Use of peroxidase Cab4 UPO or mutants thereof in the synthesis of 25-hydroxyvitamin D3 and a peroxidase Cab4 UPO mutant and use thereof

Abstract

The present application relates to the application of peroxidase Cab4UPO or its mutant in synthesis of 25-hydroxyvitamin D3 and a peroxidase Cab4UPO mutant and its use, and belongs to the field of enzyme engineering.The present application provides the application of peroxidase Cab4UPO or its mutant in synthesis of 25-hydroxyvitamin D3, wherein the amino acid sequence of Cab4UPO is shown as SEQ ID NO:4.The present application provides a peroxidase Cab4UPO mutant, a DNA molecule, an expression vector, a host and a synthesis method of 25-hydroxyvitamin D3.The synthesis method has the advantages of short reaction time, high substrate conversion rate, high enzyme selectivity, few by-products, and no need to add coenzyme and surfactant in the reaction system.

Description

Technical Field

[0001] This invention relates to the application of peroxidase Cab4UPO or its mutants in the synthesis of 25-hydroxyvitamin D3, as well as a peroxidase Cab4UPO mutant and its uses, belonging to the field of enzyme engineering. Background Technology

[0002] 25-Hydroxyvitamin D3, also known as calcidiol, is the first active metabolite of vitamin D3, possessing stronger physiological activity and being the main active form of vitamin D3 in the bloodstream. It enters the bloodstream directly without requiring liver metabolism. Also known as active vitamin D3, its biological potency is 20-40 times that of vitamin D3, promoting animal bone development, improving feed conversion rate, and enhancing immunity. Developed by American scientists, 25-hydroxyvitamin D3 received FDA approval in 1995 for use as a novel nutritional additive in livestock and poultry feed. On February 1, 2014, my country began permitting its production and use in the feed industry. 25-Hydroxyvitamin D3 is expected to replace vitamin D3 as an essential vitamin in livestock and poultry farming in the future.

[0003] Vitamin D3 is primarily synthesized using biological methods, including bio-fermentation and biocatalysis. Bio-fermentation involves the conversion of vitamin D3 into 25-hydroxyvitamin D3 by specific enzymes within microorganisms during fermentation. However, this method suffers from problems such as complex fermentation broth composition, low product concentration (less than 1 g / L), and long fermentation cycles, significantly impacting subsequent extraction efficiency. Biocatalysis, on the other hand, utilizes genetic engineering to prepare specific oxidases that specifically oxidize vitamin D3 to 25-hydroxyvitamin D3 in vitro. This method is environmentally friendly and produces fewer toxic or harmful substances.

[0004] CN115838695A reported a study on the preparation of calcidiol from vitamin D3 via selective hydroxylation catalyzed by P450 enzymes. However, this reaction requires expensive NADPH as a coenzyme, and most P450 enzymes have low expression levels (membrane proteins), low substrate concentrations, poor positional selectivity of some P450 enzyme varieties leading to the easy generation of other byproducts, and long catalytic time. Industrial production process conditions need to be optimized in many aspects.

[0005] CN202310529525.3, entitled "Enzyme Mixture and Preparation Method Thereof and Method for Preparing 25-Hydroxycholesterol or 25-Hydroxyvitamin D3," discloses an enzyme mixture and its preparation method, as well as a method for preparing 25-hydroxycholesterol or 25-hydroxyvitamin D3. The enzyme mixture comprises a self-consistent P450 enzyme and glucose dehydrogenase. The enzyme mixture, including the self-consistent P450 enzyme and glucose dehydrogenase, is used to co-catalyze the production of 25-hydroxycholesterol or 25-hydroxyvitamin D3 from the substrate cholesterol or vitamin D3. Compared to the single-enzyme method using only P450, this method allows the use of inexpensive glucose as a cofactor, achieves higher substrate conversion rates, and produces fewer byproducts. However, this method requires cell wall disruption, and the P450 enzyme has poor stability and is difficult to preserve, making it unsuitable for large-scale industrial operation.

[0006] Unspecific peroxidases (UPOs, EC 1.11.2.1.) have been increasingly discovered and characterized in recent years. They share the same active site and catalytic mechanism as P450 enzymes. UPOs are a class of fungal oxidases belonging to the heme oxidase family and possess multifunctional oxidative catalytic activity. Compared to the more widely reported P450 monooxygenases and oleic acid hydratases, UPOs have a broader substrate range, and their catalytic process can directly utilize inexpensive hydrogen peroxide as both an oxygen donor and electron acceptor, eliminating the need for complex electron transport chains and significantly reducing reaction costs. Therefore, they have greater application potential.

[0007] CN115181758A reports a method for the one-step catalytic synthesis of 25-hydroxyvitamin D3 using immobilized UPO. Using vitamin D3 as a raw material, a non-specific peroxygenase derived from *Agrocybe aegerita* is used as a catalyst, and hydrogen peroxide as an oxidant to activate the CH bond and induce hydroxylation, achieving a one-step catalytic oxidation method for calcidiol preparation. However, the selectivity of this enzyme for C-25 hydroxylation is currently only 70%, and the presence of numerous byproducts increases the difficulty of downstream processing.

[0008] CN116240246A discloses a method for synthesizing calcidiol using peroxidase. However, the reaction system of this scheme uses glucose oxidase as H2O2 donor, which does not solve the problem of peroxidase's intolerance to hydrogen peroxide and increases the economic cost.

[0009] CN118325858A, CN119242605A and CN119752829A all disclose a non-specific peroxyase mutant derived from Agrocybe aegerita and its application in catalyzing steroidal compounds. However, the above schemes generally suffer from problems such as low substrate concentration, insufficient raw material utilization, low yield of target product, and long reaction time.

[0010] Therefore, it is necessary to further explore non-specific peroxidases with good selective catalytic performance, and use enzyme engineering to modify them to improve their catalytic activity for vitamin D3 and selectivity for C-25 hydroxylation, thereby improving catalytic efficiency and enabling them to better meet the needs of production processes. Summary of the Invention

[0011] This invention provides the application of peroxidase Cab4UPO or its mutant in the synthesis of 25-hydroxyvitamin D3, as well as a peroxidase Cab4UPO mutant and its use.

[0012] This invention provides the application of peroxidase Cab4UPO or its mutant in the synthesis of 25-hydroxyvitamin D3; the amino acid sequence of said Cab4UPO is shown in SEQ ID NO:4.

[0013] The peroxidase Cab4UPO mutant of the present invention is obtained by mutating phenylalanine at position 125 of peroxidase Cab4UPO, as shown in SEQ ID No. 4, to methionine, resulting in Cab4UPO-F125M.

[0014] It uses Cab4UPO-F125M as the parent and makes mutations at positions 14 and / or 132, where phenylalanine at position 14 is mutated to tryptophan and serine at position 132 is mutated to isoleucine.

[0015] It uses Cab4UPO-F125M as the parent and mutates at positions 14 and 132 to obtain the mutant Cab4UPO-F14W-F125M-S132I.

[0016] The present invention provides a DNA molecule that encodes the peroxidase Cab4UPO mutant.

[0017] The present invention provides an expression vector containing the aforementioned DNA molecule.

[0018] The present invention provides a host cell containing the expression vector described above.

[0019] The present invention also provides the use of the peroxidase Cab4UPO mutant, the DNA molecule, the expression vector, or the host cell in the synthesis of 25-hydroxyvitamin D3.

[0020] This invention provides a method for synthesizing 25-hydroxyvitamin D3, which uses vitamin D3 as a substrate, adds the aforementioned peroxidase Cab4UPO mutant, a solubilizer, hydrogen peroxide, and a buffer solution, and reacts to generate 25-hydroxyvitamin D3.

[0021] The co-solvent is acetone; the buffer solution is disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and the synthesis conditions are: pH 7.0, 37℃, and stirring reaction for 90 min.

[0022] In existing technologies, the substrate concentration in the reaction system is low, at 5 mM. In this invention, the substrate concentration is 8 mM in 1 L of reaction, and 2.61 g of pure product is obtained after separation, with a separation yield of 81.7%. The separation yield is defined as: (mass of the purified product / theoretical mass of the substrate completely converted into the product) × 100%.

[0023] The beneficial effects of this invention are:

[0024] Compared to previously reported literature, this invention requires only one step of centrifugation after enzyme fermentation, and the reaction can proceed using the supernatant, eliminating the need for cell collection and disruption. This invention features a short reaction time, high substrate conversion rate, good enzyme selectivity, minimal byproducts, and eliminates the need for coenzymes and surfactants such as cyclodextrins in the reaction system. Under hydrogen peroxide conditions, the reaction requires only one hydroxylation step to obtain the target product, exhibiting good stereoselectivity, mild conditions, high conversion rate, and high product separation yield. Attached Figure Description

[0025] Figure 1 To investigate the activity of different wild-type peroxidases in catalyzing the production of 25-hydroxyvitamin D3 from vitamin D3;

[0026] Figure 2 Image of pPICZA-Cab4UPO plasmid;

[0027] Figure 3 The product standard curve for 25-hydroxyvitamin D3;

[0028] Figure 4 The image shows the NMR spectrum of the product from reaction 4. Detailed Implementation

[0029] Example 1: Construction of a recombinant Pichia pastoris engineered strain expressing wild-type peroxidase, and a screening test for the synthesis of 25-hydroxyvitamin D3.

[0030] The wild-type peroxidase SEQ ID NO:1 (NCBI accession number: EWC45465.1) derived from Drechslerella stenobrocha 248 has the following amino acid sequence:

[0031] MGGVVDKAAGAVLKLLVFQWDIALFVLNFVTPDRKEGEVVPKGAPGHHGAWPEYVPPTDGDSRSACPMLNAMANHGVLPHDGKNITFVDLNHSIRKTFNFAPSFCFFVPKFAADFLKRSYWKDTFNLEELSLHNAIEHDASLTRQDSAL VPDQSKPDLKLVHDLFAEATGKMPDGSPRLTIPDLSRALSKRRVDARLSNKDYSETRFHNMFGSSNSSTMLTLFAGSVEDLTPMLTEERFSENWEPRVRSRFGLTITKFNLAHVLPVERGVNTKKIEEEREAAAALAQAEATSGAAGEASK

[0032] The wild-type peroxidase derived from Cyclocybe aegerita, SEQ ID NO:2 (NCBI accession number: B9W4V6.1), has the following amino acid sequence:

[0033] MKYFPLFPTLVFAARVVAFPAYASLAGLSQQELDAIIPTLEAREPGLPPGPLENSSAKLVNDEAHPWKPLRPGDIRGPCPGLNTLASHGYLPRNGVATPVQIINAVQEGLNFDNQAAVFATYAAHLVDGNLITDLLSIGRKTRLTGPDPPPPASVGGLNEHGTFEGDASMTRGDAFFGNNHDFNE TLFEQLVDYSNRFGGGKYNLTVAGELRFKRIQDSIATNPNFSFVDFRFFTAYGETTFPANLFVDGRRDDGQLDMDAARSFFQFSRMPDFFRAPSPRSGTGVEVVIQAHPMQPGRNVGKINSYTVDPTSSDFSTPCLMYEKFVNITVKSLYPNPTVQLRKALNTNLDFFFQGVAAGCTQVFPYGRD

[0034] The wild-type peroxidase SEQ ID NO:3 (NCBI accession number: RXW14249.1) from Candolleomyces aberdarensis has the following amino acid sequence:

[0035] MVSKTFALLTALLFTLGSLSTSFAFPNLQARNGKLLRRQEVEGSTASQAGGGRPGQGVDPPPPPGPPSFTGLKLVNDRDHPWRPLRNGDIRGPCPGLNTLASHGYLPRDGVASPAQIVKAVQEGFNMDHATAVTAAYLGHILNGNLVTDLLSIGGKTPKTGPPPPPPAHAGGLNVHGTFEGDAGLTRADDFFGDNHSFNQTLFEKFVDFSNRFGGGFYNLTVAGELRYSRIQDSIATNPQFTFKNIRYLTAYGETVFPINLFVDGRQTERKLSMDHAASFFRDMKFPPDFHRAAQPSSGAGVEQVIAAYPWLPGGNADGQLNNYVVDPTSADLSDPCGLYTFVIGSVQELYPNPTGILRRNLIKNLDYWHAGGFAGCTELFPYGQ

[0036] Wild-type peroxidase from Candolleomyces aberdarensis, SEQ ID NO:4 (NCBI accession number: RXW25099.1), amino acid sequence is as follows:

[0037] MVSKPFALLTALLFTLGSLSTSFAFPNLQVPNGNLLRRQEVETASQAGGGRPGQGVDPPPPPGPISFTGTKLVNDRDHPWRPLRKGDIRGPCPGLNTLASHGYLPRDGVATPTQIINACQEGFNFDHSAAVSATYLGHILNGNLVTDLLSIGGKTPKTGPPPPPPAHAGGLNVHGTFEGDAGMTRADEFFGDNHSFNQTLFDKFVDFSNRYGGGFYNLTVAGELRYSRIQDSIATNPQFSFKNVRFLTAYGETVFPINLFVDGRQTERKLSMDHAAAFFRDMKFPPDFHRAAQPSSAEDVEKVLEAHPWLPGNNADGQLNNYVADPNSADFTDPCSLHRFVIGSVQELYPRPTGILRRNLIKNIGYWYTAAFAPAGCPELLPYGQL

[0038] The codons of the above sequences were optimized using *Pichia pastoris* as the host, and named Dst1UPO, Cae2UPO, Cab3UPO, and Cab4UPO, respectively. The whole genome was synthesized by Beijing Qingke Biotechnology Co., Ltd., and inserted between the restriction endonuclease sites EcoRI and XhoI of the expression vector pPICZA, resulting in recombinant *E. coli* strain DH5α containing the recombinant vectors pPICZA-Dst1UPO, pPICZA-Cae2UPO, pPICZA-Cab3UPO, and pPICZA-Cab4UPO. The recombinant bacteria were activated overnight in LB liquid medium (0.5% yeast extract, 1% tryptone, 1% sodium chloride) containing Zeocin antibiotic, and plasmids were extracted using a plasmid extraction kit (Beijing Tiangen Biotech). The plasmids pPICZA-Dst1UPO, pPICZA-Cae2UPO, pPICZA-Cab3UPO, and pPICZA-Cab4UPO were linearized using the SacI restriction endonuclease (Thermo Fisher), and the linearized DNA fragments were then purified using a DNA recovery kit (Beijing Tiangen Biotech).

[0039] Pichia pastoris X33 was cultured to OD using 50 mL of YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C and 250 rpm. 600 The supernatant was removed after centrifugation at 4000 rpm at 4°C. The pellet was resuspended in 50 mL of pre-chilled sterile water, centrifuged again at 4000 rpm at 4°C, and the supernatant was removed. This process was repeated once. Competent cells were prepared by resuspending the pellet in 1.5 mL of pre-chilled 1 M sorbitol. 10 μL of the linearized fragment was added to 90 μL of competent cells, and electroporation was performed using a Biorad electroporator. 1 mL of pre-chilled 1 M sorbitol was added, and the mixture was incubated at 30°C for 1 h. 100 μL of the culture medium was then evenly spread onto YPD solid medium containing Zeocin and incubated at 30°C for 3 days.

[0040] Single colonies were selected and cultured overnight in YPD medium at 30°C and 250 rpm. Colony PCR was performed using primers 5´-AOX1: GACTGGTTCCAATTGACAAGC and 3´-AOX1: GCAAATGGCATTCTGACATCC for verification. The colonies were then inoculated into 50 mL of BMGY medium [1% yeast extract, 2% peptone, 100 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 6.0), 1.34% YNB (amino acid-free yeast nitrogen source), 0.00004% biotin, and 1% glycerol]. After culturing at 30°C and 250 rpm for 48 h, the supernatant was removed by centrifugation, and 50 mL of BMMY medium [1% yeast extract, 2% peptone, 100 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 6.0), 1.34% YNB (amino acid-free yeast nitrogen source), 0.00004% biotin, and 1% methanol] was added. Cultured at 30℃ and 250 rpm, with 1% methanol added every 24 h, and the supernatant was collected by centrifugation after 7 days of induction.

[0041] Take 40 mL of the supernatant, add 30 mL of acetone, 1 g of vitamin D3, 2.5 mL of 10 M hydrogen peroxide, and 27.5 mL of 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.0). Incubate at 30 °C with stirring for 4 h. Extract with 100 mL of ethyl acetate, concentrate by rotary evaporation, and resuspend in 1 mL of anhydrous ethanol for LC-MS detection. Chromatographic column: Agilent InfinityLab Poroshell 120 EC-C18, C18, 4 μm, 4.6 × 150 mm; flow rate: 1.5 mL / min; column temperature: 40 °C; mobile phase: water:methanol = 5:95 containing 0.1% formic acid.

[0042] like Figure 1 As shown, the elution time of 25-hydroxyvitamin D3 was 1.898 min. The peak area of ​​the product obtained by LC-MS detection is shown in Table 1. The higher the peak area of ​​the product, the higher the activity of the enzyme in catalyzing the production of 25-hydroxyvitamin D3 from vitamin D3. Therefore, Cab4UPO was selected as the research object.

[0043] Table 1. Peak areas of the products from VD3 to 25-hydroxyvitamin D3 catalyzed by different wild-type peroxidases, as determined by LC-MS.

[0044] Enzyme class Product peak area Dst1 UPO 654.91 Dae2 UPO 4901.12 Cab3 UPO 69157.02 Cab4 UPO 427448.01

[0045] Example 2 Construction of peroxidase Cab4UPO mutant

[0046] The plasmid map of pPICZA-Cab4UPO is as follows:Figure 2 As shown.

[0047] Primers were designed using the NNK codon strategy, and mutations were performed at amino acid positions 14, 125, and 132.

[0048] The PCR system consisted of: 2 μL of template pPICZA-Cab4UPO plasmid, 2 μL each of primers F and R, 14 μL of deionized water, and 20 μL of 2×KOD enzyme.

[0049] The PCR program was: 98℃, 3 min;

[0050] 98℃, 15 s, 60℃, 15 s, 68℃, 30 s; this process is repeated 34 times.

[0051] 68℃, 2 min.

[0052] The linearized DNA fragments were then purified using a DNA recovery kit (Beijing Tiangen Biotech), transformed into E. coli DH5α, plated on LB solid medium containing Zeocin, and incubated overnight at 37°C. Single clones were picked and incubated in LB liquid medium containing Zeocin at 37°C for 8 hours, and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0053] Sequencing yielded the following mutants:

[0054] 1. The 14th position is mutated from phenylalanine to tryptophan;

[0055] 2. The 14th position is changed from phenylalanine to glycine;

[0056] 3. The 14th position is mutated from phenylalanine to glutamic acid;

[0057] 4. The 14th position is mutated from phenylalanine to valine;

[0058] 5. The 125th position is mutated from phenylalanine to methionine;

[0059] 6. The 125th position is mutated from phenylalanine to glycine;

[0060] 7. The 125th position is mutated from phenylalanine to lysine;

[0061] 8. The 125th position is mutated from phenylalanine to histidine;

[0062] 9. The 125th position is changed from phenylalanine to tryptophan;

[0063] 10. The 132nd position is changed from serine to alanine;

[0064] 11. The 132nd position is changed from serine to valine;

[0065] 12. The 132nd position is changed from serine to isoleucine;

[0066] 13. The 132nd position is changed from serine to glycine;

[0067] 14. The 132nd position is changed from serine to leucine;

[0068] 15. The 132nd position is mutated from serine to histidine;

[0069] 16. The 132nd position is changed from serine to arginine;

[0070] 17. The 132nd position is changed from serine to threonine.

[0071] The 17 plasmids constructed were linearized using the SacI restriction endonuclease (Thermo Fisher), and then the linearized DNA fragments were purified using a DNA recovery kit (Beijing Tiangen Biotech) for later use.

[0072] Pichia pastoris X33 was cultured to OD using 50 mL of YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C and 250 rpm. 600 The supernatant was removed after centrifugation at 4000 rpm at 4°C. The pellet was resuspended in 50 mL of pre-chilled sterile water, centrifuged again at 4000 rpm at 4°C, and the supernatant was removed. This process was repeated once. The pellet was then resuspended in 50 mL of pre-chilled 1 M sorbitol, centrifuged again at 4000 rpm at 4°C, and the supernatant was removed. This process was repeated once. Competent cells were prepared by resuspending the pellet in 1.5 mL of pre-chilled 1 M sorbitol. 10 μL of the linearized fragment was added to 90 μL of competent cells, and electroporation was performed using a Biorad electroporator. 1 mL of pre-chilled 1 M sorbitol was added, and the mixture was incubated at 30°C for 1 h. 100 μL of the culture medium was then evenly spread onto YPD solid medium containing Zeocin and incubated at 30°C for 3 days.

[0073] Single colonies were selected and cultured overnight in YPD medium at 30°C and 250 rpm. Colony PCR was performed using primers 5´-AOX1: GACTGGTTCCAATTGACAAGC and 3´-AOX1: GCAAATGGCATTCTGACATCC for verification. The colonies were then inoculated into 50 mL of BMGY medium [1% yeast extract, 2% peptone, 100 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 6.0), 1.34% YNB (amino acid-free yeast nitrogen source), 0.00004% biotin, and 1% glycerol]. After culturing at 30°C and 250 rpm for 48 h, the supernatant was removed by centrifugation, and 50 mL of BMMY medium [1% yeast extract, 2% peptone, 100 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 6.0), 1.34% YNB (amino acid-free yeast nitrogen source), 0.00004% biotin, and 1% methanol] was added. Cultured at 30℃ and 250 rpm, with 1% methanol added every 24 h, and the supernatant was collected by centrifugation after 7 days of induction.

[0074] Example 3: Determination of oxidase activity of peroxidase Cab4UPO mutant

[0075] The oxidase activity of Cab4UPO was detected using ABTS (2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid) as a substrate. The absorbance was measured over 1 minute at 420 nm using 750 μL of 0.1 M disodium hydrogen phosphate / 0.1 M citrate buffer (pH 4.4), 100 μL of 3 mM ABTS, 50 μL of 40 mM hydrogen peroxide, and 100 μL of crude enzyme solution. ABTS =36000M -1 cm -1 Enzyme activity (U / L) = Absorbance change (min) -1 )×10 / [36(mM -1 cm -1 [×1 cm], relative oxidase activity multiple is defined as: sample oxidase activity / control parent oxidase activity.

[0076] The relative oxidase activities of each single-point mutant were tested and are shown in Table 2.

[0077] Table 2. Oxidase activity results of Cab4UPO single-point mutants

[0078] Enzyme Relative oxidative enzyme activity fold Cab4 UPO 1 F14W 1.66 F14G 1.3 F14E 0.34 F14V 0.59 F125M 2.17 F125G 1.26 F125K 1.43 F125H 0.17 F125W 0.21 S132A 1.35 S132V 1.44 S132I 1.5 S132G 1.15 S132L 1.2 S132H 1.13 S132R 0.55 S132T 0.4

[0079] As shown in Table 2 above, Cab4UPO-F125M exhibited the highest activity. Using this as a template, site-directed mutagenesis PCR was employed to obtain the double mutants Cab4UPO-F125M-F14W and Cab4UPO-F125M-S132I, and finally, the triple mutant Cab4UPO-F14W-F125M-S132I was obtained. Following the experimental procedure described above, the activity results of each mutant are shown in Table 3.

[0080] Table 3. Oxidase activity results of Cab4UPO multiple mutants

[0081] Enzyme Relative oxidative enzyme activity fold Cab4 UPO 1 Cab4 UPO-F125M-F14W 4.32 Cab4 UPO-F125M-S132I 3.6 Cab4 UPO-F14W-F125M-S132I 12.10

[0082] Example 4: Screening test of reaction conditions for the synthesis of 25-hydroxyvitamin D3 catalyzed by peroxidase mutants (5 mutants).

[0083] The above process was followed to catalyze the synthesis of 25-hydroxyvitamin D3 from vitamin D3. The relative reactivity fold was defined as: mutant reactivity / control parent reactivity. The reaction results are shown in Table 4.

[0084] Table 4. Relative reactivity results of Cab4UPO mutants

[0085] Enzyme Relative reaction activity fold Cab4 UPO 1 Cab4 UPO-F125M 2.17 Cab4 UPO-F125M-F14W 5.64 Cab4 UPO-F125M-S132I 3.6 Cab4 UPO-F14W-F125M-S132I 15.79

[0086] Based on previous experiments, the basic reaction conditions were determined as follows: 400 μL of Cab4UPO-F14W-F125M-S132I supernatant, 300 μL of acetone, 2.5 mM vitamin D3, 25 mM hydrogen peroxide, 300 μL of 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.0), 6 mg cyclodextrin, 33℃, and stirring for 90 min. Extraction was performed with 1 mL of ethyl acetate, followed by rotary evaporation and concentration. The mixture was then resuspended in 1 mL of anhydrous ethanol for LC-MS analysis. Under these conditions, the reaction time, type, and content of co-solvents in the synthesis conditions of this invention were further screened. The results of the screening experiments are shown in Table 5.

[0087] Table 5 Results of the screening experiment for reaction conditions

[0088] Substrate concentration (mM) Hydrogen peroxide (mM) Reaction time (min) Co-solvent class Co-solvent content (%) Temperature (°C) Surfactant Analytical yield (%) 5 25 90 Acetone 30 33 Cyclodextrin 38 5 25 90 Ethanol 30 33 Cyclodextrin ＜1 5 25 90 Dithio sulfoxide 30 33 Cyclodextrin ＜1 5 25 90 Cyclohexane 30 33 Cyclodextrin ＜1 5 25 90 Ethyl acetate 30 33 Cyclodextrin ＜1 5 25 90 Methyl tert-butyl ether 30 33 Cyclodextrin ＜1 5 50 90 Acetone 30 33 Cyclodextrin 26 5 50 90 Acetone 30 33 Cyclodextrin 21 5 25 90 Acetone 30 33 Cyclodextrin 28 5 50 90 Acetone 30 33 Cyclodextrin 28 5 25 90 Acetone 30 37 Cyclodextrin 43 5 25 90 Acetone 40 33 Cyclodextrin 50 5 25 90 Acetone 30 33 Tween 20 36 5 25 90 Acetone 30 33 Tocopherol 28 5 25 90 Acetone 30 33 Coconut oil 27 5 25 90 Acetone 30 33 None 37

[0089] The detection conditions for the reaction results were as follows: the chromatographic column was an Agilent InfinityLab Poroshell 120 EC-C18, C18, 4 μm, 4.6 × 150 mm; the flow rate was 1.5 mL / min; the column temperature was 40℃; and the mobile phases were water containing 0.1% formic acid and methanol in a ratio of 5:95. A standard curve was prepared based on the theoretical yield to analyze the relationship between the product yield and the product peak area, as shown below.Figure 3 As shown.

[0090] The optimal reaction conditions were determined to be: 25 mM hydrogen peroxide, 90 min reaction time, 40% acetone as the co-solvent, 37℃ reaction temperature, and no surfactant used.

[0091] Pichia pastoris containing Cab4UPO-F14W-F125M-S132I was inoculated into 200 mL of YPD liquid medium and cultured at 30°C and 250 rpm for 16 h. In a 5 L fermenter, 2 L of BSM medium was prepared: 54 g 85% phosphate, 1.4 g calcium sulfate, 36 g potassium sulfate, 48 g magnesium sulfate heptahydrate, 8 g potassium hydroxide, 80 g glycerol, 10 g yeast extract, and the pH was adjusted to 6 with ammonia. After autoclaving, 17.6 mL of sterile PMT1 (Beijing Cooler Master Technology Co., Ltd.) was added, along with 200 mL of the inoculum. The fermenter was then fed with feed (50% glycerol + 6 mL / L PTM1) at 30°C until OD reached [the desired growth rate]. 600 When the dissolved oxygen level reaches 200, stop feeding until the dissolved oxygen level rises and stabilizes. Then, induce fermentation at 25°C (methanol + 6 mL / L PTM1). After 7 days, stop fermentation and collect the supernatant by centrifugation at 8000 rpm for 30 min at 4°C.

[0092] 400 mL of supernatant from Cab4UPO-F14W-F125M-S132I, 400 mL of acetone, 5-10 mM vitamin D3, 25 mM hydrogen peroxide, and 200 mL of 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.0) were added. The reaction was carried out at 37°C with stirring for 90 min. After the reaction, the product was extracted and separated. The reaction results are shown in Table 6. The reaction volume was 1 L. After the reaction, the product was separated and purified to obtain the final product. The separation yield was defined as: (mass of the purified product / theoretical mass of the substrate completely converted into the product) × 100%.

[0093] Table 6. Reaction results at different substrate concentrations

[0094] Reaction number Substrate concentration (mM) Mass of fully converted product (g) Mass of product obtained after isolation and purification (g) Isolation yield (%) 1 5 2 1.61 80.5 2 6 2.4 1.99 82.9 3 7 2.8 2.27 81.1 4 8 3.2 2.62 81.9 5 9 3.6 2.46 68.3 6 10 4 2.61 65.3

[0095] NMR spectra of the products of reaction 4 are as follows Figure 4 As shown, the reaction has few impurities and the product has high purity.

Claims

1. A peroxidase Cab4UPO mutant, characterized by: It involves mutating phenylalanine at position 125 of the peroxidase Cab4UPO, as shown in SEQ ID No. 4, to methionine, resulting in Cab4UPO-F125M.

2. A peroxidase Cab4UPO mutant, characterized by: It involves mutating phenylalanine at position 125 of the peroxidase Cab4UPO, as shown in SEQ ID No. 4, to methionine, resulting in Cab4UPO-F125M. Then, mutations are made at positions 14 and / or 132, where phenylalanine at position 14 is mutated to tryptophan and serine at position 132 is mutated to isoleucine.

3. The peroxidase Cab4UPO mutant according to claim 2, characterized in that: It uses Cab4UPO-F125M as the parent and mutates at positions 14 and 132 to obtain the mutant Cab4UPO-F14W-F125M-S132I.

4. A DNA molecule, characterized in that: The DNA molecule encodes the peroxidase Cab4UPO mutant according to any one of claims 1-3.

5. An expression vector, characterized in that: The expression vector contains the DNA molecule as described in claim 4.

6. A host cell, characterized in that: The host cell contains the expression vector as described in claim 5.

7. A method for synthesizing 25-hydroxyvitamin D3, characterized in that: It uses vitamin D3 as a substrate, adds the peroxidase Cab4UPO mutant as described in any one of claims 1-3, a solubilizer, hydrogen peroxide, and a buffer solution, and reacts to generate 25-hydroxyvitamin D3.

8. The method for synthesizing 25-hydroxyvitamin D3 according to claim 7, characterized in that: The co-solvent is acetone; the buffer solution is disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and the synthesis conditions are: pH 7.0, 37℃, and stirring reaction for 90 min.

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