Hydroxylase mutant for efficiently catalyzing synthesis of 25-hydroxyvitamin D3 in organic solvent and application of hydroxylase mutant

Abstract

The invention relates to the field of gene engineering, in particular to a hydroxylase mutant for efficiently catalyzing synthesis of 25-hydroxyvitamin D3 in an organic solvent and application. According to the present invention, hydroxylase P450-Su1 represented by SEQ ID NO.1 is adopted as a template, and single-site or double-site amino acid mutation is performed to obtain four mutants such as S82A, Q93L, D222N and G164A / V277L; compared with the wild hydroxylase of the four mutants, the stability, tolerance and enzyme activity of the enzymes in an organic solvent are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to hydroxylase mutants and their applications that efficiently catalyze the synthesis of 25-hydroxyvitamin D3 in organic solvents. Background Technology

[0002] Vitamin D3 is an important fat-soluble vitamin in living organisms and a crucial precursor for calcium and phosphorus regulation. It is inactive in the human body and must be converted into an active biological component by biological enzymes to exert its effects. 25-hydroxyvitamin D3, also known as calcidiol, is a bioactive derivative of vitamin D3. It can be converted from vitamin D3 in the liver by the action of 25-hydroxylase, and its biological potency is 3-5 times that of vitamin D3.

[0003] From a molecular structure perspective, 25-hydroxyvitamin D3 is formed by adding a hydrophilic hydroxyl group to the 25th carbon atom of vitamin D3. It can be synthesized by chemical methods, which involve a series of 20 steps such as chemical modification, substitution, and hydrogenation. However, it has disadvantages such as many reaction steps and complex separation and purification processes, which are not conducive to large-scale industrial production. Moreover, the final yield is very low, only about 1%.

[0004] Compared to chemical synthesis, biocatalysis offers advantages such as lower cost and higher reaction specificity. In recent years, the biocatalytic synthesis of 25(OH)VD3 has gradually become a research hotspot. However, the extremely low water solubility of vitamin D3 is the main limiting factor restricting the rate of vitamin D3 hydroxylation reaction.

[0005] Vitamin D3 is poorly soluble in water but soluble in organic solvents and fats. This characteristic makes it possible for vitamin D3 to undergo enzymatic reactions in organic solvents. However, the activity, stability, and catalytic efficiency of hydroxylases are significantly affected in organic solvents. By modifying the enzyme to improve its catalytic activity in organic solvents, the problem of low concentrations in the vitamin D3 hydroxylation reaction in organic solvents can be effectively solved. Summary of the Invention

[0006] The purpose of this invention is to provide hydroxylase mutants and applications for the efficient catalysis of 25-hydroxyvitamin D3 synthesis in organic solvents. Specifically, this invention provides a series of modified vitamin D3 hydroxylase mutants, which contain amino acid substitutions at four positions, thereby improving the stability, tolerance, and activity of the enzyme in organic solvents compared to wild-type hydroxylases.

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

[0008] The first objective of this invention is to provide a hydroxylase mutant that efficiently catalyzes the synthesis of 25-hydroxyvitamin D3 in organic solvents. This hydroxylase mutant comprises a hydroxylase P450-Su1 enzyme mutant formed by using the hydroxylase P450-Su1 from *Streptomyces griseolus* (SEQ ID NO. 1) as a template, with mutations occurring at amino acid residues 82, 93, 222, and / or 164 and 277 as follows:

[0009] The hydroxylase P450-Su1 mutant S82A is formed by mutating serine at position 82 to alanine, with the amino acid sequence shown in SEQ ID NO.2; and / or

[0010] The glutamine at position 93 is mutated to leucine to form the hydroxylase P450-Su1 mutant Q93L, with the amino acid sequence shown in SEQ ID NO.3; and / or

[0011] The aspartic acid at position 222 is mutated to form the asparagine-derived hydroxylase P450-Su1 mutant D222N, with the amino acid sequence shown in SEQ ID NO.4; and / or

[0012] The hydroxylase P450-Su1 enzyme mutant G164A / V277L is formed by mutating glycine at position 164 to alanine and valine at position 277 to leucine, with the amino acid sequence shown in SEQ ID NO.5.

[0013] To further clarify, the organic solvent is acetone and / or isopropanol and / or dimethyl sulfoxide.

[0014] Furthermore, the mutant exhibits enhanced structural stability and / or enzyme activity in organic solvent systems containing 10% acetone, 10% isopropanol, or 10% dimethyl sulfoxide.

[0015] A second objective of this invention is to provide a coding gene that encodes the hydroxylase mutant as described above.

[0016] The present invention also provides a recombinant expression vector comprising the genes described above.

[0017] To further clarify, the carrier is pPICZαA.

[0018] The present invention also provides a recombinant bacterium expressing the hydroxylase mutant described above.

[0019] To further clarify, the recombinant bacteria are not limited to Pichia pastoris as the expression host, but can also be Escherichia coli, Saccharomyces cerevisiae, or Yersinia lipolytica.

[0020] The present invention also provides an application of using the hydroxylase mutant described above or the recombinant bacteria described above in catalyzing the synthesis of 25-hydroxyvitamin D3 from vitamin D3.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention modifies hydroxylases through single-point and multi-site combined mutations, significantly improving the enzyme's stability and tolerance in organic solvents. Specifically, the modified enzyme exhibits a substantial increase in the efficiency of catalyzing the production of 25-hydroxyvitamin D3 from vitamin D3 in 10% acetone, 10% isopropanol, and 10% dimethyl sulfoxide solutions, effectively addressing the issue of low substrate concentration in aqueous reactions. The hydroxylase mutants discovered in this application specifically improve the enzyme's catalytic performance in organic solvents by increasing its hydrophobicity, reducing polar surface residues, or enhancing the internal stability of its structure. In summary, this invention utilizes enzyme modification technology to solve key problems in the catalytic reaction of vitamin D3 in organic solvents, providing a new technical pathway for the efficient synthesis of 25-hydroxyvitamin D3. Attached Figure Description

[0023] Figure 1 The simulation results of protein molecular dynamics in 10% acetone organic solvent in this invention are shown in the figure, where A: root mean square deviation plot; B: radius of gyration plot; S82A, Q93L, D222N and G164A / V277L all represent mutant proteins and WT represents wild-type proteins.

[0024] Figure 2 The results of protein molecular dynamics simulation in 10% isopropanol organic solvent in this invention are shown in the figure, where A: root mean square deviation plot; B: radius of gyration plot; S82A, Q93L, D222N and G164A / V277L all represent mutant proteins and WT represents wild-type proteins.

[0025] Figure 3 This is a graph comparing the relative enzyme activities of the wild-type enzyme and the mutant of this invention in a 10% acetone solution.

[0026] Figure 4 This is a graph comparing the relative enzyme activities of the wild-type enzyme and the mutant of this invention in a 10% isopropanol solution.

[0027] Figure 5 This is a graph comparing the relative enzyme activities of the wild-type enzyme and the mutant of this invention in a 10% dimethyl sulfoxide solution. Detailed Implementation

[0028] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0029] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0030] Example 1: Source of the target enzyme

[0031] The amino acid sequence of the actinomycete (Streptomyces griseolus) hydroxylase P450-SU1 was obtained from the website (https: / / www.ncbi.nlm.nih.gov / protein / P18326.2) (accession number: P18326.2), as shown in SEQ ID NO.1.

[0032] SEQ ID NO.1

[0033] MTDTATTPQTTDAPAFPSNRSCPYQLPDGYAQLRDTPGPLHRVTLYDGRQAWVVTKHEAARKLLGDPRL

[0034] SSNRTDDNFPATSPRFEAVRESPQAFIGLDPPEHGTRRRMTISEFTVKRIKGMRPEVEEVVHGFLDEMLAA

[0035] GPTADLVSQFALPPVPSMVICRLLGVPYADHEFFQDASKRLVQSTDAQSALTARNDLAGYLDGLITQFQTEP

[0036] GAGLVGALVADQLANGEIDREELISTAMLLLIAGHETTASMTSLSVITLLDHPEQYAALRADRSLVPGAVE

[0037] ELLRYLAIADIAGGRVATADIEVEGHLIRAGEGVIVVNSIANRDGTVYEDPDALDIHRSARHHLAFGFGVH

[0038] QCLGQNLARLELEVILNALMDRVPTLRLAVPVEQLVLRPGTTIQGVNELPVTW

[0039] Example 2: Screening for hydroxylase mutants

[0040] 1. Mutation strategy

[0041] The following strategies, which are beneficial for increasing protein stability in organic solvents, should be used to identify mutation sites:

[0042] (1) Increase hydrophobicity by mutating the polar or charged amino acids exposed on the surface into hydrophobic amino acids such as serine and threonine into alanine and valine.

[0043] (2) Introduce hydrogen bond donor or acceptor amino acids, or form new salt bridges to stabilize the α-helix or β-sheet structure, and mutate alanine to lysine or glutamic acid.

[0044] (3) Introduce cysteine ​​mutations at key positions of the enzyme to form new disulfide bonds that stabilize the three-dimensional structure of the enzyme.

[0045] (4) Low surface charge of enzymes, which mutates the surface charged amino acids lysine, arginine, glutamic acid and aspartic acid into neutral amino acids alanine or serine.

[0046] (5) Increase rigidity by mutating the more flexible glycine into proline.

[0047] (6) Reduce the surface polarity of the enzyme by mutating the polar amino acids serine, threonine, and aspartic acid on the enzyme surface into hydrophobic amino acids.

[0048] 2. Identify candidate mutants

[0049] Candidate mutants of the hydroxylase protein were obtained through the following steps: i. Selected amino acid residues were virtually mutated using Foldx software (https: / / foldxsuite.crg.eu / ), with each residue mutated to one of 19 additional residues. Mutants that resulted in a decrease in free energy (i.e., a more stable structure) were selected. ii. The mutants were then visualized using protein three-dimensional structure visualization software (VMD).

[0050] (See https: / / www.ks.uiuc.edu / Research / vmd / ). Based on the mutation strategy, candidate mutants that meet the strategy are screened from the mutants obtained in step i. These candidate hydroxylase mutants are then used in the next step of molecular dynamics simulations to verify their stability under solution conditions.

[0051] 3. Constructing the 3D structures of wild-type and mutant proteins.

[0052] There is currently no experimentally obtained three-dimensional structural data for hydroxylase. Therefore, based on the primary sequence of the hydroxylase protein, the three-dimensional structural coordinates of the hydroxylase were obtained using the Robbetta online prediction platform (https: / / robetta.bakerlab.org). Hydroxylase possesses a large transmembrane region, primarily composed of α-helical structures, exhibiting high structural conservation. Therefore, the predicted structure has a confidence value exceeding 90%. The predicted structure was then optimized for subsequent analysis. The three-dimensional structure of the hydroxylase mutant was obtained using Foldx software.

[0053] 4. Comparison of kinetic simulations between wild-type and mutant proteins to screen for the most stable mutants.

[0054] Molecular dynamics simulations of wild-type hydroxylases and their mutants were performed using the Gromacs package (https: / / www.gromacs.org) to characterize the stability of the hydroxylases in solvents. The hydroxylase molecule was placed in a cubic box with the box boundary 1.5 nm from the protein molecule. The box was filled with a mixture of water and an organic solvent (10% acetone or 10% isopropanol). The simulation was performed according to the guidelines provided in the Gromacs package. In short, a CHARMM force field was used with an integration step size of 2 fs. First, the hydroxylase simulation system was equilibrated at 300 K (27 °C) for 5 ns, and then equilibrated at 1 atm for 5 ns. At this point, the simulation system was under similar ambient temperature conditions (i.e., 27 °C, 1 atm). The protein simulation system was then simulated for another 100 ns under these temperature and pressure conditions. The equilibrated trajectory was then captured for analysis. By comparing indicators such as the root mean square deviation (RMSD) of wild-type hydroxylase and mutant structures during the simulation process under the same molecular dynamics simulation conditions, mutants with enhanced stability can be screened.

[0055] 5. The top 10 proteins with the best stability are paired to generate two-site mutants. Steps 3 and 4 are repeated to select the four best mutant structures: S82A, Q93L, D222N and the multi-site combination mutant G164A / V277L.

[0056] Among them, the P450-Su1 enzyme mutant S82A has the amino acid sequence shown in SEQ ID NO.2;

[0057] The P450-Su1 enzyme mutant Q93L has the amino acid sequence shown in SEQ ID NO.3;

[0058] The P450-Su1 enzyme mutant D222N has the amino acid sequence shown in SEQ ID NO.4;

[0059] The P450-Su1 enzyme mutant G164A / V277L has an amino acid sequence as shown in SEQ ID NO.5.

[0060] SEQ ID NO.2

[0061] MTDTATTPQTTDAPAFPSNRSCPYQLPDGYAQLRDTPGPLHRVTLYDGRQAWVVTKHEAARKLLGDPRLSSNRTDDNFPATAPRFEAVRESPQAFIGLDPPEHGTRRRMTISEFTVKRIKGMRPEVEEVVHGFLDEMLAAGPTADLVSQFALPVPSMVICRLLGVPYADHEFFQDASKRLVQSTDAQSALTARNDLAGYLDGLITQFQTEPGAGLVGALVADQLANGEIDREELISTAMLLLIAGHETTASMTSLSVITLLDHPEQYAALRADRSLVPGAVEELLRYLAIADIAGGRVATADIEVEGHLIRAGEGVIVVNSIANRDGTVYEDPDALDIHRSARHHLAFGFGVHQCLGQNLARLELEVILNALMDRVPTLRLAVPVEQLVLRPGTTIQGVNELPVTW

[0062] SEQ ID NO.3

[0063] MTDTATTPQTTDAPAFPSNRSCPYQLPDGYAQLRDTPGPLHRVTLYDGRQAWVVTKHEAARKLLGDPRLSSNRTDDNFPATSPRFEAVRESPLAFIGLDPPEHGTRRRMTISEFTVKRIKGMRPEVEEVVHGFLDEMLAAGPTADLVSQFALPVPSMVICRLLGVPYADHEFFQDASKRLVQSTDAQSALTARNDLAGYLDGLITQFQTEPGAGLVGALVADQLANGEIDREELISTAMLLLIAGHETTASMTSLSVITLLDHPEQYAALRADRSLVPGAVEELLRYLAIADIAGGRVATADIEVEGHLIRAGEGVIVVNSIANRDGTVYEDPDALDIHRSARHHLAFGFGVHQCLGQNLARLELEVILNALMDRVPTLRLAVPVEQLVLRPGTTIQGVNELPVTW

[0064] SEQ ID NO.4

[0065] MTDTATTPQTTDAPAFPSNRSCPYQLPDGYAQLRDTPGPLHRVTLYDGRQAWVVTKHEAARKLLGDPRLSSNRTDDNFPATSPRFEAVRESPQAFIGLDPPEHGTRRRMTISEFTVKRIKGMRPEVEEVVHGFLDEMLAAGPTADLVSQFALPVPSMVICRLLGVPYADHEFFQDASKRLVQSTDAQSALTARNDLAGYLDGLITQFQTEPGAGLVGALVANQLANGEIDREELISTAMLLLIAGHETTASMTSLSVITLLDHPEQYAALRADRSLVPGAVEELLRYLAIADIAGGRVATADIEVEGHLIRAGEGVIVVNSIANRDGTVYEDPDALDIHRSARHHLAFGFGVHQCLGQNLARLELEVILNALMDRVPTLRLAVPVEQLVLRPGTTIQGVNELPVTW

[0066] SEQ ID NO.5

[0067] MTDTATTPQTTDAPAFPSNRSCPYQLPDGYAQLRDTPGPLHRVTLYDGRQAWVVTKHEAARKLLGDPRLSSNRTDDNFPATSPRFEAVRESPQAFIGLDPPEHGTRRRMTISEFTVKRIKGMRPEVEEVVHGFLDEMLAAGPTADLVSQFALPVPSMVICRLLAVPYADHEFFQDASKRLVQSTDAQSALTARNDLAGYLDGLITQFQTEPGAGLVGALVADQLANGEIDREELISTAMLLLIAGHETTASMTSLSVITLLDHPEQYAALRADRSLLPGAVEELLRYLAIADIAGGRVATADIEVEGHLIRAGEGVIVVNSIANRDGTVYEDPDALDIHRSARHHLAFGFGVHQCLGQNLARLELEVILNALMDRVPTLRLAVPVEQLVLRPGTTIQGVNELPVTW

[0068] from Figure 1 and Figure 2It can be seen that in 10% acetone or 10% isopropanol solutions, the root mean square deviation (RMSD) and radius of gyration of the mutant proteins S82A, Q93L, D222N, and G164A / V277L were significantly lower than those of the wild-type proteins in the later stable state of the simulation, indicating that the structural stability of the mutant proteins in organic solvents is significantly better than that of the wild-type proteins. Comparatively, the structures of the two mutants, G164A / V277L and Q93L, are more stable.

[0069] The nucleotide sequence encoding the P450-Su1 enzyme mutant S82A is shown in SEQ ID NO.6:

[0070]

[0071] The nucleotide sequence encoding the P450-Su1 enzyme mutant Q93L is shown in SEQ ID NO.7:

[0072]

[0073] The nucleotide sequence encoding the P450-Su1 enzyme mutant D222N is shown in SEQ ID NO.8:

[0074]

[0075] The nucleotide sequence encoding the P450-Su1 enzyme mutant G164A / V277L is shown in SEQ ID NO.9:

[0076]

[0077] Example 3: Construction and screening of recombinant expression strains of hydroxylase mutants

[0078] Taking the mutant protein S82A as an example, the recombinant bacterial construction process is as follows:

[0079] (1) The gene sequence of the mutant enzyme S82A was designed and synthesized using the online tool https: / / www.novopro.cn / tools / codon-optimization.html based on the codon preference of Pichia pastoris. The underlined parts are the added restriction sites EcoRI and XhoI and the protective bases.

[0080] CGGAATTC ATGACAGATACAGCTACTACCCCTCAAACCACCGATGCCCCTGCTTTTCCAT

[0081] CTAACAGAAGTTGCCCATATCAGCTTCCAGACGGATATGCTCAATTAAGGGATACTCCTG

[0082] GTCATTGCATAGAGTTACCTTGTATGATGGTCGTCAAGCATGGGTTGTTACTAAGCATG

[0083] AAGCAGCTAGAAAGCTACTTGGTGATCCCCGATTGTCCTCTAACCGAACAGACGATAAT

[0084] TTCCCTGCAACCTCACCTAGGTTTGAGGCCGTTAGAGAATCACCACAGGCATTCATTGGT

[0085] TTGGATCCTCCAGAACACGGAACCCGTAGAAGAATGACTATTTCAGAGTTCACTGTTAA

[0086] GAGGATTAAGGGAATGAGGCCAGAAGTCGAGGAGGTGGTACACGGATTTCTAGATGAA

[0087] ATGCTTGCCGCTGGTCCAACTGCTGATTTGGTGTCTCAATTTGCCCTGCCTGTTCCATCCA

[0088] TGGTGATTTGTCGATTGCTTGGCGTTCCATACGCTGATCATGAGTTTTTTCAAGACGCTTC

[0089] CAAACGTCTTGTGCAGAGTACCGACGCTCAATCCGCTTTGACTGCCAGAAACGATCTAG

[0090] CTGGTTACCTGGACGGACTGATTACACAGTTTCAAACGGAACCTGGTGCAGGCTTAGTC

[0091] GGAGCTTTAGTCGCAGATCAGTTGGCTAACGGTGAAATTGATCGAGAAGAGTTGATTAG

[0092] TACGGCAATGCTACTACTGATCGCCGGTCACGAAACCACTGCATCTATGACCTCTCTATC

[0093] CGTGATTACTTTGTTGGATCATCCTGAACAATACGCCGCCTTGAGAGCCGATCGTTCTCT

[0094] GGTTCCAGGAGCAGTTGAAGAGCTGTTGCGATACTTGGCAATCGCTGATATAGCAGGTG

[0095] GTAGGGTTGCAACAGCCGACATTGAAGTGGAGGGCCATCTGATCAGAGCTGGTGAAGG

[0096] TGTCATTGTTGTGAACTCAATCGCTAATAGAGACGGTACTGTCTACGAAGATCCAGACGC

[0097] CCTAGACATTCACAGGTCTGCTAGACACCATCTAGCTTTTGGATTTGGTGTCCATCAGTG

[0098] TCTTGGACAAAACTTGGCTAGACTGGAGTTGGAAGTGATTCTTAACGCTCTGATGGATA

[0099] GAGTTCCAACCTTGAGATTGGCAGTGCCAGTTGAACAATTGGTTTTGAGGCCTGGTACT

[0100] ACAATTCAAGGTGTTAACGAATTGCCAGTTACTTGG CTCGAGCGG

[0101] (2) Design primers P1 and P2 based on the gene sequence.

[0102] P1:5-CGGAATTCATGACAGATACAGC-3; P2:5-CCGCTCGAGCCAAGTAACTGG-3

[0103] (3) Using the synthesized gene as a template, P1 and P2 were used as primers for PCR amplification. The PCR product was digested with EcoRI and XhoI, ligated into the same double digestion site as the vector pPICZαA, and then transformed into Escherichia coli BL21. Positive clones were obtained by screening with bleomycin 50 μg / mL resistance plates. After PCR, double digestion and sequencing identification, the clones were used for subsequent experiments.

[0104] (4) Culture the Escherichia coli selected in the previous step, extract plasmids, linearize the plasmids with Sac I and transform them into Pichia pastoris GS115. Use bleomycin 100 μg / mL resistant plates for screening. After obtaining positive clones, identify them by PCR, double enzyme digestion and sequencing.

[0105] Those skilled in the art can construct hydroxylase mutant recombinant expression strains -Q93L, D222N, and G164A / V277L respectively according to the construction procedure of mutant protein S82A group bacteria.

[0106] Example 4: Comparison of enzyme activity of hydroxylase mutants in organic solvents

[0107] 1. Induced expression of wild-type (WT) and mutant enzymes (Mut-S82A, Mut-Q93L, Mut-D222N, Mut-G164A / V277L)

[0108] (1) Inoculate fresh bacterial culture into 200mL BMGY medium (500ml shake flask) at an inoculation ratio of 1:10. The BMGY medium formula is as follows: 1% yeast extract, 2% peptone, 100mM phosphate buffer (pH 6.0), 1% glycerol, and add bleomycin to a final concentration of 100μg / mL.

[0109] (2) Incubate at 28℃ and 250 rpm until OD600 = 3-6

[0110] (3) Collect the cells by centrifuging at 4000 rpm for 5 min and washing them once with BMMY medium to remove glycerol (BMMY medium formula is the same as BMGY, but replace glycerol with 0.5% methanol).

[0111] (4) Resuspend the bacterial cells in 200 mL of BMMY medium and adjust OD600 to 1.0.

[0112] (5) Continue culturing at 28℃ and 250rpm, adding 100% methanol every 24h to a final concentration of 0.5% to maintain induction. After 72h of induction culture, take samples, centrifuge at 4000rpm for 5min, and collect the supernatant for enzyme activity determination.

[0113] 2. Enzyme activity assay

[0114] Prepare the reaction solution according to the following procedure:

[0115] The total reaction volume is 3 mL, including 200 μL of enzyme solution; 0.02% vitamin D3; 1 mM NADPH; 300 μL of acetone; and 50 mM Tris-HCl buffer (pH 7.5), to a final volume of 3 mL. When determining enzyme activity in 10% isopropanol and 10% dimethyl sulfoxide solutions, simply replace the acetone in the reaction solution with the corresponding organic solvent.

[0116] Place the opening of the reaction tube in a shaker and react in the dark at 200 rpm and 37°C for 4 hours.

[0117] After the reaction was complete, the product was extracted with twice the volume of ethyl acetate and the mixture was shaken to mix. The mixture was then centrifuged to separate the layers, and the upper organic phase was collected.

[0118] The mixture was dried under nitrogen, redissolved in methanol, and the 25-hydroxyvitamin D3 content was determined by high-performance liquid chromatography (HPLC). The 25-hydroxyvitamin D3 content generated by the wild-type enzyme reaction was defined as 100%, and the relative enzyme activity was calculated to compare the activities of each enzyme in organic solvents.

[0119] The results showed that the enzyme activities of mutant proteins S82A, Q93L, D222N and G164A / V277L in three organic solvents were significantly higher than those of wild-type proteins. Among them, the enzyme activity of the double-site mutant protein G164A / V277L was the best, with the relative enzyme activity reaching 200% of that of wild-type proteins.

[0120] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A hydroxylase mutant that efficiently catalyzes the synthesis of 25-hydroxyvitamin D3 in organic solvents, characterized in that, The hydroxylase mutant includes the hydroxylase P450-Su1 enzyme mutant formed by using the hydroxylase P450-Su1 from the actinomycete *Streptomyces griseolus* (SEQ ID NO. 1) as a template, with the following mutations occurring at amino acid residues 82, 93, 222, and / or 164 and 277: The hydroxylase P450-Su1 mutant S82A is formed by mutating serine at position 82 to alanine, with the amino acid sequence shown in SEQ ID NO.2; and / or The glutamine at position 93 is mutated to leucine to form the hydroxylase P450-Su1 mutant Q93L, with the amino acid sequence shown in SEQ ID NO.3; and / or The aspartic acid at position 222 is mutated to form the asparagine-derived hydroxylase P450-Su1 mutant D222N, with the amino acid sequence shown in SEQ ID NO.4; and / or The hydroxylase P450-Su1 enzyme mutant G164A / V277L is formed by mutating glycine at position 164 to alanine and valine at position 277 to leucine, with the amino acid sequence shown in SEQ ID NO.

5.

2. The hydroxylase mutant according to claim 1, characterized in that, The organic solvent is acetone and / or isopropanol and / or dimethyl sulfoxide.

3. The hydroxylase mutant according to claim 1, characterized in that, The mutant exhibits enhanced structural stability and / or enzyme activity in organic solvent systems containing 10% acetone, 10% isopropanol, or 10% dimethyl sulfoxide.

4. A coding gene encoding the hydroxylase mutant as described in claim 1.

5. A recombinant expression vector comprising the gene as described in claim 4.

6. The recombinant expression vector according to claim 5, characterized in that, The carrier is pPICZαA.

7. A recombinant bacterium expressing the hydroxylase mutant of claim 1.

8. The recombinant bacteria according to claim 7, characterized in that, The recombinant bacteria are not limited to Pichia pastoris as the expression host, but can also be Escherichia coli, Saccharomyces cerevisiae, or Yersinia lipolytica.

9. An application of the hydroxylase mutant as described in any one of claims 1-3 or the recombinant bacteria as described in claim 7 or 8 in catalyzing the synthesis of 25-hydroxyvitamin D3 from vitamin D3.

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