Non-specific peroxygenase mutant, application thereof and preparation method of 25-hydroxyvitamin D3

Abstract

The invention discloses a non-specific peroxygenase mutant and application thereof and a preparation method of 25-hydroxyvitamin D3, and particularly relates to a protein obtained by taking an amino acid sequence as shown in SEQ ID NO.1 as a starting sequence and mutating amino acid residues as shown in the 20th site and / or the 25th site and / or the 57th site. The catalyst has relatively high catalytic activity and stereocatalysis specific selectivity; according to the method, acetone, vitamin D3, hydrogen peroxide and a non-specific peroxygenase mutant are respectively added into a potassium phosphate buffer solution with the pH value of 7.0-8.0 to form a reaction mixed solution, an oxidation reaction is carried out for 10-50 hours at the temperature of 25-35 DEG C, the 25-hydroxyvitamin D3 is generated, the reaction condition is mild, the reaction operation is simple and convenient, the conversion rate is high, the cost is low, the preparation efficiency of the 25-hydroxyvitamin D3 is greatly improved, and the method is suitable for industrial production. Good industrial application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a non-specific peroxygenase mutant and application thereof in the synthesis of 25-hydroxyvitamin D3. Background Art

[0002] 25-hydroxyvitamin D3 (25-hydroxy cholecalciferol or calcifediol), also known as calcifediol, is the first active metabolite of vitamin D3. It has stronger physiological activity and is also the main active form of vitamin D3 in the blood circulation. It enters the blood directly without being metabolized by the liver. After vitamin D3 enters the human or animal body, it must first be converted into 25-hydroxyvitamin D3 by the liver, and then metabolized by the kidneys to 1α, 25-dihydroxyvitamin D3, in order to activate the calcium and phosphorus transport system in the small intestine and promote the absorption and deposition of calcium and phosphorus. Therefore, 25-hydroxyvitamin D3 is also called active vitamin D3, and 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 the effects of promoting animal bone development, improving feed conversion rate, and enhancing resistance. 25-Hydroxyvitamin D3 was developed by American scientists in the early years and was certified by the U.S. Food and Drug Administration (FDA) in 1995. It was approved by the U.S. FDA to be used as a new nutritional additive in livestock and poultry feed. On February 1, 2014, my country began to allow the feed industry to produce and use it. In the future, 25-Hydroxyvitamin D3 is expected to replace vitamin D3 and become one of the essential vitamins in livestock and poultry farming. In livestock and poultry farming, 25-Hydroxyvitamin D3 mainly plays three core functions: 1. Increase blood calcium content and enhance bone density; 2. Promote the body to produce immunoglobulins and enhance immunity; 3. Improve sperm quality, increase embryo implantation rate, and increase hatching rate and birth rate.

[0003] Traditional industrial production methods are chemical and biological. The chemical method first requires the separation of complex cholesterol in the separation workshop through separation and extraction, dehydrogenation of crude products and other processes to obtain 25-hydroxycholesterol crude product; then in the thermal workshop, the 25-hydroxycholesterol crude product is converted into 25-hydroxy-7-dehydrocholesterol through crystallization, acylation, oxidation, hydrazone preparation, dehydrazone, saponification and other processes; finally, in the photochemical workshop, 25-hydroxy-7-dehydrocholesterol is subjected to light ring opening and column separation to obtain 25-hydroxyvitamin D3 crystals. Garden Bio 25-hydroxyvitamin D3 is prepared by a special molecular distillation extraction method. The complex cholesterol obtained from the lanolin comprehensive utilization project is used as a raw material to separate a certain steroid compound from it, and further obtain high-purity 25-hydroxycholesterol. The synthesis process from 25-hydroxycholesterol to 25-hydroxyvitamin D3 is basically similar to the process route from cholesterol to vitamin D3. DSM first obtains 5,7,24-triene cholesterol through biological fermentation, then obtains the precursor of 25-hydroxyvitamin D3, 25-hydroxy-7-dehydrocholesterol, through chemical synthesis methods such as epoxidation and reduction processes, further obtains 25-hydroxyvitamin D3 through photochemical reaction, and finally obtains the final 25-hydroxyvitamin D3 product through microencapsulation technology. It can be seen that the chemical synthesis method has cumbersome steps, harsh reaction conditions, large amounts of organic solvents, and produces many toxic and harmful by-products, which pollutes the environment. There are also problems such as poor product stereoselectivity, low conversion rate, and high cost. The biological method is divided into biological fermentation and biocatalysis. The biological fermentation method is to convert vitamin D3 into 25-hydroxyvitamin D3 through specific enzymes in the microorganism during the fermentation process of microorganisms. This method has mild production conditions and is environmentally friendly, but it also has problems such as complex fermentation broth composition, low product concentration (less than 1g / L), and long fermentation cycle, which seriously affect the post-extraction efficiency. The biocatalytic principle mainly uses genetic engineering technology to prepare specific oxidases, which specifically oxidize vitamin D3 in vitro to generate 25-hydroxyvitamin D3. It only needs to undergo a one-step hydroxylation reaction, and the reaction has good stereoselectivity, short reaction steps, mild reaction conditions, high conversion rate, high product purity, and is environmentally friendly, and does not produce many toxic and harmful substances. It has been reported that multiple P450 enzymes such as CYP105A1, CYP109A2, CYP109E1, Vdh, etc. can realize the catalytic process of vitamin D3 to calcifediol, that is, hydroxylation at the C-25 position of vitamin D3. CN115838695A reported the research work of using P450 enzymes to catalyze vitamin D3 through selective hydroxylation reaction to prepare calcifediol. The P450 enzymes derived from Bacillus sonorensis were screened and improved to have higher catalytic activity, and overcome the defect of low singleness of its hydroxylation position, and exclusively generate calcifediol.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 catalytic substrate concentrations, and some P450 enzyme varieties have poor positional selectivity, which makes it easy to produce other by-products. The catalysis time is long, and the industrial production process conditions need to be optimized in many aspects.

[0004] In recent years, unspecific peroxygenase (UPO, EC 1.11.2.1.) has been continuously discovered and characterized. It has the same active center as P450 enzyme and similar catalytic mechanism. Unspecific peroxygenase is a kind of oxidase derived from fungi, belongs to the heme oxygenase family, and has multifunctional oxidation catalytic activity. Compared with oxidases such as P450 monooxygenase and oleate hydratase, which are widely reported in current research, UPO has a wider substrate range, and the catalytic process can directly use cheap hydrogen peroxide as an oxygen donor and electron acceptor, without the need for a complex electron transport chain, etc., which greatly saves the reaction cost, and therefore has greater application prospects. CN115181758A reports a method for catalytic synthesis of 25-hydroxyvitamin D3 by immobilized UPO, using vitamin D3 as a raw material, using nonspecific peroxygenase from Agrocybe olei as a catalyst, and using hydrogen peroxide as an oxidant to carry out CH bond activation and hydroxylation reaction, which can realize a one-step catalytic oxidation method to prepare calcifediol. However, the enzyme's selectivity for C-25 hydroxylation is only 70% at present, and the large number of by-products increases the difficulty of downstream processing. Therefore, it is necessary to use enzyme engineering methods to transform UPO, improve the catalytic activity for vitamin D3 and the specific selectivity for C-25 hydroxylation, improve the catalytic efficiency, and simplify the reaction system so that it can better meet the production process requirements. This has become an important research direction for the development of green synthesis technology for 25-hydroxyvitamin D3. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a non-specific peroxygenase mutant and its application and a method for preparing 25-hydroxyvitamin D3. The non-specific peroxygenase mutant has high catalytic activity and stereocatalytic specific selectivity, and can catalyze vitamin D3 to produce 25-hydroxyvitamin D3 with high selectivity. Compared with the wild-type enzyme, the mutant can significantly improve the catalytic efficiency and yield and reduce by-products. In the synthesis of 25-hydroxyvitamin D3, the reaction conditions are mild, the reaction operation is simple, the conversion rate is high, the cost is low, the preparation efficiency of 25-hydroxyvitamin D3 is greatly improved, and it has a good industrial application prospect.

[0006] In order to achieve the above objectives and technical effects, the present invention adopts the following technical solutions: The present invention provides a non-specific peroxygenase mutant, which comprises an 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 thereto, wherein the protein is obtained by mutating the amino acid residues shown at positions 20 and / or 25 and / or 57 of SEQ ID NO.1.

[0007] In some embodiments of the present invention, the non-specific peroxygenase mutant includes any of the following: (1) mutating the 20th position located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from E to A; (2) mutating position 25 located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from M to K; (3) mutating position 57 located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from S to V; (4) the 20th position located at or corresponding to 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; (5) the 25th position located or corresponding to 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; (6) the 20th position located at or corresponding to 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; (7) The 20th position 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.

[0008] The present invention provides a non-specific peroxygenase mutant, which is a protein obtained by mutating the amino acid residues at positions 20 and / or 25 and / or 57 of the amino acid sequence shown in SEQ ID NO.1.

[0009] In some embodiments of the present invention, the non-specific peroxygenase mutant includes any of the following: (1) The 20th position of the amino acid sequence shown in SEQ ID NO.1 was mutated from E to A, and the mutant was named E20A; (2) The 25th position of the amino acid sequence shown in SEQ ID NO.1 was mutated from M to K, and the mutant was named M25K; (3) The 57th position of the amino acid sequence shown in SEQ ID NO.1 was mutated from S to V, and the mutant was named S57V; (4) The amino acid sequence shown in SEQ ID NO.1 at position 20 was mutated from E to A, and the amino acid sequence at position 25 was mutated from M to K, and the mutant was named E20A / M25K; (5) The amino acid sequence shown in SEQ ID NO.1 at position 25 was mutated from M to K, and at position 57 was mutated from S to V, and the mutant was named M25K / S57V; (6) The amino acid sequence shown in SEQ ID NO.1 at position 20 was mutated from E to A, and the amino acid sequence at position 57 was mutated from S to V, and the mutant was named E20A / S57V; (7) The amino acid sequence shown in SEQ ID NO. 1 was mutated from position 20 to A, from position 25 to K, and from position 57 to V, and the mutant was named E20A / M25K / S57V.

[0010] Another object of the present invention is to provide a polynucleotide encoding the non-specific peroxygenase mutant.

[0011] Another object of the present invention is to provide a recombinant vector comprising a polynucleotide encoding the non-specific peroxygenase mutant.

[0012] Another object of the present invention is to provide a recombinant vector, wherein the recombinant vector is pET-23a, pET-28a or pPIC9k, preferably pPIC9k. Specifically, the recombinant vector can be constructed by connecting the gene encoding the non-specific peroxygenase mutant to the vector by conventional methods in the art.

[0013] In some embodiments of the present invention, the recombinant vector is prepared by the following method: the nucleic acid product obtained by artificial chemical synthesis (for example, a wild-type non-specific peroxygenase gene fragment) and the vector pPIC9k are double-digested with restriction endonucleases EcoRⅠ and NotⅠ, respectively, to form complementary sticky ends, and then connected with T4 ligase to form the recombinant vector UPO-pPIC9k.

[0014] Another object of the present invention is to provide a recombinant engineered bacterium containing the recombinant vector.

[0015] 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 obtained by transforming the above-mentioned recombinant vector or the gene encoding the non-specific peroxygenase mutant into the host cell. The host cell can be a conventional host cell in the art, but it must be able to satisfy the stable self-replication of the recombinant vector, and the gene encoding the non-specific peroxygenase mutant carried can be effectively expressed.

[0016] Another object of the present invention is to provide a use of the non-specific peroxygenase mutant in catalyzing the oxidation reaction of vitamin D3 to generate 25-hydroxyvitamin D3.

[0017] Another object of the present invention is to provide a method for preparing 25-hydroxyvitamin D3, the method comprising: Acetone, vitamin D3, hydrogen peroxide and the non-specific peroxygenase mutant are dissolved in potassium phosphate buffer to form a reaction mixture, and the mixture is stirred to react to generate 25-hydroxyvitamin D3.

[0018] In some embodiments of the present invention, in the method for preparing 25-hydroxyvitamin D3, the non-specific peroxygenase mutant is in the form of any one of the following: a1) a cell, wherein the cell contains a recombinant vector, and the recombinant vector contains a gene encoding the non-specific peroxygenase mutant; a2) crude enzyme solution, which is the fermentation solution obtained by fermenting the cells described in a1); a3) pure enzyme solution, obtained by purifying the crude enzyme solution described in a2); 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; a5) Immobilized enzyme, that is, the non-specific peroxygenase mutant is treated by physical or chemical methods to increase its stability while retaining its catalytic activity, so that it can be used repeatedly.

[0019] As a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 of the present invention, wherein: In the reaction mixture, the concentration of the nonspecific peroxygenase mutant is 1-10 U / L, the concentration of acetone is 200-500 g / L, the concentration of hydrogen peroxide is 1-10 mmol / L, and the concentration of vitamin D3 is 1-10 g / L.

[0020] As a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 of the present invention, wherein: The concentration of the potassium phosphate buffer is 10-100 mmol / L, and the pH is 7.0-8.0.

[0021] As a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 of the present invention, wherein: The stirring reaction is carried out at a temperature of 25° C. to 35° C. and for a time of 10 to 50 hours.

[0022] As a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 of the present invention, wherein: The concentration of the nonspecific peroxygenase mutant in the reaction mixture is 1 U / L, 3 U / L, 4 U / L, 5 U / L, 6 U / L, 8 U / L or 10 U / L, the concentration of acetone is 200 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L, 450 g / L or 500 g / L, the concentration of hydrogen peroxide is 1 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 8mmol / L or 10 mmol / L, and the concentration of vitamin D3 is 1 g / L, 3g / L, 5 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L.

[0023] As a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 of the present invention, wherein: The concentration of the potassium phosphate buffer is 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 the pH is 7.0, 7.5 or 8.0.

[0024] As a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 of the present invention, wherein: The stirring reaction temperature is 25°C, 28°C, 30°C, 32°C or 35°C, and the stirring reaction time is 10h, 20h, 24h, 30h, 36h, 40h, 46h or 50h.

[0025] As a preferred embodiment of the method for preparing 25-hydroxyvitamin D3 of the present invention, wherein: The concentration of the nonspecific peroxygenase mutant in the reaction mixture is 5 U / L, the concentration of acetone is 400 g / L, the concentration of hydrogen peroxide is 5 mmol / L, and the concentration of vitamin D3 is 10 g / L; The concentration of the potassium phosphate buffer is 27 mmol / L and the pH is 8.0; The stirring reaction temperature is 30° C., and the stirring reaction time is 24 h.

[0026] Definition of terms The terms "mutant" or "variant" are used interchangeably throughout the present invention and refer to a UPO variant of the present invention obtained by the method described herein and having at least 1 mutation, preferably at least 2 mutations, more preferably at least 3 mutations, which result in a higher peroxygenase activity compared to the corresponding native or wild-type enzyme.

[0027] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides.

[0028] The term "encoding" refers to the correlation between nucleotide triplets or codons in a DNA sequence and the amino acids that make up a peptide, amino acid sequence or protein. When a nucleotide sequence is described as encoding a peptide, it means that when the nucleotide sequence is transcribed into messenger RNA (mRNA) and the mRNA is translated, the peptide will be produced.

[0029] The term "homology" or "percent homology" (percent homology, identity+similarity) uses homology comparison software such as BLASP, TBLASTN or tBLASTX from the National Center for Biotechnology Information (NCBI) to determine biotechnological information, using specific parameters. For the purposes of the present invention, the term "homology" or "homolog" refers to the identity of two or more nucleic acid sequences or the identity of two or more amino acid sequences. Homologous sequences include "paralogs" and "orthologs" sequences. The term "paralogs" refers to gene duplication within a species genome, resulting in paralogous genes. The term "orthologs" refers to homologous genes produced in different organisms due to ancestral relationships.

[0030] The term "vector" or "expression vector" refers to a vector that can introduce a DNA or RNA sequence (e.g., a heterologous gene) into a host cell to transform the host and promote cell production. For example, transcription and translation of the introduced sequence. Vectors generally contain DNA of transmissible factors, into which foreign DNA encoding proteins is inserted by restriction enzyme technology. A common type of vector is a "plasmid," which is generally a double-stranded DNA molecule that can easily accept additional (foreign) DNA and can be easily introduced into a suitable host cell, including plasmids and fungal vectors, which 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 (New England 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 a host, such as antibiotic resistance, and one or more expression cassettes. Suitable vectors for inserting the polynucleotides are vectors derived from prokaryotic expression vectors, such as pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, Co1E1, pCR1, RP4, phages and "shuttle" vectors, such as pSA3 and pAT28; yeast expression vectors such as Saccharomyces cerevisiae two-micron plasmids, integration plasmids, YEP vectors, centromere plasmids; expression vectors in insect cells, such as 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 baculovirus suitable for transfecting insect cells using any commercially available baculovirus system. Other vectors can be used by those skilled in the art as needed. If different from those described in the embodiments, routine experiments in biotechnology can be used to determine which vectors are most suitable for the present invention. In general, the selection of the vector depends on the size of the polynucleotide sequence and the host cell used in the method of the present invention.

[0031] Throughout the specification and claims, the word "comprising" is not intended to exclude other technical features, additives, components or steps. For those skilled in the art, other objects, advantages and features of the present invention will appear in part from the specification and in part from the practice of the present invention. The following examples are provided to illustrate the present invention and are not intended to limit the present invention.

[0032] The beneficial effects of the present invention are: (1) The nonspecific peroxygenase mutant provided by the present invention has high catalytic activity and stereocatalytic specific selectivity, and can catalyze the oxidation reaction of vitamin D3 to generate 25-hydroxyvitamin D3 with high selectivity. Compared with the wild-type enzyme, the mutant can significantly improve the catalytic efficiency and yield and reduce by-products. The nonspecific peroxygenase mutant of the present invention can be prepared in large quantities by constructing recombinant engineering bacteria and fermentation culture, and is relatively cheap and easy to obtain.

[0033] (2) The nonspecific peroxygenase mutant provided by the present invention has mild reaction conditions, simple reaction operation, high conversion rate and low cost in catalyzing the oxidation reaction of vitamin D3 to generate 25-hydroxyvitamin D3, which 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, which has a good prospect for industrial application. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts disclosed in the present invention. Such structures and technologies are also described in many publications.

[0035] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0036] Example 1 Construction of wild-type nonspecific peroxygenase The non-specific peroxygenase fragment derived from Coprinopsis marcescibilis (gene sequence as shown in SEQ ID NO.2) was fully synthesized by Sangon Biotech (Shanghai) Co., Ltd. after being optimized for Pichia pastoris codons. After being digested with restriction endonucleases EcoRⅠ and NotⅠ (purchased from New England Biolabs, operated according to the instructions), it was ligated to the vector pPIC9k that was similarly digested with restriction endonucleases to obtain the wild-type recombinant vector UPO-pPIC9k.

[0037] The gene sequence of wild-type non-specific peroxygenase (as shown in SEQ ID NO.2): TTACCTCCTCCACCACCAGCTCCACCATCTTTTACTGGAGCTAAGTTAGTTGATGATGAAGATCATCCTTGGATGCCTTTGCAATCTGGTGACATTAGAGGTCCATGTCCTGGATTGAACACTTTGGCTTCTCACGGTTACTTGCCTAGAAATGGAGTTGCTTCTCCATCTGATATGATTAAGGCTGTTCAAGAAGGTTTTAACATGGAAACTCAAGCTGCTAGAGTTGCTGCTTACTCTGCTCACTTGTTGGAAGGTAACTTGGTTACTGATTTGTTGTCTATTGGTGGTAAAACTTCTTTGACTGGTCCAGATCCACCAGCTCCAGCTACTGTTGCTGGAATTTCTAACCATGGTTCCTTTGAAGGTGACGCTTCCATGACTAGAATTGATGCTTTCTTTGGAGATCACACTGTTTTTAACCAACCATTGTTTGATCAATTTGTTGATTTTTCTAACAGATTTGGTAACGGTTTTTATAACTATACTGTTGGTGGTGAGTTGAGATTTCATAGAATTCAACAATCTATTGAAACTAATCCTCAATTTTCTATTAGAGGTTTCAGACACTTGACTGCTTACGGTGAAGCTGCTTTTGTTGCTAACATTTTTGTTGATGGTAGAAAGACTGGTGCTGATGCTCATCATTTGGATATGGATTCTGCTTTGTCCTTCTTTAGAGATATGAGATTCCCACAAGGTTTCTTCAGACCTTCCAGTCCTACCGCTGGTGAGGGTGCTGATATTATTTTTGCTGCTCATCCAACTCAGCCAGGATCTAATAACGGTGCTGTTAACTCATTTGTTGTTGATACTTCTATGGGTGGTTTGACTGATCCATGTACTTTTTACGTTCAATTTGTTTCTACTACTATTCCAGCTTTGTACCCTAACCCAACTGGTGTTTTGAGAAGAAACTTGATTATTAACTTGGGTTTTCTTTACGATGCTTTGTCTCCACCTAACTGTCCACAATTGTTTCCATACGGTCAAAACTGA。

[0038] The amino acid sequence of wild-type non-specific peroxygenase (as shown in SEQ ID NO.1): LPPPPPAPPSFTGAKLVDDEDHPWMPLQSGDIRGPCPGLNTLASHGYLPRNGVASPSDMIKAVQEGFNMETQAARVAAYSAHLLEGNLVTDLLSIGGKTSLTGPDPPAPATVAGISNHGSFEGDASMTRIDAFFGDHTVFNQPLFDQFVDFSNRFGNGFYNYTVGG ELRFHRIQQSIETNPQFSIRGFRHLTAYGEAAFVANIFVDGRKTGADAHHLDMDSALSFFRDMRFPQGFFRPSSPTAGEGADIIFAAHPTQPGSNNGAVNSFVVDTSMGGLTDPCTFYVQFVSTTIPALYPNPTGVLRRNLIINLGFLYDALSPPNCPQLFPYGQN.

[0039] Example 2 Construction of non-specific peroxygenase mutants The amino acid sequence SEQ ID NO.1 was used as a template to construct a random mutant library using the Agilent GeneMorph II Random Mutagenesis Kit (Cat. No. 200550), with the forward primer UPO-EcoRⅠ-F 5-CCGGAATTCTTACCTCCTCCACCACCAGCTCCAC-3' and the reverse primer UPO-NotⅠ-R 5'-ATAAGAATGCGGCCGCTCAGTTTTGACCGTATGGAAACAAT-3'.

[0040] 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-NotⅠ-R (10 μM), 1 μl Mutazyme II DNA polymerase (2.5 U / μl), and 50 ng UPO-pPIC9k made up to 50 μl.

[0041] The PCR program was 95°C for 2 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; 30 cycles, 72°C for 10 min.

[0042] The 1 kb random mutation fragment was recovered from the gel, digested with restriction endonucleases EcoRⅠ and NotⅠ (purchased from New England Biolabs, according to the instructions), and then recombined into the vector pPIC9k, and transformed into Escherichia coli DH5α chemically competent cells (Quanshijin Company) to obtain a random mutation library.

[0043] Example 3 Expression and screening of wild-type and mutant non-specific peroxygenase in Pichia pastoris (1) Linearization of plasmid DNA The non-specific peroxygenase wild-type and mutant library vectors were linearized using restriction endonuclease SalI (purchased from New England Biolabs, according to the manufacturer's instructions).

[0044] (2) Electroporation into Pichia pastoris The linearized fragment was added to 80 μl of Pichia pastoris GS115 competent cells (purchased from Beijing Huayueyang) and placed 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 and coated on MD plates. After incubation at 30°C until colonies grew, single colonies were separated by streaking. Single colonies were picked into sterile water, an appropriate amount of Lyticase (purchased from Sigma) was added, and the cell walls were digested at 37°C for 1 hour. Part of the digestion product was added to the PCR system to detect positive clones.

[0045] (3) Pichia pastoris expression BMD medium: 100 ml 1 M phosphate buffer (pH 6.0), 100 ml 10% glucose, 100 ml 10× YNB, 2 ml 500× biotin, 698 ml purified water.

[0046] 1% methanol BMMY medium: 100 ml 1M phosphate buffer (pH 6.0), 10 ml methanol, 100 ml 10×YNB, 2 ml 500× biotin, add purified water to 1L.

[0047] 5% methanol BMMY medium: 100 ml 1M phosphate buffer (pH 6.0), 50 ml methanol, 100 ml 10×YNB, 2 ml 500× biotin, add purified water to 1L.

[0048] Transformants from the wild-type and mutant libraries were selected with a toothpick and inoculated into a 96-well deep-well culture plate containing 250 μl of BMD medium. The culture was shaken at 30°C and 280 rpm for 48 h, and 250 μl of 1% methanol BMMY medium was added to continue the culture. Every 24 hours, 50 μl of 5% methanol BMMY medium was added. The culture was shaken at 30°C and 280 rpm for 96 hours. After that, the supernatant was collected by centrifugation at 10,000 rpm to detect the enzyme activity.

[0049] Example 4 UPO enzyme activity determination 2,2'-Azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was used as a substrate to detect the enzyme activity of UPO: 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 to a 96-well plate in sequence. After thorough mixing, 20 μL of 20 mM hydrogen peroxide was added, and the absorbance change was detected at a wavelength of 418 nm. The enzyme activity determination results are shown in Table 1.

[0050] 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 free radical) is 1 enzyme activity unit (U). The results are shown in Table 1.

[0051] Table 1 Conclusion: Compared with wild-type UPO, the nonspecific peroxygenase mutant of the present invention has higher catalytic activity.

[0052] Example 5 Preparation of non-specific peroxygenase mutants Shake flask fermentation: (a) Preparation of shake flask seed culture medium: 10 g yeast extract, 20 g peptone, and 20 g glucose were dissolved in 800 mL distilled water, the pH was adjusted to 7, and the volume was made up to 1000 mL with distilled water. The mixture was kept at 115 °C for 30 min.

[0053] (b) Fermentation step: A single clone of mutant E20A / M25K / S57V was picked from the plate and inoculated into 50 mL seed culture medium (250 mL Erlenmeyer flask), and cultured at 30°C, 200 rpm, and OD600 grew to 4 after 24 hours. A 1% inoculum was inoculated into 300 mL seed culture medium (1 L Erlenmeyer flask), and cultured at 30°C, 200 rpm, and OD600 grew to 7 after 24 hours.

[0054] Fermentation in fermenter: The seed solution was inoculated at 10% inoculation rate and cultured at 30°C and 200 r / min for 24 h. The secondary seed solution cultured to the logarithmic growth phase was inoculated at 10% (V / V) into a 10 L fermenter sterilized by high pressure (containing 6 L basal salt medium: 26.7 mL / L 85% phosphoric acid, 0.93 g / L CaSO 4 ▪2H 2 O, 14.9 g / L MgSO 4 ▪7H 2 O, 18.2 g / L K 2 SO 4 , 7.13 g / L KOH, 40 g / L glycerol, add PTM1 trace elements to 12 mL / L after sterilization), culture at 30℃ and 800 r / min. When the glycerol in the basal salt medium is consumed, feed with 50% (V / V) glycerol (containing 12 mL / L PTM1 trace elements), and the flow rate is adjusted according to the dissolved oxygen (DO) to keep the dissolved oxygen concentration at about 40%. When the wet weight reaches 160 g / L, stop feeding and starve culture for 1 h. After the starvation culture, set the temperature to 22℃ and control the pH at about 6.0. Methanol was added for low-temperature induction to control the dissolved oxygen at about 40% for 120 h. The enzyme solution was concentrated with a 10KDa ultrafiltration membrane package and the expression of the recombinant protein and enzyme activity were detected.

[0055] Example 6 Preparation of 25-hydroxyvitamin D3 by non-specific peroxygenase mutants In a 250 ml reaction bottle, 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) were added. The final reaction system volume was about 100 ml. The concentrations of the above reagents were calculated based on the final reaction system volume (for example, the concentration of the enzyme solution = (0.5 U / ml×1 ml) / 100 ml=5 U / L). The reaction temperature was 30°C. The reaction was stirred with a magnetic stirrer for 24 hours. After the reaction was terminated, 100 μl of the reaction solution was taken, 200 μl of ethyl acetate was added, extracted, dried over anhydrous sodium sulfate, and the content of 25-hydroxyvitamin D3 was detected by HPLC. Then, the conversion rate of 25-hydroxyvitamin D3 was calculated. The results are shown in Table 2.

[0056] Table 2 Conclusion: Compared with the wild-type UPO, the nonspecific peroxygenase mutant described in the present invention has a higher yield and conversion rate in the process of catalyzing the oxidation reaction of vitamin D3 to produce 25-hydroxyvitamin D3.

[0057] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A non-specific peroxygenase mutant, characterized in that: The non-specific peroxygenase mutant comprises an amino acid sequence as 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 thereto, wherein the protein is obtained by mutation of the amino acid residues at or corresponding to positions 20 and / or 25 and / or 57 of SEQ ID NO.

1.

2. The non-specific peroxygenase mutant according to claim 1, characterized in that This includes any of the following: (1) mutating the 20th position located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from E to A; (2) mutating position 25 located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from M to K; (3) mutating position 57 located at or corresponding to the amino acid sequence shown in SEQ ID NO.1 from S to V; (4) the 20th position located at or corresponding to 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; (5) the 25th position located at or corresponding to 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; (6) the 20th position located at or corresponding to 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; (7) The 20th position 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.

3. A polynucleotide encoding the non-specific peroxygenase mutant according to claim 1 or 2.

4. A recombinant vector, characterized in that: The recombinant vector comprises the polynucleotide according to claim 3.

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

6. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contains the recombinant vector according to claim 4 or 5.

7. Use of the nonspecific peroxygenase mutant according to claim 1 or 2 in catalyzing the oxidation reaction of vitamin D3 to produce 25-hydroxyvitamin D3.

8. A method for preparing 25-hydroxyvitamin D3, characterized in that: Acetone, vitamin D3, hydrogen peroxide and the non-specific peroxygenase mutant as claimed in claim 1 or 2 are dissolved in potassium phosphate buffer to form a reaction mixture, and the mixture is stirred for reaction to generate 25-hydroxyvitamin D3.

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

0.

10. The method for preparing 25-hydroxyvitamin D3 according to claim 8, characterized in that: The stirring reaction is carried out at a temperature of 25° C. to 35° C. and for a time of 10 to 50 hours.

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