Synthetic method of L-2-amino adipic acid-delta-semialdehyde
The prior art was solved by using L-lysine 6-aminotransferase to convert L-lysine and α-ketoglutaric acid into L-2-aminoadipic acid-δ-semialdehyde by biocatalytic method. The problem of low conversion efficiency has achieved the efficient and pure synthesis of L-2-aminoadipic acid-δ-semialdehyde.
Patent Information
- Application Number
- CN202411410668.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2025-02-14
AI Technical Summary
In the prior art, the conversion efficiency of L-2-aminoadipic acid-δ-semialdehyde is low and cannot meet the industrial production needs of L-2-aminoadipic acid.
By using biocatalytic method, L-lysine and α-ketoglutaric acid are converted to L-2-aminoadipic acid-δ-semialdehyde using L-lysine 6-aminotransferase or its mutant, through Transaminase catalyzed reaction to achieve efficient synthesis.
The conversion efficiency of L-2-aminoadipic acid-δ-semialdehyde has been improved, reaching more than 72%, and the product purity has reached 99%, meeting the intermediate supply needs for drug research and development.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biosynthesis, and in particular to a method for synthesizing L-2-aminoadipic acid-δ-semialdehyde. Background Art
[0002] As a non-protein amino acid, L-2-aminoadipic acid has a wide range of uses. It is an important chiral building block for the synthesis of β-lactam antibiotic penicillin and cephalosporin precursors, an intermediate for broad-spectrum synthetic drugs, and can be used to treat rheumatism, psoriasis, and prostate cancer. It can also be used as an intermediate for the synthesis of certain special chemical substances, such as sweeteners, peptides, and chelating agents. L-2-aminoadipic acid has great market potential in the fields of medicine, chemical industry, food, and feed, but the current supply of L-2-aminoadipic acid cannot meet market demand. L-2-aminoadipic acid-δ-semialdehyde, as an important precursor for the synthesis of L-2-aminoadipic acid, is mainly derived from chemical synthesis, but this method has the disadvantages of long production route, high raw material cost, harsh reaction conditions, unsatisfactory yield, and environmental pollution.
[0003] The preparation of L-2-aminoadipic acid by biocatalysis or bioconversion has the advantages of mild conditions, low cost, less environmental pollution, etc., but it started late and has not formed an industrial production process. Miruxiang Company of Japan screened out alcaligenes, Pseudomonas, and Coulterella, which can synthesize L-2-aminoadipic acid from the substrate L-hexahydropyridine carboxylic acid. Using lysine as a substrate, Agrobacterium, alcaligenes, Brevibacterium, and Bacillus were screened to synthesize L-2-aminoadipic acid. The former substrate is expensive, and the latter reaction efficiency is extremely low, both of which are not suitable for industrial production. Kuniho Nakata et al. screened out a strain of Flavobacterium, which can directly convert the aminomethyl on lysine into a carboxyl group in one step through microbial culture, and there is no need to modify the amino group at the α position, but the conversion efficiency is only 25%. Therefore, the present invention develops an environmentally friendly and efficient method for preparing L-2-aminoadipic acid-δ-semialdehyde to meet the synthesis and application of L-2-aminoadipic acid. Summary of the invention
[0004] The invention provides a method for synthesizing L-2-aminoadipic acid-δ-semialdehyde, which solves the problem of low conversion efficiency of L-2-aminoadipic acid-δ-semialdehyde in the related art.
[0005] The technical solution of the present invention is as follows: The present invention provides a method for synthesizing L-2-aminoadipic acid-δ-semialdehyde, comprising the following steps: Using L-lysine and α-ketoglutarate as substrates, L-2-aminoadipic acid-δ-semialdehyde is obtained through the catalytic reaction of transaminase and cofactors; The transaminase is one of L-lysine 6-aminotransferase and a mutant of L-lysine 6-aminotransferase; The L-lysine 6-aminotransferase is derived from South Korea's Stenotrophomonas ( Stenotrophomonas koreensis ), the amino acid sequence is shown in SEQ ID No.2; The N-terminus and / or C-terminus of the defined protein is connected with a tag to obtain a fusion protein.
[0006] As a further technical solution, the amino acid sequence of the mutant of the L-lysine 6-aminotransferase is shown in SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20, SEQ ID No.22 or SEQ ID No.24, and the mutant of the L-lysine 6-aminotransferase has at least one of the following mutations compared with the L-lysine 6-aminotransferase: Q71K, N76L, G212A, R213G.
[0007] As a further technical solution, the sequence of the gene encoding L-lysine 6-aminotransferase is SEQ ID No. 1 or a site-directed mutagenesis sequence that retains its function and encodes the same protein.
[0008] As a further technical solution, the encoding gene sequence of the mutant of L-lysine 6-aminotransferase is any one of SEQID No.3, SEQID No.5, SEQID No.7, SEQID No.9, SEQID No.11, SEQID No.13, SEQID No.15, SEQID No.17, SEQID No.19, SEQID No.21, and SEQID No.23.
[0009] As a further technical solution, the transaminase is added in the form of crude enzyme solution or whole cells.
[0010] As a further technical solution, the method for preparing the crude enzyme solution comprises the following steps: expressing the L-lysine 6-aminotransferase in a host cell to obtain a recombinant cell; and lysing the recombinant cell to obtain the crude enzyme solution.
[0011] As a further technical solution, the method for preparing the whole cell comprises the following steps: expressing the L-lysine 6-aminotransferase in a host cell, and the obtained recombinant cell is a whole cell.
[0012] As a further technical solution, the method for preparing the recombinant cell comprises the following steps: introducing a nucleic acid molecule capable of expressing the L-lysine 6-aminotransferase into the host cell, and obtaining the recombinant cell expressing the L-lysine 6-aminotransferase after induction culture.
[0013] As a further technical solution, the nucleic acid molecule of L-lysine 6-aminotransferase is introduced into the host cell via a recombinant vector.
[0014] As a further technical solution, the host cell is a prokaryotic cell.
[0015] As a further technical solution, the prokaryotic cell is a bacterium.
[0016] As a further technical solution, the bacteria is Escherichia coli, specifically E. coli BL21 (DE3).
[0017] As a further technical solution, the recombinant vector is a bacterial plasmid carrying the coding gene of the L-lysine 6-aminotransferase.
[0018] As a further technical solution, the recombinant vector is specifically a recombinant plasmid obtained by replacing the small fragment between the restriction sites BamH 1 and Xho 1 of the pET-28a(+) vector with the coding gene of the L-lysine 6-aminotransferase or the L-lysine 6-aminotransferase mutant.
[0019] As a further technical solution, when the transaminase is added in the form of a crude enzyme solution, the concentration of the transaminase in the catalytic reaction system is 0.1-5 g / L.
[0020] As a further technical solution, when the transaminase is added in the form of a crude enzyme solution, the concentration of the transaminase in the catalytic reaction system is 0.2 g / L.
[0021] As a further technical solution, when the transaminase is added in the form of whole cells, the wet weight of the transaminase in the catalytic reaction system is 10-50 g / L.
[0022] As a further technical solution, when the transaminase is added in the form of whole cells, the wet weight of the transaminase in the catalytic reaction system is 25 g / L.
[0023] As a further technical solution, the catalytic reaction is carried out in a phosphate buffer with a concentration of 50-100 mM and a pH of 6.5-8.5.
[0024] As a further technical solution, when the transaminase is added in the form of a crude enzyme solution, the catalytic reaction is carried out in a phosphate buffer with a concentration of 50 mM and a pH value of 7.0.
[0025] As a further technical solution, when the transaminase is added in the form of whole cells, the catalytic reaction is carried out in a phosphate buffer with a concentration of 50 mM and a pH value of 7.0.
[0026] As a further technical solution, the temperature of the catalytic reaction is 10-30°C, and the time of the catalytic reaction is 12-24h.
[0027] As a further technical solution, the temperature of the catalytic reaction is 15° C., and the time of the catalytic reaction is 12 hours.
[0028] As a further technical solution, a cofactor is also added to the catalytic reaction, and the cofactor is 5-pyridoxal phosphate.
[0029] As a further technical solution, when the transaminase is added in the form of crude enzyme solution or whole cells, the concentration of pyridoxal 5-phosphate in the catalytic reaction system is 0.1-1 mM.
[0030] As a further technical solution, when the transaminase is added in the form of crude enzyme solution or whole cells, the concentration of pyridoxal 5-phosphate in the catalytic reaction system is 0.4 mM.
[0031] The working principle and beneficial effects of the present invention are: In the present invention, L-2-aminohexanic acid-δ-semialdehyde is prepared by biocatalysis, and the method has mild conditions, low cost, green environmental protection, high efficiency, and good application prospects. In the present invention, L-lysine and α-ketoglutaric acid are used as substrates, and L-2-aminohexanic acid-δ-semialdehyde is synthesized under the catalysis of L-lysine 6-aminotransferase or a mutant of L-lysine 6-aminotransferase, and its conversion efficiency can reach more than 72%, and the purity is 99%. By this efficient method for synthesizing L-2-aminohexanic acid-δ-semialdehyde, a stable intermediate supply can be provided for drug research and development, and the development and production of new drugs can be promoted. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0033] Figure 1 This is a schematic diagram of the reaction of L-lysine 6-aminotransferase catalyzing L-lysine and α-ketoglutarate to synthesize L-2-aminoadipic acid-δ-semialdehyde; Figure 2 It is the color reaction diagram of L-2-aminoadipic acid-δ-semialdehyde; Figure 3 It is the standard curve of L-2-aminoadipic acid-δ-semialdehyde concentration; Figure 4 is the liquid chromatographic peak diagram of L-lysine before the reaction; Figure 5 The figure is the liquid chromatography peak diagram of L-lysine after the reaction. DETAILED DESCRIPTION
[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0035] In the following embodiments, unless otherwise specified, all of them are conventional methods.
[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0037] Example 1 The method for preparing L-lysine 6-aminotransferase or its mutant engineering bacteria comprises the following steps: The coding genes of L-lysine 6-aminotransferase were synthesized (codon optimization was performed using Escherichia coli as the host as needed), the synthesized genes were connected to the pET28a(+) expression vector, and the recombinant vector was obtained after sequencing verification. The related gene mutants were obtained by site-directed mutagenesis.
[0038] The above recombinant expression vector verified to be correct by sequencing is transformed into a microbial host of E. coli BL21 (DE3) to obtain the genetically engineered strain of the present invention, wherein the transformation method is a conventional transformation method in the art, such as a chemical transformation method.
[0039] The gene sequence, amino acid sequence, and amino acid sequence mutation site of the L-lysine 6-aminotransferase and its mutant prepared in this example are recorded in Table 1.
[0040] Table 1 L-lysine 6-aminotransferase and its mutants
[0041] Example 2 The method for expressing L-lysine 6-aminotransferase and its mutants and preparing crude enzyme comprises the following steps: The recombinant expression vector of L-lysine 6-aminotransferase or mutant constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) competent cells and cultured at 37°C for 16 h. After the transformants grew out, a single transformant was inoculated into 5 mL LB medium containing kanamycin (50 μg / mL), cultured at 37°C and 200 rpm for 16 h, and then inoculated into LB medium at a ratio of 1% (volume percentage), cultured at 37°C and 200 rpm until the OD600 value was about 0.6-0.8, IPTG was added at a final concentration of 0.1 mM, induced culture was performed at 25°C for 14 h, and the cells were collected by centrifugation at 4°C and 6000 rpm for 20 min, resuspended and washed once with 50 mM phosphate buffer with a pH value of 7.0, and then ultrasonically broken the bacteria and prepared a crude enzyme solution.
[0042] Example 3 The method for preparing L-2-aminoadipic acid-δ-semialdehyde by using the crude enzyme solutions of L-lysine 6-aminotransferases numbered 1 to 12 and mutants thereof in Example 1 respectively comprises the following steps: Phosphate buffer with a concentration of 50 mM and a pH value of 7.0, L-lysine with a final concentration of 10 mM, 5 mM α-ketoglutarate, 0.4 mM pyridoxal-5-phosphate and crude L-lysine 6-aminotransferase enzyme solution were added to the reaction system in sequence. The reaction system was reacted at 15°C for 12 hours. The reaction formula is as follows: Figure 1 shown.
[0043] After the reaction is completed, 1 mL of the supernatant is added to 1.5 mL of 4 mM O-aminobenzaldehyde, mixed evenly, and placed in an incubator at 37°C for 60 min. The catalytic product L-2-aminoadipic acid-δ-semialdehyde can react with O-aminobenzaldehyde to produce an orange-yellow complex. The absorbance value at 465 nm can identify the output of the product. L-2-aminoadipic acid-δ-semialdehyde can react with O-aminobenzaldehyde to form a color reaction. Figure 2 As shown, the standard curve of L-2-aminoadipic acid-δ-semialdehyde concentration is as shown Figure 3 shown.
[0044] After the reaction, pre-column derivatization was performed, 200 μL of the reaction solution was added to 1000 μL of 0.4 M borate buffer (0.4 M aqueous solution, pH 10.2), shaken for 1 min, 200 μL of o-phthalaldehyde solution was added, shaken for 1 min, and filtered through a 0.45 μm aqueous filter membrane. Liquid chromatography was performed, and the results of liquid chromatography were as follows: Figure 4 and Figure 5 shown.
[0045] The conversion efficiency and purity of the crude enzyme solutions of L-lysine 6-aminotransferases No. 1 to 12 and their mutants in Example 1 for preparing L-2-aminoadipic acid-δ-semialdehyde are recorded in Table 2.
[0046] Table 2 Conversion efficiency and purity of crude enzyme solution for preparing L-2-aminoadipic acid-δ-semialdehyde
[0047] Example 4 The method for preparing L-2-aminoadipic acid-δ-semialdehyde using the whole cells of L-lysine 6-aminotransferases numbered 1 to 12 and mutants thereof in Example 1 comprises the following steps: Phosphate buffer with a concentration of 50 mM and a pH value of 7.0, L-lysine with a final concentration of 10 mM, 5 mM α-ketoglutarate, 0.4 mM pyridoxal-5-phosphate and L-lysine 6-aminotransferase whole cells were added to the reaction system in sequence, and the reaction system was reacted at 15°C for 12 hours.
[0048] The conversion efficiency and purity of the whole cells of L-lysine 6-aminotransferases No. 1 to 12 and their mutants in Example 1 for preparing L-2-aminoadipic acid-δ-semialdehyde are recorded in Table 3.
[0049] Table 3 Conversion efficiency and purity of L-2-aminoadipic acid-δ-semialdehyde prepared from whole cells
[0050] Comparative Example 1 The method for preparing L-2-aminoadipic acid-δ-semialdehyde by using crude L-lysine 6-aminotransferase solutions from different sources comprises the following steps: S1, selected from the NCBI database Amycolatopsis magusensis )、Red salt bacteria( Salinibacter ruber ), Mycobacterium avium ( Mycobacterium avium )、Bacillus subtilis( Bacillus subtilis ) was connected to the pET28a(+) vector, expressed in E. coli BL21(DE3), the whole cells were collected, and the crude enzyme solution was prepared by the method in Example 2; S2. The method for preparing L-2-aminoadipic acid-δ-semialdehyde using crude enzyme solution is the same as that in Example 3.
[0051] The gene sequences, amino acid sequences and conversion efficiencies of crude L-lysine 6-aminotransferase solutions from different sources to obtain L-2-aminoadipic acid-δ-semialdehyde are recorded in Table 4.
[0052] Table 4 Conversion efficiency of crude L-lysine 6-aminotransferase from different sources to prepare L-2-aminoadipic acid-δ-semialdehyde
[0053] Comparative Example 2 The method for preparing L-2-aminoadipic acid-δ-semialdehyde by using L-lysine 6-aminotransferase whole cells from different sources comprises the following steps: S1, selected from the NCBI database Amycolatopsis magusensis )、Red salt bacteria ( Salinibacter ruber ), Mycobacterium avium ( Mycobacterium avium ), Bacillus subtilis ( Bacillus subtilis ) was ligated into the pET28a(+) vector, expressed in E. coli BL21(DE3), and the whole cells were collected; S2. The method for preparing L-2-aminoadipic acid-δ-semialdehyde using whole cells is the same as that in Example 4.
[0054] The whole cell gene sequences and amino acid sequences of L-lysine 6-aminotransferase from different sources are the same as those in Comparative Example 1.
[0055] The conversion efficiency of L-2-aminoadipate-δ-semialdehyde prepared by whole cell L-lysine 6-aminotransferase from different sources is recorded in Table 5.
[0056] Table 5 Conversion efficiency of L-2-aminoadipic acid-δ-semialdehyde prepared by L-lysine 6-aminotransferase whole cells from different sources
[0057] By comparing Example 3 with Comparative Example 1, Example 4 with Comparative Example 2, it was found that the Stenotrophomonas koreensis The crude enzyme solution and whole cells prepared from L-lysine 6-aminotransferase and its mutants catalyzed the conversion of L-lysine to L-2-aminoadipic acid-δ-semialdehyde with higher efficiency than L-lysine 6-aminotransferase from other sources, indicating that the enzyme from Stenotrophomonas koreaensis ( Stenotrophomonas koreensis ) can effectively improve the conversion efficiency of L-2-aminoadipic acid-δ-semialdehyde, solving the problem of low conversion rate of L-2-aminoadipic acid-δ-semialdehyde.
[0058] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for synthesizing L-2-aminoadipic acid-δ-semialdehyde, characterized in that: The following steps are involved: Using L-lysine and α-ketoglutarate as substrates, L-2-aminoadipic acid-δ-semialdehyde is obtained through a transaminase-catalyzed reaction. The transaminase is one of L-lysine 6-aminotransferase and a mutant of L-lysine 6-aminotransferase; The L-lysine 6-aminotransferase is derived from South Korea's Stenotrophomonas ( Stenotrophomonas koreensis ), the amino acid sequence is shown in SEQ ID No.2; A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined by SEQ ID No.
2.
2. A method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 1, characterized in that: The amino acid sequence of the mutant of the L-lysine 6-aminotransferase is shown in SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20, SEQ ID No.22 or SEQ ID No.
24. Compared with the L-lysine 6-aminotransferase, the mutant of the L-lysine 6-aminotransferase has at least one of the following mutations: Q71K, N76L, G212A, R213G.
3. A method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 1, characterized in that: The sequence of the gene encoding L-lysine 6-aminotransferase is SEQ ID No. 1 or a site-directed mutagenesis sequence that retains its function and encodes the same protein.
4. The method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 1, characterized in that: The encoding gene sequence of the mutant of L-lysine 6-aminotransferase is any one of SEQID No.3, SEQID No.5, SEQID No.7, SEQID No.9, SEQID No.11, SEQID No.13, SEQID No.15, SEQID No.17, SEQID No.19, SEQID No.21, and SEQID No.
23.
5. The method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 1, characterized in that: The added form of the transaminase includes crude enzyme solution or whole cells.
6. The method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 5, characterized in that: When the transaminase is added in the form of a crude enzyme solution, the concentration of the transaminase in the catalytic reaction system is 0.1-5 g / L.
7. The method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 5, characterized in that: When the transaminase is added in the form of whole cells, the wet weight of the transaminase in the catalytic reaction system is 10-50 g / L.
8. The method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 1, characterized in that: The catalytic reaction is carried out in a phosphate buffer solution with a concentration of 50-100 mM and a pH value of 6.5-8.
5.
9. The method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 1, characterized in that: The temperature of the catalytic reaction is 10-30° C., and the time of the catalytic reaction is 12-24 hours.
10. The method for synthesizing L-2-aminoadipic acid-δ-semialdehyde according to claim 1, characterized in that: The catalytic reaction also adds a cofactor, which is pyridoxal 5-phosphate.