CN114875077B - Method for synthesizing alfacalcidol and calcitriol by catalyzing and hydroxylating vitamin D3 through oxidase - Google Patents

Method for synthesizing alfacalcidol and calcitriol by catalyzing and hydroxylating vitamin D3 through oxidase Download PDF

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CN114875077B
CN114875077B CN202210401260.4A CN202210401260A CN114875077B CN 114875077 B CN114875077 B CN 114875077B CN 202210401260 A CN202210401260 A CN 202210401260A CN 114875077 B CN114875077 B CN 114875077B
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peroxidase
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alfacalcidol
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CN114875077A (en
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张武元
李元滢
袁波
孙周通
苏文成
马延和
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention researches and obtains two enzyme catalytic synthesis methods for synthesizing alfacalcidol and calcitriol by hydroxylation of oxidase independent of coenzyme, which can respectively and directly prepare alfacalcidol and calcitriol by a one-pot one-step method and a two-step method, and the hydroxy derivative of vitamin D3 can be used as an intermediate or a raw material medicine of various medicines. The preparation method has the advantages of simple preparation process, high yield and simple downstream treatment process, and no solvent except water and acetone (or other organic cosolvents) is used in the preparation process. The method is a green biosynthesis method, and has higher atom economy and step economy.

Description

Method for synthesizing alfacalcidol and calcitriol by catalyzing and hydroxylating vitamin D3 through oxidase
Technical Field
The invention belongs to the technical field of biocatalysis, and particularly relates to an enzyme catalysis synthesis method of two active vitamin D3 (alfacalcidol and calcitriol) compounds.
Background
Vitamin D3 is a steroid organic compound, soluble in fat, and has multiple effects in vivo. The largest potential source of vitamin D3 (VD 3) is the endogenous two-step process of ultraviolet-B (UVB) photolysis and thermal conversion of 7-dehydrocholesterol by sunlight irradiation of the skin. However, the content of vitamin D3 as a source of solar radiation varies depending on geographical location, season, skin pigmentation, lifestyle and environmental factors. Furthermore, solar radiation is often limited due to solar safety issues. Vitamin D is under-supplied, particularly in the absence of endogenous synthesis, in a large part of the population. Thus, many health care providers recommend vitamin D supplements. Since the discovery of the metabolic pathway of vitamin D3, numerous metabolites of VD3 and their biological effects have been reported. Vitamin D3, which is formed in the skin or ingested through the diet, enters the in vivo circulation, binds to D3 binding proteins, undergoes many enzymatic changes (mainly in the liver and kidneys) and becomes a biologically active form of vitamin D3 (i.e. 25-hydroxyvitamin D3 and calcitriol). The hydroxylated form 25 (OH) D3 of vitamin D3 may have five times the biological activity of vitamin D3, with 1, 25-dihydroxyvitamin D3 (calcitriol) being the predominant form and 500-1000 times more active than 25-hydroxyvitamin D3. Calcitriol is approved as one of the first-choice medicines for treating osteoporosis by world health organization, can promote absorption of calcium and blood sugar by small intestinal mucosa and renal tubule, and simultaneously cooperates with parathyroid hormone to improve release of old bone to phosphoric acid, so as to maintain and regulate normal concentration of calcium and phosphorus in blood plasma, and can also deposit calcium on the parts where new bone is required to be formed, promote osteoblast function and maturation of bone-like tissue. At present, vitamin D3 in China is in a state of supply and demand, and an efficient green synthetic route is urgently needed to be searched for to meet market demands. The synthesis method of calcitriol is also quite numerous, and mainly comprises two major types, namely a chemical method and a biological method. Chemical synthesis of calcitriol has been reported in many cases, but although the sources of the synthetic raw materials are different, convergent synthesis is mostly adopted, and the final purpose is to obtain 7-dehydrocholesterol. The chemical synthesis method has the defects of overlong reaction period, harsh conditions, low yield, high cost and unfavorable industrial production, so that the search of a green and efficient active vitamin D3 synthesis route has important significance.
Disclosure of Invention
The invention aims to provide a method for synthesizing alfacalcidol in one step by catalyzing with oxidase from vitamin D3 and synthesizing calcitriol in two steps by catalyzing with two oxidase cascades. Hydroxylation is carried out by using hydrogen peroxide dependent oxidase as a biocatalyst, and vitamin D3 is selected as a substrate to synthesize alfacalcidol and calcitriol. The method of the invention can be used for respectively adding a certain amount of enzyme catalyst, substrate (vitamin D3) and peroxide solution into a 10-40% acetone solution system at room temperature, reacting for a period of time, extracting, and drying to obtain the alfacalcidol or calcitriol.
The peroxidase is Pada-I. To increase the expression level in the host cell, the V57A/L67F/V75I/I248V/F311L amino acid in the sequence of SEQ ID NO.1 was mutated to obtain Pada-I peroxidase. To further investigate the effect of Pada-I peroxidase on vitamin D3. The Pada-I peroxidase mutant is constructed by rationally designing amino acid residues around a peroxidase activity pocket.
The invention firstly provides a preparation method for synthesizing vitamin D3 metabolite alfacalcidol, which comprises the following steps:
vitamin D3 shown in the formula I is used as a reaction substrate, and the alfacalcidol shown in the formula II is obtained by reacting in an acetone-buffer reaction system in the presence of peroxide and Pada-I peroxidase mutant. Wherein the Pada-I peroxidase mutant is obtained by rationally designing amino acid residues around a Pada-I peroxidase activity pocket.
Wherein,, the Pada-I-peroxidase mutant is one of the following mutations F234S, F234D, G284D, R300K, F S/G284D, F S/G284K, F K/G284K, F D/G284D, F S/G284D/R300K, F S/G284S/R300K, F S/G284K/R300K, F S/G284D/R300S, F234D/G284S/R300K, F S/G284D/R300D, F K/G234D/G300D/R300K, F234S/G284K/R300S, F S/G284D/R300K/G195S, F234S/G284D/R300K/A73F/G195S, F S/G284D/R300K/A73S, F D/G300K/A73L/G195S, F S/G284D/R300K/A77S, F S/G284D/R300K/A73L/A77S, F S/G284D/R300K/A73F/G195S, F S/G284D/R300K/A73L/G195S, F S/G284D/R300K/G195W.
The invention further provides an enzyme catalysis preparation method for synthesizing vitamin D3 metabolite calcitriol, which comprises the following steps:
vitamin D3 shown in a formula I is used as a reaction substrate, and reacts in an acetone-buffer reaction system in the presence of peroxide and Pada-I peroxidase mutant to obtain alfacalcidol shown in a formula II, and Pada-I peroxidase and peroxide are continuously added to obtain calcitriol shown in a formula III, wherein the Pada-I peroxidase mutant is mutated on the basis of amino acid shown in SEQ ID NO. 2.
Wherein,, the Pada-I peroxidase mutant is one of the following mutations F234S, F234D, G284D, R S/G284D, F S/G284K, F K/G284K, F D/G284D, F S/G284D/R300K, F S/G284S/R300K, F S/G284K/R300K, F S/G284D/R300S, F D/G284S/R300K, F S/G284D/R300D, F K/G234K/G D/G300K, F S/G284K/R300S, F S/G284D/R300K/G195S, F234S/G284D/R300K/A73F/G195S, F S/G284D/R300K/A73S, F D/G300K/A73L/G195S, F S/G284D/R300K/A77S, F S/G284D/R300K/A73L/A77S, F S/G284D/R300K/A73F/G195S, F S/G284D/R300K/A73L/G195S, F S/G284D/R300K/G195W.
The Pada-I peroxidase has substitution mutation in V57, L67, V75, I248 and F311 based on the amino acid shown in SEQ ID NO.1, preferably has mutation in V57A/L67F/V75I/I248V/F311, and more preferably has the amino acid sequence shown in SEQ ID NO. 2.
In the reaction system, the concentration of the substrate vitamin D3 can be 0.5mmol/L to 50mmol/L, and can be specifically 0.5mmol/L to 20mmol/L;
the reaction cosolvent can be one or a mixture of more of acetone, methanol, ethanol and ethyl acetate;
the reaction cosolvent accounts for 20-100% of the volume of the reaction system;
the buffer solution can be phosphate buffer, citrate buffer, tris-HCl, tris-H 2 SO 4 Any one of the buffers has a pH of 7, specifically 5-10;
in a specific embodiment, the peroxide is hydrogen peroxide or an organic peroxide; the organic peroxide is specifically selected from at least one of tert-butyl peroxide, tetrahydrofuran peroxide, benzoyl peroxide, ethylene glycol dimethyl ether peroxide, methyl ethyl ketone peroxide, peracetylnitrate, triperoxide, dioxirane and derivatives thereof, diethyl ether peroxide, peracetic acid and cumene hydroperoxide.
The peroxide concentration may be 0.3-2mmol/L, more specifically 1mmol/L,2mmol/L.
The peroxidases include Pada-I peroxidase mutants and Pada-I peroxidases which do not require the provision of cofactors for catalysis, but rather use monooxygenase to catalyze the transfer of a monooxygen atom from peroxide (H 2 O 2 ROOH) selectively transferred to a different target molecule.
The peroxidase plays a catalytic role in the form of crude enzyme powder, crude enzyme liquid or pure enzyme;
the concentration of peroxidase in the reaction system may be 50nmol/L to 50umol/L, specifically 200nmol/L to 5000nmol/L (0.02 to 04U/ml,1 enzyme activity unit means an amount of enzyme capable of converting 1. Mu. Mole of substrate in 1 minute under the optimum condition (25 ℃) or an amount of enzyme capable of converting 1. Mu. Mole of the relevant group in the substrate).
The reaction time of the reaction may be 1 to 48 hours and the reaction temperature may be 15 to 60 ℃.
After the reaction, the reaction solution is extracted by an organic solvent, dried, filtered and detected and analyzed by reverse liquid chromatography.
The invention researches and obtains two enzyme catalytic synthesis methods for synthesizing alfacalcidol and calcitriol by hydroxylation of oxidase independent of coenzyme, which can respectively and directly prepare alfacalcidol and calcitriol by a one-pot one-step method and a two-step method, and the hydroxy derivative of vitamin D3 can be used as an intermediate or a raw material medicine of various medicines. The preparation method has the advantages of simple preparation process, high yield and simple downstream treatment process, and no solvent except water and acetone (or other organic cosolvents) is used in the preparation process. The method is a green biosynthesis method, and has higher atom economy and step economy.
Drawings
Figure 1 time-concentration profile of vitamin D3 hydroxylation to alfacalcidol. Reaction conditions: [ Pada-I ]]=5μm, [ vitamin D3]=2.5 mM NaPi buffer (100 mM, pH 7), 40% (v/v) acetone as cosolvent, [ H ] 2 O 2 ]＝1mM/h，24h，800rpm,Total＝1mL
Figure 2 time-concentration profile of calcitriol produced by hydroxylation of vitamin D3. Reaction conditions: [ Pada-I ]]＝25μM,[Pada-I]=5μm, [ vitamin D3]=2.5 mM NaPi buffer (100 mM, pH 7), 40% (v/v) acetone as cosolvent, [ H ] 2 O 2 ]＝1mM/h，24h，800rpm,Total＝1mL。
Figure 3 HPLC diagram of vitamin D3 hydroxylated alfacalcidol.
Figure 4 HPLC profile of vitamin D3 hydroxylation to calcitriol.
Detailed Description
The invention will be further described in connection with specific examples which are not to be construed as limiting the invention.
Example 1: pada-I peroxidase preparation
Based on Agrocybe aegerita-derived peroxidase (SEQ ID NO. 1), V57A/L67F/V75I/I248V/F311L site mutation was performed on the basis of Agrocybeaegerita oxidase to form Pada-I peroxidase (SEQ ID NO. 2), the Pada-I oxidase nucleic acid sequence was subcloned into a pPICZ alpha A vector, pPICZ alpha A-Pada-I recombinant plasmid was transformed into P.pastoris strain X-33, 100. Mu.L competent cells were taken and linearized plasmid DNA was added for electrotransformation, and the cells were plated on YPD medium plates (containing 100. Mu.g/mL bleomycin) and were cultured at 30℃for 2 days. After single colony grows on YPD plate, picking single colony, then PCR colony screening and verification, according to positive result, transferring into triangular flask containing 50mLBMGY culture medium, 30 deg.C, 200rpm, culturing to OD 600 =1-1.5; collecting bacteria at 4000rpm for 5 min; the pellet was resuspended to OD with BMMY 600 =0.3 (about 100-200 mL); transfer to 500mL Erlenmeyer flask, start induction expression culture at 30℃and 200rpm for 3 days, add 3% methanol every 24h.
The bacterial solution was centrifuged at 4000rpm at 4℃for 20 minutes, and the supernatant (i.e., pada-I crude enzyme solution) was collected. The supernatant was combined on a HisTrap HP pre-packed column pre-equilibrated with buffer A (20 mM Tris-HCl, pH=7.0) at a flow rate of 1.5mL/min. 20 column volumes (20 mM Tris-HCl,20mM Imidazole,NaCl pH =7.0) were washed with wash buffer B, and 5 column volumes were eluted with elution buffer B (20 mM Tris-HCl,400mM Imidazole,NaCl pH =7.0). Collecting and obtaining the target protein. The target protein eluate was concentrated using ultrafiltration tubing and its buffer was replaced with size exclusion chromatography buffer (NaPi buffer).
The concentrated protein is further passed through molecular sieve200 Increase 10/300 GL) at a flow rate of 0.5mL/min. And (5) collecting target proteins by separate pipes. The target protein was obtained and its activity was measured using ABTS.
ABTS assay of mada-I peroxidase activity: adding 1uL sample into 200uL ABTS mixed solution, measuring absorbance at 25deg.C and 420nm with enzyme labeling instrument, measuring every 5s, calculating slope in linear change interval, adding into formula to calculate enzyme activity,wherein V is sample Representing the volume of enzyme liquid to be detected; epsilon molar absorbance value; d is the diameter of the cuvette; v (V) total Representing the total volume of the measurement system; e (E) w The absorbance of the sample was calculated to be 0.02U/ml.
ABTS mixed solution: pH4.4 citrate-phosphate buffer, 1mM ABTS, 1mM H 2 O 2 。
Example 2: preparation of Pada-I peroxidase mutant
(1) Construction of mutants
In order to increase the substrate holding capacity of the peroxidase, two mutation units are designed aiming at amino acids in a catalytic pocket of the Pada-I peroxidase by taking a plasmid pPIC9K-Pada-I as a template, a mutation library is constructed for F234, G284 and R300, the mutation library is mutated into Ser, asp and Lys respectively, and a primer is designed as shown in Table 1.
Mutant library 2 was constructed for mutants of Ala73, ala77 and Gly195, mutated to Leu, trp and Phe, respectively. The target DNA fragments and plasmid fragments are amplified by PCR, and mutant libraries are constructed by recombinant reaction.
TABLE 1 mutant library 1 primer design
PCR reaction systems were prepared according to Table 2 and PCR was performed on the insert and linearized vector, with the insert primers mixed according to the proportions of the primers of Table 1.
TABLE 2 PCR reaction System
The insert and linearized vector were PCR-set according to the PCR conditions of Table 3.
TABLE 3PCR reaction temperature procedure
The above-mentioned insert and linearized vector were purified, formulated according to the reaction system of Table 4, and left at 37℃for 2 hours.
TABLE 4 recombination reaction System
Component (A) Recombination reactions
Linearization carrier 400ng
Insertion fragment 120ng
5 Xbuffer 2μL
Recombinant enzyme 1μL
Water and its preparation method Up to 10μL
The recombinant reaction product was transformed into E.coli competent cells TOP10, and the product was plated on a plate containing solid medium (containing 100. Mu.g/mL ampicillin) and incubated overnight at 37 ℃. The plasmid was scraped and DNA sequencing was performed to verify site mutation.
(2) Mutant screening
Linearization of mutant plasmids. 10. Mu.g of plasmid was taken, added to a final concentration of 1 Xcutmart buffer and 10U of restriction enzyme SalI, and reacted at 37℃for 4 hours. The linearized plasmid was recovered using a DNA purification kit and 0.5-1. Mu.g of the linearized plasmid was transformed into 100. Mu.L of yeast competent cells GS115 (competent cell manufacturing methods see appendix A). Cells were plated on MD plates and incubated at 30℃for 2d. Monoclonal cells were picked up to 1mL of BMGY medium (2%Tryptone,1%Yeast Extract,100mM potassium phosphate buffer pH 6.0,0.34G/L YNB, 400. Mu.g/L Biotin,1% D-glucose) (containing 1mg/mL G418) and incubated at 30℃for 24h at 800 rpm.
BMGY medium was replaced with BMMY medium (2%Tryptone,1%Yeast Extract,100mM potassium phosphate buffer pH 6.0,0.34G/L YNB, 400. Mu.g/L Biotin,3% methanol) (1 mg/mL G418, 20mg/L hemoglobin) and incubated at 30℃at 800rpm for 24 hours. 3% methanol was added every 24 hours, and the culture was continued for 72 hours. Centrifugation at 3000rpm for 10min, the fermentation broth and cells were separated. 1mL of the fermentation broth was taken, added with 2mM hydrogen peroxide and 10mM vitamin D3 (substrate) as final concentrations, and left to react at 30℃at 800rpm for 6 hours, during which 2mM hydrogen peroxide was added every 1 hour.
The conversion of the substrate to α -calcitol was detected by GC and the mutants obtained were screened and their activity changes are shown in table 5.
TABLE 5 mutant conversion
From the above results, it was found that the Pada-I-F234S/G284D/R300K conversion rate was the highest, so that mutant pool 2 was constructed based on the mutant.
(3) Construction of mutant library 2 based on the results of mutant library 1
The primers are shown in Table 6.
TABLE 6 mutant conversion
Construction of the mutant library and screening are described in examples 1 (2) and (3). The screening gave the following table 7:
TABLE 7 mutant conversion
Mutation site Conversion rate
Pada-I-F234S/G284D/R300K/G195L 59.32
Pada-I-F234S/G284D/R300K/A73F/G195F 60.38
Pada-I-F234S/G284D/R300K/A73F 56.32
Pada-I-F234S/G284D/R300K/A73L/G195L 32.52
Pada-I-F234S/G284D/R300K/G195F 28.38
Pada-I-F234S/G284D/R300K/A77F 40.23
Pada-I-F234S/G284D/R300K/A73W 12.35
Pada-I-F234S/G284D/R300K/A73L/A77L 25.25
Pada-I-F234S/G284D/R300K/A73F/A77L 14.32
Pada-I-F234S/G284D/R300K/A73F/G195F 19.56
Pada-I-F234S/G284D/R300K/A73L 35.26
Pada-I-F234S/G284D/R300K/A73L/G195F 34.21
Pada-I-F234S/G284D/R300K/G195W 32.56
The Pada-I-F234S/G284D/R300K/A73F/G195F was found to have the highest conversion.
Example 3: preparation of the compound alfacalcidol:
based on the above experimental results, pada-I-F234S/G284D/R300K/A73F/G195F peroxidase mutant was obtained in the same manner as in example 1, and oxidized to alpha calcitol using D3 as a substrate.
The structural formula of the compound alfacalcidol is as follows:
in a 1mL reaction flask, 550. Mu.l of phosphate buffer solution (pH=7) was added, 350. Mu.l of acetone was added, 50. Mu.l of vitamin D3 in acetone (2.5 mmol/L, wherein vitamin D3 stock solution was prepared at a concentration of 50 mmol/L) was added, and 50. Mu.l of peroxidase (Pada-I mutant, 500 nmol/L) was added. The concentration of hydrogen peroxide in the charged system was 1.0mmol/L.
The reaction system was reacted in a shaker at 30℃for 24 hours. After the reaction, 100. Mu.l of the reaction mixture was taken, 200. Mu.l of ethyl acetate was added thereto, followed by extraction, drying over anhydrous sodium sulfate, and detection and analysis by liquid chromatography. The supernatant was subjected to liquid chromatography (HPLC) to detect calcitriol content. The HPLC detection conditions were: chromatographic column: shim-pack GIST-C18Shim-pack GIST-C18.6X1250 mm. Times.5. Mu.M; mobile phase: water: acetonitrile=45:55; flow rate: 1mL/min; the detection wavelength is 265nm. The standard substance is alfacalcidol (available in Shanghai Ala Biochemical technology Co., ltd.)Company limited), the corresponding compound concentrations were calculated using an alfacalcidol standard curve (y=305951.7475x+15946). After the liquid phase detection is finished, the molar conversion rate is calculated according to the peak area of the liquid phase chromatogram, and the conversion rate (%) =a Alfacalcidol /(A Alfacalcidol +A Vitamin D3 ) Conversion was 45% and the product conversion versus time curve is shown in figure 1. As the reaction time increases, the concentration of alfacalcidol produced increases.
Example 4: preparation of compound alfacalcidol by changing substrate concentration and enzyme concentration
Into a 1mL reaction flask, 525. Mu.l of a phosphate buffer solution (pH=7) was added, 300. Mu.l of acetone was added, 100. Mu.l of an acetone solution of vitamin D3 (5 mmol/L in which a vitamin D3 stock solution was prepared at a concentration of 50 mmol/L) was added, and 75. Mu.l of a 500nmol/LPada-I peroxidase mutant (Pada-I-F234S/G284D/R300K/A73F/G195F) was added to give a final concentration of 500nmol/L. The concentration of hydrogen peroxide in the charged system was 1.5mmol/L.
The reaction system was reacted in a shaker at 30℃for 24 hours. After the completion of the reaction, 100. Mu.l of the reaction mixture was taken, 200. Mu.l of ethyl acetate was added, the mixture was extracted, dried over anhydrous sodium sulfate, and analyzed by liquid chromatography to detect the product and substrate in the same manner as in example 1. Conversion (%) =a Alfacalcidol /(A Alfacalcidol +A Vitamin D3 ) The conversion rate is 77%.
Example 5: preparation of calcitriol:
because the Pada-I peroxidase mutant can oxidize D3 as a substrate into alpha calcitol, but Pada-I peroxidase cannot, but Pada-I peroxidase can further oxidize alpha calcitol as a substrate to generate calcitriol, so that two enzymes form cascade connection, and the synthesis from D3 directly to calcitriol can be realized.
The structural formula of the calcitriol compound is as follows:
into a 1mL reaction flask, 550. Mu.l of a phosphate buffer solution (pH=7) was added, 350. Mu.l of acetone was added, 50. Mu.l of an acetone solution of vitamin D3 (2.5 mmol/L) was added, and 50. Mu.l of Pada-I peroxidase mutant (Pada-I-F234S/G284D/R300K/A73F/G195F) was added to give a final concentration of 500nmol/L. The concentration of hydrogen peroxide in the system was 1.0mmol/L, and the reaction was stopped for three hours. After continuing the reaction for 1 hour, 75. Mu.l of Pada-I peroxidase (500 nmol/L) was added to the reaction system, and the concentration of hydrogen peroxide in the system was continued to be 1.0mmol/L, to continue the reaction.
The reaction system was reacted in a shaker at 30℃for 24 hours. After the completion of the reaction, 100. Mu.l of the reaction mixture was taken, 200. Mu.l of ethyl acetate was added, the mixture was extracted, dried over anhydrous sodium sulfate, and analyzed by liquid chromatography, the product and the substrate were detected in the same manner as in example 1, and the yield was 59%.
The product conversion versus time curve is shown in figure 2. As the reaction time increases, the concentration of alfacalcidol produced increases.
HPLC of vitamin D3 hydroxylation to calcitriol is shown in FIG. 4.
Example 6: preparation of the Compound calcitriol in 10ml reaction System
Into a 10mL reaction flask, 600. Mu.l of a phosphate buffer solution (pH=7.8) was added, 300. Mu.l of acetone was added, 100. Mu.l of an acetone solution (5 mmol/L) of vitamin D3 was added, and 50. Mu.l of Pada-I peroxidase mutant (Pada-I-F234S/G284D/R300K/A73F/G195F) was added to give a final concentration of 500nmol/L. The concentration of hydrogen peroxide in the system was 1.5mmol/L, and the reaction was stopped for three hours. After continuing the reaction for 1 hour, 75. Mu.l of Pada-I peroxidase (500 nmol/L) was added to the reaction system, and the concentration of hydrogen peroxide in the system was continued to be 1.0mmol/L, to continue the reaction.
The reaction system was reacted in a shaker at 30℃for 24 hours. After the reaction, 100. Mu.l of the reaction mixture was taken, 200. Mu.l of ethyl acetate was added thereto, followed by extraction, drying over anhydrous sodium sulfate, and detection and analysis by liquid chromatography. The product and substrate were tested as in example 1, conversion (%) =a Calcitriol /(A Calcitriol +A Vitamin D3) ) The conversion was 63%.
Example 5: preparation of the Compound calcitriol 100mL
Into a 100mL reaction flask, 550 mL of a phosphate buffer solution (pH=8.2) was added, 350 mL of acetone was added, 50. Mu.L of an acetone solution (10 mmol/L) of vitamin D3 was added, and 75. Mu.L of Pada-I peroxidase mutant (Pada-I-F234S/G284D/R300K/A73F/G195F) was added to give a final concentration of 500nmol/L. The concentration of hydrogen peroxide in the system was 2.0mmol/L, and the reaction was stopped for three hours. After continuing the reaction for 1 hour, 100. Mu.l of Pada-I peroxidase (500 nmol/L) was added to the reaction system, and the concentration of hydrogen peroxide in the system was continued to be 2.0mmol/L, to continue the reaction.
The reaction system was reacted in a shaker at 30℃for 24 hours. After the completion of the reaction, 100. Mu.l of the reaction mixture was taken, 200. Mu.l of ethyl acetate was added, the mixture was extracted, dried over anhydrous sodium sulfate, and analyzed by liquid chromatography to detect the product and substrate in the same manner as in example 1. As can be seen from the experimental results shown in FIG. 2, the concentration of calcitriol formed increases with the increase of the reaction time. Conversion (%) =a Calcitriol /A Calcitriol +A Vitamin D3 The conversion was 57%.
While the above examples are merely preferred embodiments of the present invention, it will be understood by those skilled in the art that the examples are not intended to be exemplary, and do not limit the scope of the present invention. It is intended that all technical solutions according to the present invention and their inventive concepts, and their details or forms be equally substituted or altered within the scope of the present invention by those skilled in the art.
The invention provides an enzyme catalytic synthesis method for synthesizing alfacalcidol and calcitriol by hydroxylation of two oxidases. Vitamin D3 is used as a substrate to synthesize an alfacalcidol compound, and hydrogen peroxide and peroxidase are used for catalytic oxidation. The method comprises the steps of sequentially adding a certain amount of buffer solution, hydrogen peroxide, vitamin D3 and peroxidase into an acetone solution system at room temperature, reacting for a period of time, extracting, and drying to obtain the alfacalcidol compound.
In addition, the calcitriol compound is directly prepared by a one-pot two-step method. The method comprises the steps of sequentially adding a certain amount of buffer solution, hydrogen peroxide, vitamin D3 and peroxidase (Pada-I enzyme and Pada-I enzyme) into an acetone solution system at room temperature, reacting for a period of time, extracting, and drying to obtain the calcitriol compound. In the preparation process, no solvent is used except water and acetone. The method belongs to a green preparation method, has higher atom economy utilization, simple preparation process and high conversion rate.
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> method for synthesizing alfacalcidol and calcitriol by catalyzing and hydroxylating vitamin D3 with oxidase
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 371
<212> PRT
<213> column-shaped agrocybe cylindracea (Agrocybe aegerita)
<400> 1
MKYFPLFPTLVYAVGVVAFPDYASLAGLSQQELDAIIPTLEAREPGLPPGPLENSSAKLVNDEAHPWKPLRPGDIRGPCPGLNTLASHGYLPRNGVATPVQIINAVQEGLNFDNQAAVFATYAAHLVDGNLITDLLSIGRKTRLTGPDPPPPASVGGLNEHGTFEGDASMTRGDAFFGNNHDFNETLFEQLVDYSNRFGGGKYNLTVAGELRFKRIQDSIATNPNFSFVDFRFFTAYGETTFPANLFVDGRRDDGQLDMDAARSFFQFSRMPDDFFRAPSPRSGTGVEVVIQAHPMQPGRNVGKINSYTVDPTSSDFSTPCLMYEKFVNITVKSLYPNPTVQLRKALNTNLDFFFQGVAAGCTQVFPYGRD 371
<210> 2
<211> 371
<212> PRT
<213> artificial sequence
<400> 2
MKYFPLFPTLVYAVGVVAFPDYASLAGLSQQELDAIIPTLEAREPGLPPGPLENSSAKLVNDEAHPWKPLRPGDIRGPCPGLNTLASHGYLPRNGVATPAQIINAVQEGFNFDNQAAIFATYAAHLVDGNLITDLLSIGRKTRLTGPDPPPPASVGGLNEHGTFEGDASMTRGDAFFGNNHDFNETLFEQLVDYSNRFGGGKYNLTVAGELRFKRIQDSIATNPNFSFVDFRFFTAYGETTFPANLFVDGRRDDGQLDMDAARSFFQFSRMPDDFFRAPSPRSGTGVEVVVQAHPMQPGRNVGKINSYTVDPTSSDFSTPCLMYEKFVNITVKSLYPNPTVQLRKALNTNLDFLFQGVAAGCTQVFPYGRD 371

Claims (10)

1. A preparation method of an alfacalcidol which is a synthesized vitamin D3 metabolite, comprising the following steps: vitamin D3 is used as a reaction substrate, in an acetone-phosphate buffer solution reaction system with the pH value of 7, hydrogen peroxide and Pada-I-peroxidase mutant exist for reaction to obtain alfacalcidol,
wherein the Pada-I-peroxidase mutant is obtained by carrying out one of the following mutations F234S, F234D, G284D, R, K, F S/G284D, F S/G284K, F K/G284K, F D/G284D, F S/G284D/R300K, F S/G284S/R300K, F234S/G284K/R300K, F S/G284D/R300S, F234D/G284S/R300K, F S/G284D/R300D, F234K/G284D/R300K, F234S/G284K/R300S, F234S/G284D/R300K/A77F on the basis of the amino acid shown in SEQ ID NO. 2.
2. The enzymatic preparation method of the calcitriol as a synthesized vitamin D3 metabolite comprises the following steps:
reacting vitamin D3 serving as a reaction substrate in an acetone-phosphate buffer solution reaction system with the pH value of 7 in the presence of hydrogen peroxide and Pada-I peroxidase mutant to obtain alfacalcidol, and continuously adding Pada-I peroxidase and hydrogen peroxide to obtain calcitriol;
wherein the Pada-I peroxidase mutant is obtained by carrying out one of the following mutations F234S, F234D, G284D, R K, F S/G284D, F S/G284K, F K/G284K, F D/G284D, F S/G284D/R300K, F S/G284S/R300K, F234S/G284K/R300K, F234S/G284D/R300S, F234D/G284S/R300K, F S/G284D/R300D, F234K/G284D/R300K, F234S/G284K/R300S, F234S/G284D/R300K/A77F on the basis of the amino acid shown in SEQ ID NO. 2;
the amino acid sequence of the Pada-I peroxidase is shown as SEQ ID NO. 2.
3. The method according to claim 1 or 2, wherein the concentration of the substrate vitamin D3 in the reaction system is 0.5mmol/L to 50 mmol/L.
4. A method of preparation as claimed in claim 3 wherein the substrate vitamin D3 concentration is 0.5-20mmol/L.
5. The process according to claim 1 or 2, wherein the acetone as a reaction cosolvent is 20% to 40% of the volume of the reaction system.
6. The method of claim 1, wherein the hydrogen peroxide concentration is 0.3-2 mmol/L.
7. The process according to claim 1, wherein the Pada-I-peroxidase or the Pada-I-peroxidase mutant has a concentration of 50nmol/L to 50umol/L in the reaction system.
8. The process according to claim 7, wherein the Pada-I-peroxidase or the Pada-I-peroxidase mutant has a concentration of 200nmol/L to 5000nmol/L in the reaction system.
9. The process of claim 8, wherein the reaction time is 1-48h and the reaction temperature is 15-60 ℃.
10. The method according to claim 1 or 2, wherein the reaction mixture is subjected to reverse liquid chromatography detection analysis by extracting the reaction mixture with an organic solvent, drying, and filtering.
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CN111247234A (en) * 2017-11-13 2020-06-05 宝洁公司 Method of cleaning a surface having a fatty acid containing soil and consumer product composition for use in the method
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