Non-heme iron enzyme mutant, truncated body, DNA molecule, expression vector, host cell and application in synthesis of chiral primary amine compound

Abstract

The invention relates to a non-heme iron enzyme mutant, a truncated body, a DNA molecule, an expression vector, a host cell and application in synthesis of a chiral primary amine compound, and belongs to the field of enzyme engineering. The invention provides a non-heme iron enzyme mutant. The invention also provides a non-heme iron enzyme truncated body or a mutant thereof, and an application of the non-heme iron enzyme truncated body or the mutant thereof in synthesis of a chiral primary amine compound. The truncation can catalyze the amino functionalization reaction of olefin to synthesize a chiral primary amine compound in one step. The reaction steps are simple, the conditions are mild, and the target product can be obtained with better yield and selectivity. Meanwhile, the method has a wider substrate application range. According to the technical scheme, sodium azide and sodium cyanate can be used as anions, so that corresponding 2-aminoazide and 2-cyanic acid substitution products are obtained, and the method is widely applied to the field of medical chemistry.

Description

Technical Field

[0001] The invention relates to a non-heme iron enzyme mutant, a truncate and application thereof in synthesizing chiral primary amine compounds, and belongs to the field of enzyme engineering. Background Art

[0002] Chiral primary amines are widely present in biologically active molecules and are widely used in the synthesis of natural products, drug molecules and agrochemicals. They are important structures of common drug molecule intermediates. Chiral primary amines usually contain an amino group (NH 2 ) and a chiral center, which can be a carbon atom, a nitrogen atom or other types of atoms, usually connected to different substituents. Among them, most of the important compounds related to biological functions are heterocyclic compounds, such as nucleic acids, vitamins, antibiotics, hormones, pigments and alkaloids. Among the heterocyclic compounds containing both sulfur and nitrogen, the thiazole skeleton is one of the important characteristic structures for drug development. For example, 2-aminothiazoline is often found in choline receptor modulators, nitric oxide synthase inhibitors and antibacterial compounds ( Organic letters , 2010, 12 , 5526-5529).

[0003] At present, the synthesis of 2-aminothiazoline is mainly through the conversion of 2-hydroxyethylthiourea, but the existing methods mostly use organic catalytic synthesis routes, which have obvious shortcomings. For example, the dehydration cyclization of 2-hydroxyethylthiourea is difficult to be compatible with substrates that are unstable to acid or prone to acid-catalyzed racemization under strong acid catalysis ( Journal of Fluorine Chemistry , 2005, 126 , 297-300). Although the conditions of the Mitsunobu reaction are relatively mild, a mixed product of 2-aminoimidazoline and 1,2-ethylenethiourea is often obtained ( Bioorganic&Medicinal Chemistry Letters , 2005, 15 , 3849-3852). If sulfonyl chloride is used as an activating agent, 2-aminooxazoline ( Tetrahedron , 2004, 60 , 9883-9888).

[0004] Invention patent CN202010771036.5 discloses that the target product can be obtained by condensation / oxidation coupling reaction in an organic solvent or water in an oxygen atmosphere, using ketone and thiourea or thiourea derivatives as raw materials, and under the co-catalysis of activated carbon or metal salts. Invention patent CN202110616891.3 adopts an electrochemical synthesis method, using ketone and thiourea as raw materials, dimethyl sulfoxide and water as solvents, ammonium halide or alkali metal halide as both electrolyte and electrocatalyst, and in the presence of additives, the reaction temperature is 50-90°C to obtain 2-aminothiazole compounds. Although the above technical solutions realize the one-step construction of the thiazole ring, none of them can obtain chiral 2-aminothiazole compounds, and the reaction system used seriously pollutes the environment.

[0005] The difunctionalization of olefins has good atom and step economy and can introduce new functional groups on both sides of the carbon-carbon double bond. Morandi's group has developed a series of iron-catalyzed aminofunctionalization reactions of olefins to obtain halogen ( Science , 2018, 362 , 434-439), hydroxyl ( Angewandte Chemie International Edition ,2016, 55 , 2248-2251) and azide ( Journal of the American Chemical Society , 2020, 142 ,21548-21555) and other functional group-substituted primary amine products. This reaction has gradually become a powerful tool for increasing the complexity of molecules in organic synthesis, showing great potential for industrial application.

[0006] Enzyme-catalyzed reactions are characterized by high efficiency, high selectivity, and environmental friendliness, and have been widely used in the fields of chemical engineering, energy, and materials. Based on the relevant research on transition metal catalysis, Arnold's research group modified a heat-resistant iron-containing enzyme, cytochrome C ( Angewandte Chemie International Edition , 2019, 58 , 3138-3142), which can directly convert olefins into unprotected 2-amino-1-ols with high enantioselectivity using hydroxylamine derivatives as nitrogen sources under anaerobic conditions. This method supplements the high enantioselectivity transformation pathways lacking in traditional chemical synthesis, but this method only provides primary aminated products substituted with ortho-hydroxyl groups, and the scope of application of enzyme-catalyzed amino functionalization of olefins has not been fully explored. In addition, this method uses the heme in the active center of the enzyme to form a stable coordination structure, has strong substrate specificity, and can usually only catalyze specific reactions, thus limiting the applicability of this type of enzyme.

[0007] In summary, the present invention intends to develop an engineered enzyme that can catalyze the amino functionalization reaction of olefins, achieve a more economical and green asymmetric synthesis of chiral primary amine compounds, and further develop its industrial applicability. Summary of the invention

[0008] Non-heme iron enzymes mainly include hydroxylases and halogenases. Their natural catalytic mechanism is as follows: the iron in the active center of this type of enzyme is generally coordinated with two histidines and one glutamic acid or aspartic acid. If it is a halogenase, this position is coordinated by water. When α-KG, halogen ions and substrates are added, α-KG and halogen ions will replace water molecules and coordinate with iron. In the presence of oxygen, peroxyl radicals can be formed, and then a molecule of CO is removed. 2 , obtaining a highly reactive high-valent iron-oxygen intermediate. Subsequently, the hydroxylation reaction is achieved by grabbing the substrate hydrogen atom and rebounding the hydroxyl radical, or the halogenation reaction is achieved by capturing the halogen radical. Compared with the heme iron enzyme, the non-heme iron-containing enzyme has multiple open iron coordination sites and highly flexible and adjustable reactivity, and is expected to become a strong candidate for expanding the diversity of amino functionalization reactions of olefins.

[0009] The present invention provides a non-heme iron enzyme mutant, which is a wild-type non-heme iron enzyme with an amino acid sequence as shown in SEQ ID No.1. Bs QueD is obtained by mutating the histidine at positions 234, 236 and 275 to alanine; its amino acid sequence is shown in SEQ ID No.3.

[0010] The present invention provides a non-heme iron enzyme truncation, which is obtained by truncating the non-heme iron enzyme mutant. Bs QD 174ptt , whose amino acid sequence is shown in SEQ ID No.4.

[0011] The present invention also provides the non-heme iron enzyme truncated mutant, which is a non-heme iron enzyme truncated mutant. Bs QD 174pt As the parent, the mutant obtained by mutation is: The 71st isoleucine of the parent was mutated to valine to obtain the mutant Bs QD 174pt -I71V; The 71st isoleucine of the parent was mutated to leucine to obtain the mutant Bs QD 174pt -I71L; The 116th serine of the parent was mutated to arginine to obtain a mutant Bs QD 174pt -S116R; The 116th serine of the parent was mutated to lysine to obtain a mutant Bs QD 174pt -S116K; The threonine at position 118 of the parent was mutated to histidine to obtain a mutant Bs QD 174pt -T118H; The threonine at position 118 of the parent was mutated to glutamic acid to obtain a mutant Bs QD 174pt -T118E.

[0012] Furthermore, it is a non-heme iron enzyme truncated form Bs QD 174pt -S116R mutant is the parent, and the mutants obtained by mutation are: The parent isoleucine at position 71 was mutated to methionine to obtain a mutant Bs QD 174pt -S116R-I71M; The 71st isoleucine of the parent was mutated to leucine to obtain the mutant Bs QD 174pt -S116R-I71L; The threonine at position 118 of the parent was mutated to alanine to obtain a mutant Bs QD 174pt -S116R-T118A.

[0013] Furthermore, it is a non-heme iron enzyme truncated form Bs QD 174pt -S116R-T118A mutant is the parent, and the mutants obtained by mutation are: The 71st isoleucine of the parent was mutated to leucine to obtain the mutant Bs QD 174pt -S116R-T118A-I71L.

[0014] Furthermore, it is a non-heme iron enzyme truncated form Bs QD 174pt -S116R-T118A-I71L mutant is the parent, and the mutants obtained by mutation are: The 51st leucine of the parent was mutated to valine to obtain a mutant Bs QD 174pt -S116R-T118A-I71L-L51V; Among them, the mutant Bs QD 174pt The amino acid sequence of -S116R-T118A-I71L-L51V is shown in SEQ ID No.5.

[0015] The present invention also provides a DNA molecule, wherein the DNA molecule encodes the non-heme iron enzyme mutant, the non-heme iron enzyme truncation, or the non-heme iron enzyme truncation mutant.

[0016] The present invention also provides an expression vector, which contains the DNA molecule.

[0017] The present invention also provides a host cell, wherein the host cell contains the expression vector.

[0018] The present invention also provides the use of the non-heme iron enzyme mutant, the non-heme iron enzyme truncation, or the non-heme iron enzyme truncation mutant, DNA molecules, expression vectors, and host cells in synthesizing chiral primary amine compounds; the chiral primary amine compounds are shown in Formula I and Formula II: ; Among them, R 1 The group is an aromatic ring and its derivatives, and the structural formula is: 1 The group is an aromatic ring and its derivatives, and the structural formula is: ; Among them, R a , R b , R c , R d , R e are independently selected from hydrogen or any group other than hydrogen; Ring A is selected from: .

[0019] The present invention provides a method for synthesizing a chiral primary amine compound, which uses Formula III as a substrate, and uses the non-heme iron enzyme mutant, non-heme iron enzyme truncated body, or non-heme iron enzyme truncated body mutant, DNA molecules, expression vectors, and host cells as biocatalysts to synthesize the chiral primary amine compound; The general formula of substrate formula III is: ; The synthetic routes are: Reaction Scheme 1: , Reaction Scheme 2: , Among them, R 1 The group is an aromatic ring and its derivatives, and the structural formula is: , Among them, R a , R b , R c , R d, R e are independently selected from hydrogen or any group other than hydrogen; Ring A is selected from: .

[0020] The beneficial effects of the present invention are: The present invention provides a non-heme iron enzyme truncate, which can catalyze the amino functionalization reaction of olefins and synthesize chiral primary amine compounds in one step. The reaction steps are simple, the conditions are mild, and the target product can be obtained with good yield and selectivity. At the same time, the method has a wider substrate application range. Sodium azide and sodium cyanate can also be used as anions by using the technical solution to obtain corresponding 2-aminoazide and 2-cyanic acid substitution products. It is worth mentioning that the 2-aminoazide product can be used to obtain an important chiral adjacent diamine product in one step through the staudinger reaction. The product is a key intermediate for preparing the drug levamisole hydrochloride, a 2-cyanic acid substitution product, and 2-aminooxazoline is also a class of compounds with important medicinal value, which are widely used in the field of pharmaceutical chemistry. Therefore, the technical solution provided by the present invention has more potential in industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For pET-28a- Bs Gene map of QueD; Figure 2 is a standard curve diagram of 5-phenyl-4,5-dihydrothiazol-2-amine; Figure 3 for Bs SDS-PAGE gel images of QueD and truncated proteins; Figure 4 for Bs QD AF The chiral detection spectrum of the product 5-phenyl-4,5-dihydrothiazol-2-amine generated by the catalytic reaction of styrene under the optimal conditions (where A is the chiral detection original spectrum of the racemic 5-phenyl-4,5-dihydrothiazol-2-amine; B is Bs QD AF Catalytic reaction of styrene to produce products S- 5-phenyl-4,5-dihydrothiazol-2-amine: 94% ee chiral detection original spectrum); Figure 5 for Bs QD AF The chiral detection spectrum of the product 2-azido-2-phenylethylamine generated by the catalytic reaction of styrene under the optimal conditions (where A is the chiral detection original spectrum of the racemic 2-azido-2-phenylethylamine; B is Bs QD AF Catalyze the reaction of styrene to produce chiral productsS- 2-Azide-2-phenylethylamine: 94% ee chiral detection original spectrum). DETAILED DESCRIPTION

[0022] Example 1 Construction of expression strain and investigation of initial reaction activity 1. Wild type Bs Construction of QueD expression strain Targeting Bacillus subtilis Bacillus subtilis Wild-type non-heme iron enzyme Bs QueD (Genebank Accession: CAB16035.2), amino acid sequence as shown in SEQ ID No.1, codon optimization and full gene synthesis were performed using Escherichia coli as host cells. Nco Ⅰ and Xho I restriction site Bs The QueD gene was constructed in the pET28a(+) expression vector and Nco Add two nucleotides, CG, after the ATG start codon in the I restriction site to avoid frameshift and obtain the wild type Bs The recombinant expression vector pET28a- Bs QueD, gene map reference Figure 1 . Bs The QueD recombinant expression vector was transformed into Escherichia coli TSR2566 strain (Qingke Biotechnology) to construct Bs QueD-expressing strains.

[0023] 2. Bs Protein expression of QueD and preparation of crude enzyme solution The constructed Bs The QueD expression strain was inoculated into a shaking tube containing 3 mL of LB liquid medium containing kanamycin (hereinafter referred to as LB-Kan) and activated overnight in a shaker at 37°C and 250 rpm. 1 mL of the activated bacterial solution was inoculated into a conical flask containing 50 mL of TB medium containing kanamycin (hereinafter referred to as TB-Kan) and incubated at 37°C and 200 rpm for 2.5 h. 600 When the OD value reaches 0.6-0.8, place the bacterial solution in an ice bath for 30 min, add IPTG with a final concentration of 0.5 mM, and induce expression for 18 h at 22°C and 150 rpm. After the induction expression is completed, collect the bacteria by centrifugation and discard the supernatant. Resuspend the bacterial pellet with 100 mM potassium phosphate (KPi, pH 7.0) buffer and adjust the OD value. 600 to 30 to obtain crude enzyme solution.

[0024] 3.Bs Investigation of initial reaction activity of QueD Using styrene as substrate, Bs QueD catalyzes the reaction of styrene to form products S- 5-phenyl-4,5-dihydrothiazol-2-amine (hereinafter referred to as model reaction 1), S- 2-azido-2-phenylethyl-1-amine (hereinafter referred to as the reaction model 2) and S- Reactivity of 5-phenyl-4,5-dihydrooxazol-2-amine.

[0025] The specific steps are as follows: add 300 μL of the resuspended crude enzyme solution to a 2 mL glass reaction bottle, transfer to an anaerobic glove box, add 20 μL of L-ascorbic acid (20 mM aqueous solution), 20 μL of ammonium ferrous sulfate (0.2 mM aqueous solution), 20 μL of sodium thiocyanate, sodium azide or sodium cyanate (200 mM aqueous solution), 20 μL of styrene (200 mM ethanol solution) and 20 μL of pivaloylhydroxylamine trifluoromethanesulfonate (hereinafter referred to as nitrogen source, 200 mM aqueous solution) in turn, and react at room temperature in an anaerobic glove box for 24 h. After the reaction is completed, add 900 μL of ethanol internal standard solution (containing 0.25 mM 1,3,5-trimethoxybenzene), shake and mix, centrifuge, and take 400 μL of the supernatant and add it to a 2 mL injection bottle containing an inner insert for analysis and detection. The concentration of the product is calculated by using LC-MS to detect product standards with different concentration gradients and containing internal standards, and the peak area ratio of the standard and the internal standard is calculated to make a standard curve (hereinafter collectively referred to as analytical yield). The standard curve is as follows Figure 2 As shown. Calculated by product peak area ee value, ee The value is the ratio of the peak areas between products with different configurations of enantiomers. ee = ((S,SR,R) / (S,S+R,R))*100%. The initial reactivity results are shown in Table 1.

[0026] Table 1. Bs QueD Reactivity

[0027] 4. Bs Investigation of the active center of QueD reaction Bs QueD has a dimer structure, and each monomer contains two metal active sites. The Fe 2+ The active site 2 (hereinafter referred to as Site 2) is bound to the histidine at positions 62, 64, and 103 and the glutamic acid at position 69. 2+The histidines at positions 234, 236, and 275 are bound respectively. This means that the reaction may be catalyzed by the two active centers individually or in coordination.

[0028] Further site silencing experiments were performed to confirm the active sites that contribute substantially to the catalytic reaction. Based on different active sites Site 1 and Site 2, histidine was mutated to alanine, and the wild type Bs The amino acid sequence of QueD after silencing Site1 is shown in SEQ ID No. 2. Bs The amino acid sequence after QueD silenced Site2 is shown in SEQ ID No. 3. Protein expression and crude enzyme solution were prepared respectively. Under the conditions of model reaction 1, styrene was used as substrate to determine the reaction activity. The specific reaction results are shown in Table 2.

[0029] Table 2. Bs QueD site silencing activity

[0030] ND, not detected.

[0031] 5. Bs Investigation of QueD protein truncation and reaction activity According to the results of site silencing reaction activity, the active center of the catalytic reaction is Site 1, and after silencing Site 2, the reaction activity increased by 1.6 times compared with the wild type. ee The value increased from 37% to 51%. Therefore, we tried to identify 5 sites (V162, K174, S192, Q211, G216) Bs QueD was used for protein truncation. The truncated plasmids of the above six sites were constructed, and protein expression and crude enzyme solution were prepared. Under the conditions of model reaction 1, styrene was used as the substrate, and the reaction activity was determined. The specific reaction results are shown in Table 3. The proteins obtained by truncating from the K174 site (hereinafter collectively referred to as Bs QD 174pt ) showed enhanced reactivity, which was better than that of single silencing Site 2. Bs QD 174pt The amino acid sequence of is shown in SEQ ID No. 4. Further, select Bs QD 174pt Directed evolution was performed to construct a mutant library. The SDS-PAGE protein expression results are shown in Figure 3 shown.

[0032] Table 3. Bs QueD protein truncation-responsive activity

[0033] Example 2: Truncated Bs QD 174pt As the parent, directed evolution was carried out under the conditions of model reaction 1 1. Bs QD 174pt Mutant library construction by Bs QD 174pt As the parent, site saturation mutations were performed on amino acid residues such as I71, S116, and T118. Bs QD 174pt Plasmid was used as template, and the corresponding degenerate primers were designed by 22 codon trick method for PCR reaction. PCR reaction was carried out using high-fidelity DNA polymerase KOD One TM PCR Master Mix (TOYOBO) was used. The PCR reaction system is shown in Table 4: Table 4. PCR reaction system

[0034] PCR reaction conditions are shown in Table 5: Table 5. PCR reaction conditions

[0035] The linear DNA products obtained by PCR were subjected to 1% agarose gel electrophoresis and separated by gel excision and recovery. The nucleic acid concentration of the gel recovery products was determined by ultra-micro nucleic acid analyzer, and ligated according to the DNA seamless cloning kit (ClonExpress® II One Step Cloning Kit, Novozyme). The ligation system is shown in Table 6. The products were incubated at 37°C for 30 min, and the ligation products were transformed into TSR2566 Escherichia coli competent cells.

[0036] Table 6. PCR product ligation system

[0037] 2. Reactivity screening (1) Pick a single colony from the plate and inoculate it into a 96-well plate containing 300 μL LB-Kan liquid medium, and activate it overnight in a shaker at 37°C and 250 rpm. Take 50 μL of the activated bacterial solution and transfer it to a 96-deep-well plate containing 950 μL TB-Kan liquid medium, and culture it at 37°C and 250 rpm for 2.5 h. Place the 96-well plate on ice for 30 min, then add 50 μL IPTG stock solution (final concentration 0.5 mM) to induce expression, and culture it at 22°C and 200 rpm for 20 h. Collect the bacteria by low-temperature centrifugation, add 300 μL KPi (pH 7.0) buffer to each well, and vortex to completely resuspend the cells. Transfer to an anaerobic glove box, add 20 μL L-ascorbic acid (20 mM aqueous solution), 20 μL ammonium ferrous sulfate (0.2 mM aqueous solution), 20 μL sodium thiocyanate (200 mM aqueous solution), 20 μL styrene (200 mM ethanol solution) and 20 μL nitrogen source (200 mM aqueous solution) in sequence, and shake in the anaerobic glove box at room temperature for 24 h. After the reaction is completed, add 900 μL anhydrous ethanol, shake and mix, and then centrifuge. Take 200 μL of the supernatant to a 96-well ELISA plate for LC-MS detection.

[0038] The activity and ee The mutants with improved values ​​were cultured in shake flasks and the reaction activity was verified. The screening and detection methods for verifying the reaction activity were the same as those in Example 1-3. The relative activity, ee Values ​​(Relative activity was determined based on the calibration curve of the corresponding peak area and product concentration measured by LC-MS, relative activity = product concentration of mutant / product concentration of parent in this round). The results of the first round of directed evolution verification are shown in Table 7.

[0039] Table 7. Results of the first round of directed evolution verification

[0040] (2) Bs QD 174pt -S116R mutant was used as the parent, and site-directed saturation mutation was performed on the I71 and T118 amino acid residues. The mutant library construction, protein expression, reaction screening, and detection methods were the same as described above. The results of the second round of directed evolution verification are shown in Table 8.

[0041] Table 8. Results of the second round of directed evolution verification

[0042] (3) Bs QD 174pt-S116R-T118A mutant was used as the parent, and site-directed saturation mutagenesis was performed on L51 and I71 amino acid residues. Mutant library construction, protein expression, reaction screening, and detection methods were the same as described above. The results of the third round of directed evolution verification are shown in Table 9.

[0043] Table 9. Results of the third round of directed evolution verification

[0044] (4) Bs QD 174pt -S116R-T118A-I71L mutant was used as the parent, and site-directed saturation mutagenesis was performed on L51, Q108, L114 and L126 amino acid residues. Mutant library construction, protein expression, reaction screening, and detection methods were the same as described above. The fourth round of directed evolution verification results are shown in Table 10.

[0045] Table 10. Results of the fourth round of directed evolution verification

[0046] Under the conditions of model reaction 1, styrene is the substrate. Bs QD 174pt Four rounds of directed evolution were performed to screen the optimal mutants. Bs QD 174pt -S116R-T118A-I71L -L51V (hereinafter referred to as Bs QD AF ) is: Bs QD 174pt The parent is the serine at position 116 mutated to arginine, the threonine at position 118 mutated to alanine, the isoleucine at position 71 mutated to leucine, and the leucine at position 51 mutated to valine. The amino acid sequence is shown in SEQ ID No.5.

[0047] Example 3 Bs QD AF Optimization of mutant reaction conditions Under the condition of mode response 1, Bs QD AF Reaction conditions were optimized to improve enzyme reaction activity. The optimized conditions included substrate concentration, iron concentration, buffer type and pH value, cosolvent type, reducing agent type and concentration, equivalent ratio, reaction temperature, cosolvent volume, etc. Protein expression and crude enzyme solution preparation, reaction screening, and detection methods were the same as described above. Relative activity of the detection reaction, ee Values, where relative activity = product concentration under different conditions / product concentration under initial conditions. The results of the optimization of substrate concentration, iron concentration, buffer type and pH value are shown in Table 11.

[0048] Table 11. Results of optimization of conditions for model reaction 1

[0049] Since the substrate concentration of 5 mM, the iron concentration of 100 μM, and M9-N (pH 6.0) as the buffer are all helpful for improving the enzyme reaction activity, the above optimal conditions were combined to optimize the subsequent reaction conditions. The results of the optimization of other conditions are shown in Table 12.

[0050] Table 12. Results of optimization of conditions for model reaction 1 According to the results of the investigation, the optimized reaction conditions were determined as follows: 5 mM model substrate, M9-N (pH 6.0) as buffer, iron concentration of 100 μM, sodium dithionite (final concentration of 2 mM) as reducing agent, acetonitrile (volume ratio 15%) as cosolvent, the equivalent ratio of anion, styrene, and nitrogen source was 3:1:2, and the reaction at 8°C was the best reaction condition. Under the best reaction conditions, Bs QD AF The analytical yield of the corresponding product obtained by catalytic mode reaction 1 was 72%. ee The value is 94%. The inspection results before and after the conditions are optimized are shown in Table 13.

[0051] Table 13. Results of the model reaction 1 before and after condition optimization

[0052] The chiral separation results of the products are as follows Figure 4 The chiral detection chromatographic column used was a chiral IG column - Daicel CHIRALPAK (5 μm filler, 4.6×250 mm), the mobile phase ratio was 50% acetonitrile / 50% water (containing 20 mM ammonium acetate), and the flow rate was 0.7 mL / min.

[0053] The best of Example 2 Bs QD AF The mutants were also used for model reaction 2.

[0054] Using styrene as substrate, Bs QD AF The reaction conditions were optimized to catalyze the chiral product 2-azido-2-phenylethylamine to improve the enzyme reaction activity. The optimized conditions included substrate concentration, iron concentration, buffer type and pH value, cosolvent type, reducing agent type and concentration, equivalent ratio, reaction temperature, cosolvent volume, etc. The protein expression and crude enzyme solution preparation, reaction screening, and detection methods were the same as described above. The relative activity of the detection reaction, eeValues, where relative activity = product concentration under different conditions / product concentration under initial conditions. The results of the optimization of substrate concentration, iron concentration, buffer type and pH value are shown in Table 14.

[0055] Table 14. Results of optimization of conditions for model reaction 2

[0056] Since the substrate concentration of 5 mM, the iron concentration of 100 μM, and M9-N (pH 6.0) as the buffer are all helpful for improving the enzyme reaction activity, the above optimal conditions were combined to optimize the subsequent reaction conditions. The results of the optimization of other conditions are shown in Table 15.

[0057] Table 15. Results of optimization of conditions for model reaction 2

[0058] According to the results of the investigation, the optimized reaction conditions were determined as follows: 5 mM model substrate, M9-N (pH 6.0) as buffer, iron concentration of 100 μM, L-ascorbic acid (final concentration of 0.1 mM) as reducing agent, acetonitrile (volume ratio 15%) as cosolvent, the equivalent ratio of anion, styrene, and nitrogen source was 3:3:1, and the reaction at 8°C was the best reaction condition. Under the best reaction conditions, Bs QD AF The analytical yield of the corresponding product obtained by catalytic mode reaction 2 was 49%. ee The value is 94%. The investigation results before and after reaction optimization are shown in Table 16.

[0059] Table 16. Results of the model reaction 2 before and after condition optimization

[0060] The chiral separation structure of the product is as follows Figure 5 The chiral detection chromatographic column used was a chiral IG column - Daicel CHIRALPAK (5 μm filler, 4.6×250 mm), the mobile phase ratio was 50% acetonitrile / 50% water (containing 20 mM ammonium acetate), and the flow rate was 0.7 mL / min.

[0061] Example 4 Bs QD AF Investigation of substrate suitability of mutants Investigation of substrate suitability under route 1 Under the optimal reaction conditions, Bs QD AF The mutants were tested for their reactivity to other substrates. The protein expression and crude enzyme solution preparation, reaction screening and detection methods were the same as described above. The results of the substrate applicability test are shown in Table 17.

[0062] Table 17. Results of investigation on the suitability of the substrate for route 1

[0063] 2. Investigation of substrate suitability under route 2 Under the optimal reaction conditions, Bs QD AF The mutants were tested for their reactivity to other substrates. The protein expression and crude enzyme solution preparation, reaction screening and detection methods were the same as described above. The results of the substrate applicability test are shown in Table 18.

[0064] Table 18. Results of investigation on the suitability of the substrate for route 2

[0065] Sequence Schedule:

[0066] SEQ ID No.1 wild type Bs QueD amino acid sequence MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPPHSYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAK PACSAKLAELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDHYHEYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVHSYRLDSHYTKMVGVLVPGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKP SEQ ID No.2 wild type Bs Amino acid sequence after QueD silences Site1 MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLAVAKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPASYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDHYHEYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVHSYRLDSHYTKMVGVLVPGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKP SEQ ID No.3 Wild type Bs Amino acid sequence after silencing Site2 of QueD MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPHSYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDAYAEYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVASYRLDSHYTKMVGVLVPGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKP SEQ ID No.4 Bs QueD 174pt : Wild type Bs Amino acid sequence after truncating QueD at position K174 MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPPHSYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSA SEQ ID No.5 Bs QD AF : Amino acid sequence of the best mutant obtained by directed evolution MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLVSGGKGDAFPLHVHKDTHEGLLVLDGKLELTLDGERYLLISGDYANIPAGTPPHSYRMQSHRTRLVRYAMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSA.

Claims

1. A non-heme iron enzyme mutant, characterized in that: It is a wild-type non-heme iron enzyme with an amino acid sequence as shown in SEQ ID No.

1. Bs QueD is obtained by mutating the histidine at positions 234, 236 and 275 to alanine; its amino acid sequence is shown in SEQ ID No.

3.

2. A truncated non-heme iron enzyme, characterized in that: The non-heme iron enzyme mutant according to claim 1 is truncated to obtain the non-heme iron enzyme truncated form. Bs QD 174pt , whose amino acid sequence is shown in SEQ ID No.

4.

3. The non-heme iron enzyme truncated mutant according to claim 2, characterized in that: Non-heme iron enzyme truncated Bs QD 174pt As the parent, the mutant obtained by mutation is: The 71st isoleucine of the parent was mutated to valine to obtain the mutant Bs QD 174pt -I71V; The 71st isoleucine of the parent was mutated to leucine to obtain the mutant Bs QD 174pt -I71L; The 116th serine of the parent was mutated to arginine to obtain a mutant Bs QD 174pt -S116R; The 116th serine of the parent was mutated to lysine to obtain a mutant Bs QD 174pt -S116K; The threonine at position 118 of the parent was mutated to histidine to obtain a mutant Bs QD 174pt -T118H; The threonine at position 118 of the parent was mutated to glutamic acid to obtain a mutant Bs QD 174pt -T118E.

4. The non-heme iron enzyme truncated mutant according to claim 3, characterized in that: Non-heme iron enzyme truncated Bs QD 174pt -S116R mutant is the parent, and the mutants obtained by mutation are: The parent isoleucine at position 71 was mutated to methionine to obtain a mutant Bs QD 174pt -S116R-I71M; The 71st isoleucine of the parent was mutated to leucine to obtain the mutant Bs QD 174pt -S116R-I71L; The threonine at position 118 of the parent was mutated to alanine to obtain a mutant Bs QD 174pt -S116R-T118A.

5. The non-heme iron enzyme truncated mutant according to claim 4, characterized in that: Non-heme iron enzyme truncated Bs QD 174pt -S116R-T118A mutant is the parent, and the mutants obtained by mutation are: The 71st isoleucine of the parent was mutated to leucine to obtain the mutant Bs QD 174pt -S116R-T118A-I71L.

6. The non-heme iron enzyme truncated mutant according to claim 5, characterized in that: Non-heme iron enzyme truncated Bs QD 174pt -S116R-T118A-I71L mutant is the parent, and the mutants obtained by mutation are: The 51st leucine of the parent was mutated to valine to obtain a mutant Bs QD 174pt -S116R-T118A-I71L-L51V; Among them, the mutant Bs QD 174pt The amino acid sequence of -S116R-T118A-I71L-L51V is shown in SEQ ID No.

5.

7. A DNA molecule, characterized in that: The DNA molecule encodes the non-heme iron enzyme mutant according to claim 1, the non-heme iron enzyme truncation according to claim 2, or the non-heme iron enzyme truncation mutant according to any one of claims 3-6.

8. An expression vector, characterized in that: The expression vector contains the DNA molecule according to claim 7.

9. A host cell, characterized in that: The host cell contains the expression vector according to claim 8.

10. Use of the non-heme iron enzyme mutant according to claim 1, the non-heme iron enzyme truncated form according to claim 2, or the non-heme iron enzyme truncated form mutant according to any one of claims 3 to 6, the DNA molecule according to claim 7, the expression vector according to claim 8, and the host cell according to claim 9 in synthesizing chiral primary amine compounds; the chiral primary amine compounds are shown in formula I and formula II: ； in, The R1 group is an aromatic ring and its derivatives, and the structural formula is: ； Among them, R a , R b , R c , R d , R e are independently selected from hydrogen or any group other than hydrogen; Ring A is selected from: 。 11. A method for synthesizing a chiral primary amine compound, characterized in that: It uses formula III as a substrate, the non-heme iron enzyme mutant according to claim 1, the non-heme iron enzyme truncated body according to claim 2, or the non-heme iron enzyme truncated body mutant according to any one of claims 3 to 6, the DNA molecule according to claim 7, the expression vector according to claim 8, and the host cell according to claim 9 as a biocatalyst to synthesize a chiral primary amine compound; The general formula of substrate formula III is: ; The synthetic routes are: Reaction Scheme 1: ， Reaction Scheme 2: ， Among them, the R1 group is an aromatic ring and its derivatives, and the structural formula is: ， Among them, R a , R b , R c , R d , R e are independently selected from hydrogen or any group other than hydrogen; Ring A is selected from: 。

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