A peroxidase AniUPO or a mutant thereof, and use thereof in synthesizing α-hydroxy β-ketoester derivatives, and a method for synthesizing α-hydroxy β-ketoester derivatives

Abstract

The present invention relates to a peroxidase AniUPO or a mutant thereof, and an application of the peroxidase AniUPO in synthesizing α-hydroxy β-ketoester derivatives and a method for synthesizing α-hydroxy β-ketoester derivatives, belonging to the field of enzyme engineering. Years UPO mutant, which is a wild-type peroxidase with an amino acid sequence as shown in SEQ ID NO.1 Years UPO is the parent, and the amino acid sequence is obtained by mutation. The present invention also provides a peroxidase encoding Years DNA molecules, expression vectors, host cells of UPO mutants, and methods for synthesizing chiral α-hydroxy β-ketoester derivatives. The peroxidase mutant of the present invention has the advantages of higher catalytic activity and better selectivity compared to the wild type, and the applicable substrate types can be expanded to a variety of cyclic and non-cyclic β-ketoester substrates, does not require expensive catalysts and ligands, has mild reaction conditions, and has potential industrial application prospects.

Description

Technical Field

[0001] The present invention relates to a peroxidase Ani The invention relates to UPO and its mutants, and application in synthesizing chiral α-hydroxy β-ketoester derivatives, belonging to the field of enzyme engineering. Background Art

[0002] Chiral α-hydroxy β-ketoester structures are common and important structural units in natural products, such as the antibiotic kelmania ketone, doxycycline, marine diterpenoid natural product Hamiglan A, indole alkaloid vindoline analogs, antibacterial agent pralmanamicin and biologically active natural product Y224A. In addition, key intermediates in the synthetic routes of many drug molecules also contain this structural unit. Therefore, the research and development of the synthesis technology of chiral α-hydroxy β-ketoester structural units has always been a research hotspot in the field of new drug development.

[0003] In the synthesis of chiral α-hydroxy β-ketoester structural units, direct α-hydroxylation of 1,3-dicarbonyl compounds is the most direct and effective route, which mainly includes chemical synthesis and biocatalysis. The main methods of chemical synthesis are as follows: transition metal catalysis using Fe, Mg, Ni, Pd, Zr, etc. as catalysts (Tetrahedron Lett. 2008, 49, 5976-5979; Adv. Synth. Catal. 2013, 355, 1924-1930; Angew. Chem. Int. Ed. 2009,48, 803-806; Chem. Commun. 2009, 3925-3927; Org. Lett. 2017, 19, 448-451); organic small molecule catalytic synthesis using chiral guanidine, cinchona alkaloids and their derivatives, chiral phosphoric acid, etc. as catalysts (Org. Lett. 2016, 18, 3602-3605; Adv. Synth. Catal. 2016, 358,737 –745; J. Am.Chem. Soc. 2009, 131, 4562–4563); and, using visible light or sunlight as the driving force, combined with transition metals and organic catalysts for synergistic catalytic synthesis (J. Org. Chem. 2016, 81, 7042−7050; ACS Catal.2023, 13, 7538−7543; J. Am. Chem. Soc. 2021, 143, 1078−1087). The chemical synthesis method has cumbersome synthesis steps, high catalyst loading, high price, harsh conditions, is not green and safe, and has serious environmental pollution. Biocatalysis mainly uses cells or enzyme proteins as catalysts. Currently, there are few reports on biocatalysis methods for synthesizing enantiomerically pure α-hydroxy β-ketoester structural units. Chinese patent CN115807042A discloses a method for asymmetric hydroxylation of 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid methyl ester, which is enzymatically reacted in the presence of Baeyer-Villiger monooxygenase and cofactors to obtain (S)- or (R)-5-chloro-2,3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester. However, this technical solution only involves one substrate, and the substrate has limited universality.

[0004] In summary, developing a method for the asymmetric α-hydroxylation of 1,3-dicarbonyl compounds with mild reaction conditions, high yield, high stereoselectivity, wide substrate applicability and environmental friendliness has important value and application prospects. Summary of the invention

[0005] The present invention provides a peroxidase Ani UPO and its mutants, and their applications in the synthesis of chiral α-hydroxy β-ketoester derivatives.

[0006] Unspecific peroxygenases (UPOs) are heme thiooxygenases derived from fungi. They are a class of stable extracellular enzymes, and their heme active centers are mainly formed by the PCP motif (proline-cysteine-proline). The peroxidase provided by the present invention Ani UPO and its mutants are used as catalysts to overcome the defects of the asymmetric α-hydroxylation reaction of 1,3-dicarbonyl compounds in the prior art, such as harsh reaction conditions, long reaction time, high cost, and great environmental pressure. This biocatalytic reaction method can achieve good yield and stereoselectivity, efficiently construct chiral α-hydroxy β-ketoester structural units, and has mild conditions, green and environmentally friendly, and has greater prospects for industrial application.

[0007] The present invention provides a peroxidase Ani UPO mutant, which is a wild-type peroxidase with an amino acid sequence as shown in SEQ ID NO.1 Ani UPO is the parent, and the mutant is obtained by mutation, wherein the mutant is one of the following:

[0008] The phenylalanine at position 161 of the parent was mutated to alanine to obtain a mutant Ani UPO-F161A;

[0009] The 63rd position alanine of the parent was mutated to glycine to obtain a mutant Ani UPO-A63G;

[0010] The glutamic acid at position 69 of the parent was mutated to valine to obtain a mutant Ani UPO-E69V;

[0011] Or the 69th glutamic acid of the parent is mutated to isoleucine to obtain a mutant Ani UPO-E69I;

[0012] Or the 69th glutamic acid of the parent is mutated to leucine to obtain a mutant Ani UPO-E69L.

[0013] Furthermore, it is based on Ani UPO-F161A is the parent, and the mutant is obtained by mutation, wherein the mutant is one of the following:

[0014] The leucine at position 66 of the parent was mutated to valine to obtain a mutant Ani UPO-F161A-L66V;

[0015] The 66th leucine of the parent was mutated to phenylalanine to obtain a mutant Ani UPO-F161A-L66F;

[0016] The leucine at position 66 of the parent was mutated to alanine to obtain a mutant Ani UPO-F161A-L66A;

[0017] The parent leucine at position 66 was mutated to phenylalanine and the alanine at position 63 was mutated to serine to obtain a mutant Ani UPO-F161A-L66F-A63S;

[0018] The mutant was obtained by mutating the leucine at position 66 of the parent to phenylalanine and the glutamic acid at position 69 to methionine. Ani UPO-F161A-L66F-E69M;

[0019] The mutant was obtained by mutating the leucine at position 66 to phenylalanine and the glutamic acid at position 69 to glycine. Ani UPO-F161A-L66F-E69G;

[0020] The mutant was obtained by mutating the leucine at position 66 of the parent to phenylalanine and the leucine at position 52 to phenylalanine. Ani UPO-F161A-L66F-L52F, among which, the mutant Ani The amino acid sequence of UPO-F161A-L66F-L52F is shown in SEQ ID NO.2.

[0021] Furthermore, it is based on Ani UPO-F161A-L66F-L52F is the parent, and the mutant obtained by mutation is one of the following:

[0022] The 164th glycine of the parent was mutated to alanine to obtain the mutant Ani UPO CHO-6 -G164A;

[0023] The 217th leucine of the parent was mutated to valine to obtain a mutant Ani UPO CHO-6 -L217V;

[0024] The 160th serine of the parent was mutated to isoleucine to obtain a mutant Ani UPO CHO-6 -S160I;

[0025] Among them, the Ani UPO CHO-6 for AniUPO-F161A-L66F-L52F.

[0026] Furthermore, it is Ani UPO CHO-6 -G164A is the parent, and the mutant is obtained by mutation, wherein the mutant is one of the following:

[0027] The 160th serine of the parent was mutated to threonine to obtain a mutant Ani UPO CHO-6 -G164A-S160T;

[0028] The 160th serine of the parent was mutated to isoleucine to obtain a mutant Ani UPO CHO-6 -G164A-S160I;

[0029] The 160th serine of the parent was mutated to leucine to obtain the mutant Ani UPO CHO-6 -G164A-S160L;

[0030] The 217th leucine of the parent was mutated to valine to obtain the mutant Ani UPO CHO-6 -G164A-L217V;

[0031] Furthermore, it is Ani UPO CHO-6 -G164A-S160T is the parent, and the mutant obtained by mutation is one of the following:

[0032] The mutant was obtained by mutating the leucine at position 217 to valine and the glutamic acid at position 156 to valine. Ani UPO CHO-6 -G164A-S160T-L217V-E156V;

[0033] The mutant was obtained by mutating the leucine at position 217 to valine and the alanine at position 168 to glycine. Ani UPO CHO-6 -G164A-S160T-L217V-A168G;

[0034] The 217th leucine of the parent was mutated to valine and the 172th valine was mutated to leucine to obtain a mutant Ani UPO CHO-6 -G164A-S160T-L217V-V172L;

[0035] in, Ani UPO CHO-6The amino acid sequence of -G164A-S160T-L217V-A168G is shown in SEQ ID NO.3.

[0036] The present invention provides a DNA molecule encoding the peroxidase Ani UPO mutants.

[0037] The invention provides an expression vector, which contains the DNA molecule.

[0038] The present invention provides a host cell, wherein the host cell contains the expression vector, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0039] Wherein, the eukaryotic cell is yeast.

[0040] The present invention provides the peroxidase Ani UPO mutants or peroxidase Ani Application of UPO in the synthesis of α-hydroxy β-ketoester derivatives; the chemical formula of the α-hydroxy β-ketoester derivative is shown in Formula I:

[0041]

[0042] Where n = 0, 1, 2, 3, 4, 5; R = X, H, OMe; R 1 = alkyl, benzyl or phenyl.

[0043] More preferably, said n= 1, 2, 3; R 1 = Me, Et.

[0044] The present invention provides a method for synthesizing α-hydroxy β-ketoester derivatives, which uses formula II as a substrate, and in a reaction system containing H2O2, the peroxidase Ani UPO mutants or peroxidase Ani UPO is used as a biocatalyst to synthesize α-hydroxy β-ketoester derivatives. The synthetic route is as follows:

[0045]

[0046] Where n = 0, 1, 2, 3, 4, 5; R = X, H, OMe; R 1 = alkyl, benzyl or phenyl.

[0047] More preferably, said n= 1, 2, 3; R 1 = Me, Et.

[0048] The present invention also provides a method for synthesizing α-hydroxy β-ketoester derivatives, which uses formula II as a substrate, and reacts the peroxidase in a reaction system containing H2O2. Ani UPO mutants or peroxidase Ani UPO is used as a biocatalyst to synthesize α-hydroxy β-ketoester derivatives. The synthetic route is as follows:

[0049]

[0050] Where n=1; R= X, H, OMe; R 1 = alkyl, benzyl or phenyl.

[0051] The beneficial effects of the present invention are:

[0052] The present invention screens out Aspergillus niger Peroxidase Ani UPO, the amino acid sequence is shown in SEQ ID NO: 1, and through the directed evolution strategy of the enzyme, the wild-type peroxidase is modified by mutation to obtain the peroxidase mutant of the present invention, which can be used for the synthesis of chiral α-hydroxy β-ketoester structural units. The peroxidase mutant has the advantages of higher catalytic activity and better selectivity compared with the wild-type, and the applicable substrate types can be expanded to a variety of cyclic and non-cyclic β-ketoester substrates, does not require expensive catalysts and ligands, and has mild reaction conditions, and has potential industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Peroxidase Ani Recombinant plasmid map of UPO;

[0054] Figure 2 It is the standard curve of the model substrate 1a corresponding to the hydroxylated product 2a detected by LC-MS;

[0055] Figure 3 It is the standard curve diagram of the model substrate 1b corresponding to the hydroxylated product 2b detected by LC-MS;

[0056] Figure 4 Peroxidase Ani UPO CHO-6 The recombinant plasmid map;

[0057] Figure 5 is the standard curve of the model substrate 1a corresponding to the hydroxylated product 2a detected by HPLC;

[0058] Figure 6 for Ani UPO CHO-6The chiral detection original spectrum of the product 2a generated by the catalytic reaction under the optimal conditions (wherein A is the chiral detection original spectrum of the racemate 2a; B is the chiral detection original spectrum of the product 2a: 99% ee of the AniUPOCHO-6 catalytic reaction);

[0059] Figure 7 Peroxidase Ani UPO CHO-5 The recombinant plasmid map;

[0060] Figure 8 It is the standard curve diagram of the model substrate 1b corresponding to the hydroxylation product 2b detected by HPLC;

[0061] Fig. 9 This is the chiral detection original spectrum of the product 2b generated by the catalytic reaction of AniUPOCHO-5 under optimal conditions (where A is the chiral detection original spectrum of the racemate 2b; B is the chiral detection original spectrum of the product 2b: 94% ee of the catalytic reaction of AniUPOCHO-5). DETAILED DESCRIPTION

[0062] Example 1: Wild type Ani Construction of UPO expression strain and investigation of initial reaction activity

[0063] 1. Wild type Ani Construction of UPO expression strain

[0064] For those coming from Aspergillus niger Wild-type peroxidase Ani The amino acid sequence of UPO is SEQ ID NO.1. Codon optimization was performed using Saccharomyces cerevisiae as the host. Through full gene synthesis and vector construction, a recombinant expression vector containing the peroxidase encoding gene was obtained and named pYES2- Ani UPO, gene map reference Figure 1 . Ani The UPO recombinant expression vector was transformed into the Saccharomyces cerevisiae BJ5464 strain to construct Ani UPO expressing strains.

[0065] 2. Wild type Ani Protein expression of UPO and preparation of crude enzyme solution

[0066] Will build successfully AniThe UPO expression strain was inoculated into a shake tube containing 5 mL of SD-Ura liquid medium and cultured at 30°C and 250 rpm for 48 h. Then, the seed solution in the 3 mL shake tube was transferred to a conical flask containing 50 mL of YP expression medium (10 g of yeast extract, 20 g of peptone, 67 mM K2HPO4 / KH2PO4 (pH 6.0), 2.2 mMMgSO4, 31.6 mL of EtOH, 2% galactose, 1 mL of chloramphenicol (25 mg / mL)) in an ultra-clean workbench and cultured at 25°C and 250 rpm for 72 h. Centrifuge, collect the supernatant, and concentrate it to 420 μL by ultrafiltration as the crude enzyme solution.

[0067] 3. Wild type Ani Study on the Reactivity of UPO

[0068] Methyl 6-chloro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-carboxylate (hereinafter referred to as model substrate 1a) was used for initial reaction activity verification screening. The reaction system contained 10 μL 1a (0.1 M, CH3CN solubilization), 140 μL crude enzyme solution, 200 μL NaPi buffer (100mM, pH 7.0), and 50 μL hydrogen peroxide (0.1M, diluted in NaPo buffer). After reacting at 30°C and 800 rpm for 16 h, 800 μL of anhydrous ethanol was added to precipitate the protein, centrifuged, and the supernatant was taken for LC-MS detection.

[0069] The analytical yield of product 2a was calculated by detecting the standard sample containing internal standard at different concentration gradients by LC-MS, and the peak area ratio of the standard sample to the internal standard was calculated by making a standard curve (hereinafter collectively referred to as analytical yield). The standard curve is shown in Figure 2 As shown. The ee value was calculated by the product peak area, ee = ((RS) / (R+S))*100%. The initial reaction activity results are shown in Table 1.

[0070] Table 1: Initial reaction activity under the conditions of model substrate 1a

[0071] Enzymes Source GenBank Accession Analytical yield (%) ee (%) UPO XP_025450509.1 0.2 58

[0072] Example 2: Wild-type Ani UPO was used as the parent and directed evolution was performed using the model substrate 1a

[0073] 1. Ani Construction of UPO mutant library

[0074] Containing Ani The expression vector pYES2- Ani UPO recombinant bacteria SC (BJ5464)-pYES2- Ani UPO was used as the starting strain, and site-directed saturation mutagenesis was performed to improve the catalytic activity and stereoselectivity of peroxidase for substrate 1a. The recombinant expression vector pYES2- Ani UPO was used as a template, and forward and reverse primers containing mutation sites were designed according to the 22c-trick method, and mutations were introduced by PCR.

[0075] A 50 μL PCR reaction system includes:

[0076] 1 μL pYES2- Ani UPO plasmid template (about 100 ng / μL);

[0077] 25 μL 2×KOD DNA Polymerase;

[0078] 2.5 μL forward primer (10 μM);

[0079] 2.5 μL reverse primer (10 μM);

[0080] 19 μL sterile HO.

[0081] The forward primer and reverse primer are specific primers used in the construction process of different mutants. The conditions of the PCR reaction are as follows:

[0082] (1) Pre-denaturation at 98°C for 2 min;

[0083] (2) Denaturation at 98°C for 10 s;

[0084] (3) (Tm of primer) °C annealing for 10 s;

[0085] (4) 68°C extension for 1 min;

[0086] The above steps (2)-(4) were performed for a total of 10 cycles, and the annealing temperature was reduced by 0.5°C each cycle;

[0087] (5) Denaturation at 98°C for 10 s;

[0088] (6) (primer Tm-5) °C annealing for 10 s;

[0089] (7) 68°C extension for 1 min;

[0090] The above steps (5)-(7) were performed for 30 cycles, with a final extension at 68°C for 5 min.

[0091] The linear fragments obtained by PCR amplification were separated and recovered by 1% agarose gel electrophoresis. The purified PCR products were transformed into Saccharomyces cerevisiae BJ5464 competent cells, spread on SD-Ura solid medium, and placed in an incubator at 30°C for inverted culture for about 48 h.

[0092] 2. Using model substrate 1a Ani UPO mutant library for activity screening

[0093] Pick a single clone into a 96-well plate containing 1 mL of SD-Ura medium and culture it in a constant temperature incubator at 30°C and 250 rpm for 48 h to obtain a seed solution. Then, in an ultra-clean workbench, transfer 300 μL of the seed solution in the 96-well plate to a 24-well plate containing 2.7 mL of YP expression medium and culture it at 25°C and 250 rpm for 72 h. Centrifuge at 3900 rpm for 10 min, collect the supernatant, and obtain the peroxidase catalyst.

[0094] Using model substrate 1a, Ani The enzyme activity of the UPO mutants was preliminarily screened. 340 μL of the above supernatant was placed in a 96-well plate and precooled on ice, and then 10 μL of 1a (0.1M, DMSO solution) and 50 μL of hydrogen peroxide (0.1M, diluted in NaPi buffer) were added in sequence. After reacting at 30°C and 800 rpm for 16 h, 400 μL of anhydrous ethanol was added to precipitate the protein, centrifuged, and the supernatant was taken for LC-MS detection. The enzyme activity was preliminarily screened based on the amount of product generated.

[0095] Select the monoclones with improved enzyme activity relative to the parent in the preliminary screening results for verification. Pipette 300 μL of the seed solution in the above 96-well plate into a shake tube containing 5 mL SD-Ura medium, and culture at 30°C, 250 rpm for 48 h. Then transfer the seed solution in the 3 mL shake tube to a conical flask containing 50 mL YP expression medium in an ultra-clean workbench, and culture at 25°C, 250 rpm for 72 h. Centrifuge, collect the supernatant, filter through a 0.45 μm filter membrane, and concentrate to 420 μL by ultrafiltration. Place the reaction bottle on ice for precooling, add 200 μL NaPi buffer (100mM, pH 7.0), 140 μL concentrated enzyme solution, 10 μL 1a (0.1 M, CH3CN solubilization), 50 μL hydrogen peroxide (0.1 M, NaPi buffer dilution) in sequence, react at 30°C, 800 rpm for 16 h, add 800 μL anhydrous ethanol to precipitate protein, centrifuge, take the supernatant for LC-MS detection, and verify the enzyme activity based on the amount of product generated. The analytical yield of product 2a is based on the standard curve ( Figure 2) was calculated, and the catalytic activity of the mutants was compared by analyzing the yield. The activity of the parent in each round was taken as 1. The relative activity of the mutant = the analytical yield of the hydroxylated product generated by the mutant / the analytical yield of the hydroxylated product generated by the parent (hereinafter collectively referred to as relative activity). The ee value was calculated by the product peak area, ee = ((RS) / (R+S))*100%, and the results are shown in Table 2.

[0096] Table 2: Results of the first round of directed evolution under model substrate 1a

[0097] Mutants Relative activity ee(%) UPO 1.00 58 UPO-F161S 2.06 ND UPO-F161A 3.28 66 UPO-F161C 1.38 ND UPO-L66V 2.41 ND UPO-L66C 1.18 ND UPO-A63G 1.98 63 UPO-E69V 1.95 53 UPO-E69I 2.13 57 UPO-E69L 1.51 58 UPO-F82W 0.45 ND

[0098] ND, not detected.

[0099] choose Ani UPO-F161A was used as the second round parent for further directed evolution. The mutant library construction, protein expression, reaction screening, and detection methods were the same as described above, and the results are shown in Table 3.

[0100] Table 3: Results of the second round of directed evolution under model substrate 1a

[0101] Mutants Relative activity ee(%) UPO-F161A 1.00 66 UPO-F161A-L66V 0.86 83 UPO-F161A-L66F 0.96 92 UPO-F161A-L66A 0.74 76

[0102] choose Ani UPO-F161A-L66F was used as the third round parent for further directed evolution. The mutant library construction, protein expression, reaction screening, and detection methods were the same as described above, and the results are shown in Table 4.

[0103] Table 4: Results of the third round of directed evolution under the model substrate 1a condition

[0104] Mutants Relative activity ee(%) UPO-F161A-L66F 1.00 92 UPO-F161A-L66F-A63S 1.10 90 UPO-F161A-L66F-A63V 0.05 47 UPO-F161A-L66F-E69M 1.15 91 UPO-F161A-L66F-E69G 0.85 89 UPO-F161A-L66F-L52F 1.54 94

[0105] Under the conditions of model substrate 1a, by Ani The optimal mutants obtained by three rounds of directed evolution of UPO (hereinafter referred to as Ani UPO CHO-6 ) is: wild type Ani UPO was used as the parent, and the phenylalanine at position 161 was mutated to alanine, the leucine at position 66 was mutated to phenylalanine, and the leucine at position 52 was mutated to phenylalanine. The amino acid sequence is shown in SEQ ID NO.2. Ani UPO CHO-6 It is able to catalyze the synthesis of α-hydroxylated product 2a from model substrate 1a with high enantioselectivity, increasing the ee value from 58% to 94%.

[0106] Example 3: Using ethyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylate (hereinafter referred to as model substrate 1b) to Ani Secondary screening of the UPO mutant library

[0107] In order to further expand the scope of applicability of the substrate structure, the enzyme catalyst was obtained according to the operation method (2) in Example 1, and the model substrate 1b was used to Ani The UPO mutant library was subjected to secondary screening for reactivity.

[0108] The reaction system included: 200 μL NaPi buffer (100mM, pH 5.8), 140 μL crude enzyme solution, 10 μL 1b (0.1 M, CH3CN solution), 50 μL hydrogen peroxide (0.04 M, diluted with NaPi buffer), 30°C, 800 rpm reaction for 16 h, then 800 μL anhydrous ethanol was added to precipitate the protein, centrifuged, and the supernatant was taken for LC-MS detection. The reaction activity of the enzyme was verified based on the amount of product 2b generated. The analytical yield of product 2b was calculated based on the standard curve. The catalytic activity of the mutant was compared by analytical yield. The activity of the parent in each round was 1, and the relative activity of the mutant = analytical yield of hydroxylation product generated by the mutant / analytical yield of hydroxylation product generated by the parent (hereinafter collectively referred to as relative activity). The ee value was calculated by the product peak area, ee=((RS) / (R+S))*100%, and the results are shown in Table 5. The standard curve of LC-MS detection of the model substrate 1b corresponding to the hydroxylation product 2b is shown in Figure 3 .

[0109] Table 5: Ani Results of the screening of the UPO mutant library for reactivity

[0110] Mutants Relative activity ee(%) UPO 1.00 5 UPO-F161A 11.05 22 UPO-F161A-L66F 7.18 22 <![CDATA[ Ani UPO-F161A-L66F-L52F( Ani YES CHO-6 )]]> 6.87 40

[0111] Example 4: Peroxidase mutants Ani UPO CHO-6 Optimization of reaction conditions

[0112] Targeting peroxidase mutants Ani UPO CHO-6 The amino acid sequence of Ani UPO CHO-6 The recombinant expression vector was named pPIC3.5k- Ani UPO CHO-6 , gene map reference Figure 4 The successfully constructed recombinant plasmid was transformed into Pichia pastoris GS115 and fermented in a 10 L fermenter to obtain a concentrated solution containing the target enzyme. The enzyme concentration was determined using the pyridine method.

[0113] Using 1a as a model substrate, the peroxidase mutant Ani UPOCHO-6 The reaction conditions were optimized, including the type of cosolvent, hydrogen peroxide concentration, and enzyme concentration. The yield of the product was finally increased to 80%, and the ee value was increased to >99%. The analytical yield of product 2a was detected by HPLC with different concentration gradients of standard products containing internal standards. The standard curve was calculated by statistically analyzing the UV absorption ratio of the standard product and the internal standard. The standard curve detected by HPLC is shown in Figure 2 Figure 5 As shown. The ee was calculated by the UV absorption of different enantiomers of the product, ee = ((RS) / (R+S))*100%. The results of the investigation on the optimization of reaction conditions are shown in Table 6.

[0114] Table 6: Ani UPO CHO-6 Results of the optimization of reaction conditions

[0115] Solvent <![CDATA[H2O2(mM)]]> <![CDATA[ Ani UPO CHO-6 (nM)]]> Analytical yield (%) ± SD ee (%) <![CDATA[CH3CN]]> 12.5 650 9.7±0.3 95 DMSO 12.5 650 59.8±2.1 >99 EtOH 12.5 650 42.0±1.0 99 1,4-Dioxane 12.5 650 12.0±2.0 >99 EtOAc 12.5 650 1.8±0.3 >99 Acetone 12.5 650 26.7±1.4 98 DMSO 5 650 45.5±0.4 99 DMSO 25 650 36.0±3.0 >99 DMSO 50 650 17.4±1.0 >99 DMSO 12.5 875 70.8±7.4 >99 DMSO 12.5 1050 78.3±1.6 >99 DMSO 12.5 1300 79.1±6.6 99 DMSO 12.5 1500 80.0±2 >99

[0116] According to the optimized reaction conditions, the optimal reaction conditions were: 2.5 mM model substrate 1a, 100 mM NaPi buffer (pH 7.0), 2.5% DMSO as a cosolvent, 12.5 mM hydrogen peroxide, and 1500 nM enzyme. Figure 6 The chiral detection column used was Chiralpak IE, the mobile phase ratio was acetonitrile / water = 60:40, and the flow rate was 1.0 mL / min.

[0117] Example 5: Mutants under optimized conditions Ani UPO CHO-6 Conduct substrate suitability investigation

[0118] The reaction system was 400 μL, and NaPi buffer (100 mM, pH 7.0), 1500 nM Ani UPO CHO-6 , 2.5 mM substrate (2.5% DMSO solution), 12.5 mM hydrogen peroxide, at 30 ° C, 800 rpm shaking reaction for 16 h, add 800 μL of anhydrous ethanol (containing 0.25 mM 1,3,5-trimethoxybenzene or 1,2,3-trimethoxybenzene as internal standard) to precipitate protein, centrifuge and take the supernatant for HPLC detection, calculate and analyze the yield and ee value. The results of the investigation are shown in Table 7.

[0119] Table 7: Ani UPO CHO-6 Results of investigation on substrate suitability.

[0120]

[0121] Embodiment 6: Ani UPOCHO-5 As the parent, directed evolution was continued using model substrate 1b

[0122] According to the operation method in Example 2, the model substrate 1b was used to continue the directional and activity screening, and the enzymatic activity of the peroxidase mutant was evaluated by LC-MS detection of the product generation. The reaction system was 400 μL, including 340 μL of supernatant, 10 μL of 1b (0.1M, CH3CN solubilization), 50 μL of hydrogen peroxide (0.04M, NaPi buffer dilution), and after reacting at 30°C and 800 rpm for 16 h, 800 μL of anhydrous ethanol was added to precipitate the protein, centrifuged, and the supernatant was taken for LC-MS detection. The results are shown in Table 8.

[0123] Table 8: Results of the first round of directed evolution under model substrate 1b

[0124] Mutants Relative activity ee(%) <![CDATA[ Ani YES CHO-6 ]]> 1.00 40 <![CDATA[ Ani YES CHO-6 -G164A]]> 2.11 72 <![CDATA[ Ani YES CHO-6 -L217V]]> 1.74 35 <![CDATA[ Ani YES CHO-6 -Y65L]]> 0.73 ND <![CDATA[ Ani YES CHO-6 -Y65F]]> 0.97 ND <![CDATA[ Ani YES CHO-6 -S160A]]> 1.54 24 <![CDATA[ Ani YES CHO-6 -S160T]]> 0.48 ND <![CDATA[ Ani YES CHO-6 -S160I]]> 1.64 45

[0125] choose Ani UPO CHO-6 -G164A was used as the second round parent for further directed evolution. The construction of mutant library, protein expression, reaction screening and detection were the same as in Example 2. The experimental results are shown in Table 9.

[0126] Table 9: Results of the second round of directed evolution under model substrate 1b

[0127] Mutants Relative activity ee(%) <![CDATA[ Ani YES CHO-6 -G164A]]> 1 72 <![CDATA[ Ani YES CHO-6 -G164A-S160T]]> 1.51 82 <![CDATA[ Ani YES CHO-6 -G164A-S160I]]> 1.77 65 <![CDATA[ Ani YES CHO-6 -G164A-S160L]]> 1.92 65 <![CDATA[ Ani YES CHO-6 -G164A-L217V]]> 1.38 65 <![CDATA[ Ani YES CHO-6 -G164A-A22S]]> 0.98 ND <![CDATA[ Ani UP CHO-6 -G164A-S214Q]]> 0.50 ND <![CDATA[ Ani UP CHO-6 -G164A-S214R]]> 0.81 ND <![CDATA[ Ani YES CHO-6 -G164A-F230G]]> 0.65 ND

[0128] choose Ani UPO CHO-6 -G164A-S160T was used as the third round parent for further directed evolution. Construction of mutant library, protein expression, reaction screening and detection were the same as in Example 2. The experimental results are shown in Table 10.

[0129] Table 10: Results of the third round of directed evolution under the conditions of model substrate 1b

[0130] Mutants Relative activity ee(%) <![CDATA[ Ani YES CHO-6 -G164A-S160T]]> 1 82 <![CDATA[ Ani YES CHO-6 -G164A-S160T-L217V-E156V]]> 1.36 73 <![CDATA[ Ani YES CHO-6 -G164A-S160T-L217V-A168G]]> 1.48 92 <![CDATA[ Ani YES CHO-6 -G164A-S160T-L217V-V172L]]> 1.13 84 <![CDATA[ Ani YES CHO-6 -G164A-S160T-L217V-E233G]]> 0.90 73

[0131] Under the conditions of model substrate 1b, by Ani UPO CHO-6 The optimal mutants (hereinafter referred to as Ani UPO CHO-5 ) is: Ani UPO CHO-6 The parent, glycine at position 164 was mutated to alanine, serine at position 160 was mutated to threonine, leucine at position 217 was mutated to valine, and alanine at position 168 was mutated to glycine. The amino acid sequence is shown in SEQ ID NO.3, and the vector map is shown in Figure 7 . Ani UPO CHO-5 It was able to catalyze the synthesis of α-hydroxylated products from model substrate 1b with high enantioselectivity (92% ee).

[0132] Example 7: Peroxidase mutants Ani UPO CHO-5 Optimization of reaction conditions

[0133] According to the method of Example 4, 1b was used as the model substrate to treat the peroxidase mutant Ani UPO CHO-5 The reaction conditions were optimized, including the type of cosolvent, pH of the reaction buffer, and enzyme concentration. The yield of product 2b was finally increased to 97%, and the ee value was increased to 94%. The analytical yield of product 2b was detected by HPLC with different concentration gradients of standard products containing internal standards. The standard curve was calculated by statistically analyzing the UV absorption ratio of the standard product to the internal standard. The standard curve of HPLC detection is shown in Figure 1. Figure 8 As shown. The ee was calculated by the UV absorption of different enantiomers of the product, ee = ((RS) / (R+S))*100%. The results of the investigation on the optimization of reaction conditions are shown in Table 11.

[0134] Table 11: Ani UPO CHO-5 Results of the optimization of reaction conditions

[0135] Solvent Buffer <![CDATA[ Ani UPO CHO-5 (nM)]]> Analytical yield (%) ± SD ee (%) <![CDATA[CH3CN]]> NaPi(pH 5.8) 900 40.2±6.4 67 DMSO NaPi(pH 5.8) 900 77.9±0.7 90 1,4-Dioxane NaPi(pH 5.8) 900 85.7±4.9 73 EtOAc NaPi(pH 5.8) 900 22.4±0.5 39 DMSO citrate buffer (pH 4) 900 83.8±6.5 92 DMSO citrate buffer (pH 5) 900 92.2±5.7 94 DMSO citrate buffer (pH 6) 900 69.6±2.3 87 DMSO citrate buffer (pH 5) 585 96.8±3.1 94

[0136] The optimal reaction conditions were determined based on the optimized reaction conditions: 2.5 mM model substrate 1b, in pH 5.0 citrate buffer, 2.5% DMSO as a cosolvent, 5 mM hydrogen peroxide, and 585 nM enzyme. Figure 9 The chiral detection conditions were: Chiralpak IE, acetonitrile / water = 50:50, 1.0 mL / min.

[0137] Example 8: Mutants under optimized conditions Ani UPO CHO-5 Conduct substrate suitability investigation

[0138] The reaction system was 400 μL, and citrate buffer (100 mM, pH 5.0), 585 nM Ani UPO CHO-5, 2.5 mM substrate (2.5% DMSO solution), 5 mM hydrogen peroxide, at 30°C, 800 rpm shaking reaction for 16 h, add 800 μL of anhydrous ethanol (containing 0.25 mM 1,3,5-trimethoxybenzene or 1,2,3-trimethoxybenzene as internal standard) to precipitate protein, centrifuge, take the supernatant for HPLC detection, calculate and analyze the yield and ee value. The results of the investigation are shown in Table 12.

[0139] Table 12: Ani UPO CHO-5 Results of investigation on substrate suitability

[0140]

[0141] Sequence Schedule:

[0142] SEQ ID NO.1: Wild-type peroxidase Ani UPO

[0143] MKYFPLFPTLVYAVGVVAFPDYASLAGLSQQELDAIIPTLEAR FPQQGAPHPLPWSPPGPNDVRAPCPMLNTLANHGYLPHNGKDITEQHTINALYNALGIEEELAVYLHQEAVTTNPAPNATTFSLNDLSRHDILEHDASLSRQDAFFGDNHDFNQTIFDETRSY WTSPIIDVKQAALSRQARVNTSMATNPNYTMSELGNSFSYGETAAYIIVLGDKEKGLVNRSRVEYLFENERLPLDLGWSRAKENIITFDDLSTMLQRIINATGGESEFDRELAKRGGVHVGSWRG

[0144] SEQ ID NO.2: Mutant Ani UPO-F161A-L66F-L52F

[0145] MKYFPLFPTLVYAVGVVAFPDYASLAGLSQQELDAIIPTLEAR FPQQGAPHPLPWSPPGPNDVRAPCPMLNTLANHGYLPHNGKDITEQHTINAFYNALGIEEELAVYFHQEAVTTNPAPNATTFSLNDLSRHDILEHDASLSRQDAFFGDNHDFNQTIFDETRSY WTSPIIDVKQAALSRQARVNTSMATNPNYTMSELGNSASYGETAAYIIVLGDKEKGLVNRSRVEYLFENERLPLDLGWSRAKENIITFDDLSTMLQRIINATGGESEFDRELAKRGGVHVGSWRG

[0146] SEQ ID NO.3: Mutant Ani UPO CHO-6 -G164A-S160T-L217V-A168G

[0147] MKYFPLFPTLVYAVGVVAFPDYASLAGLSQQELDAIIPTLEAR FPQQGAPHPLPWSPPGPNDVRAPCPMLNTLANHGYLPHNGKDITEQHTINAFYNALGIEEELAVYFHQEAVTTNPAPNATTFSLNDLSRHDILEHDASLSRQDAFFGDNHDFNQTIFDETRSYWTSPIIDVKQAALSRQARVNTSMATNPNYTMSELGNTASYAETAGYIIVLGDKEKGLVNRSRVEYLFENERLPLDLGWSRAKENITFDDLSTMVQRIINATGGESEFDRELAKRGGVHVGSWRG

Claims

1. A peroxidase Ani A UPO mutant characterized by: It is a wild-type peroxidase with an amino acid sequence as shown in SEQ ID NO.1 Ani UPO is the parent, and the mutant obtained by mutation is: The phenylalanine at position 161 of the parent was mutated to alanine to obtain a mutant Ani UPO-F161A; Then Ani UPO-F161A is the parent, and the mutant is obtained by mutation, wherein the mutant is one of the following: The leucine at position 66 of the parent was mutated to valine to obtain a mutant Ani UPO-F161A-L66V; The 66th leucine of the parent was mutated to phenylalanine to obtain a mutant Ani UPO-F161A-L66F; The leucine at position 66 of the parent was mutated to alanine to obtain a mutant Ani UPO-F161A-L66A; The parent leucine at position 66 was mutated to phenylalanine and the alanine at position 63 was mutated to serine to obtain a mutant Ani UPO-F161A-L66F-A63S; The mutant was obtained by mutating the leucine at position 66 of the parent to phenylalanine and the glutamic acid at position 69 to methionine. Ani UPO-F161A-L66F-E69M; The mutant was obtained by mutating the leucine at position 66 to phenylalanine and the glutamic acid at position 69 to glycine. Ani UPO-F161A-L66F-E69G; The mutant was obtained by mutating the leucine at position 66 of the parent to phenylalanine and the leucine at position 52 to phenylalanine. Ani UPO-F161A-L66F-L52F, among which, the mutant Ani The amino acid sequence of UPO-F161A-L66F-L52F is shown in SEQ ID NO.

2.

2. The peroxidase according to claim 1 Ani A UPO mutant characterized by: It is based on Ani UPO-F161A-L66F-L52F is the parent, and the mutant obtained by mutation is one of the following: The 164th glycine of the parent was mutated to alanine to obtain the mutant Ani UPO CHO-6 -G164A; The 217th leucine of the parent was mutated to valine to obtain a mutant Ani UPO CHO-6 -L217V; The 160th serine of the parent was mutated to isoleucine to obtain a mutant Ani UPO CHO-6 -S160I; Among them, the Ani UPO CHO-6 for Ani UPO-F161A-L66F-L52F.

3. The peroxidase according to claim 2 Ani A UPO mutant characterized by: It is based on Ani UPO CHO-6 -G164A is the parent, and the mutant is obtained by mutation, wherein the mutant is one of the following: The 160th serine of the parent was mutated to threonine to obtain a mutant Ani UPO CHO-6 -G164A-S160T; The 160th serine of the parent was mutated to isoleucine to obtain a mutant Ani UPO CHO-6 -G164A-S160I; The 160th serine of the parent was mutated to leucine to obtain the mutant Ani UPO CHO-6 -G164A-S160L; The 217th leucine of the parent was mutated to valine to obtain the mutant Ani UPO CHO-6 -G164A-L217V.

4. The peroxidase according to claim 3 Ani A UPO mutant characterized by: It is based on Ani UPO CHO-6 -G164A-S160T is the parent, and the mutant obtained by mutation is one of the following: The mutant was obtained by mutating the leucine at position 217 to valine and the glutamic acid at position 156 to valine. Ani UPO CHO-6 -G164A-S160T-L217V-E156V; The mutant was obtained by mutating the leucine at position 217 to valine and the alanine at position 168 to glycine. Ani UPO CHO-6 -G164A-S160T-L217V-A168G; The 217th leucine of the parent was mutated to valine and the 172th valine was mutated to leucine to obtain a mutant Ani UPO CHO-6 -G164A-S160T-L217V-V172L; in, Ani UPO CHO-6 The amino acid sequence of -G164A-S160T-L217V-A168G is shown in SEQ ID NO.

3.

5. A DNA molecule, characterized in that: The DNA molecule encodes the peroxidase according to any one of claims 1 to 4 Ani UPO mutants.

6. An expression vector, characterized in that: The expression vector contains the DNA molecule according to claim 5.

7. A host cell, characterized in that: The host cell contains the expression vector according to claim 6.

8. The host cell according to claim 7, characterized in that: The host cell is a prokaryotic cell or a eukaryotic cell.

9. The host cell according to claim 8, characterized in that: The eukaryotic cell is yeast.

10. The peroxidase according to any one of claims 1 to 4 Ani UPO mutants or peroxidase Ani Application of UPO in the synthesis of α-hydroxy β-ketoester derivatives; the chemical formula of the α-hydroxy β-ketoester derivative is shown in Formula I: ;in, n=0, 1, 2, 3, 4, 5; R= X, H, OMe; R 1 = alkyl, benzyl or phenyl.

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