A method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis
Through the multi-enzyme cascade catalytic method, glycerol is used as the substrate, and the catalytic action of sugar alcohol dehydrogenase, aminotransferase and dehydrase, the efficient synthesis of 1,3-propanediamine is achieved, solving the problems of low conversion rate and difficult product separation in the prior art, and providing an efficient and environmentally friendly synthesis pathway.
Patent Information
- Application Number
- CN202410498273.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-24
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2044-04-24
AI Technical Summary
The existing chemical synthesis methods of 1,3-propylene diamine have problems such as high raw material costs, difficult product separation, large energy consumption and low conversion rate of biosynthesis methods. The existing biological enzyme synthesis methods have not yet achieved efficient multi-enzyme cascade reactions.
Using a multi-enzyme cascade catalytic method, using glycerol as a substrate, using the catalytic action of sugar alcohol dehydrogenase, aminotransferase and dehydrase, 1,3-propylene diamine was synthesized through four-step reactions, including the catalytic synthesis of 3-amino-1,2-propylene glycol, 3-aminopropanaldehyde and final 1,3-propylene diamine, and specific enzyme systems such as ScALDO, PsωTA and KpGDHT were selected for enzyme transformation to improve the conversion rate.
The conversion rate of 1,3-propylene diamine reached 77%, which has the advantages of simple process, mild conditions, and easy separation of products, which significantly improves the biosynthesis efficiency.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis. Background Art
[0002] 1,3-Propylenediamine is a widely used diamine compound that can be used in the synthesis of various complexes and is widely used in fields such as microelectronics and porous materials. It can also be used as a curing agent to react with epoxy resins to form practical three-dimensional structures, or as a raw material for the synthesis of the anti-radiation drug amifostine.
[0003] Currently, the chemical synthesis methods for 1,3-propylene diamine include the halocarbon ammonolysis method, the acrylonitrile ammonolysis method, and the propylene glycol ammonolysis method. The halocarbon ammonolysis method is a batch reaction in a kettle, using 1,3-dichloropropane as the raw material to synthesize 1,3-propylene diamine. This method has the disadvantages of high equipment corrosion, high waste generation, high raw material consumption, and a large number of polyamine by-products. Its synthesis route is shown below:
[0004]
[0005] The acrylonitrile ammonolysis method primarily involves reacting ammonia with acrylonitrile to produce 3-aminopropionitrile, which is then catalytically hydrogenated to produce 1,3-propylenediamine. This method offers advantages such as a simple process, mild conditions, and a wide range of raw material sources, making it a relatively common method for producing 1,3-propylenediamine. However, this method suffers from the complex two-step process, high catalyst costs, and difficulty in large-scale production. The propylene glycol ammonolysis method uses propylene glycol as a raw material to synthesize 1,3-propylenediamine. The synthesis route is as follows:
[0006]
[0007] The propylene glycol ammonolysis method offers advantages such as a simple process and a pollution-free design. However, product separation is difficult and the catalyst and raw material costs are high. Therefore, further research is needed on other synthetic methods for 1,3-propylenediamine.
[0008] The production of 1,3-propanediamine has been detected in some microorganisms such as Pseudomonas and Acinetobacter, making the biosynthesis of 1,3-propanediamine possible. Figure 1Acinetobacter baumannii synthesizes 1,3-propanediamine using the C4 pathway, which uses L-aspartate as a precursor to generate aspartate semialdehyde, and then synthesizes 1,3-propanediamine under the catalysis of 2-oxoglutarate-4-aminotransferase (DAT) and L-2,4-diaminobutyrate decarboxylase (DDC); Pseudomonas aeruginosa synthesizes 1,3-propanediamine using the C5 pathway, which uses L-glutamate as a precursor and, through a series of enzyme-catalyzed reactions, synthesizes 1,4-butanediamine via ornithine or arginine, and then synthesizes spermine under the catalysis of spermidine synthase (SpeE). Spermidine is then synthesized into 1,3-propanediamine under the action of spermidine dehydrogenase (SpdH). Acinetobacter baumannii and Pseudomonas aeruginosa are pathogenic bacteria. Therefore, some studies have utilized heterologous expression of enzyme components from related pathways to construct engineered E. coli strains that produce 1,3-propylenediamine, ultimately producing 13 g / L of 1,3-propylenediamine via fermentation. However, current methods for the biosynthesis of 1,3-propylenediamine still suffer from issues such as high energy consumption, environmental unfriendliness, and low conversion rates. Therefore, further exploration of alternative biosynthetic methods for 1,3-propylenediamine, such as bioenzymatic methods, is warranted. However, to date, there has been no research on the synthesis of 1,3-propylenediamine using multi-enzyme cascade reactions. Summary of the Invention
[0009] In view of the deficiencies of the prior art, the present invention provides a method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis.
[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:
[0011] A method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis comprises the following steps:
[0012] (1) Using glycerol as substrate, 3-amino-1,2-propanediol was catalyzed and synthesized using the catalysts of sugar alcohol dehydrogenase and transaminase;
[0013] (2) Dehydratase is used to catalyze the synthesis of 3-aminopropane aldehyde from 3-amino-1,2-propanediol, and transaminase is used to catalyze the synthesis of 1,3-propylenediamine from 3-aminopropane aldehyde.
[0014] Compared with the existing technology, the present invention uses a multi-enzyme cascade to catalyze the synthesis of 1,3-propylenediamine from glycerol, with a conversion rate of up to 77%, which is significantly higher than the fermentation method for synthesizing 1,3-propylenediamine. This provides a potential direction for using glycerol to synthesize high-value-added compounds. In addition, this method has the advantages of simple process, mild conditions, and easy product separation.
[0015] Furthermore, the amino acid sequence of the dehydratase is shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
[0016] Furthermore, the amino acid sequence of the dehydratase is shown in SEQ ID NO:9.
[0017] Furthermore, the amino acid sequence of the sugar alcohol oxidase is shown in SEQ ID NO: 1, and the amino acid sequence of the transaminase is shown in SEQ ID NO: 2.
[0018] Furthermore, the added amount of the sugar alcohol oxidase is 100-200 μg / mL, the added amount of the transaminase is 5-10 mg / mL, and the added amount of the dehydratase is 4-8 mg / mL.
[0019] Furthermore, the dehydratase is added to the reaction system of step (1) after the catalytic reaction in step (1) is carried out for 6 hours.
[0020] Furthermore, the reaction pH of the multi-enzyme cascade is 8, and the reaction temperature is 45°C.
[0021] Furthermore, the amine donor of the transaminase is alanine.
[0022] Furthermore, the multi-enzyme cascade reaction system also includes alanine dehydrogenase and phosphite dehydrogenase.
[0023] Furthermore, its amino acid sequence is shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The biosynthetic pathways of 1,3-propanediamine are shown in Figure 2, where (a) is the C5 pathway of Pseudomonas and (b) is the C4 pathway of Acinetobacter.
[0025] Figure 2 Flowchart of the multienzyme cascade reaction for the synthesis of 1,3-propylenediamine from glycerol.
[0026] Figure 3(a) is a comparison of the enzyme activities of mutant glycerol dehydratase KpGDHT / V301A, KpGDHT / S302A, KpGDHT / Q337A, and KpGDHT / F375A under 10 mM 3-amino-1,2-propanediol conditions.
[0027] Figure 3(b) is a comparison of the enzyme activities of KpGDHT / V301A, KpGDHT / S302A, KpGDHT / Q337A, and KpGDHT / F375A under 100 mM 3-amino-1,2-propanediol conditions.
[0028] Figure 3(c) shows the comparison of enzyme activities of KpGDHT 337 site saturation mutation under 10 mM 3-amino-1,2-propanediol conditions.
[0029] Figure 3(d) shows the comparison of enzyme activities of KpGDHT / Q337A 302 site saturation mutation under 10 mM 3-amino-1,2-propanediol conditions.
[0030] Figure 3(e) is a comparison of the enzyme activities of glycerol dehydratase KpGDHT and its mutants under 10 mM 3-amino-1,2-propanediol conditions.
[0031] Figure 4(a) shows the product analysis results of 1,3-propylenediamine by HPLC.
[0032] Figure 4(b) shows the product analysis results of 3-amino-1,2-propanediol by HPLC.
[0033] Figure 5(a) shows the optimized overall cascade reaction time curve.
[0034] Figure 5(b) shows the effect of different hydrogen peroxide removers on the cascade reaction yield.
[0035] Figure 5(c) shows the effect of different pH values on the yield of the cascade reaction.
[0036] Figure 5(d) shows the effect of different amine donor addition amounts on the cascade reaction yield.
[0037] Figure 5(e) shows the effect of different metal ions on the yield of the cascade reaction.
[0038] Figure 5(f) shows the yield comparison before and after the optimization of the overall cascade reaction.
[0039] Figure 6 Comparison of the yields of 1,3-propylenediamine synthesized from different glycerol substrates. DETAILED DESCRIPTION
[0040] In view of the problems of high raw material cost and difficult product separation in the existing chemical synthesis method of 1,3-propylenediamine, and the problems of low conversion rate and low substrate specificity in the biosynthesis method of 1,3-propylenediamine, the present invention intends to synthesize 1,3-propylenediamine by bioenzymatic method and screen and modify the enzymes involved in the bioenzymatic synthesis pathway. Figure 2The present invention uses the retrosynthetic analysis method to design a new four-step enzymatic synthesis pathway for synthesizing 1,3-propylenediamine from glycerol, namely, glycerol is catalyzed into glyceraldehyde by alcohol oxidase, then catalyzed into 3-amino-1,2-propanediol by transaminase, then catalyzed into 3-aminopropanealdehyde by glycerol dehydratase, and finally catalyzed into 1,3-propylenediamine by transaminase.
[0041] Based on this four-step enzymatic synthesis pathway, the present invention screened for alcohol oxidases, transaminases, and glycerol dehydratases to improve the conversion rate of this synthesis pathway. Ultimately, the sugar alcohol oxidase ScALDO from Streptomyces coelicolor A3, the transaminase PsωTA-KES2345 from Pseudomonas sp. Strain AAC, and the glycerol dehydratase KpGDHT from Klebsiella pneumoniae were selected as the glycerol dehydratase. Compared with the fermentative production of 1,3-propylenediamine, the four-step enzymatic synthesis pathway combines the advantages of biological synthesis, which is low in energy consumption and environmentally friendly. In addition, in order to improve substrate specificity and further improve the conversion rate of this synthetic pathway, the present invention uses the glycerol dehydratase crystal structure (1iwp) by Discoverystudio 2019 to select four target sites V301, S302, Q337 and F375, and mutate the amino acid residues of these four target sites. Then, the forward mutant site is saturated mutated, and the most suitable mutant is found through multiple rounds of iteration. After screening, the enzyme activity of the mutant KpGDHT / Q337A / S302D is increased to 4.4 times that of the wild type, and the four-step enzymatic synthesis pathway of the mutant KpGDHT / Q337A / S302D is used to increase the conversion rate to more than 77%. Therefore, the new pathway for glycerol synthesis of 1,3-propylenediamine constructed by the present invention provides a potential direction for the synthesis of high value-added compounds using glycerol.
[0042] The present invention will be further described below with reference to specific embodiments.
[0043] The whole gene synthesis in the examples of the present invention was completed by Suzhou Genewise Biotechnology Co., Ltd.
[0044] The reagents used in the embodiments of the present invention are as follows:
[0045] LB medium containing 1% peptone, 0.5% yeast extract and 1% sodium chloride was used for bacterial culture.
[0046] TB medium contains 1.2% peptone, 2.4% yeast extract, 0.4% glycerol, and is supplemented with 10-fold phosphate buffered saline (PBS) for protein expression.
[0047] A 10-fold concentration of PBS solution was prepared by dissolving 23.1 g of potassium dihydrogen phosphate (KH2PO4) and 125.4 g of potassium hydrogen phosphate (K2HPO4) in 1 L of water.
[0048] Glycerol, sodium phosphite, Nicotinamide adenine dinucleotide (NADH), piperazine-N ˊ-2-ethanesulfonic acid (HEPES), and PLP (pyridoxal phosphate) were purchased from MacLean. L-alanine, coenzyme B12, Nicotinamide adenine dinucleotide (NADH) + ) was purchased from Aladdin. NH4Cl and KCl were purchased from Damao (Tianjin, China). Other materials and reagents used were obtained from commercial sources unless otherwise specified.
[0049] The experimental methods in the examples of the present invention where specific conditions are not specified are generally based on the conditions recommended by the manufacturer.
[0050] The wild-type sugar alcohol oxidase ScALDO gene (Gene ID: 1101588) from Streptomyces coelicolor A3 contains mutations at four sites: V125M, A244T, V133M, and G399R, and its amino acid sequence is shown in SEQ ID No: 1.
[0051] The amino acid sequence of the transaminase PsωTA derived from Pseudomonas sp. Strain AAC is shown in SEQ ID No: 2.
[0052] The amino acid sequence of alanine dehydrogenase AfAlaDH from Archaeoglobus fulgidus VC-16 (DSM4304) is shown in SEQ ID No: 3.
[0053] The amino acid sequence of phosphite dehydrogenase RsPTXD from Ralstonia sp. strain 4506 is shown in SEQ ID No: 4.
[0054] The glycerol dehydratase KpGDHt from Klebsiella pneumoniae, wherein the α subunit and the β subunit are connected by a linker (G(PT)4T(PT)7G) (hereinafter referred to as KpGDHt), wherein the αLβ subunits are cloned into the first multiple cloning site, i.e., the BamHI and HindIII restriction sites of pACYC-duet1, and the γ subunit is cloned into the second multiple cloning site, i.e., the NdeI and KpnI restriction sites. Its amino acid sequence is shown in SEQ ID No: 5.
[0055] The host strain for the plasmid construction of all enzymes was Escherichia coli (E. coli) Top 10, and they were expressed in E. coli BL21 (DE3).
[0056] In a preferred embodiment of the present invention, the enzyme expression and purification method comprises the following steps:
[0057] The gene of the above enzyme was constructed into the pET28a expression vector to obtain a recombinant plasmid carrying the target enzyme gene, which was then transferred into the host cell to obtain the corresponding engineered strain, i.e., Escherichia coli BL21 (DE3) carrying the target enzyme gene plasmid. The engineered strain was inoculated into LB medium and cultured overnight at 37°C for 16 hours. It was then transferred to TB medium and cultured at 37°C and 220 rpm until the OD value reached 0.6-0.8. IPTG was then added at a final concentration of 0.5 mM and cultured at 16°C and 200 rpm for 16 hours.
[0058] The cells were collected by centrifugation at 6000 rpm, washed twice with 100 mM HEPES (PH = 7.4) buffer, resuspended in buffer A (100 mM HEPES and 50 mM NaCl, pH 7.4), and then disrupted using an ultrasonic disruptor. To remove cell debris and intact cells, the cell lysate was centrifuged at 4 ° C and 10000 rpm for 30 minutes. The supernatant was filtered with a 0.22 μm filter and loaded onto a 5 mL HisTrap HP column (GE Healthcare). To obtain purified protein, impurities were first eluted with buffer B (300 mM imidazole, 100 mM HEPES and 50 mM NaCl, pH 7.4) in different proportions. After the impurity elution was completed, the target protein was eluted with 100% buffer B. The purified protein was desalted using a desalting column (GE Healthcare). The desalted proteins were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantified using a Bradford protein detection kit (GENEray, China).
[0059] The present invention will be further described below with reference to specific embodiments.
[0060] Example 1: Glycerol dehydratase modification
[0061] (1) Construction of mutants
[0062] Key mutation sites in the glycerol dehydratase KpGDHT were selected using Discovery Studio 2019. Homology modeling of the enzyme was performed based on its crystal structure (1iwp). Simulation analysis was performed using protein 3D structure analysis software and molecular docking software. Taking into account the docking results between the enzyme and its substrate, the characteristics of the enzyme's substrate binding pocket, the structural characteristics of the enzyme's stereoselective substrate recognition, and the enzyme's catalytic mechanism, the researchers ultimately mutated valine at amino acid position 301, serine at position 302, glutamine at position 337, and phenylalanine at position 375 to alanine.
[0063] The enzyme activity of the mutants was measured, and then the forward mutant sites were subjected to saturation mutation, and the optimal mutant was found through multiple rounds of iterations.
[0064] (2) Determination of enzyme activity of glycerol dehydratase KpGDHT and its mutants
[0065] The reaction system was 30 μg KpGDHT or its mutant, 0.3 mM coenzyme B12, 50 mM KCl, 10 mM NAD + , 10mM~100mM 3-amino-1,2-propanediol, 30μg KSGADH, test the absorbance change of the system at 340nm in 5min, calculate the enzyme activity of the mutant towards 3-amino-1,2-propanediol, and calculate the relative enzyme activity with the enzyme activity of KpGDHT as 100%.
[0066] (3) Result analysis
[0067] By comparing the structural differences between glycerol and 3-amino-1,2-propanediol, the natural substrates of glycerol dehydratase, it was found that the carbon 3 of glycerol is a hydroxyl group, while 3-amino-1,2-propanediol is an amino group. Therefore, it is speculated that some sites in the active site of glycerol dehydratase interact with this residue in an unfavorable way, resulting in the reduction of the activity of glycerol dehydratase on the substrate 3-amino-1,2-propanediol. The amino acids docked within the range were excluded and the conserved sites were excluded. Finally, the remaining four sites, V301, S302, Q337, and F375, were scanned for alanine.
[0068] See Figures 3a-3bThe results showed that compared to the wild-type glycerol dehydratase KpGDHT, the enzyme activities of the mutants KpGDHT / S302A and KpGDHT / Q337A were increased by 1.4-fold and 1.3-fold, respectively, at a substrate concentration of 10 mM 3-amino-1,2-propanediol, and by 2.7-fold and 7.6-fold, respectively, at a substrate concentration of 100 mM 3-amino-1,2-propanediol. The amino acid sequences of the mutants KpGDHT / S302A and KpGDHT / Q337A are shown in SEQ ID No: 6 and SEQ ID No: 7.
[0069] See Figures 3c-3d To further explore potential highly active mutants, saturation mutagenesis at position Q377 was performed. The results showed that the KpGDHT / Q337S and KpGDHT / Q337A mutants significantly increased their activity, with the KpGDHT / Q337A mutant exhibiting the highest activity. Using the KpGDHT / Q337A mutant as a template, saturation mutagenesis at position S302 revealed that the KpGDHT / Q337A / S302D mutant exhibited the highest activity, with an enzyme activity 4.4-fold that of wild-type glycerol dehydratase at 10 mM 3-amino-1,2-propanediol. The amino acid sequences of the KpGDHT / Q337S and KpGDHT / Q337A / S302D mutants are shown in SEQ ID Nos. 8 and 9, respectively.
[0070] Example 2: Construction and optimization of complete pathway cascade reaction
[0071] Schematic diagram of the multi-enzyme cascade catalyzing the synthesis of 1,3-propylenediamine from glycerol Figure 2 shown.
[0072] (1) Construction of complete pathway cascade reaction
[0073] The enzymatic reaction was carried out in HEPES buffer (100 mM HEPES and 50 mM NaCl, pH 7.4) at 45°C and 800 rpm. The reaction system contained 5 mM glycerol, 0.5 mM alanine, 100 μM pyridoxal phosphate (PLP), 100 μg catalase, 50 mM NH4Cl, 50 mM sodium phosphite, 0.5 mM NADH, 200 μM coenzyme B12, 30 μg ScALDO, 1.2 mg KpGDHT / Q337A / S302D, 1.5 mg PsωTA, 30 μg AfAlaDH, and 90 μg RsPtXD at final concentrations of 200 μL.
[0074] When performing a complete pathway cascade reaction, the first two steps of the cascade reaction are carried out first, i.e., 3-amino-1,2-propanediol is catalyzed and synthesized using glycerol as a substrate. When the yield of the intermediate product 3-amino-1,2-propanediol reaches a maximum, the dehydratase mutant KpGDHT / Q337A / S302D is added to carry out the second two steps of the reaction, i.e., 1,3-propanediamine is catalyzed and synthesized using 3-amino-1,2-propanediol as a substrate.
[0075] Liquid chromatography for 1,3-propylenediamine and 3-amino-1,2-propylene glycol was performed using a Waters 2489 detector and a Waters Sunfire C18 (4.6×250mm, 5mm) column. The determination method was as follows: the amine compounds were first derivatized with dansyl chloride, and the derivative content was detected at 254nm. The mobile phase for 1,3-propylenediamine was acetonitrile:water = 75:25, and the mobile phase for 3-amino-1,2-propylene glycol was acetonitrile:water = 40:60. Figure 4a and 4b , Data represent the mean of three biological replicates, and error bars correspond to standard deviations.
[0076] See Figure 5a The yield of the first two steps of the cascade reaction can be completely reacted in 6 hours. Subsequently, the optimized second two-step cascade reaction system is added. The cascade reaction yield reaches the maximum after 2 hours, and 2.1mM 1,3-propylenediamine is obtained.
[0077] (2) Effects of hydrogen peroxide removers on the complete pathway cascade reaction
[0078] MnO2 was used instead of catalase to carry out the complete pathway cascade reaction described in step (1), and the yield of 1,3-propylenediamine was detected.
[0079] MnO2 can scavenge hydrogen peroxide and is cheaper. Figure 5b By replacing catalase with 200 μg 20 nm MnO2, not only can the reaction cost be reduced, but the yield of 1,3-propylenediamine can be increased to 3.0 mM, and the conversion rate of the complete pathway cascade reaction can be increased to 60%.
[0080] (3) Effect of pH on the complete pathway cascade reaction
[0081] The complete pathway cascade reaction of step (1) is carried out in a buffer solution with a pH value of 7.0 to 9.0, and the yield of 1,3-propylenediamine is detected.
[0082] See Figure 5c , the yield of the complete pathway cascade reaction was highest at pH 8.0.
[0083] (4) Effect of the amount of alanine added on the complete pathway cascade reaction
[0084] The amount of alanine added in the complete pathway cascade reaction of step (1) was changed to 0.25 mM to 1.25 mM, and the yield of 1,3-propylenediamine was detected.
[0085] See Figure 5d By optimizing the addition amount of amine donor alanine, it was found that the yield of 1,3-propanediamine was highest at a concentration of 0.5 mM alanine.
[0086] (5) Effect of metal ion types on the complete pathway cascade reaction
[0087] Add ethylenediaminetetraacetic acid (EDTA) solutions of different metal ions to the reaction system of step (1) respectively, so that Mg 2+ , Ni 2+ , Ca 2+ , Fe 3+ ,Co 2+ , Cu 2+ The final concentration was 0.5 mM, and the production of 1,3-propylenediamine was detected.
[0088] See Figure 5e , metal ion Mg 2+ , Ni 2+ , Ca 2+ , Fe 3+ ,Co 2+ , Cu 2+ All of them have a significant inhibitory effect on the complete pathway cascade reaction, among which the addition of Cu 2+ After 1 h, the relative production of 1,3-propylenediamine decreased to 6.5% of that in the control group.
[0089] In summary, see Figure 5f The optimal conditions for glycerol to synthesize 1,3-propylenediamine are pH 8.0, temperature 45°C, and an amount of alanine added of 0.5 mM. Under these optimal conditions, the maximum conversion rate of glycerol to synthesize 1,3-propylenediamine was measured, and it was found that the maximum yield of 1,3-propylenediamine was 3.87 mM, and the conversion rate was 77%, which was 1.8 times higher than before optimization.
[0090] Example 3: Robustness of the complete pathway cascade
[0091] To explore the robustness of this pathway, crude glycerol was used instead of pure glycerol as the substrate to perform the complete pathway cascade reaction described in Example 2 and the cascade reaction yield was determined by liquid chromatography.
[0092] See Figure 6Under optimal conditions, 3.95 mM 1,3-propylenediamine can be produced using 5 mM crude glycerol as a substrate, which is comparable to the yield of pure glycerol substrate. This result indicates that this cascade reaction system is highly robust.
[0093] In summary, the present invention's multi-enzyme cascade catalytic synthesis of 1,3-propylenediamine achieves conversion rates of 77% and 79% for pure and crude glycerol, respectively, under optimal conditions. The present invention mutates the glycerol dehydratase in the multi-enzyme cascade to obtain mutants with significantly enhanced enzymatic activity: KpGDHT / S302A, KpGDHT / Q337A, KpGDHT / Q337S, and KpGDHT / Q337A / S302D. The KpGDHT / Q337A / S302D mutant exhibits 4.4 times the enzymatic activity of the wild-type glycerol dehydratase on the substrate 3-amino-1,2-propanediol.
[0094] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.
Claims
1. A method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis, characterized in that: The method comprises sequentially performing steps (1) and (2) to obtain 1,3-propylenediamine; (1) Using glycerol as a substrate, a catalyst containing sugar alcohol oxidase and transaminase was used to catalyze the synthesis of 3-amino-1,2-propanediol; (2) using dehydratase to catalyze the synthesis of 3-aminopropane aldehyde from 3-amino-1,2-propanediol, and using transaminase to catalyze the synthesis of 1,3-propylenediamine from 3-aminopropane aldehyde; The amino acid sequence of the dehydratase is shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, the amino acid sequence of the sugar alcohol oxidase is shown in SEQ ID NO: 1, and the amino acid sequence of the transaminase is shown in SEQ ID NO:
2.
2. The method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis according to claim 1, characterized in that: The amino acid sequence of the dehydratase is shown in SEQ ID NO:
9.
3. The method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis according to any one of claims 1 to 2, characterized in that: The added amount of the sugar alcohol oxidase is 100-200 μg / mL, the added amount of the transaminase is 5-10 mg / mL, and the added amount of the dehydratase is 4-8 mg / mL.
4. The method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis according to claim 3, characterized in that: After the catalytic reaction in step (1) is carried out for 6 hours, the dehydratase is added to the reaction system of step (1).
5. The method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis according to claim 4, characterized in that: The reaction pH of the multi-enzyme cascade is 8, and the reaction temperature is 45°C.
6. The method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis according to claim 5, characterized in that: The amine donor of the transaminase is alanine.
7. The method for synthesizing 1,3-propylenediamine by multi-enzyme cascade catalysis according to claim 6, characterized in that: The multi-enzyme cascade reaction system also includes alanine dehydrogenase and phosphite dehydrogenase.