P450 enzyme, enzyme composition and application thereof

Abstract

The invention discloses a P450 enzyme, an enzyme composition and application thereof. The amino acid sequence of the P450 enzyme is as shown in SEQ ID NO: 4. According to the P450 enzyme or enzyme composition provided by the invention, 7-dehydrocholesterol is used as a substrate to synthesize an intermediate 25-hydroxy-7-dehydrocholesterol of 25-hydroxyvitamin D3, and then the 25-hydroxyvitamin D3 is generated through illumination. The 7-dehydrocholesterol is low in price, so that the cost can be effectively reduced. Moreover, by further optimizing reaction conditions, when the concentration of 7-dehydrocholesterol is 13 g / L, the conversion rate can reach 95.5%. The P450 enzyme or enzyme composition provided by the invention has a wide application prospect in the technical field of enzyme catalysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme catalysis, and specifically relates to a P450 enzyme, an enzyme composition and their applications. Background Art

[0002] Vitamin D3 is a provitamin, which plays an important role in maintaining human health, cell growth and development. When the skin is exposed to sunlight, ultraviolet rays irradiate 7-dehydrocholesterol in the skin, and through a series of complex reactions, it can be converted into vitamin D3. 25-Hydroxyvitamin D3 is a derivative of vitamin D3 and is the main form of vitamin D3 in the body. It has very important significance for human health and has a very positive therapeutic effect on certain diseases such as osteoporosis, osteomalacia, hyperglycemia, chronic kidney disease, etc. In addition, in the field of animal nutrition, 25-hydroxyvitamin D3 has a higher absorption rate than vitamin D3 and is less affected by liver and intestinal health. Its biological potency is 3-5 times that of ordinary vitamin D3, and it can quickly improve the calcium and phosphorus absorption status of livestock and poultry. Therefore, it is widely used in the feed additive industry.

[0003] With the cross-integration of multiple disciplines such as gene mining, bioinformatics, and enzyme engineering, the green enzymatic synthesis strategy has gradually developed into a powerful tool for synthesizing bioactive substances, drug molecules and other valuable organic molecules. The simplest and most direct way to synthesize 25-hydroxyvitamin D3 is to hydroxylate vitamin D3 at C25. The most popular P450 enzyme for the biosynthesis of 25-hydroxyvitamin D3. CN 116790527 B uses a dual-enzyme mixture of a self-consistent P450 enzyme and glucose dehydrogenase for catalysis, and finally produces 5.09 g / L of 25-hydroxyvitamin D3. CN 116240246 A uses a non-specific peroxidase from Coprinopsis cinerea okayama7#130 to catalyze 5 g / L of substrate, and the conversion rate can reach more than 80% within 3 h. However, the substrates used in the above methods are all vitamin D3, resulting in a relatively high cost for the conversion.

[0004] 7-Dehydrocholesterol, an important raw material for 25-hydroxyvitamin D3, is inexpensive. Synthesizing 25-hydroxy-7-dehydrocholesterol using this as a substrate and then generating 25-hydroxyvitamin D3 by light can effectively reduce the cost. However, among the currently studied P450 enzymes, few use 7-dehydrocholesterol as a substrate to synthesize 25-hydroxyvitamin D3, and the catalytic selectivity and conversion rate are unknown. Therefore, exploring a P450 enzyme that can efficiently catalyze the formation of 25-hydroxy-7-dehydrocholesterol from 7-dehydrocholesterol plays an important role in the low-cost synthesis of 25-hydroxyvitamin D3. Summary of the Invention

[0005] To solve the problem that the substrate of biocatalytic method in the prior art is generally vitamin D3, and the low conversion rate increases the cost of large-scale production of 25-hydroxyvitamin D3, the present invention provides an application of P450 enzyme in catalyzing 7-dehydrocholesterol. The present invention synthesizes 25-hydroxy-7-dehydrocholesterol from 7-dehydrocholesterol and then regenerates 25-hydroxyvitamin D3, which can effectively reduce the cost. Moreover, the present invention realizes a high conversion rate by further optimizing the reaction conditions.

[0006] The present invention solves the above technical problems through the following technical solutions:

[0007] In the first aspect of the present invention, a P450 enzyme is provided, and the amino acid sequence of the P450 enzyme is as shown in SEQ ID NO: 4.

[0008] In the second aspect of the present invention, an isolated nucleic acid is provided, and the nucleic acid encodes the P450 enzyme described in the first aspect.

[0009] In some embodiments of the present invention, the nucleic acid comprises the nucleotide sequence as shown in SEQ ID NO: 8.

[0010] In the third aspect of the present invention, a recombinant expression vector is provided, and the recombinant expression vector comprises the nucleic acid described in the second aspect.

[0011] In the fourth aspect of the present invention, a transformant is provided, and the transformant comprises the nucleic acid described in the second aspect, or the recombinant expression vector described in the third aspect; the transformant is not an animal breed or a plant breed.

[0012] In some embodiments of the present invention, the transformant is a prokaryotic cell.

[0013] In some preferred embodiments of the present invention, the prokaryotic cell is Bacillus subtilis or Escherichia coli.

[0014] In the fifth aspect of the present invention, a method for preparing P450 enzyme is provided, and the method comprises the steps of culturing the transformant described in the fourth aspect and obtaining the P450 enzyme from the culture.

[0015] In the sixth aspect of the present invention, a method for preparing 25-hydroxy-7-dehydrocholesterol is provided, and the method comprises catalyzing the conversion of a substrate in a reaction system containing the P450 enzyme described in the first aspect, and the substrate is 7-dehydrocholesterol.

[0016] In some embodiments of the present invention, the reaction system further comprises a co-solvent, an electron transfer chain protein and / or a coenzyme and its regeneration system.

[0017] In some preferred embodiments of the present invention, the co-solvent is 2-hydroxypropyl-β-cyclodextrin.

[0018] In some preferred embodiments of the present invention, the electron transfer chain protein is ferredoxin reductase and / or ferredoxin.

[0019] In some preferred embodiments of the present invention, the coenzyme is NADPH.

[0020] In some preferred embodiments of the present invention, the coenzyme regeneration system comprises glucose and glucose dehydrogenase.

[0021] In some embodiments of the present invention, the use form of the P450 enzyme is selected from pure enzyme, crude enzyme solution, enzyme solution, fermentation broth, enzyme powder and immobilized enzyme.

[0022] In the present invention, the pure enzyme refers to a single enzyme protein obtained by multi-step separation and purification techniques (such as chromatography, electrophoresis), and the purity is usually >95% (showing a single main band in electrophoresis). The crude enzyme solution refers to an enzyme solution obtained by preliminary purification (such as centrifugation, salting out) after cell disruption, containing a large amount of miscellaneous proteins and cell debris, and the purity is <60%. The enzyme solution refers to a purified enzyme solution (such as purified by chromatography), and the purity is between the crude enzyme solution and the pure enzyme. The fermentation broth refers to a mixed solution after microbial fermentation culture, containing bacteria, metabolites (such as enzymes and organic acids), culture medium residues and by-products. The enzyme powder refers to a solid powder obtained by converting an enzyme-containing solution through drying techniques (such as freeze drying or spray drying). The immobilized enzyme refers to an enzyme-carrier complex formed by immobilizing the enzyme on a carrier (such as agarose microspheres and nanomaterials) through physical adsorption, covalent binding or entrapment method, which can be reused.

[0023] In some embodiments of the present invention, when the use form of the P450 enzyme is enzyme solution, the reaction system contains 2-5 μM P450 enzyme, 5-15 g / L 7-dehydrocholesterol, 100-150 g / L 2-hydroxypropyl-β-cyclodextrin, 1-5 mM NADPH, 10-20 wt% glucose, 15-30 U / mL glucose dehydrogenase, 5-15 μM ferredoxin reductase and / or 20-60 μM ferredoxin.

[0024] In some specific embodiments of the present invention, when the use form of the P450 enzyme is enzyme solution, the reaction system contains 4 μM P450 enzyme, 10 g / L 7-dehydrocholesterol, 125 g / L 2-hydroxypropyl-β-cyclodextrin, 2 mM NADPH, 15 wt% glucose, 20 U / mL glucose dehydrogenase, 5 μM ferredoxin reductase and / or 20 μM ferredoxin.

[0025] In some specific embodiments of the present invention, when the form of the P450 enzyme used is an enzyme solution, the reaction system contains 4 μM P450 enzyme, 13 g / L 7-dehydrocholesterol, 125 g / L 2-hydroxypropyl-β-cyclodextrin, 2 mM NADPH, 15 wt% glucose, 20 U / mL glucose dehydrogenase, 5 μM ferredoxin reductase, and / or 20 μM ferredoxin.

[0026] In some embodiments of the present invention, the reaction conditions catalyzed in the method are: pH is 6 - 8, and the temperature is 30 - 40 °C.

[0027] In some specific embodiments of the present invention, the reaction conditions catalyzed in the method are: pH is 7, and the temperature is 35 °C.

[0028] In some embodiments of the present invention, 7-dehydrocholesterol is added in batches in equal amounts.

[0029] In some specific embodiments of the present invention, 7-dehydrocholesterol is added in two equal amounts, at 0 h and 3 h of the reaction respectively.

[0030] The seventh aspect of the present invention provides an enzyme composition, which comprises one or more P450 enzymes as described in the first aspect.

[0031] In some embodiments of the present invention, the enzyme composition further comprises glucose dehydrogenase and / or ferredoxin reductase.

[0032] The eighth aspect of the present invention provides an application of the P450 enzyme as described in the first aspect or the enzyme composition as described in the seventh aspect in the preparation of 25-hydroxy-7-dehydrocholesterol.

[0033] The present invention screens out the P450 enzyme and its dominant mutants that can efficiently catalyze 7-dehydrocholesterol through the exploration of enzyme resources and protein structure analysis. Through the expression, mutation and transformation of the enzyme, and the establishment and optimization of the catalytic reaction system, a P450 enzyme with high catalytic activity is obtained.

[0034] The technical solution is elaborated in four points:

[0035] 1. Exploration of P450

[0036] The inventor selected the P450 enzyme from Fictibacillus macauensis through literature research, NCBI enzyme gene mining and multiple sequence alignment.

[0037] Through structural simulation and molecular docking, the hot-spot amino acids in the active pocket and some amino acid sites that may have interactions were determined. Finally, three mutants, D125L, F256P, and R288A, which may improve its selectivity and catalytic activity, were screened out.

[0038] 2. Construction and expression of P450 and mutant vectors

[0039] The wild-type P450 sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized CYP gene was ligated between the BamH I and Hind III restriction sites of pRSFDuet-FDR_FDI, and the ligated vector was transformed into the host E. coli BL21(DE3) competent cells to obtain an engineering strain containing the CYP gene. Using the plasmid with the wild-type sequence as a template, recombinant vectors containing the P450 mutant sequences of D125L, F256P, and R288A were constructed and transformed into the host E. coli BL21(DE3) competent cells to construct engineering strains containing different mutants.

[0040] 3. P450 enzyme-catalyzed reaction (catalytic synthesis of 25-hydroxy-7-dehydrocholesterol from 7-dehydrocholesterol)

[0041] The cells expressing the wild-type and mutant P450 enzymes were lysed to obtain the cell lysate supernatant, which was mixed with the substrate 7-dehydrocholesterol, glucose, and glucose dehydrogenase, and the catalytic reaction was carried out under the same reaction conditions. After the reaction, HPLC was used for detection and analysis of the conversion rate, the yield and selectivity of 25-hydroxy-7-dehydrocholesterol, and the catalytic activities of different mutants were compared.

[0042] 4. Optimization of P450 enzyme-catalyzed reaction

[0043] The dominant mutants were optimized for pH, temperature, cosolvent, and the addition method of the substrate to obtain the optimal catalytic conditions. Finally, under the condition of 13 g / L of the substrate, the conversion rate reached 95.5%.

[0044] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain the preferred embodiments of the present invention.

[0045] The reagents and raw materials used in the present invention are all commercially available.

[0046] The positive and progressive effects of the present invention are as follows: The P450 enzyme provided by the present invention uses 7-dehydrocholesterol as a substrate to synthesize 25-hydroxy-7-dehydrocholesterol, an intermediate of 25-hydroxyvitamin D3, and then generates 25-hydroxyvitamin D3 through light irradiation. 7-dehydrocholesterol is inexpensive, which can effectively reduce costs. Moreover, by further optimizing the reaction conditions, when the concentration of 7-dehydrocholesterol is 13 g / L, the conversion rate can reach 95.5%. Detailed implementation mode

[0047] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0048] Example 1 Mining and modification of P450 enzyme

[0049] Through literature research, NCBI enzyme gene mining and multiple sequence alignment, the inventors selected a P450 enzyme that may use 7-dehydrocholesterol as a substrate to synthesize 25-hydroxy-7-dehydrocholesterol, which is derived from Fictibacillus macauensis and named WT. There may be problems such as low catalytic efficiency in the wild-type P450 enzyme. Therefore, based on structural simulation and molecular docking, the inventors screened the hot spot amino acids in the active pocket and some amino acids close to the active site in terms of spatial distance. Finally, three mutants that may improve its selectivity and catalytic activity were selected: D125L, F256P, and R288A, which were named P1, P2, and P3 respectively. The amino acid sequences of the wild type and the three mutants are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 respectively, and the sequences are shown in Table 1.

[0050] Table 1 Amino acid sequences of P450

[0051]

[0052] The nucleotide sequences of the P450 enzymes with amino acid sequences as shown in SEQ ID NO: 1-4 are shown in Table 2.

[0053] Table 2 Nucleotide sequences of P450

[0054]

[0055]

[0056] Example 2 Construction of wild-type P450 and its mutant engineering bacteria

[0057] 2.1 Construction of wild-type P450 and its mutant vectors

[0058] The wild-type P450 sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized CYP genes were respectively ligated between the BamH I and Hind III restriction enzyme sites of pRSFDuet-FDR_FDI. pRSFDuet-FDR_FDI also contains the genes required for the electron transfer chain of the P450 enzyme: coenzyme FDR (ferredoxin reductase) and FDI (ferredoxin). The preparation method of pRSFDuet-FDR_FDI is as follows: The nucleotide sequences of codon-optimized FDR and FDI were sent to Shanghai Sangon Biotech Co., Ltd. for synthesis. Then, FDR was ligated at the EcoR I and Nco I sites of the commercial plasmid pRSFDuet through a seamless cloning kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to obtain the pRSFDuet-FDR recombinant vector. Then, in the same way, FDI was cloned between the Nde I and Xho I restriction enzyme sites of pRSFDuet-FDR to obtain the pRSFDuet-FDR_FDI recombinant vector.

[0059] FDR amino acid sequence:

[0060] MQIASDVEAPPPAPAKVEKHSKKMEEGITVNKFKPKTPYVGRCLLNTKITGDDAPGETWHMVFSHEGEIPYREGQSVGVIPDGEDKNGKPHKLRLYSIASSALGDFGDAKSVSLCVKRLIYTNDAGETIKGVCSNFLCDLKPGAEVKLTGPVGKEMLMPKDPNATIIMLGTGTGIAPFRSFLWKMFFEKHDDYKFNGLAWLFLGVPTSSSLLYKEEFEKMKEKAPDNFRLDFAVSREQTNEKGEKMYIQTRMAQYAVELWEMLKKDNTYFYMCGLKGMEKGIDDIMVSLAAAEGIDWIEYKRQLKKAEQWNVAVY (SEQ ID NO: 9)

[0061] FDR nucleotide sequence:

[0062] ATGCAGATTGCGAGCGATGTGGAAGCGCCGCCGCCGGCGCCGGCGAAAGTGGAAAAACATAGCAAAAAAATGGAAGAAGGCATTACCGTGAACAAATTTAAACCGAAAACCCCGTATGTGGGCCGCTGCCTGCTGAACACCAAAATTACCGGCGATGATGCGCCGGGCGAAACCTGGCATATGGTGTTTAGCCATGAAGGCGAAATTCCGTATCGCGAAGGCCAGAGCGTGGGCGTGATTCCGGATGGCGAAGATAAAAACGGCAAACCGCATAAACTGCGCCTGTATAGCATTGCGAGCAGCGCGCTGGGCGATTTTGGCGATGCGAAAAGCGTGAGCCTGTGCGTGAAACGCCTGATTTATACCAACGATGCGGGCGAAACCATTAAAGGCGTGTGCAGCAACTTTCTGTGCGATCTGAAACCGGGCGCGGAAGTGAAACTGACCGGCCCGGTGGGCAAAGAAATGCTGATGCCGAAAGATCCGAACGCGACCATTATTATGCTGGGCACCGGCACCGGCATTGCGCCGTTTCGCAGCTTTCTGTGGAAAATGTTTTTTGAAAAACATGATGATTATAAATTTAACGGCCTGGCGTGGCTGTTTCTGGGCGTGCCGACCAGCAGCAGCCTGCTGTATAAAGAAGAATTTGAAAAAATGAAAGAAAAAGCGCCGGATAACTTTCGCCTGGATTTTGCGGTGAGCCGCGAACAGACCAACGAAAAAGGCGAAAAAATGTATATTCAGACCCGCATGGCGCAGTATGCGGTGGAACTGTGGGAAATGCTGAAAAAAGATAACACCTATTTTTATATGTGCGGCCTGAAAGGCATGGAAAAAGGCATTGATGATATTATGGTGAGCCTGGCGGCGGCGGAAGGCATTGATTGGATTGAATATAAACGCCAGCTGAAAAAAGCGGAACAGTGGAACGTGGCGGTGTAT (SEQ ID NO: 10)

[0063] FDI amino acid sequence:

[0064] MAAYKVTLVTPTGNVEFQCPDDVYILDAAEEEGIDLPYSCRAGSCSSCAGKLKTGSLNQDDQSFLDDDQIDEGWVLTCAAYPVSDVTIETHKKEELTA (SEQ ID NO: 11)

[0065] FDI nucleotide sequence:

[0066] ATGGCGGCGTATAAAGTGACCCTGGTGACCCCGACCGGCAACGTGGAATTTCAGTGCCCGGATGATGTGTATATTCTGGATGCGGCGGAAGAAGAAGGCATTGATCTGCCGTATAGCTGCCGCGCGGGCAGCTGCAGCAGCTGCGCGGGCAAACTGAAAACCGGCAGCCTGAACCAGGATGATCAGAGCTTTCTGGATGATGATCAGATTGATGAAGGCTGGGTGCTGACCTGCGCGGCGTATCCGGTGAGCGATGTGACCATTGAAACCCATAAAAAAGAAGAACTGACCGCG (SEQ ID NO: 12)

[0067] To construct the expression vectors of P1, P2 or P3, using the plasmid with wild-type P450 sequence as a template, primers were designed at the mutation sites for inverse PCR. The amplified PCR products were digested with Dpn I overnight and inactivated at 80 °C for 20 min, then transferred into E. coli DH5α competent cells. Recovery solution was added and cultured for 45 min, then spread on LB solid medium containing 100 μg / mL kanamycin and cultured overnight at 37 °C in an inverted position. Single colonies were picked and sent to the company for sequencing. The sequencing results were analyzed with SnapGene software to determine the correct mutation of amino acids at the mutation sites, and thus the recombinant vectors containing the mutated sites of P450 enzyme were obtained.

[0068] 2.2 Construction and expression of wild-type P450 and its mutant expression host bacteria

[0069] To obtain engineering bacteria expressing P450, its mutants and electron transfer chain genes, 4 recombinant plasmids were transformed into BL21(DE3) competent cells. Recovery solution was added and cultured for 45 min, then spread on LB solid medium containing 100 μg / mL kanamycin and cultured overnight at 37 °C in an inverted position. The growing single colonies were the engineering bacteria of wild-type P450 and its mutants.

[0070] Streak each engineering bacterium and inoculate single colonies into 5 mL of TB liquid medium containing 100 μg / mL kanamycin, and culture with shaking at 37°C for 12 h. Transfer the culture to 100 mL of fresh TB liquid medium containing 100 μg / mL kanamycin at an inoculation amount of 1% (v / v), and culture with shaking at 37°C until the OD600 reaches about 0.8. Then add IPTG to a final concentration of 0.1 mM and induce culture at 18°C for 16 h. After the culture is completed, centrifuge the culture solution at 10,000 rpm for 10 min, discard the supernatant, and collect the cells. These cells are the cells expressing the wild-type P450 enzyme, mutants, and proteins required for the electron transport chain.

[0071] LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0072] TB medium formula: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0 - 7.5.

[0073] Example 3 Catalytic Reactions of Wild-Type and Its Mutants

[0074] 3.1 Obtaining of P450 Enzyme and Electron Transport Chain Proteins

[0075] Wash the cells collected by induced expression twice with 0.1 M potassium phosphate buffer at pH 8.0 to obtain resting cells. Then resuspend the obtained cells with 0.1 M potassium phosphate buffer at pH 8.0, ultrasonically disrupt them in an ice bath, collect the supernatant by high-speed centrifugation, and filter it through a 0.22 μm filter membrane to obtain an enzyme solution containing P450 enzyme and electron transport chain proteins.

[0076] 3.2 Catalytic Reaction with 7-Dehydrocholesterol as Substrate

[0077] To the enzyme solution containing P450 enzyme and electron transfer chain proteins prepared in 3.1 (which contains 4 μM P450 enzyme, 5 μM FDR, and 20 μM FDI), 2-hydroxypropyl-β-cyclodextrin was added to a final concentration of 100 g / L, stirred to dissolve, then 2 mM NADPH, 15 wt% glucose, 20 U / mL glucose dehydrogenase (aladdin, G139687-1KU), and 10 g of 7-dehydrocholesterol solid were added. The total reaction volume was 100 mL. After reacting at 30 °C for 12 h, 100 μL of the reaction solution was taken with a dropper, 900 μL of the termination solution (acetonitrile:ethanol = 4:5) was added to terminate the reaction, shaken and mixed evenly, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by liquid phase for its selectivity and yield. The results are shown in Table 3.

[0078] The yield was calculated by the following formula: actual yield / theoretical yield × 100%, where the theoretical yield was calculated according to the reaction equation process.

[0079] Table 3 Reaction conditions of wild type and mutants

[0080]

[0081] Substrate conversion: In the case of 10 g / L substrate, both wild type P450 enzyme and mutants could catalyze the substrate to produce 25-hydroxy-7-dehydrocholesterol. The yields of 25-hydroxy-7-dehydrocholesterol of WT, P1, P2, and P3 were 23.6%, 32.1%, 31.3%, and 54.3% respectively. Under the condition of 10 g / L 7-dehydrocholesterol, the yield of the product of mutant P3 was 1.3 times higher than that of wild type P450.

[0082] Example 4 Optimization of the catalytic reaction conditions of mutant P3

[0083] 4.1 Optimization of pH

[0084] The inventors found that mutant P3 could significantly improve the catalytic activity of P450 enzyme. Therefore, under the conditions of mutant P3, the reaction conditions were optimized, and 4 pH values of pH = 5, pH = 6, pH = 7, and pH = 8 were selected for the catalytic reaction. Specifically, after obtaining the bacterial cells expressing P3 according to the aforementioned conditions, they were resuspended with phosphate buffer solutions of different pH values, and then catalyzed with 7-dehydrocholesterol as the substrate under the same catalytic conditions respectively. The yields of 25-hydroxy-7-dehydrocholesterol were analyzed. The results are shown in Table 4.

[0085] Table 4 Reaction conditions of P3 at different pH values

[0086]

[0087] In the pH range of 5 - 8, the yield of 25 - hydroxy - 7 - dehydrocholesterol of mutant P3 is the highest at 65.3% when pH = 7.0.

[0088] 4.2 Optimization of temperature

[0089] Under the condition of pH = 7.0, the prepared reaction system was subjected to catalytic reaction with 7 - dehydrocholesterol as the substrate at 25, 30, 35 and 40 °C (other conditions and reaction systems are the same as those in 3.2 of Example 3), and the yield of 25 - hydroxy - 7 - dehydrocholesterol was analyzed. The results are shown in Table 5.

[0090] Table 5 Reaction conditions of P3 at different temperatures

[0091]

[0092] In the range of 25 - 40 °C, the yield of 25 - hydroxy - 7 - dehydrocholesterol of mutant P3 is the highest at 78.8% when the temperature is 35 °C.

[0093] 4.3 Optimization of the concentration of 2 - hydroxypropyl - β - cyclodextrin

[0094] 2 - Hydroxypropyl - β - cyclodextrin has the effect of solubilizing the substrate 7 - dehydrocholesterol. Different concentrations of 2 - hydroxypropyl - β - cyclodextrin may affect the process of the catalytic reaction. Therefore, on the basis of the reaction conditions with the best effect in 4.2, different concentrations of cosolvent in the system were further explored, and the catalytic reaction was carried out to analyze the yield of 25 - hydroxy - 7 - dehydrocholesterol. The results are shown in Table 6.

[0095] Table 6 Reaction conditions of P3 with different concentrations of cosolvent

[0096]

[0097] Under the condition of different concentrations of 2 - hydroxypropyl - β - cyclodextrin, the yield of 25 - hydroxy - 7 - dehydrocholesterol of mutant P3 is the highest at 95.5% when the concentration is 125 g / L.

[0098] 4.4 Optimization of the addition method of 7 - dehydrocholesterol

[0099] The inventor of the present invention found that under the optimal conditions, when the substrate concentration was continuously increased, there would be a situation where the substrate could not be completely dissolved, which led to the inability to continuously increase the productivity of the product. Therefore, the substrate was added in batches, and the substrate was added in two equal amounts at 0 h and 3 h of the reaction respectively. Finally, under the optimal catalytic conditions, mutant P3 catalyzed 13 g / L of 7 - dehydrocholesterol to produce 12.86 g / L of 25 - hydroxy - 7 - dehydrocholesterol, and the production rate of the product was 95%.

Claims

1. A P450 enzyme, characterized in that, The amino acid sequence of the P450 enzyme is shown in SEQ ID NO:

4.

2. An isolated nucleic acid, characterized in that, The nucleic acid encodes the P450 enzyme as claimed in claim 1.

3. The nucleic acid according to claim 2, wherein The nucleic acid contains the nucleotide sequence shown in SEQ ID NO:

8.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as claimed in claim 2 or 3.

5. A transformant, characterized in that, The transformant contains the nucleic acid as claimed in claim 2 or 3, or the recombinant expression vector as claimed in claim 4; the transformant is not an animal variety or a plant variety.

6. The transformant according to claim 5, wherein The transformant is a prokaryotic cell.

7. The transformant according to claim 6, wherein The prokaryotic cell is Bacillus subtilis or Escherichia coli.

8. A method for preparing P450 enzyme, characterized in that, The method includes the steps of culturing the transformant as claimed in any one of claims 5 - 7 and obtaining the P450 enzyme from the culture.

9. A method for preparing 25-hydroxy-7-dehydrocholesterol, characterized in that, The method includes catalyzing the conversion of a substrate in a reaction system containing the P450 enzyme as claimed in claim 1, and the substrate is 7-dehydrocholesterol.

10. The method according to claim 9, wherein The reaction system further contains a cosolvent, an electron transfer chain protein and / or a coenzyme and its regeneration system; and / or, The usage form of the P450 enzyme is selected from pure enzyme, crude enzyme solution, enzyme solution, fermentation broth, enzyme powder and immobilized enzyme.

11. The method according to claim 10, wherein The cosolvent is 2-hydroxypropyl-β-cyclodextrin; and / or, the electron transfer chain protein is ferredoxin reductase and / or ferredoxin; and / or, the coenzyme is NADPH; and / or, the regeneration system of the coenzyme contains glucose and glucose dehydrogenase; and / or, When the usage form of the P450 enzyme is enzyme solution, the reaction system contains 2 - 5 μM P450 enzyme, 5 - 15 g / L 7-dehydrocholesterol, 100 - 150 g / L 2-hydroxypropyl-β-cyclodextrin, 1 - 5 mM NADPH, 10 - 20 wt% glucose, 15 - 30 U / mL glucose dehydrogenase, 5 - 15 μM ferredoxin reductase and / or 20 - 60 μM ferredoxin.

12. The method according to any one of claims 9-11, characterized in that, The reaction conditions catalyzed in the method are: pH is 6 - 8, and the temperature is 30 - 40°C; and / or, in the method, 7-dehydrocholesterol is added in equal amounts in batches.

13. An enzyme composition, characterized in that, The enzyme composition contains one or more P450 enzymes as claimed in claim 1.

14. The enzyme composition according to claim 13, wherein The enzyme composition further contains glucose dehydrogenase and / or ferredoxin reductase.

15. Use of the P450 enzyme as claimed in claim 1 or the enzyme composition as claimed in claim 13 or 14 in the preparation of 25-hydroxy-7-dehydrocholesterol.

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