Synthesis method and application of amino alcohol compound catalyzed by benzaldehyde lyase mutant

Abstract

The invention discloses a benzaldehyde lyase derived from Pseudomonas fluorescens, a mutant of the benzaldehyde lyase, and an application of the benzaldehyde lyase and the mutant of the benzaldehyde lyase as a catalyst in catalysis of chiral amino alcohol. The invention further discloses a preparation method of the benzaldehyde lyase and the mutant of the benzaldehyde lyase. The benzaldehyde lyase disclosed by the invention has the advantages of high reactivity, high stereoselectivity, wide substrate range and the like, the yield is as high as 99%, the ee value is as high as 99%, low-cost production is favorably realized, and the benzaldehyde lyase has an industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical fields of enzyme engineering and chemical engineering, and particularly relates to a benzaldehyde lyase mutant, and more particularly to a method for synthesizing amino alcohol compounds catalyzed by a benzaldehyde lyase mutant and its application. Background Art

[0002] Benzaldehyde Lyase (BAL) is a thiamine diphosphate (ThDP)-dependent enzyme, initially discovered from Pseudomonas fluorescens. It is a multifunctional enzyme that can catalyze C-C bond formation and cleavage reactions between aldehyde substrates, etc. Benzaldehyde lyase is a multifunctional enzyme with broad industrial and scientific research application prospects. Through enzyme engineering and synthetic biology means, its catalytic performance and application scope have been continuously expanded, providing new tools for green chemistry and chiral compound synthesis.

[0003] The amino alcohol group is an important part of pharmaceuticals and chemicals, widely existing in natural products, drugs, and active molecules. Amino alcohols are not only important structural units of pharmacologically active molecules, but also have extensive applications in the fields of synthetic chemistry, drug discovery, and chiral catalysts. The carbonyl-hydroxy-amino unit is commonly present in many natural products, pharmaceuticals, pesticides, and other fine chemicals. For example, Paclitaxel isolated from Taxus brevifolia is an anti-cancer drug that can promote tubulin polymerization. Bestatin is an aminopeptidase inhibitor with immunomodulatory activity and is used clinically as an adjuvant inhibitor for cancer chemotherapy; the lactone AI-77-B isolated from the culture broth of Bacillus sp. is a molecule with a unique structure and has gastroprotective activity. Blebbistatin is a myosin inhibitor that mainly targets myosin II and is widely used in research to inhibit cardiac myosin, non-muscle myosin II, and skeletal muscle myosin. IRAK4 inhibitor (a kinase involved in innate immune signal transduction) can block the production of pro-inflammatory cytokines in mice and can be used for the treatment of rheumatism. The amino alcohol unit is an important building block for preparing various drugs and bioactive molecules and may also be a potential anti-tumor drug. However, currently, its synthesis methods are limited and mainly rely on traditional chemical synthesis methods, which have problems such as expensive catalysts, complex processes, and difficult separation and purification. Therefore, it is necessary to explore novel benzaldehyde lyases with better performance and develop an economical, efficient, and environmentally friendly biocatalytic synthesis process. Summary of the Invention

[0004] The first aspect of the present invention is to provide a method for catalytically synthesizing a compound of formula I by a benzaldehyde lyase or its mutant, and the reaction formula is as follows:

[0005]

[0006] The benzaldehyde lyase has the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of the benzaldehyde lyase mutant is obtained by mutating the sequence shown in SEQ ID NO: 1;

[0007] wherein, R1 is selected from: C 1～6 alkyl, C 1～6 alkoxy, C 2～6 alkenyl, C 2～6 alkynyl, C3-C 10 cycloalkyl, C6-C 10 aryl, C6-C containing N, O or S atoms 10 heteroaryl or C6-C containing N, O or S atoms 10 aromatic heterocyclic group; the C 1～6 alkyl, C 1～6 alkoxy, C 2～6 alkenyl, C 2～6 alkynyl, C3-C 10 cycloalkyl, C6-C 10 aryl, C6-C containing N, O or S atoms 10 heteroaryl or C6-C containing N, O or S atoms 10 aromatic heterocyclic group may be further optionally substituted by one, two or three independent R5,

[0008] wherein R5 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, halogen-substituted C1-C6 alkoxy, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)(C1-C6 alkyl) or cyano,

[0009] R2 is selected from: hydrogen, C 1～6 alkyl, C 1～6 alkoxy, C 2～6 alkenyl or C 2～6 alkynyl,

[0010] R3 is selected from: hydrogen, C 1～6 alkyl, C 1～6 alkoxy, C 2～6 alkenyl, C 2～6 alkynyl, C3-C 10 cycloalkyl, C6-C 10 aryl, C6-C containing N, O or S atoms 10 heteroaryl or C6-C containing N, O or S atoms 10 aromatic heterocyclic group, -C(O)(C 1～6 alkyl), -C(O)(C 1～6(alkoxy), -C(O)OC3-C 10 cycloalkyl or -C(O)OC6-C 10 aryl,

[0011] R4 is selected from: hydrogen, C 1～6 alkyl, C 1～6 alkoxy, C 2～6 alkenyl, C 2～6 alkynyl, C3-C 10 cycloalkyl of [number of carbon atoms], C6-C 10 aryl, C6-C containing N, O or S atoms 10 heteroaryl or C6-C containing N, O or S atoms 10 aryl heterocyclic group.

[0012] Furthermore, the R1 is selected from: C6-C 10 aryl or C6-C containing N, O or S atoms 10 heteroaryl; the C6-C 10 aryl or C6-C containing N, O or S atoms 10 heteroaryl may be further optionally substituted by one, two or three independent R5,

[0013] the R5 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl or halogen-substituted C1-C6 alkyl,

[0014] the R2 is selected from: hydrogen or C 1～6 alkyl,

[0015] the R3 is selected from: C3-C 10 cycloalkyl, C6-C 10 aryl, C6-C containing N, O or S atoms 10 heteroaryl or C6-C containing N, O or S atoms 10 aryl heterocyclic group, -C(O)(C 1～6 alkyl), -C(O)(C 1～6 alkoxy), -C(O)OC3-C 10 cycloalkyl or -C(O)OC6-C 10 aryl,

[0016] the R4 is selected from: hydrogen, C 1～6 alkyl, C 1～6 alkoxy, C 2～6 alkenyl or C 2～6 alkynyl.

[0017] Preferably,

[0018] the R1 is selected from: C6-C 10 aryl or C6-C containing N, O or S atoms10 Heteroaryl; said C6-C 10 Aryl or C6-C containing N, O or S atoms 10 The heteroaryl may further optionally be substituted by one or two independent R5,

[0019] wherein R5 is selected from: hydrogen, hydroxy, halogen, C1-C6 alkoxy, C1-C6 alkyl or halogen-substituted C1-C6 alkyl,

[0020] wherein R2 is selected from: hydrogen,

[0021] wherein R3 is selected from: -C(O)(C 1～6 alkoxy),

[0022] Furthermore, R1 is selected from: C6-C 10 Aryl or C6-C containing N, O or S atoms 10 Heteroaryl; said C6-C 10 Aryl or C6-C containing N, O or S atoms 10 The heteroaryl may further optionally be substituted by one or two independent R5,

[0023] wherein R5 is selected from: hydrogen, hydroxy, halogen, C1-C6 alkoxy, C1-C6 alkyl or halogen-substituted C1-C6 alkyl,

[0024] wherein R2 is selected from: hydrogen,

[0025] wherein R3 is selected from: -C(O)(C 1～6 alkoxy);

[0026] Preferably,

[0027] R1 is selected from

[0028] wherein R2 is selected from: hydrogen,

[0029] wherein R3 is selected from: -Boc;

[0030] More preferably, R1 is selected from

[0031]

[0032] The second aspect of the present invention provides a ThDP-dependent enzyme, namely benzaldehyde lyase PfBAL or a mutant of benzaldehyde lyase PfBAL. By analyzing bioinformatics methods, the present invention analyzes and predicts enzyme genes that may have obvious reducing activity towards substrates, sorts them out for cloning and expression, and constructs recombinant Escherichia coli cells. By measuring the activity of the recombinantly expressed ThDP-dependent enzyme and its stereoselectivity towards catalytic substrates, etc., the cloned enzymes are screened, and finally a mutant enzyme with the best catalytic performance is obtained. It is derived from Pseudomonas fluorescens AAA50176.1 and is named benzaldehyde lyase PfBAL. The amino acid sequence of the benzaldehyde lyase is preferably as shown in SEQ ID No.1 in the sequence listing, and the amino acid sequence has been reported in the existing literature Gene, 144(1994), 137-138, 10.1016 / 0378-1119(94)90218-6.

[0033] Preferably, it is a derivative protein composed of a new amino acid sequence formed by substituting one or several amino acids for alanine at position 28, threonine at position 73, tyrosine at position 397, alanine at position 480, and threonine at position 481 in the amino acid sequence shown in SEQ ID No.1. The amino acid sequences of the further preferred mutants are as follows:

[0034] (1) Substitute arginine for alanine at position 28 in the amino acid sequence shown in SEQ ID No.1;

[0035] (2) Substitute aspartic acid for alanine at position 28 in the amino acid sequence shown in SEQ ID No.1;

[0036] (3) Substitute glutamic acid for alanine at position 28 in the amino acid sequence shown in SEQ ID No.1;

[0037] (4) Substitute phenylalanine for alanine at position 28 in the amino acid sequence shown in SEQ ID No.1;

[0038] (5) Substitute glycine for alanine at position 28 in the amino acid sequence shown in SEQ ID No.1;

[0039] (6) Substitute histidine for alanine at position 28 in the amino acid sequence shown in SEQ ID No.1;

[0040] (7) Substitute isoleucine for alanine at position 28 in the amino acid sequence shown in SEQ ID No.1;

[0041] (8) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with leucine;

[0042] (9) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with lysine;

[0043] (10) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with methionine;

[0044] (11) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with proline;

[0045] (12) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with serine;

[0046] (13) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan;

[0047] (14) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with tyrosine;

[0048] (15) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with valine;

[0049] (16) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with cysteine;

[0050] (17) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with asparagine;

[0051] (18) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with threonine;

[0052] (19) Replace alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with glutamine;

[0053] (20) Replace threonine at position 73 of the amino acid sequence shown in SEQ ID No. 1 with arginine;

[0054] (21) Replace threonine at position 73 of the amino acid sequence shown in SEQ ID No. 1 with aspartic acid;

[0055] (22) Replace threonine at position 73 of the amino acid sequence shown in SEQ ID No. 1 with glutamic acid;

[0056] (23) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with phenylalanine;

[0057] (24) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with glycine;

[0058] (25) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with histidine;

[0059] (26) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with isoleucine;

[0060] (27) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with leucine;

[0061] (28) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with lysine;

[0062] (29) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with methionine;

[0063] (30) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with proline;

[0064] (31) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with serine;

[0065] (32) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with tryptophan;

[0066] (33) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with tyrosine;

[0067] (34) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with valine;

[0068] (35) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with cysteine;

[0069] (36) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with asparagine;

[0070] (37) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with alanine;

[0071] (38) Replace the threonine at position 73 of the amino acid sequence shown in SEQ ID No.1 with glutamine;

[0072] (39) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with arginine;

[0073] (40) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with aspartic acid;

[0074] (41) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with glutamic acid;

[0075] (42) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with phenylalanine;

[0076] (43) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with glycine;

[0077] (44) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with histidine;

[0078] (45) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with isoleucine;

[0079] (46) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with leucine;

[0080] (47) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with lysine;

[0081] (48) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with methionine;

[0082] (49) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with proline;

[0083] (50) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with serine;

[0084] (51) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with tryptophan;

[0085] (52) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with alanine;

[0086] (53) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with valine;

[0087] (54) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with cysteine;

[0088] (55) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with asparagine;

[0089] (56) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with threonine;

[0090] (57) Replace the tyrosine at position 397 of the amino acid sequence shown in SEQ ID No.1 with glutamine;

[0091] (58) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with arginine;

[0092] (59) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with aspartic acid;

[0093] (60) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with glutamic acid;

[0094] (61) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with phenylalanine;

[0095] (62) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with glycine;

[0096] (63) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with histidine;

[0097] (64) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with isoleucine;

[0098] (65) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with leucine;

[0099] (66) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with lysine;

[0100] (67) Replace the alanine at position 480 of the amino acid sequence shown in SEQ ID No.1 with methionine;

[0101] (68) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with proline;

[0102] (69) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with serine;

[0103] (70) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with tryptophan;

[0104] (71) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with tyrosine;

[0105] (72) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with valine;

[0106] (73) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with cysteine;

[0107] (74) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with asparagine;

[0108] (75) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with threonine;

[0109] (76) Replace the alanine at position 480 in the amino acid sequence shown in SEQ ID No. 1 with glutamine;

[0110] (77) Replace the threonine at position 481 in the amino acid sequence shown in SEQ ID No. 1 with arginine;

[0111] (78) Replace the threonine at position 481 in the amino acid sequence shown in SEQ ID No. 1 with aspartic acid;

[0112] (79) Replace the threonine at position 481 in the amino acid sequence shown in SEQ ID No. 1 with glutamic acid;

[0113] (80) Replace the threonine at position 481 in the amino acid sequence shown in SEQ ID No. 1 with phenylalanine;

[0114] (81) Replace the threonine at position 481 in the amino acid sequence shown in SEQ ID No. 1 with glycine;

[0115] (82) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with histidine;

[0116] (83) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with isoleucine;

[0117] (84) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with leucine;

[0118] (85) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with lysine;

[0119] (86) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with methionine;

[0120] (87) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with proline;

[0121] (88) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with serine;

[0122] (89) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan;

[0123] (90) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with tyrosine;

[0124] (91) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with valine;

[0125] (92) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with cysteine;

[0126] (93) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with asparagine;

[0127] (94) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with threonine;

[0128] (95) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with glutamine;

[0129] Alternatively, the reaction is carried out under the catalysis of benzaldehyde lyase or its mutant, and the benzaldehyde lyase has the amino acid sequence shown in SEQ ID NO: 1; and / or the amino acid sequence of the benzaldehyde lyase mutant is obtained by mutating the sequence shown in SEQ ID NO: 1, and the mutations include:

[0130] The alanine at position 28 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, glutamine,

[0131] and / or, the threonine at position 73 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, alanine, glutamine,

[0132] and / or, the tyrosine at position 397 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, alanine, valine, cysteine, asparagine, threonine, glutamine,

[0133] and / or, the alanine at position 480 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, glutamine,

[0134] and / or, the threonine at position 481 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, alanine, glutamine.

[0135] Furthermore, the enzyme for the catalytic reaction is selected from the benzaldehyde lyase mutant PfBAL A28G.

[0136] Moreover, the catalytic reaction is an asymmetric reduction reaction, and the asymmetric reduction reaction is carried out in the presence of MgSO4 and ThDP.

[0137] Furthermore, the concentration of the compound of Formula 1 is 10 - 30 mmol / L, the concentration of the compound of Formula 2 is 20 - 50 mmol / L, the concentration of MgSO4 is 2.0 - 3.0 mmol / L, the concentration of ThDP is 0.1 - 0.3 mmol / L, the reaction temperature is 10 - 30 °C, and the pH value is 6.7 - 7.9.

[0138] And / or, preferably, the concentration of the compound of Formula 1 is 20 mmol / L, the concentration of the compound of Formula 2 is 40 mmol / L, the concentration of MgSO4 is 2.5 mmol / L, the concentration of ThDP is 0.15 mmol / L, the reaction temperature is 30 °C, and the pH value is 7.3.

[0139] For the substrates benzaldehyde and N - tert - butoxycarbonyl - 2 - aminoacetaldehyde, the following exemplary method can be adopted: In a phosphate buffer solution with pH 7.3, in the presence of MgSO4 and ThDP, under the action of the ThDP - dependent enzyme and its mutant strains, tert - butyl (R)-(2 - hydroxy - 3 - oxo - 3 - phenylpropyl)(methyl)carbamate is prepared. In this reaction, in order to carry out coenzyme recycling, additional MgSO4 and ThDP are added to the reaction system. The dosage of MgSO4 is 2.5 mmol / L, and the dosage of ThDP is 0.15 mmol / L. The phosphate buffer solution can be any conventional phosphate buffer solution in the art, and the concentration of the phosphate buffer solution can be 50 mmol / L. The temperature of the asymmetric reduction reaction can be 25 - 35 °C, preferably 30 °C. During the reaction, samples are taken intermittently to determine the reaction conversion rate. The reaction time is based on the time when the substrate completely reacts or the reaction terminates by itself, generally 16 hours. The yield and diastereomeric purity can be analyzed by high - performance liquid chromatography. Preferably, for the yield, a C18 - PFP column is used, the mobile phase is acetonitrile / formic acid water = 50:50 (v / v), the column temperature is 25 °C, the flow rate is 1 mL / min, and the UV detection wavelength is 250 nm. For the ee value, an AS - H column is used, the mobile phase is n - hexane / isopropanol = 80:20 (v / v), the column temperature is 25 °C, the flow rate is 0.8 mL / min, and the UV detection wavelength is 250 nm.

[0140] After the reaction is completed, the reaction solution is extracted with an equal volume of a water - insoluble organic solvent conventional in the art, such as ethyl acetate, dichloromethane, etc., and the extraction is repeated three times. The combined extraction solution is washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain a crude extract of tert - butyl (R)-(2 - hydroxy - 3 - oxo - 3 - phenylpropyl)(methyl)carbamate, which can be further purified by conventional methods, such as column chromatography, etc., to obtain a highly chemically pure product.

[0141] In the third aspect of the present invention, there is provided a separated nucleic acid, which is a nucleic acid molecule encoding the above-mentioned ThDP-dependent enzyme (benzaldehyde lyase PfBAL or benzaldehyde lyase PfBAL mutant).

[0142] In the fourth aspect of the present invention, there is provided a recombinant expression vector containing the nucleic acid sequence of the above-mentioned ThDP-dependent enzyme gene. The recombinant expression vector can be constructed by cloning the above-mentioned enzyme gene into various vectors by conventional methods in the art. The expression vector preferably includes various conventional vectors in the art, such as commercially available plasmids, cosmids, phages or viral vectors, etc., and the vector is preferably the plasmid pET28a.

[0143] In the fifth aspect of the present invention, there is provided a recombinant expression transformant containing the aforementioned ThDP-dependent enzyme gene or its recombinant expression vector. The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into a host cell. The host cell is a conventional host cell in the art, as long as it can satisfy that the recombinant expression vector can stably replicate itself, and the ThDP-dependent enzyme gene carried by it can be effectively expressed. The host cell is preferably Escherichia coli, more preferably Escherichia coli (E. coli) BL21(DE3) or Escherichia coli DH5α. Transforming the aforementioned recombinant expression vector into Escherichia coli (E. coli) BL21(DE3) can obtain the preferred genetically engineered strain of the present invention. For example, transforming the recombinant expression vector pET28a-PfBAL into Escherichia coli (E. coli) BL21(DE3) can obtain recombinant Escherichia coli E. coli BL21(DE3) / pET28a-PfBAL.

[0144] In the sixth aspect of the present invention, the preparation method of the recombinant ThDP-dependent enzyme is preferably: culturing the recombinant expression transformant as described above to obtain the recombinant expressed ThDP-dependent enzyme. The medium used for culturing the recombinant expression transformant is any medium in the art that can enable the transformant to grow and produce the recombinant ThDP-dependent enzyme of the present invention. The medium is preferably LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. There are no special limitations on the culture method and culture conditions, and appropriate selection can be made according to factors such as the type of host cell and the culture method according to the conventional knowledge in the art, as long as the transformant can grow and produce the enzyme. The specific operations for culturing the transformant can be carried out according to the conventional operations in the art. The strain culture method preferably includes: inoculating the recombinant Escherichia coli of the present invention, such as E. coli BL21(DE3) / pET28a-PfBAL, into the LB medium containing kanamycin, culturing at 37 °C, and when the optical density OD of the culture solution 600When it reaches 0.5 - 1.0 (preferably 0.6), isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 - 1.0 mmol / L (preferably 1 mmol / L) is added for enzyme production induction, and the culture is continued at 18°C for 24 h, then the ThDP-dependent enzyme described in the present invention can be highly expressed. After the culture is completed, the precipitated bacterial cells are harvested by centrifugation, which are the resting cells of the recombinant expression transformant; the obtained bacterial cell precipitate can be freeze-dried to obtain freeze-dried cells, which is beneficial for long-term storage and convenient for future use.

[0145] By detecting the change in product formation, the activity of the ThDP-dependent enzyme is determined using a high-performance liquid chromatograph. The activity of the ThDP-dependent enzyme can be determined by the following method: A 500 μL reaction system (50 mmol / L phosphate buffer, pH 7.3) containing 20 mmol / L benzaldehyde and 40 mmol / L N-tert-butoxycarbonyl-2-aminoacetaldehyde is preheated to 30°C, then an appropriate amount of the ThDP-dependent enzyme is added, and the reaction is incubated at 30°C. The formation of the product is detected by high-performance liquid chromatography, and the change value of the yield within 5 minutes is recorded.

[0146] The enzyme activity is calculated according to the following formula: Enzyme activity (U) = amount of product formed / time · amount of enzyme

[0147] In the formula, the amount of product formed is the amount of product formed within this time, with the unit of μmmol; the time is the reaction time, with the unit of min; the amount of enzyme is the amount of pure enzyme added in the reaction, with the unit of mg.

[0148] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0149] As used in the present invention, "wild-type benzaldehyde lyase" refers to the naturally occurring, unmodified benzaldehyde lyase, whose nucleotide can be obtained by genetic engineering techniques such as genomic sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be deduced from the nucleotide sequence. The amino acid sequence of the wild-type benzaldehyde lyase is shown in SEQ ID NO:1:

[0150] MAMITGGELVVRTLIKAGVEHLFGLHGAHIDTIFQACLDHDVPIIDTRHEAAAGHAAEGYARAGAKLGVALVTAGGGFTNAVTPIANAWLDRTPVLFLTGSGALRDDETNTLQAGIDQVAMAAPITKWAHRVMATEHIPRLVMQAIRAALSAPRGPVLLDLPWDILMNQIDEDSVIIPDLVLSAHGARPDPADLDQALALLRKAERPVIVLGSEASRTARKTALSAFVAATGVPVFADYEGLSMLSGLPDAMRGGLVQNLYSFAKADAAPDLVLMLGARFGLNTGHGSGQLIPHSAQVIQVDPDACELGRLQGIALGIVADVGGTIEALAQATAQDAAWPDRGDWCAKVTDLAQERYASIAAKSSSEHALHPFHASQVIAKHVDAGVTVVADGALTYLWLSEVMSRVKPGGFLCHGYLGSMGVGFGTALGAQVADLEAGRRTILVTGDGSVGYSIGEFDTLVRKQLPLIVIIMNNQSWGATLHFQQLAVGPNRVTGTRLENGSYHGVAAAFGADGYHVDSVESFSAALAQALAHNRPACINVAVALDPIPPEELILIGMDPFA。

[0151] For the mutant protein of the present invention, as used herein, the terms "mutant", "benzaldehyde lyase mutant protein", "mutant protein", "benzaldehyde lyase mutant protein", "mutant protein of the present invention", "benzaldehyde lyase mutant protein of the present invention", and "benzaldehyde lyase mutant of the present invention" are used interchangeably and all refer to a non-naturally occurring benzaldehyde lyase, and the mutant protein is a protein artificially modified based on the protein shown in SEQ ID NO:1, and the mutant protein of the present invention has the activity of efficiently catalyzing the formation of optically pure amino alcohol compounds.

[0152] Among them, the benzaldehyde lyase mutant of the present invention, PfBAL A28G, is a known mutant disclosed in the existing literature (J. Am. Chem. Soc. 2025, 147, 3102 - 3109).

[0153] Among them, as used herein, the term "AxxB" means that the amino acid A at position xx is changed to amino acid B. For example, "A28G" means that alanine A at position 28 is mutated to glycine G, and so on.

[0154] Advantageous technical effects of the present invention:

[0155] 1. The ThDP-dependent enzyme and its mutant strains provided by the present invention can be used for highly efficient catalytic production of optically pure amino alcohol compounds. Using this enzymatic catalysis technology, the yield can exceed 99%, and the ee value of the product is higher than 99%. The present invention has the advantages of high product yield and high optical purity, which is conducive to realizing the high-efficiency and low-cost production of amino alcohols and has the prospect of industrial application.

[0156] 2. The present invention has the advantages of high substrate concentration, mild reaction conditions, environmental friendliness, high yield, high optical purity of the product, etc., so it has good application prospects in industrial production. Brief Description of the Drawings

[0157] Figure 1 : Schematic diagram of the construction of the recombinant expression plasmid pET28a-PfBAL. Detailed Embodiments

[0158] The present invention is further illustrated by the following examples, which are not intended to limit the present invention.

[0159] The sources of the materials in the following examples are:

[0160] The recombinant expression plasmid pET28a-PfBAL was purchased from Nanjing Genscript Corporation.

[0161] The competent cells of E. coli DH5α and E. coli BL21(DE3), and the agarose gel DNA recovery kit were all purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0162] Molecular biology reagents such as the restriction enzyme DpnI, PCR extraction kit and plasmid mini kit were purchased from Thermo Scientific and Omega Biotek companies.

[0163] Example 1: Preparation of the recombinant expression plasmid and recombinant expression transformant of benzaldehyde lyase PfBAL

[0164] The construction process of plasmid pET28a-PfBAL is as follows: First, using the target gene PfBAL as a template, PCR amplification is carried out, and appropriate restriction enzyme sites are introduced into the primers. Then, the pET-28a(+) vector is linearized with the corresponding restriction endonuclease to generate sticky ends compatible with the inserted fragment. Next, the PfBAL fragment is ligated to the linearized vector using DNA ligase to construct the recombinant plasmid. Subsequently, the recombinant plasmid is transformed into Escherichia coli, and positive clones are screened by kanamycin resistance. Finally, restriction enzyme digestion identification and sequencing are used to verify whether the insertion is correct, ensuring that the PfBAL gene is successfully cloned into pET-28a(+), and the C-terminal His tag is retained for subsequent protein expression and purification. See the attached drawings of the specification Figure 1 .

[0165] The recombinant plasmid was transformed into E. coli DH5α, spread on an LB medium plate containing 50 μg / ml kanamycin, and cultured at 37 °C for 8 hours. Colony PCR verification was performed on the grown colonies. After sequencing verification, the corresponding plasmid was extracted and further transformed into E. coli BL21(DE3). Positive clones were picked to obtain the recombinant expression transformant E. coli BL21(DE3) / pET28a-PfBAL.

[0166] Example 2: Construction of the PfBAL A28G mutant of benzaldehyde lyase

[0167] Through molecular docking, directed evolution of the wild enzyme was carried out to construct a mutant library of benzaldehyde lyase PfBAL: Single-point mutations were introduced to non-conserved residues within the substrate-binding pocket of PfBAL, and mutant primers were designed. The sequences of the mutant primers are shown as No. 5 in Table 1. Using pET28a-PfBAL as a template, PCR was performed with the high-fidelity polymerase PrimeSTAR (Premix).

[0168] The PCR reaction conditions are as follows: In a 25-μL PCR reaction system, 0.5 - 20 ng of template, 12.5 μL of 2×PrimeSTAR (Premix), 1 μL (10 μM) of each of a pair of mutant primers are added, and sterilized distilled water is added to make up to 25 μL. The PCR reaction procedure: (1) Denaturation at 98°C for 10 sec, (2) Annealing at 60°C for 30 sec, (3) Extension at 72°C for 120 sec. Steps (1) - (3) are carried out for 30 cycles in total, and the product is stored at 4°C. After the PCR product is verified by agarose gel electrophoresis analysis, restriction endonuclease DpnI is added and digested at 37°C for 3 h. The digested product is transferred into competent E. coli BL21(DE3) cells and spread on a plate containing kanamycin antibiotic, and is statically cultured in a 37°C incubator for about 12 h. The obtained monoclonal colonies are picked into a test tube for culture, and the gene of the benzaldehyde lyase PfBAL A28G mutant is sequenced.

[0169] According to the same construction process as described above, different mutant primer sequences are designed respectively, referring to the mutant primer series numbered 1 - 4, 6 - 95, and the corresponding genes are obtained for sequencing.

[0170] Table 1 Mutant primer sequences

[0171]

[0172]

[0173]

[0174]

[0175] Example 3 Induced expression of benzaldehyde lyase PfBAL

[0176] The recombinant expression transformant E. coli BL21(DE3) / pET28a-PfBAL obtained in Example 1 is inoculated into an LB medium containing 50 μg / ml kanamycin, and cultured with shaking at 37°C for 12 hours. Then, it is inoculated into a 500-ml Erlenmeyer flask containing 100 ml of LB medium at an inoculation amount of 1% (v / v), and cultured with shaking in a 37°C, 180-rpm shaker. When the OD 600 of the culture broth reaches 0.6, IPTG is added to a final concentration of 1 mmol / L for induction. After induction at 18°C for 24 hours, the culture broth is centrifuged at 8000 rpm for 10 min to collect the cells, which are stored at -80°C.

[0177] The resting cells obtained by the above method were suspended in phosphate buffer at pH 7.3 and ultrasonically disrupted in an ice-water bath. The supernatant was collected by centrifugation, which was the crude enzyme solution of recombinant benzaldehyde lyase. The obtained crude enzyme solution was analyzed by polyacrylamide gel electrophoresis, and benzaldehyde lyase existed in a soluble form.

[0178] Example 4 Induced Expression of PfBAL A28G Mutant of Benzaldehyde Lyase

[0179] The recombinant expression transformant E. coli BL21(DE3) / pET28a-PfBAL mutants obtained in Example 2 was inoculated into LB medium containing 50 μg / ml kanamycin and cultured with shaking at 37 °C for 12 hours. Then, it was inoculated into a 500 ml Erlenmeyer flask containing 100 ml LB medium at an inoculation amount of 1% (v / v) and cultured with shaking in a shaker at 37 °C and 180 rpm. When the OD 600 of the culture broth reached 0.6, IPTG was added to a final concentration of 1 mmol / L for induction. After induction at 18 °C for 24 hours, the culture broth was centrifuged at 8000 rpm for 10 min to collect the cells, which were stored at -80 °C.

[0180] Example 5 Effects of pH and Temperature on the Catalytic Activity and Stereoselectivity of PfBAL mutants (PfBAL A28G Mutant of Benzaldehyde Lyase)

[0181] The reaction was carried out in a 2 mL centrifuge tube. In 500 μl of buffer (50 mmol / L, phosphate buffer), 400 μl of the crude enzyme solution of PfBAL mutants (PfBAL A28G mutant of benzaldehyde lyase) prepared in Example 4 was added, and benzaldehyde, N-tert-butoxycarbonyl-2-aminoacetaldehyde, MgSO4 and ThDP were added to final concentrations of 20 mmol / L, 40 mmol / L, 2.5 mmol / L and 0.15 mmol / L, respectively. Controlling variables, one variable was to set the gradient pH values as 6.7, 7.0, 7.3, 7.6, 7.9, and the other variable was the gradient temperature as 10 °C, 20 °C, 30 °C. The reaction was placed on a thermostatic mixer for 16 hours, and samples were taken to detect the reaction conversion rate and the ee value of the product. It was found that when the pH value was 7.3 and the temperature was 30 °C, the yield was as high as 90% and the ee value was as high as 96%. Therefore, pH 7.3 and temperature 30 °C were the optimal reaction conditions for this reaction.

[0182] Synthesis Preparation Example 1 Asymmetric Reduction Reaction of PfBAL mutants (PfBAL A28G Mutant of Benzaldehyde Lyase) Catalyzed Synthesis of a Series of Amino Alcohol Compounds

[0183]

[0184] In 5 mL of phosphate buffer (50 mmol / L, pH 7.3), add the crude enzyme solution of the PfBAL mutants benzaldehyde lyase PfBAL A28G mutant as described in Example 4. Add the corresponding carbonyl compound substrates as shown in Formula 1 and Formula 2 to a final concentration of 20 mmol / L and 40 mmol / L respectively, and add MgSO4 at a final concentration of 2.5 mmol / L and ThDP at 0.15 mmol / L. Place it on a thermostatic mixer and react at 30 °C and 1000 rpm for 16 hours. Terminate the reaction, extract three times with an equal volume of ethyl acetate, combine the extracts, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, rotary evaporate and then separate and purify by column chromatography. The yield is measured to be 94% and the ee value of the product is 96%. 1H NMR (400 MHz, DMSO) δ 8.02 (d, J = 7.0 Hz, 2H), 7.64 (d, J = 7.5 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 5.63 - 5.54 (m, 1H), 5.16 - 5.08 (m, 1H), 3.65 - 3.56 (m, 1H), 3.37 (s, 2H), 2.85 (s, 3H), 1.29 (d, 9H).

[0185] Table 2 Asymmetric reduction reaction of a series of carbonyl compounds catalyzed by the benzaldehyde lyase PfBAL A28G mutant

[0186]

[0187]

[0188] Synthesis Preparation Example 2

[0189] Under the same reaction conditions as in Synthesis Preparation Example 1, for the substrate structures of Formula 1 and Formula 2, see Table 2, to obtain the product 1 1H NMR (400 MHz, DMSO) δ 7.92 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 5.50 (dd, J = 27.1, 6.7 Hz, 1H), 5.15 - 5.07 (m, 1H), 3.59 (dd, J = 14.4, 4.8 Hz, 1H), 3.23 (ddd, J = 42.9, 14.2, 7.6 Hz, 1H), 2.84 (s, 3H), 2.37 (s, 3H), 1.30 (d, J = 37.9 Hz, 9H).

[0190] Synthesis Preparation Example 3

[0191] Under the same reaction conditions as in Synthesis Preparation Example 1, for the substrate structures of Formula 1 and Formula 2, see Table 2, to obtain the product 11H NMR (400 MHz, DMSO) δ 7.82 (d, J = 8.3 Hz, 2H), 7.48 - 7.37 (m, 2H), 5.53 (d, J = 18.1 Hz, 1H), 5.17 - 5.08 (m, 1H), 3.64 - 3.54 (m, 1H), 3.26 (ddd, J = 53.9, 14.2, 7.4 Hz, 1H), 2.84 (s, 3H), 2.36 (s, 3H), 1.29 (d, J = 45.4 Hz, 9H).

[0192] Synthesis Preparation Example 4

[0193] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates Formula 1 and Formula 2, see Table 2, to obtain the product 1H NMR (400 MHz, DMSO) δ 8.03 (t, J = 8.3 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 5.43 (dd, J = 37.2, 6.8 Hz, 1H), 5.05 (s, 1H), 4.03 (s, 1H), 3.85 (s, 3H), 3.61 (d, J = 19.5 Hz, 1H), 2.85 (d, J = 7.4 Hz, 3H), 1.35 (d, J = 15.4 Hz, 9H).

[0194] Synthesis Preparation Example 5

[0195] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates Formula 1 and Formula 2, see Table 2, to obtain the product 1 1H NMR (400 MHz, DMSO) δ 10.44 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 5.36 (s, 1H), 5.04 (s, 1H), 3.57 (d, J = 14.3 Hz, 1H), 3.19 (ddd, J = 47.4, 14.2, 7.8 Hz, 1H), 2.83 (s, 3H), 1.31 (d, J = 37.2 Hz, 9H).

[0196] Synthesis Preparation Example 6

[0197] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates Formula 1 and Formula 2, see Table 2, to obtain the product 1H NMR (400 MHz, DMSO) δ 9.79 (s, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.38 - 7.29 (m, 2H), 7.03 (d, J = 7.9 Hz, 1H), 5.56 (s, 1H), 5.05 (d, J = 5.7 Hz, 1H), 3.59 (dd, J = 14.5, 5.2 Hz, 1H), 3.32 - 3.14 (m, 1H), 2.84 (s, 3H), 1.30 (d, J = 44.1 Hz, 9H).

[0198] Synthesis Preparation Example 7

[0199] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates Formula 1 and Formula 2, see Table 2, to obtain the product 1H NMR (400 MHz, DMSO) δ 7.61 (d, J = 13.0 Hz, 2H), 7.28 (s, 1H), 5.48 (s, 1H), 5.10 (t, J = 6.1 Hz, 1H), 3.62 - 3.51 (m, 1H), 3.19 (dd, J = 13.9, 7.5 Hz, 1H), 2.84 (d, J = 12.0 Hz, 3H), 2.33 (s, 6H), 1.29 (d, J = 45.8 Hz, 9H).

[0200] Synthesis Preparation Example 8

[0201] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates Formula 1 and Formula 2, see Table 2, to obtain the product 1H NMR (400 MHz, DMSO) δ 8.02 (t, J = 7.3 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H), 5.68 (dd, J = 45.5, 6.5 Hz, 1H), 5.06 (dd, J = 25.1, 5.5 Hz, 1H), 3.59 (dd, J = 14.4, 5.5 Hz, 1H), 3.25 (dd, J = 14.0, 7.4 Hz, 1H), 2.85 (d, J = 4.2 Hz, 3H), 1.27 (d, J = 46.0 Hz, 9H).

[0202] Synthesis Preparation Example 9

[0203] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates Formula 1 and Formula 2, see Table 2, to obtain the product 11H NMR (400 MHz, DMSO) δ 8.01 - 7.94 (m, 2H), 7.71 (t, J = 7.3 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 5.73 (dd, J = 42.5, 6.4 Hz, 1H), 5.06 (dd, J = 12.6, 6.5 Hz, 1H), 3.58 (dd, J = 14.5, 5.7 Hz, 1H), 3.41 (d, J = 6.8 Hz, 1H), 2.85 (d, J = 7.3 Hz, 3H), 1.26 (d, J = 47.9 Hz, 9H).

[0204] Synthesis Preparation Example 10

[0205] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates of Formula 1 and Formula 2, see Table 2, to obtain the product 1 1H NMR (400 MHz, DMSO) δ 8.13 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.84 (t, J = 7.9 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 5.82 - 5.65 (m, 1H), 5.05 (dd, J = 12.5, 6.3 Hz, 1H), 3.58 (dd, J = 14.4, 5.4 Hz, 1H), 3.43 - 3.38 (m, 1H), 3.28 (dd, J = 14.1, 7.0 Hz, 1H), 2.85 (d, J = 8.6 Hz, 3H), 1.25 (d, J = 47.7 Hz, 9H).

[0206] Synthesis Preparation Example 11

[0207] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates of Formula 1 and Formula 2, see Table 2, to obtain the product 1 1H NMR (400 MHz, DMSO) δ 8.00 (d, J = 7.9 Hz, 2H), 7.64 (t, J = 7.4 Hz, 1H), 7.52 (t, J = 7.7 Hz, 2H), 6.80 (s, 1H), 5.02 (t, J = 5.9 Hz, 1H), 3.35 - 3.31 (m, 1H), 3.15 (m, 1H), 1.32 (s, 9H).

[0208] Synthesis Preparation Example 12

[0209] Under the same reaction conditions as in Synthesis Preparation Example 1, for the structures of Substrates of Formula 1 and Formula 2, see Table 2, to obtain the product 11H NMR (400 MHz, DMSO) δ 7.8 (s, 2H), 7.5 - 7.4 (m, 2H), 6.8 (t, J = 5.9 Hz, 1H), 5.4 (d, J = 7.0 Hz, 1H), 5.0 (q, J = 6.8, 5.9 Hz, 1H), 3.4 (s, 1H), 3.1 (dt, J = 13.4, 6.4 Hz, 1H), 2.4 (s, 3H), 1.3 (s, 9H).

[0210] Synthesis Preparation Example 13

[0211] Under the same reaction conditions as in Synthesis Preparation Example 1, for the substrate structures of Formula 1 and Formula 2, see Table 2, to obtain the product 1 1H NMR (400 MHz, DMSO) δ 7.9 (d, J = 8.3 Hz, 2H), 7.3 (d, J = 8.3 Hz, 2H), 6.8 (t, J = 5.9 Hz, 1H), 5.4 (d, J = 7.0 Hz, 1H), 5.0 (q, J = 5.9, 5.0 Hz, 1H), 3.3 - 3.3 (m, 1H), 3.1 (dt, J = 13.4, 6.4 Hz, 1H), 2.4 (s, 3H), 1.3 (s, 9H).

[0212] Synthesis Preparation Example 14

[0213] Under the same reaction conditions as in Synthesis Preparation Example 1, for the substrate structures of Formula 1 and Formula 2, see Table 2, to obtain the product 1 1H NMR (400 MHz, DMSO) δ 8.2 (d, J = 8.3 Hz, 2H), 7.9 (d, J = 8.4 Hz, 2H), 6.8 (t, J = 6.0 Hz, 1H), 5.7 (d, J = 6.8 Hz, 1H), 5.0 (q, J = 6.1 Hz, 1H), 3.3 - 3.1 (m, 1H), 1.3 (s, 9H).

[0214] Synthesis Preparation Example 15

[0215] Under the same reaction conditions as in Synthesis Preparation Example 1, for the substrate structures of Formula 1 and Formula 2, see Table 2, to obtain the product 1 1H NMR (400 MHz, DMSO) δ 8.1 - 8.0 (m, 2H), 7.3 (dd, J = 4.9, 3.8 Hz, 1H), 6.9 (t, JJ = 6.0 Hz, 1H), 5.7 (s, 1H), 4.7 (q, J = 6.5 Hz, 1H), 3.4 (s, 1H), 3.1 (dt, J = 13.4, 6.3 Hz, 1H), 1.3 (s, 9H).

[0216] Scaled-up reaction for the synthesis of tert-butyl (R)-(2-hydroxy-3-oxo-3-phenylpropyl)(methyl)carbamate catalyzed by the synthetic preparation example 16 PfBAL mutants (benzaldehyde lyase PfBAL A28G mutant)

[0217] Add the crude enzyme solution of PfBAL mutants as described in Example 4 to 250 mL of phosphate buffer (50 mmol / L, pH 7.3), add the corresponding carbonyl compound substrates as shown in Formula 1 and Formula 2 to a final concentration of 20 mmol / L and 40 mmol / L respectively, and add MgSO4 at a final concentration of 2.5 mmol / L and ThDP at 0.15 mmol / L. Place it in a shaker and react at 30 °C and 200 rpm for 16 hours. Terminate the reaction, extract three times with an equal volume of ethyl acetate, combine the extracts, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, spin-dry and then purify by column chromatography to obtain 1.13 g of pure product tert-butyl (R)-(2-hydroxy-3-oxo-3-phenylpropyl)(methyl)carbamate, with a separation yield of 81%.

[0218] As described above, only the preferred specific embodiments of the present invention are provided, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for synthesizing a compound of formula I by using benzaldehyde lyase or a mutant thereof, wherein the reaction formula is as follows: The benzaldehyde lyase has an amino acid sequence as shown in SEQ ID NO: 1; the amino acid sequence of the benzaldehyde lyase mutant is obtained by mutating the sequence as shown in SEQ ID NO: 1; in, R1 is selected from: C 1～6 Alkyl, C 1～6 Alkoxy, C 2～6 Alkenyl, C 2～6 Alkynyl, C3~C 10 Cycloalkyl, C6-C 10 Aryl, C6-C containing N, O or S atoms 10 Heteroaryl or C6-C 10 Aromatic heterocyclic group; said C 1～6 Alkyl, C 1～6 Alkoxy, C 2～6 Alkenyl, C 2～6 Alkynyl, C3~C 10 Cycloalkyl, C6-C 10 Aryl, C6-C containing N, O or S atoms 10 Heteroaryl or C6-C 10 The aromatic heterocyclic group may be further optionally substituted by one, two or three independent R5, The R5 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, halogen-substituted C1-C6 alkoxy, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)(C1-C6 alkyl) or cyano, R2 is selected from: hydrogen, C 1～6 Alkyl, C 1～6 Alkoxy, C 2～6 Alkenyl or C 2～6 Alkynyl, R3 is selected from: hydrogen, C 1～6 Alkyl, C 1～6 Alkoxy, C 2～6 Alkenyl, C 2～6 Alkynyl, C3~C 10 Cycloalkyl, C6-C 10 Aryl, C6-C containing N, O or S atoms 10 Heteroaryl or C6-C 10 Aromatic heterocyclic group, -C(O)(C 1～6 alkyl), -C(O)(C 1～6 Alkoxy), -C(O)OC3~C 10 Cycloalkyl or -C(O)OC6-C 10 Aryl, R4 is selected from: hydrogen, C 1～6 Alkyl, C 1～6 Alkoxy, C 2～6 Alkenyl, C 2～6 Alkynyl, C3~C 10 Cycloalkyl, C6-C 10 Aryl, C6-C containing N, O or S atoms 10 Heteroaryl or C6-C 10 Aromatic heterocyclic group.

2. The method according to claim 1, characterized in that: The R1 is selected from: C6-C 10 Aryl or C6-C containing N, O or S atoms 10 Heteroaryl; the C6-C 10 Aryl or C6-C containing N, O or S atoms 10 Heteroaryl may be further optionally substituted by one, two or three independent R5, R5 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl or halogen-substituted C1-C6 alkyl, The R2 is selected from: hydrogen or C 1～6 alkyl, The R3 is selected from: C3~C 10 Cycloalkyl, C6-C 10 Aryl, C6-C containing N, O or S atoms 10 Heteroaryl or C6-C 10 Aromatic heterocyclic group, -C(O)(C 1～6 alkyl), -C(O)(C 1～6 Alkoxy), -C(O)OC3~C 10 Cycloalkyl or -C(O)OC6-C 10 Aryl, The R4 is selected from: hydrogen, C 1～6 Alkyl, C 1～6 Alkoxy, C 2～6 Alkenyl or C 2～6 Alkynyl; Preferably, The R1 is selected from: C6-C 10 Aryl or C6-C containing N, O or S atoms 10 Heteroaryl; the C6-C 10 Aryl or C6-C containing N, O or S atoms 10 Heteroaryl may be further optionally substituted by one or two independent R5, R5 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl or halogen-substituted C1-C6 alkyl, The R2 is selected from: hydrogen, The R3 is selected from: -C(O)(C 1～6 alkoxy).

3. The method according to claim 1 or 2, characterized in that: The R1 is selected from: C6-C 10 Aryl or C6-C containing N, O or S atoms 10 Heteroaryl; the C6-C 10 Aryl or C6-C containing N, O or S atoms 10 Heteroaryl may be further optionally substituted by one or two independent R5, R5 is selected from: hydrogen, hydroxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl or halogen-substituted C1-C6 alkyl, The R2 is selected from: hydrogen, The R3 is selected from: -C(O)(C 1～6 Alkoxy); Preferably, The R1 is selected from The R2 is selected from: hydrogen, The R3 is selected from: -Boc; Further preferably, the R1 is selected from 4. The method according to claim 1 or 2, characterized in that: The amino acid sequence of the benzaldehyde lyase mutant is obtained by mutation of the sequence shown in SEQ ID NO: 1, wherein the mutation comprises: The alanine at position 28 is replaced with any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine, and / or, the threonine at position 73 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, alanine, and glutamine, and / or, the tyrosine at position 397 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, alanine, valine, cysteine, asparagine, threonine, and glutamine, and / or, the alanine at position 480 is replaced by any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, threonine, and glutamine, And / or, the threonine at position 481 is replaced with any one of arginine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tryptophan, tyrosine, valine, cysteine, asparagine, alanine, and glutamine.

5. The method according to claim 4, wherein the catalytic reaction is selected from the benzaldehyde lyase mutant PfBAL A28G.

6. The preparation method according to claim 5, characterized in that: The catalytic reaction is an asymmetric reduction reaction, and the asymmetric reduction reaction is carried out in the presence of MgSO4 and ThDP.

7. The preparation method according to claim 6, characterized in that: The concentration of the compound of formula 1 is 10-30 mmol / L, the concentration of the compound of formula 2 is 20-50 mmol / L, the concentration of MgSO4 is 2.0-3.0 mmol / L, the concentration of ThDP is 0.1-0.3 mmol / L, the reaction temperature is 10-30°C, and the pH value is 6.7-7.

9. And / or, preferably, the concentration of the compound of formula 1 is 20 mmol / L, the concentration of the compound of formula 2 is 40 mmol / L, the concentration of MgSO4 is 2.5 mmol / L, the concentration of ThDP is 0.15 mmol / L, the reaction temperature is 30°C, and the pH value is 7.

3.

8. An isolated nucleic acid, characterized in that: The nucleic acid is a nucleic acid molecule encoding a benzaldehyde lyase mutant PfBALA28G.

9. An expression vector containing the gene encoding the benzaldehyde lyase mutant PfBAL A28G.

10. A recombinant cell containing a gene encoding the benzaldehyde lyase mutant PfBAL A28G.

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