Use of an esterase in the synthesis of (r)-3-methylsuccinic acid monomethyl ester

Abstract

The application belongs to the field of biotechnology and biological catalysis, and discloses application of an esterase in synthesis of (R)-3-methyl succinic acid monomethyl ester. The esterase provided by the application has high activity and high regioselectivity on (R)-2-methyl succinic acid dimethyl ester, and the ratio of obtained products (R)-3-methyl succinic acid monomethyl ester and (R)-4-methoxy-3-methyl-4-oxobutanoic acid is 215:1. The recombinant strain constructed based on the gene of the enzyme can be used for hydrolysis of (R)-2-methyl succinic acid dimethyl ester to synthesize (R)-3-methyl succinic acid monomethyl ester, has strong substrate tolerance, can realize high-concentration feeding (50-200 g / L), has high catalytic efficiency, the conversion rate is more than 98% and no by-product is generated, the reaction cycle is short, the amount of biological catalyst is small, the preparation method is simple and convenient, the conditions are mild, the environment is friendly, and the application has good industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and biocatalysis, specifically relating to an esterase and its application in the synthesis of (R)-3-methylsuccinic acid monomethyl ester. Background Technology

[0002] 3-Methylsuccinate monomethyl ester with a chiral carbon atom is an important structural unit, commonly found in natural products, drug molecules, and synthetic intermediates. R-configuration 3-methylsuccinate monomethyl ester is a precursor for the synthesis of sacubitril, a neprilysin inhibitor that blocks the degradation of two polypeptides responsible for vasodilation, thus lowering blood pressure. Sacubitril / valsartan sodium, a 1:1 combination of sacubitril and valsartan, is Novartis' first clinically approved dual inhibitor of angiotensin receptor and neprilysin. This combination drug has vasodilatory effects, prevents and reverses cardiovascular remodeling, and promotes sodium excretion, representing a novel cardiovascular drug mechanism. Sacubitril / valsartan sodium received FDA and EMA approval in 2015 and NMPA approval in 2017, marketed under the brand name Noxinto (CN 108602785 B).

[0003] The synthesis of (R)-3-methylsuccinate monomethyl ester can be obtained by hydrolyzing (R)-2-methylsuccinate dimethyl ester. However, chemical hydrolysis often has poor regioselectivity and cannot achieve the hydrolysis of a single methyl ester.

[0004] Esterases are a class of enzymes that catalyze the hydrolysis and synthesis of ester bonds (carboxyl esters, amides, thioesters, etc.). They possess high stereoselectivity and regioselectivity and are ubiquitous in nature, widely found in fungi, bacteria, and plants. In 2012, a reaction was reported using a commercially available hydrolase to hydrolyze dimethyl (R)-2-methylsuccinate to synthesize monomethyl (R)-3-methylsuccinate, with a substrate concentration of 35 g / L and a large amount of toluene as a co-solvent (28%) [D. Mangan, A Three-Enzyme System Involving an Ene-Reductase for Generating Valuable Chiral Building Blocks, Advanced Synthesis & Catalysis 2012, 354, 2185-2190]. The reaction involved is as follows:

[0005]

[0006] Currently, reports on the biosynthesis of (R)-3-methylsuccinate monomethyl ester are limited to laboratory scales and suffer from problems such as the use of commercially available enzymes as catalysts, poor substrate tolerance, and insufficient substrate concentration or selectivity. Therefore, for the synthesis of (R)-3-methylsuccinate monomethyl ester, there is an urgent need to screen for highly efficient and regioselective esterases to meet industrial requirements. Summary of the Invention

[0007] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide an application of esterase in the synthesis of (R)-3-methylsuccinic acid monomethyl ester, so as to solve the problems of low regioselectivity of esterase for diester compounds, low substrate concentration and low conversion rate during application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides the application of an esterase in the synthesis of (R)-3-methylsuccinate monomethyl ester, wherein the amino acid sequence of the esterase is shown in SEQ ID NO: 1.

[0010] This invention provides the application of a recombinant host bacterium expressing an esterase in the synthesis of (R)-3-methylsuccinate monomethyl ester, wherein the amino acid sequence of the esterase is shown in SEQ ID NO: 1.

[0011] Preferably, the recombinant host bacterium is Escherichia coli, more preferably Escherichia coli BL21(DE3).

[0012] The present invention also provides a method for synthesizing (R)-3-methylsuccinate monomethyl ester, which involves using the esterase to catalyze the hydrolysis of (R)-2-methylsuccinate dimethyl ester to synthesize (R)-3-methylsuccinate monomethyl ester, optionally further comprising the step of separating or purifying the resulting (R)-3-methylsuccinate monomethyl ester.

[0013] Specifically, the esterase is in the form of a pure enzyme, a crude enzyme, or a whole-cell catalyst.

[0014] The whole-cell catalyst was obtained by culturing a recombinant host bacterium capable of expressing the esterase.

[0015] The recombinant host bacteria were obtained by introducing a recombinant expression vector containing the encoding gene of the esterase.

[0016] Preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.

[0017] In a preferred embodiment of the present invention, the recombinant expression vector uses pET-21a as the starting vector.

[0018] Preferably, the recombinant host bacterium is Escherichia coli, more preferably Escherichia coli BL21(DE3).

[0019] In a specific embodiment, the concentration of (R)-2-methylsuccinate dimethyl ester in the reaction system is 50-200 g / L. A buffer solution with a pH of 7.0-8.0 is used as the reaction medium. The reaction is carried out by shaking at 30℃-40℃. After the reaction is complete, (R)-3-methylsuccinate monomethyl ester is obtained by acid adjustment, extraction, and removal of solvent under reduced pressure.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Through gene mining, the inventors screened a highly active and regioselective esterase for (R)-2-methylsuccinate dimethyl ester, with a product ratio of (R)-3-methylsuccinate monomethyl ester to (R)-4-methoxy-3-methyl-4-oxobutyric acid of 215:1. This esterase shows less than 60% similarity to other esterases reported in the literature, exhibiting significant differences. This provides conditions for further research on the evolutionary relationships of this enzyme family and also provides more genetic resources for constructing genetically engineered bacteria with highly efficient esterases using genetic engineering techniques.

[0022] 2. A novel esterase gene was heterologously overexpressed in *E. coli* using genetic engineering techniques, resulting in a genetically engineered bacterium that efficiently expresses the esterase. A biotransformation process for the synthesis of (R)-3-methylsuccinate monomethyl ester by this genetically engineered bacterium was established. The process exhibits strong substrate tolerance, allows for high-concentration feeding (50-200 g / L), high catalytic efficiency (conversion rate exceeding 98%), no byproduct formation, a short reaction cycle, low biocatalyst usage, and a simple, convenient, mild, and environmentally friendly preparation method, demonstrating excellent prospects for industrialization. Attached Figure Description

[0023] Figure 1 This is an SDS-PAGE image of purified esterase E31.

[0024] Figure 2 These are the optimal reaction temperature and optimal reaction pH for esterase E31. Figure (a) shows the optimal reaction temperature, and Figure (b) shows the optimal reaction pH.

[0025] Figure 3 The figures show the temperature and pH stability of esterase E31. Figure (a) shows the temperature stability, and Figure (b) shows the pH stability.

[0026] Figure 4This is a gas chromatogram of the synthesis of (R)-3-methylsuccinate monomethyl ester by esterase E31. (a) shows the substrate (R)-2-methylsuccinate dimethyl ester; (b) shows the products (R)-3-methylsuccinate monomethyl ester and (R)-4-methoxy-3-methyl-4-oxobutyric acid; and (c) shows the conversion sample of (R)-3-methylsuccinate monomethyl ester synthesized by esterase E31. Detailed Implementation

[0027] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention.

[0028] Example 1: Synthesis of esterase (E31) gene and construction of genetically engineered bacteria

[0029] 1.1 Discovery and synthesis of esterase genes

[0030] Using bioinformatics databases such as NCBI, KEGG, Uniprot, Brenda, and Foldseek, and combined with various software such as Mega, Discovery Studio, and Cytoscape, a comprehensive analysis of the enzyme protein was conducted from multiple levels, including sequence, three-dimensional structure, evolutionary relationship, and protein-protein interaction. Ultimately, the esterase with protein sequence number MGYP001091172583 (MGnify (https: / / www.ebi.ac.uk / metagenomics)) was determined to have great potential.

[0031] The gene was synthesized by codon optimization based on the protein sequence (amino acid sequence as shown in SEQ ID NO: 1) (optimized nucleotide sequence as shown in SEQ ID No: 2), and constructed into the pET21a expression vector with gene insertion sites of NdeI and HindIII.

[0032] 1.2 Transformation of recombinant plasmids

[0033] Competent Escherichia coli cells were prepared using the calcium chloride method.

[0034] (1) Take 10 μL of recombinant plasmid into 50 μL of Escherichia coli BL21(DE3) competent cells and incubate on ice for 30 min.

[0035] (2) Heat shock in a 42°C water bath for 45 seconds, then quickly place on ice for 1–2 minutes.

[0036] (3) Add 600 μL of fresh LB liquid medium and incubate at 37°C with shaking for 45–60 min.

[0037] (4) Take 200 μL of bacterial solution and spread it on the surface of LB solid medium containing ampicillin. Incubate at 37°C for 12-16 h until single colonies appear.

[0038] Example 2: Induction and purification of esterase (E31)

[0039] Prepare 50 mL of seed culture in LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl). Use an inoculation loop to pick a single colony of the genetically engineered bacteria and inoculate it into the medium. Incubate overnight at 37°C and 200 rpm. Transfer the overnight cultured seed culture to fermentation medium (LB medium) at a 1% inoculation rate and incubate at 37°C and 200 rpm until OD reaches [the desired growth rate]. 600 Add 0.05 mM IPTG to a concentration of approximately 0.6-1.0 and incubate at 25°C and 200 rpm for 10-12 hours. Collect bacterial cells by centrifugation at 4°C and 6000 rpm, wash twice with phosphate buffer (50 mM, pH 8.0), homogenize using a high-pressure homogenizer, centrifuge at 13000 rpm, and collect the supernatant. Then, purify and recover the target protein using metal affinity chromatography (nickel column). After dialysis to remove imidazole, the target protein yields the pure enzyme solution. SDS-PAGE electrophoresis shows that the purified protein has a single band, indicating electrophoretic purity (see...). Figure 1 ).

[0040] Example 3: Determination of enzymatic properties and kinetic parameters of esterase (E31)

[0041] 3.1 Enzymatic properties of esterase (E31)

[0042] A temperature range of 25–45°C was set, and measurements were taken every 5°C. By comparing the conversion at different temperatures, the optimal reaction temperature of the enzyme was determined. Conversion was measured at 30°C under different pH conditions using citrate / sodium citrate buffer (pH 5.0, 6.0), phosphate buffer (pH 6.0, 7.0, 8.0), Tris-HCl buffer (pH 8.0, 9.0), and glycine / NaOH buffer (pH 9.0, 10.0). By comparing the conversion under different pH conditions, the optimal reaction pH of the enzyme was determined. The reaction system was as follows: total reaction volume 1 mL, substrate (R)-2-methylsuccinate dimethyl ester (62.5 mM), pure enzyme (70 μg), reacted for 1 h under different conditions, acidified, and then extracted with ethyl acetate. Gas chromatography analysis was performed as follows. Figure 4 As shown, (a) is the substrate (R)-2-methylsuccinate dimethyl ester; (b) are the products (R)-3-methylsuccinate monomethyl ester and (R)-4-methoxy-3-methyl-4-oxobutyric acid; and (c) is the conversion sample of (R)-3-methylsuccinate monomethyl ester synthesized by esterase E31.

[0043] The conversion results of the gas-phase detection reaction are as follows: Figure 2As shown in the figures, (a) shows the optimal reaction temperature, and (b) shows the optimal reaction pH. The results indicate that the optimal reaction temperature for E31 is 35°C, and its activity begins to decrease sharply when the temperature exceeds 40°C. E31 is active at pH 5.0–10.0, with maximum activity observed in phosphate buffer at pH 8.0. Notably, the activity of E31 decreases significantly below pH 7.0, with a conversion rate of only 15% at pH 5.0.

[0044] Temperature stability was determined by incubating the enzyme solution at different temperatures (30°C, 35°C, 40°C, and 45°C) for 24 hours, followed by sampling to determine the residual protein activity. pH stability was determined by incubating the enzyme solution at citrate / sodium citrate buffer (pH 5.0, 6.0), phosphate buffer (pH 7.0, 8.0), and glycine / NaOH buffer (pH 9.0, 10.0) for 24 hours, followed by sampling to determine the residual protein activity. The enzyme activity assay system consisted of a total reaction volume of 0.2 mL, 100 mM phosphate buffer (pH 8.0), 0.5 mM 4-nitrophenylbutyrate, and 7 μg of pure enzyme. Detection was initiated at 30°C, and the absorbance at 405 nm was measured. Results are as follows: Figure 3 As shown in the figures (where (a) represents temperature stability and (b) represents pH stability), E31 exhibits good thermal stability at 30-35°C, retaining more than 50% of its activity after 24 hours. Furthermore, E31 demonstrates good pH stability within a pH range of 5.0-10.0.

[0045] 3.2 Kinetic parameters of dimethyl (R)-2-methylsuccinate by esterase (E31)

[0046] Enzyme activity was determined by constructing a reaction and measuring the initial rate of the catalytic reaction using product formation. The reaction system consisted of 1 mL phosphate buffer (200 mM, pH 8.0), 35 μg of pure enzyme, and a reaction time of 20 min. Different substrate concentrations were set, and after acid adjustment, ethyl acetate was added for extraction. Product formation was detected by gas chromatography. The amount of product formed was determined using a product standard curve. Enzyme activity unit (U) was defined as the amount of enzyme required to generate 1 μmol of product per minute under the above reaction conditions. The measured data are shown in Table 1. The specific activity of E31 for (R)-2-methylsuccinate dimethyl ester was 26.3 U / mg, and its catalytic efficiency... It is 0.5 s -1 mM -1 .

[0047] Table 1. Kinetic parameters of esterase E31 for dimethyl (R)-2-methylsuccinate

[0048]

[0049] Example 4: Whole-cell synthesis of (R)-3-methylsuccinate monomethyl ester using esterase E31

[0050] Seed culture: Single colonies of the genetically engineered strain BL21-E31 were picked with an inoculation loop and inoculated into LB medium containing ampicillin. The culture was carried out overnight at 37°C and 200 rpm.

[0051] Fermentation induction culture: The overnight cultured seed culture was transferred to the fermentation medium at an inoculum rate of 1%, and cultured at 37°C and 200 rpm until OD200. 600 Add 0.1 mM IPTG to a concentration of approximately 0.6-1.0, and incubate at 25°C and 200 rpm for 16 h. Collect the cells by centrifugation at 4000 g for 10 min for subsequent reactions.

[0052] 20 mL of phosphate buffer (pH 8.0), 10 mg / mL of wet esterase E31 cells, substrate concentration of 50-200 g / L, reaction at 30 °C for 24 h, followed by acid adjustment, extraction with ethyl acetate, centrifugation, drying of the organic phase, and gas chromatography analysis. The conversion results are shown in Table 2. At substrate concentrations of 50 g / L and 80 g / L, the conversion rate reached 99% within 6 hours. When the substrate concentration was further increased to 150 g / L and 200 g / L, the conversion rate reached 98% within 24 hours.

[0053] Table 2. Whole-cell transformation of dimethyl (R)-2-methylsuccinate by esterase E31

[0054] .

Claims

1. Use of an esterase in the synthesis of (R)-3-methylbutane dioic acid monomethyl ester, wherein, The amino acid sequence of the esterase is shown as SEQ ID NO:

1.

2. Use of a recombinant host expressing an esterase for the synthesis of (R)-3- methylbutane dioic acid monomethyl ester, characterized in that, The amino acid sequence of the esterase is shown as SEQ ID NO:

1.

3. Use according to claim 2, wherein the compound is ###0002### The recombinant host bacterium is obtained by introducing a recombinant expression vector containing a gene encoding the esterase.

4. The use according to claim 2, wherein the compound is ###0002### The recombinant host bacterium is Escherichia coli.

5. A process for the synthesis of (R)-3-methylbutane dioic acid monomethyl ester, characterized in that, The esterase is used to catalyze the hydrolysis of (R)-2-methyl succinic acid dimethyl ester to synthesize (R)-3-methyl succinic acid monomethyl ester; the amino acid sequence of the esterase is shown as SEQ ID NO:

1.

6. The method of claim 5, wherein, The step of isolating or purifying the produced (R)-3-methyl succinic acid monomethyl ester is further included.

7. The method of claim 5, wherein, The esterase is in the form of a pure enzyme, a crude enzyme, or a whole cell catalyst of a recombinant host bacterium expressing the esterase.

8. The method of claim 7, wherein, The whole cell catalyst is obtained by culturing a recombinant host bacterium capable of expressing the esterase, which is obtained by introducing a recombinant expression vector containing a gene encoding the esterase.

9. The method of claim 8, wherein, The nucleotide sequence of the encoding gene is shown as SEQ ID NO: 2; the recombinant expression vector uses pET-21a as a starting vector; and the recombinant host bacterium is Escherichia coli.

10. The method of claim 5, wherein, In the reaction system, the concentration of (R)-2-methyl succinic acid dimethyl ester is 50-200 g / L, a buffer with a pH of 7.0-8.0 is used as a reaction medium, and the reaction is carried out at 30-40°C under oscillation; after the reaction is completed, (R)-3-methyl succinic acid monomethyl ester is obtained by adjusting the acid, extraction, and removal of the solvent under reduced pressure.

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