P450 enzymes, enzyme compositions and applications thereof

Abstract

The present invention discloses a P450 enzyme, an enzyme composition, and applications thereof. The amino acid sequence of the P450 enzyme is shown in SEQ ID NO: 4. The P450 enzyme or enzyme composition 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 by light. 7-dehydrocholesterol is inexpensive, which can effectively reduce costs. In addition, by further optimizing the reaction conditions, the present invention can achieve a conversion rate of 95.5% when the concentration of 7-dehydrocholesterol is 13 g / L. The P450 enzyme or enzyme composition provided by the present invention has broad application prospects in the field of enzyme catalysis technology.

Description

Technical Field

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

[0002] Vitamin D3 is a prohormone that plays a vital role in maintaining human health, cell growth, and development. When the skin is exposed to sunlight, ultraviolet rays strike 7-dehydrocholesterol in the skin, which, through a series of complex reactions, is converted into vitamin D3. 25-Hydroxyvitamin D3, a derivative of vitamin D3 and the primary form of vitamin D3 in the body, is crucial for human health and has a very positive therapeutic effect on certain diseases such as osteoporosis, osteomalacia, hyperglycemia, and chronic kidney disease. Furthermore, in animal nutrition, 25-Hydroxyvitamin D3 has a higher absorption rate than vitamin D3 and is less affected by liver and intestinal health. Its bioavailability is 3-5 times that of regular vitamin D3, and it can rapidly improve calcium and phosphorus absorption in livestock and poultry. Therefore, it is widely used in the feed additive industry.

[0003] With the cross-disciplinary integration of gene mining, bioinformatics, and enzyme engineering, green enzymatic synthesis strategies have gradually developed into powerful tools for synthesizing active substances, drug molecules, and other valuable organic molecules. The simplest and most direct way to synthesize 25-hydroxyvitamin D3 is to hydroxylate the C25 residue of vitamin D3. The most popular bioenzymatic method for synthesizing 25-hydroxyvitamin D3 is P450 enzymes. CN 116790527 B uses a self-consistent P450 enzyme and glucose dehydrogenase as catalytic agents to ultimately produce 5.09 g / L of 25-hydroxyvitamin D3. CN 116240246 A uses a nonspecific peroxidase derived from Coprinopsis cinerea okayama 7#130 to catalyze the conversion of 5 g / L of substrate to over 80% within 3 hours. However, the substrate used in these methods is vitamin D3, which results in high conversion costs.

[0004] 7-Dehydrocholesterol, a key raw material for 25-hydroxyvitamin D3, is inexpensive. Using it as a substrate to synthesize 25-hydroxy-7-dehydrocholesterol, followed by light-induced 25-hydroxyvitamin D3, can effectively reduce costs. However, few P450 enzymes currently studied use 7-dehydrocholesterol as a substrate for 25-hydroxyvitamin D3 synthesis, and the catalytic selectivity and conversion rate are unknown. Therefore, identifying P450 enzymes that efficiently catalyze the conversion of 7-dehydrocholesterol to 25-hydroxy-7-dehydrocholesterol is crucial for the low-cost synthesis of 25-hydroxyvitamin D3. Summary of the Invention

[0005] To address the problem in existing bioenzymatic methods where the substrate used is generally vitamin D3 and the conversion rate is low, resulting in high costs for scaled-up production of 25-hydroxyvitamin D3, the present invention provides the use of a P450 enzyme in catalyzing the conversion of 7-dehydrocholesterol. The present invention effectively reduces costs by synthesizing 25-hydroxy-7-dehydrocholesterol from 7-dehydrocholesterol to regenerate 25-hydroxyvitamin D3. Furthermore, the present invention achieves 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 a first aspect, the present invention provides a P450 enzyme, the amino acid sequence of which is shown in SEQ ID NO: 4.

[0008] The second aspect of the present invention provides an isolated nucleic acid encoding the P450 enzyme of the first aspect.

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

[0010] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid as described in the second aspect.

[0011] The fourth aspect of the present invention provides a transformant, which comprises the nucleic acid as described in the second aspect, or the recombinant expression vector as described in the third aspect; the transformant is not an animal variety or a plant variety.

[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] The fifth aspect of the present invention provides a method for preparing a P450 enzyme, comprising the steps of culturing the transformant as described in the fourth aspect and obtaining the P450 enzyme from the culture.

[0015] A sixth aspect of the present invention provides a method for preparing 25-hydroxy-7-dehydrocholesterol, the method comprising catalyzing the conversion of a substrate, 7-dehydrocholesterol, in a reaction system comprising the P450 enzyme as described in the first aspect.

[0016] In some embodiments of the present invention, the reaction system further comprises a cosolvent, an electron transport chain protein and / or a coenzyme and a regeneration system thereof.

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

[0018] In some preferred embodiments of the present invention, the electron transport 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 P450 enzyme is used in a form selected from the group consisting of pure enzyme, crude enzyme solution, enzyme solution, fermentation broth, enzyme powder and immobilized enzyme.

[0022] For the purposes of this invention, pure enzyme refers to a single enzyme protein obtained through multi-step separation and purification techniques (such as chromatography and electrophoresis), typically with a purity greater than 95% (electrophoresis reveals a single main band). Crude enzyme solution refers to an enzyme solution obtained after cell disruption and preliminary purification (such as centrifugation and salting out), containing a large amount of impurities and cell debris, and with a purity less than 60%. Enzyme solution refers to a purified enzyme solution (e.g., purified by chromatography), with a purity intermediate between that of crude enzyme solution and pure enzyme. Fermentation broth refers to the mixed solution obtained after microbial fermentation and culture, containing bacterial cells, metabolites (such as enzymes and organic acids), culture medium residues, and by-products. Enzyme powder refers to a solid powder obtained by drying an enzyme solution (such as freeze-drying or spray-drying). Immobilized enzyme refers to a reusable enzyme-support complex formed by immobilizing the enzyme on a support (such as agarose microspheres and nanomaterials) through physical adsorption, covalent bonding, or encapsulation.

[0023] In some embodiments of the present invention, when the P450 enzyme is used in the form of an enzyme solution, the reaction system comprises 2-5 μM P450 enzyme, 5-15 g / L 7-dehydrocholesterol, 100-150 g / L 2-hydroxypropyl-β-cyclodextrin, 1-5 mMNADPH, 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 P450 enzyme is used in the form of an enzyme solution, the reaction system comprises 4 μM P450 enzyme, 10 g / L 7-dehydrocholesterol, 125 g / L 2-hydroxypropyl-β-cyclodextrin, 2 mM NADPH, 15wt% 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 P450 enzyme is used in the form of an enzyme solution, the reaction system comprises 4 μM P450 enzyme, 13 g / L 7-dehydrocholesterol, 125 g / L 2-hydroxypropyl-β-cyclodextrin, 2 mM NADPH, 15wt% glucose, 20 U / mL glucose dehydrogenase, 5 μM ferredoxin reductase and / or 20 μM ferredoxin.

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

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

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

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

[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 a use 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 screened P450 enzymes and advantageous mutants that can efficiently catalyze 7-dehydrocholesterol through enzyme resource mining and protein structure analysis. Through enzyme expression and mutation modification, and the establishment and optimization of the catalytic reaction system, a P450 enzyme with high catalytic activity was obtained.

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

[0035] 1. Excavation of P450

[0036] The inventors selected a 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 interact were identified, and finally three mutants that may improve their selectivity and catalytic activity were screened out: D125L, F256P, and R288A.

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

[0039] The wild-type P450 sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The synthesized CYP genes were ligated between the BamHI and HindIII restriction sites of pRSFDuet-FDR_FDI. The ligated vectors were transformed into competent E. coli BL21(DE3) cells to generate engineered strains containing the CYP genes. Using the plasmid containing the wild-type sequence as a template, recombinant vectors containing the P450 mutant sequences (D125L, F256P, and R288A) were constructed and transformed into competent E. coli BL21(DE3) cells to generate engineered strains containing the different mutants.

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

[0041] The supernatant obtained by disrupting the cells expressing wild-type and mutant P450 enzymes was mixed with the substrates 7-dehydrocholesterol, glucose, and glucose dehydrogenase. The catalytic reaction was carried out under the same reaction conditions. After the reaction, HPLC was used to analyze the conversion rate, 25-hydroxy-7-dehydrocholesterol yield and selectivity, and the catalytic activity of different mutants was compared.

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

[0043] The pH, temperature, co-solvent and substrate addition method of the dominant mutant were optimized to obtain the optimal catalytic conditions. Ultimately, the conversion rate reached 95.5% at a substrate concentration of 13 g / L.

[0044] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0046] The present invention demonstrates a significant improvement in that the P450 enzyme synthesizes 25-hydroxy-7-dehydrocholesterol, an intermediate of 25-hydroxyvitamin D3, using 7-dehydrocholesterol as a substrate. This enzyme then generates 25-hydroxyvitamin D3 through light irradiation. 7-Dehydrocholesterol is inexpensive, effectively reducing costs. Furthermore, by further optimizing the reaction conditions, the present invention achieves a conversion rate of 95.5% at a 7-dehydrocholesterol concentration of 13 g / L. DETAILED DESCRIPTION

[0047] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0048] Example 1: Discovery and transformation of P450 enzymes

[0049] Through literature research, NCBI enzyme gene mining, and multiple sequence alignment, the inventors selected a P450 enzyme that may synthesize 25-hydroxy-7-dehydrocholesterol using 7-dehydrocholesterol as a substrate. This enzyme, derived from Fictibacillus macauensis, was designated WT. The wild-type P450 enzyme may have problems such as low catalytic efficiency. Therefore, based on structural modeling and molecular docking, the inventors screened for hot spots in the active pocket and some amino acids close to the active site. Ultimately, three mutants with potential for improved selectivity and catalytic activity were identified: D125L, F256P, and R288A, designated P1, P2, and P3, respectively. The amino acid sequences of the wild-type and three mutants are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and are shown in Table 1.

[0050] Table 1 Amino acid sequence of P450

[0051]

[0052] The nucleotide sequences of the P450 enzymes whose amino acid sequences are shown in SEQ ID NOs: 1 to 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 engineered bacteria

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

[0058] Wild-type P450 sequences were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The synthetic CYP genes were ligated between the BamHI and HindIII restriction sites of pRSFDuet-FDR_FDI. pRSFDuet-FDR_FDI also contains genes required for the P450 electron transport chain: the coenzymes FDR (ferredoxin reductase) and FDI (ferredoxin). pRSFDuet-FDR_FDI was prepared by sending the codon-optimized FDR and FDI nucleotide sequences to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. FDR was then ligated into the commercial plasmid pRSFDuet at the EcoRI and NcoI sites using a seamless cloning kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) to generate the pRSFDuet-FDR recombinant vector. FDI was then cloned using the same method between the NdeI and XhoI restriction sites of pRSFDuet-FDR to create the pRSFDuet-FDR_FDI recombinant vector.

[0059] FDR amino acid sequence:

[0060] MQIASDVEAPPPAPAKVEKHSKKMEEGITVNKFKPKTPYVGRCLLNTKITGDDAPGETWHMVFSHEGEIPYREGQSVGVIPDGEDKNGKPHKLRLYSIASSALGDFGDAKSVSLCVKRLIYTNDAGETIKGVCSNFLCDLKPGAEVKLTGPVGKEMLMP KDPNATIIMLGTGTGIAPFRSFLWKMFFEKHDDYKFNGLAWLFLGVPTSSSLLYKEEFEKMKEKAPDNFRLDFAVSREQTNEKGEKMYIQTRMAQYAVELWEMLKKDNTYFYMCGLKGMEKGIDDIMVSLAAAEGIDWIEYKRQLKKAEQWNVAVY (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] ATGGCGGCGTATAAAGTGACCCTGGTGACCCCGACCGGCAACGTGGAATTTCAGTGCCCGGATGATGTGTATATTCTGGATGCGGCGGAAGAAGAAGGCATTGATCTGCCGTATAGCTGCCGCGCGGGCAGCTGCAGCAGCTGCGCGGG CAAACTGAAAACCGGCAGCCTGAACCAGGATGATCAGAGCTTTCTGGATGATGATCAGATTGATGAAGGCTGGGTGCTGACCTGCGCGGCGTATCCGGTGAGCGATGTGACCATTGAAACCCATAAAAAAGAAGAACTGACCGCG (SEQ ID NO: 12)

[0067] To construct expression vectors for P1, P2, or P3, a plasmid carrying the wild-type P450 sequence was used as a template. Primers were designed at the mutation site for inverse PCR. The amplified PCR product was digested with Dpn I overnight and inactivated at 80°C for 20 min. The product was then transferred to E. coli DH5α competent cells, cultured with recovery solution for 45 min, and plated on LB solid medium containing 100 μg / mL kanamycin. The cells were incubated in an inverted position at 37°C overnight. Single clones were picked and sent to the company for sequencing. The sequencing results were analyzed using SnapGene software to confirm the correct amino acid mutation at the mutation site, thus obtaining a recombinant vector containing the P450 enzyme mutation site.

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

[0069] To obtain engineered bacteria expressing P450 and its mutants and electron transport chain genes, the four recombinant plasmids were transformed into BL21 (DE3) competent cells, incubated with resuscitation solution for 45 minutes, plated on LB solid medium containing 100 μg / mL kanamycin, and incubated inverted at 37°C overnight. Single colonies that grew were engineered bacteria expressing wild-type P450 and its mutants.

[0070] Streak a single colony from each engineered strain and inoculate it into 5 mL of TB liquid medium containing 100 μg / mL kanamycin. Cultivate with shaking at 37°C for 12 hours. Transfer a 1% (v / v) inoculum to 100 mL of fresh TB liquid medium also containing 100 μg / mL kanamycin. Cultivate with shaking at 37°C until the OD600 reaches approximately 0.8. Then, add IPTG to a final concentration of 0.1 mM and induce the culture at 18°C ​​for 16 hours. After incubation, centrifuge the culture at 10,000 rpm for 10 minutes, discard the supernatant, and collect the cells. These cells express wild-type P450 enzymes, mutants, and proteins required for the electron transport chain.

[0071] LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, and 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 reaction of wild type and its mutants

[0074] 3.1 Acquisition of P450 enzymes and electron transport chain proteins

[0075] The induced expression collected bacteria were washed twice with 0.1 M, pH 8.0 potassium phosphate buffer to obtain resting cells, and then the obtained cells were resuspended with 0.1 M, pH 8.0 potassium phosphate buffer, disrupted by ultrasonication in an ice bath, and the supernatant was collected by high-speed centrifugation and filtered with a 0.22 μm filter membrane to obtain an enzyme solution containing P450 enzymes and electron transport chain proteins.

[0076] 3.2 Catalytic reactions using 7-dehydrocholesterol as substrate

[0077] To the enzyme solution containing P450 enzymes and electron transport chain proteins prepared in 3.1 (containing 4 μM P450 enzymes, 5 μM FDR, and 20 μM FDI), 2-hydroxypropyl-β-cyclodextrin was added to a final concentration of 100 g / L and dissolved with stirring. Then, 2 mM NADPH, 15 wt% glucose, 20 U / mL glucose dehydrogenase (aladdin, G139687-1KU), and 10 g of solid 7-dehydrocholesterol were added to a total reaction volume of 100 mL. After incubation at 30°C for 12 h, 100 μL of the reaction solution was pipetted and terminated with 900 μL of stop solution (acetonitrile:ethanol = 4:5). The reaction was mixed by vortexing and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and analyzed by liquid chromatography for selectivity and yield. The results are shown in Table 3.

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

[0079] Table 3 Responses of wild type and mutants

[0080]

[0081] Substrate conversion: At 10 g / L substrate, both the wild-type P450 enzyme and the mutants catalyzed the conversion of the substrate to 25-hydroxy-7-dehydrocholesterol. The 25-hydroxy-7-dehydrocholesterol yields for WT, P1, P2, and P3 were 23.6%, 32.1%, 31.3%, and 54.3%, respectively. At 10 g / L 7-dehydrocholesterol, the mutant P3 exhibited a 1.3-fold increase in product yield compared to the wild-type P450.

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

[0083] 4.1 pH optimization

[0084] The inventors discovered that mutant P3 significantly enhances the catalytic activity of P450 enzymes. Therefore, under the conditions of mutant P3, the reaction conditions were optimized, and four pH values ​​(pH 5, pH 6, pH 7, and pH 8) were selected for the catalytic reaction. Specifically, after obtaining P3-expressing bacteria under the aforementioned conditions, they were resuspended in phosphate buffers of different pH values ​​and subjected to catalytic reactions using 7-dehydrocholesterol as a substrate under the same catalytic conditions. The yield of 25-hydroxy-7-dehydrocholesterol was analyzed, and the results are shown in Table 4.

[0085] Table 4 Reaction of P3 at different pH

[0086]

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

[0088] 4.2 Temperature Optimization

[0089] At pH 7.0, the prepared reaction system was catalyzed at 25, 30, 35, and 40°C (other conditions and reaction system were the same as in 3.2 of Example 3) using 7-dehydrocholesterol as a substrate. The yield of 25-hydroxy-7-dehydrocholesterol was analyzed, and the results are shown in Table 5.

[0090] Table 5 Reaction of P3 at different temperatures

[0091]

[0092] At 35℃, the yield of 25-hydroxy-7-dehydrocholesterol of mutant P3 was the highest, reaching 78.8%, within the temperature range of 25-40℃.

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

[0094] 2-Hydroxypropyl-β-cyclodextrin has a solubilizing effect on the substrate 7-dehydrocholesterol. Different concentrations of 2-Hydroxypropyl-β-cyclodextrin may affect the progress of the catalytic reaction. Therefore, based on the most effective reaction conditions in 4.2, we further explored different concentrations of the solubilizing agent in the system, carried out the catalytic reaction, and analyzed the yield of 25-hydroxy-7-dehydrocholesterol. The results are shown in Table 6.

[0095] Table 6 Reaction status at different concentrations of P3 cosolvent

[0096]

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

[0098] 4.4 Optimization of 7-dehydrocholesterol addition method

[0099] The inventors discovered that under optimal conditions, further increasing the substrate concentration would result in incomplete substrate dissolution. This resulted in a stagnant increase in product productivity. Therefore, the substrate was added in two equal batches, added at 0 and 3 hours of reaction time, respectively. Ultimately, under optimal catalytic conditions, mutant P3 catalyzed the conversion of 13 g / L of 7-dehydrocholesterol to 12.86 g / L of 25-hydroxy-7-dehydrocholesterol, with a product yield of 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 according to claim 1.

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

8.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 2 or 3.

5. A transformant, characterized in that The transformant comprises the nucleic acid according to claim 2 or 3, or the recombinant expression vector according to claim 4; and the transformant is not an animal species or a plant species.

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 a P450 enzyme, characterized in that: The method comprises the steps of culturing the transformant according to any one of claims 5 to 7 and obtaining the P450 enzyme from the culture.

9. A method for preparing 25-hydroxy-7-dehydrocholesterol, characterized in that: The method comprises catalyzing the conversion of a substrate in a reaction system comprising the P450 enzyme according to claim 1, wherein the substrate is 7-dehydrocholesterol.

10. The method according to claim 9, wherein The reaction system further comprises a cosolvent, an electron transport chain protein and a coenzyme and a regeneration system thereof; and / or, The P450 enzyme is used in a form 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 transport chain protein is ferredoxin reductase and ferredoxin; and / or, the coenzyme is NADPH; and / or, the coenzyme regeneration system comprises glucose and glucose dehydrogenase; and / or, When the P450 enzyme is used in the form of an 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 20-60 μM ferredoxin.

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

13. An enzyme composition, characterized in that The enzyme composition comprises the P450 enzyme of claim 1.

14. The enzyme composition according to claim 13, wherein The enzyme composition also comprises glucose dehydrogenase and ferredoxin reductase.

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

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