Application of esterase in synthesis of (R)-3-methylsuccinic acid monomethyl ester

Abstract

The invention belongs to the field of biotechnology and biocatalysis, and discloses an application of esterase in synthesis of (R)-3-methyl methylsuccinate. The esterase provided by the invention has high activity and high regioselectivity to the (R)-2-dimethyl methylsuccinate, and the ratio of the obtained product (R)-3-methyl methylsuccinate to the obtained product (R)-4-methoxy-3-methyl-4-oxobutyric acid is 215: 1. The recombinant strain constructed based on the gene of the enzyme can be used for hydrolyzing (R)-2-dimethyl methylsuccinate to synthesize (R)-3-methyl methylsuccinate, the substrate tolerance is high, high-concentration feeding (50-200 g / L) can be realized, the catalytic efficiency is high, the conversion rate reaches 98% or above, no by-product is generated, the reaction period is short, the dosage of a biocatalyst is small, and the method is suitable for industrial production. The preparation method is simple and convenient, mild in condition and environment-friendly, and has a very good industrialization prospect.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and biocatalysis, and particularly relates to an esterase and application thereof in synthesizing (R)-3-methylsuccinic acid monomethyl ester. Background Art

[0002] 3-Methylsuccinate (MS), a chiral carbon atom, is an important structural unit, commonly found in natural products, pharmaceutical molecules, and synthetic intermediates. R-configured MS is a precursor to the synthesis of sacubitril, a neprilysin inhibitor that blocks the degradation of two peptides responsible for vasodilation, thereby lowering blood pressure. Sacubitril-valsartan sodium, a 1:1 combination of sacubitril and valsartan, is the first dual-action inhibitor of angiotensin receptors and neprilysin developed and clinically approved by Novartis. This combination drug exhibits vasodilation, prevention and reversal of cardiovascular remodeling, and natriuresis, representing a novel cardiovascular drug mechanism of action. Sacubitril-valsartan sodium was approved for marketing by the US FDA and EU EMA in 2015 and by the China National Medical Products Administration (NMPA) in 2017 under the trade name Entresto (CN 108602785 B).

[0003] The synthesis of (R)-3-methylsuccinic acid monomethyl ester can be obtained by hydrolyzing (R)-2-methylsuccinic acid 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 (such as carboxylester bonds, amide bonds, and thioester bonds). They exhibit high stereo- and regio-selectivity and are ubiquitous in nature, being widely found in fungi, bacteria, and plants. In 2012, a commercial hydrolase was reported for the hydrolysis of dimethyl (R)-2-methylsuccinate to synthesize monomethyl (R)-3-methylsuccinate. The substrate concentration was 35 g / L, and a large amount of toluene (28%) was used as a cosolvent [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 are limited to laboratory-scale methods and often suffer from issues such as commercial enzyme catalysts, poor substrate tolerance, and insufficient substrate concentration or selectivity. Therefore, there is an urgent need to screen for highly efficient and regioselective esterases for the synthesis of (R)-3-methylsuccinate to meet industrial needs. Summary of the Invention

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

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides an application of an esterase in synthesizing (R)-3-methylsuccinic acid monomethyl ester, wherein the amino acid sequence of the esterase is shown as SEQ ID NO: 1.

[0010] The present invention provides an application of a recombinant host bacteria expressing an esterase in synthesizing (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 bacteria is Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0012] The present invention also provides a method for synthesizing (R)-3-methylsuccinic acid monomethyl ester, which comprises using the esterase to catalyze the hydrolysis of (R)-2-methylsuccinic acid dimethyl ester to synthesize (R)-3-methylsuccinic acid monomethyl ester, and optionally further comprising the step of separating or purifying the generated (R)-3-methylsuccinic acid 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 is obtained by culturing a recombinant host bacterium capable of expressing the esterase.

[0015] The recombinant host bacteria is obtained by introducing a recombinant expression vector containing the esterase coding gene.

[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 bacteria is Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0019] In a specific embodiment, in the reaction system, the concentration of (R)-2-methylsuccinic acid dimethyl ester is 50-200 g / L, a buffer solution with a pH of 7.0-8.0 is used as the reaction medium, and the reaction is oscillated at 30°C-40°C. After the reaction is complete, the reaction is acidified, extracted, and the solvent is removed under reduced pressure to obtain (R)-3-methylsuccinic acid monomethyl ester.

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

[0021] Through gene mining, the inventors have identified an esterase with high activity and regioselectivity for (R)-2-methylsuccinate dimethyl ester. The product, (R)-3-methylsuccinate monomethyl ester: (R)-4-methoxy-3-methyl-4-oxobutanoic acid, has a ratio of 215:1. This esterase has a similarity of less than 60% with other esterases reported in the literature, demonstrating significant differences. This esterase provides opportunities for further study of the evolutionary relationships of this enzyme family and provides additional genetic resources for the construction of genetically engineered bacteria with highly efficient esterases using genetic engineering techniques.

[0022] 2. Through genetic engineering, a novel esterase gene was heterologously overexpressed in Escherichia coli, resulting in a genetically engineered bacterium that efficiently expresses the esterase. A bioconversion process for (R)-3-methylsuccinate was established using this engineered bacterium. The process exhibits strong substrate tolerance, enabling high feed concentrations (50-200 g / L), high catalytic efficiency, with conversion rates exceeding 98% and no byproducts generated. The reaction cycle is short, requiring minimal biocatalyst dosage. The preparation method is simple, convenient, mild, and environmentally friendly, demonstrating promising prospects for industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 The optimal reaction temperature and pH value of esterase E31 are shown in Figure 1. (a) shows the optimal reaction temperature, and (b) shows the optimal reaction pH value.

[0025] Figure 3 Figure 2 shows the temperature and pH stability of esterase E31. (a) shows the temperature stability, and (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-oxobutanoic acid; and (c) shows the conversion sample of (R)-3-methylsuccinate monomethyl ester by esterase E31. DETAILED DESCRIPTION

[0027] The following examples further illustrate 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 Esterase gene mining and gene synthesis

[0030] With the help of bioinformatics databases such as NCBI, KEGG, Uniprot, Brenda, and Foldseek, 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 relationships, and protein interactions. Ultimately, it was determined that the esterase with the protein sequence number MGYP001091172583 (MGnify (https: / / www.ebi.ac.uk / metagenomics)) has great potential.

[0031] The gene was synthesized by codon optimization according to the protein sequence (amino acid sequence as shown in SEQ ID NO: 1) (the optimized nucleotide sequence is shown in SEQ ID No: 2) and constructed into the pET21a expression vector with the gene insertion sites being 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 and add it to 50 μL of E. coli BL21 (DE3) competent cells and incubate on ice for 30 minutes.

[0035] (2) Heat shock at 42°C in a 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 culture at 37°C with shaking for 45-60 min.

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

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

[0039] Prepare 50 mL of seed solution 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 at 37°C, 200 rpm overnight. Transfer the overnight cultured seed solution to the fermentation medium (LB medium) at a 1% inoculum volume and incubate at 37°C, 200 rpm until the OD 600 0.6-1.0, add 0.05mM IPTG, and induce at 25°C, 200rpm for 10-12h. Collect the cells by centrifugation at 4°C, 6000rpm, wash twice with phosphate buffer (50mM, pH 8.0), and break with a high-pressure homogenizer. Centrifuge at 13000rpm to retain the supernatant, and then use metal affinity chromatography (nickel column) to purify and recover the target protein. After the target protein is dialyzed to remove imidazole, the pure enzyme solution is obtained. The SDS-PAGE electrophoresis pattern shows that the purified protein has a single band and has reached 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] The temperature range was set at 25–45°C, and the test was performed every 5°C. The optimal reaction temperature of the enzyme was found by comparing the conversion conditions at different temperatures. The conversion conditions under different pH conditions were measured at 30°C. The buffers were citric acid / 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 conditions under different pH conditions, the optimal reaction pH of the enzyme was obtained. The reaction system is as follows: total reaction volume 1 mL, substrate (R)-2-methylsuccinic acid dimethyl ester (62.5 mM), pure enzyme (70 μg), reaction under different conditions for 1 h, ethyl acetate was added for extraction after acid adjustment, and gas chromatography detection was as follows: Figure 4 As shown, (a) is the substrate (R)-2-methylsuccinate dimethyl ester; (b) is the product (R)-3-methylsuccinate monomethyl ester and (R)-4-methoxy-3-methyl-4-oxobutanoic acid; (c) is the conversion sample of esterase E31 to synthesize (R)-3-methylsuccinate monomethyl ester.

[0043] The results of gas phase detection reaction conversion are as follows Figure 2The results show that the optimum reaction temperature of E31 is 35 °C, and when the temperature exceeds 40 °C, the activity begins to decrease sharply. E31 has activity at pH 5.0-10.0, and the maximum activity is observed in a phosphate buffer at pH 8.0. It is worth noting that the activity of E31 decreases significantly at a pH value lower than 7.0, and the conversion rate is only 15% at pH 5.0.

[0044] Temperature stability was determined by incubating enzyme solution at different temperatures (30 °C, 35 °C, 40 °C and 45 °C) for 24 h, and then sampling to determine the residual protein activity. pH stability was determined by incubating enzyme solution in citric acid / 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 h, and then sampling to determine the residual protein activity. The enzyme activity determination system: total reaction volume 0.2 mL, 100 mM phosphate buffer pH 8.0, 0.5 mM 4-nitrophenyl butyrate, after adding pure enzyme (7 μg), start testing at 30 °C, determine the change of absorbance at 405 nm. The results are shown in Figure 3 Fig. (a) is temperature stability, and (b) is pH stability), E31 shows good thermal stability at 30-35 °C, and still maintains more than 50% activity after 24 hours. In addition, E31 shows good pH stability in the pH range of 5.0-10.0.

[0045] 3.2 Kinetic parameters of esterase (E31) on (R)-dimethyl 2-methyl succinate

[0046] The initial speed of the catalytic reaction was determined by constructing a reaction to calibrate the enzyme activity with the generation of products. The reaction system: 1 mL of phosphate buffer (200 mM, pH 8.0), 35 μg of pure enzyme, reaction time 20 min, set different substrate concentrations, adjust the acid, add ethyl acetate for extraction, and detect the product generation by gas phase. The product generation amount was calibrated by the product standard curve, and the 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 data measured are shown in Table 1, the specific activity of E31 on (R)-dimethyl 2-methyl succinate is 26.3 U / mg, and the catalytic efficiency is 0.5 s -1 mM -1 .

[0047] Table 1, Kinetic parameters of esterase E31 on (R)-dimethyl 2-methyl succinate

[0048]

[0049] Example 4: Synthesis of (R)-3-methylsuccinate using esterase E31 whole cells

[0050] Seed culture: Use an inoculation loop to pick a single colony of the genetically engineered strain BL21-E31 and inoculate it into LB medium containing ampicillin. Culture it at 37°C and 200 rpm overnight.

[0051] Fermentation induction culture: transfer the overnight culture seed solution to the fermentation medium at a 1% inoculum volume and culture at 37°C and 200 rpm until the OD 600 When the concentration of the culture medium was about 0.6-1.0, 0.1 mM IPTG was added and the culture was induced at 25°C and 200 rpm for 16 h. The cells were collected 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 E31 esterase, and a substrate concentration of 50-200 g / L were added. The reaction was carried out at 30°C for 24 hours. After acidification, ethyl acetate extraction was added, and the organic phase was centrifuged and dried before gas chromatography-mass spectrometry analysis. The conversion results are shown in Table 2 below. 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. Conversion of (R)-2-methylsuccinate by whole cells using esterase E31

[0054] .

Claims

1. An 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.

2. Use of a recombinant host bacterium expressing an esterase in the synthesis of (R)-3-methylsuccinate, characterized in that: The amino acid sequence of the esterase is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that The recombinant host bacteria is obtained by introducing a recombinant expression vector containing the esterase coding gene.

4. The use according to claim 2, characterized in that The recombinant host bacteria is Escherichia coli.

5. A method for synthesizing (R)-3-methylsuccinic acid monomethyl ester, characterized in that: Esterase is used to catalyze the hydrolysis of (R)-2-methylsuccinate dimethyl ester to synthesize (R)-3-methylsuccinate monomethyl ester; the amino acid sequence of the esterase is shown in SEQ ID NO:

1.

6. The method according to claim 5, wherein The method further includes a step of isolating or purifying the produced (R)-3-methylsuccinic acid monomethyl ester.

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

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

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

10. The method according to claim 5, wherein In the reaction system, the concentration of (R)-2-methylsuccinic acid dimethyl ester is 50-200 g / L, a buffer solution with a pH of 7.0-8.0 is used as the reaction medium, and the reaction is oscillated at 30°C-40°C. After the reaction is complete, the reaction is acidified, extracted, and the solvent is removed under reduced pressure to obtain (R)-3-methylsuccinic acid monomethyl ester.

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