Non-heme iron enzyme, mutant thereof and application of non-heme iron enzyme in synthesis of chiral primary amine compound

Abstract

The invention relates to an application of a non-heme iron enzyme BsQueD or a mutant thereof in synthesis of a chiral primary amine compound. The chiral primary amine compound is shown as a formula I and a formula II, wherein the amino acid sequence of the non-heme iron enzyme BsQueD is shown as SEQ ID No. 1, and the non-heme iron enzyme BsQueD is shown as SEQ ID No. 2. The invention further provides a mutant of the non-heme iron enzyme BsQueD. The invention also provides a method for synthesizing the chiral primary amine compound. 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. The technical scheme provided by the invention has potential in industrial application.

Description

Technical Field

[0001] The present invention relates to a non-heme iron enzyme BsQueD and its mutants, and their application in the synthesis of chiral primary amine compounds. 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. Asymmetric 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 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 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 conversion 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 (Journal of Fluorine Chemistry, 2005, 126, 297-300). Although the Mitsunobu reaction conditions are relatively mild, a mixture 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 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 is 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 electrocatalyst. In the presence of an additive, at a reaction temperature of 50 - 90 °C, 2-aminothiazole compounds are obtained. Although the above technical solutions achieve the one-step construction of a 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 applied in fields such as chemical engineering, energy, and materials. Based on relevant research on transition metal catalysis, the Frances Arnold research group modified a heat-resistant iron-containing enzyme, cytochrome C (Angewandte Chemie International Edition, 2019, 58, 3138 - 3142), which 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 conversion pathways in traditional chemical synthesis. However, this method only provides primary amination products substituted with ortho-hydroxy groups, and the scope of application of enzyme-catalyzed amino-functionalization of alkenes has not been fully explored. Moreover, this method uses a heme iron enzyme and utilizes its active center heme cofactor, 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 aims to develop an engineered enzyme that can catalyze the amino-functionalization reaction of olefins to 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 two types: 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 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 reactions are achieved by abstracting a hydrogen atom from the substrate and rebound of the hydroxyl radical, or halogenation reactions are achieved by capturing halogen radicals. Compared with heme iron enzymes, non-heme iron-containing enzymes have multiple open iron coordination sites, highly flexible and tunable reactivity, and are expected to be powerful candidates for expanding the diversity of amino-functionalization reactions of olefins.

[0009] The present invention provides the use of a non-heme iron enzyme BsQueD or its mutant in the synthesis of chiral primary amine compounds; the chiral primary amine compounds are shown as Formula I and Formula II:

[0010]

[0011] The amino acid sequence of the non-heme iron enzyme BsQueD is shown as SEQ ID No.1.

[0012] The present invention also provides the mutant of the non-heme iron enzyme BsQueD, which is a mutant obtained by mutating the non-heme iron enzyme BsQueD as the parent:

[0013] The serine at position 116 of the parent is mutated to lysine to obtain the mutant BsQueD-S116K;

[0014] The phenylalanine at position 59 of the parent is mutated to tyrosine to obtain the mutant BsQueD-F59Y;

[0015] The valine at position 123 of the parent is mutated to glutamate to obtain the mutant BsQueD-V123E;

[0016] The valine at position 123 of the parent is mutated to proline to obtain the mutant BsQueD-V123P;

[0017] The valine at position 123 of the parent is mutated to glycine to obtain the mutant BsQueD-V123G;

[0018] The isoleucine at position 71 of the parent was mutated to leucine to obtain the mutant BsQueD-I71L;

[0019] The isoleucine at position 71 of the parent was mutated to valine to obtain the mutant BsQueD-I71V.

[0020] Furthermore, it is a mutant obtained by mutating the non-heme iron enzyme mutant BsQueD-S116K as the parent:

[0021] The isoleucine at position 71 of the parent was mutated to leucine to obtain the mutant BsQueD-S116K-I71L;

[0022] The threonine at position 67 of the parent was mutated to leucine to obtain the mutant BsQueD-S116K-T67L;

[0023] The threonine at position 118 of the parent was mutated to glycine to obtain the mutant BsQueD-S116K-T118G;

[0024] The threonine at position 118 of the parent was mutated to serine to obtain the mutant BsQueD-S116K-T118S;

[0025] The valine at position 123 of the parent was mutated to glutamic acid to obtain the mutant BsQueD-S116K-V123E;

[0026] The valine at position 123 of the parent was mutated to lysine to obtain the mutant BsQueD-S116K-V123K;

[0027] The valine at position 158 of the parent was mutated to aspartic acid to obtain the mutant

[0028] BsQueD-S116K-V158D;

[0029] The valine at position 158 of the parent was mutated to asparagine to obtain the mutant

[0030] BsQueD-S116K-V158N;

[0031] The valine at position 158 of the parent was mutated to threonine to obtain the mutant BsQueD-S116K-V158T.

[0032] Furthermore, it is a mutant obtained by mutating the non-heme iron enzyme mutant BsQueD-S116K-T118S as the parent:

[0033] The leucine at position 51 of the parent was mutated to isoleucine to obtain the mutant

[0034] BsQueD-S116K-T118S-L51I;

[0035] The valine at position 123 of the parent was mutated to asparagine to obtain a mutant

[0036] BsQueD-S116K-T118S-V123N;

[0037] The valine at position 123 of the parent was mutated to aspartic acid to obtain a mutant

[0038] BsQueD-S116K-T118S-V123D;

[0039] The valine at position 158 of the parent was mutated to aspartic acid to obtain a mutant

[0040] BsQueD-S116K-T118S-V158D.

[0041] Furthermore, it is a mutant obtained by mutating the non-heme iron enzyme mutant BsQueD-S116K-T118S-L51I as the parent:

[0042] The isoleucine at position 71 of the parent was mutated to leucine to obtain a mutant

[0043] BsQueD-S116K-T118S-L51I-I71L;

[0044] The valine at position 158 of the parent was mutated to alanine to obtain a mutant

[0045] BsQueD-S116K-T118S-L51I-V158A;

[0046] The valine at position 158 of the parent was mutated to glycine to obtain a mutant

[0047] BsQueD-S116K-T118S-L51I-V158G.

[0048] Furthermore, it is a mutant obtained by mutating the non-heme iron enzyme mutant BsQueD-S116K-T118S-L51I-I71L as the parent:

[0049] The proline at position 102 of the parent was mutated to threonine to obtain a mutant

[0050] BsQueD-S116K-T118S-L51I-I71L-P102T;

[0051] The proline at position 102 of the parent was mutated to lysine to obtain a mutant

[0052] BsQueD-S116K-T118S-L51I-I71L-P102K;

[0053] The proline at position 102 of the parent was mutated to serine to obtain a mutant

[0054] BsQueD-S116K-T118S-L51I-I71L-P102S;

[0055] The leucine at position 126 of the parent was mutated to asparagine to obtain a mutant

[0056] BsQueD-S116K-T118S-L51I-I71L-L126N;

[0057] The leucine at position 126 of the parent was mutated to proline to obtain a mutant

[0058] BsQueD-S116K-T118S-L51I-I71L-L126P;

[0059] The leucine at position 126 of the parent was mutated to tyrosine to obtain a mutant

[0060] BsQueD-S116K-T118S-L51I-I71L-L126Y;

[0061] The valine at position 158 of the parent was mutated to asparagine to obtain a mutant

[0062] BsQueD-S116K-T118S-L51I-I71L-V158N;

[0063] The valine at position 158 of the parent was mutated to aspartic acid to obtain a mutant

[0064] BsQueD-S116K-T118S-L51I-I71L-V158D;

[0065] The alanine at position 159 of the parent was mutated to glutamic acid to obtain a mutant

[0066] BsQueD-S116K-T118S-L51I-I71L-A159E;

[0067] The alanine at position 159 of the parent was mutated to histidine to obtain a mutant

[0068] BsQueD-S116K-T118S-L51I-I71L-A159H.

[0069] Furthermore, it is a non-heme iron enzyme mutant

[0070] BsQueD-S116K-T118S-L51I-I71L-P102T is used as the parent, and mutants are obtained by mutation:

[0071] Leucine at position 126 of the parent is mutated to asparagine to obtain a mutant

[0072] BsQueD-S116K-T118S-L51I-I71L-P102T-L126N;

[0073] Leucine at position 126 of the parent is mutated to serine to obtain a mutant

[0074] BsQueD-S116K-T118S-L51I-I71L-P102T-L126S;

[0075] Tyrosine at position 127 of the parent is mutated to alanine to obtain a mutant

[0076] BsQueD-S116K-T118S-L51I-I71L-P102T-Y127A;

[0077] Valine at position 158 of the parent is mutated to aspartic acid to obtain a mutant

[0078] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D;

[0079] Alanine at position 159 of the parent is mutated to asparagine to obtain a mutant

[0080] BsQueD-S116K-T118S-L51I-I71L-P102T-A159N;

[0081] Alanine at position 159 of the parent is mutated to aspartic acid to obtain a mutant

[0082] BsQueD-S116K-T118S-L51I-I71L-P102T-A159D.

[0083] Furthermore, it is a mutant obtained by mutating the non-heme iron enzyme mutant

[0084] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D is used as the parent, and mutants are obtained by mutation:

[0085] Leucine at position 126 of the parent is mutated to asparagine to obtain a mutant

[0086] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N;

[0087] The leucine at position 126 of the parent was mutated to proline to obtain a mutant

[0088] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126P;

[0089] The leucine at position 126 of the parent was mutated to glycine to obtain a mutant

[0090] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126G;

[0091] The tyrosine at position 127 of the parent was mutated to serine to obtain a mutant

[0092] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-Y127S;

[0093] The tyrosine at position 127 of the parent was mutated to glycine to obtain a mutant

[0094] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-Y127G.

[0095] It is a mutant obtained by mutating the non-heme iron enzyme mutant

[0096] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N as the parent:

[0097] The valine at position 49 of the parent was mutated to methionine to obtain a mutant

[0098] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-V49M;

[0099] The tyrosine at position 127 of the parent was mutated to aspartic acid to obtain a mutant

[0100] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-Y127D;

[0101] The tyrosine at position 127 of the parent was mutated to threonine to obtain a mutant

[0102] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-Y127T;

[0103] The alanine at position 157 of the parent was mutated to leucine to obtain a mutant

[0104] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-A157L;

[0105] The phenylalanine at position 295 of the parent was mutated to proline to obtain a mutant

[0106] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-F295P;

[0107] Among them, the amino acid sequence of BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-V49M is shown in SEQ ID No.4.

[0108] The present invention also provides a non-heme iron enzyme BsQueD mutant, which is obtained by mutating the histidines at positions 234, 236 and 275 of the non-heme iron enzyme BsQueD mutant to alanine.

[0109] The present invention also provides a DNA molecule, and the DNA molecule encodes the non-heme iron enzyme BsQueD mutant.

[0110] The present invention also provides an expression vector, and the expression vector contains the DNA molecule.

[0111] The present invention also provides a host cell, and the host cell contains the expression vector.

[0112] The present invention also provides a method for synthesizing a chiral primary amine compound, which uses styrene as a substrate and the non-heme iron enzyme BsQueD or mutant, DNA molecule, expression vector, host cell as a biocatalyst to synthesize a chiral primary amine compound;

[0113] The synthesis routes are respectively:

[0114] Reaction route 1:

[0115]

[0116] Reaction route 2:

[0117]

[0118] The present invention provides a non-heme iron-containing enzyme and its mutants, which can catalyze the amino-functionalization reaction of olefins to synthesize chiral primary amine compounds in one step. This reaction has simple steps and mild conditions, and can obtain the target product with good yield and selectivity.

[0119] Meanwhile, 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-aminoazide and 2-cyano-substituted products. It is worth mentioning that the 2-aminoazide product can be used to obtain an important chiral ortho-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

[0120] Figure 1 Gene map of pET-28a-BsQueD;

[0121] Figure 2 SDS-PAGE gel map of BsQueD;

[0122] Figure 3 Standard curve of 5-phenyl-4,5-dihydrothiazol-2-amine;

[0123] Figure 4 S-BsQueD KM Chiral 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 racemic 5-phenyl-4,5-dihydrothiazol-2-amine; B is the original chiral detection spectrum of S-BsQueD KM catalyzing styrene reaction to form the product S-5-phenyl-4,5-dihydrothiazol-2-amine: the original chiral detection spectrum with 96% ee). Detailed Embodiments

[0124] Example 1 Construction of the expression strain and investigation of the initial reaction activity

[0125] 1. Construction of the wild-type BsQueD expression strain

[0126] For the wild-type non-heme iron enzyme BsQueD (Genebank Accession: CAB16035.2) derived from Bacillus subtilis, the amino acid sequence is shown in SEQ ID No. 1. Codon optimization and gene synthesis were performed using Escherichia coli as the host cell. The BsQueD gene was constructed into the pET28a(+) expression vector using NcoⅠ and XhoⅠ restriction sites, and two nucleotides CG were added after the ATG start codon in the Nco I restriction site to avoid frameshift, obtaining the recombinant expression vector pET28a-BsQueD of wild-type BsQueD. The gene map is referred to Figure 1 . The recombinant expression vector pET28a-BsQueD was transformed into Escherichia coli strain TSR2566 (Tsingke Biological), and the BsQueD expression strain was constructed. The SDS-PAGE protein expression is shown in Figure 2 .

[0127] 2. Protein expression of BsQueD and preparation of crude enzyme solution

[0128] The constructed BsQueD 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 cultured at 37 °C and 200 rpm for 2.5 h. When the OD of the bacterial solution 600 reached 0.6 - 0.8, the bacterial solution was ice-bathed for 30 min, and IPTG with a final concentration of 0.5 mM was added, and induced expression was carried out at 22 °C and 150 rpm for 18 h. After the induction expression was completed, the cells were collected by centrifugation, and the supernatant was discarded. The cell pellet was resuspended with 100 mM potassium phosphate (KPi, pH 7.0) buffer, and the OD 600 was adjusted to 30 to obtain the crude enzyme solution.

[0129] 3. Investigation of the initial reaction activity of BsQueD

[0130] Using styrene as the substrate, the initial reaction activities of BsQueD-catalyzed styrene reactions to produce products 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, and S-5-phenyl-4,5-dihydrooxazol-2-amine were investigated. The specific steps were as follows: 300 μL of the resuspended crude enzyme solution was added to a 2 mL glass reaction flask, transferred to an anaerobic glove box, and 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) were added in sequence. The reaction was shaken at room temperature in the anaerobic glove box for 24 h. After the reaction was completed, 900 μL of ethanol internal standard solution (containing 0.25 mM 1,3,5-trimethoxybenzene) was added, shaken and mixed evenly, and then centrifuged. 400 μL of the supernatant was taken and added to a 2 mL injection vial containing an inner cannula for analysis and detection. The concentration of the product was calculated by using LC-MS to detect the product standard with different concentration gradients and containing an internal standard, and making a standard curve by statistically analyzing the ratio of the peak areas of the standard and the internal standard (hereinafter collectively referred to as the analytical yield). The standard curve is as Figure 3 shown. The ee value was calculated from the product peak area. The ee value is the ratio of the peak areas between the enantiomeric products of different configurations, ee = ((S,S-R,R) / (S,S+R,R))*100%. The results of the initial reaction activities are shown in Table 1.

[0131] Table 1. Initial reaction activities of BsQueD

[0132]

[0133] Example 2 Using wild-type BsQueD as the parent, directed evolution was carried out under the conditions of model reaction 1

[0134] 1. Construction of the BsQueD mutant library

[0135] Using BsQueD as the parent, site-saturation mutagenesis was performed on amino acid residues such as F59, I71, I97, S116, T118, V123, and Y127. First, using the pET28a-BsQueD plasmid as a template, degenerate primers were designed by 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 2:

[0136] Table 2. PCR reaction system

[0137]

[0138] The PCR reaction conditions are shown in Table 3:

[0139] Table 3. PCR reaction conditions

[0140]

[0141]

[0142] The linear DNA products obtained by PCR were 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 ligated according to the DNA seamless cloning kit ( IIOne Step Cloning Kit, Novoprotein), and the ligation system is shown in Table 4. It was incubated at 37 °C for 30 min, and the ligation product was transformed into TSR2566 Escherichia coli competent cells.

[0143] Table 4. Ligation system of PCR products

[0144]

[0145] 2. Reaction activity screening

[0146] (1) Pick a single colony on the plate and inoculate it into a 96-well plate containing 300 μL of LB-Kan liquid medium, and activate it overnight in a shaker at 37 °C and 250 rpm. Transfer 50 μL of the activated bacterial solution to a 96-deep well plate containing 950 μL of TB-Kan liquid medium, and culture it at 37 °C and 250 rpm for 2.5 h. Ice-bath the 96-well plate for 30 min, then add 50 μL of IPTG mother liquor (final concentration 0.5 mM) to induce expression, and culture it at 22 °C and 200 rpm for 20 h. Centrifuge at low temperature to collect the bacterial cells, add 300 μL of KPi (pH 7.0) buffer to each well, and vortex 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 is completed, add 900 μL of absolute ethanol, mix well by shaking and then centrifuge. Take 200 μL of the supernatant to a 96-well enzyme-linked immunosorbent assay plate for LC-MS detection.

[0147] The mutants that were initially detected to have improved activity and ee value in the 96-well plate were cultured in shake flasks and their reaction activities were verified. The screening and detection methods for verifying the reaction activities were the same as those in Examples 1-3. The relative activity and ee value of the reaction were measured (the relative activity was determined according to the standard 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 5.

[0148] Table 5. Verification Results of the First Round of Directed Evolution

[0149]

[0150] (2) Using the BsQueD-S116K mutant as the parental strain, site-directed saturation mutagenesis was performed on amino acid residues such as T67, I71, T118, V123, and V158. 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 6.

[0151] Table 6. Verification Results of the Second Round of Directed Evolution

[0152]

[0153]

[0154] (3) Using the BsQueD-S116K-T118S mutant as the parental strain, site-directed saturation mutagenesis was performed on amino acid residues such as L51, V123, Y127, and V158. 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 7.

[0155] Table 7. Verification Results of the Third Round of Directed Evolution

[0156]

[0157] (4) Using the BsQueD-S116K-T118S-L51I mutant as the parental strain, site-directed saturation mutagenesis was performed on amino acid residues I71 and V158. 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 8.

[0158] Table 8. Verification Results of the Fourth Round of Directed Evolution

[0159]

[0160] (5) Using the BsQueD-S116K-T118S-L51I-I71L mutant as the parent, site-directed saturation mutagenesis was performed on amino acid residues such as P102, L126, L114, V158, and A159. The construction of the mutant library, protein expression, reaction screening, and detection methods were the same as described above. The verification results of the fifth round of directed evolution are shown in Table 9.

[0161] Table 9 Verification Results of the Fifth Round of Directed Evolution

[0162]

[0163]

[0164] (6) Using the BsQueD-S116K-T118S-L51I-I71L-P102T mutant as the parent, site-directed saturation mutagenesis was performed on amino acid residues such as Q108, L126, Y127, V158, and A159. The construction of the mutant library, protein expression, reaction screening, and detection methods were the same as described above. The verification results of the sixth round of directed evolution are shown in Table 10.

[0165] Table 10. Verification Results of the Sixth Round of Directed Evolution

[0166]

[0167] (7) Using the BsQueD-S116K-T118S-L51I-I71L-P102T-V158D mutant as the parent, site-directed saturation mutagenesis was performed on amino acid residues such as I71, S116, T118, L126, and Y127. The construction of the mutant library, protein expression, reaction screening, and detection methods were the same as described above. The verification results of the seventh round of directed evolution are shown in Table 11.

[0168] Table 11. Verification Results of the Seventh Round of Directed Evolution

[0169]

[0170] (8) Using the BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N mutant as the parent, site-directed saturation mutagenesis was performed on amino acid residues such as V49, Y127, A157, F295, etc. The construction of the mutant library, protein expression, reaction screening, and detection methods were the same as described above. The verification results of the eighth round of directed evolution are shown in Table 12.

[0171] Table 12. Verification Results of the Eighth Round of Directed Evolution

[0172]

[0173]

[0174] Under the conditions of Model Reaction 1, wild-type BsQueD was subjected to eight rounds of directed evolution, and the optimal mutant obtained by screening

[0175] BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-V49M (hereinafter collectively referred to as BsQueD KM ): It uses BsQueD as the parent, mutates serine at position 116 to lysine, threonine at position 118 to serine, leucine at position 51 to isoleucine, isoleucine at position 71 to leucine, proline at position 102 to threonine, valine at position 158 to aspartic acid, leucine at position 126 to asparagine, and valine at position 49 to methionine. The amino acid sequence is shown in SEQ ID No. 4.

[0176] Investigation and comparison of the reactivity in the site-silencing experiment of Example 3

[0177] 1. Investigation of the reactivity of BsQueD site-silencing

[0178] BsQueD has a dimer structure, and each monomer contains two metal active centers. The Fe in Active Center 1 (hereinafter briefly referred to as Site 1) 2+ binds to histidines at positions 62, 64, and 103 and glutamate at position 69 respectively. The Fe in Active Center 2 (hereinafter briefly referred to as Site 2) 2+ binds to histidines at positions 234, 236, and 275 and glutamate at position 241 respectively. This means that the catalysis of the reaction may be carried out independently or synergistically by the two active centers.

[0179] 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 Site 1 and Site 2 respectively, histidine was mutated to alanine. The amino acid sequence of wild-type BsQueD after silencing Site 1 is shown in SEQ ID No. 2, and the amino acid sequence of wild-type BsQueD after silencing Site 2 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 13.

[0180] Table 13. Reactivity of BsQueD site-silencing

[0181]

[0182] Note: N.D., not detected.

[0183] 2. Comparison of the reactivity after site-silencing of the dominant mutant sites

[0184] According to the results of silencing the wild-type BsQueD locus, it was found that after silencing Site 2, the enzyme reaction activity increased. Therefore, Site 2 was silenced in the dominant mutants obtained in each round of the eight-round directed evolution process, that is, the histidines at positions 234, 236, and 275 in Site 2 were mutated to alanines, plasmids for silencing Site 2 of the dominant mutants were constructed, protein expression was carried out, and crude enzyme solutions were prepared. Under the conditions of Model Reaction 1, the reaction activity was measured, and the specific reaction results are shown in Table 14.

[0185] Table 14. Comparison of reaction activities of dominant mutant site silencing

[0186]

[0187] From the reaction results, it can be seen that after silencing Site 2 in the eight dominant mutants, the enzyme reaction activities were further improved, and the best dominant mutant (BsQueD KM ) had the best reaction activity after silencing Site 2, that is, S-BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-V49M (hereinafter collectively referred to as S-BsQueD KM ), and its amino acid sequence is shown in SEQ ID No. 5. Therefore, S-BsQueD KM was selected to further optimize the reaction conditions of Model Reaction 1.

[0188] Example 4 Optimization of reaction conditions of S-BsQueD KM Optimization of mutant reaction conditions

[0189] 1. Optimization of the conditions of Model Reaction 1

[0190] Select S-BsQueD KM , under the conditions of Model Reaction 1, using styrene as the substrate, the reaction conditions were optimized to improve the enzyme reaction activity. The optimized conditions included the type of cosolvent, equivalent ratio, iron concentration, OD 600 value, type of buffer solution and pH value, reaction temperature, etc. Protein expression, preparation of crude enzyme solution, reaction screening, and detection methods were 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 the reaction condition optimization are shown in Tables 15 - 16.

[0191] Table 15. Investigation results of the optimization of the conditions of Model Reaction 1

[0192]

[0193] Table 16. Investigation results of the optimization of the conditions of Model Reaction 1

[0194]

[0195] According to the investigation results, when the cosolvent is isopropyl alcohol, the equivalent ratio is 2:3:1, and the reaction temperature is 8 °C, the enzyme reaction activity is improved. The optimized reaction conditions are determined as follows: KPi (pH 7.0) is used as the buffer solution, the iron concentration is 10 μM, isopropyl alcohol (5%) is used as the cosolvent, and the equivalent ratio of styrene, anion, and nitrogen source is 2:3:1. The OD of the bacterial solution 600 is 30, and the reaction at 8 °C is the optimal reaction condition. Under the optimal reaction conditions, the analytical yield of the corresponding product obtained by the catalytic mode reaction 1 of S-BsQueD KM is 41%, and the ee value is 96%. The investigation results before and after the condition optimization are shown in Table 17.

[0196] Table 17. Investigation results before and after the optimization of the reaction conditions of model reaction 1

[0197]

[0198] The chiral separation structure of the product is as Figure 4 shown. The chiral detection chromatographic column uses a chiral IG column - Daicel CHIRALPAK (5 μm packing, 4.6×250 mm), the mobile phase ratio is 60% acetonitrile / 40% water (containing 20 mM ammonium acetate), and the flow rate is 1 mL / min.

[0199] Figure 4 for S-BsQueD KM The chiral detection chromatogram of the product 5-phenyl-4,5-dihydrothiazol-2-amine formed by the catalytic reaction of styrene under the optimal conditions (where A is the original chiral detection chromatogram of the racemic 5-phenyl-4,5-dihydrothiazol-2-amine; B is the original chiral detection chromatogram of the product S-5-phenyl-4,5-dihydrothiazol-2-amine with 96% ee formed by the catalytic reaction of styrene by S-BsQueD KM ).

[0200] Example 5 S-BsQueD KM Investigation on the substrate applicability of mutants

[0201] Under the optimal reaction conditions described in Specific Example 4, using styrene as the substrate, the substrate applicability of the S-BsQueD KM mutant was further investigated. The protein expression, preparation of crude enzyme solution, reaction screening, and detection methods were the same as those described above. The investigation results of the substrate applicability are shown in Table 18.

[0202] Table 18. Investigation results of the substrate applicability

[0203]

[0204] SEQ ID No.1

[0205] Wild-type BsQueD amino acid sequence

[0206] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDA

[0207] FPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPHSYRMQSHRTRLVSYT

[0208] MKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAE

[0209] LTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDHYH

[0210] EYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVHSYRLDSHYTKMVGVLVPG

[0211] LFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKPSEQ ID No.2

[0212] Amino acid sequence of wild-type BsQueD after silencing Site1

[0213] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDA

[0214] FPLAVAKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPASYRMQSHRTRLVSYT

[0215] MKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAE

[0216] LTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDHYH

[0217] EYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVHSYRLDSHYTKMVGVLVPG

[0218] LFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKPSEQ ID No.3

[0219] Amino acid sequence after silencing Site2 of wild-type BsQueD

[0220] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIVLLSGGKGDA

[0221] FPLHVHKDTHEGILVLDGKLELTLDGERYLLISGDYANIPAGTPHSYRMQSHRTRLVSYT

[0222] MKGNVAHLYSVIGNPYDHAEHPPYASEEVSNERFAEAAAVADIVFLDEAKPACSAKLAE

[0223] LTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDAYA

[0224] EYHTETFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVASYRLDSHYTKMVGVLVPG

[0225] LFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKPSEQ ID No.4

[0226] Optimal advantageous mutant BsQueD of directed evolution KM Amino acid sequence of

[0227] MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIMLISGGKGDA

[0228] FPLHVHKDTHEGLLVLDGKLELTLDGERYLLISGDYANIPAGTTHSYRMQSHRTRLVKY

[0229] SMKGNVAHNYSVIGNPYDHAEHPPYASEEVSNERFAEAAADADIVFLDEAKPACSAKL

[0230] AELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDH

[0231] YHEYHTATFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVHSYRLDSHYTKMVGVLV

[0232] PGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKPSEQ ID No.5

[0233] S-BsQueD KM : Optimal dominant mutant (BsQueD KM ) Amino acid sequence after silencing Site 2: MKTLCTHSLPKEKMPYLLRSGEGERYLFGRQVATVMANGRSTGDLFEIMLISGGKGDAFPLHVHKDTHEGLLVLDGKLELTLDGERYLLISGDYANIPAGTTHSYRMQSHRTRLVKYSMKGNVAHNYSVIGNPYDHAEHPPYASEEVSNERFAEAAADADIVFLDEAKPACSAKLAELTELPDGAVPYVLESGEGDRLLTGDQLHRIVAAQKNTDGQFIVVSSEGPKGDRIVDAYAEYHTATFYCLEGQMTMWTDGQEIQLNPGDFLHVPANTVASYRLDSHYTKMVGVLVPGLFEPFFRTLGDPYEGHIFPCEPQALRFDRILQNIEALDLKVMKP

Claims

1. Use of a non-heme iron enzyme BsQueD or a mutant thereof in the synthesis of a chiral primary amine compound; the chiral primary amine compound is as shown in Formula I and Formula II: The amino acid sequence of the non-heme iron enzyme BsQueD is shown in SEQ ID No.

1.

2. The non-heme iron enzyme BsQueD mutant according to claim 1, characterized in that: It is a mutant obtained by mutation of the non-heme iron enzyme BsQueD as a parent: The serine at position 116 of the parent was mutated to lysine to obtain the mutant BsQueD-S116K; The phenylalanine at position 59 of the parent was mutated to tyrosine to obtain the mutant BsQueD-F59Y; The valine at position 123 of the parent was mutated to glutamic acid to obtain the mutant BsQueD-V123E; The valine at position 123 of the parent was mutated to proline to obtain the mutant BsQueD-V123P; The valine at position 123 of the parent was mutated to glycine to obtain the mutant BsQueD-V123G; The isoleucine at position 71 of the parent was mutated to leucine to obtain the mutant BsQueD-I71L; The isoleucine at position 71 of the parent was mutated to valine to obtain the mutant BsQueD-I71V; Preferably, it is a mutant obtained by taking the non-heme iron enzyme mutant BsQueD-S116K as a parent and causing mutation: The isoleucine at position 71 of the parent was mutated to leucine to obtain the mutant BsQueD-S116K-I71L; The threonine at position 67 of the parent was mutated to leucine to obtain the mutant BsQueD-S116K-T67L; The threonine at position 118 of the parent was mutated to glycine to obtain the mutant BsQueD-S116K-T118G; The threonine at position 118 of the parent was mutated to serine to obtain the mutant BsQueD-S116K-T118S; The valine at position 123 of the parent was mutated to glutamic acid to obtain the mutant BsQueD-S116K-V123E; The valine at position 123 of the parent was mutated to lysine to obtain the mutant BsQueD-S116K-V123K; The valine at position 158 of the parent was mutated to aspartic acid to obtain the mutant BsQueD-S116K-V158D; The valine at position 158 of the parent was mutated to asparagine to obtain the mutant BsQueD-S116K-V158N; The valine at position 158 of the parent was mutated to threonine to obtain the mutant BsQueD-S116K-V158T; More preferably, it is a mutant obtained by taking the non-heme iron enzyme mutant BsQueD-S116K-T118S as a parent and causing mutation: The leucine at position 51 of the parent was mutated to isoleucine to obtain the mutant BsQueD-S116K-T118S-L51I; The valine at position 123 of the parent was mutated to asparagine to obtain the mutant BsQueD-S116K-T118S-V123N; The valine at position 123 of the parent was mutated to aspartic acid to obtain the mutant BsQueD-S116K-T118S-V123D; The valine at position 158 of the parent was mutated to aspartic acid to obtain the mutant BsQueD-S116K-T118S-V158D; More preferably, it is a non-heme iron enzyme mutant BsQueD-S116K-T118S-L51I 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-S116K-T118S-L51I-I71L; The valine at position 158 of the parent was mutated to alanine to obtain the mutant BsQueD-S116K-T118S-L51I-V158A; The valine at position 158 of the parent was mutated to glycine to obtain the mutant BsQueD-S116K-T118S-L51I-V158G.

3. The non-heme iron enzyme BsQueD mutant according to claim 2, characterized in that: It is a mutant obtained by taking the non-heme iron enzyme mutant BsQueD-S116K-T118S-L51I-I71L as the parent: The proline at position 102 of the parent was mutated to threonine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T; The proline at position 102 of the parent was mutated to lysine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102K; The proline at position 102 of the parent was mutated to serine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102S; The leucine at position 126 of the parent was mutated to asparagine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-L126N; The leucine at position 126 of the parent was mutated to proline to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-L126P; The leucine at position 126 of the parent was mutated to tyrosine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-L126Y; The valine at position 158 of the parent was mutated to asparagine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-V158N; The valine at position 158 of the parent was mutated to aspartic acid to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-V158D; The alanine at position 159 of the parent was mutated to glutamic acid to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-A159E; The alanine at position 159 of the parent was mutated to histidine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-A159H.

4. The non-heme iron enzyme BsQueD mutant according to claim 3, characterized in that: It is a mutant obtained by taking the non-heme iron enzyme mutant BsQueD-S116K-T118S-L51I-I71L-P102T as the parent: The leucine at position 126 of the parent was mutated to asparagine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-L126N; The leucine at position 126 of the parent was mutated to serine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-L126S; The tyrosine at position 127 of the parent was mutated to alanine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-Y127A; The valine at position 158 of the parent was mutated to aspartic acid to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D; The alanine at position 159 of the parent was mutated to asparagine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-A159N; The alanine at position 159 of the parent was mutated to aspartic acid to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-A159D.

5. The non-heme iron enzyme BsQueD mutant according to claim 4, characterized in that: It is a mutant obtained by taking the non-heme iron enzyme mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D as a parent: The leucine at position 126 of the parent was mutated to asparagine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N; The leucine at position 126 of the parent was mutated to proline to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126P; The leucine at position 126 of the parent was mutated to glycine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126G; The tyrosine at position 127 of the parent was mutated to serine, resulting in the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-Y127S; The tyrosine at position 127 of the parent was mutated to glycine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-Y127G; Preferably, it is a non-heme iron enzyme mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N is the parent, and the mutants obtained by mutation are: The valine at position 49 of the parent was mutated to methionine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-V49M; The tyrosine at position 127 of the parent was mutated to aspartic acid to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-Y127D; The tyrosine at position 127 of the parent was mutated to threonine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-Y127T; The alanine at position 157 of the parent was mutated to leucine to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-A157L; The phenylalanine at position 295 of the parent was mutated to proline to obtain the mutant BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-F295P; Among them, the amino acid sequence of BsQueD-S116K-T118S-L51I-I71L-P102T-V158D-L126N-V49M is shown in SEQ ID No.

4.

6. A non-heme iron enzyme BsQueD mutant, characterized in that: It is obtained by mutating the histidine at positions 234, 236 and 275 to alanine in the non-heme iron enzyme BsQueD mutant described in any one of claims 1 to 5.

7. A DNA molecule, characterized in that: The DNA molecule encodes the non-heme iron enzyme BsQueD mutant according to any one of claims 2-5.

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. A method for synthesizing a chiral primary amine compound, characterized in that: It uses styrene as a substrate, the non-heme iron enzyme BsQueD according to claim 1 or the mutants according to claims 2-6, 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 synthetic routes are: Reaction Scheme 1: Reaction Scheme 2:

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