Iron-containing sulfur cluster and efficient antenna coenzyme photorepair enzyme and preparation method thereof

Abstract

The application belongs to the field of enzymes, and discloses a preparation method of iron-containing sulfur cluster and high-efficiency antenna coenzyme photorepair enzyme, wherein the method is realized by constructing recombinant engineering bacteria for simultaneously expressing Synechococcus elongatus 6-4 photorepair enzyme SePhrB and Streptomyces coelicolor 8-hydroxy deazaflavin synthase ScFbiC, and by using low rotation speed to induce culture in an iron-containing salt culture medium, so as to realize the assembly of [4Fe-4S] iron sulfur cluster in the photorepair enzyme and high-efficiency antenna coenzyme 8-hydroxy deazaflavin, and to obtain the target protein through affinity chromatography and molecular sieve chromatography purification, wherein the obtained photorepair enzyme has the structure of simultaneously combining flavin adenine dinucleotide coenzyme, high-efficiency antenna coenzyme 8-hydroxy deazaflavin and [4Fe-4S] iron sulfur cluster, and has high 6-4 photoproduct repair activity.

Description

Technical Field

[0001] This invention belongs to the field of enzymes, specifically relating to a method for preparing a photorepair enzyme containing iron-sulfur clusters and a highly efficient antenna coenzyme. Background Technology

[0002] Ultraviolet (UV) radiation is a significant environmental stressor threatening the survival and evolution of life on Earth. When organisms are exposed to UV radiation, their genomic DNA forms two main damage products: cyclobutanepyrimidine dimer (CPD) and pyrimidine-pyrimidone 6-4 photoproduct (6-4PP, or simply 6-4 photoproduct). This damage hinders the normal functioning of DNA and RNA polymerases, interferes with DNA replication and transcription, disrupts the stability of genetic information transmission, and ultimately leads to cellular environmental disturbances. In mammals, this can ultimately result in abnormal skin function and even promote the development of skin cancer.

[0003] Photorepair enzymes are DNA repair proteins widely found in various organisms. Based on their substrates, they are mainly classified into CPD photorepair enzymes and 6-4 photorepair enzymes. Photorepair enzymes repair DNA damage caused by ultraviolet radiation by absorbing light energy. They function using flavin adenine dinucleotide (FAD) coenzymes, which exist in three redox forms: fully oxidized (OX); semiquinone (SQ) with one electron; and fully reduced (HQ) with two electrons. Photorepair enzymes rely on the two-electron reduced FAD coenzyme to perform their function. Some photorepair enzymes also bind secondary coenzymes such as 8-hydroxy-5-deazaflavin (8-HDF), which can absorb additional light energy and transfer energy to the two-electron reduced FAD, thereby enhancing the repair activity of the photorepair enzyme.

[0004] Cyanobacteria possess highly efficient photorepair capabilities, and *Synechococcus elongatus*, a representative species of cyanobacteria, has long been a model organism for photorepair research. Previous studies only identified one CPD photorepair enzyme (SePhrA) in *Synechococcus elongatus*, capable of repairing CPD damage. *Synechococcus elongatus* also contains a prokaryotic 6-4 photorepair enzyme (SePhrB) to repair 6-4 photoproducts. Research indicates that SePhrB contains FAD as a catalytic coenzyme, 8-HDF as an antenna coenzyme, and a [4Fe-4S] iron-sulfur cluster, which assists in enzyme catalysis. SePhrB exhibits extremely high catalytic repair efficiency for 6-4 photoproducts; its addition to cosmetic formulations can prevent pathological changes induced by ultraviolet radiation on human skin, protecting the body from sunburn, mutations, and skin cancer.

[0005] Currently, there are three major challenges in expressing the SePhrB protein: 1. The SePhrB gene originates from Synechococcus slenderus, and due to codon bias, its expression efficiency in the Escherichia coli (E. coli) expression system is difficult to achieve at its maximum; 2. The E. coli expression system lacks the 8-HDF synthase system, meaning the expressed SePhrB cannot bind 8-HDF, resulting in low activity; 3. Under high dissolved oxygen conditions, the stability of the iron-sulfur cluster is threatened, often disrupting the integrity of the SePhrB protein. Overcoming these technical challenges is of great significance for the practical application of SePhrB. Summary of the Invention

[0006] The purpose of this invention is to provide an iron-sulfur cluster-containing, high-efficiency antenna coenzyme photorepair enzyme and its preparation method.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme (SePhrB monomeric protein), the preparation method comprising the following steps:

[0009] 1) Inoculate the above-mentioned recombinant engineered bacteria into a culture medium containing iron salts;

[0010] 2) After the recombinant engineered bacteria grow to the logarithmic growth phase, IPTG is added to induce expression under low-speed stirring and cultured to obtain the culture;

[0011] 3) The culture was disrupted, and the supernatant was purified by Ni-IDA affinity chromatography, followed by molecular sieve chromatography to obtain the photorepair enzyme containing iron-sulfur clusters and high-efficiency antenna coenzyme.

[0012] In step 1), the iron salt is a divalent iron salt; the Fe of the iron salt... 2+The ion concentration is 0.4~0.6 mmol / L.

[0013] According to a specific embodiment of the present invention, preferably, the iron salt is one of ferrous ammonium sulfate or ferrous ammonium citrate.

[0014] According to a specific embodiment of the present invention, preferably, the logarithmic growth phase refers to OD. 600 It ranges from 0.6 to 0.8.

[0015] According to a specific embodiment of the present invention, preferably, in step 1), the culture medium contains 10 ± 0.2 g / L tryptone, 5 ± 0.1 g / L yeast extract and 10 ± 0.2 g / L sodium chloride.

[0016] According to a specific embodiment of the present invention, preferably, in step 1), the culture medium does not contain cysteine.

[0017] In step 2), the final concentration of IPTG is 0.4~0.6 mmol / L.

[0018] In step 2), the speed of the low-speed stirring is 90~110 rpm; the temperature during the cultivation process is 19.5~20.5℃, and the time is 19.5~20.5 h.

[0019] According to a specific embodiment of the present invention, preferably, in step 3), the molecular sieve is Superdex 200Increase 10 / 300 GL molecular sieve.

[0020] The recombinant engineered bacteria is Escherichia coli BL21(DE3) containing the recombinant expression plasmid.

[0021] The recombinant expression plasmid contains both the modified Synechococcus slenderus 6-4 photoreptolytic enzyme gene and the 8-HDF synthase gene, abbreviated as pCDFSePhrBScFbiC.

[0022] The recombinant expression plasmid was constructed using pCDFDuet-1 as a vector.

[0023] The modified Synechococcus slenderus 6-4 photorepair enzyme gene (SePhrB) has the nucleotide sequence shown in SEQ ID NO.1, including a codon-optimized Synechococcus slenderus 6-4 photorepair enzyme coding sequence and a 6×His tag coding sequence located at the 3' end.

[0024] The 8-HDF synthase gene (SePhrC) has the nucleotide sequence shown in SEQ ID NO.2 and is a codon-optimized 8-HDF synthase encoding gene from Streptomyces azureense.

[0025] This invention provides an iron-sulfur cluster and a high-efficiency antenna coenzyme photorepair enzyme prepared using the above preparation method. The iron-sulfur cluster and the high-efficiency antenna coenzyme photorepair enzyme simultaneously contain flavin adenine dinucleotide coenzyme, [4Fe-4S] iron-sulfur cluster and antenna coenzyme 8-hydroxydeoxyflavin (8-HDF).

[0026] The preparation method of this invention involves obtaining the prokaryotic 6-4 photorepair enzyme gene sequence from existing Synechococcus slenderus, performing codon optimization, and adding a 6 × His tag coding sequence to the 3' end to obtain the gene SePhrB with the SEQ ID NO.1 sequence; performing codon optimization on the 8-HDF synthase gene from Streptomyces azureus to obtain the gene SePhrC with the SEQ ID NO.2 sequence; and ligating the gene fragments SePhrB and SePhrC with the pCDFDuet-1 vector to obtain the recombinant expression plasmid pCDFSePhrBScFbiC.

[0027] pCDFSePhrBScFbiC was transformed into Escherichia coli BL21(DE3) competent cells to construct the recombinant engineered strain BL21(DE3) / pCDFSePhrBScFbiC. Under conditions of 19.5–20.5℃, 0.4–0.6 mmol / L IPTG, and 90–110 rpm, SePhrB and ScFbiC proteins could be expressed. The SePhrB monomer protein expressed under these conditions contained FAD coenzyme, [4Fe-4S] iron-sulfur cluster, and antenna coenzyme 8-hydroxydeoxyflavin (8-HDF).

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. Compared with the wild-type genes of *Synechococcus slenderus* and *Streptomyces azureus*, the modified SePhrB and SePhrC genes of this invention have higher expression efficiency in *Escherichia coli* after codon optimization, which is beneficial to improving protein yield.

[0030] 2. Compared with the method in Comparative Example 8, which uses two plasmids to co-express SePhrB and ScFbiC proteins to obtain SePhrB protein containing 8-HDF, this invention uses a recombinant expression plasmid (pCDFSePhrBScFbiC) to achieve the same purpose, and the operational efficiency is effectively improved.

[0031] 3. Regarding the optimization of culture conditions, this invention uses 90-110 rpm instead of the commonly used 200-250 rpm to culture the recombinant engineered bacteria BL21(DE3) / pCDFSePhrBScFbiC. At the same time, ferrous salts are added to the culture medium to supplement the iron source, which can achieve the optimal balance between the bacterial growth requirements for dissolved oxygen and the promotion of iron-sulfur cluster synthesis and maintenance of its stability, thereby realizing the expression of SePhrB monomer protein containing complete iron-sulfur clusters.

[0032] 4. The SePhrB monomeric protein produced by this invention contains complete iron-sulfur clusters and 8-HDF, and has a higher specific activity than the AfPhrB photorepair enzyme described in the comparative example, which is beneficial for its application in preventing ultraviolet damage and treating ultraviolet-induced diseases. Attached Figure Description

[0033] Figure 1 This is a map of the pCDFSePhrBScFbiC recombinant expression plasmid described in Example 2;

[0034] Figure 2 This is a colony PCR detection diagram of the recombinant engineered bacteria BL21(DE3) / pCDFSePhrBScFbiC in Example 3;

[0035] Figure 3 The graph shows the separation effect of the Superdex 200 Increase 10 / 300 GL molecular sieve column on the protein after crude purification of cultures at different rotation speeds of 50-200 rpm by Ni-IDA affinity chromatography column in Example 4 and Comparative Examples 1-3. The lower part of the horizontal axis represents the elution volume, and the upper part represents the number of fraction elution tubes.

[0036] Figure 4 The graph shows the separation effect of the Superdex 200 Increase 10 / 300 GL molecular sieve column on the proteins of different culture media after crude purification by Ni-IDA affinity chromatography column in Example 4 and Comparative Examples 4-6. The lower part of the horizontal axis represents the elution volume, and the upper part represents the number of fraction elution tubes.

[0037] Figure 5 This is an SDS-PAGE electrophoresis image of the protein purified by the Superdex 200 Increase 10 / 300 GL molecular sieve column in Example 4. Lane M: Standard protein marker; "7-9", "11-15": Fractional elution tube number, representing aggregate and monomer components, respectively.

[0038] Figure 6The image shows the spectral changes in the activity of the photorepair enzyme SePhrB monomer protein in Example 4. The absorption spectra of the repair system at mins 0, 1, 2, 4, and 8 are measured. The decrease in absorption at 325 nm and the increase in absorption at 265 nm indicate that the photoproducts 6-4 are repaired.

[0039] Figure 7 The graphs show the changes in absorbance at 325 nm for the SePhrB monomer at 13.5 mL elution time in Example 4, the SePhrB aggregate at 9 mL elution time, the SePhrB protein expressing 8-HDF-free protein in Comparative Example 7, and the AfPhrB protein expressing AfPhrB protein in Comparative Example 9 in repairing 6-4 photoproducts. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0042] The LB medium used in the following examples and comparative examples includes the following ingredients:

[0043] Tryptone 10 g / L;

[0044] Yeast extract 5 g / L;

[0045] Sodium chloride 10 g / L.

[0046] The Start buffer described in the following embodiments and comparative examples consists of the following components:

[0047] Tris-HCl 50 mM;

[0048] NaCl 500 mM;

[0049] Imidazole 50 mM;

[0050] 10 wt% glycerol

[0051] The remainder is water.

[0052] The Elution buffer consists of:

[0053] Tris-HCl 50 mM;

[0054] NaCl 500 mM;

[0055] Imidazole 500 mM;

[0056] 10 wt% glycerol

[0057] The remainder is water.

[0058] The protein buffer consists of:

[0059] Tris-HCl 50 mM;

[0060] NaCl 50 mM;

[0061] 10 wt% glycerol

[0062] The remainder is water.

[0063] Example 1

[0064] The method for obtaining the SePhrB and SePhrC genes specifically includes the following steps:

[0065] The gene sequence of the photorepair enzyme 6-4 from *Synechococcus slenderus* was searched in Genbank, optimized based on codon bias in *Escherichia coli*, and a nucleotide sequence encoding 6×His was added to the 3' end to artificially synthesize the SePhrB gene. The gene sequence of the 8-HDF synthase from *Streptomyces coli* was searched, optimized based on codon bias in *Escherichia coli*, and artificially synthesized the SePhrC gene.

[0066] The nucleotide sequence of the modified Synechococcus slenderus 6-4 photorepair enzyme gene (SePhrB) is shown below:

[0067]

[0068] The nucleotide sequence of the 8-HDF synthase gene (SePhrC) is shown below:

[0069]

[0070] Example 2

[0071] The method for constructing recombinant expression plasmids specifically includes the following steps:

[0072] The SePhrB and SePhrC genes were inserted into the latter part of the two T7 promoter sequences of the pCDFDuet-1 plasmid vector to obtain the recombinant expression plasmid pCDFSePhrBScFbiC, as shown in the diagram. Figure 1 As shown.

[0073] Example 3

[0074] The method for constructing recombinant engineered bacteria specifically includes the following steps:

[0075] The recombinant expression plasmid pCDFSePhrBScFbiC was transformed into Escherichia coli BL21(DE3) competent cells to obtain the recombinant engineered strain BL21(DE3) / pCDFSePhrBScFbiC.

[0076] Colony PCR detection of recombinant engineered bacteria BL21(DE3) / pCDFSePhrBScFbiC was performed using primers SePhrBF (5'-ATGACCGTTGGTATCTGGAT-3') and SePhrBR (5'-GTCCCAAGAAGCCTGCCA-3'), as well as ScFbiCF (5'-ATGACCACCTCTGCTACC-3') and ScFbiCR (5'-AGCAGCGTCCCAAGAAG-3'). The results are as follows: Figure 2 As shown, lane M is the DNA Marker; lanes 1-3 are three parallel PCR samples, in which 1500 bp and 3000 bp bands can be detected, which are the amplification products of the SePhrB and SePhrC genes, respectively.

[0077] Example 4

[0078] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0079] 1) Pick the recombinant engineered bacteria BL21(DE3) / pCDFSePhrBScFbiC, inoculate it into 5 mL of LB liquid medium containing 50 μg / mL streptomycin, incubate at 37℃ and 200 rpm overnight, and the next day inoculate the bacterial solution into 500 mL of medium containing 50 μg / mL streptomycin for further expansion culture;

[0080] 2) OD of the bacterial culture600 When the ratio was 0.7, 0.5 mmol / L IPTG was added to induce expression when the temperature was cooled from room temperature to 20℃. The induction temperature was 20℃, the culture speed was 100 rpm, and the induction time was 20 h. The bacterial cells were collected by centrifugation at 5000g and 4℃ for 500 mL of expanded culture.

[0081] 3) Add 50 mL of Start buffer to the bacterial cells from step 2), resuspend and mix well, and sonicate in an ice-water bath using an ultrasonic homogenizer. The sonication parameters are: 1 second of sonication followed by a 2-second pause as one cycle, for a total sonication time of 30 min. After homogenization, centrifuge at 4°C and 10000 g for 10 min, collect the supernatant, take the Ni-IDA affinity chromatography column, equilibrate with Start buffer, add the supernatant, mix thoroughly, and place on a horizontal shaker for 30 min to ensure complete binding of the protein with the Ni-IDA medium. After binding, discard the permeate. First, wash the Ni-IDA affinity chromatography column with Start buffer to remove impurities. Then, elute with Elution buffer to remove the target protein and collect the eluent. Use an Amicon Ultra-15 concentration tube with a molecular weight cutoff of 30 kDa to concentrate the eluent to obtain concentrated protein, and then use Superdex 200 Increase 10 / 300. The GL molecular sieve column was used for further purification, and fractions containing the target protein were collected. The product SePhrB protein included SePhrB monomeric protein and SePhrB aggregate protein. The monomeric protein with an elution volume of 13.5 mL contained iron-sulfur clusters and highly efficient antenna coenzyme photorepair enzyme.

[0082] Step 1) The culture medium used for the scale-up culture includes the following raw materials:

[0083] Tryptone 10 g / L;

[0084] Yeast extract 5 g / L;

[0085] Sodium chloride 10 g / L;

[0086] Ferrous ammonium sulfate hexahydrate 0.2 g / L.

[0087] Example 5

[0088] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0089] The only difference between this embodiment and embodiment 4 is that the cultivation speed of 100 rpm in step 2) is replaced with 90 rpm, and the rest is the same as in embodiment 4.

[0090] Example 6

[0091] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0092] The only difference between this embodiment and embodiment 4 is that the cultivation rotation speed of 100 rpm in step 2) is replaced with 110 rpm, and the rest is the same as in embodiment 4.

[0093] Example 7

[0094] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0095] The only difference between this embodiment and Embodiment 4 is that the culture medium used in step 1) for the expanded culture is replaced with:

[0096] Step 1) The culture medium used for the scale-up culture includes the following raw materials:

[0097] Tryptone 10 g / L;

[0098] Yeast extract 5 g / L;

[0099] Sodium chloride 10 g / L;

[0100] 0.13 g / L of divalent ferric ammonium citrate;

[0101] Comparative Example 1

[0102] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0103] The only difference between this comparative example and Example 4 is that the culture rotation speed of 100 rpm in step 2) is replaced with 50 rpm, and the rest is the same as Example 4.

[0104] Comparative Example 2

[0105] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0106] The only difference between this comparative example and Example 4 is that the culture rotation speed in step 2) is replaced with 150 rpm instead of 100 rpm; otherwise, it is the same as Example 4.

[0107] Comparative Example 3

[0108] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0109] The only difference between this comparative example and Example 4 is that the culture rotation speed of 100 rpm in step 2) is replaced with 200 rpm, and the rest is the same as Example 4.

[0110] Comparative Example 4

[0111] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0112] The only difference between this comparative example and Example 4 is that the culture medium used for the expansion culture in step 1) is replaced with LB medium.

[0113] Comparative Example 5

[0114] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0115] The only difference between this comparative example and Example 4 is that the culture medium used in step 1) for the expansion culture is replaced with:

[0116] Step 1) The culture medium used for the scale-up culture includes the following raw materials:

[0117] Tryptone 10 g / L;

[0118] Yeast extract 5 g / L;

[0119] Sodium chloride 10 g / L;

[0120] Cysteine ​​0.06 g / L.

[0121] Comparative Example 6

[0122] A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme specifically includes the following steps:

[0123] The only difference between this comparative example and Example 4 is that the culture medium used in step 1) for the expansion culture is replaced with:

[0124] Step 1) The culture medium used for the scale-up culture includes the following raw materials:

[0125] Tryptone 10 g / L;

[0126] Yeast extract 5 g / L;

[0127] Sodium chloride 10 g / L;

[0128] Ferrous ammonium sulfate hexahydrate 0.2 g / L;

[0129] Cysteine ​​0.06 g / L.

[0130] Comparative Example 7

[0131] This comparative example is the SePhrB protein expressed alone in Escherichia coli.

[0132] Comparative Example 8

[0133] This comparative example uses the dual plasmid expression system described in the literature (Chen, Nucleic Acids Research, 2022) to co-express SePhrB and ScFbiC proteins in Escherichia coli, and the SePhrB protein is purified by chromatography.

[0134] Comparative Example 9

[0135] This comparative example uses the 6-4 photorepair enzyme AfPhrB derived from Agrobacterium fabrum.

[0136] Test Example 1

[0137] Composition analysis:

[0138] The purified SePhrB protein was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aggregate and monomer components purified by molecular sieve in Example 4 were denatured and then loaded for detection. The protein molecular weight was calibrated by protein molecular weight standards. At the same time, the coenzyme and iron-sulfur cluster composition of the separated aggregate and monomer components were analyzed to detect the presence of FAD coenzyme, antenna coenzyme 8-HDF and [4Fe-4S] iron-sulfur cluster in the protein.

[0139] Test results as follows Figure 5 As shown, both the aggregate and monomer components exhibit single and clear protein bands with an apparent molecular weight of approximately 50 kDa, which is consistent with the theoretical molecular weight of SePhrB protein, indicating that the obtained proteins are all SePhrB proteins.

[0140] The results showed that the monomeric component contained FAD coenzyme, 8-HDF, and the [4Fe-4S] iron-sulfur cluster, indicating that the SePhrB protein in this component had completed cofactor assembly and formed a structurally complete active protein. In contrast, only iron and sulfur were detected in the aggregate component, and FAD coenzyme and the highly efficient antenna coenzyme 8-HDF were not detected, indicating that the SePhrB protein in the aggregate component had not completed cofactor assembly and belonged to an inactive aggregate state.

[0141] Test Example 2

[0142] In vitro photorepair activity test:

[0143] The repair activity of SePhrB protein on the 6-4 photoproduct was evaluated using a UV-induced oligonucleotide damage model. UV irradiation of DNA causes adjacent thymine groups to polymerize, forming the 6-4 photoproduct, which exhibits a characteristic absorption peak at 325 nm. Under visible light irradiation, the 6-4 photorepair enzyme catalyzes the repair of the 6-4 photoproduct, causing a gradual decrease in absorbance at 325 nm and a gradual increase in absorbance at 265 nm. Therefore, the repair activity of the photorepair enzyme can be evaluated by monitoring changes in absorbance at 325 nm.

[0144] At room temperature, an oligonucleotide solution containing 16 thymine bases was irradiated with a 254 nm ultraviolet lamp at a distance of 10 cm for 20 min to obtain an oligonucleotide solution containing 6-4 photoproducts.

[0145] A photorepair reaction system was prepared, containing 10 μmol / L of the UV-damaged oligonucleotide, 0.1 μmol / L of the SePhrB monomer protein described in Example 4, 1 mmol / L of dithiothreitol, and 4 mmol / L of magnesium chloride, and brought to a final volume of 600 μL using Protein Buffer. The reaction system was placed in a quartz cuvette and irradiated with a light-emitting diode light source with an emission wavelength of 417 nm at a distance of 5 cm. Changes in absorbance around 325 nm were measured periodically during the irradiation process to evaluate the photorepair enzyme's ability to repair the 6-4 photoproducts.

[0146] Test results are as follows Figure 6 As shown, the SePhrB monomeric protein described in Example 4 can effectively catalyze the repair of 6-4 photoproducts. With prolonged illumination time, the characteristic absorption peak at 325 nm continuously decreased, while the absorption peak at 265 nm gradually increased. The curves correspond to the absorption spectra at illumination times of 0 min, 1 min, 2 min, 4 min, and 8 min, respectively, indicating that 6-4 photoproducts are continuously repaired.

[0147] Further comparisons were made of the photorepair activity of SePhrB monomeric protein, SePhrB aggregate protein, SePhrB protein expressing 8-HDF in Comparative Example 7, and AfPhrB protein expressing in Comparative Example 9. The results are as follows: Figure 7 As shown in Table 1, the absorbance of SePhrB monomeric protein decreased significantly faster at 325 nm than that of AfPhrB and SePhrB protein without 8-HDF, indicating that it has a higher repair efficiency for 6-4 photoproducts. In contrast, the absorbance of SePhrB aggregate protein changed very little at 325 nm during the test, showing almost no photorepair activity.

[0148] Based on the results of test example 1, it can be seen that the SePhrB monomer protein contains FAD coenzyme, 8-HDF and [4Fe-4S] iron-sulfur cluster, forming a structurally complete active photorepair enzyme; while the SePhrB aggregate protein lacks FAD coenzyme and 8-HDF, resulting in its near loss of photorepair activity.

[0149] Table 1

[0150]

[0151] Table 1 shows that in Comparative Example 1, adjusting the culture speed to 50 rpm significantly reduced the SePhrB monomeric protein yield to only 2.1 mg / L, indicating that excessively low culture speeds are detrimental to the expression of SePhrB monomeric proteins. In Comparative Example 2, adjusting the culture speed to 150 rpm reduced the SePhrB monomeric protein yield to 3.5 mg / L, while increasing the aggregate protein yield, indicating that higher culture speeds are not conducive to the formation of SePhrB monomeric proteins. In Comparative Example 3, adjusting the culture speed to 200 rpm further reduced the SePhrB monomeric protein yield, while further increasing the aggregate protein yield, indicating that excessively high culture speeds tend to promote protein aggregation, which is not conducive to obtaining SePhrB active monomers. In Example 4, the SePhrB monomeric protein yield obtained at 100 rpm was 16.1 mg / L, which was approximately 7.7 times, 4.6 times, and 8.9 times that of Comparative Examples 1, 2, and 3, respectively, indicating that a culture speed of 100 rpm can significantly improve the efficiency of SePhrB monomeric protein acquisition.

[0152] When LB medium was used for culture in Comparative Example 4, the yield of SePhrB monomeric protein was lower than that in Example 4, indicating that supplementing the medium with an iron source is beneficial to improving the yield of SePhrB monomeric protein.

[0153] When the culture medium with added cysteine ​​was used in Comparative Example 5, the yield of SePhrB monomeric protein was further reduced, indicating that supplementing cysteine ​​alone is not effective in improving the expression level of SePhrB monomeric protein.

[0154] When Comparative Example 6 was cultured in a medium containing both ferrous ammonium sulfate hexahydrate and cysteine, the yield of SePhrB monomeric protein was slightly lower than that in Example 4. This indicates that under the conditions of the present invention, a high yield of SePhrB monomeric protein can be obtained by simply supplementing an appropriate amount of iron source, and further addition of cysteine ​​did not bring about a significant improvement.

[0155] In Comparative Example 7, when only SePhrB protein was expressed, 8-HDF coenzyme was not detected, and the 6-4 photoproduct conversion number was only 0.03 s. -1The results were significantly lower than in Example 4, indicating that ScFbiC co-expression can effectively promote the formation of 8-HDF coenzyme, thereby significantly improving the activity of photorepair enzyme.

[0156] When the SePhrB protein was prepared using the dual plasmid co-expression system reported in the literature in Comparative Example 8, its monomeric protein yield was lower than that in Example 4, and the 8-HDF ratio was also slightly lower than that in Example 4. This indicates that the present invention uses a single plasmid co-expression system, which improves the yield of SePhrB monomeric protein while maintaining the assembly level and catalytic activity of photorepair enzyme cofactors.

[0157] When Comparative Example 9 used AfPhrB photorepair enzyme derived from Agrobacterium, its 6-4 photoproduct switching number was only 0.05 s. -1 The results were significantly lower than those in Example 4, indicating that the SePhrB photorepair enzyme prepared in this invention has higher photorepair activity.

[0158] In summary, the results show that by constructing a co-expression system of SePhrB and ScFbiC single plasmids and combining it with a culture speed of 90-110 rpm and an iron-containing medium, this invention can effectively improve the yield of SePhrB active monomer protein while maintaining a high level of 8-HDF coenzyme and [4Fe-4S] iron-sulfur cluster assembly, thus obtaining a SePhrB photorepair enzyme with high photorepair activity.

[0159] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0160] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing an iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme, characterized in that, The preparation method includes the following steps: 1) Inoculate the recombinant engineered bacteria into a culture medium containing iron salts; 2) After the recombinant engineered bacteria grow to the logarithmic growth phase, IPTG is added to induce expression under low-speed stirring and cultured to obtain the culture; 3) The culture was disrupted, and the supernatant was purified by Ni-IDA affinity chromatography, followed by molecular sieve chromatography to obtain the photorepair enzyme containing iron-sulfur clusters and high-efficiency antenna coenzyme.

2. The preparation method according to claim 1, characterized in that, In step 1), the iron salt is a divalent iron salt; the Fe of the iron salt... 2+ The ion concentration is 0.4~0.6 mmol / L.

3. The preparation method according to claim 1, characterized in that, In step 2), the final concentration of IPTG is 0.4~0.6 mmol / L.

4. The preparation method according to claim 1, characterized in that, In step 2), the speed of the low-speed stirring is 90~110 rpm; the temperature during the cultivation process is 19.5~20.5℃, and the time is 19.5~20.5 h.

5. The preparation method according to claim 1, characterized in that, The recombinant engineered bacteria is Escherichia coli BL21(DE3) containing the recombinant expression plasmid.

6. The preparation method according to claim 5, characterized in that, The recombinant expression plasmid contains both the modified Synechococcus slenderus 6-4 photorepair enzyme gene and the 8-HDF synthase gene.

7. The preparation method according to claim 6, characterized in that, The recombinant expression plasmid was constructed using pCDFDuet-1 as a vector.

8. The preparation method according to claim 6, characterized in that, The modified Synechococcus slenderus 6-4 photorepair enzyme gene has the nucleotide sequence shown in SEQ ID NO.1, including a codon-optimized Synechococcus slenderus 6-4 photorepair enzyme coding sequence and a 6×His tag coding sequence located at the 3' end.

9. The preparation method according to claim 6, characterized in that, The 8-HDF synthase gene has the nucleotide sequence shown in SEQ ID NO.2 and is a codon-optimized gene encoding the 8-HDF synthase from Streptomyces azureense.

10. An iron-sulfur cluster and a highly efficient antenna coenzyme photorepair enzyme prepared by the preparation method according to any one of claims 1-9, characterized in that, The iron-sulfur cluster and the highly efficient antenna coenzyme photorepair enzyme simultaneously contain flavin adenine dinucleotide coenzyme, [4Fe-4S] iron-sulfur cluster and antenna coenzyme 8-hydroxydeoxyflavin.

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