Non-heme iron enzyme mutants, truncated forms, DNA molecules, expression vectors, host cells, and applications in synthesizing chiral primary amine compounds

Abstract

The present invention relates to a non-heme iron enzyme mutant, a truncate, a DNA molecule, an expression vector, a host cell, and an application in synthesizing a chiral primary amine compound, and belongs to the field of enzyme engineering. The present invention provides a non-heme iron enzyme mutant. The present invention also provides a non-heme iron enzyme truncate or a mutant thereof, and an application in synthesizing a chiral primary amine compound. The truncate of the present invention can catalyze the amino functionalization reaction of olefins 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 good yield and selectivity. At the same time, the method has a wider scope of application of substrates. Sodium azide and sodium cyanate can also be used as anions using the technical solution to obtain corresponding 2-amino azide and 2-cyanic acid substitution products, which are widely used in the field of pharmaceutical chemistry.

Description

Technical Field

[0001] The present invention relates to a non-heme iron enzyme mutant, a truncated form, and their applications in the synthesis of chiral primary amine compounds, belonging to the field of enzyme engineering. Background Art

[0002] Chiral primary amines are widely present in bioactive 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, and is usually connected to different substituents. Among them, most important compounds related to biological functions are heterocyclic compounds, such as nucleic acids, vitamins, antibiotics, hormones, pigments, and alkaloids. In 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 present in cholinergic receptor modulators, nitric oxide synthase inhibitors, and antibacterial compounds ( Organic letters , 2010, 12 , 5526-5529).

[0003] Currently, the synthesis of 2-aminothiazoline is mainly through the transformation of 2-hydroxyethylthiourea, but existing methods mostly adopt organic catalytic synthesis routes, which have obvious deficiencies. For example, the dehydration cyclization of 2-hydroxyethylthiourea is difficult to be compatible with substrates that are acid-labile or prone to acid-catalyzed racemization under strong acid catalysis conditions ( Journal of Fluorine Chemistry , 2005, 126 , 297-300). Although the Mitsunobu reaction conditions are relatively mild, a mixture of 2-aminoimidazoline and 1,2-ethylene thiourea is often obtained ( Bioorganic&Medicinal Chemistry Letters , 2005, 15 ,3849-3852). If sulfonyl chloride is used as an activator, 2-aminooxazoline is often preferentially formed ( Tetrahedron , 2004, 60 , 9883-9888).

[0004] The invention patent CN202010771036.5 discloses that in an organic solvent or water, under an aerobic atmosphere, using a ketone and thiourea or a thiourea derivative as raw materials, and under the co - catalysis of activated carbon or a metal salt, the target product can be obtained through a condensation / oxidative coupling reaction. The invention patent CN202110616891.3 adopts an electrochemical synthesis method, using a ketone and thiourea as raw materials, dimethyl sulfoxide and water as solvents, and ammonium halide or alkali metal halide as both an electrolyte and an electro - catalyst. In the presence of an additive, at a reaction temperature of 50 - 90 °C, 2 - aminothiazole compounds are obtained. Although the above technical solutions have achieved the one - step construction of the thiazole ring, chiral 2 - aminothiazole compounds have not been obtained, and the reaction systems used cause serious environmental pollution.

[0005] The difunctionalization reaction of alkenes has good atom and step economy, and can introduce new functional groups on both sides of the carbon - carbon double bond simultaneously. The Morandi research group has developed a series of iron - catalyzed amino - functionalization reactions of alkenes to obtain primary amine products substituted with functional groups such as halogens ( Science , 2018, 362 , 434 - 439), hydroxyl groups ( Angewandte Chemie International Edition ,2016, 55 , 2248 - 2251) and azides ( Journal of the American Chemical Society , 2020, 142 ,21548 - 21555). This reaction has gradually become a powerful tool for increasing molecular complexity in organic synthesis, showing great potential for industrial applications.

[0006] Enzyme - catalyzed reactions have the characteristics of high efficiency, high selectivity and environmental friendliness, and have been widely used in fields such as chemical engineering, energy and materials. Based on relevant research on transition - metal catalysis, the Arnold research group modified a heat - resistant iron - containing enzyme, cytochrome C ( Angewandte Chemie International Edition , 2019, 58 , 3138 - 3142). It can directly convert alkenes into highly enantioselective and unprotected 2 - amino - 1 - alcohols using hydroxylamine derivatives as a nitrogen source under anaerobic conditions. This method complements the lack of highly enantioselective transformation pathways in traditional chemical synthesis. However, this method only provides primary amination products substituted with ortho - hydroxyl groups, and the scope of application of enzyme - catalyzed amino - functionalization of alkenes has not been fully explored. Moreover, this method uses the heme in the enzyme active center to form a stable coordination structure, with strong substrate specificity, and usually can 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 alkenes, achieve more economical and green asymmetric synthesis of chiral primary amine compounds, and further develop its applicability in industry. Summary of the Invention

[0008] Non-heme iron enzymes mainly include two types: hydroxylase and halogenase. 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 glutamate or aspartate. If it is a halogenase, this position is coordinated by water. When α-KG, halide ions, and the substrate are added, α-KG and halide ions will replace the water molecule and coordinate with the iron. In the presence of oxygen, a peroxyl radical can be formed, and then a molecule of CO 2 is removed to obtain a highly reactive high-valent iron-oxo intermediate. Subsequently, hydroxylation reaction is achieved by abstracting the substrate hydrogen atom and hydroxyl radical rebound, or halogenation reaction is achieved by capturing halogen radicals. Compared with heme iron enzymes, non-heme iron-containing enzymes have multiple open iron coordination sites, highly flexible and adjustable reactivity, and are expected to be powerful candidates for expanding the diversity of amino functionalization reactions of olefins.

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

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

[0011] The present invention also provides a mutant of the above-mentioned truncated non-heme iron enzyme, which is a mutant obtained by mutating the truncated non-heme iron enzyme Bs QueD 174pt as the parent:

[0012] Mutating the isoleucine at position 71 of the parent into valine to obtain the mutant Bs QueD 174pt -I71V;

[0013] Mutating the isoleucine at position 71 of the parent into leucine to obtain the mutant Bs QueD 174pt -I71L;

[0014] Mutating the serine at position 116 of the parent into arginine to obtain the mutant Bs QueD 174pt -S116R;

[0015] Mutating the serine at position 116 of the parent into lysine to obtain the mutantBs QueD 174pt -S116K;

[0016] The threonine at position 118 of the parent was mutated to histidine to obtain a mutant Bs QueD 174pt -T118H;

[0017] The threonine at position 118 of the parent was mutated to glutamic acid to obtain a mutant Bs QueD 174pt -T118E.

[0018] Furthermore, it is a truncated non-heme iron enzyme Bs QueD 174pt -S116R mutant as the parent, and the mutants obtained by mutation are:

[0019] The isoleucine at position 71 of the parent was mutated to methionine to obtain a mutant Bs QueD 174pt -S116R-I71M;

[0020] The isoleucine at position 71 of the parent was mutated to leucine to obtain a mutant Bs QueD 174pt -S116R-I71L;

[0021] The threonine at position 118 of the parent was mutated to alanine to obtain a mutant Bs QueD 174pt -S116R-T118A.

[0022] Furthermore, it is a truncated non-heme iron enzyme Bs QueD 174pt -S116R-T118A mutant as the parent, and the mutants obtained by mutation are:

[0023] The isoleucine at position 71 of the parent was mutated to leucine to obtain a mutant Bs QueD 174pt -S116R-T118A-I71L.

[0024] Furthermore, it is a truncated non-heme iron enzyme Bs QueD 174pt -S116R-T118A-I71L mutant as the parent, and the mutants obtained by mutation are:

[0025] The leucine at position 51 of the parent was mutated to valine to obtain a mutant Bs QueD 174pt -S116R-T118A-I71L-L51V;

[0026] Among them, the mutant Bs QueD 174pt -S116R-T118A-I71L-L51V has an amino acid sequence as shown in SEQ ID No. 5.

[0027] The present invention also provides a DNA molecule encoding the non-heme iron enzyme mutant, the non-heme iron enzyme truncation, or the non-heme iron enzyme truncation mutant.

[0028] The present invention also provides an expression vector containing the DNA molecule.

[0029] The present invention also provides a host cell containing the expression vector.

[0030] 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, the DNA molecule, the expression vector, and the host cell in the synthesis of chiral primary amine compounds; the chiral primary amine compounds are as shown in Formula I and Formula II:

[0031] ;

[0032] Among them, R 1 group is an aromatic ring and its derivatives, and the structural formula is: Among them, R 1 group is an aromatic ring and its derivatives, and the structural formula is:

[0033] ;

[0034] Among them, R a , R b , R c , R d , R e are each independently selected from hydrogen or any group other than hydrogen; ring A is selected from:

[0035] .

[0036] 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, the non-heme iron enzyme truncation, or the non-heme iron enzyme truncation mutant, the DNA molecule, the expression vector, and the host cell as biocatalysts to synthesize the chiral primary amine compound;

[0037] The general formula of the substrate Formula III is: ;

[0038] The synthesis routes are respectively:

[0039] Reaction Route 1:

[0040] ,

[0041] Reaction route 2:

[0042] ,

[0043] wherein, R 1 group is an aromatic ring and its derivatives, and the structural formula is:

[0044] ,

[0045] wherein, R a , R b , R c , R d , R e are each independently selected from hydrogen or any group other than hydrogen; ring A is selected from: .

[0046] The beneficial effects of the present invention are as follows:

[0047] The present invention provides a non-heme iron enzyme truncation body, which can catalyze the amino functionalization reaction of olefins and synthesize chiral primary amine compounds in one step. This reaction has simple steps and mild conditions, and the target product can be obtained with good yield and selectivity. At the same time, this method has a wider substrate scope. Using this technical solution, sodium azide and sodium cyanate can also be used as anions to obtain the corresponding 2-amino azide and 2-cyano-substituted products. It is worth mentioning that the 2-amino azide product can obtain an important chiral o-diamine product through the Staudinger reaction in one step. This product is a key intermediate for the preparation of the drug levamisole hydrochloride. The 2-cyano-substituted product, 2-aminooxazoline is also a class of compounds with important medicinal value and is widely used in the field of medicinal chemistry. Therefore, the technical solution provided by the present invention has greater potential in industrial applications. Description of the Drawings

[0048] Figure 1 is the gene map of pET-28a- Bs QueD;

[0049] Figure 2 is the standard curve graph of 5-phenyl-4,5-dihydrothiazol-2-amine;

[0050] Figure 3 is Bs the SDS-PAGE gel graph of QueD and the truncated protein;

[0051] Figure 4 is Bs QueD AFChiral detection spectrum of the product 5-phenyl-4,5-dihydrothiazol-2-amine formed by catalyzing styrene reaction under the optimal conditions (where A is the original chiral detection spectrum of the racemate 5-phenyl-4,5-dihydrothiazol-2-amine; B is Bs QueD AF Catalyze the styrene reaction to produce the product S- 5-phenyl-4,5-dihydrothiazol-2-amine: 94% ee of the original chiral detection spectrum);

[0052] Figure 5 is Bs QueD AF Chiral detection spectrum of the product 2-azido-2-phenylethylamine formed by catalyzing styrene reaction under the optimal conditions (where A is the original chiral detection spectrum of the racemate 2-azido-2-phenylethylamine; B is Bs QueD AF Catalyze the styrene reaction to produce the chiral product S- 2-azido-2-phenylethylamine: 94% ee of the original chiral detection spectrum). Detailed implementation mode

[0053] Example 1 Construction of expression strain and investigation of initial reaction activity

[0054] 1. Wild type Bs Construction of QueD expression strain

[0055] For the wild-type non-heme iron enzyme Bacillus subtilis QueD (Genebank Accession: CAB16035.2) derived from Bacillus subtilis, the amino acid sequence is shown in SEQ ID No.1, and codon optimization and gene synthesis were carried out using Escherichia coli as the host cell. Using Bs QueD(Genebank Accession: CAB16035.2), the amino acid sequence is shown in SEQ ID No.1, and codon optimization and gene synthesis were carried out using Escherichia coli as the host cell. Using Nco Ⅰ and Xho Ⅰ restriction enzyme sites to Bs Construct the QueD gene in the pET28a(+) expression vector, and add two nucleotides CG after the ATG start codon in the Nco I restriction enzyme site to avoid frameshift, and obtain the recombinant expression vector pET28a- Bs QueD of wild-type Bs QueD, and the gene map is referred to Figure 1 . Transform the pET28a- Bs QueD recombinant expression vector into Escherichia coli TSR2566 strain (GenScript), and construct Bs QueD expression strain.

[0056] 2. BsProtein Expression of QueD and Preparation of Crude Enzyme Solution

[0057] Inoculate the constructed Bs QueD-expressing strain into a shaking tube containing 3 mL of LB liquid medium containing kanamycin (hereinafter referred to as LB-Kan), and activate it overnight in a shaker at 37 °C and 250 rpm. Inoculate 1 mL of the activated bacterial solution into a conical flask containing 50 mL of TB medium containing kanamycin (hereinafter referred to as TB-Kan), and culture it at 37 °C and 200 rpm for 2.5 h. When the OD 600 of the bacterial solution reaches 0.6 - 0.8, ice-bath the bacterial solution for 30 min, add IPTG with a final concentration of 0.5 mM, and induce expression at 22 °C and 150 rpm for 18 h. After the induction of expression is completed, collect the bacterial cells by centrifugation and discard the supernatant. Resuspend the bacterial cell pellet with 100 mM potassium phosphate (KPi, pH 7.0) buffer, and adjust the OD 600 to 30 to obtain the crude enzyme solution.

[0058] 3. Bs Investigation of the Initial Reaction Activity of QueD

[0059] Using styrene as the substrate, investigate Bs the reaction activity of QueD to catalyze styrene to produce the product S- 5-phenyl-4,5-dihydrothiazol-2-amine (hereinafter collectively referred to as this reaction as Model Reaction 1), S- 2-azido-2-phenylethyl-1-amine (hereinafter collectively referred to as this reaction as Model Reaction 2), and S- 5-phenyl-4,5-dihydrooxazol-2-amine.

[0060] The specific steps are as follows: Add 300 μL of the resuspended crude enzyme solution to a 2 mL glass reaction bottle, transfer it to an anaerobic glove box, and sequentially 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 the nitrogen source, 200 mM aqueous solution), and react with shaking at room temperature in the 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 well, then centrifuge, and pipette 400 μL of the supernatant into a 2 mL injection vial containing an inner cannula for analysis and detection. The concentration of the product is calculated by using LC-MS to detect the product standards with different concentration gradients and containing internal standards, and statistically calculating the ratio of the peak areas of the standards to the internal standard to make a standard curve (hereinafter collectively referred to as the analysis yield). The standard curve is asFigure 2 As shown. Calculate by the product peak area ee value ee The value is the ratio of the peak areas between the products of enantiomers with different configurations ee = ((S,S-R,R) / (S,S + R,R)) * 100%. The initial reaction activity results are shown in Table 1

[0061] Table 1 Bs QueD reaction activity

[0062]

[0063] 4. Bs Investigation of the reaction active center of QueD

[0064] Bs QueD has a dimer structure, and each monomer contains two metal active centers. The Fe in active center 1 (hereinafter referred to as Site 1) 2+ binds to the histidines at positions 62, 64, 103 and the glutamate at position 69 respectively. The Fe in active center 2 (hereinafter referred to as Site 2) 2+ binds to the histidines at positions 234, 236, 275 respectively. This means that the reaction may be catalyzed by either of the two active centers alone or in cooperation

[0065] Further site-silencing experiments were carried out to confirm the active centers that make a substantial contribution to the catalytic reaction. Based on different active centers Site1 and Site2 respectively, histidine was mutated to alanine. The amino acid sequence of wild-type Bs QueD after silencing Site1 is shown in SEQ ID No.2, and the amino acid sequence of wild-type Bs QueD after silencing Site2 is shown in SEQ ID No.3. Protein expression and crude enzyme solution preparation were carried out respectively. Under the conditions of Model Reaction 1, using styrene as the substrate, the reaction activity was measured. The specific reaction results are shown in Table 2

[0066] Table 2 Bs QueD site-silencing reaction activity

[0067]

[0068] N.D., not detected

[0069] 5. Bs Investigation of QueD protein truncation and reaction activity

[0070] According to the site-silencing reaction activity results, the active center of the catalytic reaction is Site 1, and the reaction activity after silencing Site 2 is 1.6 times higher than that of the wild typeee The value increased from 37% to 51%. Therefore, attempts were made to truncate the protein QueD at five sites (V162, K174, S192, Q211, G216). Bs Truncated plasmids at the above six sites were constructed respectively, and protein expression and crude enzyme solution preparation were carried out. Under the conditions of Model Reaction 1, using styrene as the substrate, the reaction activity was measured. The specific reaction results are shown in Table 3. The proteins truncated from the K174 site (hereinafter collectively referred to as QueD Bs QueD 174pt ) all showed enhanced reaction activity, which was better than that of single silent Site 2. The amino acid sequence of QueD Bs QueD 174pt is shown in SEQ ID No. 4. Further, QueD Bs QueD 174pt was selected for directed evolution to construct a mutant library. The SDS-PAGE protein expression results are as Figure 3 shown.

[0071] Table 3. Bs Reaction activity of QueD protein truncation

[0072]

[0073] Example 2 Using the truncated body Bs QueD 174pt as the parent, directed evolution was carried out under the conditions of Model Reaction 1

[0074] 1. Bs QueD 174pt Construction of mutant library

[0075] Using Bs QueD 174pt as the parent, site-saturation mutagenesis was carried out on amino acid residues such as I71, S116, T118, etc. First, using the pET28a- Bs QueD 174pt plasmid as the template, degenerate primers were designed using the 22 codon trick method for PCR reaction. The PCR reaction was carried out using the high-fidelity DNA polymerase KOD One TM PCR Master Mix (TOYOBO). The PCR reaction system is shown in Table 4:

[0076] Table 4. PCR reaction system

[0077]

[0078] The PCR reaction conditions are shown in Table 5:

[0079] Table 5. PCR reaction conditions

[0080]

[0081] The linear DNA product obtained by PCR was subjected to 1% agarose gel electrophoresis. After electrophoresis separation, the gel was cut and recovered. The gel recovery product was measured for nucleic acid concentration using a ultra-micro nucleic acid analyzer, and ligation was carried out according to the DNA seamless cloning kit (ClonExpress® II One Step Cloning Kit, Novoprotein). The ligation system is shown in Table 6 and incubated in a 37 °C environment for 30 min. The ligation product was transformed into TSR2566 Escherichia coli competent cells.

[0082] Table 6. Ligation system of PCR products

[0083]

[0084] 2. Reaction activity screening

[0085] (1) Single colonies on the plate were picked and inoculated into a 96-well plate containing 300 μL of LB-Kan liquid medium and activated overnight in a shaker at 37 °C and 250 rpm. 50 μL of the activated bacterial solution was transferred to a 96-deep well plate containing 950 μL of TB-Kan liquid medium and cultured at 37 °C and 250 rpm for 2.5 h. The 96-well plate was ice-bathed for 30 min, and then 50 μL of IPTG mother liquor (final concentration 0.5 mM) was added to induce expression, and cultured at 22 °C and 200 rpm for 20 h. The cells were collected by low-temperature centrifugation, and 300 μL of KPi (pH 7.0) buffer was added to each well and vortexed to completely resuspend the cells. Transfer to an anaerobic glove box, and sequentially 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 (200 mM aqueous solution), 20 μL of styrene (200 mM ethanol solution), and 20 μL of nitrogen source (200 mM aqueous solution), and react with shaking at room temperature in the anaerobic glove box for 24 h. After the reaction, 900 μL of absolute ethanol was added, shaken well and centrifuged. 200 μL of the supernatant was taken to a 96-well microplate for LC-MS detection.

[0086] The mutants with initially detected activity and ee value improvement in the 96-well plate were subjected to shake flask culture and reaction activity verification. The screening and detection methods for verifying the reaction activity were the same as those in Examples 1-3, and the relative activity of the reaction was measured. eeValue (the relative activity was determined according to the calibration curve of the corresponding peak area measured by LC-MS and the product concentration, relative activity = product concentration of the mutant / product concentration of the parental strain in this round). The verification results of the first round of directed evolution are shown in Table 7.

[0087] Table 7. Verification Results of the First Round of Directed Evolution

[0088]

[0089] (2) Using Bs QueD 174pt -S116R mutant as the parental strain, site-directed saturation mutagenesis was performed on amino acid residues I71 and T118. The construction of the mutant library, protein expression, reaction screening, and detection methods were the same as described above. The verification results of the second round of directed evolution are shown in Table 8.

[0090] Table 8. Verification Results of the Second Round of Directed Evolution

[0091]

[0092] (3) Using Bs QueD 174pt -S116R-T118A mutant as the parental strain, site-directed saturation mutagenesis was performed on amino acid residues L51 and I71. The construction of the mutant library, protein expression, reaction screening, and detection methods were the same as described above. The verification results of the third round of directed evolution are shown in Table 9.

[0093] Table 9. Verification Results of the Third Round of Directed Evolution

[0094]

[0095] (4) Using Bs QueD 174pt -S116R-T118A-I71L mutant as the parental strain, site-directed saturation mutagenesis was performed on amino acid residues L51, Q108, L114, and L126. The construction of the mutant library, protein expression, reaction screening, and detection methods were the same as described above. The verification results of the fourth round of directed evolution are shown in Table 10.

[0096] Table 10. Verification Results of the Fourth Round of Directed Evolution

[0097]

[0098] Under the conditions of Model Reaction 1, using styrene as the substrate, through Bs QueD 174pt 4 rounds of directed evolution were carried out, and the optimal mutant Bs QueD 174pt -S116R-T118A-I71L -L51V (hereinafter referred to as BsQueD AF is: Using Bs QueD 174pt as the parent, serine at position 116 was mutated to arginine, threonine at position 118 was mutated to alanine, isoleucine at position 71 was mutated to leucine, and leucine at position 51 was mutated to valine. The amino acid sequence is as shown in SEQ ID No.5.

[0099] Example 3 Bs QueD AF Optimization of Mutant Reaction Conditions

[0100] Under the conditions of Model Reaction 1, the reaction conditions of Bs QueD AF were optimized to improve the enzyme reaction activity. The optimized conditions include 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, crude enzyme solution preparation, reaction screening, and detection methods are the same as described above. The relative activity and ee value of the reaction were detected, where the relative activity = product concentration under different conditions / product concentration under the initial conditions. The investigation results of optimizing the substrate concentration, iron concentration, buffer type and pH value are shown in Table 11.

[0101] Table 11. Investigation Results of Optimizing the Conditions of Model Reaction 1

[0102]

[0103] Since a substrate concentration of 5 mM, an 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 condition combination was used for subsequent optimization of the reaction conditions. The investigation results of optimizing other conditions are shown in Table 12.

[0104] Table 12. Investigation Results of Optimizing the Conditions of Model Reaction 1

[0105]

[0106] According to the investigation results, the optimized reaction conditions were determined as follows: 5 mM of the model substrate, M9-N (pH 6.0) as the buffer, an iron concentration of 100 μM, sodium dithionite (final concentration of 2 mM) as the reducing agent, acetonitrile (volume ratio of 15%) as the cosolvent, and the equivalent ratio of anion, styrene, and nitrogen source being 3:1:2. The reaction at 8 °C was the optimal reaction condition. Under the optimal reaction conditions, Bs QueD AF catalyzed Model Reaction 1 to obtain an analytical yield of 72% of the corresponding product, ee and the value was 94%. The investigation results before and after condition optimization are shown in Table 13.

[0107] Table 13. Investigation results before and after optimization of the conditions for Model Reaction 1

[0108]

[0109] The chiral separation results of the product are as Figure 4 shown. The chiral detection chromatographic column used was a chiral IG column - Daicel CHIRALPAK (5 μm packing material, 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.

[0110] The best Bs QueD AF mutant of Example 2 was also used in Model Reaction 2.

[0111] Using styrene as the substrate, the Bs QueD AF catalyzed formation of the chiral product 2-azido-2-phenylethylamine was subjected to reaction condition optimization to improve the enzyme reaction activity. The optimized conditions included substrate concentration, iron concentration, buffer type and pH value, co-solvent type, reducing agent type and concentration, equivalent ratio, reaction temperature, co-solvent volume, etc. Protein expression, preparation of crude enzyme solution, reaction screening, and detection methods were the same as described above. The relative activity of the detection reaction, ee value, where relative activity = product concentration under different conditions / product concentration under initial conditions. The investigation results of the optimization of substrate concentration, iron concentration, buffer type and pH value are shown in Table 14.

[0112] Table 14. Investigation results of the optimization of the conditions for Model Reaction 2

[0113]

[0114] Since a substrate concentration of 5 mM, an iron concentration of 100 μM, and M9-N (pH 6.0) as the buffer were all helpful for improving the enzyme reaction activity, the above optimal condition combination was used for the subsequent optimization of reaction conditions. The investigation results of the optimization of other conditions are shown in Table 15.

[0115] Table 15. Investigation results of the optimization of the conditions for Model Reaction 2

[0116]

[0117] According to the investigation results, the optimized reaction conditions were determined as follows: 5 mM of the model substrate, M9-N (pH 6.0) as the buffer, an iron concentration of 100 μM, L-ascorbic acid (final concentration of 0.1 mM) as the reducing agent, acetonitrile (volume ratio of 15%) as the cosolvent, and the equivalent ratio of anion, styrene, and nitrogen source being 3:3:1. The reaction at 8 °C was the optimal reaction condition. Under the optimal reaction conditions, Bs QueD AF the analytical yield of the corresponding product obtained by the catalytic mode reaction 2 was 49%, ee with a value of 94%. The investigation results before and after the reaction optimization are shown in Table 16.

[0118] Table 16. Investigation Results before and after the Optimization of the Conditions for Model Reaction 2

[0119]

[0120] The chiral separation structure of the product is as Figure 5 shown. The chiral detection chromatographic column used was a chiral IG column - Daicel CHIRALPAK (5 μm packing, 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.

[0121] Example 4 Bs QueD AF Investigation of the Substrate Applicability of the Mutant

[0122] Investigation of the Substrate Applicability under Route 1

[0123] Under the optimal reaction conditions, using Bs QueD AF the mutant to conduct the reaction activity investigation on other substrates. The protein expression, preparation of the crude enzyme solution, reaction screening, and detection methods were the same as described above. The investigation results of the substrate applicability are shown in Table 17.

[0124] Table 17. Investigation Results of the Substrate Applicability under Route 1

[0125]

[0126] 2. Investigation of the Substrate Applicability under Route 2

[0127] Under the optimal reaction conditions, using Bs QueD AF the mutant to conduct the reaction activity investigation on other substrates. The protein expression, preparation of the crude enzyme solution, reaction screening, and detection methods were the same as described above. The investigation results of the substrate applicability are shown in Table 18.

[0128] Table 18. Investigation Results of Substrate Applicability of Route 2

[0129]

[0130] Sequence Schedule:

[0131] SEQ ID No.1

[0132] Wild type Bs Amino Acid Sequence of QueD

[0133] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPHSYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDHYHEYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVHSYRLDSHYTKMVGVLVPGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKP

[0134] SEQ ID No.2

[0135] Wild type Bs Amino Acid Sequence after Silencing Site1 of QueD

[0136] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLAVAKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPASYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDHYHEYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVHSYRLDSHYTKMVGVLVPGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKP

[0137] SEQ ID No.3

[0138] Wild type Bs Amino acid sequence after QueD silencing at Site 2

[0139] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPHSYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDAYAEYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVASYRLDSHYTKMVGVLVPGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKP

[0140] SEQ ID No.4

[0141] Bs QueD 174pt : Wild type Bs Amino acid sequence after QueD truncated at position K174

[0142] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDAFPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPHSYRMQSHRTRLVSYTMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSA

[0143] SEQ ID No.5

[0144] Bs QueD AF : Amino acid sequence of the best dominant mutant obtained by directed evolution

[0145] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLVSGGKGDAFPLHVHKDTHEGLLVLDGKLELTLDGERYLLISGDYANIPAGTPHSYRMQSHRTRLVRYAMKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSA.

Claims

1. A truncated non-heme iron enzyme, characterized in that: It is a non-heme iron enzyme mutant that is truncated to obtain the non-heme iron enzyme truncated form BsQueD 174pt , whose amino acid sequence is shown in SEQ ID No.

4.

2. The non-heme iron enzyme truncated mutant according to claim 1, characterized in that: BsQueD 174pt As the parent, the mutant obtained by mutation is: The mutant BsQueD was obtained by mutating the isoleucine at position 71 of the parent to valine. 174pt -I71V; The isoleucine at position 71 of the parent was mutated to leucine to obtain the mutant BsQueD 174pt -I71L; The 116th serine of the parent was mutated to arginine to obtain the mutant BsQueD 174pt -S116R; The 116th serine of the parent was mutated to lysine to obtain the mutant BsQueD 174pt -S116K; The threonine at position 118 of the parent was mutated to histidine to obtain the mutant BsQueD 174pt -T118H; The threonine at position 118 of the parent was mutated to glutamic acid to obtain the mutant BsQueD 174pt -T118E.

3. The non-heme iron enzyme truncated mutant according to claim 2, characterized in that: BsQueD 174pt -S116R mutant is the parent, and the mutants obtained by mutation are: The isoleucine at position 71 of the parent was mutated to methionine to obtain the mutant BsQueD 174pt -S116R-I71M; The isoleucine at position 71 of the parent was mutated to leucine to obtain the mutant BsQueD 174pt -S116R-I71L; The threonine at position 118 of the parent was mutated to alanine to obtain the mutant BsQueD 174pt -S116R-T118A.

4. The non-heme iron enzyme truncated mutant according to claim 3, characterized in that: BsQueD 174pt -S116R-T118A mutant is the parent, and the mutants obtained by mutation are: The isoleucine at position 71 of the parent was mutated to leucine to obtain the mutant BsQueD 174pt -S116R-T118A-I71L.

5. The non-heme iron enzyme truncated mutant according to claim 4, characterized in that: BsQueD 174pt -S116R-T118A-I71L mutant is the parent, and the mutants obtained by mutation are: The leucine at position 51 of the parent was mutated to valine to obtain the mutant BsQueD 174pt -S116R-T118A-I71L-L51V; among them, the mutant BsQueD 174pt The amino acid sequence of -S116R-T118A-I71L-L51V is shown in SEQ ID No.

5.

6. A DNA molecule, characterized in that: The DNA molecule encodes the non-heme iron enzyme truncated form according to claim 1, or the non-heme iron enzyme truncated form mutant according to any one of claims 2-5.

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

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

9. Use of the non-heme iron enzyme truncated form according to claim 1, or the non-heme iron enzyme truncated form mutant according to any one of claims 2 to 5, the DNA molecule according to claim 6, the expression vector according to claim 7, and the host cell according to claim 8 in synthesizing a chiral primary amine compound; the chiral primary amine compound is:

10. A method for synthesizing a chiral primary amine compound, characterized in that: It uses a substrate and the non-heme iron enzyme truncated body described in claim 1 or the non-heme iron enzyme truncated body mutant described in any one of claims 2 to 5 as a biocatalyst to synthesize a chiral primary amine compound; The substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is: Or the substrate is: The chiral primary amine compound is:

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