A method for synthesizing S, S-EDDS by using EDDS cleavage enzyme as catalyst
By screening and cloning highly active EDDS lyases, the problem of low efficiency in the synthesis of EDDS by biological enzymatic catalysis has been solved, achieving high chiral selectivity and high enzyme activity, making it suitable for industrial production of high-purity S,S-EDDS.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TAIZHOU LINGFENG BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-05-27
- Publication Date
- 2026-06-30
AI Technical Summary
Existing bio-enzymatic methods for synthesizing ethylenediamine disuccinic acid (EDDS) suffer from problems such as a limited variety of enzymes and poor enzyme activity, resulting in low synthesis efficiency and hindering large-scale production.
EDDS lyases with specific amino acid sequences were screened and cloned, and genetically engineered bacteria were constructed. These bacteria catalyze the reaction of fumaric acid and ethylenediamine to generate highly chiral and selective S,S-EDDS, with enzyme activity more than 1.4 times that of the control and ee value exceeding 99%.
It achieves highly efficient catalytic generation of S,S-EDDS with high chiral purity, avoids flocculation, simplifies reaction operation, and is suitable for industrial production.
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Abstract
Description
Technical Field
[0001] This invention relates to a method for synthesizing S,S-EDDS using EDDS lyase catalysis, belonging to the field of enzyme catalysis technology. Background Technology
[0002] Ethylenediamine disuccinic acid (EDDS) is an aminopolycarboxylic acid chelating agent and a green metal chelating agent. Compared with traditional chelating agents, EDDS is not only biodegradable but also highly selective for metal ions. For example, it has a strong affinity for metal ions such as copper and iron, and can precisely lock and complex harmful metal ions. In addition to its role as a metal chelating agent for environmental remediation, its mild molecular structure and green and safe properties also allow for various other applications, such as serving as a carrier of trace elements and as an easily absorbed and residue-free agricultural fertilizer.
[0003] Currently, EDDS is synthesized using chemical methods, microbial fermentation, and enzymatic methods. Chemical synthesis of EDDS primarily uses fumaric acid or aspartic acid as raw materials. For example, patent literature (authorization announcement number: CN101808980B) reports the chemical synthesis of EDDS using ethylenediamine and fumaric acid as raw materials. However, the product in this report is a mixed isomer, and subsequent processing is complex. Regardless of the raw material used, the chemical synthesis of EDDS requires harsh conditions and large amounts of toxic reagents. Another example is the biosynthesis method using cloned EDDS synthesis gene clusters from *Amycolatpsis japnicum*, constructing recombinant expression vectors, and fermenting for 7-10 days, achieving an EDDS yield of 9.8 g / L. However, the yield of this method is not ideal. This demonstrates that microbial fermentation is time-consuming and involves cumbersome separation and extraction processes, making it unsuitable for large-scale production.
[0004] Currently, bio-enzymatic catalysis is gaining popularity in the synthetic field due to its good environmental compatibility. It is becoming a key research method both domestically and internationally, not only meeting the industrial demands for sustainable development but also offering unique advantages in atom economy and energy conservation. For example, existing literature reports that studies on the structure and catalytic mechanism of EDDS lyase derived from Chelativorans sp. BNC1 have found that (S,S)-EDDS can be synthesized using this enzyme. The enzyme activity is approximately 1307 U / L.
[0005]
[0006] However, the induction conditions for this enzyme during fermentation are quite demanding, and it is extremely unstable during industrial-scale fermentation. Furthermore, due to the current pursuit of green and sustainable development, the bio-enzymatic synthesis of EDDS has received widespread attention, and more highly active and chiral EDDS lysins should be screened for biocatalytic synthesis. Summary of the Invention
[0007] To address the shortcomings of the existing technologies, this invention provides a method for synthesizing S,S-EDDS using EDDS lyase catalysis, in order to solve the problems of limited types of biological enzymes and poor specific enzyme activity in existing technologies.
[0008] The objective of this invention is achieved through the following technical solution: a method for synthesizing S,S-EDDS using EDDS lyase catalysis, characterized in that the method comprises using fumaric acid and ethylenediamine as substrates, and carrying out a catalytic reaction to generate S,S-EDDS under the catalysis of EDDS lyase, wherein the amino acid sequence of the EDDS lyase is selected from the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 or SEQ ID NO.10.
[0009] By screening for protein sequences similar to the amino acid sequence of the control enzyme WP_011579909.1 (hereinafter referred to as SEQ ID NO.1), and cloning them into a vector and transforming them into *E. coli* to construct genetically engineered bacteria, enzymes with similar amino acid sequences to the control enzyme were screened from multiple cloned and constructed genetically engineered bacteria. Enzyme activity was then confirmed, and the aforementioned ethylenediamine disuccinate lyase (or EDDS lyase) with good enzyme activity was innovatively screened. This screening method increases the success rate of newly screened lyases exhibiting good S,S-isomer selectivity. Furthermore, the lyase has a high specific enzyme activity, effectively catalyzing the substrate to generate S,S-EDDS with high chiral purity. The optimal specific enzyme activity is more than 1.4 times that of the control, and the product ee value can reach over 99%. This effectively catalyzes the synthesis of the highly selective product (S,S)-ethylenediamine disuccinate from the substrate, without flocculation during the reaction, which is more conducive to the reaction operation.
[0010] In the above-described method for synthesizing S,S-EDDS using EDDS lyase, preferably, the amino acid sequence of the EDDS lyase is selected from amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, or SEQ ID NO.10. Lyases with one of the above-mentioned amino acid sequences also exhibit high specific enzyme activity for catalyzing the production of (S,S)-ethylenediaminedisuccinate from fumarate and ethylenediamine substrates, reaching more than 1.4 times the specific enzyme activity of the control, and producing a product with an ee value of over 99%.
[0011] In the above method for synthesizing S,S-EDDS using EDDS lyase catalysis, the construction of the genetically engineered bacteria involves converting the obtained protein sequence into a nucleotide sequence, inserting the nucleotide sequence of the target gene into the Pet-24a vector, and then transforming it into E. coli Top10 for stable culture. Next, a plasmid containing the target gene is extracted from E. coli Top10 and transformed into E. coli BL21(DE3) using a heat shock method. Protein expression is induced with IPTG to obtain wet cells, which are then broken to obtain the corresponding crude enzyme solution. Further analysis can be performed to test the enzyme activity and ee value of the lysin, all of which demonstrate excellent performance.
[0012] In the above method for synthesizing S,S-EDDS using EDDS lyase, preferably, the specific enzyme activity of the EDDS lyase is more than 1.4 times that of the specific enzyme activity of the control enzyme with the amino acid sequence shown in SEQ ID NO.1.
[0013] In the above method for synthesizing S,S-EDDS using EDDS lyase catalysis, preferably, the molar ratio of ethylenediamine to fumaric acid is 1:2-2.5.
[0014] In summary, compared with the prior art, the present invention has the following advantages:
[0015] 1. The lyase selected in this invention has high enzyme catalytic activity and can effectively catalyze the generation of the required (S,S)-ethylenediamine disuccinic acid. Compared with the control, the specific enzyme activity of some enzymes can reach more than 1.4 times.
[0016] 2. The lyases of this invention can catalyze the production of ethylenediamine disuccinic acid from raw materials with fumaric acid and ethylenediamine as substrates, and have high chiral selectivity, with the product ee value reaching over 99%. Detailed Implementation
[0017] The technical solution of the present invention will be further described in detail below through specific embodiments, but the present invention is not limited to these embodiments.
[0018] Example 1
[0019] Based on the selected amino acid sequence lysins, plasmids from the corresponding EDDS lysin Top10 engineered bacteria were transformed into BL21(DE3) engineered bacteria. Glycerol-containing *E. coli* Top10 / pE1-27 (containing EDDS lysins from different sources) engineered bacteria were inoculated into 5 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C and 200 rpm for 12 h. After overnight culture, 4-5 mL of cell culture medium was used to extract plasmids using the SanPrep column-based plasmid DNA mini-extraction kit from Sangon Biotech.
[0020] The plasmid containing the target gene was transformed into Escherichia coli BL21(DE3) by heat shock. The transformed plasmid was then spread on a solid culture medium and incubated overnight at 37°C with the medium inverted.
[0021] Example 2
[0022] EDDS lysin engineered bacteria fermented in 1L shake flasks, enzyme activity assay
[0023] Single colonies of suitable shape and size were picked from the solid plates successfully transformed in Example 1 and transferred to 5 mL of LB medium (containing 50 μg / mL kanamycin). The plates were then incubated overnight at 37°C with a shaker at 200 rpm. After overnight incubation, the colonies were transferred to 200 mL of TB medium. The plates were incubated at 37°C with a shaker at 200 rpm for approximately 5 hours. Then, 0.5 mM IPTG was added, and the plates were incubated at 15°C for 12-15 hours to induce fermentation. After fermentation, the fermentation broth was centrifuged at 4°C and 10,000 g, and the cells were collected and stored at -20°C.
[0024] Take about 0.5g of bacterial cells, dissolve the bacterial cells in water at a ratio of 1:3, and break them up in an ultrasonic cell disruptor for 10 minutes. Centrifuge at 10000g for 10 minutes. The supernatant is the crude enzyme solution, which is used for enzyme activity testing.
[0025] Methods for determining the activity of EDDS lysin:
[0026] Prepare a reaction solution containing 50 mM Tris-HCl, 50 mM sodium dihydrogen phosphate, 300 mM ethylenediamine hydrochloride, and 600 mM fumaric acid, with a pH of 8.0.
[0027] Add 1 mL of the reaction solution to 0.05 mL of the supernatant after centrifugation of the broken enzyme solution. Incubate the reaction at 30°C and 200 rpm in a water bath for 30 min. After the reaction, dilute the reaction solution 50-fold with the mobile phase, centrifuge at 1000 g for 5 min, and determine the amount of (S,S)-EDDS generated by HPLC. One enzyme activity unit is defined as the amount of enzyme solution required to catalyze the production of 1 μmol of EDDS per minute.
[0028] A total of 27 strains were tested for enzyme activity. The first 11 strains (E1-E11) all had EDDS lyase activity. Among them, the lyases in the first 11 strains, whose amino acid sequences are as shown in SEQ ID NO.2-SEQ ID NO.13, had specific enzyme activity. E7 had the highest enzyme activity, which was 4477.09 U / L, showing superior enzyme activity performance, about three times that of the control strain W. The specific analysis results are shown in Table 1 below.
[0029] Table 1:
[0030]
[0031] In Table 1 above, labels E1-E12 correspond to the amino acid sequences shown in SEQ ID NO.2-SEQ ID NO.13; label W corresponds to the control amino acid sequence shown in SEQ ID NO.1. The enzyme activity data in the table above show that not all lyase pairs similar to W, using fumarate and ethylenediamine as substrates, exhibit high enzyme activity in the synthesis of S,S-EDDS. Rather, obtaining these requires extensive technological innovation and improved screening.
[0032] Example 3
[0033] EDDS Lysis Enzyme Engineered Bacteria ee Value Determination
[0034] Take 0.3g of each of the corresponding strains E1-11 and the control strain W, add 1mL of water to resuspend them evenly, and then sonicate them in a cell sonicator for 10min. Centrifuge at 10000g, and take 0.5mL of the supernatant after centrifugation. Add it to 5mL of enzyme activity reaction solution, place it in a 30℃ steam bath shaker, and shake at 200 rpm for 5 hours. After 5 hours of reaction, centrifuge the reaction solution and take the supernatant to determine the ee value. The specific measurement results are shown in the corresponding ee value column of Table 1 above.
[0035] Example 4
[0036] Purification and Immobilization of Highly Active EDDS Lysis Enzyme
[0037] The lyase corresponding to the amino acid sequence of E4 was selected for purification and immobilization studies. Specifically, 3g of wet bacterial cells were weighed, and 10mL of buffer (50mM disodium hydrogen phosphate, pH 8.0) was added to dissolve the cells. The cells were then sonicated in a cell sonicator using a No. 6 amplitude transducer at 50% power for 3.0s on and 6.0s off, for a total of 15min. The cells were then centrifuged at 10000g for 10min.
[0038] Pack 10 mL of nickel-IDA resin into a column, and pass 10 mL of the supernatant through the column at a 1:1 ratio. After passing through the column, wash with washing buffer equal to 3 column volumes, and then elute the pure enzyme solution with elution buffer containing 500 mM imidazole. Immobilize the eluted pure enzyme solution using a LATE-707 immunosorbent assay (IPA) at room temperature (15-20℃) for 4-15 hours. The enzyme activity remaining in the supernatant is 10-15%, and the recovery rate of immobilized enzyme activity is 70%-85%.
[0039] Example 5
[0040] The lyase corresponding to E4 was selected to catalyze the production of (S,S)-ethylenediaminedisuccinate.
[0041] Preparation of reaction solution: The reaction solution contains 50 mM Tris-HCl, 50 mM sodium dihydrogen phosphate, 300 mM ethylenediamine hydrochloride, 600 mM fumaric acid, and has a pH of 8.0.
[0042] Add 1 mL of the reaction solution to 0.05 mL of the supernatant after centrifugation of the enzyme-broken solution (corresponding to the supernatant of E4). Control the temperature at 30℃ and react in a water bath at 200 rpm for 30 min. After the reaction, dilute the reaction solution 50 times with the mobile phase and centrifuge at 10000 rpm for 5 min to obtain the desired (S,S)-ethylenediamine disuccinate with an ee value of 99.62%. This indicates high enzyme activity.
[0043] The specific embodiments described in this invention are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.
[0044] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.
Claims
1. A method for synthesizing S,S-EDDS using EDDS lyase catalyzed by enzyme, characterized in that... The method includes using fumaric acid and ethylenediamine as substrates, and carrying out a catalytic reaction under the catalysis of EDDS lyase to generate S,S-EDDS, wherein the amino acid sequence of the EDDS lyase is selected from the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 or SEQ ID NO.
10.
2. The method for synthesizing S,S-EDDS using EDDS lyase catalyzed according to claim 1, characterized in that... The amino acid sequence of the EDDS lyase is selected from amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 or SEQ ID NO.
10.
3. The method for synthesizing S,S-EDDS using EDDS lyase catalyzed according to claim 1, characterized in that... The specific enzyme activity of the EDDS lyase is more than 1.4 times that of the control enzyme with the amino acid sequence shown in SEQ ID NO.
1.
4. The method for synthesizing S,S-EDDS using EDDS lyase catalyzed according to claim 1, 2, or 3, characterized in that... The molar ratio of ethylenediamine to fumaric acid is 1:2-2.5.