CN114507648B - P450 enzyme mutant and application thereof - Google Patents
P450 enzyme mutant and application thereof Download PDFInfo
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- CN114507648B CN114507648B CN202210289700.1A CN202210289700A CN114507648B CN 114507648 B CN114507648 B CN 114507648B CN 202210289700 A CN202210289700 A CN 202210289700A CN 114507648 B CN114507648 B CN 114507648B
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- C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
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Abstract
The invention discloses a catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 The P450 enzyme mutant is formed by respectively mutating 75 th amino acid of P450 enzyme with an amino acid sequence shown as SEQ ID NO.1 from leucine to alanine or glycine or threonine or histidine, and the amino acid sequence is shown as SEQ ID NO.2-NO. 5. The invention also discloses a preparation method of the P450 enzyme mutant and a preparation method of the P450 enzyme mutant in the production of 25-hydroxy vitamin D 3 Is used in the field of applications. Experiments prove that the P450 enzyme mutant catalyzes vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 The highest efficiency of (2) is 3.1 times as high as that of P450BM3F 87A. And, the whole production of 25-hydroxyvitamin D 3 The method has the advantages of simple operation process, mature process, environmental friendliness and wide application prospect.
Description
Technical Field
The invention belongs to the technical fields of enzyme engineering, biocatalysis, biochemistry and molecular biology, and relates to a catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 P450 enzyme mutants of (c) and uses thereof.
Background
Vitamin D 3 (vitamin D 3 Cholecalciferol) is a steroid derivative, and is a fat-soluble vitamin playing an important role in the vital activities such as growth and development, calcium-phosphorus metabolism, immunity, cell proliferation and differentiation of human beings and mammals. In the human body, vitamin D 3 Is prepared from 7-dehydrocholesterol in skin by exposing to ultraviolet. Vitamin D 3 The product has no bioactivity, and is required to be converted into a physiologically active form of 25-hydroxy vitamin D by oxidation of cytochrome P450 enzyme in human body 3 (calcitol) and 1 alpha, 25-dihydroxyvitamin D 3 (calcitriol) and thus plays a role in regulating the above-mentioned important vital activities. Due to 25-hydroxy vitamin D 3 Can promote the absorption of the organism to calcium and phosphorus and regulate immunity, so that the medicine is also an important medicine for treating rickets, osteoporosis, thyroid gland function decline and other diseases in clinic. Meanwhile, the method is widely applied to the fields of food health care, livestock breeding and the like, and has high economic value.
Industrial production of 25-hydroxy vitamin D 3 The method mainly adopts a chemical synthesis strategy, and the yield is lower than 1% due to the complex multi-step reactions such as group protection and deprotection, illumination, ring opening, isomerization and the like in a synthesis route, so that the method has high cost and is not environment-friendly. In contrast, the bioconversion method has simple reaction steps, mild reaction conditions and specific product configuration, and is a technical means with great application prospect. Wherein the microorganism strain obtained by screening from natural environment has been successfully applied to vitamin D 3 To the active form 25-hydroxyvitamin D 3 Is produced by the method. However, the wild type microorganism strain often has the limitations of unknown genetic background, difficult transformation, poor cell permeability, organic solvent intolerance and the like. Because cytochrome P450 enzyme can catalyze the selective oxidation of inert carbon-hydrogen bonds of a substrate under mild conditions, the catalyst has the advantage that a chemical catalyst is difficult to compare, and is an important source of the biocatalyst for catalyzing the oxidation reaction industrially at present. Currently, catalytic vitamin D has been found 3 The P450 catalytic system for realizing 25-position hydroxylation reaction mainly comprises a two-component system consisting of eukaryotic P450 enzyme and independent reductase (CPR) and a three-component system consisting of bacterial P450 enzyme and a pair of redox chaperones, but both systems have the restriction factors of low electron transfer efficiency, low protein expression level, complex components and the like.
Applicants' studies have found that if expressed inMonocomponent P450 monooxygenase with high content, known crystal structure and high catalytic efficiency is used as parent enzyme, and directed evolution technology is adopted to screen out catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 Is expected to realize the improvement of the conversion efficiency and the reduction of 25-hydroxy vitamin D 3 And (5) production cost. In P450BM3 from Bacillus megaterium (Bacillus megaterium), its P450 domain is naturally fused to its CPR domain, being a catalytic autonomous P450 enzyme. The crystal structure of P450BM3 is known and has high catalytic efficiency, and the kcat value of the catalytic natural substrate hydroxylation reaction is as high as 17000min -1 . However, the literature and patent search shows that no P450BM3 wild or mutant is used for catalyzing vitamin D at home and abroad 3 Synthesis of 25-hydroxy vitamin D 3 Is reported in (3).
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 P450 enzyme mutants of (c) and uses thereof.
The invention relates to a catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 The P450 enzyme mutant of (2), characterized in that: the P450 enzyme mutants are named as P450BM3F 87A/L75A, P BM3F 87A/L75G, P BM3F87A/L75T, P BM3F 87A/L75H; wherein the P450BM3F 87A/L75A is formed by mutating the 75 th amino acid of P450 enzyme with an amino acid sequence shown as SEQ ID NO.1 from leucine to alanine, and the amino acid sequence is shown as SEQ ID NO. 2; the P450BM3F 87A/L75G is formed by mutating the 75 th amino acid of P450 enzyme with an amino acid sequence shown as SEQ ID NO.1 from leucine to glycine, and the amino acid sequence is shown as SEQ ID NO. 3; the P450BM3F 87A/L75T is formed by mutating the 75 th amino acid of P450 enzyme with an amino acid sequence shown as SEQ ID NO.1 from leucine to threonine, and the amino acid sequence is shown as SEQ ID NO. 4; the P450BM3F 87A/L75H is formed by mutating the 75 th amino acid of P450 enzyme with an amino acid sequence shown as SEQ ID NO.1 from leucine to histidine, and the amino acid sequence of the P450 enzyme is shown as SEQ ID NO. 5.
The invention is described inCatalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 The preparation method of the P450 enzyme mutant comprises the following steps:
(1) PCR amplification is carried out by taking a P450BM3F87A gene with a nucleotide sequence shown as SEQ ID NO.6 as a template and taking L75A-F/L75A-R or L75G-F/L75G-R or L75T-F/L75T-R or L75H-F/L75H-R as primers, so as to sequentially obtain PCR products named P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H; then, taking an escherichia coli expression vector pET30a as a vector, respectively constructing expression vectors carrying the PCR product genes P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H; the constructed expression vector pET30a-P450 BM3F 87A/L75A or pET30a-P450 BM3F 87A/L75G or pET30a-P450 BM3F87A/L75T or pET30a-P450 BM3F87A/L75H is respectively transformed into escherichia coli BL21 (DE 3) chemocompetence, and the correspondingly obtained transformant is named BL21 (DE 3) -pET30a-P450 BM3F 87A/L75A or BL21 (DE 3) -pET30a-P450 BM3F 87A/L75G or BL21 (DE 3) -pET30a-P450 BM3F87A/L75T or BL21 (DE 3) -pET30a-P450 BM3F87A/L75H respectively;
wherein, the nucleotide sequences of the above primers are respectively:
L75A-F：CAAGCGGCTAAATTTGTACGTGATTTT
L75A-R：AAATTTAGCCGCTTGACTTAAGTTTTT
L75G-F：CAAGCGGGGAAATTTGTACGTGATTTT
L75G-R：AAATTTCCCCGCTTGACTTAAGTTTTT
L75T-F：CAAGCGACGAAATTTGTACGTGATTTT
L75T-R：AAATTTCGTCGCTTGACTTAAGTTTTT
L75H-F：CAAGCGCATAAATTTGTACGTGATTTT
L75H-R：AAATTTATGCGCTTGACTTAAGTTTTT
(2) The four obtained transformants were inoculated into LB liquid medium containing 50mg/L kanamycin, and cultured overnight at 37℃and 220rpm to prepare four fermentation seed solutions; inoculating the obtained four fermentation seed solutions into TB culture medium, and culturing at 37deg.C and 220rpm to OD 600 After=1.0±0.1, IPTG of 0.2mM was added to the fermentation broth,fermenting and culturing at 18 ℃ and 180rpm for 24 hours; respectively centrifuging the cultured bacterial liquid, discarding the supernatant, collecting the bacterial cells to obtain four protein-producing bacterial cells, namely BL21 (DE 3) -pET30a-P450 BM3F 87A/L75A or BL21 (DE 3) -pET30a-P450 BM3F 87A/L75G or BL21 (DE 3) -pET30a-P450 BM3F87A/L75T or BL21 (DE 3) -pET30a-P450 BM3F87A/L75H, and freezing at the temperature of minus 80 ℃;
(3) Respectively carrying out ultrasonic crushing on the four protein-producing thalli, then carrying out nickel column affinity chromatography purification to obtain P450 enzyme mutant purified proteins, respectively named P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H, freezing with liquid nitrogen, and preserving at-80 ℃.
The invention provides a group of recombinant expression vectors capable of expressing the P450 enzyme mutant as claimed in claim 1, which is characterized in that: the recombinant expression vector is pET30a-P450 BM3F 87A/L75A, pET a-P450 BM3F 87A/L75G, pET a-P450 BM3F87A/L75T or pET30a-P450 BM3F87A/L75H respectively; or pET28b-P450 BM3F 87A/L75A, pET b-P450 BM3F 87A/L75G, pET b-P450 BM3F87A/L75T or pET28b-P450 BM3F 87A/L75H.
The invention provides a group of genetically engineered bacteria capable of expressing the P450 enzyme mutant as claimed in claim 1, which is characterized in that: the genetically engineered bacteria are escherichia coli respectively transformed by recombinant expression vectors of the P450 enzyme mutants.
Wherein: the genetic engineering bacteria are preferably BL21 (DE 3) -pET30a-P450 BM3F 87A/L75A, BL (DE 3) -pET30a-P450 BM3F 87A/L75G, BL (DE 3) -pET30a-P450 BM3F87A/L75T or BL21 (DE 3) -pET30a-P450 BM3F87A/L75H respectively.
The invention relates to a catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 P450 enzyme mutants (P450 BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H) in the production of 25-hydroxyvitamin D 3 Is used in the field of applications.
Wherein: p450 enzyme mutants and vitamin D in said applications 3 Reaction of (a) to produce 25-hydroxyvitamin D 3 The 100. Mu.L enzyme reaction system of (2) was: 3+ -0.5 mu M P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H,50020 mu M vitamin D 3 5.+ -. 1. Mu.M glucose dehydrogenase GDH, 1.+ -. 0.2mM reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), 10.+ -. 2mM glucose, and reacted at 30.+ -. 2 ℃ for 12.+ -. 2 hours. The preferred embodiments are: p450 enzyme mutants and vitamin D in said applications 3 Reaction of (a) to produce 25-hydroxyvitamin D 3 The 100. Mu.L enzyme reaction system of (2) was: 3 mu M P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H 500 mu M vitamin D 3 mu.M glucose dehydrogenase GDH,1mM reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), 10mM glucose, and at 30℃for 12 hours.
The invention discloses a catalytic vitamin D 3 Specific synthesis of 25-hydroxyvitamin D 3 P450 enzyme mutants of (c) and uses thereof. The inventor obtains a kind of catalytic vitamin D by modifying P450BM3F87A through a directed evolution strategy 3 Specific synthesis of 25-hydroxyvitamin D 3 Is a mutant of (2): p450BM3F 87A/L75A, P BM3F 87A/L75G, P BM3F87A/L75T and P450BM3F 87A/L75H. Experiments prove that the vitamin D catalyst 3 Synthesis of 25-hydroxy vitamin D 3 The catalytic efficiency of (a) was 3.1, 2.6, 1.9 and 1.8 times that of the starting enzyme P450BM3F87A, respectively. The P450 enzyme mutant has the advantages of high catalytic efficiency, high protein expression, low industrial cost and the like. Whole catalytic vitamin D 3 Specific synthesis of 25-hydroxyvitamin D 3 The method has the advantages of simple operation process, mature process, low cost, no harmful impurities, no toxicity and environmental friendliness. The invention provides a catalytic vitamin D 3 Specific synthesis of 25-hydroxyvitamin D 3 The P450 enzyme mutant of (C) is in 25-hydroxy vitamin D 3 Has wide application prospect in the preparation industry, and the product is expected to be applied to the fields of medicine, food and agriculture.
Drawings
FIG. 1 shows vitamin D according to the invention 3 And 25-hydroxy vitamin D 3 Is a chemical structure diagram of (a).
FIG. 2 shows that the P450BM3F87A, P BM3F 87A/L75A, P BM3F 87A/L75G, P450BM3F 87A/L75T or P450BM3F 87A/L75HCatalytic vitamin D 3 The resulting product 25-hydroxyvitamin D 3 And 1 alpha, 25-dihydroxyvitamin D 3 HPLC diagram of (2).
FIG. 3 shows that the P450BM3F87A, P BM3F 87A/L75A, P BM3F 87A/L75G, P450BM3F 87A/L75T or P450BM3F 87A/L75H catalyzes vitamin D 3 Production of 25-hydroxyvitamin D 3 Is a graph of the catalytic activity analysis of (a).
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
In the examples described below, materials, reagents, plasmids, strains, kits and the like were obtained commercially unless otherwise specified.
Among them, the strain Escherichia coli BL (DE 3) competent cells used in the examples of the present invention were purchased from Peking's family, and the E.coli expression vector pET30a was purchased from Invitrogen. P450BM 3/F87A (P450 BM3F 87A) is a single mutant F87A of P450BM3, and the nucleotide sequence of the gene P450BM3F87A is shown in SEQ ID NO. 6.
The medium used in the examples:
LB medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl;
TB medium: 12g/L tryptone, 24g/L yeast extract, 40g/L glycerol, K 2 HPO 4 9.4g/L，KH 2 PO 4 2.2g/L。
Protein purification buffer used in the examples:
lysis buffer (ph=8.0): naH (NaH) 2 PO 4 6g/L, 17.532g/L NaCl, 100g/L glycerol and 0.6808g/L imidazole.
Wash buffer (ph=8.0): naH (NaH) 2 PO 4 6g/L NaCl 17.532g/L glycerol100g/L of oil and 1.3616g/L of imidazole.
Elution buffer (ph=8.0): naH (NaH) 2 PO 4 6g/L, 17.532g/L NaCl, 100g/L glycerol and 17.02g/L imidazole.
Desalination buffer (ph=7.4) NaH 2 PO 4 6g/L, glycerin 100g/L.
Reagents used in the examples:
the one-step cloning kit used in the examples of the present invention was purchased from the company Nanjinouzan; agarose gel DNA recovery kits and plasmid extraction kits were all purchased from Omega company; high fidelity DNA polymerase was purchased from Takara; restriction enzymes were purchased from sirolimus; PCR primer synthesis and DNA sequencing were completed by Shanghai workers; vitamin D 3 25-hydroxy vitamin D 3 And 1 alpha, 25-dihydroxyvitamin D 3 Purchased from Solarbio and Sigma-Aldrich.
The apparatus used in the examples:
PCR amplification apparatus (Eppendorf), high-speed refrigerated centrifuge (Eppendorf), agarose gel electrophoresis apparatus (BIO-RAD), agarose gel imaging system (Shanghai Technical Co., ltd.), constant temperature shaker (Jingqi), spectraMAX M2 multifunctional enzyme labeling apparatus (Molecular Devices Co.).
EXAMPLE 1 construction of P450BM3 mutant library
According to the crystal structure of P450BM3 complex with natural fatty acid substrate N-palmitoylglycine (PDB: 1 JPZ), 11 key amino acid residues (L75, V78, F81, A82, A180, L181, A184, L188, A328, A330, I263) within 5 angstrom of the active pocket of the substrate are selected and allocated into 6 groups according to the nearest principle, in turn A (L75, V78), B (F81, A82), C (A180, L181), D (A184, L188), E (A328, A330) and F (I263). The P450BM3F87A is used as a starting template, and the mature degenerate NDT codon technology is adopted to carry out the combined mutation of 12 different amino acids, so as to construct a mutant library with the coverage rate of more than 95% and the library capacity of more than 2000.
The method comprises the following specific steps: the gene with the nucleotide sequence of SEQ ID NO.6 is used as a template, and A-F/A-R or B-F/B-R or C-F/C-R or D-F/D-R or E-F/E-R or F-F/F-R is respectively used as a primer for PCR amplification. PCR procedure: 5X PrimeSTAR GXL Buffer. Mu.L, 200. Mu.M dNTPss, upstream and downstream primers are 0.3 mu M respectively, a proper amount of DNA template (10-100 ng), high-fidelity polymerase (PrimeSTAR GXL DNA polymerase) 2.5U, ddH 2 O is added to 50 mu L; the reaction conditions are as follows: pre-denaturation at 98℃for 5min, denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 68℃for 4min, reaction for 30 cycles, and extension at 68℃for 10min. After the PCR product was purified by the nucleic acid purification kit, the recovered PCR product was digested with restriction enzyme DpnI at 37℃for 3 hours to remove template DNA, and an expression plasmid was obtained using the one-step cloning kit and then directly electrotransformed into competent cells of E.coli BL21 (DE 3) to construct a mutant library.
Randomly selecting 400-450 single colonies of A, B, C, D, E or F libraries respectively, and inoculating into a sterile 96-deep well plate in 300 mu L LB liquid medium containing 50 mu g/mL kanamycin. Single colony cultures of the A library, the B library, the C library, the D library, the E library or the F library are cultured overnight at 37 ℃ and 220rpm, 40% glycerol with equal volume and no bacteria is added, and the mixture is frozen at-80 ℃ to complete the construction of the P450BM3 mutant library.
Table 1: primer sequences used in the examples
Example 2 screening of mutant library and confirmation of highly active transformed vitamin D 3 Production of 25-hydroxyvitamin D 3 Mutant of (2)
The P450BM3 mutant bacterial solutions of example 1 stored in 96 deep well plates, either pool A or B or C or pool D or E or F, were inoculated into sterile 96 deep well plates in 300. Mu.L LB liquid medium containing 50. Mu.g/mL kanamycin, respectively. The cultures were incubated overnight at 37℃and 220rpm, and 40. Mu.L of the bacterial liquid per well was transferred as seed liquid into a new sterile 96-well plate containing 400. Mu.L of TB liquid medium containing 50. Mu.g/mL kanamycin.
After the medium was cultured at 37℃and 220rpm for a while (OD 600 =1.0 or so), isopropyl- β -dithiogalactopyranoside (IPTG) at a final concentration of 0.2mM and 5-aminolevulinic acid (5-ALA) at 0.5mM were added to each individual well and inCulturing at 18deg.C and 230rpm for 24 hr to obtain mutant protein thallus of library A, B, C, D, E or F. The supernatant was decanted by centrifugation at 3700 Xg for 10 minutes and stored at-80℃for later use.
Mutant protein thalli of either library A or B or C or D or E or F were thawed and resuspended in 200. Mu.L of 50mM sodium phosphate buffer (pH=7.4) containing 100mg/L lysozyme, 300U/mL DNaseI and 10% Triton X-100. After 30 min incubation at 30℃centrifugation was performed and the supernatant was transferred to a new shallow 96-well plate containing 200. Mu.M vitamin D 3 1mM reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), 10mM glucose, 5. Mu.M GDH. The reaction was carried out at 30℃for 12 hours. The reaction was then quenched by the addition of 300 μl of ethyl acetate and the mixture was shaken at 30deg.C for 5min for organic extraction. The mixture was centrifuged at 3700 Xg for 10min, 200. Mu.L of the organic phase was pipetted into a new 96 shallow well plate, dried with nitrogen, redissolved in 100. Mu.L of methanol, centrifuged at high speed and the samples were analyzed by HPLC or LC-MS.
HPLC analysis procedure: 80% methanol-100% methanol, 25min, 1mL/min flow rate, and 265nm detection wavelength.
By comparison with standard substance and control group (P450 BM3F 87A), high activity transformed vitamin D is obtained 3 Production of 25-hydroxyvitamin D 3 The P450 enzyme mutant is named P450BM3F 87A/L75H, is formed by mutating the 75 th amino acid of P450 enzyme with an amino acid sequence shown as SEQ ID NO.1 from leucine to histidine, and has an amino acid sequence shown as SEQ ID NO. 5. The acquisition of the active mutant P450BM3F 87A/L75H means that the 75 th amino acid catalyzes vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 Has important functions.
EXAMPLE 3 construction of L75 saturated mutant library
In the screening process of the mutant library of example 2, the mutant P450BM3F 87A/L75H at the L75 position is found to have higher catalytic activity, and the site (L75) is subjected to saturation mutation in consideration of the fact that only 12 amino acid residues can be derived from the NDT degenerate codon, so that the catalytic activity comparison of 20 amino acids at the site is realized.
Saturation mutagenesis is performed by using mature degenerate NNK codon technology, and the specific steps are as follows: PCR amplification was performed using the gene of which nucleotide sequence was SEQ ID NO.6 as a template and L75-F/L75-R as a primer (Table 2). PCR procedure: 5X PrimeSTAR GXL Buffer. Mu.L, 200. Mu.M dNTPs, 0.3. Mu.M each for upstream and downstream primers, appropriate amount of DNA template (10-100 ng), 2.5U for high-fidelity polymerase (PrimeSTAR GXL DNA polymerase), ddH 2 O is added to 50 mu L; the reaction conditions are as follows: pre-denaturation at 98℃for 5min, denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 68℃for 4min, reaction for 35 cycles, and extension at 68℃for 10min. The PCR product was purified by a nucleic acid purification kit, and the recovered PCR product was digested with DpnI at 37℃for 3 hours to remove the template DNA, and an expression plasmid was obtained using a one-step cloning kit and then directly transformed into competent cells of E.coli BL21 (DE 3) to construct a mutant library.
Randomly selecting 40-50 single colonies, inoculating the single colonies into 3mL LB liquid medium containing 50 mug/mL kanamycin for overnight culture, collecting the single colony thalli selected above for sequencing, adding the mutant bacterial liquid of 20 different amino acids at the L75 position with accurate sequencing into sterilized 40% glycerol with equal volume, freezing at-80 ℃ and completing the construction of the L75 position saturated mutant library.
Table 2: primer sequences used in example 3
EXAMPLE 4 fermentative expression of mutant proteins, nickel column affinity chromatography purification and protein concentration determination
The L75 saturated mutant strain frozen at-80℃in example 3 was removed from the glycerol tube, subjected to three-compartment streak activation on a kana-resistant LB plate, and cultured in a 37℃incubator for 12-16 hours; picking single colony, inoculating into 50mL LB liquid medium containing kanamycin, culturing at 37 ℃ and 220rpm for 12-16h; transferring the cultured seed liquid into 500mL of TB liquid culture medium containing kanamycin according to the volume ratio of 1:100, and culturing for 3-4h at 37 ℃ and 220 rpm; waiting for OD 600 At 0.8-1.0, IPTG was added to a final concentration0.15mM, 5-ALA and VB1 were added to a final concentration of 0.5mM, and protein expression was induced at 18℃and 180rpm for 20-24h; centrifugation is carried out at 6000rpm at 4 ℃ for 10min, L75 saturated mutant thalli are respectively collected and frozen at-80 ℃.
Protein purification
Taking out frozen L75 saturated mutant thalli from the temperature of-80 ℃, melting at room temperature, adding 35mL of lysis buffer solution into the thalli collected according to each 1L of fermentation liquid, and re-suspending on ice; ultrasonic crushing on ice: 25% power, 5s working, 5s intermittent, about 30-35min working until the solution is clear, centrifuging the crushed suspension at 4 ℃ and 10000rpm for 60min, collecting supernatant, adding 1-2mL nickel column (Ni-NTA) resin, and incubating at 4 ℃ for 1-2h; placing the uniformly mixed solution into a protein separation column, and adding about 5 column volumes of cleaning buffer solution for cleaning until no protein flows out by using G250 staining solution; eluting the target protein from the nickel column by adopting 5-10mL of elution buffer solution and collecting the target protein; concentrating with Millipore ultrafiltration tube (30 kDa) at 4deg.C and 5500rpm until the volume of target protein eluate is 1-2mL; firstly, balancing a PD-10 desalting column of GE Healthcare by using 5 times of desalting buffer solution, then adding concentrated protein eluent into the desalting column, adding the desalting buffer solution for eluting after the sample liquid completely enters the column, collecting eluted protein, and concentrating to 1-2mL by centrifugal ultrafiltration; mixing the concentrated proteins uniformly, subpackaging 100 mu L of the proteins in each tube, quick-freezing the proteins by liquid nitrogen, and respectively storing the L75 saturated mutants at-80 ℃.
Protein concentration determination
The corresponding P450 protein concentration determination was performed with reference to the reported CO differential spectroscopy.
To prepare a ferrous complex of each enzyme reduced with sodium dithionite, 50-80. Mu. L P450 enzyme (purified L75 saturated mutant protein above) was separately taken and desalted with buffer (pH 7.4, 50mM NaH) 2 PO 4 10% glycerol) was diluted to 900 μl and CO gas was slowly passed through the sample in a fume hood. Transferring the P450 sample saturated by CO into a cuvette, and placing the cuvette in a spectrophotometer for full-wavelength scanning at 350-500 nm; the cuvette was removed and a suitable amount of sodium hydrosulfite (Na 2 S 2 O 4 ) Scanning the whole wavelength again; according toThe absorbance values measured twice are used for obtaining the P450 active concentration according to the absorbance difference between 450nm and 490nm, and the formula is as follows: dilution factor X (. DELTA.A450-. DELTA.A490)/0.091 in. Mu.M.
Example 5 quantitative analysis and application of L75 saturated mutant
The L75 saturated mutant proteins obtained in example 4 were each reacted according to the following reaction system: 3 mu M P450 enzyme, 500 mu M vitamin D 3 1mM reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), 10mM glucose, 5. Mu.M GDH, 30.+ -. 2 ℃ for 12.+ -. 2 hours. Adding equal volume of methanol to terminate reaction, centrifuging at high speed, collecting supernatant, performing HPLC analysis, and determining vitamin D 3 Consumption of 25-hydroxy vitamin D 3 The amount of the produced product was quantitatively analyzed.
Catalyzing vitamin D by an initial template enzyme (P450 BM3F 87A) 3 Synthesis of 25-hydroxy vitamin D 3 Is used for obtaining the catalytic vitamin D 3 Specific production of 25-hydroxyvitamin D 3 P450 enzyme mutants with activities increased to 3.1, 2.6, 1.9, 1.8-fold, respectively, designated as P450BM3F 87A/L75A, P450BM3F 87A/L75G, P450BM3F 87A/L75T, P450BM3F 87A/L75H, respectively (FIGS. 2 and 3). Experiments prove that the obtained P450 enzyme mutant can actually catalyze vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 Meaning that the invention provides this class of catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 P450 enzyme mutant of (C) in the production of 25-hydroxy vitamin D 3 Has great application prospect.
Example 6 preparation of P450 enzyme mutant P450BM3F 87A/L75A, P450BM3F 87A/L75G, P450BM3F 87A/L75T, P450BM3F 87A/L75H by:
(1) PCR amplification is carried out by taking a P450BM3F87A gene with a nucleotide sequence shown as SEQ ID NO.6 as a template and taking L75A-F/L75A-R or L75G-F/L75G-R or L75T-F/L75T-R or L75H-F/L75H-R as primers, so as to sequentially obtain PCR products named P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H; then, taking an escherichia coli expression vector pET30a as a vector, respectively constructing expression vectors carrying the PCR product genes P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H; the constructed expression vector pET30a-P450 BM3F 87A/L75A or pET30a-P450 BM3F 87A/L75G or pET30a-P450 BM3F87A/L75T or pET30a-P450 BM3F87A/L75H is respectively transformed into escherichia coli BL21 (DE 3) chemocompetence, and the correspondingly obtained transformant is named BL21 (DE 3) -pET30a-P450 BM3F 87A/L75A or BL21 (DE 3) -pET30a-P450 BM3F 87A/L75G or BL21 (DE 3) -pET30a-P450 BM3F87A/L75T or BL21 (DE 3) -pET30a-P450 BM3F87A/L75H respectively;
wherein, the nucleotide sequences of the above primers are respectively:
L75A-F：CAAGCGGCTAAATTTGTACGTGATTTT
L75A-R：AAATTTAGCCGCTTGACTTAAGTTTTT
L75G-F：CAAGCGGGGAAATTTGTACGTGATTTT
L75G-R：AAATTTCCCCGCTTGACTTAAGTTTTT
L75T-F：CAAGCGACGAAATTTGTACGTGATTTT
L75T-R：AAATTTCGTCGCTTGACTTAAGTTTTT
L75H-F：CAAGCGCATAAATTTGTACGTGATTTT
L75H-R：AAATTTATGCGCTTGACTTAAGTTTTT
(2) Four transformants BL21 (DE 3) -pET30a-P450 BM3F 87A/L75A or BL21 (DE 3) -pET30a-P450 BM3F 87A/L75G or BL21 (DE 3) -pET30a-P450 BM3F87A/L75T or BL21 (DE 3) -pET30a-P450 BM3F87A/L75H obtained as described above were inoculated into 50mL LB liquid medium (containing 50mg/L kanamycin) respectively, cultured overnight at 37℃and 220rpm, respectively, to obtain four fermentation seed solutions; the four seed solutions obtained were inoculated in an inoculum size of 1:50 volume ratio into a medium containing 1L TB medium (containing 50mg/L kanamycin, 1mM vitamin B) 1 ) In a 3L fermentation flask at 37℃and 220rpm to OD 600 After about 1.0, 0.2mM IPTG was added to the fermentation broth, and the fermentation broth was incubated at 18℃for 24 hours at 180 rpm; centrifuging the cultured bacterial solutions at 600 rpm and 4deg.C for 10min, pouring out the supernatant, and collecting bacterial bodies to obtain four protein-producing bacterial bodies, which are BL21 (DE 3) -pET30a-P450 BM3F 87A/L75A or BL21 (DE 3) -pET30a-P450 BM3F 87A/L75G or BL21 (DE 3) -pET30a-P450 BM3F87A/L75T or B respectivelyL21 (DE 3) -pET30a-P450 BM3F87A/L75H was frozen at-80 ℃.
(3) Respectively carrying out ultrasonic crushing on four protein-producing thalli, and then carrying out nickel column affinity chromatography purification to obtain P450 enzyme mutant purified proteins, wherein the purified proteins are P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H respectively, and then carrying out split charging, liquid nitrogen freezing and preserving at the temperature of minus 80 ℃.
(4) Protein concentration measurement of P450 enzyme 50-80 mu L P enzyme P450BM3F 87A/L75A or P450BM3F 87A/L75G or P450BM3F 87A/L75T or P450BM3F 87A/L75H was diluted to 900. Mu.L with desalting buffer and CO gas was slowly passed through the sample in a fume hood; transferring the P450 sample saturated by CO into a cuvette, and placing the cuvette in a spectrophotometer for full-wavelength scanning at 350-500 nm; the cuvette was removed and a suitable amount of sodium hydrosulfite (Na 2 S 2 O 4 ) Scanning the whole wavelength again; and according to the absorbance values measured twice, the P450 active concentration is obtained according to the absorbance difference between 450nm and 490nm, and the formula is as follows: dilution factor X (. DELTA.A450-. DELTA.A490)/0.091 in. Mu.M.
Sequence list
<110> university of Shandong
<120> P450 enzyme mutant and application thereof
<141> 2022-03-17
<160> 6
<210> 1
<211> 1049
<212> PRT
<213> Bacillus megaterium (Bacillus megaterium)
<221> amino acid sequence of Gene P450BM3F87A
<222> （1）…（1049）
<400> 1
MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK 60
EACDESRFDK NLSQALKFVR DFAGDGLATS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM 120
VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR 180
ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN 240
GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV 300
DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ 360
LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK 420
HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN 480
TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH 540
PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD 600
RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH 660
GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG 720
LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE 780
LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE 840
KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI 900
MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT 960
LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 1020
VHQVSEADAR LWLQQLEEKG RYAKDVWAG 1049
<210> 2
<211> 1049
<212> PRT
<213> Bacillus megaterium (Bacillus megaterium)
<221> amino acid sequence of Gene P450BM3F 87A/L75A
<222> （1）…（1049）
<400> 2
MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK 60
EACDESRFDK NLSQAAKFVR DFAGDGLATS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM 120
VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR 180
ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN 240
GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV 300
DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ 360
LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK 420
HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN 480
TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH 540
PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD 600
RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH 660
GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG 720
LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE 780
LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE 840
KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI 900
MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT 960
LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 1020
VHQVSEADAR LWLQQLEEKG RYAKDVWAG 1049
<210> 3
<211> 1049
<212> PRT
<213> Bacillus megaterium (Bacillus megaterium)
<221> amino acid sequence of Gene P450BM3F 87A/L75G
<222> （1）…（1049）
<400> 3
MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK 60
EACDESRFDK NLSQAGKFVR DFAGDGLATS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM 120
VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR 180
ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN 240
GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV 300
DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ 360
LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK 420
HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN 480
TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH 540
PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD 600
RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH 660
GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG 720
LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE 780
LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE 840
KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI 900
MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT 960
LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 1020
VHQVSEADAR LWLQQLEEKG RYAKDVWAG 1049
<210> 4
<211> 1049
<212> PRT
<213> Bacillus megaterium (Bacillus megaterium)
<221> amino acid sequence of Gene P450BM3F 87A/L75T
<222> （1）…（1049）
<400> 4
MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK 60
EACDESRFDK NLSQATKFVR DFAGDGLATS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM 120
VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR 180
ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN 240
GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV 300
DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ 360
LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK 420
HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN 480
TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH 540
PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD 600
RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH 660
GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG 720
LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE 780
LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE 840
KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI 900
MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT 960
LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 1020
VHQVSEADAR LWLQQLEEKG RYAKDVWAG 1049
<210> 5
<211> 1049
<212> PRT
<213> Bacillus megaterium (Bacillus megaterium)
<221> amino acid sequence of Gene P450BM3F 87A/L75H
<222> （1）…（1049）
<400> 5
MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK 60
EACDESRFDK NLSQAHKFVR DFAGDGLATS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM 120
VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR 180
ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN 240
GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV 300
DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ 360
LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK 420
HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN 480
TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH 540
PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD 600
RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH 660
GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG 720
LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE 780
LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE 840
KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI 900
MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT 960
LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 1020
VHQVSEADAR LWLQQLEEKG RYAKDVWAG 1049
<210> 6
<211> 3150
<212> DNA
<213> Bacillus megaterium (Bacillus megaterium)
<221> nucleotide sequence of Gene P450BM3F87A
<222>（1）…（3150）
<400> 6
atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60
ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120
tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180
gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240
gattttgcag gagacgggtt agcaacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300
cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360
gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420
gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480
tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540
gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600
gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660
attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720
ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780
acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840
ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900
gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaaac 960
gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020
gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080
cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140
gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200
tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260
cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320
aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380
tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440
acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500
ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560
gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620
ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680
aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740
aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800
cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860
atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920
tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980
ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040
agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100
ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160
ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220
ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280
acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340
cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400
atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460
cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520
aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580
tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640
tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700
atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760
ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820
catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880
cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940
gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000
ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060
gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120
cgatacgcaa aagacgtgtg ggctgggtaa 3150
Claims (3)
1. Catalytic vitamin D 3 Synthesis of 25-hydroxy vitamin D 3 P450 enzyme mutant of (C) in the production of 25-hydroxy vitamin D 3 Is applied to the application of the system; wherein the amino acid sequence of the P450 enzyme mutant is shown as SEQ ID NO.2, or as SEQ ID NO.3, or as SEQID No.4, or SEQ ID No. 5.
2. The use according to claim 1, characterized in that: p450 enzyme mutants and vitamin D in said applications 3 Reaction of (a) to produce 25-hydroxyvitamin D 3 The 100. Mu.L enzyme reaction system of (2) was: 3. 0.5 mu M of the P450 enzyme mutant, 500 + -20 mu M vitamin D 3 5.+ -. 1. Mu.M glucose dehydrogenase GDH, 1.+ -. 0.2. 0.2mM reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), 10.+ -. 2. 2mM glucose, and at 30.+ -. 2 ℃ for 12.+ -. 2 hours.
3. The use according to claim 2, characterized in that: p450 enzyme mutants and vitamin D in said applications 3 Reaction of (a) to produce 25-hydroxyvitamin D 3 The 100. Mu.L enzyme reaction system of (2) was: 3. mu M of the P450 enzyme mutant, 500 mu M vitamin D 3 5. Mu.M glucose dehydrogenase GDH,1mM reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), 10mM glucose, and reacted at 30℃for 12 hours.
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