Synthesis method and application of chromone 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 serving as a catalyst in catalytic synthesis of 3-hydroxychromone. The benzaldehyde lyase disclosed by the invention has the advantages of high reactivity, high selectivity, wide substrate range and the like, the conversion rate is up to 99%, low-cost production is favorably realized, and the benzaldehyde lyase has an industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and chemical engineering technology, specifically relating to a benzaldehyde lyase mutant, and more particularly to a method for synthesizing chromone compounds catalyzed by the benzaldehyde lyase mutant and its application. Background Technology

[0002] Benzaldehyde lyase (BAL) is a thiamine diphosphate (ThDP)-dependent enzyme, originally derived from *Pseudomonas fluorescens*. Pseudomonas fluorescens It was discovered in [the study]. It is a multifunctional enzyme capable of catalyzing the formation and cleavage of C-C bonds between aldehyde substrates. Benzaldehyde lyase is a multifunctional enzyme with broad industrial and scientific research applications. Through enzyme engineering and synthetic biology, its catalytic performance and application range are continuously expanding, providing new tools for green chemistry and the synthesis of chiral compounds.

[0003] Chromones are the structural backbones of flavonoid natural products and functional molecules, abundant in plants, and play various roles, including maintaining growth, inhibiting dormancy, and stimulating oxygen uptake. Chromones have wide applications in clinical medicine and functional materials, such as anti-cancer, anti-allergic, anti-inflammatory, and antiviral applications. Among all chromone backbones, 3-hydroxy-chromones and their derivatives are an important class. Due to the excited state of their intramolecular proton transfer process, they exhibit fluorescence properties. These significant fluorescence properties make their derivatives fluorescent sensors for normal and supercritical liquids, lipid membranes, DNA, and proteins. Furthermore, 3-hydroxy-chromones possess certain biological activities and potential pharmacological activities. For example, diosmin, a sugar-substituted chromone derivative, is most commonly used to treat hemorrhoids and leg ulcers caused by poor blood flow, and is used to treat various vascular problems, including hemorrhoids, varicose veins, venous stasis, and bleeding from the eyes or gums. Apigenin, a plant-derived chromone derivative, is used to treat cancer. Flavonoids, known as antimuscarinic esters, are muscle relaxants used to treat bladder and urinary system disorders. Khellin is a furan-cyclized chromone derivative, known as furan pigment, whose main function is as a vasodilator. Chromen-5-one is a folk remedy used to treat various ailments, including kidney stones, psoriasis, vitiligo, bronchial asthma, coronary artery disease, and renal colic. However, current synthetic methods for 3-hydroxy-chromone are limited, mainly relying on the oxidation of chromone-type substrates or the oxidative cyclization of o-hydroxyphenylenamine ketones. These oxidation methods suffer from lengthy substrate synthesis routes, require organic peracids or hydrogen peroxide as oxidants, and are carried out in organic solvents.

[0004] Therefore, there is a need to explore biocatalytic synthesis processes that are highly efficient, selective, and more environmentally friendly. In view of this, the present invention is proposed. Summary of the Invention

[0005] The first aspect of this invention is to provide a method for synthesizing compound of formula 2 catalyzed by benzaldehyde lyase or a mutant thereof, the reaction formula of which is as follows: ; 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 mutation of the sequence shown in SEQ ID NO:1; Where X is selected from O, S, or N; n is selected from 1, 2, or 3; R1 is selected from the following groups: hydrogen, halogen, C1~C6 alkyl, C1~C6 alkoxy, halogen-substituted C1~C6 alkyl, -COOC1~C6 alkyl, C2~C6 alkenyl or C2~C6 alkenyl C1-C6 alkyl; when n is selected from 2 and the substituent R1 group is adjacent, the adjacent R1 group and the C atom attached to it can form a C6-C10 aryl group; R2 is selected from the following groups: hydrogen, halogen-substituted C1~C6 alkyl or C1~C6 alkyl.

[0006] Furthermore, where X is selected from: O or S; n is selected from 1 or 2; R1 is selected from the following groups: hydrogen, halogen, C1~C6 alkyl, C1~C6 alkoxy, halogen-substituted C1~C6 alkyl, -COOC1~C6 alkyl, C2~C6 alkenyl or C2~C6 alkenyl C1-C6 alkyl; when n is selected from 2 and the R1 groups are adjacent, the adjacent R1 groups and the C atoms attached to them can form a phenyl group. R2 is selected from the following groups: hydrogen or C1~6 alkyl.

[0007] Furthermore, n is selected from 1 or 2; R1 is selected from methyl, methoxy, ethoxy, allyl, phenylcycloyl, methyl ester, fluorine, chlorine, bromine, or trifluoromethyl. The R2 is selected from: hydrogen or methyl; X is selected from: O or S; Further preferably, Formula I is selected from the following compounds: , , , , , , , , , , , , , , , , , , , , or .

[0008] A second aspect of the present invention is to provide a ThDP-dependent enzyme, namely benzaldehyde lyase. Pf BAL or benzaldehyde lyase Pf This invention utilizes bioinformatics methods to analyze and predict enzyme genes that may exhibit significant reducing activity against substrates, then selects and clones these genes for expression, constructing recombinant *E. coli* cells. By measuring the activity and stereoselectivity of the recombinant ThDP-dependent enzymes, the cloned enzymes are screened, ultimately obtaining the mutant enzyme with the best catalytic performance, derived from *Pseudomonas fluorescens* (…). Pseudomonas fluorescens AAA50176.1, named benzaldehyde lyase Pf BAL. 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 existing literature Gene, 144 (1994), 137-138, 10.1016 / 0378-1119(94)90218-6.

[0009] Preferably, the derived protein is composed of a novel amino acid sequence formed by substituting one or more amino acids of alanine at position 28, leucine at position 112, glutamine at position 113, and threonine at position 481 in the amino acid sequence shown in SEQ ID No. 1. Further preferred mutant amino acid sequences are as follows: (1) Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine; (2) Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with glycine; (3) Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with serine; (4) Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan; (5) Replace the alanine at position 28 of the amino acid sequence shown in SEQ ID No. 1 with tyrosine; (6) Replace the leucine at position 112 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine; (7) Replace the leucine at position 112 of the amino acid sequence shown in SEQ ID No. 1 with glycine; (8) Replace the leucine at position 112 of the amino acid sequence shown in SEQ ID No. 1 with serine; (9) Replace leucine at position 112 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan; (10) Replace the leucine at position 112 of the amino acid sequence shown in SEQ ID No. 1 with tyrosine; (11) Replace the leucine at position 112 of the amino acid sequence shown in SEQ ID No. 1 with alanine; (12) Replace glutamine at position 113 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine; (13) Replace glutamine at position 113 of the amino acid sequence shown in SEQ ID No. 1 with glycine; (14) Replace glutamine at position 113 of the amino acid sequence shown in SEQ ID No. 1 with serine; (15) Replace glutamine at position 113 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan; (16) Replace glutamine at position 113 of the amino acid sequence shown in SEQ ID No. 1 with alanine; (17) Replace glutamine at position 113 of the amino acid sequence shown in SEQ ID No. 1 with tyrosine; (18) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine; (19) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with glycine; (20) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with serine; (21) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan; (22) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with tyrosine; (23) Replace the threonine at position 481 of the amino acid sequence shown in SEQ ID No. 1 with alanine; Alternatively, the reaction may be carried out under the catalytic conditions of benzaldehyde lyase or its mutant, wherein 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 mutation of the sequence shown in SEQ ID NO:1, wherein the mutation includes: The alanine at position 28 should be replaced with any one of phenylalanine, glycine, serine, tryptophan, or tyrosine. And / or, the tyrosine at position 112 may be replaced with any one of phenylalanine, glycine, tyrosine, serine, tryptophan, or alanine. And / or, the threonine at position 113 may be replaced with any one of phenylalanine, glycine, serine, tryptophan, tyrosine, or alanine. And / or, the threonine at position 481 is replaced with any one of phenylalanine, glycine, serine, tryptophan, tyrosine, or alanine.

[0010] Furthermore, the enzyme catalyzing the reaction is selected from a benzaldehyde lyase mutant. Pf BAL Q113S / A28G, the aforementioned Pf The BAL Q113S / A28G amino acid sequence is selected from the amino acid sequence shown in SEQ ID No:2.

[0011] Furthermore, the catalytic reaction is an asymmetric reduction reaction, which is carried out in the presence of MgSO4 and ThDP.

[0012] Preferably, the concentration of the compound of Formula 1 is 30-90 mmol / L, the concentration of MgSO4 is 2.0-3.0 mmol / L, the concentration of ThDP is 0.1-0.3 mmol / L, and the reaction temperature is 10-30 °C.

[0013] And / or, more preferably, the concentration of the compound of Formula 1 is 75 mmol / L, the concentration of MgSO4 is 2.5 mmol / L, the concentration of ThDP is 0.15 mmol / L, and the reaction temperature is 20 °C.

[0014] The substrate 2-(2-formylphenoxy)acetonitrile can be prepared by the following exemplary method: 3-hydroxy-4H-1-benzopyran-4-one is prepared in phosphate buffer at pH 6.0 in the presence of MgSO4 and ThDP, using the ThDP-dependent enzyme and its mutant strain. In this reaction, MgSO4 and ThDP are added to the reaction system in the presence of MgSO4 (2.5 mmol / L) and ThDP (0.15 mmol / L) to facilitate coenzyme cycling. The phosphate buffer can be any phosphate buffer conventional in the art, and its concentration can be 50 mmol / L. The reduction reaction temperature can be 15–35 °C, preferably 20 °C. During the reaction, the conversion rate is determined intermittently, and the reaction time is based on the time it takes for the substrate to completely react or for the reaction to terminate spontaneously, typically 24 hours. The yield can be analyzed by nuclear magnetic resonance (NMR), preferably at a wavelength of 400 MHz.

[0015] After the reaction is complete, the reaction solution is extracted with an equal volume of a conventional water-insoluble organic solvent, such as ethyl acetate or dichloromethane. The extraction is repeated three times. The extracts are combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain a crude extract of 3-hydroxy-4H-1-benzopyran-4-one. Further purification by conventional methods, such as column chromatography, yields a highly chemically pure product.

[0016] In a third aspect, the present invention provides a benzaldehyde lyase mutant, wherein the amino acid sequence of the benzaldehyde lyase mutant is selected from the amino acid sequence shown in SEQ ID No:2.

[0017] Provided is an isolated nucleic acid that encodes the aforementioned ThDP-dependent enzyme (benzaldehyde lyase). Pf BAL or benzaldehyde lyase Pf The nucleic acid molecule of the BAL mutant, wherein the amino acid sequence of the benzaldehyde lyase mutant is selected from the amino acid sequence shown in SEQ ID No:2 below.

[0018] In a fourth aspect, this invention provides a recombinant expression vector containing the above-mentioned ThDP-dependent enzyme gene nucleic acid sequence. The recombinant expression vector can be constructed by cloning the above-mentioned enzyme gene into various vectors using conventional methods in the art. Preferably, the expression vector includes various vectors conventional in the art, such as commercially available plasmids, granules, bacteriophages, or viral vectors, etc., and the vector is preferably the pET28a plasmid. The amino acid sequence of the benzaldehyde lyase mutant is selected from the amino acid sequence shown in SEQ ID No:2 below.

[0019] In a fifth aspect, the present invention provides a recombinant expression transformant comprising the aforementioned ThDP-dependent enzyme gene or its recombinant expression vector. The amino acid sequence of the benzaldehyde lyase mutant is selected from the amino acid sequence shown in SEQ ID No:2. 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 satisfies the requirement that the recombinant expression vector can stably replicate spontaneously and that the ThDP-dependent enzyme gene it carries can be effectively expressed. The host cell is preferably *Escherichia coli*, more preferably *Escherichia coli* (…). E. coli BL21 (DE3) or Escherichia coli DH5α. The aforementioned recombinant expression vector was transformed into Escherichia coli (BL21 (DE3) or E. coli DH5α). E. coli The preferred genetically engineered strain of this invention can be obtained from BL21 (DE3). For example, the recombinant expression vector pET28a- Pf BAL is converted to Escherichia coli ( E. coli Recombinant Escherichia coli was obtained from BL21 (DE3). E. coli BL21(DE3) / pET28a- Pf BAL.

[0020] In a sixth aspect, the present invention provides a preferred method for preparing the recombinant ThDP-dependent enzyme: culturing the recombinant expression transformant as described above to obtain the recombinant ThDP-dependent enzyme. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art capable of enabling the transformant to grow and produce the recombinant ThDP-dependent enzyme of the present invention. The culture medium is preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl. There are no special limitations on the culture method and conditions; appropriate selections can be made according to the host cell type and culture method, based on conventional knowledge in the art, as long as the transformant can grow and produce the enzyme. Specific operations for culturing the transformant can be performed according to conventional operations in the art. The strain culture method preferably includes: culturing the recombinant *Escherichia coli* described in the present invention, for example... E. coli BL21 (DE3) / pET28a- Pf BAL was inoculated into LB medium containing kanamycin and cultured at 37 °C. When the optical density OD of the culture medium reached a certain level... 600 When the concentration reaches 0.5~1.0 (preferably 0.6), add isopropyl- to a final concentration of 0.1~1.0 mmol / L (preferably 1 mmol / L). βEnzyme production was induced by β-D-thiogalactopyranoside (IPTG), and the cells were cultured at 18 °C for 24 h to efficiently express the ThDP-dependent enzyme described in this invention. After culture, the bacterial cells were harvested by centrifugation, which are the resting cells of the recombinant expression transformant. The obtained bacterial cell pellet was freeze-dried to obtain frozen stem cells, which are beneficial for long-term storage and convenient for future use.

[0021] The activity of ThDP-dependent enzymes was determined by high-performance liquid chromatography (HPLC) by detecting changes in product formation. The activity of the ThDP-dependent enzymes was determined as follows: a 400 μL reaction system containing 75 mmol / L 2-(2-formylphenoxy)acetonitrile (50 mmol / L phosphate buffer, pH 6.0) was preheated to 20 °C, then an appropriate amount of ThDP-dependent enzyme was added, and the reaction was incubated at 20 °C. Product formation was detected by HPLC, and the yield change over 30 minutes was recorded.

[0022] Enzyme activity is calculated using the following formula: Enzyme activity (U) = Product production amount / Time Enzyme amount In the formula, the product amount is the amount of product generated within that time period, in μmmol; the time is the reaction time, in min; and the enzyme amount is the amount of pure enzyme added during the reaction, in mg.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] As used in this invention, "wild-type benzaldehyde lyase" refers to naturally occurring benzaldehyde that has not been artificially modified. The nucleotides of the lyase can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR), and their amino acid sequences can be deduced from the nucleotide sequences. The amino acid sequence of the wild-type benzaldehyde lyase is shown in SEQ ID NO:1. .

[0025] The mutant protein of this invention, as used herein, is referred to by the terms "mutant," "benzaldehyde lyase mutant protein," "mutant protein," "benzaldehyde lyase mutant protein," "mutant protein of this invention," "benzaldehyde lyase mutant protein of this invention," and "benzaldehyde lyase mutant of this invention," which are used interchangeably and all refer to benzaldehyde lyase that is not naturally occurring. The mutant protein is an artificially modified protein based on the protein shown in SEQ ID NO:1, and the mutant protein of this invention has highly efficient catalytic activity for the generation of optically pure ketone compounds.

[0026] The benzaldehyde lyase mutant described in this invention is... Pf BAL Q113S / A28G is an unknown mutant; the amino acid sequence is shown in SEQ ID NO:2. .

[0027] In this context, as used herein, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, "A28G" indicates that alanine A at position 28 is mutated to glycine G, and so on.

[0028] "Saturation mutagenesis" is a method that modifies the gene encoding a target protein to obtain mutants in a short period of time, where the target amino acid is replaced by one of 19 other amino acids. This method is not only a powerful tool for targeted protein modification but also an important means of studying protein structure-function relationships. Saturation mutagenesis often yields more desirable evolutionary variants than single-point mutagenesis. Furthermore, saturation mutagenesis excels at addressing problems that site-directed mutagenesis cannot solve. The mutants obtained through saturation mutagenesis are sequenced and identified, and their activity against different substrates and tolerance to high temperatures are tested.

[0029] Site-directed mutagenesis refers to the introduction of desired changes (usually changes indicating a favorable direction) into a target DNA fragment (which can be genomic or plasmid) using methods such as polymerase chain reaction (PCR). These changes include base addition, deletion, and point mutations. Site-directed mutagenesis can rapidly and efficiently improve the characteristics and characterization of the target protein expressed by the DNA, making it a very useful tool in gene research. The method of introducing site-directed mutagenesis using whole plasmid PCR is simple and effective, and is currently widely used. The principle is that a pair of primers (forward and reverse) containing the mutation site, along with a template plasmid, are annealed and then subjected to "cyclic extension" using polymerase. (Cyclic extension refers to the polymerase extending the primers according to the template, returning to the 5' end of the primer after one cycle, and then repeating the cycle of heating and annealing. This reaction differs from rolling circle amplification and does not form multiple tandem copies.) The extension products of the forward and reverse primers are annealed and paired to form a notched open circular plasmid. The Dpn I digestion extension product, since the original template plasmid is derived from conventional E. coli and is modified by dam methylation, is sensitive to Dpn I and is cleaved. However, the in vitro synthesized plasmid with the mutant sequence is not methylated and is not cleaved. Therefore, it can be successfully transformed in the subsequent transformation, and the clone of the mutant plasmid can be obtained.

[0030] The mutants screened from the mutant library were subjected to third-generation sequencing. Based on the sequencing results, suitable mutants were selected for amplification reaction activity testing. After multiple rounds of evolution, the inventors obtained mutants with significantly improved activity and selectivity for chromones. These mutants can be used for industrial production, resulting in a significant improvement in catalytic efficiency.

[0031] "Amino acid residues" can be represented by three-letter or one-letter amino acid codes according to standards known and agreed upon in the art. In this invention, the abbreviations for amino acid residues are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0032] Beneficial technical effects of the present invention: 1. The ThDP-dependent enzyme and its mutant strains provided by this invention can be used for the efficient catalytic production of chromone compounds. Using this enzymatic catalysis technology, the conversion rate can exceed 99%, and the product selectivity is greater than 80%. This invention has the advantage of high conversion rate, which is conducive to the efficient and low-cost production of chromones and has promising prospects for industrial application.

[0033] 2. This invention has advantages such as high substrate concentration, mild reaction conditions, environmental friendliness, and high yield, and therefore has good application prospects in industrial production. Attached Figure Description

[0034] Figure 1 Recombinant expression plasmid pET28a- Pf A schematic diagram of the BAL construction. Detailed Implementation

[0035] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0036] The materials used in the following embodiments are sourced from: Recombinant expression plasmid pET28a- Pf BAL was purchased from Nanjing Genscript Company.

[0037] E. coli DH5α and E. coli BL21(DE3) competent cells and agarose gel DNA recovery kit were purchased from Beijing Tiangen Biotech Co., Ltd.

[0038] F. Molecular biology reagents such as the restriction enzyme DpnI, PCR extraction kits, and plasmid mini kits were purchased from ThermoScientific and Omega Biotek.

[0039] Example 1: Benzaldehyde lyase Pf Preparation of BAL recombinant expression plasmid and recombinant expression transformant plasmid pET28a- Pf The construction process of BAL is as follows: First, using the target gene... Pf Using BAL as a template, PCR amplification was performed, and suitable restriction enzyme sites were introduced into the primers. Then, the pET-28a(+) vector was linearized with the appropriate restriction endonuclease to give it sticky ends compatible with the insert fragment. Next, DNA ligase was used to ligate... Pf The BAL fragment was ligated into a linearized vector to construct a recombinant plasmid. Subsequently, the recombinant plasmid was transformed into *E. coli*, and positive clones were selected by kanamycin resistance screening. Finally, restriction enzyme digestion and sequencing were used to verify the correct insertion, ensuring... PfThe BAL gene was successfully cloned into pET-28a(+), retaining the C-terminal His tag for subsequent protein expression and purification. See the attached diagram in the instruction manual. Figure 1 .

[0040] Transform the recombinant plasmid into E. coli DH5α was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated at 37 ºC for 8 hours. Colony growth was verified by colony PCR and sequencing. The corresponding plasmid was then extracted and further transformed into... E. coli BL21 (DE3) was used to select positive clones, thus obtaining the recombinant expression transformants. E. coli BL21(DE3) / pET28a- Pf BAL.

[0041] Example 2: Benzaldehyde lyase Pf Construction of BAL Q113S / A28G mutant Benzaldehyde lyase was constructed by directed evolution of wild-type enzymes through molecular docking. Pf BAL's mutation library: selection Pf Single-point mutation of non-conserved residues within the BAL substrate binding pocket was performed. Mutation primers were designed, and their sequences are shown in Table 1, number 5. The primers used were pET28a- Pf Using BAL as a template, PCR was performed using the high-fidelity polymerase PrimeSTAR (Premix).

[0042] The PCR reaction conditions were as follows: In a PCR reaction system with a total volume of 25 μL, 0.5–20 ng of template, 12 μL of 2×PrimeSTAR (Premix), 0.5 μL of each of the two mutant primers, and 0.5 μL of DMSO were added to sterile distilled water to a final volume of 25 μL. The PCR reaction procedure was as follows: (1) denaturation at 98 °C for 10 sec, (2) annealing at 60 °C for 30 sec, and (3) extension at 72 °C for 120 sec. Steps (1) to (3) were repeated for a total of 30 cycles. The product was stored at 4 °C. After verification by agarose gel electrophoresis, the PCR product was digested with restriction endonuclease DpnI at 37 °C for 2 h. The digested product was then transferred to… E. coli BL21 (DE3) competent cells were plated on agar plates containing kanamycin and incubated statically at 37 °C for approximately 12 h. The resulting monoclonal colonies were then picked and cultured in test tubes for benzaldehyde lyase activity. Pf The genes of the BAL Q113S / A28G mutant were sequenced.

[0043] Based on the same construction process described above, different mutant primer sequences were designed, as shown in the mutant primer series numbered 1-5, 6-11, 12-17, and 18-23, and the corresponding genes were sequenced.

[0044] Table 1. Mutant primer sequences ; Example 3 Benzaldehyde lyase Pf BAL inducible expression The recombinant expression transformant obtained in Example 1 E. coli BL21 (DE3) / pET28a- Pf BAL was inoculated into LB medium containing 50 μg / ml kanamycin and cultured at 37 °C with shaking for 12 hours. Then, 1% (v / v) was inoculated into 500 mL Erlenmeyer flasks containing 100 mL of LB medium and cultured at 37 °C with shaking at 220 rpm. When the OD of the culture medium... 600 When the concentration 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 medium was centrifuged at 8000 rpm for 10 min, the cells were collected, and stored at -80 °C.

[0045] The resting cells obtained by the above method were suspended in phosphate buffer at pH 6.0 and sonicated in an ice-water bath. The supernatant was collected by centrifugation, which was the crude enzyme solution of recombinant benzaldehyde lyase. Analysis of the crude enzyme solution by polyacrylamide gel electrophoresis showed that the benzaldehyde lyase existed in a soluble form.

[0046] Example 4 Benzaldehyde lyase Pf Inducible expression of BAL Q113S / A28G mutant The recombinant expression transformant obtained in Example 2 E. coli BL21 (DE3) / pET28a- Pf BALmutants were inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37 °C with shaking for 12 hours. Then, at a 1% (v / v) inoculation rate, they were transferred to 500 mL Erlenmeyer flasks containing 100 mL of LB medium and cultured at 37 °C with shaking at 180 rpm. The culture medium was then... 600 When the concentration 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 medium was centrifuged at 8000 rpm for 10 min, the cells were collected, and stored at -80 °C.

[0047] Example 5 pH PfBAL mutants (benzaldehyde lyase) Pf Effect of BAL Q113S / A28G mutant on catalytic activity The reaction was carried out in a 2 mL centrifuge tube, with 400 μl of the solution prepared as in Example 4 added to 500 μl of buffer (50 mmol / L, phosphate buffer). Pf BAL mutants pure enzyme solution (benzaldehyde lyase) Pf The BAL Q113S / A28G mutant was added, along with 2-(2-formylphenoxy)acetonitrile, MgSO4, and ThDP to final concentrations of 30 mmol / L, 2.5 mmol / L, and 0.15 mmol / L, respectively. A pH gradient of 6.0, 6.5, 7.0, 7.5, and 8.0 was controlled. The mixture was placed in a constant-temperature mixer and reacted for 16 hours. Samples were taken to detect the reaction conversion rate and the ratio of main and by-products. The results showed that the yield of the main product was as high as 80% at pH 6.0; therefore, pH 6.0 is the optimal reaction condition for this reaction.

[0048] Synthesis and Preparation Example 1 Pf BAL mutants (benzaldehyde lyase) Pf BAL Q113S / A28G mutant) catalyzes the synthesis of chromone compounds via cyano conversion. ; Add the following to 0.4 mL of phosphate buffer (50 mmol / L, pH 6.0) as described in Example 4. Pf BALmutants benzaldehyde lysin Pf The purified enzyme solution of BAL Q113S / A28G mutant was mixed with the corresponding cyano derivative substrate (Formula 1) to a final concentration of 75 mmol / L, followed by the addition of MgSO4 to a final concentration of 2.5 mmol / L and ThDP to a final concentration of 0.15 mmol / L. The mixture was placed in a constant-temperature mixer and reacted at 20 °C and 1000 rpm for 16 hours. The reaction was then terminated, and the mixture was extracted three times with an equal volume of ethyl acetate. The extracts were combined, dried over anhydrous sodium sulfate, and an equal volume of extract was evaporated to dryness. The yield was determined by 1H NMR spectroscopy, showing a main product yield of 80% and a byproduct yield of 10%. 1 H NMR (400 MHz, CDCl3) d 8.26 (dd, J = 8.1, 1.7Hz, 1H), 8.01 (s, 1H), 7.70 - 7.66 (m, 1H), 7.50 (d, J = 7.5 Hz, 1H), 7.41 (t, J= 7.6 Hz, 1H). Table 2 Benzaldehyde lyase Pf The BAL Q113S / A28G mutant catalyzes the cyano-catalyzed reaction of chromone compounds. ; Synthesis and Preparation Example 2 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, CDCl3) d 8.10 (d, J = 6.5 Hz, 1H), 8.06 (s, 1H), 7.51 (d, J = 7.1 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 2.49 (s, 3H). Synthesis and Preparation Example 3 Under the same reaction conditions as in Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, CDCl3) d 8.13 (d, J = 8.2 Hz, 1H), 7.96 (s,1H), 7.27 (s, 1H), 7.21 (d, J = 8.2 Hz, 1H), 2.48 (s, 3H). Synthesis and Preparation Example 4 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, CDCl3) d 8.03 (s, 1H), 7.99 (s, 1H), 7.48 (dd, J = 8.7, 2.2 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 2.46 (s, 3H). Synthesis and Preparation Example 5 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, CDCl3) d 8.08 (s, 1H), 7.81 (d, J= 8.1 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 7.9 Hz, 1H), 4.01 (s, 3H). Synthesis and Preparation Example 6 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, DMSO- d 6) d 9.01 (s, 1H), 8.13 (s, 1H), 7.99(d, J = 8.9 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 8.9, 2.4 Hz, 1H), 3.87(s, 3H). Synthesis and Preparation Example 7 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, DMSO- d 6) d 9.11 (s, 1H), 8.20 (s, 1H), 7.55(d, J = 9.1 Hz, 1H), 7.43 (d, J = 3.1 Hz, 1H), 7.33 (dd, J = 9.1, 3.1 Hz, 1H), 3.84(s, 3H). Synthesis and Preparation Example 8 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, CDCl3) d 8.14 (d, J = 9.6 Hz, 1H), 8.05 (s, 1H), 7.53 (d, J = 7.2 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 6.06 – 5.96 (m, 1H), 5.15 – 5.09 (m, 2H), 3.64(d, J = 6.5 Hz, 2H). Synthesis and Preparation Example 9 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, CDCl3) d 8.09 (s, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 7.9 Hz, 1H), 4.21 (q, J = 7.0 Hz, 2H), 1.54 (t, J = 7.0 Hz, 3H). Synthesis and Preparation Example 10 Under the same reaction conditions as in Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, DMSO- d 6) d 9.40 (s, 1H), 8.34 (s, 1H), 8.05 (dd, J = 8.1, 1.5 Hz, 1H), 7.91 (dd, J = 7.7, 1.5 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H). Synthesis and Preparation Example 11 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, CDCl3) d 8.23 (d, J = 8.0 Hz, 1H), 8.11 (s, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H). Synthesis and Preparation Example 12 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, DMSO- d 6) d9.31 (s, 1H), 8.23 ​​(s, 1H), 8.09 (d, J = 8.6 Hz, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.47 (dd, J = 8.6, 2.0 Hz, 1H). Synthesis and Preparation Example 13 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, CDCl3) d 8.28 (dd, J = 8.7, 6.2 Hz, 1H), 7.99 (s, 1H), 7.19 –7.14 (m, 2H), 6.23 (s, 1H). Synthesis and Preparation Example 14 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, CDCl3) d 8.03 (s, 1H), 7.88 (dd, J = 8.1, 3.1 Hz, 1H), 7.52 (dd, J = 9.2, 4.2 Hz, 1H), 7.45 – 7.40 (m, 1H), 6.22 (s, 1H). Synthesis and Preparation Example 15 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, CDCl3) d 8.58 (s, 1H), 8.06 (s, 1H), 7.90 (dd, J = 8.9, 2.3 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H). Synthesis and Preparation Example 16 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 H NMR (400 MHz, DMSO- d 6) d 9.43 (s, 1H), 8.67 (d,J = 2.2Hz, 1H), 8.29 (s, 1H), 8.23 ​​(dd, J = 8.9, 2.2 Hz, 1H), 7.75 (d, J = 8.9 Hz, 1H), 3.91 (s, 3H). Synthesis and Preparation Example 17 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, DMSO- d 6) d 9.15 (s, 1H), 8.18 (s, 1H), 7.74 – 7.69 (m, 1H), 7.43(d, J = 8.5 Hz, 1H), 7.18 (dd, J = 11.4, 8.0 Hz, 1H). Synthesis and Preparation Example 18 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, DMSO- d 6) d 9.07 (s, 1H), 8.23 ​​(s, 1H), 7.71 (s, 1H), 7.41 (s,1H), 2.38 (s, 3H), 2.36 (s, 3H). Synthesis and Preparation Example 19 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, DMSO- d 6) d 9.35 (s, 1H), 8.45 (d, J = 9.5 Hz, 1H), 8.43 (s, 1H), 8.10 – 8.08 (m, 1H), 8.05 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.81 –7.74 (m, 2H). Synthesis and Preparation Example 20 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. ,1 HNMR (400 MHz, DMSO- d 6) d 8.83 (s, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.72 (t, J = 7.7Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 2.39 (s, 3H). Synthesis and Preparation Example 21 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, DMSO- d 6) d 8.84 (s, 1H), 7.60 – 7.57 (m, 1H), 7.32 (d, J = 4.7 Hz, 2H), 3.93 (s, 3H), 2.40 (s, 3H). Synthesis and Preparation Example 22 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, DMSO- d 6) d 8.60 (s, 1H), 7.58 (dd, J = 8.2, 1.5 Hz, 1H), 7.06 (d, J =8.2 Hz, 1H), 6.88 – 6.84 (m, 1H), 6.77 – 6.73 (m, 1H), 6.70 (s, 1H). Synthesis and Preparation Example 23 Under the same reaction conditions as in Synthetic Preparation Example 1, and with the substrate structure shown in Table 2, the product was obtained. , 1 HNMR (400 MHz, DMSO- d 6) d 9.23 (s, 1H), 8.37 (d, J = 6.8 Hz, 1H), 7.85 (d, J = 8.1Hz, 1H), 7.69 (t, J= 6.9 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 2.36 (s, 3H). Synthesis and Preparation Example 24 Pf BAL mutants (benzaldehyde lyase) Pf Scale-up reaction of 3-hydroxy-4H-1-benzopyran-4-one synthesized catalyzed by BAL Q113S / A28G mutant Add the following to 40 mL of phosphate buffer (50 mmol / L, pH 6.0) as described in Example 4. Pf The crude BALmutants enzyme solution was mixed with the corresponding cyano compound substrate (Formula 1) at a final concentration of 75 mmol / L, and MgSO4 at a final concentration of 2.5 mmol / L and ThDP at a final concentration of 0.15 mmol / L were added. The mixture was placed in a shaker and reacted at 20 °C and 200 rpm for 24 hours. The reaction was then terminated, and the mixture was extracted three times with an equal volume of ethyl acetate. The extracts were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain 0.386 g of pure product 3-hydroxy-4H-1-benzopyran-4-one, with a yield of 80%.

[0049] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for synthesizing compound 2 by benzaldehyde lyase or a mutant thereof, wherein the reaction formula is as follows: ； 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 mutation of the sequence shown in SEQ ID NO:1; in, X is selected from O, S, or N; n is selected from 1, 2, or 3; R1 is selected from the following groups: hydrogen, halogen, C1~C6 alkyl, C1~C6 alkoxy, halogen-substituted C1~C6 alkyl, -COOC1~C6 alkyl, C2~C6 alkenyl or C2~C6 alkenyl C1-C6 alkyl; when n is selected from 2 and the substituent R1 group is adjacent, the adjacent R1 group and the C atom attached to it can form a C6-C10 aryl group; R2 is selected from the following groups: hydrogen, halogen-substituted C1~C6 alkyl or C1~C6 alkyl.

2. The method according to claim 1, characterized in that: in, X is selected from: O or S; n is selected from 1 or 2; R1 is selected from the following groups: hydrogen, halogen, C1~C6 alkyl, C1~C6 alkoxy, halogen-substituted C1~C6 alkyl, -COOC1~C6 alkyl, C2~C6 alkenyl or C2~C6 alkenyl C1-C6 alkyl; when n is selected from 2 and the R1 groups are adjacent, the adjacent R1 groups and the C atoms attached to them can form a phenyl group. R2 is selected from the following groups: hydrogen or C1~6 alkyl.

3. The method according to claim 1 or 2, characterized in that: n is selected from 1 or 2; R1 is selected from methyl, methoxy, ethoxy, allyl, phenylcycloyl, methyl ester, fluorine, chlorine, bromine, or trifluoromethyl. The R2 is selected from: hydrogen or methyl; X is selected from: O or S; Further preferably, Formula I is selected from the following compounds: , , , , , , , , , , , , , , , , , , , , or .

4. The method according to claim 1 or 2, characterized in that: The amino acid sequence of the benzaldehyde lyase mutant was obtained by mutation of the sequence shown in SEQ ID NO:1, wherein the mutation includes: The alanine at position 28 should be replaced with any one of phenylalanine, glycine, serine, tryptophan, or tyrosine. And / or, glutamine at position 113 may be replaced with any one of phenylalanine, glycine, serine, tryptophan, tyrosine, or alanine. And / or, the leucine at position 112 may be replaced with any one of phenylalanine, glycine, tyrosine, serine, tryptophan, or alanine. And / or, the threonine at position 481 is replaced with any one of phenylalanine, glycine, serine, tryptophan, tyrosine, or alanine.

5. The method according to claim 4, wherein the catalytic reaction is selected from a benzaldehyde lyase mutant. Pf BAL Q113S / A28G, the aforementioned Pf The BAL Q113S / A28G amino acid sequence is selected from the amino acid sequence shown in SEQ ID No:

2.

6. The preparation method according to claim 5, characterized in that, The catalytic reaction is a cyano conversion reaction, which is carried out in the presence of MgSO4 and ThDP. Preferably, the concentration of the compound of Formula 1 is 30-90 mmol / L, the concentration of MgSO4 is 2.0-3.0 mmol / L, the concentration of ThDP is 0.1-0.3 mmol / L, and the reaction temperature is 10-30 °C. And / or, more preferably, the concentration of the compound of Formula 1 is 75 mmol / L, the concentration of MgSO4 is 2.5 mmol / L, the concentration of ThDP is 0.15 mmol / L, and the reaction temperature is 20 °C.

7. A benzaldehyde lyase mutant, characterized in that: The amino acids of the benzaldehyde lyase mutant are selected from the amino acid sequence shown in SEQ ID No:

2.

8. An isolated nucleic acid, characterized in that: The nucleic acid described encodes a mutant such as benzaldehyde lyase. Pf The nucleic acid molecule BALQ113S / A28G, the benzaldehyde lyase mutant Pf The BAL Q113S / A28G amino acid sequence is selected from the amino acid sequence shown in SEQ ID No:

2.

9. Contains a benzaldehyde lyase mutant. Pf The expression vector encoding the BAL Q113S / A28G gene, the benzaldehyde lyase mutant Pf The BAL Q113S / A28G amino acid sequence is selected from the amino acid sequence shown in SEQ ID No:

2.

10. Contains a benzaldehyde lyase mutant. Pf Recombinant cells encoding the BAL Q113S / A28G gene, the benzaldehyde lyase mutant Pf The BAL Q113S / A28G amino acid sequence is selected from the amino acid sequence shown in SEQ ID No:2.

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