A method for the enzymatic synthesis of gamma-hydroxy keto acids

Abstract

The present disclosure provides a method for the enzyme-catalyzed synthesis of gamma-hydroxy keto acid, which comprises reacting an aldehyde compound represented by formula (A) or a ketone compound represented by formula (D) with a keto acid compound represented by formula (B) in the presence of an aldolase to obtain a gamma-hydroxy keto acid represented by formula (C) or formula (E). The method has the remarkable characteristics of mild conditions, no pollution, wide universality, and high product conversion rate.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis, specifically relating to a method for enzyme-catalyzed synthesis of γ-hydroxyketo acids, and their applications. Background Technology

[0002] γ-hydroxyketo acids and their 2-hydroxy-4-butyrolactone derivatives are bioactive components widely found in natural products, synthetic pharmaceuticals, and biodegradable polymers. C-C bond formation reactions are widely considered a key strategy for the synthesis of organic molecules. Among the many C-C bond formation methods, the biosynthesis of γ-hydroxyketo acids via the condensation of aldehydes with pyruvate catalyzed by aldolases is a promising approach.

[0003] However, aldolases typically exhibit high substrate selectivity, enhancing the broad spectrum of industrial enzymes and enabling them to accept a wider range of substrates, thus expanding their substrate spectrum and playing a significant role in their application in industrial biocatalysis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this disclosure provides a method for synthesizing various types of γ-hydroxyketo acids via enzyme catalysis. The aldolase biocatalysis method used in this disclosure offers relatively mild reaction conditions, high reaction efficiency, and significant advantages such as environmental friendliness, lack of pollution, and simple process route. In particular, it possesses a broader substrate spectrum, offering a wider substrate range compared to traditional synthetic methods, accepting not only aldehydes but also ketones as substrates.

[0005] According to one aspect of this disclosure, the use of aldolases in the enzymatic synthesis of γ-hydroxyketo acids having the structure shown in formula (C) or (E) is provided.

[0006]

[0007] R1, R3 and R4 are each independently selected from C1-C10 straight-chain or branched alkyl groups, C6-C10 aryl groups or C3-C10 heteroaryl groups;

[0008] R2 is selected from H or C1-C10 straight-chain or branched alkyl groups;

[0009] The alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from hydroxyl, halogen, nitro, C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, CH3S-substituted C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy.

[0010] In some embodiments, R1 is selected from C1-C6 straight-chain or branched alkyl, C6-C10 aryl, or C3-C6 heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted by one or more substituents selected from nitro, C1-C3 alkyl, hydroxyl-substituted C1-C3 alkyl, or C1-C3 alkoxy.

[0011] In some embodiments, R2 is selected from H or C1-C6 straight-chain or branched alkyl groups, which are optionally substituted with one or more substituents selected from C1-C3 alkyl groups, halogen-substituted C1-C3 alkyl groups, or hydroxyl-substituted C1-C3 alkyl groups.

[0012] In some embodiments, R3 and R4 are each independently selected from C1-C6 straight-chain or branched alkyl groups, which are optionally substituted by one or more substituents selected from C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, or C1-C3 alkoxy groups.

[0013] In some embodiments, the heteroatom in the heteroaryl group is selected from N, O, or S.

[0014] In some embodiments, the halogen is selected from F, Cl, or Br.

[0015] In some embodiments, the aldolase comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with it.

[0016] In some embodiments, the aldolase is derived from fungal yeast, preferably from Candida humicola.

[0017] In some embodiments, the aldolase is obtained by expression in a host cell containing a recombinant vector that contains the coding sequence of the aldolase.

[0018] In some embodiments, the host cell is selected from bacteria, fungi, insect cells, plant cells or animal cells, and more preferably, the cell is selected from Escherichia coli, Bacillus subtilis or yeast.

[0019] According to another aspect of this disclosure, a method for the enzyme-catalyzed synthesis of γ-hydroxyketo acid is provided, the method comprising reacting an aldehyde compound of formula (A) with a keto acid compound of formula (B) in the presence of an aldolase to obtain the γ-hydroxyketo acid of formula (C).

[0020]

[0021]

[0022] Wherein, R1 is selected from C1-C10 straight-chain or branched alkyl, C6-C10 aryl, or C3-C10 heteroaryl; R2 is selected from H or C1-C10 straight-chain or branched alkyl; the alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from hydroxyl, halogen, nitro, C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, CH3S-substituted C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy.

[0023] In some embodiments, R1 is selected from C1-C6 straight-chain or branched alkyl, C6-C10 aryl, or C3-C6 heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted by one or more substituents selected from nitro, C1-C3 alkyl, hydroxyl-substituted C1-C3 alkyl, or C1-C3 alkoxy.

[0024] In some embodiments, R2 is selected from H or C1-C6 straight-chain or branched alkyl groups, which are optionally substituted with one or more substituents selected from C1-C3 alkyl groups, halogen-substituted C1-C3 alkyl groups, or hydroxyl-substituted C1-C3 alkyl groups.

[0025] In some embodiments, the heteroatom in the heteroaryl group is selected from N, O, or S.

[0026] In some embodiments, the halogen is selected from F, Cl, or Br.

[0027] In some embodiments, the aldehyde compound represented by formula (A) is selected from the following compounds:

[0028]

[0029] In some embodiments, the keto acid compound represented by formula (B) is selected from the following compounds:

[0030]

[0031] In some embodiments, the molar ratio of the aldehyde compound represented by formula (A) to the keto acid compound represented by formula (B) is (0.1-10):1, preferably (0.5-2):1. In some specific embodiments, the molar ratio of the aldehyde compound represented by formula (A) to the keto acid compound represented by formula (B) is 0.1:1, 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 4:1, 6:1, 8:1, 10:1, or any value between them.

[0032] In some embodiments, the molar concentration of the aldehyde compound represented by formula (A) is 0.5-500 mM, preferably 10-100 mM. In some specific embodiments, the molar concentration of the aldehyde compound represented by formula (A) is 0.5 mM, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or any value between them.

[0033] In some embodiments, the molar concentration of the keto acid compound represented by formula (B) is 0.5-500 mM, preferably 10-100 mM. In some specific embodiments, the molar concentration of the keto acid compound represented by formula (B) is 0.5 mM, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or any value between them.

[0034] In some embodiments, the molar concentration of the aldolase is 0.01-2 mg / mL, preferably 0.1-1 mg / mL. In some specific embodiments, the molar concentration of the aldolase is 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or any value between them.

[0035] In some embodiments, the reaction system further comprises divalent metal ions. Preferably, the divalent metal ions include Mg. 2+ Ni 2+ Co 2+ Zn 2+ or Ca 2+ One or more of the following. In some specific embodiments, the reaction system of the reaction also contains MgCl2.

[0036] In some embodiments, the concentration of the divalent metal ion is 0.1-10 mM, for example, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM or any value between them.

[0037] In some embodiments, the reaction temperature is 20-65°C, preferably 35-50°C, and more preferably 38-42°C. In some specific embodiments, the reaction temperature is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or any value between them.

[0038] In some embodiments, the reaction time is 2-24 hours, preferably 8-12 hours. In some specific embodiments, the reaction time is 2 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 20 hours, 24 hours, or any value between them.

[0039] In some embodiments, the pH of the reaction is 5.0-9.0, preferably 7.0-8.5, and more preferably 7.5-8.0. In some specific embodiments, the pH of the reaction is 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or any value between them.

[0040] In some embodiments, the buffer used for the reaction includes one or more of phosphate buffer, Tris hydrochloride buffer, and HEPES buffer, preferably phosphate buffer.

[0041] In some embodiments, the solvent used to dissolve the substrate aldehyde or ketone compound in the reaction is one or more of water, dimethyl sulfoxide, acetonitrile, or ethanol, preferably water or acetonitrile.

[0042] According to another aspect of this disclosure, a method for the enzyme-catalyzed synthesis of γ-hydroxyketo acid is provided, the method comprising reacting a ketone compound of formula (D) with a keto acid compound of formula (B) in the presence of an aldolase to obtain the γ-hydroxyketo acid of formula (E).

[0043]

[0044] R3 to R4 are each independently selected from C1-C10 straight-chain or branched alkyl, C6-C10 aryl, or C3-C10 heteroaryl; R2 is selected from H or C1-C10 straight-chain or branched alkyl; the alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from hydroxyl, halogen, nitro, C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, CH3S-substituted C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy.

[0045] In some embodiments, R2 is selected from H or C1-C6 straight-chain or branched alkyl groups, which are optionally substituted with one or more substituents selected from C1-C3 alkyl groups, halogen-substituted C1-C3 alkyl groups, or hydroxyl-substituted C1-C3 alkyl groups.

[0046] In some embodiments, R3 and R4 are each independently selected from C1-C6 straight-chain or branched alkyl groups, which are optionally substituted by one or more substituents selected from C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, or C1-C3 alkoxy groups.

[0047] In some embodiments, the heteroatom in the heteroaryl group is selected from N, O, or S.

[0048] In some embodiments, the halogen is selected from F, Cl, or Br.

[0049] In some embodiments, the ketone compound represented by formula (D) is selected from the following compounds:

[0050]

[0051] In some embodiments, the keto acid compound represented by formula (B) is selected from the following compounds:

[0052]

[0053] In some embodiments, the aldolase comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with it.

[0054] In some embodiments, the aldolase is derived from fungal yeast, preferably from Candida humicola.

[0055] In some embodiments, the aldolase is obtained by expression in a host cell containing a recombinant vector that contains the coding sequence of the aldolase.

[0056] In some embodiments, the host cell is selected from bacteria, fungi, insect cells, plant cells or animal cells, and more preferably, the cell is selected from Escherichia coli, Bacillus subtilis or yeast.

[0057] In some embodiments, the molar ratio of the ketone compound represented by formula (D) to the keto acid compound represented by formula (B) is (0.1-10):1, preferably (0.5-2):1. In some specific embodiments, the molar ratio of the ketone compound represented by formula (D) to the keto acid compound represented by formula (B) is 0.1:1, 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 4:1, 6:1, 8:1, 10:1, or any value between them.

[0058] In some embodiments, the molar concentration of the ketone compound represented by formula (D) is 0.5-500 mM, preferably 10-100 mM. In some specific embodiments, the molar concentration of the ketone compound represented by formula (D) is 0.5 mM, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or any value between them.

[0059] In some embodiments, the molar concentration of the keto acid compound represented by formula (B) is 0.5-500 mM, preferably 10-100 mM. In some specific embodiments, the molar concentration of the keto acid compound represented by formula (B) is 0.5 mM, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or any value between them.

[0060] In some embodiments, the molar concentration of the aldolase is 0.01-2 mg / mL, preferably 0.1-1 mg / mL. In some specific embodiments, the molar concentration of the aldolase is 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or any value between them.

[0061] In some embodiments, the reaction system further comprises divalent metal ions. Preferably, the divalent metal ions include Mg. 2+ Ni 2+ Co 2+ Zn 2+ or Ca 2+ One or more of the following. In some specific embodiments, the reaction system of the reaction also contains MgCl2.

[0062] In some embodiments, the concentration of the divalent metal ion is 0.1-10 mM, for example, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM or any value between them.

[0063] In some embodiments, the reaction temperature is 20-65°C, preferably 35-50°C, and more preferably 38-42°C. In some specific embodiments, the reaction temperature is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or any value between them.

[0064] In some embodiments, the reaction time is 2-24 hours, preferably 8-12 hours. In some specific embodiments, the reaction time is 2 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 20 hours, 24 hours, or any value between them.

[0065] In some embodiments, the pH of the reaction is 5.0-9.0, preferably 7.0-8.5, and more preferably 7.5-8.0. In some specific embodiments, the pH of the reaction is 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or any value between them.

[0066] In some embodiments, the buffer used for the reaction includes one or more of phosphate buffer, Tris hydrochloride buffer, and HEPES buffer, preferably phosphate buffer.

[0067] In some embodiments, the solvent used to dissolve the substrate aldehyde or ketone compound in the reaction is one or more of water, dimethyl sulfoxide, acetonitrile, or ethanol, preferably water or acetonitrile.

[0068] In some embodiments, the preparation method includes a step of catalyzing the production of γ-hydroxyketo acids using an aldolase derived from Vanrija humicola. The reaction product is a bioactive component widely found in natural products, synthetic pharmaceuticals, and biodegradable polymers, and has high synthetic value.

[0069] In some specific embodiments, the preparation method includes:

[0070] (1) The gene of aldolase was constructed into genetically engineered bacteria and induced to express aldolase.

[0071] (2) Add the obtained aldolase to the reaction system and carry out the reaction under appropriate conditions.

[0072] The aldolase used to prepare this application is a separated product obtained by breaking, separating, and purifying bacterial cells obtained by centrifuging the culture medium using a nickel column.

[0073] In some embodiments, the aldolase is derived from Vanrija humicola, and after codon optimization, the gene is fully synthesized and introduced into a corresponding plasmid vector. In some embodiments, the aldolase can be expressed in Escherichia coli, for example, but not limited to, BL21, Rosetta, etc.

[0074] According to another aspect of this application, the use of γ-hydroxyketo acids obtained by the methods described in this disclosure in the production of plastics, food, or pharmaceuticals is provided.

[0075] According to another aspect of this application, the use of hydroxyl lactones formed by acid treatment and cyclization of γ-hydroxyketo acids obtained by the methods of this disclosure in the production of plastics, food or pharmaceuticals is provided.

[0076] This disclosure describes a method for forming carbon-carbon bonds by aldehyde or ketone compounds undergoing aldol condensation with keto acids in the presence of aldolase, ultimately yielding γ-hydroxyketo acids. This method is characterized by mild conditions, lack of pollution, wide applicability, and high product conversion rate. Attached Figure Description

[0077] Figure 1 The SDS-PAGE image after VhAld expression is shown. M: marker; 1: VhAld precipitate after lysis and centrifugation; 2: VhAld supernatant; 3: VhAld flow-through after column loading; 4: 15% B wash 1; 5: 15% B wash 2; 6: VhAld obtained after final desalting.

[0078] Figure 2 The optimal pH for the VhAld aldolase reaction was shown.

[0079] Figure 3 The optimal temperature for the VhAld aldolase reaction was shown.

[0080] Figure 4 The HPLC chromatograms after the representative substrate reaction are shown (reaction of donor substrate 1 and acceptor substrate 21). Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications. The compounds, reagents, and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0082] Example 1. Construction of gene cloning and expression vectors

[0083] Codon optimization was performed on the pyruvate aldolase VhAld (GenBank: TXT13602.1) from Vanrija humicola, enabling the expression of this fungal gene in the Escherichia coli host. The encoded amino acid sequence is shown in SEQ ID NO:1 (MASALASKLHLKNALAAGEKGIGFWLTFPGPHVVRAVSGIKGF NWACVDGEHGQIADPDYYELCNALVANGVSPIIRVPNAEEWMVKRALDAGAHGVMTPMCHTADDARKIVSWNKYPPNGTRGFGPIYAPHAFGYAAEAEYGAAADDSLVVMVQIESRLGVENVEEIAKVDGLDVLLIGPFDLSKSMGVAFGGDEHQAAIARILAAAKAAGKAAAIFCVNGDQAATRLAQGFEVVSIGTDIGSLVADMTRQVAAATGVSAQKKAGGYS). Simultaneously, NdeI and BamHI restriction sites were added to both ends of the gene, and the recombinant plasmid was constructed into the pET-16b vector to prepare the recombinant plasmid.

[0084] The prepared recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells by chemical transformation. The transformation conditions were: heat shock at 42°C for 60 seconds, ice bath for 3 minutes, followed by recovery in an LB culture medium at 37°C for 1 hour. Finally, the plasmid was plated on an LB agar plate containing 100 μg / mL ampicillin and incubated overnight at 37°C to obtain the recombinant transformant.

[0085] The LB medium (1L) consisted of 10g peptone, 10g sodium chloride, 5g yeast extract, and pH 7.

[0086] Example 2. Expression and purification of pyruvate aldolase

[0087] Single colonies of *E. coli* containing the pyruvate aldolase expression plasmid were picked from agar plates and inoculated into 6 mL of LB broth containing 100 μg / mL ampicillin. The culture was incubated overnight at 37°C with shaking as a seed culture. This seed culture was then inoculated into Erlenmeyer flasks containing 600 mL of LB broth at a 1% (v / v) inoculation rate. The flasks were incubated at 37°C until the OD value reached approximately 0.6-0.8. Then, 0.1 mM IPTG was added to induce pyruvate aldolase expression. Incubation was performed overnight at 16°C and 230 rpm for 16-20 h. The bacterial cells were collected after incubation at 6000 rpm for 15 min.

[0088] The collected bacterial cells were resuspended in buffer A (50 mM phosphate buffer, 500 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0) and disrupted using conventional methods such as sonication or autoclaving. The cells were then centrifuged at low temperature, filtered, and the supernatant was retained. The supernatant was transferred to a nickel column equilibrated with buffer A, and then the column was washed with wash buffer (50 mM phosphate buffer, 500 mM NaCl, 75 mM imidazole, pH 8.0, 10% glycerol) to remove contaminating proteins. After elution, the target protein was eluted with buffer C (50 mM phosphate buffer, 500 mM NaCl, 500 mM imidazole, 10% glycerol, pH 8.0) and collected. Finally, pyruvate aldolase was desalted and stored using buffer D (20 mM HEPES, 300 mM NaCl, pH 7.5, 10% glycerol). Electrophoresis gel images of the purification process are shown below. Figure 1 .

[0089] Example 3. Optimization of enzyme reaction conditions

[0090] Optimize the optimal pH and reaction temperature of VhAld enzyme using pyruvate and glyceraldehyde as substrates.

[0091] (1) Optimal pH screening, reaction conditions: 50 mM pyruvate (dissolved in water to prepare a 2 M stock solution) and 50 mM glyceraldehyde (dissolved in water to prepare a 0.2 M stock solution), 2 mM MgCl2, 0.05 mg / mL VhAld, the reaction was carried out in different pH buffers (phosphate solution at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, HEPES buffer at pH 7.0, 7.5, 8.0, Tris buffer at pH 7.0, 7.5, 8.0, 8.5, 9.0), and the reaction was carried out at room temperature for 5 min.

[0092] (2) Optimal reaction temperature and reaction conditions: 50mM pyruvate and 50mM glyceraldehyde, 50mM phosphate buffer pH7.5, 2mM MgCl2, 0.05mg / mL VhAld, reacted at different temperatures (between 20℃ and 65℃) for 5min.

[0093] Quantification and identification of reaction products: After the enzyme reaction, 10 μL of the enzyme reaction product was mixed with O-benzylhydroxylamine hydrochloride solution (20 μL, pyridine:methanol:water = 33:15:2 in 130 mM stock solution). After reacting at room temperature for 10 min, the sample was diluted with 200 μL of methanol, centrifuged, filtered through a 0.22 μm filter membrane, and analyzed by HPLC using an Agilent C18 column (5 μm, 5.0 × 240 mm). Mobile phase A consisted of deionized water with 0.1% (v / v) trifluoroacetic acid (TFA); mobile phase B consisted of acetonitrile with 0.1% (v / v) trifluoroacetic acid (TFA). The flow rate was 1 mL / min, detection was at 215 nm, and the column temperature was 30 °C. Elution conditions: 0–5 min, 10% B–35% B; 5–38 min, 35% B–90% B; 38 min–40 min, 90% B–10% B.

[0094] like Figure 2 VhAld exhibits higher activity in phosphate and HEPES buffers, while its activity significantly decreases in Tris buffer. The optimal enzyme reaction conditions are phosphate buffer at 7.5-8.0. Figure 3 The enzyme activity was significantly increased at 35℃, reached its maximum at 40℃, and then decreased slightly.

[0095] Example 4. Synthesis of γ-hydroxyketo acid-acceptor substrate using enzymatic catalysis

[0096] Experimental conditions: 50 mM of pyruvate as the donor substrate and 50 mM of aldehydes or ketones of different substrates, 50 mM phosphate buffer (pH 8.0), 2 mM MgCl2, 0.5 mg / ml VhAld, reaction at 40℃ for 8 h. Aldehydes or ketones were dissolved in water or acetonitrile to prepare a 1 M stock solution. Compounds 7, 8, 9, 10, 15, 16, 17, 18, 21, and 23 were dissolved in acetonitrile; the remaining compounds were dissolved in water. After the enzyme reaction, 10 μL of the enzyme product was mixed with O-benzylhydroxylamine hydrochloride solution (20 μL, pyridine:methanol:water = 33:15:2 stock solution). After reacting at room temperature for 10 min, the sample was diluted with 200 μL of methanol, centrifuged, filtered through a 0.22 μm filter, and analyzed by HPLC using an Agilent C18 column (5 μm, 5.0 × 240 mm). Mobile phase A consisted of deionized water with 0.1% (v / v) trifluoroacetic acid (TFA) added; mobile phase B consisted of acetonitrile with 0.1% (v / v) trifluoroacetic acid (TFA) added. The flow rate was 1 mL / min, detection was performed at 215 nm, and the column temperature was 30 °C. Elution conditions were: 0–5 min, 10% B–35% B; 5–38 min, 35% B–90% B; 38 min–40 min, 90% B–10% B.

[0097] The spectra and activity data of the different aldehyde or ketone substrates measured in this embodiment are shown in Table 1.

[0098] HPLC chromatograms, represented by donor substrate 1 (pyruvate) and acceptor substrate 21 (2,2-dimethoxyacetaldehyde), are as follows: Figure 4 As shown, the control group had the same components as the reaction group, except that VhAld enzyme was not added. Results showed that after derivatization, both the substrate and product exhibited absorption peaks at 215 nm. With changes in mobile phase polarity, peaks appeared at different time points: pyruvate at 14.1 min, 2,2-dimethoxyacetaldehyde at 20 min, and a distinct absorption peak at 10.9 min in the reaction group (i.e., the experimental group), representing the final product. The substrate conversion rate was quantified based on the consumption of pyruvate. Other substrate identification and calculation methods were the same as above.

[0099] Table 1 shows that when the donor substrate is pyruvate, the aldolase VhAld exhibits catalytic activity for most aldehydes or ketones. For aldehydes, those with pyridine heterocyclic substitutions show higher activity than those with thiophene, pyrrole, or benzene rings. Compared to acceptor substrates 5 and 7, adding a nitro group to the thiophene ring significantly improves enzyme activity. Overall, all six aldehydes exhibit high conversion rates, including acceptor substrates 5, 9-10, and 19-21, with acceptor substrates 19 (glyceraldehyde), 20 (lactic acid), and 21 (2,2-dimethoxyacetaldehyde) all exceeding 80% conversion. The selected ketone substrates showed the highest conversion rates between 50-60%, with 24 (1,3-dihydroxyacetone) and 25 (1,3-difluoroacetone) being the preferred substrates. Therefore, in most cases, nitro, hydroxyl, fluorine, or methoxy substitutions on aldehyde or ketone substrates improve substrate conversion.

[0100] Table 1

[0101]

[0102]

[0103]

[0104]

[0105] Example 5. Synthesis of γ-hydroxyketo acids using enzymatic catalysis – donor substrate profile

[0106] Experimental conditions: 50 mM of different donor substrates (keto acids and glyceraldehyde), 50 mM phosphate buffer (pH 8.0), 2 mM MgCl2, 0.5 mg / ml VhAld, reaction at 40℃ for 8 h. After the enzyme reaction, 10 μL of the enzyme product was mixed with O-benzylhydroxylamine hydrochloride solution (20 μL, pyridine:methanol:water = 33:15:2 stock solution). After reacting at room temperature for 10 min, the sample was diluted with 200 μL of methanol, centrifuged, filtered through a 0.22 μm filter, and analyzed by HPLC using an Agilent C18 column (5 μm, 5.0 × 240 mm). Mobile phase A: 0.1% (v / v) trifluoroacetic acid (TFA) in deionized water; mobile phase B: 0.1% (v / v) trifluoroacetic acid (TFA) in acetonitrile; flow rate: 1 mL / min; detection at 215 nm; column temperature: 30℃. Elution conditions: 0-5 min, 10% B-35% B; 5-38 min, 35% B-90% B; 38 min-40 min, 90% B-10% B.

[0107] The different donor substrate spectra and their activity data measured in this embodiment are shown in Table 2.

[0108] As shown in Table 2, the aldolase VhAld has certain catalytic activity for most keto acids. Among them, donor substrate 1 (pyruvate) has the highest activity and conversion rate. The activity gradually decreases with the increase of the chain length of the substituent at the C3 position of pyruvate. At the same time, the hydroxyl and fluorine substitution on the donor substrate pyruvate will increase the substrate conversion rate.

[0109] Table 2

[0110]

[0111]

[0112] In summary, the method of the present invention has a broad substrate spectrum, accepting different substituted pyruvates as donor substrates and various types of aldehydes or ketones as acceptor substrates, ultimately synthesizing various types of γ-hydroxyketo acid products.

[0113] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Use of aldolases in the enzymatic synthesis of γ-hydroxyketo acids having the structure shown in formula (C) or (E), R1, R3 and R4 are each independently selected from C1-C10 straight-chain or branched alkyl groups, C6-C10 aryl groups or C3-C10 heteroaryl groups; R2 is selected from H or C1-C10 straight-chain or branched alkyl groups; The alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from hydroxyl, halogen, nitro, C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, CH3S-substituted C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy.

2. The use according to claim 1, characterized in that, The aldolase comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 75% sequence identity with it; and / or The aldolase is derived from fungi, preferably from Candida humicola.

3. The use according to claim 1 or 2, characterized in that... ； R1 is selected from C1-C6 straight-chain or branched alkyl groups, C6-C10 aryl groups, or C3-C6 heteroaryl groups, wherein the alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from nitro, C1-C3 alkyl, hydroxyl-substituted C1-C3 alkyl, or C1-C3 alkoxy groups; and / or R2 is selected from H or C1-C6 straight-chain or branched alkyl groups, wherein the alkyl group is optionally substituted by one or more substituents selected from C1-C3 alkyl groups, halogen-substituted C1-C3 alkyl groups, or hydroxyl-substituted C1-C3 alkyl groups; Preferably, R3 and R4 are each independently selected from C1-C6 straight-chain or branched alkyl groups, wherein the alkyl group is optionally substituted by one or more substituents selected from C1-C3 alkyl groups, C1-C3 haloalkyl groups, hydroxyl-substituted C1-C3 alkyl groups, or C1-C3 alkoxy groups; and / or The heteroatom in the heteroaryl group is selected from N, O, or S; and / or The halogen is selected from F, Cl or Br.

4. A method for the enzyme-catalyzed synthesis of γ-hydroxyketo acid, comprising reacting an aldehyde compound of formula (A) with a keto acid compound of formula (B) in the presence of an aldolase to obtain the γ-hydroxyketo acid of formula (C). in, R1 is selected from C1-C10 straight-chain or branched alkyl, C6-C10 aryl, or C3-C10 heteroaryl; R2 is selected from H or C1-C10 straight-chain or branched alkyl groups; The alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from hydroxyl, halogen, nitro, C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, CH3S-substituted C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy.

5. A method for the enzyme-catalyzed synthesis of γ-hydroxyketo acid, comprising reacting a ketone compound of formula (D) with a keto acid compound of formula (B) in the presence of an aldolase to obtain the γ-hydroxyketo acid of formula (E). in, R3 to R4 are each independently selected from C1-C10 straight-chain or branched alkyl groups, C6-C10 aryl groups, or C3-C10 heteroaryl groups; R2 is selected from H or C1-C10 straight-chain or branched alkyl groups; The alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from hydroxyl, halogen, nitro, C1-C3 alkyl, C1-C3 haloalkyl, hydroxy-substituted C1-C3 alkyl, CH3S-substituted C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkoxy.

6. The method according to claim 4 or 5, characterized in that, R1 is selected from C1-C6 straight-chain or branched alkyl groups, C6-C10 aryl groups, or C3-C6 heteroaryl groups, wherein the alkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from nitro, C1-C3 alkyl, hydroxyl-substituted C1-C3 alkyl, or C1-C3 alkoxy groups; and / or R2 is selected from H or a C1-C6 straight-chain or branched alkyl group, wherein the alkyl group is optionally substituted by one or more substituents selected from C1-C3 alkyl groups, halogen-substituted C1-C3 alkyl groups, or hydroxyl-substituted C1-C3 alkyl groups; and / or R3 and R4 are each independently selected from C1-C6 straight-chain or branched alkyl groups, wherein the alkyl group is optionally substituted by one or more substituents selected from C1-C3 alkyl, C1-C3 haloalkyl, hydroxyl-substituted C1-C3 alkyl, or C1-C3 alkoxy groups; and / or The heteroatom in the heteroaryl group is selected from N, O, or S; and / or The halogen is selected from F, Cl, or Br; Preferably, the aldehyde compound represented by formula (A) is selected from the following compounds: Preferably, the keto acid compound represented by formula (B) is selected from the following compounds: Preferably, the ketone compound represented by formula (D) is selected from the following compounds:

7. The method according to any one of claims 4-6, characterized in that, The aldolase comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 75% sequence identity with it; and / or The aldolase is derived from fungi and yeasts, preferably from terrestrial Candida (Vanrija humicola); Preferably, the aldolase is obtained by expression in a host cell containing a recombinant vector, the recombinant vector containing the coding sequence of the aldolase; More preferably, the host cell is selected from bacteria, fungi, insect cells, plant cells or animal cells, and even more preferably, the cell is selected from Escherichia coli, Bacillus subtilis or yeast.

8. The method according to any one of claims 4-7, characterized in that, The molar ratio of the aldehyde compound of formula (A) or the ketone compound of formula (D) to the keto acid compound of formula (B) is (0.1-10):1, preferably (0.5-2):1; and / or The molar concentrations of the aldehyde compounds of formula (A), the keto acid compounds of formula (B), and / or the ketone compounds of formula (D) are 0.5-500 mM, preferably 10-100 mM; and / or The molar concentration of the aldolase is 0.01-2 mg / mL, preferably 0.1-1 mg / mL; and / or The reaction system further includes divalent metal ions, preferably Mg. 2+ Ni 2 + Co 2+ Zn 2+ or Ca 2+ One or more of the following, more preferably, the concentration of the divalent metal ion is 0.1-10 mM.

9. The method according to any one of claims 4-8, characterized in that, The reaction temperature is 20-65°C, preferably 35-50°C, more preferably 38-42°C; and / or The reaction time is 2-24 hours, preferably 8-12 hours; and / or The pH of the reaction is 5.0-9.0, preferably 7.0-8.5, more preferably 7.5-8.0; and / or Preferably, the buffer used in the reaction includes one or more of phosphate buffer, Tris hydrochloride buffer, and HEPES buffer, and preferably includes phosphate buffer; and / or The solvent used to dissolve the substrate aldehyde or ketone compounds in the reaction is one or more of water, dimethyl sulfoxide, acetonitrile, or ethanol, preferably water or acetonitrile.

10. Use of the γ-hydroxyketo acid obtained by any one of claims 4-9, or the hydroxyl lactone formed by acid treatment and cyclization of the γ-hydroxyketo acid, in the production of plastics, food, or pharmaceuticals.

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