A p450 mutant and its use in the synthesis of calcifediol

Abstract

The application discloses a P450 mutant and application thereof in synthesis of calcifediol, and belongs to the technical field of bioengineering. The P450 mutant enzyme is constructed, and a single-plasmid three-enzyme co-expression system of an oxidation-reduction partner and a cofactor circulation enzyme is constructed, so that sufficient electron transfer chains are provided for the P450 enzyme, and calcifediol can be synthesized with higher efficiency under mild conditions and with vitamin D3 as a substrate. The conversion rate of the calcifediol can reach 63.1%, and the conversion rate of the reaction is greatly improved. The method disclosed by the application improves the catalytic efficiency of the catalyst and reduces the cost of the reaction.

Description

TECHNICAL FIELD

[0001] The present application relates to a P450 mutant and its application in the synthesis of calcifediol, belonging to the field of bioengineering technology. BACKGROUND

[0002] Calcifediol (also known as 25-hydroxyvitamin D3), with the molecular formula C 27 H 44 O, is the main form of vitamin D3 in the human body. Calcifediol not only can treat vitamin D deficiency, but also can maintain liver homeostasis and maintain intestinal immune function. Calcifediol preparation products can be used in clinical treatment of senile osteoporosis, and also have preventive effects on chronic kidney disease, hyperglycemia and cholestatic liver disease.

[0003] At present, the main methods for industrial production of calcifediol include total synthesis and semi-synthesis. The total synthesis method uses cholestanol as the raw material to gradually pass through 25-hydroxycholesterol, 25-hydroxy-7-ketocholsterol, to generate 25-hydroxy-7-dehydrocholesterol, and finally cleave the ring by light to obtain the target product. The semi-synthesis method uses cholestanol as the raw material to obtain 7-dehydrocholesterol through multi-step fermentation of yeast, and then generates 25-hydroxy-7-dehydrocholesterol through chemical method, and then cleaves the ring by light to obtain the target product. The two types of reaction paths are long, need to frequently use double bond protecting agents and hydroxyl protecting agents, and finally need to go through the step of cleaving the ring by light. The reaction has many by-products, the yield of the product is low, and the time and intensity of light need to be strictly controlled, so the cost of industrial production is high. The biological synthesis method is favored by many scholars due to its high selectivity, few by-products and mild reaction conditions. With the development of synthetic biology, enzyme engineering and other biological technologies, the synthesis pathway of calcifediol has been gradually constructed in engineering bacteria, and preliminary progress has been made. Using relatively cheap vitamin D3 as a substrate for one-step enzymatic production of calcifediol is a potential biological synthesis method, which only needs one-step enzymatic reaction, does not need to add protecting agents, and can realize product synthesis without strict conditions such as strong light. However, the reported biological synthesis methods have generally low catalytic efficiency for the substrate vitamin D3, which cannot meet the requirements of industrial production. Therefore, there is an urgent need for a biological method for preparing calcifediol with low-cost vitamin D3 as a substrate and high efficiency. SUMMARY

[0004] The present application provides a P450 enzyme mutant, which is obtained by mutating one or more of the 73rd and 81st amino acids in the starting sequence.

[0005] In one embodiment, the mutant is any one of the following (a) to (c):

[0006] (a) mutating glutamine Q at position 73 of P450 enzyme CYP109E1 with amino acid sequence as shown in SEQ ID NO. 1 into methionine M, and the obtained mutant is named as Q73M;

[0007] (b) mutating glycine G at position 81 of P450 enzyme CYP109E1 with amino acid sequence as shown in SEQ ID NO. 1 into tryptophan W, and the obtained mutant is named as G81W;

[0008] (c) mutating glutamine Q at position 73 of P450 enzyme CYP109E1 with amino acid sequence as shown in SEQ ID NO. 1 into methionine M, and mutating glycine G at position 81 into tryptophan W, and the obtained mutant is named as Q73M / G81W.

[0009] In an embodiment, the amino acid sequence of the starting sequence CYP109E1 of the P450 enzyme is as shown in SEQ ID NO. 1, and the nucleotide sequence encoding the P450 enzyme is as shown in SEQ ID NO. 5.

[0010] The present application also provides a gene encoding the P450 enzyme mutant.

[0011] In an embodiment, the nucleotide sequence of the P450 enzyme mutant is as shown in SEQ ID NO. 6.

[0012] The present application also provides a recombinant vector carrying the gene.

[0013] In an embodiment, the recombinant vector uses pET-28a as the expression vector.

[0014] The present application also provides a microbial cell carrying the above gene or the above recombinant vector.

[0015] In an embodiment, the microbial cell uses bacteria or fungi as the expression host.

[0016] In an embodiment, the microbial cell uses Escherichia coli BL21 (DE3) as the expression host.

[0017] The present application also provides a recombinant Escherichia coli expressing the above P450 enzyme CYP109E1 mutant.

[0018] In an embodiment, the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as the expression host, and pET-28a as the expression vector.

[0019] The application also provides a method for obtaining the P450 enzyme CYP109E1 mutant, comprising the following steps:

[0020] (1) determining a mutation site based on the amino acid sequence of the P450 enzyme CYP109E1; designing a mutation primer for site-directed mutagenesis, and performing site-directed mutagenesis with a carrier carrying the P450 enzyme CYP109E1 gene as a template; and constructing a plasmid vector containing the mutant;

[0021] (2) transforming the plasmid vector containing the mutant into a host cell;

[0022] (3) selecting a positive clone for fermentation culture, and purifying the P450 enzyme CYP109E1.

[0023] In an embodiment, the host cell is Escherichia coli.

[0024] The application also provides a construction method of a single-plasmid three-enzyme co-expression system, comprising the following steps:

[0025] (1) respectively amplifying gene sequences of ferredoxin (SelFdx) and ferredoxin reductase (SelFdR) derived from Synechococcus elongatus PCC 7942 (Synechococcus elongatus PCC 7942) and a gene sequence of glucose dehydrogenase (BsGDH) derived from Bacillus subtilis (Bacillus subtilis), and inserting the gene sequences into an expression plasmid pRSFDuet-1 to respectively obtain a plasmid pRSFDuet-SelFdx, a plasmid pRSFDuet-SelFdR and a plasmid pRSFDuet-BsGDH;

[0026] (2) using an endonuclease to respectively cut the plasmid pRSFDuet-SelFdx, the plasmid pRSFDuet-SelFdR and the plasmid pRSFDuet-BsGDH, sequentially connecting, transforming a host Escherichia coli BL21 (DE3), and constructing a single-plasmid three-enzyme co-expression system.

[0027] The application also provides a recombinant Escherichia coli containing a three-enzyme co-expression system, wherein the three-enzyme co-expression system is used for co-expressing ferredoxin (SelFdx), ferredoxin reductase (SelFdR) and glucose dehydrogenase (BsGDH) coding genes by using the same plasmid; the nucleotide sequence for coding the ferredoxin (SelFdx) is shown in SEQ ID NO. 7; the nucleotide sequence for coding the ferredoxin reductase (SelFdR) is shown in SEQ ID NO. 8; and the nucleotide sequence for coding the glucose dehydrogenase (BsGDH) is shown in SEQ ID NO. 9.

[0028] In one embodiment, the expression vector is pRSFDuet, and the genes expressed are SelFdx, SelFdR and BsGDH.

[0029] The present application also provides a method for preparing a crude enzyme solution, characterized in that the bacterial suspension of the recombinant E. coli containing the three-enzyme co-expression system is subjected to ultrasonic disruption and centrifugation to obtain the supernatant.

[0030] In one embodiment, the crude enzyme solution is prepared by culturing the recombinant E. coli for a period of time, collecting the bacterial cells, preparing a bacterial suspension with a concentration of 100 g / L, and then subjecting the bacterial suspension to ultrasonic disruption and centrifugation to obtain the supernatant.

[0031] The present application also provides a method for preparing calcifediol, which comprises reacting the crude enzyme solution with any one of (a) to (c) in a reaction system containing the co-factor NADP + , glucose and vitamin D3; wherein:

[0032] (a) the P450 enzyme mutant;

[0033] (b) the microbial cell;

[0034] (c) the recombinant E. coli.

[0035] In one embodiment, when the microbial cell expressing the P450 enzyme mutant or the recombinant E. coli is used for catalysis, lysozyme is added to the reaction system so that the P450 enzyme is released from the cell and fully contacts with the enzymes and substrates in the reaction system.

[0036] In one embodiment, the reaction system contains hydroxypropyl-β-cyclodextrin to reduce the wall-hanging phenomenon of vitamin D3 and calcifediol when mixed with water.

[0037] In one embodiment, the crude enzyme solution is added in an amount of 12.5% of the total volume of the reaction solution.

[0038] In one embodiment, the final concentration of the recombinant E. coli added to the reaction system is 10-50 g / L.

[0039] In one embodiment, the mass / volume percentage of hydroxypropyl-β-cyclodextrin in the reaction system is 1.13-2.83%.

[0040] In one embodiment, the lysozyme is added in an amount of 1500 U / mg of cells.

[0041] In an embodiment, the amount of the crude enzyme solution added is 25% of the total volume.

[0042] In an embodiment, the vitamin D3 is pre-solubilized with a co-solvent; the co-solvent includes but is not limited to one or more of DMSO, methanol, ethanol and DMF.

[0043] In an embodiment, the final concentration of the vitamin D3 in the reaction system is 0.5-5 mM.

[0044] In an embodiment, the volume percentage of the co-solvent in the reaction system is 2-5%.

[0045] In an embodiment, the molar ratio of NADP + to the vitamin D3 is 0.05-1.2:1.

[0046] In an embodiment, the molar ratio of glucose to the vitamin D3 is 25-75:1.

[0047] In an embodiment, the reaction conditions are: pH 6.0-8.0, 37°C, and reaction time of 5-8 h.

[0048] The present application also provides use of the above-mentioned P450 enzyme CYP109E1 mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant E. coli in preparing calcifediol or a product containing calcifediol.

[0049] In an embodiment, the product is a chemical.

[0050] Advantages

[0051] (1) The present application provides a P450 enzyme CYP109E1 mutant, which can be used for catalyzing production of calcifediol, and the catalytic ability is significantly improved relative to the starting sequence.

[0052] (2) The present application provides a method for co-catalyzing production of calcifediol by using a single-plasmid three-enzyme co-expression system and a P450 enzyme, wherein the single-plasmid three-enzyme co-expression system is used for providing electrons required for CYP109E1 catalysis.

[0053] (3) The P450 enzyme CYP109E1 mutant improves the yield of vitamin D3 converted to calcifediol by 28.2% to 104.7% relative to the P450 enzyme CYP109E1. The P450 enzyme CYP109E1 mutant shows high hydroxylation activity, and the highest yield of calcifediol is 631.7 mg / L and the yield reaches 63.1% under the conditions of 37°C, pH 7.0 and reaction for 8h. The method improves the catalytic efficiency of the catalyst, reduces the cost of the reaction, and accelerates the industrialization process of the enzyme conversion method for producing calcifediol. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 : The synthesis method for synthesizing calcifediol by catalyzing vitamin D3.

[0055] Figure 2 : SDS-PAGE diagram of induced expression of glucose dehydrogenase BsGDH and oxidoreductase partners SelFdR and SelFdx.

[0056] Figure 3 : Liquid chromatogram of reaction substrate and product; i: liquid chromatogram of calcifediol standard, the retention time of calcifediol is 6.2 min; ii: detection of reaction conversion of vitamin D3 to produce calcifediol, the retention time of calcifediol is 6.2 min; iii: liquid chromatogram of vitamin D3 standard, the retention time is 12.1 min. DETAILED DESCRIPTION

[0057] The pET-28a(+) and pRSFDuet-1 plasmids involved in the following examples are purchased from Novagen (Madison, WI, U.S.A.), the restriction endonuclease, T4 DNA ligase, PrimeSTAR, etc. are purchased from TaKaRa (Dalian, China). The calcifediol standard is purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., the vitamin D3 is purchased from Tianjin Xinsisheng Biochemical Technology Co., Ltd., and the rest of the reagents are purchased from the market.

[0058] The culture medium involved in the following examples is as follows:

[0059] LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, sterilized at 121°C for 20 min.

[0060] LB solid medium: 2% agar is added to the LB liquid medium.

[0061] TB liquid medium: KH2PO4 2.31 g / L, K2HPO4·3H2O 16.42 g / L, yeast powder 24 g / L, proteose peptone 12 g / L, glycerol 4 g / L.

[0062] The detection method involved in the following examples is as follows:

[0063] Detection of the content of vitamin D3 and calcifediol:

[0064] 0.1M PBS buffer solution was used as the aqueous phase, and vitamin D3 with a final concentration of 961.6 mg / L was used as the substrate. The system also contained a single-plasmid three-enzyme co-expression system crude enzyme solution of glucose dehydrogenase BsGDH and redox partners SelFdR and SelFdx (the crude enzyme solution accounted for 12.5% of the total volume), and coenzyme NADP + 0.5 mM, hydrogen donor glucose 50 mM, cosolvent 4.375%, hydroxypropyl-β-cyclodextrin 2.25%, lysozyme 1500 U / mg 表达P450酶细胞 The whole cell was used to transform vitamin D3 to produce calcifediol. The whole cell concentration of the P450 enzyme CYP109E1 mutant was 20 g / L. After reaction at 37°C, 400 rpm constant temperature shaker for 8 h, the reaction solution was sampled, a small amount of the sample was placed in an EP tube, extracted with an equal volume of ethyl acetate for three times, the combined organic phase was removed under reduced pressure, an equal volume of methanol was added for redissolution, and 0.22 μm organic membrane was used for high performance liquid chromatography (HPLC) analysis.

[0065] The specific HPLC analysis method is as follows:

[0066] C18 (5 μm, 250 x 4.6 mm) was used as the chromatographic column, methanol filtered and degassed by ultrasonic was used as the organic phase, ultrapure water was used as the aqueous phase, and the gradient elution conditions are shown in Table 1. The injection amount was 20 μL, the column temperature was 40°C, the wavelength of the ultraviolet detector was 264 nm, the flow rate was 0.8 mL / min, and the sample processing time was 20 min.

[0067] Table 1 Gradient elution conditions

[0068]

[0069] Under the detection conditions, the retention times of calcifediol and vitamin D3 were 6.2 min and 12.1 min, respectively.

[0070]

[0071] wherein: m 骨化二醇 represents the mass of calcifediol generated; m 维生素D3 represents the initial mass of vitamin D3; 384.63 and 400.64 represent the relative molecular mass of vitamin D3 and calcifediol, respectively.

[0072] Example 1: Construction of genetically engineered bacteria expressing CYP109E1

[0073] The nucleotide sequence of the gene encoding the target protein in Bacillus megaterium (shown in SEQ ID NO. 5) was used as a template to synthesize the plasmid pET-28a(+)-CYP109E1 by Shengong Bioengineering Co., Ltd., in which the target gene fragment was connected between BamHI and HindIII of the vector. The plasmid pET-28a(+)-CYP109E1 was transformed into E. coli BL21 to obtain the engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1.

[0074] The engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1 was inoculated into LB liquid medium, and after 12 h of culture, a seed liquid was obtained. The seed liquid was inoculated into fresh TB liquid medium at an inoculation amount of 5% (v / v), and after 2 h of culture, 0.4 mM IPTG was added to induce the expression of the recombinant target protein at 16°C for 18 h. 150 mL of induced fermentation broth was centrifuged at 8000 r / min to collect the bacterial cells, which were stored at -40°C.

[0075] Example 2: Construction of a single-plasmid three-enzyme co-expression system

[0076] The gene sequences of ferredoxin (SelFdx) (shown in SEQ ID NO. 7) and ferredoxin reductase (SelFdR) (shown in SEQ ID NO. 8) derived from Synechococcus elongatus PCC 7942 and the gene sequence of glucose dehydrogenase (BsGDH) (shown in SEQ ID NO. 9) derived from Bacillus subtilis were amplified, respectively, and inserted between BamHI and HindIII of the expression plasmid pRSFDuet-1 to obtain plasmids pRSFDuet-SelFdx, pRSFDuet-SelFdR and pRSFDuet-BsGDH, respectively.

[0077] The plasmid pRSFDuet-SelFdx, the plasmid pRSFDuet-SelFdR and the plasmid pRSFDuet-BsGDH are cut by endonuclease respectively, and are connected in turn to obtain the plasmid pRSFDuet-SelFdx-SelFdR-BsGDH, which is transformed into the host E. coli BL21 (DE3) to construct the single-plasmid three-enzyme co-expression strain E. coli BL21 / pRSFDuet-SelFdx-SelFdR-BsGDH.

[0078] Example 3: Expression of the single-plasmid three-enzyme co-expression system and preparation of the crude enzyme solution

[0079] The engineered strain E. coli BL21 / pRSFDuet-SelFdx-SelFdR-BsGDH constructed in Example 2 is inoculated into LB liquid medium, and a seed solution is obtained after 12 h of culture. The seed solution is inoculated into fresh TB liquid medium at an inoculation amount of 5% (v / v), and after 2 h of culture, 0.4 mM of IPTG is added to induce expression of the recombinant target protein at 16°C for 18 h. 150 mL of the induced fermentation broth is centrifuged at 8000 r / min to collect the bacterial cells, which are stored at -40°C.

[0080] The centrifuged bacterial cells are resuspended in 0.1 M PBS buffer to a concentration of 100 g / L, and then the centrifuge tube is placed in an ice bath and put into an ultrasonic cell disruptor. The ultrasonic disruption conditions are as follows: working time 2.5 s, interval time 1.5 s, and a total of 20 min. The obtained disruption solution is subjected to low-temperature and low-speed centrifugation at 4200 rpm for 15 min at 4°C, and the supernatant is collected to obtain the crude enzyme solution.

[0081] Example 4: Preparation of calcifediol by conversion of vitamin D3

[0082] The CYP109E1 protein and the crude enzyme solution of the three-enzyme co-expression system constructed in Example 3 are used to convert vitamin D3 to prepare calcifediol. A reaction system containing 2.5 mM of vitamin D3 is prepared, in which the molar ratio of NADPH to the vitamin D3 is 0.1:1, the molar ratio of glucose to the vitamin D3 is 50:1, the wet bacterial cell concentration of CYP109E1 is 20 g / L, and the volume ratio of the crude enzyme solution to the total system is 12.5%. The specific steps are as follows: + The molar ratio of NADPH to the vitamin D3 is 0.1:1, the molar ratio of glucose to the vitamin D3 is 50:1, the wet bacterial cell concentration of CYP109E1 is 20 g / L, and the volume ratio of the crude enzyme solution to the total system is 12.5%. The specific steps are as follows:

[0083] In 20 mL small brown bottle, respectively add 0.1 g of whole cell E. coli BL21 / pET-28a(+)-CYP109E1 expressing CYP109E1 protein after induction culture according to the method of Example 1, 2.5 mL of 0.05M PBS buffer, 0.625 mL of 5 g / L final concentration of lysozyme (enzyme activity ≥20000 U / mg, China Reagent Co., Ltd., Catalog No: 64006060) aqueous solution, 37℃ lysis for 90 min, then add 1.9 mg NADP + , 45.0 mg of hydrogen donor glucose, 112.5 mg of hydroxypropyl-β-cyclodextrin, add 0.1M PBS to 4781.3 μL, finally add 218.7 μL of vitamin D3 mother liquor pre-dissolved in DMF, make the final concentration of vitamin D3 in 5 mL reaction system 961.6 mg / L, start the reaction, adjust the pH of the system to 7.0, react at 37℃, 400 rpm constant temperature shaker for 8h. After conversion, add an equal volume of ethyl acetate to the reaction liquid sample and shake vigorously in a vortex shaker, collect the upper organic phase by high speed centrifugation, repeat the extraction twice, combine the organic phase, volatilize the ethyl acetate in a reduced pressure centrifuge, add an equal volume of methanol, shake to promote dissolution, filter through a 0.22 μm organic filter membrane, and analyze by high performance liquid chromatography (HPLC). The analysis results are as follows: the yield of recombinant bacteria E. coli BL21 / pET-28a(+)-CYP109E1 for conversion to prepare calcidiol is 308.6 mg / L, and the conversion rate is 30.8%.

[0084] Example 5: Construction and screening of single mutant

[0085] Using pET-28a(+)-CYP109E1 constructed in Example 1 as the template, primers for Q73M and G81W mutation sites were designed as shown in Table 2, and mutant construction was performed by whole plasmid PCR.

[0086] Table 2 Single mutant mutation primer sequence

[0087]

[0088] The PCR amplification system for the construction reaction was as follows: PrimeSTAR enzyme 25 μL, 1 μL of each primer for each mutation site, 1 μL of template (CYP109E1 WT)1 μL, water 22 μL; reaction conditions: ① 98℃ 5 min; ② 94℃ 30 s; ③ 55℃ 30 s; ④ 72℃ 3 min 20 s; ⑤ cycle 30 times for the three steps of ②-④; ⑥ 72℃ 10 min; ⑦ 12℃ incubation. The above reaction system was incubated at 37℃ for 1 h to digest the plasmid template (digestion system: DpnI 1 μL, the above reaction PCR product 8 μL, 10 x T Buffer 1 μL), and the digestion product obtained after digestion was introduced into E. coli BL21 competent cells by chemical transformation method. The specific steps of chemical transformation method are as follows:

[0089] (1) 10 μL digestion product was introduced into 100 μL E. coli BL21(DE3) competent cells;

[0090] (2) ice bath for 10 min;

[0091] (3) 42℃ water bath heat shock for 90 s, and then quickly placed in ice for 4 min;

[0092] (4) 800 μL of non-resistant LB medium was added and mixed, and then incubated at 37℃, 220 rpm for 1 h;

[0093] (5) centrifuged at 5000 rpm for 2 min to collect the bacteria;

[0094] (6) remove the supernatant, and then 100-200 μL of the remaining liquid was blown and mixed and then inoculated on a LB resistance plate containing 0.05 mg / mL kanamycin, and then incubated at 37℃ for 12 h or so;

[0095] (7) a single colony was picked and inoculated in LB containing 0.05 mg / mL kanamycin, and then incubated at 37℃, 200 rpm for 12 h, and then sent for sequencing. The correct sequencing result was the positive transformant.

[0096] The genetically engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1 Q73M , E. coli BL21 / pET-28a(+)-CYP109E1 G81W were prepared respectively.

[0097] Then, the above genetically engineered bacteria were prepared according to the method of Example 4, and the better mutant was screened out. The results are shown in Table 3. The engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1 Q73M , E. coli BL21 / pET-28a(+)-CYP109E1 G81WAll have good effect of vitamin D3 hydroxylation, relative to wild type E. coli BL21 / pET-28a(+)-CYP109E1 production is increased by 28.2% to 84.3%.

[0098] Table 3 Wild type and single mutant calcifediol production

[0099]

[0100] Example 6: Construction and screening of double mutants

[0101] The double mutant of this example is constructed by whole plasmid PCR according to the primers in Table 4 on the basis of the corresponding single mutant, for example, on the basis of mutant CYP109E1 G81W , the double mutant CYP109E1 G81W / Q73M is constructed by whole plasmid PCR using mutant primers Q73M-R and Q73M-F (Table 4).

[0102] The method for preparing genetically engineered bacteria is described in Example 5, and the primers used are shown in Table 4. According to the method of Example 5, the double mutant genetically engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1 G81W / Q73M is prepared.

[0103] Table 4 Double mutant primer sequences

[0104]

[0105] The mutant strain that has been sequenced correctly is inoculated into LB seed culture medium and cultured at 220 rpm and 37°C for 10 h. The seed liquid is inoculated into fresh TB liquid culture medium at an inoculation amount of 5% (v / v), and cultured at 220 rpm and 37°C until the OD 600 is about 0.8. IPTG is added at a final concentration of 0.4 mM, and the induction conditions are 220 rpm and 16°C for 18 h.

[0106] Subsequently, the genetically engineered bacteria obtained above are transformed to prepare calcifediol according to the method of Example 4.

[0107] After the reaction is completed, the yield of calcifediol is determined by HPLC method. The double mutant genetically engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1 G81W / Q73M has good hydroxylation effect on calcifediol, and the conversion rate of the prepared calcifediol is 63.1%, and the corresponding yield is 631.7 mg / L.

[0108] Comparative Example:

[0109] A specific embodiment is similar to Example 5, except that the following mutants were also constructed: G81L, Q73E, Q77F, Q73D, P71L, N74C, A291C, G81W / A291C, G81W / Q77F, G81W / Q73E, G81W / Q73D, G81W / N74C, G81W / P71L, L292T, F391W, N7L, N7I, N7F, Q73D / N74M, Q73M / N74Y, etc. Calcifediol was prepared according to the procedure of Example 4, and the results are shown in Table 5.

[0110] Table 5. Yields of wild type and single mutant calcifediol

[0111]

[0112] Although the present application has been disclosed with reference to the preferred embodiments, it is to be understood that various other adaptations and modifications are to be considered as falling within the scope of the present application, as defined in the appended claims.

Claims

1. A P450 enzyme mutant, characterized in that, The mutant is formed by mutating glutamine at position 73 of the P450 enzyme CYP109E1, as shown in SEQ ID NO.1, to methionine, and glycine at position 81 to tryptophan.

2. The gene encoding the P450 enzyme mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Microbial cells carrying the gene of claim 2 or the recombinant vector of claim 3.

5. A recombinant Escherichia coli, characterized in that, The P450 enzyme mutant of claim 1 was expressed.

6. A method for preparing calcidiol, characterized in that, The crude enzyme solution containing ferroreductin, ferroreductin reductase, and glucose dehydrogenase was mixed with any one of substances (a) to (c) in a solution containing the cofactor NADP. + The reaction occurs in a system of glucose, hydroxypropyl-β-cyclodextrin, and vitamin D3; where: (a) The P450 enzyme mutant of claim 1; (b) The microbial cell of claim 4; (c) The recombinant Escherichia coli of claim 5; The method for preparing the crude enzyme solution containing ferroreductin, ferroreductin reductase and glucose dehydrogenase is as follows: recombinant Escherichia coli containing a three-enzyme co-expression system is cultured for a period of time, and the crude enzyme solution containing ferroreductin, ferroreductin reductase and glucose dehydrogenase is collected. The recombinant Escherichia coli containing the three-enzyme co-expression system is a recombinant Escherichia coli that co-expresses ferroreductin, ferroreductin reductase and glucose dehydrogenase using pRSFDuet-1 as the expression vector.

7. The use of the P450 enzyme mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, the microbial cell of claim 4, or the recombinant Escherichia coli of claim 5 in the preparation of calcidiol.

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