Beta-amino acid dehydrogenase mutant and application thereof in preparation of (S)-3-aminobutyric acid

Abstract

The invention discloses a beta-amino acid dehydrogenase mutant and application of the beta-amino acid dehydrogenase mutant in preparation of (S)-3-aminobutyric acid. The beta-amino acid dehydrogenase mutant disclosed by the invention can be used for selectively reducing and aminating 3-oxobutyric acid to obtain (S)-3-aminobutyric acid, the substrate concentration can reach 20g / L, the conversion rate is greater than or equal to 99%, the ee value is greater than or equal to 99%, and the method is simple to operate and high in reaction efficiency and can be used for industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological catalysis, and particularly relates to a beta-amino acid dehydrogenase mutant and application thereof in preparation of (S)-3-aminobutyric acid. BACKGROUND

[0002] (S)-3-aminobutyric acid, as shown in formula I, has a CAS number of 3775-72-2, is an important pharmaceutical intermediate, and can be used for synthesis of a spirocyclic apolipoprotein APOL1 inhibitor and the like. In the future, the compound can be used for preparation of more new drug compounds.

[0003]

[0004] The preparation route of the compound I disclosed in the patent CN104370755B is shown in Scheme 1. The route takes ethyl acetoacetate as a starting material, first condenses with acetamide to generate compound 2, then generates intermediate 3 through asymmetric hydrogenation, then reacts with concentrated hydrochloric acid at 95-100 DEG C to hydrolyze the ester group, and finally reacts with sodium borohydride to reduce the amide to generate (S)-3-aminobutyric acid with an ee value of 99.2%. The route uses an expensive metal catalyst Rh(ScRp-DuanPhos)(COD)BF4, and the hydrogenation reaction in the route needs to be strictly avoided of oxygen, and the hydrolysis reaction needs high temperature.

[0005]

[0006] The preparation route of the compound I reported in the document Tetrahedron: Asymmetry (1997), 8 (1), 37-40. is shown in Scheme 2. The route takes racemic 3-aminobutyric acid ethyl ester as a starting material, first generates intermediate 5 through lipase (CAL) resolution, then generates (S)-3-aminobutyric acid through reactions such as amino protection, ester hydrolysis and amino deprotection. The route involves resolution, has poor atom economy, high production cost and is difficult to be industrialized.

[0007]

[0008] The preparation route of the compound I reported in the document ACS Catalysis (2015), 5 (4), 2220-2224. is shown in Scheme 3. The route takes ethyl acetoacetate as a starting material, first hydrolyzes the ester group with lipase, then catalyzes the asymmetric reductive amination reaction of compound II with beta-amino acid dehydrogenase (beta-AADH), and the yield of the product (S)-3-aminobutyric acid is only 22.1%.

[0009]

[0010] Therefore, it is necessary to develop a preparation method with low cost, simple operation, high atom economy, high catalytic efficiency and suitability for industrialization. SUMMARY

[0011] The present application aims at overcoming the deficiencies of the prior art and provides a β-amino acid dehydrogenase mutant and a method for preparing (S)-3-aminobutyric acid.

[0012] In one aspect, the present application provides a new β-amino acid dehydrogenase mutant, which is obtained by site mutation with the wild-type β-amino acid dehydrogenase amino acid sequence shown in SEQ ID NO. 1 as the reference sequence.

[0013] Further, the wild-type β-amino acid dehydrogenase is derived from Candidatus Cloacimonas acidaminovorans str. Evry, and the wild-type template NCBI accession number is 7DL1_A, and the amino acid sequence and nucleotide sequence are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0014] Further, the wild-type β-amino acid dehydrogenase is derived from Candidatus Cloacimonas acidaminovorans str. Evry, and the wild-type template NCBI accession number is 7DL1_A, and the amino acid sequence and nucleotide sequence are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0015] Further, the amino acid sequence and nucleotide sequence of the β-amino acid dehydrogenase mutant are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0016] Further, the β-amino acid dehydrogenase is expressed in a genetically engineered bacterium.

[0017] Further, the β-amino acid dehydrogenase expression strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis, preferably Escherichia coli.

[0018] In another aspect, the present application provides a method for preparing (S)-3-aminobutyric acid using a β-amino acid dehydrogenase mutant. The method uses 3-oxobutyric acid as a substrate, which is converted to (S)-3-aminobutyric acid under the catalysis of the β-amino acid dehydrogenase mutant, and the route is shown in Scheme 4.

[0019]

[0020] Further, the β-amino acid dehydrogenase is selected from the β-amino acid dehydrogenase mutant Mut1.

[0021] Further, the beta-amino acid dehydrogenase mutant participates in catalytic reaction in the form of beta-amino acid dehydrogenase enzyme powder, beta-amino acid dehydrogenase enzyme liquor, beta-amino acid dehydrogenase homogeneous liquid, cells containing beta-amino acid dehydrogenase, etc., and is preferably beta-amino acid dehydrogenase cells.

[0022] Further, coenzyme can be added to the reaction system to promote the reaction. When cells containing beta-amino acid dehydrogenase are used, the cells contain a small amount of coenzyme, and in this case, the coenzyme can also not be added. In some cases, the beta-amino acid dehydrogenase enzyme powder prepared also contains a small amount of coenzyme, and in this case, the coenzyme can also not be added. However, coenzyme can also be added to the reaction system to further promote the reaction. When coenzyme is added to the reaction system to promote the reaction, the coenzyme is selected from NAD + , NADH, NADP + , NADPH or a combination thereof, and is preferably NADP + .

[0023] Further, the concentration of the added coenzyme is 0.02-0.4 g / L, and is preferably 0.04-0.2 g / L.

[0024] Further, the coenzyme used in the technical solution is selected from the coenzyme products sold by Shangke Biomedicine (Shanghai) Co., Ltd.

[0025] Further, the reaction temperature is 20-45℃, and is preferably 25-35℃.

[0026] Further, the reaction pH is 6.5-9.0, and is preferably 7.0-8.0.

[0027] The beneficial effects of the present application are that the present application provides a new beta-amino acid dehydrogenase mutant, which can efficiently catalyze the conversion of 3-oxobutyric acid to (S)-3-aminobutyric acid, the substrate concentration can reach 20 g / L, the conversion rate is ≥99%, and the product ee value is ≥99%. The method is simple to operate, has high atom economy, and high catalytic efficiency, and can be used for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 1 is a protein expression diagram of wild-type beta-amino acid dehydrogenase. DETAILED DESCRIPTION

[0029] The technical content of the present application will be further described below in conjunction with specific examples, and the purpose is to better understand the content of the present application, but the protection scope of the present application is not limited thereto.

[0030] Example 1 Expression test of wild-type beta-amino acid dehydrogenase

[0031] The gene sequence of β-amino acid dehydrogenase from Candidatus Cloacimonas acidaminovoransstr. Evry was mined from the NCBI database. The gene sequence was synthesized and transformed into BL21(DE3) cells to construct an expression strain. Transformed single clones were then transferred to cells containing Amp + In resistant LB medium, incubated overnight at 37°C, when OD 600 When the OD value reaches approximately 10, the seed culture is transferred to 2YT liquid medium containing resistance at an inoculation ratio of 2% for expression culture. The culture is first incubated at 37°C for 3 hours, and then... 600 When the protein expression level reached 0.6–0.8, the temperature was lowered to 25°C, and 0.1 mM IPTG was added to induce expression. After 12 hours of expression, the bacterial cells were collected and centrifuged at 12,000 rpm for 10 minutes. The collected bacterial cells were then subjected to low-temperature sonication to disrupt the protein expression. The protein expression level was analyzed by SDS-PAGE electrophoresis. The results are shown in the figure. Figure 1 The protein size was consistent with expectations, and it showed good soluble expression.

[0032] Add 0.11 g of solid NH4Cl and 2 mg of NADP to the reaction system. + The reaction mixture consisted of powder, 0.1 g glucose solid, 20 mg GDH enzyme powder (Shanghai Shangke Biomedical Co., Ltd.), 0.2 g 3-oxobutyric acid, 10 mL 0.1 M Na2CO3 / NaHCO3 (pH=9) buffer, and 50 mg wild-type β-amino acid dehydrogenase cells. The reaction temperature was 25℃, and samples were taken for HPLC analysis after 24 h of reaction. The results showed that the conversion rate was 27.9% and the ee value was 89.2%.

[0033] Example 2 Construction of mutants

[0034] Based on the simulated structure of β-amino acid dehydrogenase, mutations were performed at sites 123 and 328, respectively. Mutation primers were designed using Primer 5 software, and mutants were obtained by whole plasmid PCR. The specific primer design is shown in Table 1.

[0035] Table 1 Primer sequences

[0036]

[0037]

[0038] Using whole plasmid amplification technology, site-directed mutagenesis was performed at site 123, and saturation mutagenesis was performed at site 328 to construct a mutant library. 380 single clones from the site 328 mutant library were selected and cultured in 96-well plates containing LB medium at 37°C for 16 h. The seed culture was then transferred to 96-well deep-layer plates containing 2YT medium and cultured with shaking for 14 h. After centrifugation, the bacterial cells were collected, the supernatant was discarded, and the collected cells were frozen at -40°C. After 4 h, the cells were removed, and the substrate 3-oxobutyric acid was added to each well for screening. TLC analysis was used to determine the initial screening mutants based on the amount of substrate reduction. Finally, the selected positive mutants were combined to obtain the mutant Mut 1 (L123S / H328D), which exhibited significantly improved catalytic efficiency and chiral selectivity. This mutant could convert 20 g / L of substrate to compound I with a conversion rate of 60.9% and an ee value of 99.9%.

[0039] Example 3 Reaction System Optimization - Temperature

[0040] Add 0.11g of NH4Cl solid and 2mg of NADP to the reaction vessel in sequence. + Powder, 0.1g glucose solid, 20mg GDH enzyme powder (Shanghai Shangke Biopharmaceutical Co., Ltd.), 0.2g 3-oxobutyric acid, 10mL 0.1M Na2CO3 / NaHCO3

[0041] (pH=9) buffer, 50 mg mutant cells, and then the reaction was carried out with shaking at different temperatures, with the temperature range set at 20-45℃. After 24 h of reaction, samples were taken for HPLC analysis. The results are shown in Table 2. The conversion rate and ee value were the best when the reaction temperature was 30℃.

[0042] Table 2 Optimization of reaction temperature

[0043]

[0044]

[0045] Example 4 Reaction System Optimization - pH

[0046] Add 0.11g of NH4Cl solid and 2mg of NADP to the reaction vessel in sequence. +Powder, 0.1 g glucose solid, 20 mg GDH enzyme powder (Shanghai Shangkai Biomedicine Co., Ltd.), 0.2 g 3-oxobutyric acid, 50 mg mutant cells, 10 mL 0.1 M Na2CO3 / NaHCO3buffer, pH range set to 6.5-9.0, oscillation reaction at different pH, reaction temperature 30°C, reaction for 24 h, sample for HPLC analysis, results shown in Table 3, when the reaction pH is 7.5, the conversion rate and ee value results are best.

[0047] Table 3 pH optimization of reaction

[0048] Serial number pH Conversion rate (%) ee value (%) 1 6.5 85.33 99.95 2 7.0 91.89 99.98 3 7.5 99.96 99.98 4 8.0 97.44 99.97 5 8.5 86.79 99.93 6 9.0 79.47 99.91

[0049] Example 5 Preparation of (S)-3-aminobutyric acid

[0050] Into the reaction kettle, 1.1 g NH4Cl solid, 20 mg NADP + Powder, 1 g glucose solid, 0.2 g GDH enzyme powder (Shanghai Shangkai Biomedicine Co., Ltd.), 2 g 3-oxobutyric acid, 100 mL 0.1 M Na2CO3 / NaHCO3(pH=7.5) buffer, 0.5 g mutant cells, temperature set to 30°C, sample for HPLC analysis after reaction for 24 h, conversion rate 99.98%, ee value 99.98%.

Claims

1. A β-amino acid dehydrogenase mutant, characterized in that, The amino acid and nucleotide sequences of the β-amino acid dehydrogenase mutant are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

2. The β-amino acid dehydrogenase mutant as described in claim 1, characterized in that, The β-amino acid dehydrogenase mutant was expressed in genetically engineered bacteria.

3. A method for preparing (S)-3-aminobutyric acid, characterized in that, 3-O-butyric acid is converted to (S)-3-aminobutyric acid under the catalysis of a β-amino acid dehydrogenase mutant, wherein the β-amino acid dehydrogenase mutant is selected from the β-amino acid dehydrogenase mutant of claim 1.

4. The preparation method according to claim 3, characterized in that, The β-amino acid dehydrogenase participates in the catalytic reaction in the form of β-amino acid dehydrogenase powder, β-amino acid dehydrogenase clear solution, β-amino acid dehydrogenase homogenate, and cells containing β-amino acid dehydrogenase.

5. The preparation method according to claim 3, characterized in that, The coenzyme is selected from NAD. + NADH, NADP + NADPH or combinations thereof.

6. The preparation method according to claim 3, characterized in that, The reaction is carried out at a pH of 6.5 to 9.

0.

7. The preparation method according to claim 3, characterized in that, The reaction temperature is 20℃~45℃.

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