Method for synthesizing calcifediol using peroxidase

Abstract

The present invention provides a method for synthesizing calcifediol by using peroxidase, comprising the following steps: using peroxidase SEQ ID NO: 1 or SEQ ID NO: 3 to catalyze the hydroxylation reaction of the 25th position of vitamin D3 in the presence of a H2O2 donor to obtain calcifediol.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to a method for synthesizing calcifediol by utilizing peroxidase catalysis. Background Art

[0002] Calcifediol, also known as 25-OH vitamin D3 (abbreviated as 25(OH)VD3), L-2-calcidiol, L-piperidinic acid or L-2-piperidinic acid, is the first metabolically active substance of vitamin D3 (abbreviated as VD3) and the main metabolic form of VD3. Calcifediol is of great significance to human health and has a very positive therapeutic effect on certain diseases such as osteoporosis, osteomalacia, hyperglycemia, chronic kidney disease, etc. In addition, it also performs well as a feed additive for poultry and some other farm animals, which can strengthen animal bones and reduce the mortality rate of newborn animals.

[0003] The preparation methods of calcifediol are divided into chemical synthesis and biocatalysis. The chemical synthesis method has complicated steps, harsh reaction conditions, and produces many toxic and harmful by-products. It also has problems such as poor product stereoselectivity, low conversion rate (yield of about 30%), and high cost. On the contrary, the biocatalysis method has the advantages of good reaction stereoselectivity, short steps, mild reaction conditions, and low cost, and has gradually become the focus of research and development.

[0004] At present, the simplest and most direct way to synthesize calcifediol is to hydroxylate at the C25 position of vitamin D3, that is, to add an O atom to the CH bond. It is difficult to achieve the position-specific hydroxylation reaction of this specific position by chemical methods, while biological methods can achieve this process through certain enzyme catalysis, among which cytochrome P450 monooxygenase is a type of hydroxylase, and it has been reported that multiple P450 enzymes such as CYP105A1, CYP109A2, CYP109E1, Vdh, etc. can achieve the catalytic process of VD3 to calcifediol. The inventor has reported in patent document CN202111098209.2 the research work of preparing calcifediol by selective hydroxylation reaction of VD3 using P450 enzymes. However, the P450 enzyme catalysis method also has certain limitations. The reaction process requires expensive NADPH as a coenzyme, and the catalytic substrate concentration is low, and the poor position selectivity of some P450 enzyme varieties leads to the easy production of other by-products, and the catalysis time is long, and the industrial production process conditions need to be optimized in many aspects. Summary of the invention

[0005] In order to explore new enzyme-catalyzed reaction pathways for converting VD3 to calcifediol, the inventors further studied the feasibility of using peroxidase (abbreviated as UPO) to catalyze the synthesis of calcifediol. The UPO family generally has two major defects: one is that the heterologous expression level is not high or even not expressed, and the other is that it is intolerant to H2O2. Through a large number of analyses and comparisons, the inventors screened out a non-specific peroxidase CciUPO from Coprinopsis cinerea from peroxidases from many sources. Experiments have found that it has high position specificity for the C25 hydroxylation of VD3, and relatively high tolerance to H2O2. Further, through genetic engineering technology, the heterologous expression of CciUPO in Pichia pastoris was achieved, thus laying the foundation for the catalytic synthesis of calcifediol by CciUPO.

[0006]

[0007] In order to improve the catalytic activity of peroxidase CciUPO in hydroxylation of VD3, an attempt was made to mutate it, and a mutant with improved enzyme activity was obtained. Accordingly, the present invention includes the following technical solutions.

[0008] A method for preparing calcifediol by enzyme catalysis comprises the following steps: using peroxidase SEQ ID NO: 1 (NCBI accession number: XM_001831858) or its (L64A, V82I, T255V) mutant SEQ ID NO: 3 to catalyze the hydroxylation reaction of vitamin D3 at position 25 to obtain calcifediol.

[0009] Wherein, SEQ ID NO: 1 is the amino acid sequence of the wild-type peroxidase (CciUPO) from Coprinopsis cinerea:

[0010] MISTSKHLFVLLPLFLVSHLSLVLGFPAYASLGGLTERQVEEYTSKLPIVFPPPPPEPIKDPWLKLVNDRAHPWRPLRRGDVRGPCPGLNTLASHGYLPRDGVATPAQIITAVQEGFNMEYGIATFVTYAAHLVDGNPLTNLISIGGKTRKTGPDPPPPAIVGGLNTHAVFEGDASMTRGDFHLGDNFNFNQTLWEQFKDYSNRYGGGRYNLTAAAELRWARIQQSMATNGQFDFTSPRYFTAYAESVFPINFFTDGRLFTSNTTAPGPDMDSALSFFRDHRYPKDFHRAPVPSGARGLDVVAAAYPIQPGYNADGKVNNYVLDPTSADFTKFCLLYENFVLKTVKGLYPNPKGFLRKALETNLEYFYQSFPGSGGCPQVFPWGKSD(SEQ ID NO:1);

[0011] The amino acid sequence of its (L64A, V82I, T255V) mutant is SEQ ID NO:3:

[0012] MISTSKHLFVLLPLFLVSHLSLVLGFPAYASLGGLTERQVEEYTSKLPIVFPPPPPEPIKDPW A KLVNDRAHPWRPLRRGD I RGPCPGLNTLASHGYLPRDGVATPAQIITAVQEGFNMEYGIATFVTYAAHLVDGNPLTNLISIGGKTRKTGPDPPPPAIVGGLNTHAVFEGDASMTRGDFHLGDNFNFNQTLWEQFKDYSNRYGGGRYNLTAAAELRWARIQQSMATNGQFDFTSPRYFTAYAESVFPINFF V DGRLFTSNTTAPGPDMDSALSFFRDHRYPKDFHRAPVPSGARGLDVVAAAYPIQPGYNADGKVNNYVLDPTSADFTKFCLLYENFVLKTVKGLYPNPKGFLRKALETNLEYFYQSFPGSGGCPQVFPWGKSD(SEQ ID NO:3).

[0013] Preferably, an H2O2 donor may also be present in the reaction system. The H2O2 donor may be glucose and glucose oxidase.

[0014] More preferably, the above method uses peroxidase SEQ ID NO: 3 as a biocatalyst to catalyze the reaction of calcifediol.

[0015] In an optional embodiment, the peroxidase SEQ ID NO: 1 or its mutant SEQ ID NO: 3 may be in the form of a microbial cell expressing the peroxidase.

[0016] The microorganism is any microorganism suitable for expressing the peroxidase SEQ ID NO: 1 or its mutant SEQ ID NO: 3, including bacteria and yeast. The yeast is preferably Pichia pastoris.

[0017] The reaction system is preferably a buffer solution, for example, a phosphate buffer solution with a pH of 7.0-9.5, preferably a phosphate buffer solution with a pH of 8.0-9.2, and more preferably a phosphate buffer solution with a pH of 8.2-9.0.

[0018] The reaction temperature may be 25-45°C, preferably 30-12°C, more preferably about 40°C.

[0019] Preferably, a co-solvent may be added to the above reaction system to promote the dissolution of vitamin D3 in an aqueous solution, such as DMSO, acetone, ethanol, and 2-hydroxypropyl-β-cyclodextrin.

[0020] The second aspect of the present invention provides a peroxidase, whose amino acid sequence is SEQ ID NO:3.

[0021] The third aspect of the present invention provides a gene encoding the above peroxidase.

[0022] Preferably, the nucleotide sequence of the gene encoding the peroxidase SEQ ID NO:3 is SEQ ID NO:4.

[0023] The fourth aspect of the present invention provides a microorganism, which expresses the above-mentioned peroxidase SEQ ID NO: 1 or SEQ ID NO: 3. The microorganism can be directly used as a biocatalyst for the production of calcifediol.

[0024] The peroxidase SEQ ID NO:1 and its mutant SEQ ID NO:3 screened by the present invention can selectively promote the hydroxylation reaction of vitamin D3 at position 25 to synthesize calcifediol, providing a new route for producing calcifediol. The fermentation liquid of strain CM4 expressing SEQ ID NO:3 can catalyze 5g / L of substrate to achieve a conversion rate of more than 80% within 3 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1HPLC profiles showing the effects of different temperatures on the peroxidase-catalyzed VD3 reaction.

[0026] Figure 2 HPLC profiles showing the effects of different pH on the peroxidase-catalyzed VD3 reaction.

[0027] Figure 3 HPLC profiles showing the effect of different H2O2 donor concentrations on the peroxidase-catalyzed VD3 reaction.

[0028] Figure 4 The HPLC profile of the substrate cosolvent 2-hydroxypropyl-β-cyclodextrin promoting the enzyme-catalyzed VD3 reaction is shown.

[0029] Figure 5 It is a bar chart comparing the enzyme activities of fermentation broths of different strains.

[0030] Figure 6 The NBD activity curve of the fermentation broth of strain CM4 during the induction process is shown.

[0031] Figure 7 The figure shows the conversion of substrate VD3 catalyzed by the fermentation broth of strain CM4 at different induction times. DETAILED DESCRIPTION

[0032] The wild-type peroxidase CciUPO (SEQ ID NO: 1) screened by the present invention is derived from Coprinopsis cinerea. In order to realize its industrial development and application, it needs to be heterologously expressed in industrial microorganisms that grow quickly and are suitable for the construction of genetically engineered bacteria. However, a problem of the peroxidase UPO family including CciUPO is that the heterologous expression amount is not high or even not expressed. We tried heterologous expression of the wild enzyme SEQ ID NO: 1 from some bacteria commonly used as genetically engineered bacteria, such as Escherichia coli, and yeasts, such as Pichia pastoris.

[0033] Due to its eukaryotic cell characteristics and strong protein secretion ability, Pichia pastoris has rapidly become a widely used eukaryotic expression system for heterologous proteins in recent years. The Pichia pastoris expression system has become increasingly mature and has now become an important tool for heterologous protein expression. So far, many proteins from different sources that are widely used in biomedicine and industry have been well secreted and expressed in the Pichia pastoris expression system.

[0034] We achieved high expression of wild enzyme SEQ ID NO:1 in Pichia pastoris; and achieved mutation of wild enzyme in Pichia pastoris. In the constructed mutant library, a mutant strain with high enzyme activity was screened, referred to as CM4. After gene sequencing analysis, it was determined that the wild enzyme had undergone (L64A, V82I, T255V) mutations, and mutant SEQ ID NO:3 was obtained. After 96 hours of fermentation induction in a 5L fermenter, the enzyme activity of strain CM4 measured by NBD exceeded 1700U / L. Directly using fermentation broth to catalyze 3g / L of VD3 can achieve a conversion rate of more than 99% within 3 hours, and 5g / L of VD3 can achieve a conversion rate of more than 80%.

[0035] When evaluating the enzyme activity of peroxidase, the reaction substrate is selected as NBD (5-nitro-1,3-benzodioxole). In this case, the enzyme activity can be referred to as "NBD activity" in this article. The enzyme activity unit is defined as: under the conditions of pH 7.0 and temperature 25°C, the amount of enzyme consumed to convert 1 μmol of substrate (NBD) within 1 minute is 1 unit (U).

[0036] The above-mentioned terms "wild (type)" and "wild enzyme" have the same meaning, and both refer to the peroxidase with the amino acid sequence of SEQ ID NO: 1. Correspondingly, the terms "peroxidase mutant", "mutant peroxidase" and "mutant enzyme" have the same meaning, and both refer to mutants of peroxidase. For the sake of brevity, sometimes for the sake of convenience of expression, in the present invention, the wild-type peroxidase and its mutant SEQ ID NO: 3 can be collectively referred to as "peroxidase".

[0037] Since the amino acid sequences of the peroxidase and its mutants screened by the present invention are clear, it is easy for those skilled in the art to obtain the coding genes, expression cassettes and plasmids containing these genes, and transformants containing the plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

[0038] When used as a biocatalyst for preparing calcifediol, the peroxidase of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, enzymes immobilized on carriers, etc.; the bacterial cell forms include living bacterial cells and dead bacterial cells.

[0039] It is easy for those skilled in the art to understand that the bacteria itself is a natural enzyme immobilization form, which can be used as an enzyme preparation for catalytic reactions. Since the reaction substrate and reaction product can easily pass through the biological barrier of the bacteria - the cell membrane, there is no need to lyse the bacteria or even extract and purify them, which is economically advantageous.

[0040] On the other hand, compared with the catalysis of isolated enzymes, the present invention can provide a continuous and inexhaustible supply of enzymes or by simple fermentation of microorganisms, without the need for further extraction, purification and separation of enzymes, etc., with obvious economic benefits, creating conditions for industrial applications.

[0041] Example

[0042] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0043] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0044] Materials and methods

[0045] In the examples, the whole gene synthesis, primer synthesis and sequencing were all commissioned to Sangon Biotechnology Co., Ltd.

[0046] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0047] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0048] BMGY medium: peptone: 20g / L, yeast powder: 10g / L, KP 6.0: 0.1M, YNB: 13.4g / L, glycerol: 10g / L. Peptone, yeast powder, phosphate buffer, and glycerol were mixed together and sterilized at 121℃ for 30min. After YNB was prepared, it was filtered and sterilized separately. YNB was added to the medium and mixed before use.

[0049] BMMY medium: peptone: 20g / L, yeast powder: 10g / L, KP 6.0: 0.1M, YNB: 13.4g / L, methanol: 1% (v / v). Peptone, yeast powder, and phosphate buffer are mixed together and sterilized at 121℃ for 30min. After YNB is prepared, filter and sterilize it separately. Before use, add YNB to the culture medium and mix well. Methanol is added separately at the end.

[0050] HPLC determination conditions of substrate VD3 and calcifediol:

[0051] Instrument model: SHIMADZU LC-2030Plus; column model: YMC J'sphere ODS-H80; mobile phase: acetonitrile, ultrapure water; 0-12min: acetonitrile concentration increased from 50% to 100%; 12-18min: 100% acetonitrile; 18.01-20min: 50% acetonitrile; flow rate: 1.5mL / min; column temperature: 40℃; detection wavelength: 265nm; detection time: 20min; injection volume: 10μL; VD3 retention time: 14.983min; calcifediol retention time: 7.833min.

[0052] It should be noted that, for the sake of convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme encoding gene number may share one number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0053] Example 1: Construction of a recombinant Pichia pastoris expressing wild-type peroxidase

[0054] 1.1 For the wild-type peroxidase SEQ ID NO: 1 from Coprinopsis cinerea (NCBI accession number: XM_001831858), an optimized codon sequence SEQ ID NO: 2 suitable for Pichia pastoris expression was designed, and Sangon Biotechnology Co., Ltd. was commissioned to perform full gene synthesis, and restriction sites EcoR I and Not I were designed at both ends. It was subcloned into the corresponding sites on the Pichia pastoris expression vector pPICZα-A (ThermoFisher), thereby obtaining the recombinant plasmid pPICZα-A-cci, which was placed in a refrigerator for use.

[0055] 1.2 The pPICZα-A-cci plasmid was linearized using PmeI restriction endonuclease, and 9 μL of the linearized recovery product was added to 100 μL of Pichia pastoris X33 competent cells (Invitrogen) for electroporation. After the electroporation was completed, 400 μL of recovery medium (YPD: sorbitol = 1:1) was added and placed in a 28°C shaker for incubation for 1.5-2 h. 200 μL of the culture solution was evenly spread on YPD solid medium containing 300 μg / mL bleomycin, and inverted in a 28°C incubator for incubation for 2 days. A single clone was picked and inoculated into BMGY medium (50 mL medium / 250 mL Erlenmeyer flask) after colony PCR verification. After incubation at 28°C for 48 h, the bacteria were collected by centrifugation, and the BMMY medium was changed to induce protein expression. 1 v / v% methanol was added every 24 h. After induction for 5 days, the supernatant was collected by centrifugation to detect the level of peroxidase expressed by the strain, that is, the enzyme activity.

[0056] 1.3 The enzyme activity of the fermentation broth was tested based on the ability to catalyze the reaction of the substrate NBD. The method is as follows:

[0057] Reaction system:

[0058]

[0059]

[0060] The absorbance change of the reaction system at a wavelength of 425 nm was detected.

[0061] The enzyme activity concentration was calculated as follows:

[0062]

[0063] Where: V is the volume of the reaction system (mL), ε is the molar absorption coefficient (9700M -1 cm -1 ), v is the sample volume (mL), L is the cuvette diameter (cm), △A is the absorbance change, 10 6 To convert mol to μmol.

[0064] By comparing the expression level of the wild enzyme SEQ ID NO: 1 with that of the transformants, a recombinant Pichia pastoris engineered strain with higher enzyme activity in the fermentation broth was screened out and named strain C1.

[0065] Example 2: Optimization of reaction conditions of wild enzyme CciUPO catalyzing VD3

[0066] The effects of temperature, pH, H2O2 donor and substrate concentration on peroxidase-catalyzed VD3 reaction were investigated.

[0067] 2.1 Effect of temperature on reaction

[0068] The enzyme-catalyzed reaction of VD3 was carried out in the following reaction system, and the reaction temperatures were set to 30°C, 35°C, 40°C and 45°C, respectively.

[0069] Reaction system:

[0070]

[0071] After 2 hours of reaction, 100 μL of the reaction solution was taken, 900 μL of acetonitrile was added to terminate the reaction, the mixture was shaken and mixed, and centrifuged at 12000 rpm for 1 min. The supernatant was filtered through a 0.22 μm filter membrane and then detected by liquid chromatography. Figure 1 shown.

[0072] Depend on Figure 1 It can be seen that the yield of the product increases gradually from 30℃ to 45℃. The yield at 35℃ is slightly higher than that at 30℃, and that at 45℃ is slightly higher than that at 40℃, but not significantly so. The yields at 45℃ and 40℃ are significantly higher than those at 35℃ and 30℃. Taking all factors into consideration, the optimal reaction temperature should be 40℃.

[0073] 2.2 Investigating the effect of pH on the reaction

[0074] The enzyme-catalyzed VD3 reaction was carried out in the following reaction system, using phosphate buffer solutions with pH values ​​of 5.0, 6.0, 7.0, 8.0 and 9.0, respectively, and the reaction temperature was 40°C.

[0075] Reaction system:

[0076]

[0077] After 2 hours of reaction, 100 μL of the reaction solution was taken, 900 μL of acetonitrile was added to terminate the reaction, the mixture was shaken and mixed, and centrifuged at 12000 rpm for 1 min. The supernatant was filtered through a 0.22 μm filter membrane and then detected by liquid chromatography. Figure 2 shown.

[0078] Depend on Figure 2 It can be seen that the yield of the product gradually increases from pH 5.0 to 9.0, but the increase between 9.0 and 8.0 is not very obvious. Taking all factors into consideration, the optimal pH should be between 8.0 and 9.0.

[0079] 2.3 Investigating the effect of H2O2 donor dosage on the reaction

[0080] H2O2 donors are glucose and glucose oxidase (abbreviated as GO). Glucose is set at two concentrations of 20 and 50 mM. Correspondingly, glucose oxidase is set at three concentrations of 20, 50, and 80 U / mL. The enzyme-catalyzed VD3 reaction is carried out in the following reaction system at a reaction temperature of 40°C.

[0081] Reaction system:

[0082]

[0083] After 12 hours of reaction, 100 μL of the reaction solution was taken, 900 μL of acetonitrile was added to terminate the reaction, the mixture was shaken and mixed, and centrifuged at 12000 rpm for 1 min. The supernatant was filtered through a 0.22 μm filter membrane and then analyzed by liquid chromatography. Figure 3 shown.

[0084] from Figure 3 It can be seen that peroxidase plays an important role in the reaction, and the higher the peroxidase concentration, the higher the yield of calcifediol. Comparing the glucose concentrations of 20mM and 50mM, the glucose concentration has almost no effect on the reaction under the premise of being sufficient; comparing the concentrations of glucose oxidase (20, 50, 80U / mL), the higher the concentration, the better the reaction effect.

[0085] 2.4 Optimization of substrate solubility

[0086] 2-Hydroxypropyl-β-cyclodextrin (PMCD) was dissolved in phosphate buffer solution at a concentration of 0.05 g / mL to increase the solubility of substrate VD3 in the reaction solution and promote the reaction. The enzyme-catalyzed VD3 reaction was carried out in the following reaction system at a reaction temperature of 40°C.

[0087] Reaction system:

[0088]

[0089] After 2 hours of reaction, 100 μL of the reaction solution was taken, 900 μL of acetonitrile was added to terminate the reaction, the mixture was shaken and mixed, and centrifuged at 12000 rpm for 1 min. The supernatant was filtered through a 0.22 μm filter membrane and then detected by liquid chromatography. Figure 4 shown.

[0090] from Figure 4 It can be seen that the addition of substrate cosolvent 2-hydroxypropyl-β-cyclodextrin enables the substrate VD3 to be completely converted into calcifediol without any residue, thus solving the solubility problem of the substrate in the reaction solution.

[0091] Example 3: Construction of peroxidase CciUPO mutant

[0092] In order to increase the expression level of wild-type peroxidase, a computer-simulated 3D protein model of wild-type peroxidase CciUPO was constructed using bioinformatics methods. With the help of protein structure computer-aided design, site-directed saturation mutagenesis was attempted as follows.

[0093] PCR template: pPICZα-A-cci

[0094] Primers for some mutants:

[0095] L64NNK-F: CCTATTAAGGACCCATGGNNKAAGTTGGTCAACG,

[0096] V82NNK-F: TTGAGAAGAGGTGATNNKAGAGGTCCATGTC,

[0097] T255NNK-F: ATCAACTTCTTCNNKGACGGTAGACTGTTCAC,

[0098] cciUPO-R:TTAGTCGGACTTACCCCATGGGAAAACT.

[0099] PCR reaction system (20 μL): 1-50 ng of template, 0.5 μL (10 μM) of a pair of upstream and downstream primers, 10 μL of PrimeSTAR Max DNA polymerase (2×), and sterile water to make up to 20 μL.

[0100] The PCR amplification procedure was as follows: (1) pre-denaturation at 98°C for 3 min; (2) denaturation at 98°C for 10 sec, (3) annealing at 56°C for 5 sec, and (4) extension at 72°C for 70 sec. Steps (2) to (4) were repeated 30 times, and the final step was extension at 72°C for 3 min. The PCR product was stored at 12°C.

[0101] The amplified PCR product was digested with Dpn I overnight in a 37°C water bath, inactivated at 80°C for 20 min, transferred to E. coli DH5α competent cells, added with culture medium and resuscitated at 37°C for 45-60 min, spread on low-salt LB solid culture medium containing zeocin resistance, inverted and cultured at 37°C overnight, picked a single clone and sent it to the company for sequencing, and used SnapGene bioanalysis software to view the peak graph of the sequencing results to determine the correct mutation of the amino acid at the mutation site.

[0102] A total of 720 mutant clones were obtained, including mutants M1-M5.

[0103] Example 4: Screening of mutant strains

[0104] According to the methods of steps 1.1 and 1.2 in Example 1, the mutant genes including M1-M5 constructed in Example 3 were respectively transferred into Pichia pastoris X33 to obtain transformants expressing these mutants.

[0105] Using the wild-type strain C1 as a control, the transformants expressing each mutant were cultured and induced to express according to the method of step 1.3 in Example 1, and the supernatant was collected. The enzyme activity was determined using NBD as a substrate to compare whether the enzyme activity of the fermentation broth of each mutant expression strain was improved compared with that of the C1 fermentation broth. The enzyme activity of the fermentation broth of the mutant expression strain with significantly improved enzyme activity was as follows: Figure 5 shown.

[0106] Figure 5 The results show a comparison of the enzyme activities of the fermentation broths of mutants M1-M5 with significantly improved partial enzyme activities and the wild-type strain C1, among which the enzyme activity of the fermentation broth of M4 is the highest.

[0107] Through gene sequencing, the nucleotide sequence of M4 is SEQ ID NO: 4, and its corresponding amino acid sequence is SEQ ID NO: 3, which is a L64A / V82I / T255V mutant of the wild enzyme CciUPO. In the following examples, the expression strain of this mutant can be named CM4.

[0108] Example 5: 5L fermentation tank fermentation and catalytic experiment of strain CM4

[0109] 5.1 Strain fermentation

[0110] Use the stored strain CM4 to streak on a YPD plate for activation and place it in an incubator at 28°C for 2 days;

[0111] Pick a single colony and inoculate it into BMGY medium (200mL / 1L) as the seed liquid for fermentation in the upper tank, and culture it in a shaking incubator at 28℃ for 48h;

[0112] 200 mL of the seed solution was inoculated into a 5 L fermenter containing 2.5 L of BSM basal salt medium (8 v / v% inoculation amount), 28 ° C, 500 rpm, ventilation rate of 4 NL / min, and pH was controlled at about 5.5 by ammonia. 4.35 mL / L of PTM1 and 1 mL of defoamer were also added to the BSM medium;

[0113] When the glycerol in the BSM medium is exhausted, start adding glycerol (containing 12mL / L PTM1), adjust the feeding rate to control the dissolved oxygen content at about 30%, and take samples to monitor OD 600 Change, waiting for OD 600 =180, stop feeding, and starve the C source for 1 hour;

[0114] After 1 hour of starvation of the C source, methanol was added to induce expression. The methanol addition rate and the rotation speed were linked to control the dissolved oxygen at around 20%. The tank was stopped after 109 hours of induction of expression.

[0115] During the induction process, samples were taken at intervals to measure NBD activity. Figure 6 shown.

[0116] 5.2 Fermentation broth catalytic reaction

[0117] The fermentation broth induced at different times was used to carry out catalytic reaction with VD3 as substrate. The reaction temperature was 40°C and the reaction system (2 mL) was as follows:

[0118]

[0119]

[0120] The amount of PMCD is 5wt% relative to VD3.

[0121] After 2 h of reaction, 100 μL of the reaction solution was taken and added to 900 μL of the reaction solution, mixed, centrifuged at 12000 rpm, filtered through a 0.22 μm filter membrane, and the conversion rate of substrate VD3 was detected by HPLC. The reaction results were as follows: Figure 7 shown.

[0122] from Figure 7 It can be seen that the use of fermentation broth can almost completely react 3 g / L of substrate.

[0123] The above experiments show that peroxidase SEQ ID NO: 1 and its mutant SEQ ID NO: 3 can catalyze the conversion of vitamin D3 to obtain calcifediol in the presence of H2O2 donor, among which mutant SEQ ID NO: 3 has higher catalytic efficiency and has development and application prospects. Sequence Listing <110> Zhejiang Jinlangbo Pharmaceutical Co., Ltd. <120> Method for synthesizing calcifediol using peroxidase <130> SHPI2110535 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 387 <212> PRT <213> Coprinopsis cinerea <400> 1 Met Ile Ser Thr Ser Lys His Leu Phe Val Leu Leu Pro Leu Phe Leu 1 5 10 15 Val Ser His Leu Ser Leu Val Leu Gly Phe Pro Ala Tyr Ala Ser Leu 20 25 30 Gly Gly Leu Thr Glu Arg Gln Val Glu Glu Tyr Thr Ser Lys Leu Pro 35 40 45 Ile Val Phe Pro Pro Pro Pro Pro Glu Pro Ile Lys Asp Pro Trp Leu 50 55 60 Lys Leu Val Asn Asp Arg Ala His Pro Trp Arg Pro Leu Arg Arg Gly 65 70 75 80 Asp Val Arg Gly Pro Cys Pro Gly Leu Asn Thr Leu Ala Ser His Gly 85 90 95 Tyr Leu Pro Arg Asp Gly Val Ala Thr Pro Ala Gln Ile Ile Thr Ala 100 105 110 Val Gln Glu Gly Phe Asn Met Glu Tyr Gly Ile Ala Thr Phe Val Thr 115 120 125 Tyr Ala Ala His Leu Val Asp Gly Asn Pro Leu Thr Asn Leu Ile Ser 130 135 140 Ile Gly Gly Lys Thr Arg Lys Thr Gly Pro Asp Pro Pro Pro Pro Ala 145 150 155 160 Ile Val Gly Gly Leu Asn Thr His Ala Val Phe Glu Gly Asp Ala Ser 165 170 175 Met Thr Arg Gly Asp Phe His Leu Gly Asp Asn Phe Asn Phe Asn Gln 180 185 190 Thr Leu Trp Glu Gln Phe Lys Asp Tyr Ser Asn Arg Tyr Gly Gly Gly 195 200 205 Arg Tyr Asn Leu Thr Ala Ala Ala Glu Leu Arg Trp Ala Arg Ile Gln 210 215 220 Gln Ser Met Ala Thr Asn Gly Gln Phe Asp Phe Thr Ser Pro Arg Tyr 225 230 235 240 Phe Thr Ala Tyr Ala Glu Ser Val Phe Pro Ile Asn Phe Phe Thr Asp 245 250 255 Gly Arg Leu Phe Thr Ser Asn Thr Thr Ala Pro Gly Pro Asp Met Asp 260 265 270 Ser Ala Leu Ser Phe Phe Arg Asp His Arg Tyr Pro Lys Asp Phe His 275 280 285 Arg Ala Pro Val Pro Ser Gly Ala Arg Gly Leu Asp Val Val Ala Ala 290 295 300 Ala Tyr Pro Ile Gln Pro Gly Tyr Asn Ala Asp Gly Lys Val Asn Asn 305 310 315 320 Tyr Val Leu Asp Pro Thr Ser Ala Asp Phe Thr Lys Phe Cys Leu Leu 325 330 335 Tyr Glu Asn Phe Val Leu Lys Thr Val Lys Gly Leu Tyr Pro Asn Pro 340 345 350 Lys Gly Phe Leu Arg Lys Ala Leu Glu Thr Asn Leu Glu Tyr Phe Tyr 355 360 365 Gln Ser Phe Pro Gly Ser Gly Gly Cys Pro Gln Val Phe Pro Trp Gly 370 375 380 Lys Ser Asp 385 <210> 2 <211> 1164 <212> DNA <213> Artificial Sequence () <400> 2 atgatctcca cctccaagca cttgttcgtc ttgttgccac tgttcttggt ttcccacttg 60 tccttggttt tgggtttccc agcttacgct tctcttggtg gtttgactga gagacaggtt 120 gaagagtaca cttccaagct gccaatcgtt tttccaccac cacctccaga acctattaag 180 gacccatggt tgaagttggt caacgacaga gcacatccat ggcgtccatt gagaagaggt 240 gatgttagag gtccatgtcc aggtttgaac actttggctt ctcacggtta cttgccaaga 300 gatggtgttg ctactccagc tcagatcatc actgctgttc aagagggttt caacatggaa 360 tacggtatcg ccaccttcgt tacttacgct gctcatttgg ttgacggtaa cccattgact 420 aacctgatct ccatcggtgg taagaccaga aagactggtc cagatccacc tcctccagct 480 atagttggtg gattgaacac tcacgctgtt ttcgaaggtg acgcttccat gactagaggt 540 gatttccact tgggtgataa cttcaacttc aatcagaccc tgtgggagca gttcaaggac 600 tactctaaca gatacggtgg tggtagatac aacttgactg ctgctgctga attgagatgg 660 gctagaatcc aacaatccat ggctaccaac ggtcagttcg acttcacttc cccaagatac 720 ttcactgctt acgccgagtc tgtgttccca atcaacttct tcactgacgg tagactgttc 780 acctccaaca caactgctcc aggtccagat atggactccg ctttgtcatt cttcagggac 840 cacagatacc caaaggattt tcacagagcc ccagttccat ctggtgctag aggtttggat 900 gttgttgctg ctgcataccc aattcagcca ggttacaacg ctgacggtaa ggtcaacaac 960 tacgttttgg acccaacttc cgctgacttc accaagttct gtttgctgta cgagaacttc 1020 gtccttaaga ccgtcaaggg tctgtaccca aatccaaagg gtttcttgag aaaggccttg 1080 gagactaact tggagtactt ctaccagtcc tttccaggtt ctggtggttg tccacaagtt 1140 ttcccatggg gtaagtccga ctaa 1164 <210> 3 <211> 387 <212> PRT <213> Artificial sequence () <400> 3 Met Ile Ser Thr Ser Lys His Leu Phe Val Leu Leu Pro Leu Phe Leu 1 5 10 15 Val Ser His Leu Ser Leu Val Leu Gly Phe Pro Ala Tyr Ala Ser Leu 20 25 30 Gly Gly Leu Thr Glu Arg Gln Val Glu Glu Tyr Thr Ser Lys Leu Pro 35 40 45 Ile Val Phe Pro Pro Pro Pro Pro Glu Pro Ile Lys Asp Pro Trp Ala 50 55 60 Lys Leu Val Asn Asp Arg Ala His Pro Trp Arg Pro Leu Arg Arg Gly 65 70 75 80 Asp Ile Arg Gly Pro Cys Pro Gly Leu Asn Thr Leu Ala Ser His Gly 85 90 95 Tyr Leu Pro Arg Asp Gly Val Ala Thr Pro Ala Gln Ile Ile Thr Ala 100 105 110 Val Gln Glu Gly Phe Asn Met Glu Tyr Gly Ile Ala Thr Phe Val Thr 115 120 125 Tyr Ala Ala His Leu Val Asp Gly Asn Pro Leu Thr Asn Leu Ile Ser 130 135 140 Ile Gly Gly Lys Thr Arg Lys Thr Gly Pro Asp Pro Pro Pro Pro Ala 145 150 155 160 Ile Val Gly Gly Leu Asn Thr His Ala Val Phe Glu Gly Asp Ala Ser 165 170 175 Met Thr Arg Gly Asp Phe His Leu Gly Asp Asn Phe Asn Phe Asn Gln 180 185 190 Thr Leu Trp Glu Gln Phe Lys Asp Tyr Ser Asn Arg Tyr Gly Gly Gly 195 200 205 Arg Tyr Asn Leu Thr Ala Ala Ala Glu Leu Arg Trp Ala Arg Ile Gln 210 215 220 Gln Ser Met Ala Thr Asn Gly Gln Phe Asp Phe Thr Ser Pro Arg Tyr 225 230 235 240 Phe Thr Ala Tyr Ala Glu Ser Val Phe Pro Ile Asn Phe Phe Val Asp 245 250 255 Gly Arg Leu Phe Thr Ser Asn Thr Thr Ala Pro Gly Pro Asp Met Asp 260 265 270 Ser Ala Leu Ser Phe Phe Arg Asp His Arg Tyr Pro Lys Asp Phe His 275 280 285 Arg Ala Pro Val Pro Ser Gly Ala Arg Gly Leu Asp Val Val Ala Ala 290 295 300 Ala Tyr Pro Ile Gln Pro Gly Tyr Asn Ala Asp Gly Lys Val Asn Asn 305 310 315 320 Tyr Val Leu Asp Pro Thr Ser Ala Asp Phe Thr Lys Phe Cys Leu Leu 325 330 335 Tyr Glu Asn Phe Val Leu Lys Thr Val Lys Gly Leu Tyr Pro Asn Pro 340 345 350 Lys Gly Phe Leu Arg Lys Ala Leu Glu Thr Asn Leu Glu Tyr Phe Tyr 355 360 365 Gln Ser Phe Pro Gly Ser Gly Gly Cys Pro Gln Val Phe Pro Trp Gly 370 375 380 Lys Ser Asp 385 <210> 4 <211> 1164 <212> DNA <213> Artificial Sequence () <400> 4 atgatctcca cctccaagca cttgttcgtc ttgttgccac tgttcttggt ttcccacttg 60 tccttggttt tgggtttccc agcttacgct tctcttggtg gtttgactga gagacaggtt 120 gaagagtaca cttccaagct gccaatcgtt tttccaccac cacctccaga acctattaag 180 gacccatggg ctaagttggt caacgacaga gcacatccat ggcgtccatt gagaagaggt 240 gatattagag gtccatgtcc aggtttgaac actttggctt ctcacggtta cttgccaaga 300 gatggtgttg ctactccagc tcagatcatc actgctgttc aagagggttt caacatggaa 360 tacggtatcg ccaccttcgt tacttacgct gctcatttgg ttgacggtaa cccattgact 420 aacctgatct ccatcggtgg taagaccaga aagactggtc cagatccacc tcctccagct 480 atagttggtg gattgaacac tcacgctgtt ttcgaaggtg acgcttccat gactagaggt 540 gatttccact tgggtgataa cttcaacttc aatcagaccc tgtgggagca gttcaaggac 600 tactctaaca gatacggtgg tggtagatac aacttgactg ctgctgctga attgagatgg 660 gctagaatcc aacaatccat ggctaccaac ggtcagttcg acttcacttc cccaagatac 720 ttcactgctt acgccgagtc tgtgttccca atcaacttct tcgttgacgg tagactgttc 780 acctccaaca caactgctcc aggtccagat atggactccg ctttgtcatt cttcagggac 840 cacagatacc caaaggattt tcacagagcc ccagttccat ctggtgctag aggtttggat 900 gttgttgctg ctgcataccc aattcagcca ggttacaacg ctgacggtaa ggtcaacaac 960 tacgttttgg acccaacttc cgctgacttc accaagttct gtttgctgta cgagaacttc 1020 gtccttaaga ccgtcaaggg tctgtaccca aatccaaagg gtttcttgag aaaggccttg 1080 gagactaact tggagtactt ctaccagtcc tttccaggtt ctggtggttg tccacaagtt 1140 ttcccatggg gtaagtccga ctaa 1164

Claims

1. A method for preparing calcifediol by enzyme catalysis, comprising the following steps: Peroxidase SEQ ID NO: 1 or its mutant SEQ ID NO: 3 is used to catalyze the hydroxylation reaction at position 25 of vitamin D3 to obtain calcifediol.

2. The method according to claim 1, characterized in that There is a H2O2 donor in the reaction system.

3. The method according to claim 2, characterized in that The H2O2 donors are glucose and glucose oxidase.

4. The method according to claim 1, characterized in that The peroxidase SEQ ID NO: 1 or its mutant SEQ ID NO: 3 is in the form of a microbial cell expressing the peroxidase.

5. The method according to claim 1, characterized in that A solvent is added to the reaction system to promote the dissolution of vitamin D3.

6. A peroxidase, characterized in that The amino acid sequence is SEQ ID NO:

3.

7. A gene encoding the peroxidase according to claim 6.

8. The gene according to claim 7, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

4.

9. A microorganism expressing the peroxidase SEQ ID NO: 3 as described in claim 6.

10. The microorganism according to claim 9, characterized in that It's Pichia pastoris.

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