A carboxylate reductase catalyzing the conversion of acetic acid and ammonia to acetamide, and a preparation method and application thereof

Abstract

The application belongs to the field of enzyme engineering and biological catalysis technology, and particularly relates to a carboxylate reductase SeCAR capable of efficiently catalyzing conversion of acetic acid and ammonia water into acetamide, and a preparation method and application of the enzyme. The application discloses a carboxylate reductase SeCAR capable of catalyzing conversion of acetic acid and ammonia water into acetamide, wherein an amino acid sequence is shown as SEQ ID NO. 1, and a nucleotide sequence is shown as SEQ ID NO. 2. The application also discloses a recombinant engineering bacterium containing the carboxylate reductase SeCAR, a construction method, and a method for inducing expression of the carboxylate reductase SeCAR by the recombinant engineering bacterium. The application discloses, for the first time, the SeCAR enzyme capable of directly catalyzing conversion of acetic acid and ammonia water into acetamide, and the preparation method is simple, which fills the blank of no special enzyme for catalyzing the reaction in the prior art. The prepared SeCAR enzyme has high catalytic efficiency, short reaction period, and high acetamide yield, and meets the development trend of green chemical industry.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biocatalysis technology, specifically relating to a carboxylic acid reductase SeCAR that can efficiently catalyze the conversion of acetic acid and ammonia into acetamide, as well as the preparation method and application of the enzyme. Background Technology

[0002] Acetamide is an important organic chemical raw material with high dielectric constant and low toxicity. It is an excellent solvent for many organic and inorganic substances and is widely used in dyeing wetting agents, plasticizers, pharmaceutical intermediates, antacids and other fields. It is also the main component of antidote for organofluorine pesticide poisoning (defluorin), and has extremely high application value in industrial production and the pharmaceutical field.

[0003] Currently, the traditional industrial methods for preparing acetamide are mainly chemical synthesis methods, including two main process routes: One is to pass ammonia into glacial acetic acid to produce ammonium acetate, which is then dehydrated by high-temperature thermal processes and separated by distillation to obtain acetamide. This process requires gradient distillation at 110℃, 140℃, and 210℃, resulting in harsh reaction conditions, high energy consumption, and subsequent purification through complex steps such as solvent recrystallization, making the operation cumbersome. The second method involves reacting ethyl acetate with ammonia water, followed by evaporation, crystallization, and drying to obtain the finished product. This process suffers from high raw material costs and long reaction cycles. Furthermore, existing chemical synthesis methods generally suffer from drawbacks such as harsh reaction conditions, difficulty in removing byproducts, limited yields, and high environmental pollution risks, failing to meet the development needs of green chemistry and low-carbon production.

[0004] Biocatalysis, with its advantages of being mild, efficient, green, and environmentally friendly, has become an important alternative to traditional chemical synthesis. In the synthesis of amide compounds, enzyme catalysis offers advantages such as mild reaction conditions, high specificity, and few byproducts. Existing technologies have utilized the α-β serine hydrolase family to catalyze the intramolecular dehydration of ammonium carboxylate compounds to synthesize amides; however, these enzymes require ammonium carbamate as the amino donor, resulting in high raw material costs and hindering industrial application. Ammonium carbamate is expensive, while acetic acid and ammonia are cheaper. Using formic acid and ammonia to catalyze the synthesis of acetamide can effectively reduce costs. Currently, no enzyme has been found that can directly catalyze the conversion of acetic acid and ammonia into acetamide. Therefore, there is an urgent need to develop a highly efficient, inexpensive, and easily prepared dedicated enzyme to solve the problems of existing acetamide synthesis processes. Summary of the Invention

[0005] To address the technical problems of existing acetamide synthesis processes, such as harsh reaction conditions, high energy consumption, high raw material costs, and poor environmental performance, this invention provides a SeCAR enzyme that catalyzes the conversion of acetic acid and ammonia into acetamide. This enzyme has advantages such as high catalytic efficiency, mild reaction conditions, strong specificity, and ease of preparation. The invention also provides a method for preparing this enzyme and its applications, enabling the green, efficient, and low-cost synthesis of acetamide.

[0006] The amino acid sequence of this SeCAR enzyme is shown in SEQ ID NO.1, or is a homologous sequence (homology ≥90%) that still has the activity of catalyzing the conversion of acetic acid and ammonia to acetamide after substitution, deletion or mutation of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1, including but not limited to S628L, D111G, etc.

[0007] The SeCAR enzyme belongs to the carboxyl reductase class (EC 1.2.1.30). Its adenylation domain (A domain) catalyzes the reaction of carboxylic acid substrate with ATP to generate an acyl-AMP intermediate, and then transfers the acyl group to a peptidyl carrier protein (PCP) to form a covalent acyl-PCP thioester intermediate. This intermediate is then reduced to acetamide by the reductase domain (R domain). Furthermore, the optimal reaction temperature of the SeCAR enzyme is 25-50℃, and the optimal reaction pH is 7.0-8.5. Under these conditions, the enzyme's catalytic efficiency is the highest, and the acetamide yield can reach over 92%, which is far higher than the yield of traditional chemical synthesis processes.

[0008] Furthermore, the nucleotide sequence of the SeCAR enzyme is shown in SEQ ID NO.2. This nucleotide sequence can be obtained by reverse translation of the amino acid sequence shown in SEQ ID NO.1 and optimized in combination with the codon preference of the host cell to improve its expression level in the host cell.

[0009] The preparation method of SeCAR enzyme includes the following steps: (1) Construction of SeCAR enzyme expression vector: Based on the nucleotide sequence shown in SEQ ID NO.2, design specific primers and obtain the SeCAR enzyme gene by PCR amplification; insert the amplified SeCAR enzyme gene into the expression vector to construct a recombinant expression vector; the expression vector is a prokaryotic expression vector (such as pET-28a) or a eukaryotic expression vector (such as pPIC9K), preferably a prokaryotic expression vector, so as to achieve efficient and low-cost expression of SeCAR enzyme.

[0010] (2) Transformation of host cells: The recombinant expression vector constructed in step (1) is transformed into host cells to obtain recombinant engineered bacteria; the host cells are Escherichia coli (such as BL21(DE3)), Pichia pastoris or Bacillus subtilis, preferably Escherichia coli BL21(DE3), which has the advantages of fast growth rate, high expression level and low culture cost.

[0011] (3) Induction of expression: The recombinant engineered bacteria were inoculated into the culture medium and cultured at 37℃ with shaking at 180-220 r / min until OD 600 =0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) to induce expression, the induction temperature is 25-30℃, the induction time is 8-12h, and the final IPTG concentration is 0.1-1.0mmol / L.

[0012] (4) Separation and purification: After induction of expression, the bacterial culture was centrifuged to collect the bacterial cells, resuspended in buffer and then sonicated. The supernatant was collected by centrifugation. The SeCAR enzyme in the supernatant was purified by nickel column affinity chromatography to obtain pure SeCAR enzyme with a purity of ≥95%. The buffer was a pH 7.5 Tris-HCl buffer (20 mmol / L) containing 500 mmol / L NaCl.

[0013] The SeCAR enzyme prepared above is used to catalyze the conversion of acetic acid and ammonia into acetamide. The specific application method includes the following steps: Prepare the reaction system: Add acetic acid, ammonia, and SeCAR enzyme to the reaction vessel. The molar ratio of acetic acid to ammonia is 1:1.0-1.2, and the amount of SeCAR enzyme added is 0.01%-0.1% of the total mass of the reaction system. Add an appropriate amount of buffer solution (pH 7.0-8.5) to the reaction system to maintain the pH stability of the reaction system. PBS buffer (50 mmol / L) is preferred as the buffer solution.

[0014] Catalytic reaction: The reaction system is placed at 25-50℃ and 150-200r / min and shaken for 2-6 hours to complete the conversion of acetic acid and ammonia into acetamide. The reaction does not require high temperature or high pressure, nor does it require the addition of additional dehydrating reagents. The reaction is mild and environmentally friendly.

[0015] Product separation: After the reaction is completed, the reaction solution is centrifuged to remove the precipitate. The supernatant is purified by distillation (collecting the fraction at 210-216℃) or by solvent recrystallization (using a mixture of benzene and ethyl acetate as the solvent) to obtain pure acetamide with a purity ≥99%.

[0016] Furthermore, the application can be used for industrial-scale production of acetamide, or for small-scale laboratory preparation of acetamide; it can be catalyzed by free enzymes or by immobilizing SeCAR enzymes for catalysis. The immobilized enzymes can be reused, reducing production costs.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses for the first time a SeCAR enzyme capable of directly catalyzing the conversion of acetic acid and ammonia into acetamide, filling the gap in existing technologies where no dedicated enzyme exists to catalyze this reaction. The SeCAR enzyme catalyzes the reaction under mild conditions, with an optimal reaction temperature of 25-50℃ and an optimal pH of 7.0-8.5. It eliminates the need for harsh conditions such as high-temperature pyrolysis and high-pressure distillation, reducing energy consumption by over 60% and solving the problem of high energy consumption in traditional chemical synthesis processes. Furthermore, the reaction raw materials are acetic acid and ammonia, which are inexpensive and readily available, reducing raw material costs by over 70% compared to existing enzyme catalysis processes, making it easy to implement industrially.

[0018] The SeCAR enzyme prepared by this invention has high catalytic efficiency, short reaction cycle (2-6h), and acetamide yield can reach over 92%, which is far higher than traditional chemical synthesis processes (87%-90%) and existing enzyme catalytic processes; moreover, the reaction process only produces acetamide and water, with no toxic or harmful byproducts, making it environmentally friendly and pollution-free, in line with the trend of green chemical development.

[0019] The preparation method of the SeCAR enzyme of the present invention is simple and efficient. It is expressed in large quantities in prokaryotic host cells, and the separation and purification steps are simple. The obtained enzyme has high purity and good stability. After being stored at 4°C for 30 days, the enzyme activity is still more than 90%, which makes it easy to store and use. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE electrophoresis image of SeCAR enzyme; lane 1 is the protein molecular weight standard, lane 2 is the supernatant of recombinant bacterial lysis after induction, and lane 3 is the purified SeCAR enzyme after nickel column purification, showing that the molecular weight of the purified SeCAR enzyme is approximately 130 kDa and the purity is ≥95%. Figure 2 The curves show the effect of temperature on the catalytic activity of SeCAR enzyme; the horizontal axis represents the reaction temperature (°C) and the vertical axis represents the relative enzyme activity (%), showing that SeCAR enzyme has high activity in the range of 25-50°C, and the optimal temperature is 30°C. Figure 3 The curves show the effect of pH on the catalytic activity of SeCAR enzyme; the horizontal axis represents the reaction pH, and the vertical axis represents the relative enzyme activity (%). The curves show that SeCAR enzyme has high activity in the pH range of 7.0-8.5, with the optimal pH being 7.5. Figure 4 The relative enzyme activities of wild-type SeCAR and enzymes modified at the S628L and D111G mutant sites were compared. The enzyme activities of both mutants were increased by more than 20% compared with wild-type SeCAR. Detailed Implementation

[0021] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0022] Example 1: Cloning and expression vector construction of the SeCAR enzyme gene Primer design: Based on the SeCAR enzyme nucleotide sequence shown in SEQ ID NO.2, upstream primer F: 5'-GCACATATGatgacccagtctcacacccag-3' (containing NdeⅠ restriction site) and downstream primer R: 5'-CGCCTCGAGttacagcaggcccagctg-3' (containing XhoⅠ restriction site) were designed. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0023] PCR amplification: PCR amplification was performed using genomic DNA containing the SeCAR enzyme gene as a template; PCR reaction system (50 μL): template DNA 1 μL, upstream primer F 2 μL, downstream primer R 2 μL, 2×Taq PCR MasterMix 25 μL, ddH2O 20 μL; PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ final extension for 10 min; PCR products were verified by 1% agarose gel electrophoresis to obtain a fragment (approximately 1500 bp) consistent with the size of the target gene.

[0024] Vector construction: The target gene fragment and pET-28a vector obtained by PCR amplification were double-digested with NdeⅠ and XhoⅠ, respectively. The digestion products were recovered by agarose gel electrophoresis and ligated with T4 DNA ligase. The ligation system (20 μL) was: 6 μL target gene fragment, 2 μL pET-28a vector, 1 μL T4 DNA ligase, 2 μL 10×T4 DNA ligation buffer, and 9 μL ddH2O. The ligation was carried out overnight at 16℃ to obtain the recombinant expression vector pET-28a-SeCAR.

[0025] Example 2: Construction of recombinant engineered bacteria and induction of SeCAR enzyme expression Transformation: The recombinant expression vector pET-28a-SeCAR was transformed into Escherichia coli BL21(DE3) competent cells using the heat shock method. The specific steps were as follows: the competent cells were thawed in an ice bath, 10 μL of the recombinant expression vector was added, and the cells were incubated on ice for 30 min; the cells were then heat-shocked at 42℃ for 90 s, and immediately incubated on ice for 2 min; 800 μL of LB medium was added, and the cells were cultured at 37℃ with shaking at 200 rpm for 1 h; 100 μL of the bacterial culture was spread on LB solid medium containing 50 μg / mL kanamycin and cultured overnight at 37℃ to obtain the recombinant engineered bacteria.

[0026] Induction of expression: Single colonies of the recombinant engineered bacteria were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated overnight at 37°C with shaking at 200 rpm to obtain the seed culture. The seed culture was then inoculated at a ratio of 1:100 into 100 mL of LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C with shaking at 200 rpm until OD500 was reached. 600 =0.7, add IPTG to a final concentration of 0.5 mmol / L, and induce expression at 30℃ and 200 r / min for 10 h.

[0027] Example 3: Isolation and purification of SeCAR enzyme Cell disruption: Centrifuge the induced bacterial culture at 8000 r / min, 4℃ for 10 min and collect the cells; resuspend the cells in 20 mmol / L Tris-HCl buffer (containing 500 mmol / L NaCl) at pH 7.5 and sonicate in an ice bath (300W power, 3s working time, 5s interval, total time 30 min); after disruption, centrifuge at 12000 r / min, 4℃ for 20 min and collect the supernatant, which is the crude enzyme solution.

[0028] Nickel-NTA affinity chromatography purification: The crude enzyme solution was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with equilibration buffer (pH 7.5, 20 mmol / L Tris-HCl buffer, containing 500 mmol / L NaCl) at a flow rate of 1 mL / min; the column was washed with equilibration buffer to remove unbound contaminants; the target protein was eluted with elution buffer (pH 7.5, 20 mmol / L Tris-HCl buffer, containing 500 mmol / L NaCl and 200 mmol / L imidazole), and the elution peak was collected as the crude purified SeCAR enzyme; the crude purified enzyme was dialyzed and desalted (dialysis buffer was pH 7.5, 20 mmol / L Tris-HCl buffer) to obtain SeCAR enzyme with a purity ≥95%, verified by SDS-PAGE electrophoresis (e.g., ...). Figure 1 (As shown).

[0029] Example 4: SeCAR enzyme-catalyzed reaction of acetic acid and ammonia to acetamide Table 1 shows the configuration of the reaction system: The catalytic reaction was carried out at 30°C.

[0030] Product detection and separation: After the reaction was completed, 1 mL of the reaction solution was taken, centrifuged to remove the precipitate, and the acetamide content was detected by gas chromatography. Detection conditions: column type 30mX0.320mmX0.50um 40 to 260 / 270C HP-INNOWAX 19091N-2131; (1) Initial stage: The column temperature is maintained at 70 ℃ for 4 minutes.

[0031] (2) Heating stage: The temperature is increased from 70 ℃ to 240 ℃ at a rate of 10 ℃ / min.

[0032] (3) Final stage: The column temperature is maintained at 240 ℃ for 5 minutes.

[0033] (4) Total running time: The entire program runs for 26 minutes from start to finish.

[0034] Example 5: Effect of temperature on the catalytic activity of SeCAR enzyme The reaction system was prepared according to the method in Example 4, and the catalytic reaction was carried out at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃, respectively, with other conditions remaining unchanged. After the reaction, the acetamide yield was measured, and the relative enzyme activity was calculated (with the enzyme activity at the optimum temperature as 100%). The results are as follows: Figure 2 As shown in the figure. The results indicate that SeCAR enzyme has high catalytic activity in the range of 25-50℃, with the optimal reaction temperature being 30℃. When the temperature is below 25℃ or above 50℃, the enzyme activity decreases significantly.

[0035] Example 6: Effect of pH on the catalytic activity of SeCAR enzyme The reaction system was prepared according to the method in Example 4, and the pH of the reaction system was adjusted to 6.0, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, and 9.0, respectively. The catalytic reaction was carried out at 35°C, with other conditions remaining constant. After the reaction, the acetamide yield was measured, and the relative enzyme activity was calculated (with the enzyme activity at the optimal pH as 100%). The results are as follows: Figure 3 As shown in the figure. The results indicate that the SeCAR enzyme has high catalytic activity in the pH range of 7.0-8.5, with the optimal reaction pH being 7.5. When the pH is below 7.0 or above 8.5, the enzyme activity decreases significantly.

[0036] Example 7: Stability detection of SeCAR enzyme The purified SeCAR enzyme was stored at 4℃, 25℃, and 37℃. Enzyme activity was measured after 7, 14, and 30 days of storage, following the method in Example 4 (with initial enzyme activity as 100%). The results showed that after 30 days of storage at 4℃, 90.5% of the enzyme activity was retained; after 30 days at 25℃, 78.3% was retained; and after 30 days at 37℃, 65.1% was retained. This indicates that the SeCAR enzyme has good stability, making it easy to store and apply in practice. The enzyme activity stability results are shown in Table 1 below. Table 2 shows the results of enzyme activity stability testing. Example 8: Point Mutation of SeCAR Enzyme and Enzyme Activity Detection Primer design: Based on the SeCAR enzyme nucleotide sequence shown in SEQ ID NO.2, upstream primer F for D111G was designed as 5'-CCATCGCATCCggcCTGGCGCATCACCCTC-3', and downstream primer R as 5'-GTGATGCGCCAGgccGGATGCGATGGCGGTTG-3'. For S628L, upstream primer F was designed as 5'-CGCTCGCAAAttgCTGCGTCCCAAACT-3', and downstream primer R as 5'-GGGACGCAGcaaTTTGCGAGCG-3'. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0037] PCR amplification: PCR amplification was performed using plasmid DNA containing the SeCAR enzyme gene as a template. The PCR reaction system (50 μL) consisted of: 1 μL template DNA, 2 μL upstream primer F, 2 μL downstream primer R, 12.5 μL high-fidelity DNA polymerase, and 32.5 μL ddH2O. PCR reaction conditions were: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 68℃ annealing for 5 s, and 72℃ extension for 1 min, for a total of 28 cycles; and a final extension at 72℃ for 1 min. The PCR product was verified by 1% agarose gel electrophoresis, yielding a linearized mutant product of the expected size.

[0038] DpnI digestion and transformation: Take 9 μL of the above PCR product, add 1 μL of DpnI, 2 μL of DpnI buffer, and 8 μL of ddH2O, mix well, and incubate at 37℃ for 30 min to remove methylated template plasmid. Add 20 μL of the demethylated product to 50 μL of thawed DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 45 s, incubate on ice for 2 min, add 800 μL of LB medium, and culture with shaking at 37℃ for 1 h. After centrifugation and concentration, spread on LB plates containing the corresponding antibiotic, and incubate upside down at 37℃ overnight to screen for positive mutants.

[0039] Enzyme activity assay of the mutant SeCAR: The recombinant expression vector containing the SeCAR enzyme mutant gene was transformed into E. coli BL21 competent cells. The enzyme was induced and purified according to the methods of Examples 2 and 3 to obtain pure SeCAR mutant enzyme. A 1 mL reaction system was prepared according to the method of Example 4. After reacting at 30°C for an appropriate time, the precipitate was removed by centrifugation. The acetamide content was detected by gas chromatography, and the relative enzyme activity of the mutant was calculated and compared with that of the wild-type SeCAR enzyme. The relative enzyme activity of the mutant was increased by more than 20%.

Claims

1. A carboxylic acid reductase, SeCAR, that catalyzes the conversion of acetic acid and ammonia into acetamide, characterized in that, The amino acid sequence is as shown in SEQ ID NO.1 or the amino acid sequence shown in SEQ ID NO.1 has been substituted, deleted or mutated one or more amino acid residues.

2. The carboxylic acid reductase SeCAR according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

3. The carboxylic acid reductase SeCAR according to claim 1, characterized in that, The reaction temperature of the enzyme is 25-50℃, and the pH is 7.0-8.

5.

4. The carboxylic acid reductase SeCAR according to claim 1, characterized in that, The mutation sites are S628L or D111G.

5. A recombinant engineered bacterium containing the carboxylic acid reductase SeCAR as described in claim 1.

6. A method for constructing the recombinant engineered bacteria of claim 5, characterized in that, Includes the following steps: (1) Based on the nucleotide sequence shown in SEQ ID NO.2, design upstream primer F: 5'-GCACATATGatgacccagtctcacacccag-3' and downstream primer R: 5'-CGCCTCGAGttacagcaggcccagctg-3'; (2) Using genomic DNA containing the SeCAR enzyme gene as a template, PCR amplification was performed; the PCR product was verified by 1% agarose gel electrophoresis to obtain a fragment with the same size as the target gene. (3) The target gene fragment and pET-28a vector obtained by PCR amplification were double digested with NdeⅠ and XhoⅠ, respectively. The digestion products were recovered by agarose gel electrophoresis and ligated with T4 DNA ligase to obtain the recombinant expression vector pET-28a-SeCAR. (4) Transformation: The recombinant expression vector pET-28a-SeCAR was transformed into Escherichia coli BL21 competent cells by heat shock; 800 μL of LB medium was added and cultured at 37℃ and 200 r / min for 1 h with shaking; 100 μL of bacterial culture was spread on LB solid medium containing 50 μg / mL kanamycin and cultured at 37℃ overnight to obtain recombinant engineered bacteria.

7. A method for preparing a carboxylic acid reductase SeCAR that catalyzes the conversion of acetic acid and ammonia into acetamide, characterized in that, Includes the following steps: (1) Pick a single colony of the recombinant engineered bacteria constructed according to claim 6, inoculate it into LB liquid medium, shake and culture overnight to obtain seed culture; Seed culture was inoculated into liquid culture medium and cultured with shaking to induce expression. (2) Centrifuge the bacterial culture after induction and collect the bacterial cells; resuspend the bacterial cells, break them up and collect the supernatant, which is the crude enzyme solution; (3) Load the crude enzyme solution onto a Ni-NTA affinity chromatography column that has been pre-equilibrated with equilibration buffer; rinse the column with equilibration buffer to remove unbound contaminants; elute the target protein with elution buffer and collect the elution peak, which is the crude pure SeCAR enzyme; dialyze the crude pure product to desalt and obtain a pure SeCAR enzyme with a purity ≥95%.

8. The use of the carboxylic acid reductase SeCAR according to any one of claims 1-4 or the engineered bacteria according to claim 5 or 6 in catalyzing the conversion of acetic acid and ammonia into acetamide.

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