Phenylalanine ammonialyase mutant and application thereof in synthesis of trans-cinnamic acid

Abstract

The invention relates to the field of biochemical engineering and enzyme engineering, discloses a phenylalanine ammonialyase mutant and application thereof in synthesis of trans-cinnamic acid, and provides the phenylalanine ammonialyase mutant, and the amino acid sequence of the phenylalanine ammonialyase mutant is obtained by single-point substitution mutation on the basis of a specific wild-type sequence. The invention also provides a DNA molecule for coding the mutant, a recombinant vector containing the DNA molecule and a recombinant host cell containing the recombinant vector. The invention further provides an application of the mutant enzyme or the recombinant host cell in catalyzing L-phenylalanine to synthesize trans-cinnamic acid. Compared with a wild enzyme, the phenylalanine ammonialyase mutant obtained by the invention shows improved catalytic activity and obviously enhanced thermal stability; in application, the mutant can keep efficient catalysis in a wide temperature range, and the molar conversion rate of a substrate and the yield of a target product trans-cinnamic acid are effectively improved.

Description

Technical Field

[0001] This invention relates to the fields of biochemical engineering and enzyme engineering, specifically to a phenylalanine ammonia-lyase mutant and its application in the synthesis of trans-cinnamic acid. Background Technology

[0002] Trans-cinnamic acid, as an important organic synthesis intermediate, has a wide range of applications, covering many important industrial fields such as fragrances, pharmaceuticals, food additives, and cosmetics. For example, in the fragrance industry, it can be used as a precursor for the synthesis of fragrances such as cinnamic esters; in the pharmaceutical field, it is a key raw material for the synthesis of certain drugs; and in the food industry, it can also be used as a preservative and freshness-preserving agent.

[0003] Currently, the main industrial production methods for trans-cinnamic acid include chemical synthesis and biotransformation. Chemical synthesis typically uses benzaldehyde and acetic anhydride as raw materials, proceeding via the Perkin reaction under catalysis. However, this method has inherent drawbacks. The reaction conditions are usually quite demanding, requiring high temperature and pressure environments, and often using toxic catalysts and organic solvents. This not only generates large amounts of industrial waste, burdening the environment, but also results in relatively low purity of the product, increasing the difficulty of subsequent separation and purification, which is inconsistent with the development direction of green chemistry.

[0004] Compared with chemical methods, bioconversion uses biocatalysts (such as enzymes or microorganisms) to catalyze reactions. Due to its advantages such as high efficiency, specificity and mild reaction conditions, it has become an ideal technology choice for the production of trans-cinnamic acid.

[0005] However, the inherently low catalytic activity and poor stability of naturally derived phenylalanine ammonia-lyase when directly applied to industrial production constitute a major technical bottleneck. Low catalytic activity means that large amounts of enzyme preparations are required to achieve the desired conversion rate in actual production, directly leading to a significant increase in production costs. Furthermore, the stability of the enzyme during biotransformation is directly affected by reaction conditions such as temperature and pH. Natural phenylalanine ammonia-lyase is easily deactivated at higher temperatures or under suboptimal pH conditions, preventing the catalytic reaction from proceeding continuously and efficiently, thus limiting overall production efficiency.

[0006] Therefore, molecularly modifying natural phenylalanine ammonia-lyase to obtain mutants with both high catalytic activity and high stability is a key technical problem that urgently needs to be solved in the field of producing trans-cinnamic acid using biotransformation. Summary of the Invention

[0007] The technical problem to be solved by this invention is that the natural phenylalanine ammonia-lyase (PAL) used in the prior art for the production of cinnamic acid by bioconversion has low catalytic activity and poor thermal stability, resulting in high production costs and limiting its industrial application.

[0008] The first aspect of this invention provides a phenylalanine ammonia-lyase mutant. The amino acid sequence of the phenylalanine ammonia-lyase mutant is obtained by performing a single-point substitution mutation selected from E125S, A308M, A390T, S407M, A416M, L463Y, and V531M on the amino acid sequence shown in SEQ ID NO.1. Through this single-point mutation, the catalytic performance of the phenylalanine ammonia-lyase is improved.

[0009] In a preferred embodiment, the single-point substitution mutation is S407M. Compared to wild-type phenylalanine ammonia-lyase, the S407M mutant exhibits significantly improved catalytic activity and thermal stability in the reaction catalyzing the production of trans-cinnamic acid from L-phenylalanine, thereby increasing the yield and productivity of trans-cinnamic acid.

[0010] A second aspect of the present invention provides a DNA molecule. The DNA molecule is a nucleotide sequence encoding the aforementioned phenylalanine ammonia-lyase mutant. The nucleotide sequence encoding the mutant can be obtained by site-directed mutagenesis of the phenylalanine ammonia-lyase gene.

[0011] In a preferred embodiment, the nucleotide sequence is shown in SEQ ID NO.2.

[0012] A third aspect of the present invention provides a recombinant vector. The recombinant vector carries the aforementioned DNA molecule. The vector can be used to clone, amplify, or express the DNA molecule.

[0013] In a preferred embodiment, the vector is a cloning vector or an expression vector. The cloning vector is preferably pUC57-Kan, used for gene amplification and preservation; the expression vector is preferably pET-28a, used for inducing expression of the phenylalanine ammonia-lyase mutant in host cells.

[0014] A fourth aspect of the present invention provides a recombinant host cell. The recombinant host cell contains the aforementioned recombinant vector. By introducing a recombinant vector carrying an encoding a phenylalanine ammonia-lyase mutant into the host cell, the host cell is able to express the phenylalanine ammonia-lyase mutant.

[0015] In a preferred embodiment, the host cell is *Escherichia coli*. The *E. coli* strain is preferably *E. coli* JM109 for cloning, which has high efficiency in plasmid transformation and amplification; the *E. coli* strain is preferably *E. coli* BL21(DE3) for expression, which has the ability to efficiently express exogenous proteins, thereby enabling the large-scale production of phenylalanine ammonia-lyase mutants.

[0016] The fifth aspect of this invention provides the application of a phenylalanine ammonia-lyase mutant in the synthesis of trans-cinnamic acid.

[0017] The application refers to the use of the aforementioned phenylalanine ammonia-lyase mutant or the aforementioned recombinant host cell in the catalytic synthesis of trans-cinnamic acid from L-phenylalanine. The phenylalanine ammonia-lyase mutant provided by this invention can selectively catalyze the deamination of L-phenylalanine to produce trans-cinnamic acid. Compared to the natural enzyme, the mutant exhibits higher catalytic efficiency and stability, which helps reduce reaction costs and improve conversion efficiency, thereby promoting the application of biotransformation in the production of trans-cinnamic acid and reducing environmental pollution.

[0018] In a preferred embodiment, the catalytic reaction conditions are as follows: using L-phenylalanine and ammonia as substrates, the reaction is carried out in Tris-HCl buffer at a temperature of 15-50°C and a pH of 8.5-9.0. These conditions are optimized based on the enzymatic properties of the mutant and can effectively support the catalytic activity and stability of the enzyme, achieving efficient conversion of L-phenylalanine to trans-cinnamic acid.

[0019] This invention provides a phenylalanine ammonia-lyase mutant and its application in the synthesis of trans-cinnamic acid. It has the following beneficial effects: 1. The phenylalanine ammonia-lyase mutant provided by this invention is obtained by single-point substitution mutation of the amino acid sequence shown in SEQ ID NO.1. For example, the S407M mutant exhibits increased relative enzyme activity compared to the wild-type enzyme. This increased catalytic activity improves the efficiency of converting the substrate L-phenylalanine to the product trans-cinnamic acid per unit time, thereby reducing the amount of enzyme preparation used or shortening the reaction time in industrial production, thus improving production economics.

[0020] 2. The phenylalanine ammonia-lyase mutant (e.g., S407M) provided by this invention exhibits enhanced stability at higher reaction temperatures. Experimental results show that after incubation at 45°C, the residual activity of the S407M mutant is higher than that of the wild-type enzyme. This technical feature allows the mutant to withstand longer operation times or maintain catalytic efficiency at higher reaction temperatures during biotransformation, reducing the enzyme inactivation rate and ensuring that the reaction can proceed continuously and efficiently.

[0021] 3. This invention applies a phenylalanine ammonia-lyase mutant to the preparation of trans-cinnamic acid, thereby improving the substrate conversion efficiency. Under the same reaction conditions, the S407M mutant catalyzes L-phenylalanine, increasing the conversion rate and thus improving the yield of the target product, trans-cinnamic acid. Attached Figure Description

[0022] Figure 1 This is a protein electrophoresis diagram of the phenylalanine ammonia-lyase of the present invention; Figure 2 This is a standard curve showing the relationship between the mass concentration and absorbance value of trans-cinnamic acid at 290 nm, as described in this invention. Figure 3 This is the HPLC elution chromatogram of trans-cinnamic acid according to the present invention. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Preparation example: (1) Gene synthesis and plasmid construction: Based on the amino acid sequence (SEQ ID NO. 1) and the encoded nucleotide sequence (SEQ ID NO. 3) of phenylalanine ammonia-lyase derived from Rhodotorula rubrum, a gene synthesis company was commissioned to synthesize the whole gene, and the gene was cloned into the pUC57-Kan vector plasmid.

[0025] Using the synthesized plasmid as a template, primers PAL-F and PAL-R (sequences shown in Table 1) were designed, and the wild-type phenylalanine ammonia-lyase gene (RtPAL) was amplified by PCR. Simultaneously, primers pET28a-F and pET28a-R (sequences shown in Table 1) were designed, and the pET28a linearized vector was amplified by PCR. The purified RtPAL gene fragment was ligated to the linearized vector pET28a using homologous recombination technology, and then transformed into *E. coli* JM109 competent cells. Positive clones were screened on resistant plates containing 50 μg / mL kanamycin (Kan), and colony PCR was performed using validation primers T-PAL-F and T-PAL-R (sequences shown in Table 1) to verify the results, successfully obtaining the recombinant plasmid pET28a-rtpalrtplal containing the wild-type gene.

[0026] (2) Construction of the S407M mutant: Using the recombinant plasmid pET28a-rtpalrtplal as a template, mutant primers S407M:4_FWD and S407M:4_REV (sequences shown in Table 2) were designed targeting the S407M site. A plasmid with a single-point mutation in S407M was constructed using PCR amplification and seamless cloning technology. The constructed mutant plasmid was transformed into E. coli JM109 competent cells for large-scale amplification. Kans resistance (50 μg / mL) was used for selection, and the selected clones were verified by gene sequencing to ensure successful mutation and the absence of other base mutations, ultimately obtaining the mutant plasmid pET28a-RtPAL (S407M).

[0027] (3) Construction and expression of recombinant strain The mutant plasmid pET28a-RtPAL(S407M), which was verified by sequencing, was transformed into Escherichia coli BL21(DE3) competent cells for protein expression. Screening was performed on medium containing 50 μg / mL kanamycin and the final recombinant expression strain BL21(DE3) / pET28a-RtPAL(S407M) was obtained by colony PCR verification.

[0028] The recombinant strain was inoculated into shake tubes and cultured with shaking at 37°C and 220 rpm for 12-16 hours. Subsequently, 3% of the strain was inoculated into 100 mL of LB medium and cultured with shaking at 37°C and 220 rpm for approximately 6 hours. When the optical density (OD600) reached 3-4, the culture was transferred to a 20 L fermenter for scale-up. After culturing at 37°C for 8 hours, when the OD600 reached 10, IPTG was added to the medium at a final concentration of 0.1 g / L, and the culture temperature was lowered to 25°C for induced expression for 25 hours.

[0029] After induction of expression, the fermentation broth was filtered through a ceramic membrane to remove the fermentation medium, and the bacterial cells were collected. The collected cells were homogenized under high pressure to release intracellular proteins. The supernatant was collected after centrifugation, which was the crude enzyme solution containing the phenylalanine ammonia-lyase mutant S407M. The crude enzyme solution was analyzed by SDS-PAGE protein electrophoresis, and the results are as follows: Figure 1 As shown, this indicates that the target protein, phenylalanine ammonia-lyase, was successfully expressed.

[0030] Table 1. Primer sequences for recombinant plasmids Table 2. Primer sequences of mutants Examples 1-3: Example 1: This embodiment illustrates the synthesis of trans-cinnamic acid from L-phenylalanine using the phenylalanine ammonia-lyase mutant S407M of the present invention under the lower temperature and lower pH conditions described in the claims.

[0031] In a 50 mL reaction system, 32 g / L L-phenylalanine, 20 mM Tris-HCl buffer, and 350 U of crude enzyme solution of the phenylalanine ammonia-lyase mutant S407M prepared in Preparation Example 1 were added. The pH of the reaction system was adjusted to 8.5, and the reaction was carried out at 15 °C with stirring for 24 hours. After the reaction was completed, the amount of trans-cinnamic acid produced was determined by the HPLC method described in Test Example 2, and the molar conversion rate was calculated.

[0032] Example 2: This embodiment illustrates the synthesis of trans-cinnamic acid from L-phenylalanine using the phenylalanine ammonia-lyase mutant S407M of the present invention under the medium temperature and medium pH conditions described in the claims.

[0033] In a 50 mL reaction system, 32 g / L L-phenylalanine, 20 mM Tris-HCl buffer, and 350 U of crude enzyme solution of the phenylalanine ammonia-lyase mutant S407M prepared in Preparation Example 1 were added. The pH of the reaction system was adjusted to 8.75, and the reaction was carried out at 37 °C with stirring for 24 hours. After the reaction was completed, the amount of trans-cinnamic acid produced was detected by HPLC method as described in Test Example 2, and the molar conversion rate was calculated. The experimental results showed that the molar conversion rate of L-phenylalanine reached 97%.

[0034] Example 3: This embodiment illustrates the synthesis of trans-cinnamic acid from L-phenylalanine using the phenylalanine ammonia-lyase mutant S407M of the present invention under the higher temperature and higher pH conditions described in the claims.

[0035] In a 50 mL reaction system, 32 g / L L-phenylalanine, 20 mM Tris-HCl buffer, and 350 U of crude enzyme solution of the phenylalanine ammonia-lyase mutant S407M prepared in Preparation Example 1 were added. The pH of the reaction system was adjusted to 9.0, and the reaction was carried out at 50 °C with stirring for 24 hours. After the reaction was completed, the amount of trans-cinnamic acid produced was determined by the HPLC method described in Test Example 2, and the molar conversion rate was calculated.

[0036] Comparative Example 1: Compared with Example 2, the difference is that the crude enzyme solution of the 350U phenylalanine ammonia-lyase mutant S407M was replaced with crude enzyme solution of wild-type phenylalanine ammonia-lyase (RtPAL) with the same activity, while the other reaction conditions and detection methods are the same.

[0037] Test Example 1-2: Test Example 1: Screening and stability assessment of phenylalanine ammonia-lyase mutants: Experimental description: To screen for phenylalanine ammonia-lyase mutants with enhanced catalytic performance and to evaluate their stability, the following experiments were conducted.

[0038] Induction and expression of recombinant strains and preparation of crude enzyme solution: Recombinant Escherichia coli strains containing wild-type (WT) and various mutant plasmids (E125S, A308M, A390T, S407M, A416M, L463Y, V531M) were inoculated into LB medium and cultured with shaking at 37℃ and 220 rpm. When the optical density (OD600) of the bacterial culture reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the culture was transferred to 25℃ for further induction of expression for 20 hours.

[0039] After the induction of expression was completed, the bacterial cells were collected by centrifugation at 8000 rpm for 10 minutes, and the supernatant was discarded. The bacterial cells were resuspended in 10 mL of water and the crude enzyme solution of each mutant was prepared by cell disruption.

[0040] Enzyme activity assay: Enzyme activity of each mutant was determined by ultraviolet spectrophotometry. The total reaction volume was 1 mL, containing 50 mM Tris-HCl buffer (pH 8.5), 0.5 g / L phenylalanine, and 0.00365 U of the crude enzyme solution to be tested. The reaction was carried out at 40 °C for 30 min, followed by boiling for 5 min to terminate the reaction. After centrifugation, the supernatant was collected, and its absorbance at 290 nm was measured and compared with... Figure 2 The standard curve for trans-cinnamic acid is shown to determine enzyme activity units.

[0041] Thermal stability testing is used to evaluate the thermal stability of the selected superior mutants. Wild-type phenylalanine ammonia-lyase and the crude enzyme solution of the screened S407M mutant were incubated at 45°C for 24 hours. After treatment, the residual activity was detected according to the enzyme activity assay method described above and compared with the initial activity before treatment.

[0042] Experimental data: With the activity of wild-type enzymes as 100%, the relative enzyme activity data of each mutant are summarized as follows.

[0043] Table 3. Enzyme activity of each mutant relative to wild type in conclusion: The innovative mechanism of this technical solution lies in the fact that by performing site-directed mutagenesis on wild-type phenylalanine ammonia-lyase, the amino acid residues at specific sites are changed, thereby optimizing the molecular structure of the enzyme in order to obtain higher catalytic activity and stability.

[0044] Test results show that among the series of mutants constructed in this invention, the S407M mutant exhibits significantly enhanced catalytic activity compared to the wild-type (WT), with a relative enzyme activity reaching 122.28%. Further stability assessment data shows that the S407M mutant retains 85% residual activity after incubation at 45°C for 24 hours, far exceeding the 64% of the wild-type enzyme. These data confirm that replacing serine at position 407 of the amino acid sequence of phenylalanine ammonia-lyase SEQ ID NO. 1 with methionine can effectively improve the enzyme's catalytic efficiency and thermal stability, laying the foundation for its subsequent application in the efficient production of trans-cinnamic acid.

[0045] Test Example 2: Performance evaluation of the synthesis of trans-cinnamic acid catalyzed by the phenylalanine ammonia-lyase mutant S407M: Experimental description: To verify the practical application effect of the phenylalanine ammonia-lyase mutant S407M in the catalytic reaction of L-phenylalanine to trans-cinnamic acid, this experiment was designed and compared with the wild-type enzyme.

[0046] The enzyme-catalyzed reactions were analyzed using the reaction systems from Example 2 (using the S407M mutant) and Comparative Example 1 (using wild-type RtPAL). The total reaction volume was 50 mL, containing 32 g / L L-phenylalanine, 20 mM Tris-HCl buffer (pH 8.5), and 350 U of crude enzyme solution. The reaction was carried out at 37°C with stirring for 24 hours. After the reaction, the reaction solution was boiled for 5 minutes to terminate the enzymatic reaction, followed by centrifugation, and the supernatant was used for subsequent analysis.

[0047] HPLC detection method: The supernatant from the above reaction was diluted 300 times and then quantitatively analyzed by high performance liquid chromatography (HPLC).

[0048] Column: EclipseXDB-C18 (250mm×4.6mm, 5μm); Column temperature: 40℃; Detection wavelength: 254nm; Mobile phase: Gradient elution was performed using 0.01% aqueous acetic acid and acetonitrile. The elution program was as follows: for 0-8 minutes, the volume ratio of 0.01% acetic acid to acetonitrile was maintained at 70:30; for 8-15 minutes, the volume ratio of 0.01% acetic acid to acetonitrile was linearly and gradually changed from 70:30 to 55:45. Flow rate: 1 mL / min; Under these chromatographic conditions, the target product, trans-cinnamic acid, eluted at 15.71 minutes. The HPLC chromatogram of its standard is shown below. Figure 3 As shown.

[0049] Experimental data: The reaction products were quantitatively analyzed by HPLC, and the molar conversion rate of the substrate L-phenylalanine was calculated. The results are as follows: Phenylalanine ammonia-lyase mutant S407M: molar conversion rate of 97%; Wild-type phenylalanine ammonia-lyase (RtPAL): molar conversion rate 57%; in conclusion: This technical solution aims to improve the efficiency of phenylalanine synthesis of trans-cinnamic acid by site-directed mutagenesis (S407M) of phenylalanine ammonia-lyase.

[0050] Sequence Listing SEQ ID NO.1-2: SEQ ID NO.1: ; SEQ ID NO.2:

[0051] The experimental data from this test case clearly show that, under the same reaction conditions, the substrate molar conversion rate catalyzed by the S407M mutant reached 97%, while that of the wild-type enzyme was only 57%. This indicates that the catalytic activity of the S407M mutant is 75% higher than that of the wild type. This result confirms that the S407M mutation significantly enhances the catalytic performance of phenylalanine ammonia-lyase, which can greatly improve the production efficiency of trans-cinnamic acid, demonstrating clear advantages for industrial applications.

Claims

1. A phenylalanine ammonia-lyase mutant, characterized in that, The amino acid sequence of the phenylalanine ammonia-lyase mutant was obtained by performing a single-point substitution mutation selected from E125S, A308M, A390T, S407M, A416M, L463Y and V531M on the amino acid sequence shown in SEQ ID NO.

1.

2. The phenylalanine ammonia-lyase mutant according to claim 1, characterized in that, The single-point substitution mutation is S407M.

3. A DNA molecule, characterized in that, The DNA molecule is a nucleotide sequence encoding a phenylalanine ammonia-lyase mutant, and the nucleotide sequence is shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, The recombinant vector carries a DNA molecule of a phenylalanine ammonia-lyase mutant.

5. A recombinant vector according to claim 4, characterized in that, The vector is a cloning vector or an expression vector, wherein the cloning vector is pUC57-Kan and the expression vector is pET-28a.

6. A recombinant host cell, characterized in that, The recombinant host cell contains the recombinant vector as described in claim 4 or 5.

7. A recombinant host cell according to claim 6, characterized in that, The host cell is Escherichia coli, and the Escherichia coli is either Escherichia coli JM109 used for cloning or Escherichia coli BL21(DE3) used for expression.

8. The application of a phenylalanine ammonia-lyase mutant in the synthesis of trans-cinnamic acid, characterized in that, The application of the phenylalanine ammonia-lyase mutant according to claim 1 or 2, or the recombinant host cell according to claim 6 or 7, in the catalytic synthesis of trans-cinnamic acid from L-phenylalanine.

9. The application of the phenylalanine ammonia-lyase mutant according to claim 8 in the synthesis of trans-cinnamic acid, characterized in that, The catalytic reaction conditions are as follows: using L-phenylalanine and ammonia as substrates, in Tris-HCl buffer, at a temperature of 15-50℃ and a pH of 8.5-9.0.

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