CN115181713B - Artificial photo-enzyme whole-cell biocatalyst and preparation method and application thereof - Google Patents
Artificial photo-enzyme whole-cell biocatalyst and preparation method and application thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B15/00—Peroxides; Peroxyhydrates; Peroxyacids or salts thereof; Superoxides; Ozonides
- C01B15/01—Hydrogen peroxide
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- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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Abstract
The invention discloses an artificial light enzyme whole-cell biocatalyst and a preparation method and application thereof, and belongs to the technical field of biocatalysis. The whole cell catalyst used in the invention has the advantages of simple culture medium, low cost, no need of breaking cells and separating and purifying enzyme, simple operation process, economy and environmental protection compared with free enzyme and chemical catalysis. The photochemical in-situ hydrogen peroxide generation method can continuously and stably generate low-concentration hydrogen peroxide without adding high-concentration hydrogen peroxide. The combination of photochemical in-situ hydrogen peroxide generation and a whole cell system has realized the P450 peroxygenase reaction in whole cells, and has good practical application value.
Description
Technical Field
The invention belongs to the technical field of biocatalysis, and relates to an artificial light enzyme whole-cell biocatalyst, a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Cytochrome P450 monooxygenases (cytochromes P450 monooxygenases, P450 or CYP) are iron-containing oxidase superfamilies with cysteine as the axial ligand and heme as the active center. The natural P450 can catalyze various reactions including hydroxylation, epoxidation, heteroatom oxidation and the like with high selectivity under mild conditions, but the catalytic application is limited by an expensive cofactor NAD (P) H and a complex electron transfer system, and the limitation limits the industrial application of the P450 enzyme to a certain extent, especially the in-vitro catalytic application.
In chinese patent application No. 201810126862.7, a bifunctional small molecule for activating the catalytic activity of cytochrome P450 peroxygenase is disclosed, which disclosed bifunctional small molecule is capable of activating P450 peroxygenase to utilize H 2 O 2 The ability to catalyze a substrate shows better catalytic activity for various reactions such as epoxidation of styrene, sulfination of dimethyl sulfide, etc., than the reported P450 peroxygenase. A specific reaction mechanism diagram is shown in FIG. 1.
Although this strategy allows catalytic reactions to be carried out without the additional addition of the cofactor NAD (P) H and the reduction chaperonin (enzyme) during the reaction, the inventors have found that there are certain limitations in its application to the practical industry. Firstly, for the preparation reaction, the purification process of a large amount of free enzymes is complex, the purification cost is high, and the stability of the free enzymes is poor; secondly, the concentration of the hydrogen peroxide required in the reaction system is higher and can reach 20mM, the high concentration hydrogen peroxide can excite the activity of the enzyme, but the stability of the enzyme can be reduced, and the cost of the reaction can be increased by adding the high concentration hydrogen peroxide.
Disclosure of Invention
Aiming at the problems, the invention provides an artificial light enzyme whole-cell biocatalyst and a preparation method and application thereof.
To solve the above technical problems, the following one or more embodiments of the present invention provide the following technical solutions:
in a first aspect, the invention provides an artificial photo-enzyme whole-cell biocatalyst, which comprises a photochemical in-situ hydrogen peroxide generation system and a whole-cell catalyst, wherein the photochemical in-situ hydrogen peroxide generation system comprises a photocatalyst and a reducing agent, low-concentration hydrogen peroxide can be continuously and stably generated through reducing oxygen in air, and the whole-cell catalyst is a cell for realizing P450 peroxygenase reaction in a whole cell by utilizing hydrogen peroxide under the assistance of a difunctional small molecule.
The bifunctional small molecule may be all bifunctional small molecule compounds disclosed in application number 201810126862.7. In one embodiment of the present invention, the bifunctional small molecule compound may be Im-C6-Phe, which has the following structural formula:
the cells in the whole cell catalyst are resting cells which recombinantly express the oxidation domain (BMP) of the P450 enzyme and mutants thereof.
More specifically, the cells are prepared by:
constructing an expression vector containing a target gene, and transforming the recombinant expression vector into a host (competent cell) to obtain recombinant cells expressing the P450 enzyme BMP and mutants thereof. The recombinant cells are cultured to induce the expression of BMP and its mutants.
In a second aspect, the present invention provides a method for preparing the artificial light enzyme whole cell biocatalyst, comprising:
constructing a photochemical in-situ hydrogen peroxide generation system and preparing a whole-cell catalyst.
The method for constructing the photochemical in-situ hydrogen peroxide generation system specifically comprises the following steps: under the condition of illumination, the photocatalyst and the reducing agent are added into a buffer solution system for reaction, so that oxygen in the reducing air continuously and stably generates low-concentration hydrogen peroxide.
The specific method for preparing the whole-cell catalyst comprises the following steps: constructing an expression vector containing a target gene, and transforming the recombinant expression vector into a host (competent cell) to obtain recombinant cells expressing the P450 enzyme BMP and mutants thereof.
In a third aspect, the present invention provides the use of the artificial light enzyme whole cell biocatalyst described above in any one or more of the following:
1) Catalyzing the reaction based on the whole cell internal peroxygenase;
2) And (3) preparing a medical intermediate.
Wherein the types of catalysis include, but are not limited to, epoxidation of olefins of different substituents, sulfination of sulfides of different substituents, hydroxylation of aliphatic hydrocarbons, hydroxylation of carbon-hydrogen bonds on aromatic hydrocarbons, heteroaromatic compounds, or hydroxylation on side chain substituents, and the like;
the substrate comprises
In a fourth aspect, there is provided a method of performing a reaction based on a substrate catalysed by a peroxygenase within a whole cell, the method comprising: adding bifunctional micromolecules and substrates into the artificial photo-enzyme whole-cell biocatalyst, and reacting under the illumination condition.
Compared with the prior art, the above technical scheme of the invention has the following beneficial effects:
the whole cell catalyst used in the invention has the advantages of simple culture medium, low cost, no need of breaking cells and separating and purifying enzyme, simple operation process, economy and environmental protection compared with free enzyme and chemical catalysis. The photochemical in-situ hydrogen peroxide generation method can continuously and stably generate low-concentration hydrogen peroxide without adding high-concentration hydrogen peroxide. The combination of photochemical in situ hydrogen peroxide generation and a whole cell system has realized the P450 peroxygenase reaction in whole cells.
Generating photochemical in-situ hydrogen peroxide and fully refiningCells combine to effect catalytic reactions within whole cells. The in-situ hydrogen peroxide generation technology refers to the method of reducing O in the air 2 Generation of H 2 O 2 . The catalytic reaction is maintained by continuously generating low-concentration hydrogen peroxide, and meanwhile, the damage to enzyme is reduced so as to improve the stability of the enzyme. The method has the advantages of economy, environmental protection, no pollution, low cost, mild reaction conditions and the like.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a diagram showing the activity of a bifunctional small molecule activating P450BM3 peroxygenase in the background of the invention;
FIG. 2 is a schematic diagram of photochemical in-situ hydrogen peroxide generation and whole cell catalytic combination reaction according to the invention; wherein a is a photochemical in-situ hydrogen peroxide generation schematic diagram, and b is a photochemical in-situ hydrogen peroxide generation and whole cell catalysis combined reaction schematic diagram;
FIG. 3 is a graph showing the concentration optimization of sodium ascorbate in photochemical in-situ hydrogen peroxide generation in an embodiment of the invention;
FIG. 4 is a graph showing the optimal concentration of fluorescein in photochemical in-situ hydrogen peroxide generation in an embodiment of the invention;
FIG. 5 shows the in situ H of the whole cell photocatalysis of HX ΔEG in accordance with an embodiment of the present invention 2 O 2 Is described.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In a specific embodiment of the invention, an artificial photo-enzyme whole-cell biocatalyst is provided, which comprises a photochemical in-situ hydrogen peroxide generation system and a whole-cell catalyst, wherein the photochemical in-situ hydrogen peroxide generation system comprises a photocatalyst and a reducing agent, low-concentration hydrogen peroxide can be continuously and stably generated by reducing oxygen in air, and the whole-cell catalyst is a cell for realizing the whole-cell P450 peroxygenase reaction by utilizing hydrogen peroxide under the assistance of a difunctional micromolecule.
The photocatalyst and the reducing agent are not particularly limited as long as the continuous and stable generation of low-concentration hydrogen peroxide by the oxygen in the reducing air can be realized. In one embodiment of the present invention, the photocatalyst may be fluorescein and the reducing agent may be sodium ascorbate.
The bifunctional small molecules are all bifunctional small molecule compounds described in application number 201810126862.7. In one embodiment of the present invention, the bifunctional small molecule compound may be Im-C6-Phe, which has the following structural formula:
in yet another embodiment of the invention, the cells in the whole-cell catalyst are resting cells recombinantly expressing the oxidation domain (BMP) of the P450 enzyme and mutants thereof.
More specifically, the cells are prepared by:
constructing an expression vector containing a target gene, and transforming the recombinant expression vector into a host (competent cell) to obtain recombinant cells expressing the P450 enzyme BMP and mutants thereof. The recombinant cells are cultured to induce the expression of BMP and its mutants.
Wherein, the mutant of the P450 enzyme BMP can be single-point mutation or multi-point combination mutation, such as BMP-F87A, BMP-T268V or BMP-F87A-T268V, so long as the final obtained hydrogen peroxide dependent P450 peroxygenase through the mutation of the BMP, the construction of the corresponding target gene is easy to be realized by the person skilled in the art, and the description is omitted here.
The expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F cosmid, a phage or an artificial chromosome; viral vectors may include adenovirus vectors, retrovirus vectors, or adeno-associated virus vectors, artificial chromosomes including Bacterial Artificial Chromosomes (BAC), phage P1-derived vectors (PAC), yeast Artificial Chromosomes (YAC), or Mammalian Artificial Chromosomes (MAC); further preferred are plasmids; even more preferred is the pET-28a plasmid;
the host includes, but is not limited to, bacteria, fungi and eukaryotic cells, further selected from the group consisting of Escherichia coli, bacillus subtilis, saccharomyces cerevisiae, trichoderma reesei and Penicillium oxalate; more preferably, E.coli BL21 (DE 3) is used. In order to increase the recombinant cell P450 peroxygenase activity towards H 2 O 2 By using the same, the catalase gene (e.g., katA, katB, katE and katG) present in the host may be first silenced or knocked out. Thus, in one embodiment of the invention, the host cell is E.coli BL21 (DE 3) from which the endogenous catalase genes katE and katG have been knocked out.
In still another embodiment of the present invention, there is provided a method for preparing the artificial light enzyme whole cell biocatalyst described above, comprising:
constructing a photochemical in-situ hydrogen peroxide generation system and preparing a whole-cell catalyst.
The method for constructing the photochemical in-situ hydrogen peroxide generation system specifically comprises the following steps: under the condition of illumination, the photocatalyst and the reducing agent are added into a buffer solution system for reaction, so that oxygen in the reducing air continuously and stably generates low-concentration hydrogen peroxide.
The photocatalyst and the reducing agent are not particularly limited as long as the continuous and stable generation of low-concentration hydrogen peroxide by the oxygen in the reducing air can be realized. In one embodiment of the present invention, the photocatalyst may be fluorescein and the reducing agent may be sodium ascorbate.
The buffer system may be a phosphate buffer (pH 7.4) in which the concentration of fluorescein is controlled to be 1-1000. Mu.M; the concentration of sodium ascorbate is 0.001-10mM. The generation rate and the generation amount of the hydrogen peroxide can be regulated and controlled by controlling the concentrations of the photocatalyst and the reducing agent.
The illumination may be performed using a white light lamp (25W), and the above reaction may be performed at a low temperature (e.g., 4 ℃).
The specific method for preparing the whole-cell catalyst comprises the following steps: constructing an expression vector containing a target gene, and transforming the recombinant expression vector into a host (competent cell) to obtain recombinant cells expressing the P450 enzyme BMP and mutants thereof.
Wherein, the mutant of the P450 enzyme BMP can be single-point mutation or multi-point combination mutation, such as BMP-F87A, BMP-T268V or BMP-F87A-T268V, so long as the final obtained hydrogen peroxide dependent P450 peroxygenase by mutating the BMP, the construction of the corresponding target gene is easy to realize for the person skilled in the art.
The expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F cosmid, a phage or an artificial chromosome; viral vectors may include adenovirus vectors, retrovirus vectors, or adeno-associated virus vectors, artificial chromosomes including Bacterial Artificial Chromosomes (BAC), phage P1-derived vectors (PAC), yeast Artificial Chromosomes (YAC), or Mammalian Artificial Chromosomes (MAC); further preferred are plasmids; even more preferred is the pET-28a plasmid;
the host includes, but is not limited to, bacteria, fungi and eukaryotic cells, further selected from the group consisting of Escherichia coli, bacillus subtilis, saccharomyces cerevisiae, trichoderma reesei and Penicillium oxalate; more preferably, E.coli BL21 (DE 3) is used. In order to increase the recombinant cell P450 peroxygenase activity towards H 2 O 2 By using the above-described gene, the catalase gene (e.g., katA, katB, katE, katG, etc.) present in the host may be first silenced or knocked out. Thus, in one embodiment of the invention, the host cell is E.coli BL21 (DE 3) from which the endogenous catalase genes katE and katG have been knocked out.
In yet another embodiment of the present invention, there is provided the use of the artificial light enzyme whole cell biocatalyst described above in any one or more of the following:
1) Catalyzing the reaction based on the whole cell internal peroxygenase;
2) And (3) preparing a medical intermediate.
Wherein the types of catalysis include, but are not limited to, epoxidation of olefins of different substituents, sulfination of sulfides of different substituents, hydroxylation of aliphatic hydrocarbons, hydroxylation of carbon-hydrogen bonds on aromatic hydrocarbons, heteroaromatic compounds, or hydroxylation on side chain substituents, and the like;
the substrate comprises
In yet another embodiment of the present invention, there is provided a method for catalyzing a reaction based on a substrate with a peroxygenase within a whole cell, the method comprising: adding bifunctional micromolecules and substrates into an artificial light enzyme whole cell biocatalyst, and reacting under the illumination condition.
The illumination may be performed using a white light lamp (25W), and the above reaction may be performed at a low temperature (e.g., 4 ℃).
The photochemical in-situ hydrogen peroxide generation principle and the photochemical in-situ hydrogen peroxide generation and whole cell catalysis combined reaction principle diagram are shown in fig. 2.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The test methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions.
Example 1: construction of HX delta EG-F87A/T268V whole cell catalyst
1. Plate culture: the pET-28a-F87A-T268V plasmid was transferred into HX delta EG-BL21 competent cells (rejecting endogenous H) 2 O 2 Coli BL21 (DE 3) competent cells of the scavenger genes katE and katG) and plated on LB solid medium (containing 50. Mu.g/mLKana) and cultured overnight at 37 ℃.
The method for obtaining the pET-28a-F87A-T268V plasmid comprises the following steps:
the nucleotide sequence of the P450BM3 heme domain (BMP) wild type is SEQ ID NO.1, and the primer sequence of the mutation site is designed according to a vector pET-28a-BMP (Ma et al, 2018) vector containing the BMP wild type gene. Then, taking the wild type as a template, and carrying out PCR amplification by using primer sequences of different designed mutation sites to obtain a single mutant; and then obtaining double mutation by taking the single mutant as a template.
SEQ ID NO.1：
ATGACAATTAAAGAAATGCCTCAGCCAAAAACGTTTGGAGAGCTTAAAAATTTACCGTTATTAAACACAGATAAACCGGTTCAAGCTTTGATGAAAATTGCGGATGAATTAGGAGAAATCTTTAAATTCGAGGCGCCTGGTCGTGTAACGCGCTACTTATCAAGTCAGCGTCTAATTAAAGAAGCATGCGATGAATCACGCTTTGATAAAAACTTAAGTCAAGCGCTTAAATTTGTACGTGATTTTGCAGGAGACGGGTTATTTACAAGCTGGACGCATGAAAAAAATTGGAAAAAAGCGCATAATATCTTACTTCCAAGCTTCAGTCAGCAGGCAATGAAAGGCTATCATGCGATGATGGTCGATATCGCCGTGCAGCTTGTTCAAAAGTGGGAGCGTCTAAATGCAGATGAGCATATTGAAGTACCGGAAGACATGACACGTTTAACGCTTGATACAATTGGTCTTTGCGGCTTTAACTATCGCTTTAACAGCTTTTACCGAGATCAGCCTCATCCATTTATTACAAGTATGGTCCGTGCACTGGATGAAGCAATGAACAAGCTGCAGCGAGCAAATCCAGACGACCCAGCTTATGATGAAAACAAGCGCCAGTTTCAAGAAGATATCAAGGTGATGAACGACCTAGTAGATAAAATTATTGCAGATCGCAAAGCAAGCGGTGAACAAAGCGATGATTTATTAACGCATATGCTAAACGGAAAAGATCCAGAAACGGGTGAGCCGCTTGATGACGAGAACATTCGCTATCAAATTATTACATTCTTAATTGCGGGACACGAAACAACAAGTGGTCTTTTATCATTTGCGCTGTATTTCTTAGTGAAAAATCCACATGTATTACAAAAAGCAGCAGAAGAAGCAGCACGAGTTCTAGTAGATCCTGTTCCAAGCTACAAACAAGTCAAACAGCTTAAATATGTCGGCATGGTCTTAAACGAAGCGCTGCGCTTATGGCCAACTGCTCCTGCGTTTTCCCTATATGCAAAAGAAGATACGGTGCTTGGAGGAGAATATCCTTTAGAAAAAGGCGACGAACTAATGGTTCTGATTCCTCAGCTTCACCGTGATAAAACAATTTGGGGAGACGATGTGGAAGAGTTCCGTCCAGAGCGTTTTGAAAATCCAAGTGCGATTCCGCAGCATGCGTTTAAACCGTTTGGAAACGGTCAGCGTGCGTGTATCGGTCAGCAGTTCGCTCTTCATGAAGCAACGCTGGTACTTGGTATGATGCTAAAACACTTTGACTTTGAAGATCATACAAACTACGAGCTGGATATTAAAGAAACTTTAACGTTAAAACCTGAAGGCTTTGTGGTAAAAGCAAAATCGAAAAAAATTCCGCTT。
1) The primers for the mutation of pET-28a-F87A are shown in Table 1:
TABLE 1
| Primer(s) | Sequence (5 '-3') | |
| 1 | F87A-F | GCGACAAGCTGGACGCATGAAAAAAATTG(SEQ ID NO.2) |
| 2 | F87A-R | TAACCCGTCTCCTGCAAAATCACGTACAA(SEQ ID NO.3) |
2) On the basis of pET-28a-F87A, a double mutant at the T268 position is constructed. Specific primers are shown in Table 2:
TABLE 2
| Primer(s) | Sequence (5 '-3') | |
| 1 | T268V-F | GTGACAAGTGGTCTTTTATCATTTGC(SEQ ID NO.4) |
| 2 | T268V-R | TTCGTGTCCCGCAATTAAGAATG(SEQ ID NO.5) |
TABLE 3 PCR System
TABLE 4 PCR reaction conditions
| Step (a) | Temperature (. Degree. C.) | Time |
| 1 | 94 | 5min |
| 2 | 94 | 15s |
| 3 | 57 | 15s |
| 4 | 72 | 4min |
| 5 | Step 2-4 | 27 cycles |
| 6 | 72 | 10min |
And (3) performing agarose gel electrophoresis after PCR, cutting off target bands under an ultraviolet lamp after electrophoresis, and then recycling target fragments by using a gel recycling kit.
The sequence ends of the different mutants recovered from the gel were subjected to phosphorylation, and the phosphorylation system is shown in Table 5;
TABLE 5 sequence end phosphorylation System
The phosphorylated mutant sequences were ligated, and the ligation system is shown in table 6:
table 6 connection system
| Name of the name | Volume of |
| Phosphorylation system | 20μL |
| T4DNA ligase | 1μL |
| Total volume of | 21μL |
The ligation was carried out overnight at 16 ℃.
Transferring 10 mu L of the connecting system into competent cells of escherichia coli DH5 alpha, lightly mixing, and carrying out ice bath for 30min; heat shock at 42 ℃ for 90s, and ice bath for 5min; 600 μl of fresh LB liquid medium is added, shake-cultured at 37deg.C and 200rpm for 1h; 200. Mu.L of the bacterial liquid was plated on LB plates containing 50. Mu.g/ml Kana, and cultured overnight at 37 ℃. And (3) determining and analyzing the gene sequence when a single colony grows on the plate. The plasmid which is successfully sequenced is pET-28a-F87A-T268V plasmid.
2. Activating strains: single colonies on plates were picked on an ultra clean bench and inoculated into 50mL LB liquid medium (containing 50. Mu.g/mL Kana), activated at 37℃overnight at 200 rpm.
3. Induction of expression: inoculating the bacterial liquid obtained in the previous step into 500mL of LB liquid medium (containing 50 mug/mL Kana) under aseptic condition, culturing at 37 ℃ and 200rpm until OD is 0.6-0.8, adding FeCl with final concentration of 0.5mM 3 And 5 aminolevulinic acid, culturing at 30℃and 200rpm for 30min, and adding 1mM final concentration of iso-formPropylthiogalactoside was induced at 30℃for 20h at 200 rpm.
4. And (3) collecting thalli: centrifuging the induced culture obtained in the last step for 10min at the rotation speed of 6000rpm, and separating to obtain bacterial precipitate; washing the bacterial cell precipitate with physiological saline for 3 times, centrifuging at 7000rpm for 10min, and placing the bacterial cell precipitate in a refrigerator at-80deg.C for preservation or directly using.
5. Suspending the bacterial precipitate in a buffer solution with the pH of 7.4 to obtain the recombinant escherichia coli whole-cell biocatalyst.
Example 2: concentration optimization of sodium ascorbate in photochemical in-situ hydrogen peroxide generation
1mL of the reaction system: and adding 50 mu M fluorescein, sodium ascorbate with different concentrations, a 25W white light lamp and a hydrogen peroxide test paper into a phosphate buffer solution with the pH of 7.4, and reacting at the temperature of 4 ℃ for 1h to determine the content of hydrogen peroxide. The condition optimization shows that: at 50. Mu.M fluorescein, different concentrations of H can be obtained by varying the concentration of sodium ascorbate 2 O 2 . As can be seen from FIG. 3, H was found at 4mM sodium ascorbate 2 O 2 The highest yield of (3) can reach 735 mu M.
Example 3: concentration optimization of fluorescein in photochemical in-situ hydrogen peroxide generation
1mL of the reaction system: adding 4mM sodium ascorbate, luciferin with different concentrations, a 25W white light lamp and a hydrogen peroxide test paper into a phosphate buffer solution with pH of 7.4, and reacting at 4 ℃ for 1h to determine the hydrogen peroxide content. The condition optimization shows that: at 4mM sodium ascorbate, the concentration of fluorescein affects H 2 O 2 Is produced in the same way as the production amount of the catalyst. As can be seen from FIG. 4, H is found at 100. Mu.M fluorescein 2 O 2 The maximum amount of (2) can reach 1150. Mu.M.
Example 4: in-situ photochemical hydrogen peroxide generation amount in HX delta EG-F87A/T268V whole cell
1mL of the reaction system: OD 9.0 HX delta EG-F87A/T268V bacterial solution, 4mM sodium ascorbate, 100 mu M fluorescein, 25W white light,and (3) reacting for 1h at 4 ℃, and measuring the content of hydrogen peroxide by using hydrogen peroxide test paper. From FIG. 5, it can be seen that photocatalytic in situ H 2 O 2 Production methods capable of producing H in whole cells 2 O 2 The concentration was 1.15mM. H can be determined by comparing the color change of the hydrogen peroxide test paper with a standard color spectrogram 2 O 2 Is produced in the same way as the production amount of the catalyst.
2 2 Example 5: in situ HO generation technique combined with whole cell catalytic system
1mL of the reaction system: OD 9.0 HX ΔEG-F87A/T268V Whole cell catalyst, fluo7:100 μm, sodium ascorbate: 4mM, indan: 10mM; im-C6-Phe:0.5mM,25W white light, 4℃for 1h. After the completion of the reaction, 1mL of ethyl acetate was added to 1mL of the reaction system for extraction, the organic phase was filtered through a 0.22 μm filter, water was removed with anhydrous sodium sulfate, and 1mM benzophenone was added to the organic phase as an internal standard for gas phase analysis. The hydroxylation reaction formula of indane is as follows:
as shown in Table 7, it is evident from Table 7 that 1-indenol was produced when Im-C6-Phe was added, but 1-indenol was not produced when Im-C6-Phe was not added, and this also demonstrates the importance of the bifunctional small molecule Im-C6-Phe for activating the whole-cell P450 peroxygenase reaction activity. The generation of 1-indenol suggests that we have successfully achieved an intracellular P450 peroxygenase reaction using the constructed artificial photo-enzyme whole cell catalyst.
TABLE 7
2 2 Example 6: long-term reaction of in situ HO generation technique combined with whole cell catalytic system
1mL of the reaction system: OD 9.0 HX ΔEG-F87A/T268V Whole cell catalyst, fluo7:100 μm, sodium ascorbate: 4mM, indan: 10mM; im-C6-Phe:0.5mM,25W white light lamp, reaction at 4℃for 1, 2, 3h. After the completion of the reaction, 1mL of ethyl acetate was added to 1mL of the reaction system for extraction, the organic phase was filtered through a 0.22 μm filter membrane, water was removed by anhydrous sodium sulfate, 1mM benzophenone was added to the organic phase as an internal standard, and gas phase analysis was performed, and the reaction results are shown in Table 8. As shown in Table 8, the yield of 1-indenol increases with the increase of the reaction time, which indicates that the artificial photo-enzyme whole-cell catalyst constructed by us can continuously catalyze the hydroxylation of indane, and further demonstrates the stability of the artificial photo-enzyme whole-cell catalyst.
TABLE 8
2 2 Example 7: in situ HO generation technology combined with whole cell catalytic system with different cell densities
Photocatalytic reaction conditions: OD 10, 20, 30 HX ΔEG-F87A/T268V whole cell catalyst, fluo7:100 μm, sodium ascorbate: 4mM, indan: 10mM; im-C6-Phe:0.5mM,25W white light, 4℃for 1h. After the completion of the reaction, 1mL of ethyl acetate was added to 1mL of the reaction system for extraction, the organic phase was filtered through a 0.22 μm filter, water was removed with anhydrous sodium sulfate, and 1mM benzophenone was added to the organic phase as an internal standard for gas phase analysis. The results are shown in Table 9. As is clear from Table 9, the cell density affects the catalytic efficiency of the artificial photo-enzyme whole-cell catalyst, and as the cell density increases, the concentration of 1-indenol produced by the artificial photo-enzyme whole-cell catalyst increases.
TABLE 9
Example 8: artificial photo-enzyme whole cell catalyst for catalyzing other peroxidation reactions
Ethylbenzene hydroxylation: 1mL of the reaction system: OD 9.0 HX ΔEG-F87A/T268V Whole cell catalyst, fluo7:100 μm, sodium ascorbate: 4mM, ethylbenzene: 10mM; im-C6-Phe:0.5mM,25W white light, 4℃for 1h.
Epoxidation of styrene: 1mL of the reaction system: OD 8.0 HX Δeg-F87A whole cell catalyst, fluo7:100 μm, sodium ascorbate: 4mM, styrene: 4mM; im-C6-Phe:0.5mM,25W white light, 4℃for 1h.
After the completion of the reaction, 1mL of ethyl acetate was added to 1mL of the reaction system for extraction, the organic phase was filtered through a 0.22 μm filter, water was removed with anhydrous sodium sulfate, and 1mM benzophenone was added to the organic phase as an internal standard for gas phase analysis.
As can be seen from tables 10 and 11, the catalytic substrate of the artificial photo-enzyme whole-cell catalyst is not limited to indane, and can realize the effective catalysis in whole cells for ethylbenzene hydroxylation, styrene epoxidation and other reactions.
Table 10
TABLE 11
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Qingdao bioenergy and Process institute of China academy of sciences
<120> an artificial light enzyme whole cell biocatalyst, its preparation method and application
<130>
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Claims (10)
1. The artificial photo-enzyme whole-cell biocatalyst is characterized by comprising a photochemical in-situ hydrogen peroxide generation system and a whole-cell catalyst, wherein the photochemical in-situ hydrogen peroxide generation system comprises a photocatalyst and a reducing agent, and the whole-cell catalyst is a cell for realizing the P450 peroxygenase reaction in a whole cell by utilizing hydrogen peroxide under the assistance of a difunctional micromolecule;
the photocatalyst is fluorescein with the concentration of 4mM, the reducing agent is sodium ascorbate with the concentration of 100 mu M;
the difunctional small molecule compound is Im-C6-Phe, and the structural formula is shown as follows:
the whole cell catalyst is used for eliminating endogenous H 2 O 2 Coli BL21 of the scavenger genes katE and katG, wherein the escherichia coli BL21 expresses a mutant of the P450 enzyme BMP encoded by SEQ ID NO.1, and the mutant amino acid of the mutant is specifically F87A and T268V.
2. The artificial light enzyme whole cell biocatalyst of claim 1, wherein the cell is prepared by:
constructing an expression vector containing a target gene, and transforming the recombinant expression vector into a host to obtain a recombinant cell for expressing the P450 enzyme BMP mutant.
3. The method for preparing the artificial light enzyme whole cell biocatalyst according to any one of claims 1-2, comprising:
constructing a photochemical in-situ hydrogen peroxide generation system and preparing a whole-cell catalyst.
4. The preparation method of claim 3, wherein the specific method for constructing the photochemical in-situ hydrogen peroxide generation system is as follows: under the condition of illumination, the photocatalyst and the reducing agent are added into a buffer solution system to react.
5. The method of claim 4, wherein the photocatalyst is fluorescein and the reducing agent is sodium ascorbate.
6. The method of claim 5, wherein the buffer system is phosphate buffer in which the concentration of fluorescein is controlled to be 4mM; the concentration of sodium ascorbate was 100. Mu.M.
7. The method of claim 4, wherein the illumination is performed using a white light lamp, and the reaction is performed at a low temperature;
the specific method for preparing the whole-cell catalyst comprises the following steps: constructing an expression vector containing a target gene, and transforming the recombinant expression vector into a host to obtain a recombinant cell for expressing the P450 enzyme BMP mutant.
8. Use of the artificial light enzyme whole cell biocatalyst according to any one of claims 1-2 and/or the artificial light enzyme whole cell biocatalyst prepared by the method according to any one of claims 3-7 in any one or more of the following:
1) Catalyzing the reaction based on the whole cell internal peroxygenase;
2) And (3) preparing a medical intermediate.
9. The use according to claim 8, wherein the catalytic species comprises epoxidation of olefins of different substituents, sulfination of sulfides of different substituents, hydroxylation of aliphatic hydrocarbons, hydroxylation of carbon-hydrogen bonds on aromatic hydrocarbons, heteroaromatic compounds or hydroxylation on side-chain substituents;
the substrate comprises
10. A method of catalyzing a reaction based on a whole-cell peroxygenase reaction, the method comprising: adding a bifunctional small molecule and a substrate into the artificial light enzyme whole-cell biocatalyst prepared by any one of claims 1-2 and/or the artificial light enzyme whole-cell biocatalyst prepared by any one of claims 3-7, and reacting under the illumination condition.
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