A method for synthesizing (S)-2-amino-3-(thiazol-4-yl)propionic acid by biological enzyme

Abstract

The application provides a method for catalytically synthesizing (S)-2-amino-3-(thiazole-4-yl)propionic acid, taking 2-acetylamino-3-(thiazole-4-yl)propionic acid as a substrate, wherein the catalyst contains aminoacylase, and the aminoacylase comprises an amino acid sequence as shown in at least one sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5. In the method, the catalyst has the advantages of simple preparation process, low cost, mild reaction condition, short technological process, high product yield and pure stereoisomer, and has high industrial application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biocatalysis and biopharmaceutical technology, and particularly relates to a novel aminoacylase and its sequence, and a method for preparing chiral amino acids and derivatives thereof by catalysis. BACKGROUND

[0002] Spinocerebellar ataxia is a group of chronic degenerative diseases of the central nervous system with ataxia and dysmetria as the main clinical manifestations. At present, the commonly used drug for improving ataxia symptoms is tanaproget. In 2019, lovastatin, a new generation of drug for treating ataxia, entered the third phase of clinical trials. Lovastatin is a thyrotropin releasing hormone analogue, which has high bioavailability. At the same dose, the effect of lovastatin is 30 times that of tanaproget. In the development process of lovastatin drug, (S)-2-amino-3-(thiazol-4-yl) propionic acid is an important pharmaceutical intermediate. The development of a green and economical synthesis route of (S)-2-amino-3-(thiazol-4-yl) propionic acid is of great significance to the research and development of lovastatin drug.

[0003] At present, there are few reports on the efficient, specific and green synthesis of this chiral intermediate. Patent WO9945000 discloses a process for synthesizing (S)-2-amino-3-(thiazol-4-yl) propionic acid using 4-methylthiazole as a starting material and a combination of biological and chemical methods. In this process, acyltransferase is used to synthesize (S)-2-amino-3-(thiazol-4-yl) propionic acid. However, the biological source of acyltransferase in this patent is unknown, the amino acid sequence is unknown, and the specific catalytic efficiency is not clear. Aminoacylase can stereoselectively hydrolyze the amide bond of acylated amino acid or its derivative, and can be used for the synthesis of chiral amino acid and its derivative. However, due to the different sources of aminoacylase, the activity, stability, substrate selectivity and stereoselectivity are different, which to a large extent limits the industrial application of enzyme synthesis of (S)-2-amino-3-(thiazol-4-yl) propionic acid. Therefore, it is of great value in medical production to continue to find new aminoacylase resources with high catalytic activity, strong stereoselectivity and good stability, and to develop a green, efficient and economical method for synthesizing related chiral amino acid and its derivative compounds by using aminoacylase. SUMMARY

[0004] In view of the problems of insufficient biological enzyme resources, unknown sources and unclear characteristics in the prior art, the purpose of the present application is to provide several novel aminoacylases and their related sequences, and to provide a technical method for efficiently, greenly, economically and specifically catalyzing the synthesis of (S)-2-amino-3-(thiazol-4-yl) propionic acid by using the aminoacylases.

[0005] The method has the advantages of simple catalyst preparation process, low cost, mild reaction condition, short technological process, high product yield and high stereoscopic configuration purity, and has high industrial application potential.

[0006] The application provides a method for catalytically synthesizing (S)-2-amino-3-(thiazol-4-yl) propionic acid, and the method takes 2-acetylamino-3-(thiazol-4-yl) propionic acid as a substrate, and the catalyst contains an aminoacylase which comprises an amino acid sequence as shown in at least one sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5.

[0007] The aminoacylase in the application can be prepared by the following preferred methods, but is not limited to the following preferred methods: preferably prepared by a biological engineering method, preferably obtained by preparing a recombinant bacterium expressing the aminoacylase, and preferably the coding gene sequence of the aminoacylase is prepared by a chemical synthesis method; another preferred method for obtaining the aminoacylase is to prepare a target polypeptide sequence by a chemical synthesis method to obtain the catalyst. The enzyme preparation methods capable of preparing the aminoacylase with the amino acid sequences as shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5 in the prior art are all suitable for the application.

[0008] Preferably, the catalyst is derived from a recombinant bacterium expressing the aminoacylase, and preferably the catalyst is one of a whole cell of the recombinant bacterium expressing the aminoacylase (also referred to as a whole cell catalyst in the application), a crude enzyme solution (also referred to as a crude enzyme solution catalyst in the application), a pure enzyme (also referred to as a pure enzyme catalyst in the application) or a dry powder enzyme preparation.

[0009] The application provides a method for one-step chiral resolution of a substrate 2-acetylamino-3-(thiazol-4-yl) propionic acid to synthesize (S)-2-amino-3-(thiazol-4-yl) propionic acid by using the aminoacylase as a catalyst in the form of a whole cell of a recombinant bacterium, a crude enzyme solution or a pure enzyme, Figure 1 The application provides a method for one-step chiral resolution of a substrate 2-acetylamino-3-(thiazol-4-yl) propionic acid to synthesize (S)-2-amino-3-(thiazol-4-yl) propionic acid by using the aminoacylase as a catalyst in the form of a whole cell of a recombinant bacterium, a crude enzyme solution or a pure enzyme,

[0010] The method provided by the application has the advantages of clear aminoacylase source, simple catalyst preparation process, low cost, simple and mild reaction process, environmental protection, high stereoselectivity, and can realize a theoretical maximum conversion rate of 50% and an e.e. value of the product of more than 99%.

[0011] Preferably, the recombinant bacterium expressing the aminoacylase is at least one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum and yeast.

[0012] Preferably, the coding sequence of the aminoacylase comprises the nucleotide sequence shown in at least one of SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11.

[0013] The aminoacylase of the present application is derived from bacteria, fungi or mammals. The aminoacylase can be derived from, but is not limited to, N-acyl-L-amino acid amidohydrolase from Geobacillus stearothermophilus (SEQ ID NO: 1), N-acyl-L-amino acid amidohydrolase from Dictyostelium discoideum (SEQ ID NO: 2), acylamidase from Rhodococcus erythropolis (SEQ ID NO: 3), N-acyl-aromatic-L-amino acid amidohydrolase from Homo sapiens (SEQ ID NO: 4), N-acyl-aromatic-L-amino acid amidohydrolase from Mus musculus (SEQ ID NO: 5), N-acyl-L-amino acid amidohydrolase from Sus scrofa (SEQ ID NO: 6).

[0014] Based on the amino acid sequences of the six aminoacylases, the coding sequences of the aminoacylases are optimized for the codon usage bias of the host bacteria (e.g., Escherichia coli) and the corresponding gene fragments (SEQ ID NO: 7 to SEQ ID NO: 12) are synthesized. The synthesized genes are cloned into the Escherichia coli expression vector pET-28b(+) by standard gene cloning methods, and the recombinant plasmid is introduced into the Escherichia coli BL21(DE3) strain. Isopropyl-β-D-thiogalactoside (IPTG) is used to induce the expression of the aminoacylase in the recombinant bacteria. The wet bacterial cells with catalytic activity of the aminoacylase are obtained by direct centrifugation, and are used as the whole-cell catalyst for the reaction; or the cell lysate with catalytic activity of the aminoacylase is obtained by cell disruption, and is used as the crude enzyme solution catalyst for the reaction; or the purified aminoacylase is obtained by nickel column affinity chromatography purification, and is used as the pure enzyme catalyst for the reaction.

[0015] The heterologous expression host bacteria suitable for the present application include, but are not limited to, commonly used heterologous expression microbial systems such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, yeast, etc.

[0016] Any of the above preferred is that the whole cell catalyst is preferably fresh wet bacteria obtained by centrifugation after induction, or is preferably the above wet bacteria stored at low temperature (such as -80℃ to -20℃), or is preferably the whole cell dry powder after dehydration and drying.

[0017] Any of the above preferred is that the crude enzyme liquid catalyst is preferably the crude enzyme liquid obtained directly by centrifugation after ultrasonic or high pressure crushing of the cells, or is preferably the above crude enzyme liquid stored at low temperature (such as -80℃ to -20℃), or is preferably the crude enzyme dry powder after dehydration and drying.

[0018] Any of the above preferred is that the pure enzyme catalyst is preferably the pure enzyme obtained directly after purification by nickel column affinity chromatography of the above obtained crude enzyme liquid, or is preferably the above pure enzyme stored at low temperature (such as -80℃ to -20℃), or is preferably the pure enzyme dry powder after dehydration and drying.

[0019] Any of the above preferred is that the substrate is dissolved in a buffer system, the catalyst is added, and the reaction is shaken at 25-42℃. Preferably, after the reaction is completed, the reaction is terminated by heating at 95℃ for 20 min.

[0020] Any of the above preferred is that the catalytic reaction temperature is between 25-42℃, preferably 30-42℃.

[0021] Any of the above preferred is that the substrate 2-acetylamino-3-(thiazol-4-yl) propionic acid is dissolved in a buffer system, and the obtained aminoacylase recombinant bacteria whole cell, crude enzyme liquid or pure enzyme catalyst is added to the buffer system containing the substrate in a certain proportion, and the reaction is shaken (220 rpm) at 25-42℃. After the reaction is completed, the reaction is terminated by heating at 95℃ for 20 min. The reaction sample is centrifuged, the supernatant is diluted by an appropriate multiple and filtered, and the substrate and product are detected and analyzed by HPLC.

[0022] Any of the above is preferably, the buffer system is a common buffer such as phosphate buffer, HEPES or Tris with pH 7.0-pH 8.0; further, the buffer system is preferably potassium phosphate buffer with pH 7.0-pH 8.0; preferably potassium phosphate buffer with pH 7.0; preferably potassium phosphate buffer with pH 7.2; preferably potassium phosphate buffer with pH 8.0; preferably K2HPO4 / KH2PO4 buffer with pH 7.0-pH 8.0 and concentration of 0.02-0.5M; preferably Na2HPO4 / NaH2PO4 buffer with pH 7.0-pH 8.0; preferably HEPES buffer with pH 7.0-pH 8.0; preferably Tris-HCl buffer with pH 7.0-pH 8.0; preferably K2HPO4 / KH2PO4 buffer with pH 7.0-pH 8.0 and concentration of 0.1M; preferably K2HPO4 / KH2PO4 buffer with pH 7.2 and concentration of 0.1M. The above-mentioned buffers are all conventional buffers in the prior art, and their preparation methods and compositions are described in the prior art documents such as journals, papers, and reference books.

[0023] Any of the above is preferably, in the catalytic system, the substrate concentration is 0-90g / L, and the catalytic system contains at least one of the following a, b or c catalysts:

[0024] a. The catalyst is whole cells of recombinant bacteria expressing the aminoacylase, and the mass ratio of the amount of whole cells of recombinant bacteria expressing the aminoacylase to the amount of substrate is 0-35%;

[0025] b. The catalyst is a crude enzyme solution with a protein concentration of 0-5.4g / L;

[0026] c. The catalyst is a pure enzyme with a protein concentration of 0-5.4g / L.

[0027] Preferably, the substrate concentration of the catalytic system is 0-90g / L, and the substrate concentration is further preferably 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90g / L.

[0028] Preferably, the amount of whole cell of the recombinant bacteria expressing the aminoacylase used per amount of substrate is 0-35% (mass ratio), and the amount of whole cell of the recombinant bacteria expressing the aminoacylase used per amount of substrate is further preferably 0, 5%, 10%, 15%, 20%, 25%, 30%, 35% (mass ratio). In a preferred embodiment of the present application, the amount of whole cell of the recombinant bacteria expressing the aminoacylase used per amount of substrate is further preferably 3.75%, 7.5% or 15% (mass ratio); in another preferred embodiment of the present application, the amount of whole cell of the recombinant bacteria expressing the aminoacylase used per amount of substrate is further preferably 10%, 15%, 20%, 25% (mass ratio); in a preferred embodiment of the present application, the amount of whole cell of the recombinant bacteria expressing the aminoacylase used per amount of substrate is further preferably 17% or 35% (mass ratio).

[0029] Preferably, the protein concentration of the crude enzyme solution used is 0-5.4 g / L, and the catalyst protein concentration of the crude enzyme solution used is further preferably 0, 1, 2, 3, 4, 5, 5.4 g / L.

[0030] Preferably, the protein concentration of the crude enzyme solution used is 0-5.4 g / L, and the catalyst protein concentration of the crude enzyme solution used is further preferably 0, 1, 2, 3, 4, 5, 5.4 g / L.

[0031] Preferably, the protein concentration of the crude enzyme solution used is 0-5.4 g / L, and the catalyst protein concentration of the crude enzyme solution used is further preferably 0, 1, 2, 3, 4, 5, 5.4 g / L.

[0032] The substrate 2-acetylamino-3-(thiazol-4-yl)propanoic acid and the product 2-amino-3-(thiazol-4-yl)propanoic acid are detected by HPLC, and the detection method is as follows: Agilent 1260 HPLC analysis system equipped with an Agilent Eclipse Plus C18 chromatographic column (250 mm x 4.6 mm x 5 μm); the mobile phase is 5% acetonitrile / 95% water (containing 0.1% phosphoric acid) - 95% acetonitrile / 5% water (containing 0.1% phosphoric acid) for 0-15 min, 95% acetonitrile / 5% water (containing 0.1% phosphoric acid) - 5% acetonitrile / 95% water (containing 0.1% phosphoric acid) for 15-15.1 min, 5% acetonitrile / 95% water (containing 0.1% phosphoric acid) for 15.1-17 min; the flow rate is 1.0 ml / min; the detection wavelength is 210 nm; and the column temperature is 30°C. The product has a peak time of 3.2 min, and the substrate has a peak time of 5.2 min.

[0033] The product 2-amino-3-(thiazol-4-yl)propanoic acid is subjected to chiral purity detection by HPLC, and the detection method is as follows: an Agilent 1100 HPLC analysis system is equipped with a CROWNPAK CR-I(+) chiral chromatographic column (150mmx4.6mmx5μm); the mobile phase is 10% acetonitrile / 90% water (containing 16.5g / L of perchloric acid) for 0-15min; the flow rate is 0.2ml / min; the detection wavelength is 240nm; the column temperature is 25℃; the peak time of the R configuration product is 5.4min, and the peak time of the S configuration product is 7.3min.

[0034] The substrate 2-acetylamino-3-(thiazol-4-yl)propanoic acid and the product 2-amino-3-(thiazol-4-yl)propanoic acid are subjected to mass spectrometric qualitative analysis by high performance liquid chromatography-mass spectrometry. The detection method is as follows: an Agilent 6130 high performance liquid chromatography-mass spectrometry analysis system is equipped with an Agilent SB-C18 chromatographic column (50mmx3.0mmx1.8μm); the mobile phase is 10% acetonitrile (containing 0.05% formic acid) / 90% water (containing 0.05% formic acid) for 0-7min, 95% acetonitrile (containing 0.05% formic acid) / 5% water (containing 0.05% formic acid) for 7-9min, 95% acetonitrile (containing 0.05% formic acid) / 5% water (containing 0.05% formic acid) for 9-9.1min, 10% acetonitrile (containing 0.05% formic acid) / 90% water (containing 0.05% formic acid) for 9.1-12min; the flow rate is 0.6ml / min; the detection wavelength is 210nm, and the mass spectrometry is in a positive ion mode.

[0035] The application further provides application of an aminoacylase with an amino acid sequence as shown in at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5 in preparation of a lovastatin drug or preparation of a lovastatin drug intermediate (S)-2-amino-3-(thiazol-4-yl)propanoic acid.

[0036] The application further provides application of a coding sequence of the aminoacylase in any of the above aspects in preparation of a lovastatin drug or preparation of a lovastatin drug intermediate (S)-2-amino-3-(thiazol-4-yl)propanoic acid. The coding sequence of the aminoacylase is a nucleotide sequence as shown in at least one of SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:11.

[0037] The application also provides a recombinant expression vector for preparing lovastatin or an intermediate (S)-2-amino-3-(thiazol-4-yl)propionic acid in the preparation of lovastatin, and cloning the aminoacylase coding sequence in any of the above into an E. coli expression vector. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The application provides a method for biocatalytic synthesis of (S)-2-amino-3-(thiazol-4-yl)propionic acid.

[0039] Figure 2 The mass spectrum of the substrate and product identified in the preferred embodiment 3 of the application.

[0040] Figure 3 The HPLC chromatogram for identifying the chiral purity of the product in the preferred embodiment 5 of the application. DETAILED DESCRIPTION

[0041] The substrate 2-acetylamino-3-(thiazol-4-yl)propionic acid in examples 1-8 is synthesized according to the process reported in the literature (Kobayashi N, Sato N, Sugita K, et al. Synthesis and evaluation of in vivo anti-hypothermic effect of all stereoisomers of the thyrotropin-releasing hormone mimetic: Rovatirelin Hydrate. J Pept Sci, 2019, 25(12): e3228.). The form of the self-synthesized substrate used in the catalytic system is divided into two types: one is the solid crude product obtained by concentrating the 2-acetylamino-3-(thiazol-4-yl)propionic acid synthesis reaction liquid at 50°C (also referred to as a solid crude substrate in the application), and the other is the 2-acetylamino-3-(thiazol-4-yl)propionic acid synthesis reaction liquid (also referred to as a liquid crude substrate in the application).

[0042] The preferred embodiments of the application for one-step chiral resolution of the substrate 2-acetylamino-3-(thiazol-4-yl)propionic acid to synthesize (S)-2-amino-3-(thiazol-4-yl)propionic acid using the aminoacylase as a catalyst in the form of whole cells of recombinant bacteria, crude enzyme liquid, and pure enzyme are provided in the examples, Figure 1 The application provides a method for biocatalytic synthesis of (S)-2-amino-3-(thiazol-4-yl)propionic acid.

[0043] Example 1

[0044] Construction of recombinant aminoacylase expression strains

[0045] The aminoacylase gene fragments (SEQ ID NO: 7 to SEQ ID NO: 12) for heterologous expression were synthesized by codon optimization of DNA sequences against the E. coli host, using the aminoacylase amino acid sequences (SEQ ID NO: 1 to SEQ ID NO: 6) as templates, and the target genes were cloned into the pET-28b(+) vector through NdeI and XhoI enzyme cutting sites to obtain the aminoacylase recombinant expression plasmid. The E. coli BL21(DE3) competent cells were taken out from the -80°C refrigerator in advance, placed on ice to thaw, 1 μl of the recombinant plasmid was added to 100 μl of the competent cells, and the cells were gently stirred several times, then placed on ice for 30 min, 42°C water bath or metal bath heat shock for 90 sec, placed on ice for 2-3 min, added with 900 μl of LB medium, and incubated at 37°C for 1 hour. 100 μl of the cell suspension was taken and spread on the LB solid medium containing 50 μg / ml kanamycin sulfate, and incubated at 37°C overnight to obtain the single colony of the aminoacylase recombinant expression strain.

[0046] Example 2

[0047] Preparation of different forms of aminoacylase catalysts

[0048] The single colony of the aminoacylase recombinant expression strain was picked and inoculated in 20 ml of LB liquid medium (containing 50 μg / ml kanamycin sulfate), and incubated at 37°C overnight. The overnight culture was inoculated into 400 ml of LB liquid medium (containing 50 μg / ml kanamycin sulfate) at a 1% inoculation amount, and incubated at 37°C until the OD 600 was 0.6-1.0. IPTG was added to a final concentration of 0.15 mM, and the culture was incubated at 20°C to induce the expression of the target aminoacylase. After 20 h of induction, the obtained wet bacteria were collected by centrifugation as a whole-cell catalyst; the cells were broken by ultrasonic crushing, and the cell lysate was taken after centrifugation as a crude enzyme liquid catalyst; and the pure enzyme catalyst was obtained by nickel column affinity chromatography protein purification and ultrafiltration concentration. The above whole-cell catalyst, crude enzyme liquid catalyst, and pure enzyme catalyst were used to catalyze the reaction.

[0049] Example 3

[0050] Screening of aminoacylase using whole-cell catalyst

[0051] The whole-cell catalysts obtained above were stored at -20℃ or -80℃. Before use, the whole-cell catalysts were removed and allowed to return to room temperature before being used for aminoacylase catalytic reactions. The catalytic reaction was carried out in a 1 ml reaction system consisting of a 0.1 M potassium phosphate buffer solution (pH 7.2) containing 88 g / L of the solid crude substrate 2-acetamido-3-(thiazol-4-yl)propionic acid, and a whole-cell catalyst containing recombinant aminoacylases (SEQ ID NO: 1 to SEQ ID NO: 5) at a cell concentration of 50 OD. The reaction was carried out at 37℃ with shaking (220 rpm) for 24 h. The substrate and product were detected by HPLC, and the substrate conversion rate was calculated. The results are shown in the table below. The aminoacylases SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5 showed higher substrate conversion rates. Furthermore, aminoacylase SEQ ID NO: 6 was compared with aminoacylase SEQ ID NO: 2. The catalytic reaction was carried out in a 1 ml reaction system, which was a 0.1 M potassium phosphate buffer solution (pH 7.2) containing 15 g / L of liquid crude substrate 2-acetamido-3-(thiazol-4-yl)propionic acid, and a whole-cell catalyst of recombinant aminoacylase (SEQ ID NO: 2, SEQ ID NO: 6) at a cell concentration of 2.25 OD. The reaction was carried out at 37 °C with shaking (220 rpm) for 24 h. The substrate and product were detected by HPLC, and the substrate conversion rate was calculated. The substrate conversion rate of aminoacylase SEQ ID NO: 2 was 47.97%, and the substrate conversion rate of aminoacylase SEQ ID NO: 6 was 3.99%.

[0052] The substrate conversion rate is calculated as: (substrate peak area at 0h of reaction - substrate peak area at 24h of reaction) / substrate peak area at 0h of reaction × 100%. The sample preparation method for HPLC detection is completely consistent. Unless otherwise specified, the calculation method for the substrate conversion rate in this invention is as described above.

[0053]

[0054] "—" indicates that no product was detected.

[0055] The substrate and product were identified using mass spectrometry, and the results are as follows: Figure 2 As shown. The substrate 2-acetamido-3-(thiazol-4-yl)propionic acid has a relative molecular mass of 214.24, and its characteristic mass spectrometry peak is m / z 215.00 [M+H]. + and m / z 450.90[2M+Na] + The product, 2-amino-3-(thiazol-4-yl)propionic acid, has a relative molecular mass of 172.20, and its characteristic mass spectrometry peak is at m / z 173.00 [M+H]. +and m / z 366.90 [2M+Na] + .

[0056] Example 4

[0057] Screening of aminoacylase with crude enzyme catalyst

[0058] The crude enzyme catalyst obtained above was stored at -80°C, and when used, the crude enzyme was taken out and thawed on ice for use in the aminoacylase catalytic reaction. The catalytic reaction was carried out in a 1 ml reaction system, which was a 0.1 M potassium phosphate buffer solution (pH 7.2) containing 88 g / L of solid crude substrate 2-acetylamino-3-(thiazol-4-yl) propanoic acid, and the protein concentration of the crude enzyme catalyst (SEQ ID NO: 1 to SEQ ID NO: 5) was 5.4 g / L. The reaction was carried out at 37°C with shaking (220 rpm) for 24 h, and the substrate and product were detected by HPLC to calculate the substrate conversion rate, and the results are shown in the following table. The substrate conversion rates of aminoacylases SEQ ID NO: 2 and SEQ ID NO: 4 were higher.

[0059]

[0060] “—” indicates that the product was not detected.

[0061] Example 5

[0062] Purified enzyme catalysis and chiral purity identification of product

[0063] The purified enzyme catalyst obtained above was stored at -80°C, and when used, the purified enzyme was taken out and thawed on ice for use in the aminoacylase catalytic reaction. The catalytic reaction was carried out in a 1 ml reaction system, which was a 0.1 M potassium phosphate buffer solution (pH 7.2) containing 88 g / L of solid crude substrate 2-acetylamino-3-(thiazol-4-yl) propanoic acid, and the protein concentration of the purified enzyme catalyst (SEQ ID NO: 2, SEQ ID NO: 4) was 5.4 g / L. The reaction was carried out at 37°C with shaking (220 rpm) for 24 h, and the substrate and product were detected by HPLC to calculate the substrate conversion rate, and the obtained substrate conversion rates were all ≥50% (SEQ ID NO: 2, SEQ ID NO: 4). The chiral purity of the target product was detected by HPLC, and as shown in Table 2, the e.e. values of (S)-2-amino-3-(thiazol-4-yl) propanoic acid were 99.29% (SEQ ID NO: 2) and 97.48% (SEQ ID NO: 4), respectively. Figure 3

[0064] The e.e. value of the S-type product (i.e., the enantiomeric excess percentage, i.e., (S-R) / (S+R) x 100%)

[0065] ​Example 6

[0066] Optimization of whole-cell catalytic system

[0067] The whole-cell catalyst obtained above was stored at -20°C or -80°C, and was used for aminoacylase catalyzed reaction after being taken out and restored to room temperature.

[0068] Reaction system 1: Catalytic solid crude substrate was used to analyze the amount of whole-cell catalyst, and the catalytic reaction was carried out in a 1 ml reaction system, which was 0.1 M potassium phosphate buffer solution (pH 7.2) containing 88 g / L of solid crude substrate 2-acetylamino-3-(thiazol-4-yl) propanoic acid, and the amount of recombinant aminoacylase (SEQ ID NO: 2) whole-cell catalyst per substrate was 15%, 7.5%, 3.75% (mass ratio), respectively, and the reaction was carried out at 37°C with oscillation (220 rpm) for 24 h. HPLC was used to detect the substrate and product, and the substrate conversion rate was calculated. The results are shown in the following table, and the substrate conversion rate was higher when the amount of whole-cell catalyst per substrate was 15% (mass ratio).

[0069]

[0070] Reaction system 2: Catalytic liquid crude substrate was used to analyze the amount of whole-cell catalyst, and the catalytic reaction was carried out in a 1 ml reaction system, which was 0.1 M potassium phosphate buffer solution (pH 7.2) containing 15 g / L, 30 g / L, 45 g / L, 60 g / L, 75 g / L, 90 g / L of liquid crude substrate 2-acetylamino-3-(thiazol-4-yl) propanoic acid, respectively, and the amount of recombinant aminoacylase (SEQ ID NO: 2) whole-cell catalyst per substrate was 10%, 15%, 20%, 25% (mass ratio), respectively, and the reaction was carried out at 37°C with oscillation (220 rpm) for 24 h. HPLC was used to detect the substrate and product, and the substrate conversion rate was calculated. The results are shown in the following table.

[0071] In reaction system 2, the substrate was a liquid crude substrate, and when the amount of recombinant aminoacylase (SEQ ID NO: 2) whole-cell catalyst per substrate was 15% (mass ratio), the substrate conversion rate was > 40% when the substrate concentration was ≤ 30 g / L, and the substrate conversion rate was < 30% when the substrate concentration was ≥ 45 g / L. By increasing the amount of whole-cell catalyst per substrate (mass ratio), the substrate conversion rate can be improved (as shown in the following table).

[0072] Compared with the solid crude substrate in reaction system 1, reducing the substrate concentration or increasing the amount of whole-cell catalyst per substrate (mass ratio) in reaction system 2 can ensure a substrate conversion rate of > 40% or even higher.

[0073] Example 6 The catalytic method and the catalyst according to the present application are suitable for both solid crude substrate and liquid crude substrate, the liquid crude substrate is directly the synthesis reaction solution of 2-acetylamino-3-(thiazol-4-yl)propionic acid, and is more suitable for industrial production. Therefore, the catalytic method and the aminoacylase provided by the present application provide a technical basis for the industrial production of (S)-2-amino-3-(thiazol-4-yl)propionic acid.

[0074]

[0075] Reaction system 3: analysis of the optimum reaction temperature, the catalytic reaction was carried out in 1 ml reaction system, which was 0.1 M potassium phosphate buffer solution (pH 7.2) containing 15 g / L of liquid crude substrate 2-acetylamino-3-(thiazol-4-yl)propionic acid, and the amount of whole-cell catalyst of recombinant aminoacylase (SEQ ID NO: 2) was 15% (mass ratio) of the substrate. The reaction was carried out at 25°C, 30°C, 37°C and 42°C respectively, and the substrate and product were detected by HPLC after 24 h of oscillation (220 rpm), and the substrate conversion rate was calculated. The results are shown in the following table, and the substrate conversion rate is higher when the catalytic reaction temperature is between 30-42°C.

[0076] Reaction temperature 25℃ 30℃ 37℃ 42℃ Substrate conversion 43.82％ 46.03％ 47.69％ 47.71％

[0077] Example 7

[0078] Scale-up of the target catalytic reaction system

[0079] The whole cell catalyst obtained above is stored at -20°C or -80°C, and when used, the whole cell catalyst is taken out and restored to room temperature for use in the aminoacylase catalytic reaction. The catalytic reaction is carried out in a 14.5 L reaction system, which is a 0.1 M potassium phosphate buffer solution (pH 7.2) containing 15 g / L and 30 g / L of liquid crude substrate 2-acetylamino-3-(thiazol-4-yl) propanoic acid, respectively, and the amount of recombinant aminoacylase (SEQ ID NO: 2) whole cell catalyst used per substrate is 15% and 30% (mass ratio), respectively, and the reaction is stirred at 37°C for 48 h, and then the amount of recombinant aminoacylase (SEQ ID NO: 2) whole cell catalyst used per substrate is supplemented to 18% and 35% (mass ratio), respectively, and the reaction is continued to 72 h. After the reaction is completed, the product 2-amino-3-(thiazol-4-yl) propanoic acid in the catalytic system is reacted with di-tert-butyl dicarbonate (BOC anhydride) to introduce a BOC protecting group, and then it is separated and purified, and the BOC protecting group is removed, and finally the mass of the target product (S)-2-amino-3-(thiazol-4-yl) propanoic acid is 45.5 g and 84.0 g, respectively, and the final molar yield of the product is 27% and 25%, respectively, and the e.e. value is 99.95% for both.

[0080] Note: The product yield described in Example 7 does not refer to the substrate conversion rate of the enzyme catalytic reaction step, but to the product yield of the final product.

[0081] Example 8

[0082] Preparation and catalysis of aminoacylase dry powder catalyst

[0083] The whole cell catalyst obtained above is stored at -20°C or -80°C, and when used, the whole cell catalyst is taken out and restored to room temperature for use in the aminoacylase catalytic reaction. The catalytic reaction is carried out in a 14.5 L reaction system, which is a 0.1 M potassium phosphate buffer solution (pH 7.2) containing 15 g / L and 30 g / L of liquid crude substrate 2-acetylamino-3-(thiazol-4-yl) propanoic acid, respectively, and the amount of recombinant aminoacylase (SEQ ID NO: 2) whole cell catalyst used per substrate is 15% and 30% (mass ratio), respectively, and the reaction is stirred at 37°C for 48 h, and then the amount of recombinant aminoacylase (SEQ ID NO: 2) whole cell catalyst used per substrate is supplemented to 18% and 35% (mass ratio), respectively, and the reaction is continued to 72 h. After the reaction is completed, the product 2-amino-3-(thiazol-4-yl) propanoic acid in the catalytic system is reacted with di-tert-butyl dicarbonate (BOC anhydride) to introduce a BOC protecting group, and then it is separated and purified, and the BOC protecting group is removed, and finally the mass of the target product (S)-2-amino-3-(thiazol-4-yl) propanoic acid is 45.5 g and 84.0 g, respectively, and the final molar yield of the product is 27% and 25%, respectively, and the e.e. value is 99.95% for both.

[0084] The obtained aminoacylase crude enzyme dry powder catalyst or whole cell dry powder catalyst is used to catalyze the reaction. The catalytic reaction is carried out in a 1 ml reaction system, which is a 0.1 M potassium phosphate buffer solution (pH 7.2) containing 15 g / L of liquid crude substrate 2-acetylamino-3-(thiazol-4-yl) propanoic acid, and the mass ratio of the crude enzyme dry powder catalyst to the substrate or the mass ratio of the whole cell wet bacteria corresponding to the whole cell dry powder catalyst to the substrate is both 15%, and the reaction is carried out at 37°C with oscillation (220 rpm) for 24 h. HPLC is used to detect the substrate and the product, and the substrate conversion rate is calculated. The substrate conversion rate in the aminoacylase crude enzyme dry powder catalyst or whole cell dry powder catalyst catalytic system is all ≥50%.

[0085] The above examples are only examples for clearly illustrating the present application, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. All the embodiments cannot be enumerated here, and the changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for catalytic synthesis of (S)-2-amino-3-(thiazol-4-yl)propionic acid, with 2-acetylamino-3-(thiazol-4-yl)propionic acid as a substrate, characterized in that, The catalyst contains an aminoacylase, and the amino acid sequence of the aminoacylase is at least one of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:

5.

2. The method of claim 1, wherein, The catalyst is at least one of a whole cell of a recombinant bacterium expressing the aminoacylase, a crude enzyme solution, or a pure enzyme.

3. The method of claim 2, wherein, The catalyst is a dry powder enzyme preparation.

4. The method of claim 2, wherein, The recombinant bacterium expressing the aminoacylase is at least one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or a yeast.

5. The method of claim 4, wherein, The nucleotide sequence of the aminoacylase includes at least one of SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO:

11.

6. The method of claim 5, wherein, The substrate is dissolved in a buffer system, the catalyst is added, and the reaction is shaken at 25-42℃; after the reaction is completed, the reaction is terminated by heating at 95℃ for 20 min.

7. The method of claim 6, wherein, The buffer system has a pH of 7.0-8.

0.

8. Use of an aminoacylase having an amino acid sequence as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5 in the preparation of loxoribine drug or (S)-2-amino-3-(thiazol-4-yl)propanoic acid, an intermediate of loxoribine drug, using 2-acetylamino-3-(thiazol-4-yl)propanoic acid as a substrate.

9. Use according to claim 8, wherein the compound is ###0002### The nucleotide sequence of the aminoacylase is at least one of SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO:

11.

10. Use of a recombinant expression vector for the preparation of loxoribulin or an intermediate of loxoribulin, (S)-2-amino-3-(thiazol-4-yl)propanoic acid, from 2-acetylamino-3-(thiazol-4-yl)propanoic acid, characterized in that, The nucleotide sequence of the aminoacylase of claim 9 is cloned into an Escherichia coli expression vector.

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