Nonspecific peroxygenase mutants and their applications and methods for preparing 25-hydroxyvitamin D3

Abstract

This invention discloses a nonspecific peroxygenase mutant and its application, as well as a method for preparing 25-hydroxyvitamin D3. Specifically, it relates to a protein obtained by mutating amino acid residues at positions 20 and / or 25 and / or 57, using the amino acid sequence shown in SEQ ID NO.1 as the starting sequence. This protein exhibits high catalytic activity and stereocatalytic specificity and selectivity. In a potassium phosphate buffer solution at pH 7.0–8.0, acetone, vitamin D3, hydrogen peroxide, and the nonspecific peroxygenase mutant are added to form a reaction mixture. The oxidation reaction is carried out at 25°C–35°C for 10–50 h to generate 25-hydroxyvitamin D3. The reaction conditions are mild, the operation is simple, the conversion rate is high, and the cost is low, significantly improving the preparation efficiency of 25-hydroxyvitamin D3 and showing great promise for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a non-specific peroxygenase mutant and its application in the synthesis of 25-hydroxyvitamin D3. Background Technology

[0002] 25-Hydroxyvitamin D3 (25-hydroxy cholecalciferol or calcifediol), 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. After entering the human or animal body, vitamin D3 must first be converted to 25-hydroxyvitamin D3 by the liver, and then metabolized by the kidneys to 1α,25-dihydroxyvitamin D3. Only then can it activate the calcium and phosphorus transport system in the small intestine, promoting the absorption and deposition of calcium and phosphorus. Therefore, 25-hydroxyvitamin D3 is also known as active vitamin D3, while 1α,25-dihydroxyvitamin D3 is called fully active vitamin D3. The biological potency of 25-hydroxyvitamin D3 is 20 to 40 times that of vitamin D3, and it has effects such as promoting animal bone development, improving feed conversion rate, and enhancing immunity. 25-Hydroxyvitamin D3 was initially researched and developed by American scientists and received FDA approval in 1995, permitting its use as a novel nutritional additive in livestock and poultry feed. On February 1, 2014, my country began allowing its production and use in the feed industry. 25-Hydroxyvitamin D3 is expected to replace vitamin D3 as one of the essential vitamins in livestock and poultry farming. In livestock and poultry farming, 25-Hydroxyvitamin D3 primarily performs three core functions: 1. Increasing blood calcium levels and bone density; 2. Promoting the production of immunoglobulins and enhancing immunity; 3. Improving sperm quality, increasing embryo implantation rate, hatching rate, and birth rate.

[0003] Traditional industrial production methods include chemical and biological methods. The chemical method first requires separating and extracting complex cholesterol in a separation workshop to obtain crude 25-hydroxycholesterol. Then, in a thermal processing workshop, the crude 25-hydroxycholesterol undergoes crystallization, acylation, oxidation, hydrazone formation, dehydrazone removal, and saponification to convert it into 25-hydroxy-7-dehydrocholesterol. Finally, in a photochemical workshop, the 25-hydroxy-7-dehydrocholesterol undergoes photo-induced ring-opening and column separation to obtain 25-hydroxyvitamin D3 crystals. Garden Biotechnology's 25-hydroxyvitamin D3 is prepared using a special molecular distillation extraction method. Using complex cholesterol obtained from a lanolin comprehensive utilization project as raw material, a certain steroid compound is separated from it, and high-purity 25-hydroxycholesterol is further obtained. The synthesis process from 25-hydroxycholesterol to 25-hydroxyvitamin D3 is basically similar to the process route from cholesterol to vitamin D3. DSM first obtained 5,7,24-trienol cholesterol through bio-fermentation, then obtained 25-hydroxy-7-dehydrocholesterol, a precursor of 25-hydroxyvitamin D3, through chemical synthesis methods such as epoxidation and reduction processes. Further, 25-hydroxyvitamin D3 was obtained through photochemical reactions, and finally, the final 25-hydroxyvitamin D3 product was obtained through microencapsulation technology. It is evident that chemical synthesis methods are cumbersome, require harsh reaction conditions, consume large amounts of organic solvents, produce numerous toxic and harmful byproducts, cause significant environmental pollution, and suffer from poor stereoselectivity, low conversion rates, and high costs. Bio-methods are divided into bio-fermentation and biocatalysis. Bio-fermentation involves the conversion of vitamin D3 into 25-hydroxyvitamin D3 by specific enzymes within microorganisms during fermentation. This method offers mild production conditions and is environmentally friendly, but currently, it also suffers from complex fermentation broth composition, low product concentration (less than 1 g / L), and long fermentation cycles, which severely affect subsequent extraction efficiency. Biocatalysis primarily utilizes genetic engineering to prepare specific oxidases that specifically oxidize vitamin D3 to 25-hydroxyvitamin D3 in vitro. This process requires only one hydroxylation reaction, exhibiting good stereoselectivity, short reaction steps, mild reaction conditions, high conversion rate, high product purity, and environmental friendliness, without producing many toxic or harmful substances. Several P450 enzymes, such as CYP105A1, CYP109A2, CYP109E1, and Vdh, have been reported to catalyze the conversion of vitamin D3 to calcidiol, specifically by hydroxylation at the C-25 position of vitamin D3. CN115838695A reports a study on the selective hydroxylation of vitamin D3 to calcidiol using P450 enzymes. The study screened and improved a P450 enzyme derived from *Bacillus desertica*, enhancing its catalytic activity and overcoming its limitation of low hydroxylation site specificity, thus achieving the exclusive production of calcidiol.However, the P450 enzyme catalysis method also has certain limitations. The reaction process requires expensive NADPH as a coenzyme, and most P450 enzymes have low expression levels (membrane proteins), low substrate concentrations, poor position selectivity of some P450 enzyme varieties leading to the easy generation of other byproducts, and long catalysis time. Industrial production process conditions need to be optimized in many aspects.

[0004] In recent years, unspecific peroxygenases (UPOs, EC 1.11.2.1.) have been continuously discovered and characterized. They share the same active site as P450 enzymes and exhibit similar catalytic mechanisms. UPOs are a class of fungal oxidases belonging to the heme oxidase family, possessing multifunctional oxidative catalytic activity. Compared to the more frequently reported P450 monooxygenases and oleic acid hydratases, UPOs have a broader substrate range, and the 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, thus demonstrating greater application potential. 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 nonspecific peroxygenase derived from *Agrocybe aegerita* is used as a catalyst, and hydrogen peroxide as an oxidant to activate CH bonds and perform hydroxylation reactions, enabling the one-step catalytic oxidation process to prepare calcidiol. However, the selectivity of this enzyme for C-25 hydroxylation is currently only 70%, and the large number of byproducts increases the difficulty of downstream processing. Therefore, it is necessary to use enzyme engineering to modify UPO to improve its catalytic activity for vitamin D3 and its specificity and selectivity for C-25 hydroxylation, improve catalytic efficiency, and simplify the reaction system so that it can better meet the needs of production processes. This has become an important research direction for developing green synthesis technology of 25-hydroxyvitamin D3. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a non-specific peroxygenase mutant, its applications, and a method for preparing 25-hydroxyvitamin D3. The non-specific peroxygenase mutant exhibits high catalytic activity and stereocatalytic specificity and selectivity, enabling it to selectively catalyze the oxidation of vitamin D3 to 25-hydroxyvitamin D3. Compared to the wild-type enzyme, this mutant significantly improves catalytic efficiency and yield while reducing byproducts. Furthermore, the synthesis of 25-hydroxyvitamin D3 utilizes mild reaction conditions, simple operation, high conversion rate, and low cost, greatly improving the efficiency of 25-hydroxyvitamin D3 preparation and demonstrating promising industrial application prospects.

[0006] To achieve the above objectives and technical effects, the present invention adopts the following technical solution:

[0007] The present invention provides a nonspecific peroxygenase mutant comprising the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology thereon, wherein the protein is obtained by mutating the amino acid residues located at or corresponding to the 20th and / or 25th and / or 57th positions of SEQ ID NO.1.

[0008] In some embodiments of the present invention, the nonspecific peroxygenase mutant includes any of the following:

[0009] (1) Mutate position 20 of the amino acid sequence located at or corresponding to the position shown in SEQ ID NO.1 from E to A;

[0010] (2) Mutate position 25 of the amino acid sequence located at or corresponding to the position shown in SEQ ID NO.1 from M to K;

[0011] (3) Mutate position 57 of the amino acid sequence located at or corresponding to that shown in SEQ ID NO.1 from S to V;

[0012] (4) Mutate position 20 of the amino acid sequence located at or corresponding to SEQ ID NO.1 from E to A, and position 25 from M to K;

[0013] (5) Mutate position 25 of the amino acid sequence located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from M to K, and position 57 from S to V;

[0014] (6) Mutate the 20th position of the amino acid sequence located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from E to A, and the 57th position from S to V;

[0015] (7) The 20th position of the amino acid sequence located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 is mutated from E to A, the 25th position is mutated from M to K, and the 57th position is mutated from S to V.

[0016] The present invention provides a nonspecific peroxygenase mutant, which is a protein obtained by mutating the amino acid residues shown at positions 20 and / or 25 and / or 57 of the amino acid sequence shown in SEQ ID NO.1.

[0017] In some embodiments of the present invention, the nonspecific peroxygenase mutant includes any of the following:

[0018] (1) The 20th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from E to A, and named mutant E20A;

[0019] (2) The 25th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from M to K, and named as mutant M25K;

[0020] (3) The 57th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from S to V, and named the mutant S57V;

[0021] (4) The 20th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from E to A, and the 25th position is mutated from M to K, and named the mutant E20A / M25K;

[0022] (5) The 25th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from M to K, and the 57th position is mutated from S to V, and named the mutant M25K / S57V;

[0023] (6) The 20th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from E to A, and the 57th position is mutated from S to V, named the mutant E20A / S57V;

[0024] (7) The 20th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from E to A, the 25th position is mutated from M to K, and the 57th position is mutated from S to V, and named the mutant E20A / M25K / S57V.

[0025] Another object of the present invention is to provide a polynucleotide encoding the aforementioned nonspecific peroxygenase mutant.

[0026] Another object of the present invention is to provide a recombinant vector comprising a polynucleotide encoding the aforementioned nonspecific peroxygenase mutant.

[0027] Another object of the present invention is to provide a recombinant vector, wherein the vector is pET-23a, pET-28a, or pPIC9k; preferably pPIC9k. Specifically, it can be constructed by linking the gene encoding the nonspecific peroxygenase mutant to the vector using conventional methods in the art.

[0028] In some embodiments of the present invention, the recombinant vector is prepared by the following method: a nucleic acid product obtained by artificial chemical synthesis (e.g., wild-type nonspecific peroxygenase gene fragment) and the vector pPIC9k are digested with restriction endonucleases EcoRI and NotI, respectively, to form complementary sticky ends, and then ligated with T4 ligase to form the recombinant vector UPO-pPIC9k.

[0029] Another object of the present invention is to provide a recombinant engineered bacterium containing the aforementioned recombinant vector.

[0030] In some embodiments of the present invention, the host cell of the recombinant engineered bacteria is preferably, but not limited to, Escherichia coli DH5α chemically competent cells; specifically, it can be prepared by transforming the above-mentioned recombinant vector or the gene encoding the nonspecific peroxygenase mutant into the host cell. The host cell can be a conventional host cell in the art, but it must be able to stably replicate on its own as the recombinant vector, and the gene encoding the nonspecific peroxygenase mutant carried by it must be effectively expressed.

[0031] Another object of the present invention is to provide an application of the aforementioned nonspecific peroxygenase mutant in catalyzing the oxidation of vitamin D3 to produce 25-hydroxyvitamin D3.

[0032] Another object of the present invention is to provide a method for preparing 25-hydroxyvitamin D3, the method comprising:

[0033] Acetone, vitamin D3, hydrogen peroxide, and the aforementioned nonspecific peroxygenase mutant were dissolved in potassium phosphate buffer to form a reaction mixture. The mixture was stirred to generate 25-hydroxyvitamin D3.

[0034] In some embodiments of the present invention, the method for preparing 25-hydroxyvitamin D3, wherein the nonspecific peroxygenase mutant is in any of the following forms:

[0035] a1) A cell containing a recombinant vector, wherein the recombinant vector contains a gene encoding the nonspecific peroxygenase mutant described above;

[0036] a2) Crude enzyme solution, the fermentation broth obtained from the cell fermentation described in a1);

[0037] a3) Pure enzyme solution, obtained by purifying the crude enzyme solution described in a2);

[0038] a4) Enzyme powder, obtained by drying the crude enzyme solution described in a2) or the pure enzyme solution described in a3), wherein the non-specific peroxygenase mutant in the enzyme powder still retains catalytic activity;

[0039] a5) Immobilized enzyme, that is, the non-specific peroxygenase mutant described above is treated by physical or chemical methods to increase its stability while retaining its catalytic activity, so that it can be used repeatedly.

[0040] In a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 according to the present invention, wherein:

[0041] The concentrations of nonspecific peroxygenase mutants, acetone, hydrogen peroxide, and vitamin D3 in the reaction mixture were 1–10 U / L, 200–500 g / L, 1–10 mmol / L, and 1–10 g / L, respectively.

[0042] In a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 according to the present invention, wherein:

[0043] The concentration of the potassium phosphate buffer solution is 10–100 mmol / L, and the pH is 7.0–8.0.

[0044] In a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 according to the present invention, wherein:

[0045] The temperature of the stirring reaction is 25℃~35℃, and the stirring reaction time is 10~50h.

[0046] In a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 according to the present invention, wherein:

[0047] The concentrations of nonspecific peroxygenase mutants in the reaction mixture are 1 U / L, 3 U / L, 4 U / L, 5 U / L, 6 U / L, 8 U / L, or 10 U / L; the concentrations of acetone are 200 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L, 450 g / L, or 500 g / L; the concentrations of hydrogen peroxide are 1 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 8 mmol / L, or 10 mmol / L; and the concentrations of vitamin D3 are 1 g / L, 3 g / L, 5 g / L, 7 g / L, 8 g / L, 9 g / L, or 10 g / L.

[0048] In a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 according to the present invention, wherein:

[0049] The potassium phosphate buffer solution has a concentration of 10 mmol / L, 20 mmol / L, 25 mmol / L, 27 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 80 mmol / L, or 100 mmol / L, and a pH of 7.0, 7.5, or 8.0.

[0050] In a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 according to the present invention, wherein:

[0051] The temperature of the stirring reaction is 25℃, 28℃, 30℃, 32℃ or 35℃, and the stirring reaction time is 10h, 20h, 24h, 30h, 36h, 40h, 46h or 50h.

[0052] In a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 according to the present invention, wherein:

[0053] The concentrations of the nonspecific peroxygenase mutant in the reaction mixture were 5 U / L, acetone 400 g / L, hydrogen peroxide 5 mmol / L, and vitamin D3 10 g / L.

[0054] The concentration of the potassium phosphate buffer solution is 27 mmol / L, and the pH is 8.0;

[0055] The stirring reaction was carried out at a temperature of 30°C for 24 hours.

[0056] Terminology Definition

[0057] The terms “mutant” or “variant” are used interchangeably throughout the present invention and refer to a UPO of the present invention obtained by the methods described herein and having at least one mutation, preferably at least two mutations, more preferably at least three mutations, which result in higher peroxygenase activity compared to the corresponding natural or wild-type enzyme.

[0058] The term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides and deoxyribonucleotides.

[0059] The term "encoding" refers to the relationship between a nucleotide triplet or codon in a DNA sequence and the amino acids that make up a peptide, amino acid sequence, or protein. When describing a nucleotide sequence as encoding a peptide, it means that the peptide is produced when the nucleotide sequence is transcribed into messenger RNA (mRNA) and the mRNA is translated.

[0060] The terms "homology" or "percentage homology" (percentage homology, identity + similarity) are used to determine biotechnological information using homology comparison software from the National Center for Biotechnology Information (NCBI), such as BLASP, TBLASTN, or tBLASTX, using specific parameters. For the purposes of this invention, the term "homology" or "homologous sequence" refers to the identity of two or more nucleic acid sequences or the identity of two or more amino acid sequences. Homologous sequences include "paralogous homologs" and "orthologous homologs." The term "paralogous homolog" refers to gene duplication within a species' genome, producing a paralogous gene. The term "orthologous homolog" refers to homologous genes arising from ancestral relationships in different organisms.

[0061] The term "vector" or "expression vector" refers to a means by which a DNA or RNA sequence (e.g., a heterologous gene) can be introduced into a host cell to transform the host and promote cell production. This includes, for example, the transcription and translation of the introduced sequence. Vectors typically contain DNA containing a transmissible factor, into which foreign DNA encoding a protein is inserted using restriction enzyme techniques. A common type of vector is the "plasmid," which is usually a double-stranded DNA molecule that readily accepts additional (foreign) DNA and can be easily introduced into a suitable host cell. Plasmids and fungal vectors have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, WI), pRSET or PrEP plasmid (Invitrogen, San Diego, CA), pMAL plasmid (NewEngland Biolabs, Beverly, MA), pGAPZaA, pPIC9k, pcWori+, pET-26b (+), pXTD14, pYEX-S1, pMAL, and pET22-b (+). Recombinant cloning vectors typically include one or more replication systems for cloning or expression, one or more markers for selection in the host, such as antibiotic resistance, and one or more expression cassettes. Suitable vectors for inserting the polynucleotide are vectors derived from prokaryotic expression vectors, such as pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, Co1E1, pCR1, RP4, bacteriophages, and shuttle vectors such as pSA3 and pAT28; yeast expression vectors such as Saccharomyces cerevisiae two-micron plasmids, integrative plasmids, YEP vectors, centromere plasmids; expression vectors in insect cells, such as the pAC series vectors and pVL expression vectors; plant cell expression vectors such as piBi, pEarleyGate, PAVA, pCAMBIA, PGSA, PGWB, PMDC, PMY, pore series, etc.; and other eukaryotic cell expression vectors, including baculoviruses suitable for transfecting insect cells using any commercially available baculovirus system. Those skilled in the art may use other vectors as needed. If different from those described in the examples, conventional biotechnological experiments can be used to determine which vectors are best suited for use in this invention. Generally, the choice of vector depends on the size of the polynucleotide sequence and the host cell used in the method of this invention.

[0062] Throughout the specification and claims, the word "comprising" is not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will become apparent to those skilled in the art, partly from the specification and partly from practice of the invention. The following examples are provided to illustrate the invention and are not intended to limit it.

[0063] The beneficial effects of this invention are as follows:

[0064] (1) The non-specific peroxygenase mutant provided by the present invention has high catalytic activity and stereocatalytic specificity and selectivity. It can selectively catalyze the oxidation reaction of vitamin D3 to generate 25-hydroxyvitamin D3. Compared with wild-type enzyme, the mutant can significantly improve catalytic efficiency and yield and reduce by-products. The non-specific peroxygenase mutant of the present invention can be prepared in large quantities by constructing recombinant engineered bacteria and fermentation culture, and is relatively inexpensive and readily available.

[0065] (2) The non-specific peroxygenase mutant provided by the present invention has mild reaction conditions, simple reaction operation, high conversion rate and low cost in the process of catalyzing the oxidation reaction of vitamin D3 to generate 25-hydroxyvitamin D3. It greatly improves the preparation efficiency of 25-hydroxyvitamin D3. Moreover, it is a "one-pot" reaction, in which all raw materials are added at the same time, and the final product 25-hydroxyvitamin D3 is directly obtained after the reaction. It has good prospects for industrial application. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts disclosed in this invention. Such structures and techniques have also been described in many publications.

[0067] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0068] Example 1 Construction of wild-type nonspecific peroxygenase

[0069] The non-specific peroxygenase fragment (gene sequence shown in SEQ ID NO.2) derived from Coprinopsis marcescibilis, synthesized in its entirety, was optimized for Pichia pastoris codons and then synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd. After digestion with restriction endonucleases EcoRI and NotI (purchased from New England Biolabs, and operated according to the instructions), the fragment was ligated into the similarly digested vector pPIC9k to obtain the wild-type recombinant vector UPO-pPIC9k.

[0070] The gene sequence of wild-type nonspecific peroxygenase (as shown in SEQ ID NO.2):

[0071] TTACCTCCTCCACCACCAGCTCCACCATCTTTTACTGGAGCTAAGTTAGTTGATGATGAAGATCATCCTTGGATGCCTTTGCAATCTGGTGACATTAGAGGTCCATGTCCTGGATTGAACACTTTGGCTTCTCACGGTTACTTGCCTAGAAATGGAGTTGCTTCTCCATCTGATATGATTAAGGCTGTTCAAGAAGGTTTTAACATGGAAACTCAAGCTGCTAGAGTTGCTGCTTACTCTGCTCACTTGTTGGAAGGTAACTTGGTTACTGATTTGTTGTCTATTGGTGGTAAAACTTCTTTGACTGGTCCAGATCCACCAGCTCCAGCTACTGTTGCTGGAATTTCTAACCATGGTTCCTTTGAAGGTGACGCTTCCATGACTAGAATTGATGCTTTCTTTGGAGATCACACTGTTTTTAACCAACCATTGTTTGATCAATTTGTTGATTTTTCTAACAGATTTGGTAACGGTTTTTATAACTATACTGTTGGTGGTGAGTTGAGATTTCATAGAATTCAACAATCTATTGAAACTAATCCTCAATTTTCTATTAGAGGTTTCAGACACTTGACTGCTTACGGTGAAGCTGCTTTTGTTGCTAACATTTTTGTTGATGGTAGAAAGACTGGTGCTGATGCTCATCATTTGGATATGGATTCTGCTTTGTCCTTCTTTAGAGATATGAGATTCCCACAAGGTTTCTTCAGACCTTCCAGTCCTACCGCTGGTGAGGGTGCTGATATTATTTTTGCTGCTCATCCAACTCAGCCAGGATCTAATAACGGTGCTGTTAACTCATTTGTTGTTGATACTTCTATGGGTGGTTTGACTGATCCATGTACTTTTTACGTTCAATTTGTTTCTACTACTATTCCAGCTTTGTACCCTAACCCAACTGGTGTTTTGAGAAGAAACTTGATTATTAACTTGGGTTTTCTTTACGATGCTTTGTCTCCACCTAACTGTCCACAATTGTTTCCATACGGTCAAAACTGA。

[0072] The amino acid sequence of the wild-type nonspecific peroxygenase (as shown in SEQ ID NO.1):

[0073] LPPPPPAPPSFTGAKLVDDEDHPWMPLQSGDIRGPCPGLNTLASHGYLPRNGVASPSDMIKAVQEGFNMETQAARVAAYSAHLLEGNLVTDLLSIGGKTSLTGPDPPAPATVAGISNHGSFEGDASMTRIDAFFGDHTVFNQPLFDQFVDFSNRFGNGFYNYTVGG ELRFHRIQQSIETNPQFSIRGFRHLTAYGEAAFVANIFVDGRKTGADAHHLDMDSALSFFRDMRFPQGFFRPSSPTAGEGADIIFAAHPTQPGSNNGAVNSFVVDTSMGGLTDPCTFYVQFVSTTIPALYPNPTGVLRRNLIINLGFLYDALSPPNCPQLFPYGQN.

[0074] Example 2 Construction of nonspecific peroxygenase mutants

[0075] Using the amino acid sequence SEQ ID NO.1 as a template, a random mutant library was constructed using the Agilent GeneMorph II RandomMutagenesis Kit (catalog number 200550). The forward primer was UPO-EcoRⅠ-F 5-CCGGAATTCTTACCTCCTCCACCACCAGCTCCAC-3', and the reverse primer was UPO-NotⅠ-R 5'-ATAAGAATGCGGCCGCTCAGTTTTGACCGTATGGAAACAAT-3'.

[0076] The 50 μl PCR system includes: 5 μl 10× Mutazyme II reaction buffer, 1 μl 40 mM dNTP mix (200 μM each final), 1 μl each of primers UPO-EcoRI-F and UPO-NotRI-R (10 μM), 1 μl Mutazyme II DNA polymerase (2.5 U / μl), and 50 ng UPO-pPIC9k, with water added to a final volume of 50 μl.

[0077] The PCR program was 95℃ for 2 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min; 30 cycles, 72℃ for 10 min.

[0078] A 1kb random mutant fragment was recovered from the gel, digested with restriction endonucleases EcoRI and NotI (purchased from New England Biolabs, operated according to the instructions), and recombined into the vector pPIC9k. The vector was then transformed into E. coli DH5α chemocompetent cells (TransGen) to obtain a random mutant library.

[0079] Example 3: Screening for expression of wild-type and mutant nonspecific peroxygenases in Pichia pastoris

[0080] (1) Plasmid DNA linearization

[0081] The nonspecific peroxygenase wild-type and mutant library vectors were linearized using the restriction endonuclease SalⅠ (purchased from New England Biolabs, operated according to the instructions).

[0082] (2) Electrotransfer to Pichia pastoris

[0083] The linearized fragment was added to 80 μl of Pichia pastoris GS115 competent cells (purchased from Beijing Huayueyang) and incubated on ice for 5 minutes. After electroporation, 800 μl of sorbitol was added to wash the cells into a 1.5 ml sterile centrifuge tube. After incubation at 25°C for 2 hours, the cells were centrifuged, plated on MD plates, and cultured at 30°C until colonies appeared. Single colonies were then streaked to isolate them. A single colony was picked into sterile water, and an appropriate amount of Lyticase (purchased from Sigma) was added. The cell wall was then digested at 37°C for 1 hour. A portion of the digestion product was added to a PCR system to detect positive clones.

[0084] (3) Pichia pastoris expression

[0085] BMD medium: 100 ml 1M phosphate buffer (pH 6.0), 100 ml 10% glucose, 100 ml 10×YNB, 2 ml 500×Biotin, 698 ml purified water.

[0086] 1% methanol BMMY medium: 100 ml 1M phosphate buffer (pH 6.0), 10 ml methanol, 100 ml 10×YNB, 2 ml 500×Biotin, and purified water to 1L.

[0087] 5% methanol BMMY medium: 100 ml 1M phosphate buffer (pH 6.0), 50 ml methanol, 100 ml 10×YNB, 2 ml 500×Biotin, and purified water to 1L.

[0088] Transformants from wild-type and mutant libraries were selected using toothpicks and inoculated into 96-well deep-well plates containing 250 μl of BMD medium. The plates were incubated at 30°C and 280 rpm for 48 hours with shaking. Then, 250 μl of 1% methanol BMMY medium was added and the plates were incubated for another 24 hours. Every 24 hours, 50 μl of 5% methanol BMMY medium was added and the plates were incubated at 30°C and 280 rpm for 96 hours with shaking. Finally, the supernatant was collected by centrifugation at 10,000 rpm to detect enzyme activity.

[0089] Example 4 UPO enzyme activity assay

[0090] The enzyme activity of UPO was detected using 2,2'-adiazon-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate: 100 μL of 100 mM phosphate buffer (pH 4.0), 30 μL of 2 mM ABTS, 30 μL of deionized water, and 20 μL of crude enzyme solution were added sequentially to a 96-well plate. After thorough mixing, 20 μL of 20 mM hydrogen peroxide was added, and the absorbance was measured at a wavelength of 418 nm. The enzyme activity results are shown in Table 1.

[0091] At 25°C and pH 4.0, the amount of enzyme required to hydrolyze 1 μmol of substrate (ABTS) per minute to generate the corresponding product (ABTS radical) was 1 enzyme activity unit (U). The results are shown in Table 1.

[0092] Table 1

[0093]

[0094] Conclusion: Compared with wild-type UPO, the nonspecific peroxygenase mutant described in this invention has higher catalytic activity.

[0095] Example 5 Preparation of nonspecific peroxygenase mutants

[0096] Shake-flask fermentation:

[0097] (a) Preparation of shake flask seed culture medium: 10 g yeast extract, 20 g peptone, and 20 g glucose were dissolved in 800 mL of distilled water, the pH was adjusted to 7, and the volume was brought up to 1000 mL with distilled water. The mixture was kept at 115 °C for 30 min.

[0098] (b) Fermentation steps: Single clones of the mutant E20A / M25K / S57V were picked from the plate and inoculated into 50 mL of seed medium (250 mL Erlenmeyer flask). After culturing at 30°C with shaking at 200 rpm for 24 hours, the OD600 reached 4. Then, 1% of the culture was inoculated into 300 mL of seed medium (1 L Erlenmeyer flask) and cultured at 30°C with shaking at 200 rpm for 24 hours, after which the OD600 reached 7.

[0099] Fermentation in fermentation tanks:

[0100] The seed culture was inoculated at a 10% inoculum and cultured at 30℃ and 200 r / min for 24 h. The secondary seed culture, cultured to the logarithmic growth phase, was then inoculated at 10% (V / V) into a 10 L autoclaved fermenter (containing 6 L of basal salt medium: 26.7 mL / L 85% phosphate, 0.93 g / L CaSO4▪2H2O, 14.9 g / L MgSO4▪7H2O, 18.2 g / L K2SO4, 7.13 g / L KOH, 40 g / L glycerol; after sterilization, PTM1 trace element was added to a final concentration of 12 mL / L) and cultured at 30℃ and 800 r / min. When the glycerol in the basal salt medium was depleted, 50% (V / V) glycerol was added as feed (containing 12 mL / L PTM1 trace element), with the flow rate adjusted according to dissolved oxygen (DO) to maintain a dissolved oxygen concentration of approximately 40%. Feeding was stopped when the wet weight reached 160 g / L, and the enzyme was starved for 1 h. After starvation, the temperature was set to 22℃, and the pH was controlled at around 6.0. Methanol was added for low-temperature induction to maintain dissolved oxygen at around 40% for 120 h. The enzyme solution was concentrated using a 10 kDa ultrafiltration membrane, and the expression of recombinant protein and enzyme activity were then detected.

[0101] Example 6: Preparation of 25-hydroxyvitamin D3 from a nonspecific peroxygenase mutant

[0102] In a 250 ml reaction flask, add 54 ml of 50 mmol / L potassium phosphate buffer solution (pH=8.0), 40 ml of acetone, 1 g of vitamin D3, 5 ml of 0.1 mol / L hydrogen peroxide (final concentration 5.0 mmol / L), and 1 ml of enzyme solution (0.5 U / ml). The final reaction volume is approximately 100 ml. The concentrations of the added reagents are calculated based on the final reaction volume (e.g., enzyme concentration = (0.5 U / ml × 1 ml) / 100 ml = 5 U / L). The reaction is carried out at 30℃ for 24 h with magnetic stirring. After the reaction is completed, 100 μl of the reaction solution is taken, 200 μl of ethyl acetate is added, and the mixture is extracted and dried over anhydrous sodium sulfate. The content of 25-hydroxyvitamin D3 is detected by HPLC, and the conversion rate of 25-hydroxyvitamin D3 is calculated. The results are shown in Table 2.

[0103] Table 2

[0104]

[0105] Conclusion: Compared with wild-type UPO, the nonspecific peroxygenase mutant described in this invention has higher yield and conversion rate in the process of catalyzing the oxidation of vitamin D3 to 25-hydroxyvitamin D3.

[0106] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A nonspecific peroxygenase mutant, characterized in that, It is a protein obtained by mutating the amino acid sequence shown in SEQ ID NO.1, wherein the amino acid mutation is selected from any of the following: (1) Mutate the 57th position of the amino acid sequence shown in SEQ ID NO.1 from S to V; (2) Mutate the 25th position of the amino acid sequence shown in SEQ ID NO.1 from M to K, and the 57th position from S to V; (3) Mutate the 20th position of the amino acid sequence shown in SEQ ID NO.1 from E to A, and the 57th position from S to V; (4) The 20th position of the amino acid sequence shown in SEQ ID NO.1 is mutated from E to A, the 25th position is mutated from M to K, and the 57th position is mutated from S to V.

2. A polynucleotide encoding the nonspecific peroxygenase mutant of claim 1.

3. A recombinant vector, characterized in that: The recombinant vector comprises the polynucleotide of claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector is pET-23a, pET-28a, or pPIC9k.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contain the recombinant vector as described in claim 3 or 4.

6. The application of the nonspecific peroxygenase mutant according to claim 1 in catalyzing the oxidation of vitamin D3 to produce 25-hydroxyvitamin D3.

7. A method for preparing 25-hydroxyvitamin D3, characterized in that: Acetone, vitamin D3, hydrogen peroxide, and the nonspecific peroxygenase mutant as described in claim 1 were dissolved in potassium phosphate buffer to form a reaction mixture. The mixture was stirred to generate 25-hydroxyvitamin D3.

8. The method for preparing 25-hydroxyvitamin D3 according to claim 7, characterized in that: The concentrations of nonspecific peroxygenase mutants, acetone, hydrogen peroxide, and vitamin D3 in the reaction mixture were 1–10 U / L, 200–500 g / L, 1–10 mmol / L, and 1–10 g / L, respectively. The concentration of the potassium phosphate buffer solution is 10–100 mmol / L, and the pH is 7.0–8.

0.

9. The method for preparing 25-hydroxyvitamin D3 according to claim 7, characterized in that: The temperature of the stirring reaction is 25℃~35℃, and the stirring reaction time is 10~50h.

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