACCTTTACCGTTAACGAACAGGGCCAGGTTACCGTTAATGGTAAAGCCACCAAAGGTGATGCCCATATT.
3. EutM expression and preparation of Spycatcher protein
The recombinant plasmid pET-22b-EutM-SpyCatcher was extracted and transformed into E.coli BL21 (DE 3), and the obtained transformant was inoculated into 50mL of LB liquid medium containing 50. Mu.g/mL ampicillin (Amp), and cultured overnight at 37℃and 180rpm to obtain a seed solution of recombinant E.coli. The seed solution was transferred to 0.15L of fresh LB liquid medium containing 50. Mu.g/mL ampicillin at a volume concentration of 3% by volume, and the culture was fermented at 37℃and 180rpm until the optical density (OD 600) at 600nm was 0.8, and isopropyl-. Beta. -thiogalactoside (IPTG) at a final concentration of 0.5mM was added to induce gene expression, and then the culture was transferred to a shaker at 180rpm at 18℃to induce expression for 16 hours. The culture broth was placed in a centrifuge cup and centrifuged at 8000rpm for 10min at 4℃to collect the over-expressed cell pellet, which was washed 3 times with 0.9% (v/w) physiological saline. The harvested wet cells were resuspended in 100mM sodium phosphate buffer (pH 7.0) in an amount of 50g/L and the mixture sonicated for 15min (power 360W, work 3s, disruption 7 s). Collecting the crushed liquid in a 50mL centrifuge tube, centrifuging at a speed of 8000rpm and a temperature of 4 ℃ for 10min, taking supernatant, loading into an ultrafiltration tube (10000 MWCO) for ultrafiltration concentration treatment, and obtaining the trapped liquid which is 4mL of concentrated EutM-SpyCatcher crude enzyme liquid.
The EutM-SpyCatcher crude enzyme solution and a purification medium of a reduction-resistant chelating nickel column (BeyoGold TM His-tag) are mixed according to a volume ratio of 8:1, uniformly mixing, slowly shaking in ice water for 1h, and then loading the mixture on a nickel column; washing the column with non-denaturing washing liquid for 5 times to remove the impurity protein, wherein 1 column volume is washed each time; then eluting with non-denaturing eluent 8 times, each eluting 1 column volume. Dialyzing with 0.01M sodium phosphate buffer solution with pH of 8.0 (dialysis bag molecular weight cut-off 8000-14000) for 24 hr, changing the dialysate three times, and collecting the cut-off. Then pre-freezing for 8 hours in a refrigerator at the temperature of minus 80 ℃, and then placing the pre-frozen product into a freeze dryer for vacuum drying for 24 hours at the temperature of minus 65 ℃ to obtain 8mg of pure enzyme of the fusion protein EutM-SpyCatcher. The results of protein purification are shown in FIG. 1 (A) as determined by 12% SDS-PAGE.
Non-denaturing wash: 50mM NaH 2PO4, 300mM NaCl,2mM imidazole, pH8.0.
Non-denaturing eluent: 50mM NaH 2PO4, 300mM NaCl,50mM imidazole, pH8.0.
In the same manner, a pure enzyme of self-assembled protein EutM was prepared, and 3mg of pure enzyme of protein EutM was obtained. The results of protein purification are shown in FIG. 1 (B) as detected by 10% SDS-PAGE.
Example 2: expression and preparation of SpyTag-GFP fusion protein
1. Construction of E.coli DH5 alpha/pET 28a-GFP
GFP fluorescent protein gene (nucleotide sequence is shown as SEQ ID NO:7, and is synthesized by Beijing engine family biological company) is inserted between BamH I and Xho I restriction sites of pET-28a (+) plasmid, and is transferred into DH5 alpha competence of escherichia coli to construct engineering bacterium E.coli DH5 alpha/pET 228a-GFP, and the amino acid sequence is shown as SEQ ID NO: shown at 8.
2. Construction of recombinant plasmid pET28 a-SpyTag:
the SpyTag short peptide protein gene (the nucleotide sequence is shown as SEQ ID NO:9, and is synthesized by Beijing engine family biological company) is inserted between the NdeI and BamH I restriction sites of pET-28a (+) plasmids, and is transferred into the DH5 alpha competence of escherichia coli to construct engineering bacteria E.coli DH5 alpha/pET 28a-SpyTag, and the amino acid sequence is shown as SEQ ID NO: shown at 10.
3. Construction of recombinant plasmid pET28 a-SpyTag-GFP:
(1) Extracting plasmid pET28a-SpyTag of engineering bacteria E.coli DH5 alpha/pET 28a-SpyTag constructed in the step 2; (2) Extracting plasmid pET28a-GFP of engineering bacteria E.coli DH5 alpha/pET 28a-GFP constructed in the step 1 as a PCR amplification template, and amplifying GFP genes; forward primer F TGGTGTAGACGGATTCATGGTTTCCAAAGGTGAAGAACTGT; reverse primer R TGGTGGTGGTGGTGCTCGAGTTACTTGTACAGTTCATCCATACCC; carrying out agarose gel electrophoresis on the PCR product, and recovering and purifying the gel; (3) Plasmid pET28a-SpyTag was digested with restriction enzymes BamH I and Xho I, 37℃for 3h; performing agarose gel electrophoresis on the enzyme-digested product, and recovering and purifying the carrier fragment; (4) Uniformly mixing the large fragment of the linearized pET28a-SpyTag vector after enzyme digestion with the GFP gene fragment recovered by glue according to the molar ratio of 1:3, and adding the mixture into a seamless cloning system to carry out enzyme-linked reaction on ice for 15min; (5) E.coli DH5 alpha competent cells are transformed by the enzyme-linked product, activated for 60min and then coated on LB solid medium containing 50 mug/mL kanamycin, and after half an hour at 37 ℃, the cells are inverted and cultured overnight at 37 ℃; (6) And (3) selecting single bacterial colony on the plate to perform colony PCR to screen positive clones, and obtaining plasmid pET28a-SpyTag-GFP after sequencing verification is correct, wherein the nucleotide sequence of the plasmid pET28a-SpyTag-GFP is shown as SEQ ID NO:11, the amino acid sequence is shown as SEQ ID NO: shown at 12.
4. Expression and preparation of SpyTag-GFP protein
Recombinant plasmid pET28a-SpyTag-GFP was extracted and introduced into E.coli BL21 (DE 3) competence, activated for 1h at 37℃in a 180rpm shaker, plated with LB plate containing 50. Mu.g/mL kanamycin (Kan) antibiotic, inverted and incubated at 37℃overnight. Single colonies were picked up in 50mL of LB liquid medium containing 50. Mu.g/mL Kan antibiotics, and placed in a shaking table at 37℃and 180rpm for overnight culture to obtain seed solution. The inoculated amount of 3% by volume was transferred to 0.15L of a liquid medium containing 50. Mu.g/mL Kan, and the culture was continued at 37℃and 180rpm until OD 600 was 0.8, and then IPTG was added at a final concentration of 0.5mM, and the induction was performed at 23℃and 180rpm for 16 hours. After the induction is finished, the culture solution is split into centrifugal cups and placed in a centrifugal machine at the temperature of 4 ℃ and the speed of 8000rpm for 10min. The collected cell pellet was washed with 10mL of 0.9% (v/w) physiological saline each time, for a total of 3 times. Wet cells were collected by centrifugation at 8000rpm at 4℃for 10min. The harvested wet cells were resuspended in 100mM sodium phosphate buffer (pH 7.0) in an amount of 50g/L and the mixture sonicated for 15min under ice bath conditions (power 360W, work 3s, disruption 7 s). Collecting the ultrasonic crushed liquid, centrifuging at the speed of 8000rpm and the temperature of 4 ℃ for 10min, taking supernatant, loading the supernatant into an ultrafiltration tube (10000 MWCO) for ultrafiltration concentration treatment, and obtaining the trapped liquid which is 4mL of concentrated SpyTag-GFP protease crude enzyme liquid.
The SpyTag-GFP crude enzyme solution and a purification medium of a reduction-resistant chelating nickel column (BeyoGold TM His-tag) are mixed according to a volume ratio of 8:1, uniformly mixing, slowly shaking in ice water for 1h, and then loading the mixture on a nickel column; washing the column with non-denaturing washing liquid for 5 times to remove the impurity protein, wherein 1 column volume is washed each time; then eluting with non-denaturing eluent 8 times, each eluting 1 column volume. Dialyzing with 0.01M sodium phosphate buffer solution with pH of 8.0 (dialysis bag molecular weight cut-off 8000-14000) for 24 hr, changing the dialysate three times, and collecting the cut-off. Then pre-freezing for 8 hours in a refrigerator at the temperature of minus 80 ℃, and then placing the mixture into a freeze dryer for vacuum drying for 24 hours at the temperature of minus 65 ℃ to obtain 15mg of pure enzyme of the fusion protein SpyTag-GFP. The results of protein purification are shown in FIG. 2 (A) as determined by 12% SDS-PAGE.
Non-denaturing wash: 50mM NaH 2PO4, 300mM NaCl,2mM imidazole, pH8.0.
Non-denaturing eluent: 50mM NaH 2PO4, 300mM NaCl,50mM imidazole, pH8.0.
In the same manner, a pure enzyme of fluorescent protein GFP was prepared, and 12mg of the pure enzyme of fluorescent protein GFP was obtained. The results of protein purification are shown in FIG. 2 (B) as determined by 12% SDS-PAGE.
Example 3: fluorescent microscopy verifies EutM-SpyCatcher and SpyTag-GFP covalent attachment
The SpyCatcher/SpyTag system, derived from the CnaB2 domain, is capable of spontaneously forming stable isopeptidic linkages under a variety of conditions. Lysine (Lys) in SpyPatcher and aspartic acid (Asp) in SpyTag spontaneously form covalent bonds under various conditions of temperature (4-37 ℃) and pH (5-8) buffer. To verify whether or not the EutM-SpyCatcher and SpyTag-GFP spontaneously interact specifically, the EutM-SpyCatcher and SpyTag-GFP purified enzymes prepared in examples 1 and 2 were prepared into 2. Mu.M solutions with pure water, respectively, mixed together in a molar ratio of 1:1, incubated for 1h at 4℃with gentle shaking at half an interval of half an hour to accelerate assembly. Except for this, the purified enzyme EutM prepared in example 1 was mixed with the purified enzyme GFP, eutM-SpyCatcher and GFP purified enzyme prepared in example 2 as described above to prepare a control. The protein samples were denatured by heating to 95℃for 5min, loaded onto 12% SDS-PAGE proteins for electrophoresis, stained with Coomassie brilliant blue, decolorized, and analyzed as shown in FIG. 3 (A). EutM-SpyCatcher and SpyTag-GFP fusion proteins had molecular weights of about 29kDa and 32kDa, respectively, and EutM and GFP proteins had molecular weights of about 15kDa and 30kDa, respectively. The results showed that only in Lane EutM-SpyCatcher and SpyTag-GFP mixed sample (Lane 8) a larger band appeared between the standard proteins 55kDa-65kDa, whereas the other two control groups did not appear the third band, indicating successful covalent coupling between the two assembly proteins EutM-SpyCatcher and the SpyTag-GFP protein subunits.
In order to more intuitively observe the assembly characteristics of the self-assembled protein and to examine whether the enzyme fused with SpyTag affects the covalent coupling between SpyCatcher/SpyTag, the aggregation of EutM-SpyCatcher and SpyTag-GFP was observed under a fluorescence microscope by the developability of the fluorescent protein under fluorescence, and the results are shown in FIG. 3 (B). The specific operation is as follows: the reaction mixture was incubated at room temperature for 30min, 10. Mu.L of the sample was added dropwise to the slide glass, and the results were observed under bright field and fluorescence, respectively, which showed that the self-assembled complex of EutM-SpyCatcher and SpyTag-GFP showed significant fluorescence under fluorescence, whereas the polymers of the control group EutM-SpyCatcher and GFP showed no significant fluorescence, indicating that GFP could not spontaneously form covalent coupling with EutM-SpyCatcher, and the side also demonstrated that the SpyTag-fused enzyme did not affect covalent coupling between SpyCatcher/SpyTag.
Example 4: expression and preparation of SpyTag-ATTA fusion protein
5. Construction of E.coli DH5 alpha/pET 28a-ATTA
The ATTA aminotransferase gene (the nucleotide sequence is shown as SEQ ID NO:13 and is synthesized by Beijing engine family biological company) is inserted between BamH I and Xho I restriction sites of pET-28a (+) plasmids, and is transferred into DH5 alpha competence of escherichia coli to construct engineering bacteria E.coli DH5 alpha/pET 228a-ATTA, and the amino acid sequence is shown as SEQ ID NO: 14.
6. Construction of recombinant plasmid pET28 a-SpyTag-ATTA:
(1) Extracting plasmid pET28a-SpyTag of engineering bacteria E.coli DH5 alpha/pET 28a-SpyTag constructed in example 2; (2) Extracting plasmid pET28a-ATTA of engineering bacteria E.coli DH5 alpha/pET 28a-ATTA constructed in the step 1 as a PCR amplification template, and amplifying an ATTA gene; forward primer F TGGTGTAGACGGATTCGCGAGCATGGATAAAGTTTTCG; reverse primer R TGGTGGTGGTGGTGCTCGAGTTAGTTACGTTCGTTGTAGTCAATT; carrying out agarose gel electrophoresis on the PCR product, and recovering and purifying the gel; (3) Plasmid pET28a-SpyTag was digested with restriction enzymes BamH I and Xho I, 37℃for 3h; performing agarose gel electrophoresis on the enzyme-digested product, and recovering and purifying the carrier fragment; (4) Uniformly mixing the large fragment of the linearized pET28a-SpyTag vector after enzyme digestion with the ATTA gene fragment recovered by the gel according to the molar ratio of 1:3, and adding the mixture into a seamless cloning system to carry out enzyme-linked reaction on ice for 15min; (5) E.coli DH5 alpha competent cells are transformed by the enzyme-linked product, activated for 60min and then coated on LB solid medium containing 50 mug/mL kanamycin, and after half an hour at 37 ℃, the cells are inverted and cultured overnight at 37 ℃; (6) And (3) selecting a single colony on the plate for colony PCR screening positive clones, and obtaining plasmid pET28a-SpyTag-ATTA after sequencing verification is correct, wherein the nucleotide sequence of the plasmid pET28a-SpyTag-ATTA is shown as SEQ ID NO:15, the amino acid sequence is shown as SEQ ID NO: shown at 16.
SEQ ID NO：15
ATGGGTTCTTCTGGTGCTCACATCGTTATGGTTGATGCGTACAAACCAACTAAAGGTAGCGGTGGTTCTGGTGTAGACGGATCCATGGCGAGCATGGATAAAGTTTTCGCGGGTTACGCGGCGCGTCAGGCGATCCTGGAAAGCACCGAAACCACCAACCCGTTCGCGAAAGGTATCGCGTGGGTTGAAGGCGAACTGGTTCCGCTGGCGGAAGCGCGTATCCCGCTGCTGGATCAGGGCTTCATGCACTCTGATCTGACCTACGATGTTCCGAGCGTTTGGGATGGCCGCTTCTTCCGTCTGGATGATCACATCACCCGTCTGGAAGCGTCTTGCACCAAACTGCGTCTGCGTCTGCCGCTGCCGCGTGATCAGGTTAAACAGATCCTGGTTGAAATGGTGGCGAAATCCGGTATTCGCGATGCGTTCGTTGAACTGATCGTTACCCGTGGCCTGAAAGGCGTTCGTGGCACCCGTCCGGAAGATATCGTTAACAACCTGTACATGTTCGTTCAGCCGTACGTTTGGGTTATGGAACCGGATATGCAGCGTGTTGGTGGCAGCGCGGTGGTTGCGCGTACCGTTCGCCGTGTTCCGCCGGGTGCGATCGACCCGACCGTTAAAAACCTGCAGTGGGGCGATCTGGTTCGTGGCATGTTCGAAGCTGCTGATCGTGGCGCGACCTACCCGTTCCTGACCGACGGTGACGCACACCTGACCGAAGGTAGCGGCTTCAACATCGTTCTGGTTAAAGACGGTGTTCTGTACACCCCGGATCGTGGTGTGCTGCAGGGCGTTACCCGTAAATCCGTGATCAACGCGGCGGAAGCGTTCGGTATTGAAGTTCGTGTTGAATTCGTGCCGGTGGAACTGGCGTACCGCTGCGATGAAATCTTCATGTGCACCACCGCGGGTGGCATCATGCCGATCACCACCCTGGATGGCATGCCGGTTAACGGTGGCCAGATCGGCCCGATCACCAAGAAAATCTGGGACGGCTACTGGGCGATGCACTACGATGCGGCGTACAGCTTCGAAATTGACTACAACGAACGTAACTAA.
7. Expression and preparation of SpyTag-ATTA protein
The procedure was as in step (3) of example 2, except that the recombinant plasmid used was pET28a-SpyTag-ATTA, and 12mg of the pure enzyme fusion protein SpyTag-ATTA was obtained. The results of protein purification are shown in FIG. 4, as determined by 12% SDS-PAGE.
8. Measurement of enzyme Activity
Transaminase enzyme activity assay (2 mL) composition: 100mM isopropyl amine hydrochloride (IPA, amine donor), 1mM pyridoxal 5' -phosphate (PLP, cofactor), 0.4mL dimethyl sulfoxide (DMSO, co-solvent), 75mM 1- (4-methoxyphenyl) acetone (4-MFA, substrate), 1mL of a 12mg/mL purified enzyme aqueous solution of SpyTag-ATTA prepared, and the mixture was supplemented to 2mL with 100mM sodium phosphate buffer (pH 7.0). The reaction was stopped by adding 1. Mu.L of trifluoroacetic acid ester (TFA, 1%) at 23℃and 180rpm for 2 h. The reaction solution was subjected to Gas Chromatography (GC) to determine the peak area of (R) -1- (4-methoxyphenyl) propan-2-amine ((R) -4-MPPA) and the (R) -4-MPPA content was obtained according to a standard curve, and the method was the same as in example 8.
Under the above reaction conditions, the enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. Mu. Mol (R) -4-MPPA per minute under standard assay conditions. The enzyme activity of the prepared SpyTag-ATTA pure enzyme is 1.8U/mg.
Example 5: expression and preparation of SpyTag-ADH fusion protein
9. Construction of E.coli DH 5. Alpha./pET 28a-ADH
ADH alcohol dehydrogenase gene (nucleotide sequence is shown in SEQ ID NO:17, and is synthesized by Beijing engine family biological company) is inserted between BamH I and Xho I restriction sites of pET-28a (+) plasmid, and is transferred into DH5 alpha competence of escherichia coli to construct engineering bacterium E.coli DH5 alpha/pET 228a-ADH, and the amino acid sequence is shown in SEQ ID NO: shown at 18.
10. Construction of recombinant plasmid pET28 a-SpyTag-ADH:
(1) Extracting plasmid pET28a-SpyTag of engineering bacteria E.coli DH5 alpha/pET 28a-SpyTag constructed in example 2; (2) Extracting plasmid pET28a-ADH of constructed engineering bacteria E.coli DH5 alpha/pET 28a-ADH, and amplifying ADH genes by taking the plasmid pET28a-ADH as a PCR amplification template; forward primer F TGGTGTAGACGCATTCATGAGCATTCCGGAAACCCAGA; reverse primer R TGGTGGTGGTGGTGCTCGAGTTTGCTGGTATCAACAACATAACGA; carrying out agarose gel electrophoresis on the PCR product, and recovering and purifying the gel; (3) Plasmid pET28a-SpyTag was digested with restriction enzymes BamH I and Xho I, 37℃for 3h; performing agarose gel electrophoresis on the enzyme-digested product, and recovering and purifying the carrier fragment; (4) Uniformly mixing the large fragment of the linearized pET28a-SpyTag vector after enzyme digestion with the ADH gene fragment recovered by the gel according to the molar ratio of 1:3, and adding the mixture into a seamless cloning system to carry out enzyme-linked reaction on ice for 15min; (5) E.coli DH5 alpha competent cells are transformed by the enzyme-linked product, activated for 60min and then coated on LB solid medium containing 50 mug/mL kanamycin, and after half an hour at 37 ℃, the cells are inverted and cultured overnight at 37 ℃; (6) And (3) selecting a single colony on the plate for colony PCR screening positive clones, and obtaining plasmid pET28a-SpyTag-ADH after sequencing verification is correct, wherein the nucleotide sequence of the plasmid pET28a-SpyTag-ADH is shown as SEQ ID NO:19, the amino acid sequence is shown as SEQ ID NO: shown at 20.
SEQ ID NO：19
ATGGGTTCTTCTGGTGCTCACATCGTTATGGTTGATGCGTACAAACCAACTAAAGGTAGCGGTGGTTCTGGTGTAGACGGATCCATGAGCATTCCGGAAACCCAGAAAGGGGTGATTTTTTACGAAAGCCACGGCAAACTGGAATACAAAGACATTCCGGTTCCGAAACCGAAAGCGAACGAACTGCTGATTAATGTGAAATATAGCGGCGTCTGTCATACCGATCTGCATGCCTGGCACGGTGATTGGCCGCTGCCGGTTAAACTGCCGCTGGTCGGGGGTCACGAAGGAGCAGGGGTGGTTGTTGGGATGGGTGAAAATGTTAAAGGTTGGAAAATTGGGGATTATGCGGGGATTAAATGGCTGAATGGTAGCTGTATGGCATGTGAATATTGTGAACTGGGAAATGAAAGTAATTGTCCGCATGCAGATCTGAGCGGTTATACACATGATGGTAGCTTTCAGCAGTATGCAACAGCGGATGCGGTTCAAGCCGCACATATTCCGCAGGGTACCGATCTGGCACAAGTGGCACCGATTCTGTGTGCGGGTATTACAGTTTACAAAGCGCTGAAAAGCGCAAATCTGATGGCCGGTCATTGGGTTGCAATTAGTGGTGCGGCAGGTGGGCTGGGTAGTCTGGCAGTTCAGTATGCAAAAGCAATGGGTTACCGTGTTCTGGGCATTGATGGCGGAGAAGGCAAAGAAGAACTGTTTCGTAGCATTGGGGGTGAGGTTTTTATTGATTTTACAAAAGAGAAAGACATCGTGGGTGCAGTTCTGAAAGCAACCGATGGTGGTGCACATGGAGTGATTAATGTGAGTGTGAGCGAGGCAGCAATTGAAGCAAGCACCCGCTACGTTCGTGCAAATGGAACCACAGTTCTGGTTGGTATGCCTGCAGGTGCAAAATGTTGTAGTGATGTGTTTAACCAGGTTGTGAAAAGCATTAGCATTGTGGGAAGTTACGTTGGTAATCGTGCTGATACCCGTGAAGCACTGGATTTTTTTGCACGTGGTCTGGTTAAAAGTCCGATTAAAGTTGTGGGTCTGAGTACCCTGCCGGAAATTTATGAAAAAATGGAAAAAGGTCAGATTGTTGGTCGTTATGTTGTTGATACCAGCAAA.
11. Expression and preparation of SpyTag-ADH protein
The procedure was as in step (3) of example 2, except that the recombinant plasmid used was pET28a-SpyTag-ADH, and 10mg of the pure enzyme fusion protein SpyTag-ADH was obtained. The results of protein purification are shown in FIG. 5, as determined by 12% SDS-PAGE.
12. Measurement of enzyme Activity
5ML of ethanol dehydrogenase enzyme activity assay system comprises: 2.5mL of glycine-sodium hydroxide buffer (pH 8.6), final concentration 1X 10 -3mol/L NAD+ and final concentration 5X 10 -3 mol/L ethanol, after warming in a water bath at 25℃for 20min, 1mL of the ethanol dehydrogenase pure enzyme aqueous solution prepared in step 2 warmed under the same conditions was added at 15 mg/mL. The time was then immediately counted, and the absorbance at 340nm was read every 1min for 5 consecutive min. The amount of enzyme required to increase at A340 by 0.001 per minute was one unit (U) at 25℃and pH 8.6. The enzyme activity was determined to be 1.2U/mg.
Example 6: self-assembled protein scaffold immobilized double-enzyme complex
The EutM-SpyCatcher pure enzyme prepared in the method of example 1, spyTag-ATTA and SpyTag-ADH pure enzyme prepared in the methods of examples 4 and 5 are respectively prepared into 2 mu M solution by pure water, and are mixed according to the molar ratio of 3:1:1, incubated for 2 hours at the temperature of 4 ℃, gently swaying at intervals of half an hour, and accelerating assembly. After the assembly, ultrafiltration (molecular weight cut-off 50000 MW) is carried out, the cut-off is pre-frozen for 8 hours in a refrigerator at the temperature of minus 80 ℃, and then the frozen cut-off is put into a freeze dryer for vacuum drying at the temperature of minus 65 ℃ for 24 hours, so as to obtain 2.7mg of self-assembled compound.
Example 7: self-assembled protein scaffold co-immobilized double-enzyme cascade TEM characterization
From the above, a complete self-assembled protein scaffold system has been constructed, and applied to chiral amine synthesis of double enzyme cascade, thereby achieving double enzyme cascade co-immobilization and improving enzyme stability and catalytic efficiency. To verify whether a dual enzyme cascade co-immobilization system was formed, morphological characterization was performed by transmission electron microscope TEM.
The specific operation is as follows: the EutM-SPYCATCHER, SPYTAG-ATTA and SpyTag-ADH pure enzymes prepared in examples 1, 4 and 5 are respectively prepared into 2 mu M solutions by pure water, the solutions are mixed together according to the volume ratio of 3:1:1, incubated for 1h at the temperature of 4 ℃, slightly swaying at intervals of half an hour, and the assembly is accelerated. To clarify the structural morphology of the self-assembled protein scaffold catalytic system, TEM characterization was performed on self-assembled protein mix samples. The specific operation is as follows: the scaffold protein solution was dropped on a200 μm carbon coated copper mesh surface and allowed to stand for 2 minutes. Excess liquid was then blotted from the grid with filter paper and 10 μl of 1% negative phosphotungstic acid staining solution was added to stain the proteins on the grid. The grid was visualized using a transmission electron microscope and the protein structure was imaged (as shown in fig. 6). The results showed that the scaffold protein formed a three-dimensional network with channels in between, approximately 20 μm in size.
Example 8: influence of the ratio of self-assembled proteases on the catalytic System of the self-assembled protein scaffold
To explore the best assembly ratio and achieve the best catalytic efficiency, eutM-SpyCatcher pure enzyme prepared by the method of example 1 and SpyTag-ATTA and SpyTag-ADH pure enzymes prepared by the methods of examples 4 and 5 are respectively prepared into 2 mu M solution by pure water according to the volume ratio of 1:1:1;1:2:1;1:1:2;3:1:1;5:1:1; mixing the materials in six different proportions of 7:1:1, incubating for 2 hours at the temperature of 4 ℃ for half an hour, gently shaking at intervals, accelerating assembly, concentrating by ultrafiltration (molecular weight cut-off 50000 MW) after assembly is finished, pre-freezing the cut-off liquid in a refrigerator at the temperature of-80 ℃ for 8 hours, and then placing the pre-frozen liquid in a freeze dryer at the temperature of-65 ℃ for vacuum drying for 24 hours to obtain the self-assembled compound.
In 5 different 2mL reaction systems, 125mM substrate 1- (4-methoxyphenyl) acetone (dissolved by dimethyl sulfoxide as a cosolvent), 100mM isopropyl amine hydrochloride (IPA), 1mM pyridoxal phosphate (PLP) as a final concentration, 50mM ethanol as a final concentration, NAD + as a final concentration and 20% dimethyl sulfoxide (cosolvent) as a final concentration were added, 2mg of the five groups of self-assembled complexes in different proportions were added, and the mixture was supplemented to 2mL with 100mM phosphate buffer pH7.0, and the reaction mechanism is shown in FIG. 7. After the system was reacted at 30℃and 180rpm for 24 hours, centrifuged at 8000rpm for 10 minutes at 4℃and the supernatant was collected and added with an equal volume of ethyl acetate to extract it, and after repeating the extraction three times, the collected extract was dried over anhydrous sodium sulfate and volatilized at normal temperature to remove ethyl acetate, and the peak areas of the substrates 1- (4-methoxyphenyl) propanone (4-MFA), (R) -1- (4-methoxyphenyl) propan-2-amine ((R) -4-MPPA), (S) -1- (4-methoxyphenyl) propan-2-amine ((S) -4-MPPA) were detected by gas chromatography, and the content was calculated by an internal standard method (dodecane was added as an internal standard) and the yield and enantiomeric excess (ee) of (R) -4-MPPA) were calculated by the formula (1) and the formula (2), and the results are shown in Table one.
Gas Chromatography (GC) detection conditions: chiral chromatographic column CP7502 (25 m×0.25mm×0.25 μm); sample inlet temperature 250 ℃, column temperature 160 ℃, detector 250 ℃, flow rate 1mL/min, split ratio 1:15, sample injection amount is 1 mu L. The conversion of product (R) -4-MPPA and the enantiomeric excess (ee) are calculated by the formula (1): In formula (1), M S: molecular weight of the substrate; m P: molecular weight of the product; q: the mass of the substrate at the beginning of the reaction; p: the quality of the product at the end of the reaction.
In formula (2), C R: concentration of R-type product, C S: s-type product concentration.
TABLE 1 influence of the ratio of self-assembled proteases on the catalytic System of self-assembled protein scaffolds
From the results, the self-assembled protease ratio has a certain influence on the self-assembled protein scaffold catalysis, and when the ratio is 3:1:1, the conversion rate reaches the highest; with increasing ratios, the conversion decreases and there may be an excess of protein scaffolds to sterically hinder the catalytic centers of ATTA and ADH from the substrate. Compared with free enzyme, the self-assembled complex is stable and has high conversion rate.
Example 9: self-assembled protein scaffold catalytic system applied to synthesis of 4-MPA
In a 2mL reaction system, 125mM substrate 1- (4-methoxyphenyl) acetone (added after being dissolved by dimethyl sulfoxide as a cosolvent), 100mM isopropyl amine hydrochloride (IPA), 1mM pyridoxal phosphate (PLP) as a final concentration, 50mM ethanol as a final concentration, 1.0mM NAD + as a final concentration, 2mg of the self-assembled complex prepared in the method of example 6, and 20% dimethyl sulfoxide (cosolvent) as a final concentration were added, and the mixture was made up to 2mL with 100mM phosphate buffer pH 7.0. 2mg of the prepared self-assembled complex was changed to 1mg of the pure enzyme SpyTag-ATTA prepared in example 4 and 1mg of the pure enzyme SpyTag-ADH prepared in example 5, and the other conditions were identical, as a control group. After the reaction was carried out at 30℃and 180rpm for 24 hours, centrifugation was carried out at 8000rpm for 10 minutes at 4℃to collect the supernatant, and an equal volume of ethyl acetate was added thereto for extraction, and after repeating the extraction three times, the ethyl acetate was volatilized at ordinary temperature after the collected extract was dried over anhydrous sodium sulfate, and the peak area of (R) -1- (4-methoxyphenyl) propan-2-amine ((R) -4-MPPA) was detected by Gas Chromatography (GC) to obtain the (R) -4-MPPA content according to a standard curve, and the detection method was the same as in example 8. From the above calculation, the yield of R-4-MPPA produced from the control group free enzyme catalytic substrate was 84.7% and the enantiomeric excess was 98.0%; the yield of R-4-MPPA produced by catalyzing the substrate with the assembly complex is 97.7%, and the enantiomeric excess is 99.5%.
Example 10: influence of the reaction temperature on the self-assembled protein scaffold catalytic System
In a 2mL reaction system, 125mM substrate 4-MFA (added after being dissolved in dimethyl sulfoxide), 100mM amino donor IPA, 1mM cofactor pyridoxal phosphate (PLP), 1.0mM NAD +, 50mM ethanol, 2mg self-assembled protein complex pure enzyme prepared in the method of example 6 were added, and the total volume of the final concentration was 20% dimethyl sulfoxide as a cosolvent, and 100mM sodium phosphate buffer pH 7.0 was made up to 2mL. 2mg of the prepared self-assembled complex was changed to 1mg of the pure enzyme SpyTag-ATTA prepared in example 4 and 1mg of the pure enzyme SpyTag-ADH prepared in example 5, and the other conditions were identical, as a control group. Nine temperatures are taken as reaction variables at 25-45 ℃ and reacted for 24 hours under the condition of 180rpm, the reaction is carried out for 10 minutes at 4 ℃, the supernatant is collected and added with equal volume of ethyl acetate for extraction, the extraction is repeated three times, the collected extract is dried by anhydrous sodium sulfate and volatilized from ethyl acetate at normal temperature, the (R) -1- (4-methoxyphenyl) propan-2-amine ((R) -4-MPPA) peak area is detected by Gas Chromatography (GC), the (R) -4-MPPA content is obtained according to a standard curve, and the detection method is the same as that of example 8, and the results are shown in Table 2.
TABLE 2 influence of reaction temperature on self-assembled protein scaffold catalytic systems
As can be seen from table 2, the temperature significantly affects the catalytic activity of the enzyme, and its activity changes with the change of temperature. Due to the denaturation of proteins, enzymatic activity is lost or inhibited at high temperatures. In a certain temperature range (25-35 ℃), the conversion rate of the self-assembled compound is in an ascending trend, and reaches the maximum value at 32 ℃; when the temperature reaches 37℃or higher, the conversion and ee value gradually decrease, and the temperature may become too high, and the enzyme activity may be inhibited. The conversion rate of the free double enzyme in the control group reaches the maximum value at 27 ℃, the conversion rate and the ee value decrease in the subsequent process are larger than those of the self-assembled compound along with the temperature rise, the conversion rate is reduced to 67.0% at the high temperature of 45 ℃, the conversion rate of the self-assembled compound is more than 85%, and the ee value is about 90%. From the above, the self-assembled complex not only improves the catalytic ability of the enzyme, but also provides a good microenvironment for the enzyme, improves the thermal stability of the enzyme, improves the optimal temperature from 27 ℃ to 32 ℃, and further improves the catalytic ability of the enzyme.
Claims (10)
1. A self-assembled protein scaffold co-immobilized enzyme, characterized in that: the self-assembled protein scaffold co-immobilized enzyme is obtained by immobilizing R-omega-aminotransferase and alcohol dehydrogenase by adopting EutM-SpyCatcher/SpyTag system; the amino acid sequence of the R-omega-aminotransferase is shown in SEQ ID NO:14, the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO: shown at 18.
2. The self-assembled protein scaffold co-immobilized enzyme of claim 1, wherein: the self-assembled protein scaffold co-immobilized enzyme is constructed by the following method:
mixing recombinant protein SpyTag-R-omega-aminotransferase, recombinant protein SpyTag-alcohol dehydrogenase and recombinant protein EutM-SpyCatcher in water, incubating for 1-12h at 0-37 ℃, ultrafiltering, and freeze-drying the obtained trapped fluid to obtain the self-assembled protein scaffold co-immobilized enzyme.
3. The self-assembled protein scaffold co-immobilized enzyme of claim 2, wherein: recombinant protein SpyTag-R-omega-aminotransferase, recombinant protein SpyTag-alcohol dehydrogenase and recombinant protein EutM-SpyCatcher were each prepared as 2. Mu.M aqueous solutions and mixed.
4. The self-assembled protein scaffold co-immobilized enzyme of claim 2, wherein: the amino acid sequence of the recombinant protein SpyTag-R-omega-aminotransferase is shown in SEQ ID NO: shown at 16; the amino acid sequence of the recombinant protein SpyTag-ethanol dehydrogenase is shown as SEQ ID NO: shown at 20; the amino acid sequence of the recombinant protein EutM-SpyCatcher is shown in SEQ ID NO: shown at 6.
5. The self-assembled protein scaffold co-immobilized enzyme of claim 2, wherein: the mole ratio of the recombinant protein SpyTag-R-omega-aminotransferase to the recombinant protein SpyTag-alcohol dehydrogenase to the recombinant protein EutM-SpyCatcher is 1:0.5-2:1-7.
6. Use of the self-assembled protein scaffold immobilization enzyme of any one of claims 1-5 in the asymmetric reduction of 1- (4-methoxyphenyl) acetone to synthesize (R) -1- (4-methoxyphenyl) propan-2-amine.
7. The application of claim 6, wherein the application is: the self-assembled protein scaffold immobilized enzyme is used as a catalyst, 1- (4-methoxyphenyl) acetone is used as a substrate, dimethyl sulfoxide is used as a cosolvent, isopropylamine hydrochloride is used as an ammonia donor, pyridoxal phosphate is used as a coenzyme, NAD + is used as an auxiliary factor, ethanol is used as an auxiliary substrate, a buffer solution with pH of 6-10 is used as a reaction medium to form a reaction system, the reaction system is mixed and stirred in a shaking table at 25-45 ℃ and 150-200rpm for 12-36 hours, and the obtained reaction solution is separated and purified to obtain (R) -1- (4-methoxyphenyl) propan-2-amine.
8. The use according to claim 7, characterized in that in the reaction system the final concentration of the catalyst is 0.5-5g/L, the final concentration of the 1- (4-methoxyphenyl) acetone is 25-125mM, the final concentration of the isopropylamine hydrochloride is 25-125mM, the final concentration of pyridoxal phosphate is 0.5-1.5mM, the final concentration of NAD + is 0.5-1.5mM, the final concentration of the co-substrate is 25-125mM, the volume of the co-solvent is 10% -30% of the volume of the reaction system.
9. The use according to claim 7, wherein: the buffer is a pH 7.0, 100mM sodium phosphate buffer.
10. The use according to claim 7, wherein:
The separation and purification are as follows: and centrifuging the reaction liquid, collecting supernatant, adding ethyl acetate with the same volume as the supernatant for extraction, collecting an organic phase, drying by anhydrous sodium sulfate, and volatilizing the solvent at normal temperature to obtain (R) -1- (4-methoxyphenyl) propan-2-amine.
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