CN107236753A - The method that the construction method and conversion isoeugenol of isoeugenol monooxygenase activity aggregation produce vanillic aldehyde - Google Patents

Abstract

The invention discloses a method for constructing an isoeugenol monooxygenase active aggregate and a method for converting isoeugenol to produce vanillin. The construction method includes S1, constructing an active aggregate for producing isoeugenol monooxygenase Recombinant Escherichia coli: Link the short peptide 18A with amphipathic self-assembly function to the target gene isoeugenol monooxygenase to construct the active aggregate of isoeugenol monooxygenase-18A, and express it in Escherichia coli to obtain Recombinant Escherichia coli; S2, using the constructed recombinant Escherichia coli to produce isoeugenol monooxygenase active aggregates: culture, optimize and induce the constructed recombinant Escherichia coli, collect the induced cells by centrifugation, and break the cells The crude enzyme liquid is obtained, and the obtained crude enzyme liquid is the active aggregate of isoeugenol monooxygenase. The invention realizes efficient production of vanillin and is convenient for industrial production and application.

Description

Construction method of active aggregates of isoeugenol monooxygenase and conversion of isoeugenol Method for producing vanillin

technical field

The present invention relates to the construction of genetically engineered bacteria of isoeugenol monooxygenase and the technical field of biocatalysis, in particular to a method for constructing active aggregates of isoeugenol monooxygenase and a method for converting isoeugenol to produce vanillin .

Background technique

Vanillin is one of the most demanded spices in the world and is widely used in food, beverage, medicine, cosmetics and other industries. The vanillin currently on the market is divided into two types: natural vanillin and chemically synthesized vanillin. Among them, chemically synthesized vanillin accounts for more than 90% of the market share. Studies have found that large doses of chemically synthesized vanillin can cause dizziness, vomiting, and even damage internal organs. Natural vanillin is extracted from the pods of the vanilla plant. Because the planting of vanilla is affected by climate, place of origin, and local policies, the annual output is less than 20 tons, which is far from meeting people's needs, and the price is relatively expensive. The price per kilogram It is dozens of times that of chemically synthesized vanillin. The vanillin obtained by the biotransformation method belongs to natural vanillin, and its fragrance is the same as that of natural vanillin. It has the advantages of short production cycle and low production cost, and is one of the most promising alternative methods. Isoeugenol has a wide range of sources and low price, and is an ideal precursor substance for obtaining vanillin by microbial transformation. Isoeugenol monooxygenase is the only key enzyme that efficiently catalyzes isoeugenol to vanillin.

One of the key problems to be solved for the biocatalytic production of vanillin is how to efficiently prepare isoeugenol monooxygenase. The core of enzyme catalysis technology is the efficient production, separation, preparation and application development of enzymes. However, the production process of enzyme preparations is complicated and the cost is high, which has become a key problem restricting the wide application of enzyme catalysis. In recent years, it has been found that the aggregation of active enzymes can be effectively induced by linking some short peptides (Self-assembly peptides, SAPs, generally 8-18 amino acid residues) with amphipathic self-assembly function to the end of soluble enzymes. body formation. The application of active enzyme aggregates combined with embedding method or cross-linked enzyme aggregates (cross-linked enzyme aggregates, CLEA) to prepare immobilized enzymes has the advantages of simple operation and high activity, which can significantly reduce the cost of protein immobilization and simplify the process. It can be successfully applied to the preliminary purification of the target protein, which is the basis for the industrial scale-up and high-throughput purification of active enzyme preparation. At present, there are no publications or reports on the construction of active aggregates of isoeugenol monooxygenase.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for constructing active aggregates of isoeugenol monooxygenase and a method for converting isoeugenol to produce vanillin.

The technical solution adopted by the present invention to solve the technical problems is: provide a method for constructing active aggregates of isoeugenol monooxygenase and immobilization thereof, comprising the following steps:

S1. Construction of recombinant Escherichia coli for producing isoeugenol monooxygenase active aggregates:

The short peptide 18A (EWLKAFYEKVLEKLKELF) with amphipathic self-assembly function was connected with the target gene isoeugenol monooxygenase to construct the active aggregate of isoeugenol monooxygenase-18A, which was expressed in Escherichia coli to obtain recombinant Escherichia coli;

S2, using the constructed recombinant Escherichia coli to produce isoeugenol monooxygenase activity aggregates:

The constructed recombinant Escherichia coli is cultured, optimized and induced, and the induced bacteria are collected by centrifugation, and the cells are broken to obtain a crude enzyme solution, and the obtained crude enzyme solution is centrifuged and/or filtered to obtain a precipitate that is isoeugenol mono-added Oxygenase active aggregates.

Preferably, step S1 includes the following steps:

S1-1. Culture strains E.coli BL21(DE3)IEM and E.coli BL21(DE3)pET30a-LipA-18A to obtain Escherichia coli E.coli BL21(DE3)IEM and Escherichia coli BL21( DE3) pET30a-LipA-18A;

S1-2, according to the gene sequence of isoeugenol monooxygenase, design four pairs of PCR primers, including upstream primer A, downstream primer B1, downstream primer B2, downstream primer B3 and downstream primer B4; wherein upstream primer A and downstream primer B4 is a specific primer containing restriction enzyme sites;

S1-3. Use four pairs of PCR primers in sequence, and perform PCR amplification with E. coli BL21(DE3) IEM as the first PCR template. The obtained PCR product is diluted 1000 times and 1 μL is used as the next PCR template. Template; the next PCR is amplified with the previous PCR product as a template, and finally the PCR product target gene isoeugenol monooxygenase is obtained, named IEM-1;

S1-4, purifying the target gene IEM-1;

S1-5. Extract plasmid pET30a-LipA-18A from E. coli BL21(DE3)pET30a-LipA-18A;

S1-6. Carry out double digestion of the target gene IEM-1 and the plasmid pET30a-LipA-18A respectively;

S1-7. Perform nucleic acid electrophoresis on the double-digestion product of the target gene IEM-1 and the plasmid pET30a-LipA-18A, and recover the double-digestion product;

S1-8. Ligate the target gene IEM-1 and the product after double enzyme digestion and recovery of the plasmid pET30a-LipA-18A to obtain the active aggregate of the ligated product isoeugenol monooxygenase-18A, which is named pET30a-IEM- 18A;

S1-9. Transform Escherichia coli E.coli BL21(DE3) with the ligation product pET30a-IEM-18A to obtain the recombinant strain E.coli BL21(DE3)pET30a-IEM-18A.

Preferably, in step S1-2:

The sequence of the upstream primer A is as follows: 5'-tagctacatatgatggcaacgtttaccgcaatgatc-3'

The sequence of the downstream primer B1 is as follows: 5'-gcggccgccttatctctcgaggttcttagactgccaac-3'

The sequence of the downstream primer B2 is as follows: 5'-gtgcaacgctgctcggagacggaagggtagctt-3'

The sequence of the downstream primer B3 is as follows: 5'-gactgccaacaaccgtgcaacgctgctcggaga-3'

The sequence of the downstream primer B4 is as follows: 5'-tagctaaagctttcagttcttagactgccaacaaccgtgc-3'.

Preferably, step S2 includes the following steps:

S2-1, inoculating the constructed recombinant Escherichia coli in a liquid LB medium containing kanamycin and culturing to obtain a seed liquid;

S2-2, taking the obtained seed solution and inoculating it in a solid LB medium containing kanamycin for cultivation;

S2-3, adding an inducer (IPTG) to the solid LB medium for induction;

S2-4. Collect the induced bacteria by low-temperature high-speed centrifugation, resuspend the bacteria with glycine-sodium hydroxide buffer solution, make a cell suspension, and break the cells;

S2-5. Centrifuge, add an equal volume of glycine-sodium hydroxide or sodium carbonate-sodium hydroxide buffer solution to the precipitate, and gently resuspend (use a pipette for 2-3 times), and obtain the crude enzyme solution , centrifuged and/or filtered to obtain a precipitate that is the active aggregate of isoeugenol monooxygenase.

Preferably, the construction method also includes the following steps:

S3. Immobilization of active aggregates of isoeugenol monooxygenase:

The isoeugenol monooxygenase active aggregates obtained in step S2 were immobilized without a carrier by cross-linking enzyme aggregate technology, using glutaraldehyde as a cross-linking agent.

Preferably, step S3 includes the following steps:

S3-1. Add glutaraldehyde solution (concentration: 100 mmol/L; cross-linking time: 60-240 minutes) dropwise to the crude enzyme solution obtained in step S2 at low temperature, and stir;

S3-2. High-speed centrifugation at low temperature, and the obtained precipitate is an active aggregate of immobilized isoeugenol monooxygenase, that is, an immobilized enzyme;

S3-3, resuspending the immobilized enzyme in a sodium carbonate-sodium hydroxide buffer solution, and cleaning the glutaraldehyde attached to the surface of the immobilized enzyme;

S3-4. Centrifuge at low temperature and high speed, and resuspend the immobilized enzyme in sodium carbonate-sodium hydroxide buffer solution.

The present invention also provides a method for producing vanillin by converting isoeugenol monooxygenase active aggregates into isoeugenol, comprising the following steps:

T1. Construction of recombinant Escherichia coli for producing isoeugenol monooxygenase active aggregates:

The short peptide 18A (EWLKAFYEKVLEKLKELF) with amphipathic self-assembly function was connected with the target gene isoeugenol monooxygenase to construct the active aggregate of isoeugenol monooxygenase-18A, which was expressed in Escherichia coli to obtain recombinant Escherichia coli;

T2, using the recombinant Escherichia coli constructed to transform isoeugenol into vanillin:

The constructed recombinant Escherichia coli is cultured, optimized and induced, the induced bacteria are collected by centrifugation, the bacteria are resuspended to form a cell suspension, and isoeugenol is added for transformation to obtain a reaction solution containing vanillin.

Preferably, step S1 includes the following steps:

T1-1, the strains E.coli BL21(DE3)IEM and E.coli BL21(DE3)pET30a-LipA-18A were cultured to obtain Escherichia coli E.coli BL21(DE3)IEM and Escherichia coli E.coli BL21( DE3) pET30a-LipA-18A cells;

T1-2, according to the gene sequence of isoeugenol monooxygenase, design four pairs of PCR primers, including upstream primer A, downstream primer B1, downstream primer B2, downstream primer B3 and downstream primer B4; wherein upstream primer A and downstream primer B4 is a specific primer containing restriction enzyme sites;

T1-3. Use four pairs of PCR primers in sequence, and use E. coli BL21(DE3) IEM as the first PCR template to carry out PCR amplification. The obtained PCR product is diluted 1000 times, and 1 μL is used as the next PCR template. Template; the next PCR is amplified with the previous PCR product as a template, and finally the PCR product target gene isoeugenol monooxygenase is obtained, named IEM-1;

T1-4, purifying the target gene IEM-1;

T1-5. Extract plasmid pET30a-LipA-18A from E. coli BL21(DE3)pET30a-LipA-18A;

T1-6. Carry out double digestion of the target gene IEM-1 and the plasmid pET30a-LipA-18A respectively;

T1-7. Perform nucleic acid electrophoresis on the double-digestion product of the target gene IEM-1 and the plasmid pET30a-LipA-18A, and recover the double-digestion product;

T1-8. Ligate the target gene IEM-1 and the product after double digestion and recovery of the plasmid pET30a-LipA-18A, and obtain the active aggregate of the ligated product isoeugenol monooxygenase-18A, which is named pET30a-IEM- 18A;

T1-9. The ligation product pET30a-IEM-18A was used to transform Escherichia coli E.coli BL21(DE3) to obtain the recombinant strain E.coli BL21(DE3)pET30a-IEM-18A.

Preferably, in step T1-2:

The sequence of the upstream primer A is as follows: 5'-tagctacatatgatggcaacgtttaccgcaatgatc-3'

The sequence of the downstream primer B1 is as follows: 5'-gcggccgccttatctctcgaggttcttagactgccaac-3'

The sequence of the downstream primer B2 is as follows: 5'-gtgcaacgctgctcggagacggaagggtagctt-3'

The sequence of the downstream primer B3 is as follows: 5'-gactgccaacaaccgtgcaacgctgctcggaga-3'

The sequence of the downstream primer B4 is as follows: 5'-tagctaaagctttcagttcttagactgccaacaaccgtgc-3'.

Preferably, step T2 includes the following steps:

T2-1, inoculating the constructed recombinant Escherichia coli in a seed medium containing kanamycin and culturing to obtain a seed liquid;

T2-2, the obtained seed liquid is inoculated in the fermentation medium containing kanamycin and cultivated;

T2-3, adding an inducer (IPTG) to the fermentation medium for induction;

T2-4. Centrifuge the induced fermentation broth at low temperature, collect the bacteria, resuspend the bacteria with glycine-sodium hydroxide buffer solution, and make a cell suspension;

T2-5. Take the cell suspension, add isoeugenol for transformation, and obtain a reaction solution containing vanillin.

Beneficial effects of the present invention: the isoeugenol monooxygenase gene and the amphipathic short peptide 18A with self-assembly function are used to construct isoeugenol monooxygenase active aggregates, and the aggregates are obtained by centrifugation and crushing without cumbersome Purification step. The carrier-free immobilized isoeugenol monooxygenase can be directly used to convert isoeugenol to produce vanillin, reducing enzyme purification steps, saving production costs, realizing efficient production of vanillin, and facilitating industrial production and application.

detailed description

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The construction method of the isoeugenol monooxygenase activity aggregate of the present invention, the steps include as follows:

1. Construction of recombinant bacteria: construction of recombinant Escherichia coli for the production of isoeugenol monooxygenase active aggregates

Among them, bacterial strains: E.coli BL21(DE3)IEM preserved by Shenzhen University (preservation number is CGMCCNo.8918) and E.coli BL21(DE3)pET30a-IEM-18A; Escherichia coli E.coli BL21(DE3) was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. E.coli BL21(DE3)pET30a-IEM-18A belongs to Escherichia coli (Escherichiacoli), and was preserved in the General Microorganism Center of China Committee for Microbial Culture Collection (CGMCC for short) on December 26, 2016, the address is: Beijing No. 3, No. 1 Yard, Beichen West Road, Chaoyang District), the preservation number is CGMCC No.13493.

Preparation of commonly used solutions and media:

(1) Ampicillin (100mg/mL): Weigh 5g of ampicillin, dissolve it in sterile water, dilute to volume in a 50mL volumetric flask, filter through a disposable filter, aliquot into 1.5mL EP tubes, and store at -40°C.

(2) Kanamycin (50mg/mL): Weigh 2.5g of Kanamycin, dissolve it in sterile water, dilute it in a 50mL volumetric flask, filter it with a disposable filter, and dispense it into a 1.5mL EP tube. Store at -40°C.

(3) IPTG (isopropylthiogalactopyranoside, 0.8mol/L): Weigh 0.2g IPTG, dissolve it in sterile water, dilute it in a 10mL volumetric flask, filter it with a disposable filter, and pack it in 1.5mL EP tube, store at -40°C.

(4) Nucleic acid electrophoresis buffer solution (1×TAE): Dilute the 50×TAE stock solution 50 times with ultrapure water, and store it at room temperature until use.

(5) Preparation of 1% agarose gel: Weigh 0.25g of agarose and dissolve it in 25mL 1×TAE, heat in a microwave oven for 1min until the gel dissolves evenly, cool at room temperature until it is not hot, add 3μL of nucleic acid dye, mix well, pour into the preparation The rubber plate was cooled at room temperature for later use (the agarose used was purchased from Beijing Quanshijin Biotechnology Co., Ltd.).

(6) 0.1 mol/L CaCl ₂ : Prepare 0.1 mol/L CaCl ₂ solution, filter it with a disposable filter, aliquot into 1.5 mL EP tubes, and store at -20°C.

(7) Preparation of inner solution for protein electrophoresis (1*SDS-PAGE electrophoresis buffer): Dilute 5*SDS-PAGE electrophoresis buffer with deionized water into 1*SDS-PAGE electrophoresis buffer for use.

(8) 50% glycerin: Glycerin was mixed with sterile water at a ratio of 1:1, autoclaved at 121°C for 20 minutes, and stored at room temperature for later use.

(9) Liquid LB medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, the medium was adjusted to pH 7.0 with 5mol/L NaOH; autoclaved at 121°C for 20min.

(10) Preparation method of solid LB medium:

① Preparation: Add 2g agar powder to 100mL LB medium, heat to dissolve, and then autoclave at 121°C for 20min.

② Add antibiotics: After the solid medium is autoclaved, put the medium in a sterile operating table, add antibiotics when the medium is cooled until it is not hot to the hands, mix well and pour into plates, pour 15-20mL of solid medium into each plate .

③Preservation: Seal the plate with parafilm, invert it, and store it in a refrigerator at 4°C.

④ Antibiotic concentration: the final concentration of ampicillin in the medium is 100 μg/mL, and the final concentration of kanamycin is 50 μg/mL.

1. The cultivation of strains E.coli BL21(DE3)IEM and E.coli BL21(DE3)pET30a-LipA-18A:

(1) Dip a small amount of E.coli BL21(DE3) IEM into 5 mL liquid LB medium (containing 100 μg/mL ampicillin) with a pipette tip, and culture overnight at 37°C and 200 rpm.

(2) Dip a small amount of E.coli BL21(DE3)pET30a-LipA-18A with a pipette tip and inoculate it in 5 mL solid LB medium (containing 50 μg/mL kanamycin), and culture overnight at 37°C and 200 rpm.

2. According to the design, it is necessary to introduce two restriction sites, NdeⅠ and HindⅢ, at both ends of the isoeugenol monooxygenase (IEM) gene by PCR. Since the IEM gene contains a HindⅢ restriction site, the Under the condition of the encoded amino acid, a certain base of HindⅢ in IEM is mutated, that is, site-directed mutation. According to the IEM gene sequence, four pairs of PCR primers were designed, including upstream primer A, downstream primer B1, downstream primer B2, downstream primer B3 and downstream primer B4; upstream primer A and downstream primer B4 are specific primers containing restriction enzyme sites .

Wherein, the sequence of the upstream primer A is as follows: 5'-tagctacatatgatggcaacgtttaccgcaatgatc-3';

The sequence of the downstream primer B1 is as follows: 5'-gcggccgccttatctctcgaggttcttagactgccaac-3';

The sequence of the downstream primer B2 is as follows: 5'-gtgcaacgctgctcggagacggaagggtagctt-3';

The sequence of the downstream primer B3 is as follows: 5'-gactgccaacaaccgtgcaacgctgctcggaga-3';

The sequence of the downstream primer B4 is as follows: 5'-tagctaaagctttcagttcttagactgccaacaaccgtgc-3'.

3. Use the above four pairs of primers in sequence, and use the cultured Escherichia coli E.coliBL21(DE3) IEM as the first PCR template to carry out PCR amplification. After the obtained PCR product is diluted 1000 times, take 1 μL as the template for the next PCR. template, and the next PCR will be amplified with the previous PCR product as the template. The final amplified sequence (PCR product target gene isoeugenol monooxygenase) was named IEM-1.

Among them, the PCR reaction system is: the total amount of the system is 25 μL, in which 0.75 μL of upstream and downstream primers (both at a concentration of 100 μmol/L), 1 μL of template, 12.5 μL of 2×Pfu polymerase, and finally 10 μL of sterilized water are added to make up. The principle of sample addition is: first add a small amount of reagent, and then add a large amount of reagent, the purpose is to mix the molecular reagents in the system; the template and polymerase are added last, and the whole process is kept on ice to reduce the impact of excessive temperature on the enzyme. damage (the polymerase PrimeSTAR Max DNAPolymerase used was purchased from Takara Company).

The PCR reaction conditions are: pre-denaturation at 98°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 10 seconds, extension at 72°C for 10 minutes, and finally lowering to 4°C before taking out. This reaction condition was used for four times of PCR.

After the completion of the four PCRs, the last PCR product was analyzed by agarose gel electrophoresis (the nucleic acid molecular weight marker and Loading Buffer used were purchased from Beijing Kangwei Century Technology Co., Ltd.), and the gel image was observed with a gel imaging system. The results showed that the molecular weight of the site-directed mutation IEM obtained by PCR was about 1.5kb, and the PCR product obtained in the fourth step was verified by agarose electrophoresis as a bright single band.

4. Using a DNA fragment purification kit (purchased from Takara Company) to purify the target gene IEM-1 of the PCR product.

The plasmid pET30a-LipA-18A was extracted from Escherichia coli E.coliBL21(DE3)pET30a-LipA-18A with a mini-plasmid extraction kit (purchased from Shanghai Bioengineering Technology Co., Ltd.).

5. The purified product (target gene IEM-1) and the extracted vector plasmid pET30a-LipA-18A were double-digested with NdeI and HindIII restriction enzymes (purchased from Beijing NEB Co., Ltd.), respectively.

The total double digestion system is 50 μL: 1 μL of NdeⅠ, 1 μL of HindⅢ, 1 μL of DNA or plasmid, 5 μL of 10x NEBuffer, and make up to 50 μL with sterile water. Digestion conditions: double digestion overnight, digestion temperature 37 ° C.

6. Perform nucleic acid electrophoresis on the double-digestion product of the target gene IEM-1 and the plasmid pET30a-LipA-18A, and recover the double-digestion product with a DNA gel recovery kit (purchased from Takara Company).

7. Ligate the target gene IEM-1 and the product after double enzyme digestion and recovery of the plasmid pET30a-LipA-18A, and name the resulting ligation product isoeugenol monooxygenase-18A active aggregate pET30a-IEM- 18A.

The total ligation system is 20 μL: target gene IEM-11 μL, plasmid pET30a-LipA-18A 8 μL, 10x Buffer 2 μL, T4 DNA ligase (purchased from Beijing NEB Co., Ltd.) 1 μL, sterilized water 8 μL. The molar ratio of the target gene IEM-1 to the plasmid pET30a-LipA-18A is about 3:1, and the total mass of the vector and the target fragment is about 0.01 μg. The ligation conditions were 16°C, 4h, 4°C overnight.

8. Transform E.coli BL21(DE3) competent cells with the ligation product pET30a-IEM-18A to obtain the recombinant strain E.coli BL21(DE3)pET30a-IEM-18A.

9. Verify the recombinant bacteria by colony PCR and double enzyme digestion of the recombinant plasmid, and select positive clones for gene sequencing.

E.coli BL21(DE3)pET30a-IEM-18A sequencing results:

2. Production of active aggregates of isoeugenol monooxygenase using constructed recombinant bacteria

For the preparation of commonly used solutions and culture media, refer to the preparation of solutions and culture media in the construction of recombinant bacteria mentioned above.

1. Inoculate 50 μL of E.coli DL21(DE3)pET30a-IEM-18A in 50 mL of liquid LB medium (containing 100 μg/mL kanamycin), and culture overnight at 37°C and 200 rpm to obtain the seed solution.

2. Inoculate 1mL of the seed liquid from step 1 into 100mL solid LB medium containing 100μg/mL kanamycin, culture at 37°C and 200rpm until the OD600 is 0.8, add 0.8mmol/IPTG, 25°C, 200rpm, induce 16h.

3. Centrifuge at 10,000rpm at low temperature (below 4°C) to collect the induced bacteria, resuspend the bacteria with 0.05mol/L pH 10.5 glycine-sodium hydroxide buffer solution, and make a 60g/L cell suspension (bacteria suspension), Break the cells with a high-pressure homogenizer, set the pressure at 800 bar, and break 40mL of the bacterial suspension for three minutes.

4. Centrifuge at 7000rpm for 5min, save the supernatant separately, add an equal volume of 0.05mol/L pH 10.5 glycine-sodium hydroxide buffer solution to the precipitate, gently resuspend (pipe 2-3 times with a pipette gun), and dissolve the obtained The crude enzyme solution is centrifuged and/or filtered to obtain a precipitate which is the active aggregate of isoeugenol monooxygenase.

3. Immobilization of active aggregates of isoeugenol monooxygenase

Wherein, the preparation of culture medium:

Seed medium (g/L): tryptone 10, yeast extract 5, NaCl 10; pH 7.0, liquid volume 200mL/500mL Erlenmeyer flask.

Fermentation medium (g/L): tryptone 10, yeast extract 5, NaCl 10; pH 7.0, liquid volume 400mL/1000mL Erlenmeyer flask.

1. Cell culture

Seed culture: under sterile conditions, add 200 μL E.coliBL21(DE3)pET30a-IEM-18A to the seed medium (containing 50 μg/mL kanamycin), shake overnight at 37°C and 200 rpm to obtain seeds liquid.

Fermentation culture: under sterile conditions, inoculate 80mL of seed liquid into fermentation medium (containing 50μg/mL kanamycin), shake and cultivate at 37°C and 200rpm until OD600 is 0.8, add 0.8mmol/L IPTG , 30 ℃ induction 8h.

2. Preparation of immobilized enzyme (immobilized active aggregate of isoeugenol monooxygenase)

After induction, centrifuge the bacterial solution at 10,000 rpm for 15 min at low temperature (below 4°C), collect the wet bacterial cells, add 0.05 mol/L glycine-sodium hydroxide buffer solution with pH 10.5 to prepare a 65 g/L cell suspension, and use The homogenizer was used to crush the cell suspension under high pressure, and the pressure of the homogenizer was set to 800 bar, and 40 mL of the cell suspension was circulated for 3 minutes.

After crushing, the bacterial liquid was collected. Centrifuge at 10,000 rpm for 15 minutes at 4°C, discard the supernatant, add 10 mL of 0.05 mol/L pH 10.5 sodium carbonate-sodium hydroxide buffer solution to the precipitate, gently resuspend the precipitate, and the obtained crude enzyme solution is isoeugenol mono Oxygenase activity aggregates.

Slowly add 121 μL of 50% glutaraldehyde solution dropwise to the completely resuspended crude enzyme solution at low temperature (below 4°C), and keep magnetic stirring uninterrupted while adding dropwise, and keep low temperature during stirring to prevent enzyme inactivation caused by high temperature. After stirring for 2 hours, take out the solution; centrifuge at 10,000 rpm for 15 minutes at 4°C, discard the supernatant, and resuspend twice in 20 mL of 0.05 mol/L pH 10.5 sodium carbonate-sodium hydroxide buffer solution to wash the pentadiene attached to the surface of the immobilized enzyme. Aldehydes, etc.; centrifuge at low temperature (below 4° C.) and high speed (10000 rpm) to discard the supernatant, and prepare the immobilized enzyme of isoeugenol monooxygenase.

4. Conversion of isoeugenol to vanillin using active aggregates of isoeugenol monooxygenase

Example 1

Take out 50 μL from the glycerol tube E.coli BL21(DE3)pET30a-IEM-18A stored at -80°C, cultivate overnight according to the above-mentioned seed culture method, and inoculate 1 mL of seed liquid into the fermentation culture containing 50 μg/mL kanamycin Base, when OD600 is 0.8, add 0.8mmol/L IPTG, induction temperature is 30°C, 200rpm, induce for 16h; centrifuge the induced fermentation broth at 10,000rpm at low temperature (below 4°C) for 15min, discard the supernatant, and collect the cells , with 0.05mol/L glycine-sodium hydroxide buffer solution of pH 10.5 to resuspend the bacteria to prepare 80g/L cell suspension.

Take 10 mL of bacteria (cell suspension), add 260 mmol/L isoeugenol, transform at 25°C and 200 rpm for 48 hours, and measure the concentration of vanillin in the final reaction solution to be 2.24 g/L.

Example 2

Take out 50 μL from the glycerol tube E.coli BL21(DE3)pET30a-IEM-18A stored at -80°C, culture it for 14 hours according to the above-mentioned seed culture method, and inoculate 1 mL seed liquid into the fermentation culture containing 50 μg/mL kanamycin Base, when OD600 is 0.8, add 0.8mmol/L IPTG, induce at 30°C, 200rpm, induction time is 8h, centrifuge the induced fermentation broth at 10 000rpm at low temperature (below 4°C) for 15min, discard the supernatant, and collect the bacteria cells; resuspend the cells with 0.05mol/L pH 10.5 glycine-sodium hydroxide buffer solution, crush the cell suspension under high pressure, collect the precipitate, add 10mL 0.05mol/L pH10.5 sodium carbonate-sodium hydroxide buffer solution, gently Resuspend, slowly add 121 μL of 50% glutaraldehyde solution dropwise at low temperature (below 4° C.), keep magnetic stirring for 2 hours, and then centrifuge to collect the precipitate to prepare isoeugenol monooxygenase-immobilized enzyme.

Take 9mL of the solution containing immobilized enzyme, add 1mL DMSO, 100mmol/L isoeugenol, 25°C, 200rpm, transform for 36h, centrifuge the reaction solution after the reaction, discard the supernatant, wash the cross-linked enzyme twice with buffer solution , then use 9mL 0.05mol/L pH 10.5 glycine-sodium hydroxide buffer solution to resuspend the cross-linked enzyme, continue the next enzyme-catalyzed reaction, use the cross-linked enzyme repeatedly, measure the concentration of vanillin in each reaction solution; the results show It has good operational stability, and the enzyme activity remains above 60% after repeated use for 7 times.

Determination method of vanillin:

Determination of vanillin and isoeugenol by high performance liquid chromatography (HPLC).

Sample treatment: Add the same volume of ethanol to the reaction solution to precipitate the protein (isoeugenol monooxygenase activity aggregate), dissolve the substrate (isoeugenol) and product (vanillin), and centrifuge at 10000r/min for 5min , and then diluted 10-20 times with ethanol (10 times in the embodiment), filtered through a filter membrane, and tested.

Chromatographic column: Thermo BDS HYPERSIL C18 reverse-phase chromatographic column 250×4.6mm;

Mobile phase: A=0.01% glacial acetic acid aqueous solution, B=methanol;

Gradient elution program: start at 35% B, keep for 7 minutes, increase to 70% B at 15 minutes, and drop to 35% B at 17 minutes;

Column temperature: 30°C;

The flow rate is 1mL/min, the ultraviolet detection is at a wavelength of 280nm, and the injection volume is 20μL. The peak eluting times of vanillin and isoeugenol were about 7.8 min and 17.4 min, respectively.

The above is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process conversion made by using the content of the description of the present invention, or directly or indirectly used in other related technical fields, shall be The same reasoning is included in the patent protection scope of the present invention.

<110> Shenzhen University

<120> Construction method of active aggregate of isoeugenol monooxygenase and method of converting isoeugenol to produce vanillin

<130> CN1618492YZ

<160> 6

<210> 1

<211> 36

<212>DNA

<213> Artificial sequence

<400> 1

tagctacata tgatggcaac gtttaccgca atgatc 36

<210> 2

<211> 38

<212>DNA

<213> Artificial sequence

<400> 2

gcggccgcct tatctctcga ggttcttaga ctgccaac 38

<210> 3

<211> 33

<212>DNA

<213> Artificial sequence

<400>3

gtgcaacgct gctcggagac ggaagggtag ctt 33

<210> 4

<211> 33

<212>DNA

<213> Artificial sequence

<400>4

gactgccaac aaccgtgcaa cgctgctcgg aga 33

<210> 5

<211>40

<212>DNA

<213> Artificial sequence

<400>5

tagctaaagc tttcagttct tagactgcca acaaccgtgc 40

<210> 6

<211>40

<212>DNA

<213> Artificial sequence

<400>6

atggcaacgt ttgaccgcaa tgatccgcag ttggcaggaa cgatgttccc cacccgaata

gaggcgaatg tctttgacct tgaaattgag ggcgagatcc cacgtgcaat caacgggagc

ttcttccgca acacccccga acctcaggtc accacgcaac ctttccacac cttcatcgat

ggggatggtt tggcgtctgc ttttcatttc gaagatggcc aggtcgactt tgtcagccgt

tgggtatgta ctcctcgctt tgaagctgag cggtcggctc gtaaatcact cttcggtatg

taccgcaatc cgttcactga tgatccatcg gtagaaggta ttgatcgtac agtcgccaac

accagtatca tcactcatca cgggaaagta ctggccgcaa aggaagatgg actaccttat

gagcttgacc cccaaaccct ggaaacccga ggtcgttatg attacaaggg gcaggtaacc

agccataacac atacagcgca ccctaagttc gacccccaga caggtgaaat gttactcttc

ggctccgctg ctaaaggcga acgaacgctt gatatggcgt actatattgt tgatcgctac

ggcaaggtga cacatgagac ctggtttaag cagccttacg gtgcattcat gcacgacttt

gctgtcacgc gcaactggtc aatctttccg atcatgcctg cgacaaatag ccttgagcgt

cttaaagcaa agcagcccat ttacatgtgg gagcctgagc gaggaagcta tataggagta

cttcctcgtc gtggtcaggg caaggacatt cgttggttcc gtgccccggc gttgtgggtt

ttccatgtcg tgaatgcttg ggaggaaggg aatagaattc tgattgactt gatggaaagt

gagattttgc cgttcccatt cccgaactcg cagaaccttc catttgatcc ctccaaggct

gttccgcgtc taacccgttg ggaaattgat ctcaatagtg gtaacgatga gatgaaacgt

acgcagctac acgaatattt tgcagaaatg cctatcatgg atttccgttt tgcgctccag

gatcatcgct acgcctacat gggggttgac gatcctcgtc gccccttagc tcatcagcaa

gctgaaaaaa tctttgccta caattcgtta ggggtttggg acaaccatcg taaagattat

gaactttggt ttacgggaaa aatgtctgca gcgcaggaac cggcgtttgt tcctagaagc

ccagatgcgc ctgagggcga tggctaccta ctcagtgtag tagggcggct cgatgaagat

cgtagcgatc tagttatcct tgatacgcaa tgtttggcag ctgggcctgt ggccactgtc

aagctaccct tccgtctccg agcagcgttg cacggttgtt ggcagtctaa gaactgaaag

cttccgaccc caccgaccac gccaacgcca ccaacccaccc caaccccgac gccgctggaa

cttgaactga agttaaaact ggaattagaa ttaaagctga aa 1542

Claims (10)

1. a kind of construction method of isoeugenol monooxygenase activity aggregation, it is characterised in that comprise the following steps：

S1, build recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation：

Small peptide 18A with amphipathic self-assembling function is connected with target gene isoeugenol monooxygenase, isobutyl is built fragrant Phenol monooxygenase -18A activity aggregations, and in expression in escherichia coli, obtain recombination bacillus coli；

S2, the recombination bacillus coli production isoeugenol monooxygenase activity aggregation using structure：

The recombination bacillus coli of structure is subjected to culture optimization, induction, the thalline after induction is collected by centrifugation, smudge cells with Crude enzyme liquid is obtained, gained crude enzyme liquid is centrifuged and/or filtered, it is isoeugenol monooxygenase activity aggregation to obtain precipitation.

2. the construction method of isoeugenol monooxygenase activity aggregation according to claim 1, it is characterised in that step S1 comprises the following steps：

S1-1, by bacterial strain E.coli BL21（DE3）IEM and E.coli BL21（DE3）PET30a-LipA-18A is cultivated, E. coli BL21 is obtained respectively（DE3）IEM and E. coli BL21（DE3）pET30a-LipA-18A Cell；

S1-2, the gene order according to isoeugenol monooxygenase, design four pairs of PCR primers, including sense primer A, downstream draw Thing B1, anti-sense primer B2, anti-sense primer B3 and anti-sense primer B4；Wherein sense primer A and anti-sense primer B4 is comprising digestion position The specific primer of point；

S1-3, in sequence respectively using four pairs of PCR primers, with E. coli BL21（DE3）IEM is first time PCR Template enters performing PCR amplification, and obtained PCR primer takes 1 μ L as the template of PCR next time after diluting 1000 times；Next time PCR is expanded using last PCR primer as template, finally gives PCR primer target gene isoeugenol monooxygenase, life Entitled IEM-1；

S1-4, the target gene IEM-1 is purified；

S1-5, from E. coli BL21（DE3）Plasmid pET30a-LipA-18A is extracted in pET30a-LipA-18A；

S1-6, double digestion is carried out to the target gene IEM-1 and plasmid pET30a-LipA-18A respectively；

S1-7, the double digestion product progress nucleic acid electrophoresis by target gene IEM-1 and plasmid pET30a-LipA-18A, reclaim double Digestion products；

S1-8, the product after target gene IEM-1 and plasmid pET30a-LipA-18A double digestion recyclings is attached, obtained To the active aggregations of connection product isoeugenol monooxygenase -18A, pET30a-IEM-18A is named as；

S1-9, with the connection product pET30a-IEM-18A convert E. coli BL21 (DE3), obtain recombinant bacterium E.coli BL21(DE3) pET30a-IEM-18A。

3. the construction method of isoeugenol monooxygenase activity aggregation according to claim 2, it is characterised in that step In S1-2：

Sense primer A sequence is as follows：5’-tagctacatatgatggcaacgtttaccgcaatgatc-3’

Anti-sense primer B1 sequence is as follows：5’-gcggccgccttatctctcgaggttcttagactgccaac-3’

Anti-sense primer B2 sequence is as follows：5’-gtgcaacgctgctcggagacggaagggtagctt-3’

Anti-sense primer B3 sequence is as follows：5’-gactgccaacaaccgtgcaacgctgctcggaga-3’

Anti-sense primer B4 sequence is as follows：5’-tagctaaagctttcagttcttagactgccaacaaccgtgc-3’.

4. the construction method of isoeugenol monooxygenase activity aggregation according to claim 2, it is characterised in that step S2 comprises the following steps：

S2-1, the recombination bacillus coli of structure is inoculated in received containing card mycin liquid LB medium in cultivate to be planted Sub- liquid；

S2-2, take the seed liquor of acquisition be inoculated in received containing card mycin solid-state LB culture mediums in cultivate；

S2-3, in the solid-state LB culture mediums add derivant induced；

The thalline after induction is collected by centrifugation in S2-4, low-temperature and high-speed, and thalline is resuspended with Glycine-NaOH cushioning liquid, is made into Cell suspension, smudge cells；

S2-5, centrifugation, add isometric Glycine-NaOH or sodium carbonate-sodium hydroxide buffer solution in sediment, Soft to be resuspended, the crude enzyme liquid of acquisition is centrifuged and/or filtered, and it is isoeugenol monooxygenase activity aggregation to obtain precipitation.

5. the construction method of isoeugenol monooxygenase activity aggregation according to claim 1, it is characterised in that the structure Construction method is further comprising the steps of：

S3, isoeugenol monooxygenase activity aggregation immobilization：

By cross-linked enzyme aggregate technology, using glutaraldehyde as cross linker, the isoeugenol monooxygenase that step S2 is obtained is lived Property aggregation carry out carrier-free immobilization.

6. the construction method of isoeugenol monooxygenase activity aggregation according to claim 5, it is characterised in that step S3 comprises the following steps：

Glutaraldehyde solution is added dropwise in S3-1, the crude enzyme liquid obtained at low temperature toward step S2, stirs；

S3-2, at low temperature high speed centrifugation, the sediment of acquisition are the isoeugenol monooxygenase activity aggregation of immobilization, i.e., Immobilised enzymes；

S3-3, by sodium carbonate-sodium hydroxide buffer solution to immobilised enzymes be resuspended, cleaning immobilised enzymes surface attachment penta 2 Aldehyde；

S3-4, low-temperature and high-speed centrifugation, immobilised enzymes is resuspended using sodium carbonate-sodium hydroxide buffer solution.

7. a kind of method that isoeugenol monooxygenase activity aggregation conversion isoeugenol produces vanillic aldehyde, it is characterised in that Comprise the following steps：

T1, build recombination bacillus coli for producing isoeugenol monooxygenase activity aggregation：

Small peptide 18A with amphipathic self-assembling function is connected with target gene isoeugenol monooxygenase, isobutyl is built fragrant Phenol monooxygenase -18A activity aggregations, and in expression in escherichia coli, obtain recombination bacillus coli；

T2, the recombination bacillus coli conversion isoeugenol production vanillic aldehyde using structure：

The recombination bacillus coli of structure is subjected to culture optimization, induction, the thalline after induction is collected by centrifugation, thalline is resuspended and matches somebody with somebody Into cell suspension, add isoeugenol and converted, obtain the reaction solution containing vanillic aldehyde.

8. isoeugenol monooxygenase activity aggregation conversion isoeugenol according to claim 7 produces the side of vanillic aldehyde Method, it is characterised in that step S1 comprises the following steps：

T1-1, by bacterial strain E.coli BL21（DE3）IEM and E.coli BL21（DE3）PET30a-LipA-18A is cultivated, E. coli BL21 is obtained respectively（DE3）IEM and E. coli BL21（DE3）pET30a-LipA-18A Cell；

T1-2, the gene order according to isoeugenol monooxygenase, design four pairs of PCR primers, including sense primer A, downstream draw Thing B1, anti-sense primer B2, anti-sense primer B3 and anti-sense primer B4；Wherein sense primer A and anti-sense primer B4 is comprising digestion position The specific primer of point；

T1-3, in sequence respectively using four pairs of PCR primers, with E. coli BL21（DE3）IEM is first time PCR Template enters performing PCR amplification, and obtained PCR primer takes 1 μ L as the template of PCR next time after diluting 1000 times；Next time PCR is expanded using last PCR primer as template, finally gives PCR primer target gene isoeugenol monooxygenase, life Entitled IEM-1；

T1-4, the target gene IEM-1 is purified；

T1-5, from E. coli BL21（DE3）Plasmid pET30a-LipA-18A is extracted in pET30a-LipA-18A；

T1-6, double digestion is carried out to the target gene IEM-1 and plasmid pET30a-LipA-18A respectively；

T1-7, the double digestion product progress nucleic acid electrophoresis by target gene IEM-1 and plasmid pET30a-LipA-18A, reclaim double Digestion products；

T1-8, the product after target gene IEM-1 and plasmid pET30a-LipA-18A double digestion recyclings is attached, obtained To the active aggregations of connection product isoeugenol monooxygenase -18A, pET30a-IEM-18A is named as；

T1-9, with the connection product pET30a-IEM-18A E. coli BL21 (DE3) is transferred to, obtains recombinant bacterium E.coli BL21(DE3) pET30a-IEM-18A。

9. isoeugenol monooxygenase activity aggregation conversion isoeugenol according to claim 8 produces the side of vanillic aldehyde Method, it is characterised in that in step T1-2：

Sense primer A sequence is as follows：5’-tagctacatatgatggcaacgtttaccgcaatgatc-3’

Anti-sense primer B1 sequence is as follows：5’-gcggccgccttatctctcgaggttcttagactgccaac-3’

Anti-sense primer B2 sequence is as follows：5’-gtgcaacgctgctcggagacggaagggtagctt-3’

Anti-sense primer B3 sequence is as follows：5’-gactgccaacaaccgtgcaacgctgctcggaga-3’

Anti-sense primer B4 sequence is as follows：5’-tagctaaagctttcagttcttagactgccaacaaccgtgc-3’.

10. isoeugenol monooxygenase activity aggregation conversion isoeugenol production vanillic aldehyde according to claim 8 Method, it is characterised in that step T2 comprises the following steps：

T2-1, the recombination bacillus coli of structure is inoculated in received containing card mycin seed culture medium in cultivate to obtain seed Liquid；

T2-2, obtain seed liquor be inoculated in received containing card mycin fermentation medium in culture；

T2-3, in the fermentation medium add derivant induced；

T2-4, by the zymotic fluid low-temperature centrifugation after induction, collect thalline, thalline be resuspended with Glycine-NaOH cushioning liquid, It is made into cell suspension；

T2-5, take cell suspension, add isoeugenol and converted, reaction solution of the acquisition containing vanillic aldehyde.