Process for preparing 2,4-diaminobutyric acid by enzyme catalysis method
阅读说明：本技术 酶催化法制备2,4-二氨基丁酸的工艺 (Process for preparing 2,4-diaminobutyric acid by enzyme catalysis method ) 是由 于铁妹 樊卫 林立峰 何平 潘俊锋 刘建 于 2019-12-04 设计创作，主要内容包括：本发明涉及生化技术领域,公开了突酶催化法制备2,4-二氨基丁酸的工艺。本发明以L-天冬氨酸为起始,通过一系列的酶促反应生成天冬氨酸-4-磷酸、天冬氨酸半醛中间体,最后转化成目标产物2,4-二氨基丁酸；整个反应体系还可增加四种辅酶再生体系,降低ATP、NADPH、NADH以及丙氨酸的用量,有效推动该反应至高转化率。本发明反应体系多种酶催化反应相互不干扰,反应操作简便,其最终产品转化率高,如采用固定化酶方式则可进一步提高其工业应用可行性。(The invention relates to the technical field of biochemistry, and discloses a process for preparing 2,4-diaminobutyric acid by a mutase catalytic method. The method takes L-aspartic acid as an initial, generates an intermediate of aspartic acid-4-phosphoric acid and aspartic acid semialdehyde through a series of enzymatic reactions, and finally converts the intermediate into a target product of 2,4-diaminobutyric acid; the whole reaction system can also increase four coenzyme regeneration systems, reduce the dosage of ATP, NADPH, NADH and alanine, and effectively push the reaction to high conversion rate. The invention has the advantages of non-interference of a plurality of enzyme catalytic reactions of the reaction system, simple and convenient reaction operation, high conversion rate of the final product, and capability of further improving the industrial application feasibility of the product by adopting an immobilized enzyme mode.)
1. A process for preparing 2,4-diaminobutyric acid by enzyme catalysis is characterized in that reaction raw materials of L-aspartic acid, ATP or salt thereof, pyridoxal phosphate, alanine, NADPH or salt thereof, and aspartokinase ASK, aspartate semialdehyde oxidase ASADH and transaminase AMT are subjected to enzyme catalysis reaction in a reaction medium with a pH value of 6.0-9.0 to generate the 2,4-diaminobutyric acid.
2. The process according to claim 1, wherein each enzyme is involved in the enzyme-catalyzed reaction in the form of a host cell expressing each enzyme, an enzyme solution of each enzyme, or an immobilized enzyme of each enzyme.
3. The process according to claim 2, wherein the host cell expressing each enzyme is E.coli containing a vector expressing each enzyme.
4. The process of claim 2, wherein the enzyme solution of each enzyme is an enzyme solution extracted from a host cell expressing each enzyme.
5. The process of claim 1, further comprising a step of purifying 2,4-diaminobutyric acid:
removing salt through a chromatographic column, removing a phosphate compound in the solution through anion exchange resin, and finally, freeze-drying and recrystallizing the collected crude 2,4-diaminobutyric acid.
6. The process of claim 1, wherein the reaction medium is tris.hcl.
7. The process according to claim 1, wherein the aspartokinase ASK, aspartate semialdehyde oxidase ASADH and transaminase AMT have the sequences shown in SEQ ID No. 1-3.
8. The process of any one of claims 1 to 7, further comprising: the reaction raw materials are added with polyphosphoric acid, phosphorous acid or its salt, ammonium formate, magnesium chloride, potassium chloride, NADH or its salt, and ATP regenerating enzyme PPK, NADP regenerating enzyme PDH, alanine dehydrogenase ADH and formate dehydrogenase FDH to carry out enzyme catalysis reaction.
9. The process of claim 8, wherein the sequences of the ATP regenerating enzyme PPK, the NADP regenerating enzyme PDH, the alanine dehydrogenase ADH and the formate dehydrogenase FDH are shown as SEQ ID No. 4-7.
Technical Field
The invention relates to the technical field of biochemistry, in particular to a process for preparing 2,4-diaminobutyric acid by an enzyme catalysis method.
Background
2,4-diaminobutyric acid is an unnatural amino acid occurring in nature, which is a diamino amino acid like lysine, ornithine; the molecular formula is C4H10N2O2Molecular weight 118, CAS No: 305-62-4. 2,4-diaminobutyric acid is abundantly distributed in plants, flowers, yeasts and some bacteria, and although it is not an essential constituent of proteins, it is widely involved in the biosynthesis of various natural polypeptide antibiotics, such as circulin (polypeptin), cosmistin (Comirin), Polymyxin (Polymyxin) and the like. 2,4-diaminobutyric acid has higher industrial application value at the same time, for example, it is the basic raw material for producing snake venom peptide (snake venom peptide is a substance secreted by snake gland, and has pharmacological actions of stopping pain, stopping bleeding, inhibiting thrombosis and resisting tumor, etc., snake venom peptide is now widely used in medical and beauty industry, and is an effective component for removing wrinkle and resisting wrinkle), so it is more important to develop a simpler method capable of producing 2,4-diaminobutyric acid in batches to reduce its cost.
The 2,4-diaminobutyric acid is usually prepared by a separation method or a chemical synthesis method. Although the natural 2,4-diaminobutyric acid is widely distributed, the separation and purification of the natural 2,4-diaminobutyric acid are very difficult due to the characteristics of low abundance, small molecular weight, high water solubility and the like; most Of THE commercially available 2,4-DIAMINOBUTYRIC ACID is prepared by chemical synthesis, and compared with THE classical PREPARATION process using direct conversion Of L-aspartic ACID into 2,4-DIAMINOBUTYRIC ACID under sodium azide, concentrated sulfuric ACID and chloroform conditions (up to 90% yield, g.i. tesser and j.w. van nissen, "NOTE ON this PREPARATION Of L-2, 4-diaminostatic ACID, Synthetic communication,1971,1,285-287), THE process continues to THE present, however, THE safety risk Of mass production is very high because THE reaction involves many toxic, explosive chemicals. Under the circumstance of advocating safe and green production at present, the search for a cheap and environment-friendly method for producing 2,4-diaminobutyric acid is urgent.
Disclosure of Invention
In view of the above, the present invention aims to provide a process for preparing 2,4-diaminobutyric acid by an enzyme catalysis method, such that the process has characteristics of high conversion yield, high target product concentration, low product impurity residue, and simple purification process.
In order to achieve the purpose, the invention provides the following technical scheme:
a process for preparing 2,4-diaminobutyric acid by enzyme catalysis method comprises carrying out enzyme catalysis reaction on raw materials of L-aspartic acid, ATP or its salt, pyridoxal phosphate, alanine, NADPH or its salt, aspartokinase ASK, aspartate semialdehyde oxidase ASADH and transaminase AMT in a reaction medium with pH value of 6.0-9.0 to generate 2,4-diaminobutyric acid.
Aiming at the defects of the current chemical synthesis method, the invention provides another process for synthesizing 2,4-diaminobutyric acid by starting from L-aspartic acid by utilizing an enzymatic reaction technology. L-aspartate kinase (ASK, EC 2.7.2.4), which phosphorylates L-aspartate to L-aspartate-4-phosphate; aspartate semialdehyde oxidase (ASADH, EC 1.2.1.11) converts L-aspartate-4-phosphate to L-aspartate semialdehyde in the presence of reduced nicotinamide adenine dinucleotide phosphate, NADPH; last non-specificity
Wherein each enzyme is involved in the enzyme-catalyzed reaction in the form of a host cell expressing each enzyme, an enzyme solution of each enzyme, or an immobilized enzyme of each enzyme.
In a specific embodiment of the present invention, the host cell expressing each enzyme is escherichia coli containing a vector expressing each enzyme; the specific preparation process is as follows:
PCR amplifying ASK, ASADH, AMT, PPK, PDH, ADH and FDH gene segments by using extracted Escherichia coli (Escherichia coli K12), Pseudomonas stutzeri (Pseudomonas stutzeri) chromosomes, ATCC purchased Mycobacterium smegmatis mc 2155 (ATCC 700084), Burkholderia (ATCC 16) chromosomes and the like as templates 176176176, carrying out corresponding enzyme digestion, and carrying out enzyme connection to a carrier plasmid;
the correct plasmid was verified by gene sequencing and then transferred into e.coli BL21(DE3) strain, cultured in LB broth, then expressed under IPTG induction, and wet cells of each enzyme were collected.
More specifically, the vector plasmid is a commercially available pET28a plasmid, and each enzyme amplification primer is shown in Table 1:
TABLE 1
Verifying correct plasmids through gene sequencing, transferring the plasmids into E.coli BL21(DE3) strains, carrying out small-scale culture in 5ml LB culture solution containing 50uM Kanamycin (Kanamycin) at 37 ℃, adding 0.4mM isopropyl- β -D-thiogalactopyranoside (IPTG) when cells grow to OD 0.5-0.8, inducing protein expression for 4 hours at 37 ℃, finally collecting the cells, carrying out cell disruption by a freeze-thaw method, carrying out high-speed centrifugation, confirming protein expression by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on collected supernate, culturing the strains with correct protein expression to a 5-liter fermentation tank step by step, inducing expression for 6 hours at 37 ℃ under the condition of 0.5mM IPTG, and collecting wet cells of each enzyme, wherein the LB culture medium comprises 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate and 5% glycerol.
In a specific embodiment of the invention, the aspartate kinase ASK, aspartate semialdehyde oxidase ASADH and transaminase AMT have the sequence shown in SEQ ID NO. 1-3.
In a specific embodiment of the present invention, the enzyme solution of each enzyme is an enzyme solution extracted from a host cell expressing each enzyme; the method comprises the steps of crushing the collected wet cells under high pressure, centrifuging at high speed, collecting supernatant containing crude protein, namely enzyme liquid containing enzyme, and further purifying the supernatant.
Preferably, the present invention further comprises a step of purifying 2,4-diaminobutyric acid:
removing salt through a chromatographic column, removing a phosphate compound in the solution through anion exchange resin, and finally, freeze-drying and recrystallizing the collected crude 2,4-diaminobutyric acid. Wherein the chromatographic column is a G25 chromatographic column, and deionized water is used as eluent; the anion exchange resin is D201 anion exchange resin; the recrystallization adopts ethanol water solution, and the volume ratio of ethanol to water is preferably 3: 1.
Preferably, the reaction medium is tris (hydroxymethyl) aminomethane hydrochloride (tris. hcl), more preferably 100mm ph 8.0 tris (hydroxymethyl) aminomethane hydrochloride (tris. hcl). Meanwhile, the pH value of the system is maintained to be 6-9, preferably 6.5-8.5 in the process of enzyme catalysis reaction.
In the reaction of the invention, because the coenzyme Adenosine Triphosphate (ATP) and NADPH are used, the cyclic utilization of the ATP regenerative enzyme (PPK, EC 2.7.4.1) and the polyphosphoric acid, the NADP regenerative enzyme (PDH, EC 1.20.11.1) and the phosphorous acid can be realized by introducing the ATP regenerative enzyme (PPK, EC 2.7.4.1) and the polyphosphoric acid, and the usage amount of the ATP regenerative enzyme and the phosphorous acid is greatly reduced. The transaminase (AMT) reaction is an equilibrium reaction, and by introducing ammonium formate, alanine dehydrogenase (ADH, EC 1.4.1.1) and formate dehydrogenase (FDH, EC 1.17.1.9) into the system, alanine can be recycled in the presence of catalytic amount of reduced coenzyme Nicotinamide Adenine Dinucleotide (NADH), so that the dosage of alanine can be reduced, the catalytic amount of alanine is only needed, and the transaminase reaction can be effectively promoted to a high conversion rate. Meanwhile, the enzyme related to the invention can be immobilized and applied once or for multiple times, so that the stability and the operability of large-scale production can be further improved.
Therefore, the process of the invention also comprises adding polyphosphoric acid, phosphorous acid or salts thereof, ammonium formate, potassium chloride, magnesium chloride (potassium chloride and magnesium chloride participate in the reaction of the ATP regenerating enzyme PPK), NADH or salts thereof, and adding the ATP regenerating enzyme PPK, the NADP regenerating enzyme PDH, the alanine dehydrogenase ADH and the formate dehydrogenase FDH to the reaction raw materials for enzyme-catalyzed reaction; the overall reaction principle of the invention is schematically shown in figure 1.
In the present invention, the salt of ATP is ATP sodium salt, such as adenosine disodium triphosphate; the NADPH salt is NADPH sodium salt, such as NADPH monosodium salt of reduced nicotinamide adenine dinucleotide phosphate; the NADH salt is NADH sodium salt, such as NADH disodium salt of reduced coenzyme nicotinamide adenine dinucleotide; the phosphite is a sodium phosphite, such as sodium phosphite; salts of each provide ATP, NADPH, NADH, and phosphorous acid.
In a specific embodiment of the invention, the sequences of the ATP regenerating enzyme PPK, the NADP regenerating enzyme PDH, the alanine dehydrogenase ADH and the formate dehydrogenase FDH are shown as SEQ ID No.4-7 in sequence.
The 2,4-diaminobutyric acid prepared by the process has the final yield of the pure product of more than 70 percent and the highest yield of 87 percent, is close to the yield of a chemical synthesis method, but is safer, more environment-friendly and lower in cost compared with the classical chemical synthesis method.
According to the technical scheme, L-aspartic acid is used as the starting material, and a series of enzymatic reactions are carried out to generate aspartic acid-4-phosphate and aspartate semialdehyde intermediates, and finally the intermediates are converted into the target product 2,4-diaminobutyric acid; the whole reaction system can also increase four coenzyme regeneration systems, reduce the dosage of ATP, NADPH, NADH and alanine, and effectively push the reaction to high conversion rate. The invention has the advantages of non-interference of a plurality of enzyme catalytic reactions of the reaction system, simple and convenient reaction operation, high conversion rate of the final product, and capability of further improving the industrial application feasibility of the product by adopting an immobilized enzyme mode.
Drawings
FIG. 1 shows a schematic diagram of the reaction principle of the present invention.
Detailed Description
The invention discloses a process for preparing 2,4-diaminobutyric acid by an enzyme catalysis method, and a person skilled in the art can realize the preparation by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the present technology has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the technology disclosed herein, as well as other suitable variations and combinations of parts, may be made to practice or use the present technology without departing from the spirit and scope of the invention.
The steps of the process of the present invention are intended to clearly describe the reaction scheme at the core and do not limit whether the entire reaction is carried out in a one-step or multi-step process.
The enzymes used in the invention can be artificially synthesized according to sequences, or can be expressed by induction of host cells by carrying the expression genes of the enzymes through plasmid vectors according to the method provided by the invention.
For immobilization, the conventional preparation method of immobilized enzyme in the art can be referred to, and in the specific embodiment of the present invention, the present invention utilizes LX-1000EP epoxy resin (xian blue, dawn company) according to the crude enzyme ASK, ASADH, AMT, PPK, PDH, ADH, FDH with the activity ratio of 1: 2.2:4.5:2.5:4.0:6.0:5.0, and the one-time mixing and fixing is carried out by dissolving 2000-4000U mixed enzyme in 1L25mM potassium phosphate solution with pH 8.0, adding 25-65mM phenoxyacetic acid and 300-500 g LX-1000EP epoxy resin into buffer solution, stirring for 4-8 hours at room temperature, filtering out immobilized enzyme, washing with clear water and 25mM phosphate buffer solution with pH 8.0 for three times, and storing at low temperature for later use; the immobilized mixed enzyme has 30-70% of the activity of the liquid enzyme respectively.
According to the reaction route of the process of the invention, the dosage of each reactant can be adjusted according to actual conditions, and for the maximum efficiency, the invention provides the following mole ratios of each reactant:
l-aspartic acid, polyphosphoric acid, ATP, alanine, phosphorous acid, magnesium chloride, potassium chloride, ammonium formate, NADPH, ASK, ASADH, AMT, NADH, PPK, PDH, ADH, FDH, PLP ═ 1 (1.1): (0.02): 1.1):2:5 (1.1): 0.02): 0.0002): 0.0003): 0.0005): 0.02): 0.001): 0.0005): 0.001): 0.0015): 0.00005;
in a particular embodiment of the invention, each enzyme participates in an enzymatic reaction in a wet cell fashion, according to a ratio of 12 g: 24 g: 48 g: 24 g of wet cells overexpressing ASK, ASADH, AMT, PPK, PDH, ADH and FDH enzymes were added in a ratio of 48 g to 60 g to 48 g;
adding ASK, ASADH, AMT, PPK, PDH, ADH and FDH enzyme solution into enzyme-containing solution for enzyme catalytic reaction at a volume ratio of 1:2:4:2:4:6:4, and adding at one time;
the ratio of each enzyme in the above immobilization method was referred to when each enzyme was used in the immobilized enzyme reaction.
The invention is further illustrated by the following examples.
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