A novel phenolic acid decarboxylase and its preparation method and application

Abstract

This application discloses a novel phenolic acid decarboxylase, its preparation method, and its applications, relating to the field of enzyme engineering technology. The novel enzyme was extracted from *Mycobacterium avium*, and its amino acid sequence is shown in SEQ ID NO: 3. This enzyme can efficiently catalyze the decarboxylation of hydroxycinnamic acid substrates such as ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid to generate corresponding 4-vinylphenol compounds, with yields of 93.37%, 98.12%, 85.24%, and 11.21%, respectively. The catalytic activity against sinapic acid is significantly superior to most reported phenolic acid decarboxylases. Furthermore, under pH 8.5 and CO2 conditions, this enzyme can also reverse-catalyze the carboxylation reaction of 4-vinylphenol compounds to generate the corresponding hydroxycinnamic acid, with yields between 12% and 20%. It shows promising applications in the high-value utilization of lignin, food flavoring synthesis, and CO2 bioimmobilization.

Description

Technical Field

[0001] This application relates to the field of enzyme engineering technology, and in particular to a novel phenolic acid decarboxylase, its preparation method, and its application. Background Technology

[0002] Phenolic acid decarboxylases (PADs) are enzymes that specifically catalyze the non-oxidative decarboxylation of unsaturated aromatic acids containing para-hydroxyl substitutions (such as hydroxycinnamic acid compounds), producing mainly 4-vinylphenol compounds. This catalytic process does not rely on any cofactors or metal ions, operates under mild conditions, and the resulting 4-vinylphenol compounds are widely used in food flavorings, pharmaceuticals, bioenergy, and environmental remediation. Currently, reported phenolic acid decarboxylases are mainly derived from microorganisms such as *Pseudomonas fluorescens*, *Bacillus subtilis*, *Lactobacillus brevis*, and *Bacillus amyloliquefaciens*, as well as some bryophytes. Among these, the *Escherichia coli* (E. coli) heterologous expression system is widely used for the preparation of recombinant PADs due to its safety and high expression efficiency.

[0003] However, most reported phenolic acid decarboxylases can only efficiently catalyze two substrates: ferulic acid and p-coumaric acid, exhibiting a significant drawback of a narrow substrate range. For example, the relative specific activity of BlPAD derived from Bacillus licheniformis for sinapic acid is only 0.29% of that for p-coumaric acid, and the relative activity of PAD derived from Bacillus amyloliquefaciens for sinapic acid is only about 10.51%. Some PADs even completely fail to recognize caffeic acid or sinapic acid. Furthermore, natural PADs generally suffer from poor stability and low yields due to heterologous expression, making it difficult to meet the comprehensive requirements of industrial applications for enzyme catalyst yield, stability, and substrate breadth. Lignin, as the most abundant renewable aromatic resource on Earth, contains a large number of hydroxycinnamic acid monomers such as ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid in its depolymerization products. However, existing PADs, due to their narrow substrate range, cannot achieve efficient and comprehensive conversion of these monomers, severely restricting the high-value utilization of lignin.

[0004] Therefore, there is an urgent need to develop a novel phenolic acid decarboxylase with a broader substrate spectrum, high catalytic activity for various hydroxycinnamic acids (especially sinapic acid and caffeic acid), good stability, and efficient heterologous expression capability. This would enrich the PAD enzyme resource library, enable the efficient decarboxylation conversion of various phenolic acid monomers derived from lignin and the green synthesis of corresponding 4-vinylphenol products, and provide a new enzymatic tool for the application of reversible carboxylation reactions in CO2 bioimmobilization and the synthesis of phenolic acid compounds. Summary of the Invention

[0005] In view of this, embodiments of this application provide a novel phenolic acid decarboxylase, its preparation method, and its application.

[0006] The first aspect of this application provides a novel phenolic acid decarboxylase, which is any one of the following:

[0007] A1) A protein with the amino acid sequence shown in SEQ ID NO: 3;

[0008] A2) Proteins that share amino acid residue homology greater than 99%, 95%, 90%, 85%, or 80% with the proteins shown in A1) and have the same function;

[0009] A3) A protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins shown in A1)-A2).

[0010] In some embodiments, the novel phenolic acid decarboxylase is a protein that has greater than 99% homology with the amino acid residues of the protein shown in A1) and has the same function.

[0011] In some embodiments, the label includes a purification label and / or a solubilization label.

[0012] A second aspect of this application provides a polynucleotide encoding a novel phenolic acid decarboxylase as described in any of the first aspects.

[0013] In some embodiments, the sequence of the polynucleotide is shown in SEQ ID NO: 2.

[0014] A third aspect of this application provides a recombinant expression vector comprising the polynucleotides described in the second aspect.

[0015] In some embodiments, the backbone vector of the recombinant expression vector is pETDuet-1.

[0016] A fourth aspect of this application provides a recombinant bacterial strain comprising the recombinant expression vector described in the third aspect.

[0017] In some embodiments, the recombinant strain is Escherichia coli.

[0018] In some embodiments, the recombinant strain is a recombinant strain that can produce the phenolic acid decarboxylase MavPAD, constructed using pETDuet-1 as the starting vector and Escherichia coli (E. coli) as the heterologous expression system.

[0019] The fifth aspect of this application provides a method for preparing a novel phenolic acid decarboxylase, which includes converting an expression vector encoding the novel phenolic acid decarboxylase described in the first aspect into a host cell for expression, followed by purification.

[0020] In some embodiments, the host cell comprises E. coli TOP10 competent cells.

[0021] The sixth aspect of the embodiments of this application provides the use of the novel phenolic acid decarboxylase as described in any one of the first aspects in any of the following;

[0022] B1) Catalyzes the decarboxylation of hydroxycinnamic acid compounds;

[0023] B2) Reverse catalysis of carboxylation of 4-vinylphenol compounds.

[0024] In some embodiments, the hydroxycinnamic acid compound includes at least one selected from ferulic acid, p-coumaric acid, caffeic acid, and / or sinapic acid. Its chemical structural formula is shown below:

[0025] .

[0026] In some embodiments, the 4-vinylphenolic compounds include at least one of 4-vinylguaiacol, 4-vinylphenol, 4-vinylcatechol, and 4-vinyleugenol.

[0027] In some embodiments, the chemical structural formulas of 4-vinylguaiacol, 4-vinylphenol, 4-vinylcatechol, and 4-vinyleugenol are shown below:

[0028] .

[0029] In some embodiments, the carboxylation reaction system comprises 2-8 mM 4-vinylphenol compound, 0.05-0.5 mg / mL of the novel phenolic acid decarboxylase, 0.5-2 M NaHCO3 solution, 40-60 mM Tris-HCl buffer (pH 8.5), and continuously purged with CO2.

[0030] In some embodiments, the carboxylation reaction is performed at 35±2°C for 30~90 min.

[0031] The seventh aspect of this application provides a method for catalytic decarboxylation of hydroxycinnamic acid compounds, which includes mixing the novel phenolic acid decarboxylase described in the first aspect with the hydroxycinnamic acid compound in a reaction system, and then reacting.

[0032] In some embodiments, the hydroxycinnamic acid compounds include at least one of ferulic acid, p-coumaric acid, caffeic acid, and / or sinapic acid.

[0033] In some embodiments, the concentration of the novel phenolic acid decarboxylase is 1-90 mM; preferably 30-50 mM.

[0034] In some embodiments, the concentration of the hydroxycinnamic acid compound is 1-40 mM, preferably 10-30 mM.

[0035] In some embodiments, the reaction time is 30 to 90 minutes.

[0036] In some embodiments, the reaction temperature is 30~37°C, preferably 35°C.

[0037] In some embodiments, the reaction system further comprises 45-55 mM phosphate buffer (pH 6.0).

[0038] This application includes at least the following beneficial effects:

[0039] This application provides a novel phenolic acid decarboxylase, MavPAD, its preparation method, and its applications. Specifically, the encoding gene Mavpad was discovered and synthesized from *Mycobacterium avium*, and a recombinant strain was constructed using an *E. coli* heterologous expression system. High-purity recombinant enzyme (yield of 141.5 mg / L) was obtained after induced expression and affinity purification. This enzyme efficiently catalyzes the decarboxylation of four hydroxycinnamic acid substrates—ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid—at 35°C and pH 6.0, generating the corresponding 4-vinylphenol compounds with yields of 93.37%, 98.12%, 85.24%, and 11.21%, respectively. The catalytic activity against sinapic acid is significantly superior to most reported phenolic acid decarboxylases. Meanwhile, under pH 8.5 and CO2 conditions, this enzyme can also reversely catalyze the carboxylation of 4-vinylguaiacol, 4-vinylphenol, 4-vinylcatechol and 4-vinyleugenol to generate the corresponding hydroxycinnamic acid, with a yield between 12% and 20%.

[0040] Furthermore, the novel phenolic acid decarboxylase MavPAD of this application also exhibits good temperature and pH stability (maintaining high activity after 12 h of storage within the range of 25-45℃ and pH 5.0-10.0), providing a new enzyme tool to solve the problems of narrow substrate spectrum, low sinapic acid conversion rate and insufficient stability of existing phenolic acid decarboxylases, and has application prospects in the fields of high-value utilization of lignin, food flavor synthesis and CO2 bioimmobilization. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0042] Figure 1 SDS-PAGE gel electrophoresis analysis of MavPAD in this application;

[0043] Figure 2 The results of the optimal temperature and stability test for the MavPAD catalytic decarboxylation reaction in this application are shown, where A is the optimal temperature test result and B is the temperature stability test result.

[0044] Figure 3 The results show the optimal pH and stability of the MavPAD catalytic decarboxylation reaction in this application, where A represents the optimal temperature and pH test results, and B represents the pH stability test results.

[0045] Figure 4 This is a substrate concentration-relative enzyme activity curve for MavPAD-catalyzed decarboxylation in this application;

[0046] Figure 5 This is a graph showing the enzyme concentration-relative enzyme activity for MavPAD-catalyzed decarboxylation in this application.

[0047] Figure 6 This is a reaction time-relative enzyme activity curve for MavPAD-catalyzed decarboxylation in this application;

[0048] Figure 7 This application provides a yield analysis of MavPAD-catalyzed decarboxylation of different hydroxycinnamic acid substrates.

[0049] Figure 8 These are high-performance liquid chromatography (HPLC) chromatograms of the synthesis of 4-vinylphenol compounds from hydroxycinnamic acid catalyzed by MavPAD, as described in this application. A: HPLC chromatogram of the formation of 4-vinylguaiacol from ferulic acid catalyzed by MavPAD; B: HPLC chromatogram of the formation of 4-vinylphenol from p-coumaric acid catalyzed by MavPAD; C: HPLC chromatogram of the formation of 4-vinylcatechol from caffeic acid catalyzed by MavPAD; D: HPLC chromatogram of the formation of 4-vinyleugenol from sinapic acid catalyzed by MavPAD.

[0050] Figure 9 This application provides a yield analysis of the carboxylation of different 4-vinylphenol substrates catalyzed by MavPAD.

[0051] Figure 10 These are high-performance liquid chromatography (HPLC) chromatograms of the synthesis of p-hydroxycinnamic acid compounds from p-vinylphenol catalyzed by MavPAD in this application. A: HPLC chromatogram of the formation of ferulic acid from 4-vinylguaiacol catalyzed by MavPAD; B: HPLC chromatogram of the formation of p-coumaric acid from 4-vinylphenol catalyzed by MavPAD; C: HPLC chromatogram of the formation of caffeic acid from 4-vinylcatechol catalyzed by MavPAD; D: HPLC chromatogram of the formation of sinapic acid from 4-vinyleugenol catalyzed by MavPAD. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0053] In this specification, E. coli TOP10 and E. coli BL21(DE3)pLysS competent cells were purchased from Tiangen Biotech (Beijing) Co., Ltd.; the plasmid pETDuet-MavPAD was constructed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Furthermore, all catalysis-related reagents used in this application were commercial standards, purchased from Shanghai Aladdin Biotech Co., Ltd. and Shanghai McLean Biotech Co., Ltd.

[0054] In this instruction manual, the culture media used in the experiment are LB liquid medium, LB solid medium, and ZYM-5052 self-induction medium, with the specific formulations as follows:

[0055] LB liquid medium: Each liter of medium contains 10 g peptone, 5 g yeast extract and 5 g NaCl.

[0056] LB solid medium: Each liter of medium contains 10 g peptone, 5 g yeast extract, 5 g NaCl, 15 g agar powder and 100 mg ampicillin.

[0057] ZYM-5052 self-induction medium: Each liter of medium contains 10 g peptone, 5 g yeast extract, 5 g glycerol, 2 g lactose, 0.5 g glucose, 2.67 g NH4Cl, 6.70 g Na2HPO4·7H2O, 3.40 g KH2PO4, 0.71 g Na2SO4 and 0.24 g MgSO4.

[0058] Unless otherwise specified in this instruction manual, standard test conditions or the test conditions recommended by the reagent company are generally followed. Unless otherwise specified, all materials and reagents used are commercially available.

[0059] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0060] Example 1: Screening and obtaining the sequence of the novel phenolic acid decarboxylase MavPAD

[0061] First, the complete amino acid sequence of Bacillus coagulans phenolic acid decarboxylase BcPAD was obtained (GenBank: WP_017550974.1), and its amino acid sequence is shown in SEQ ID NO:1.

[0062] MGSSHHHHHHSQDPNSMKTLEEFLGTHMIYTYENGWEYEFYVKNQNTVDYRIHSGMVGGRWVRGQKADIVKITDGVFKVSWTEPTGTDVSLNFMPDDKRMHGVIFFPKWVHEHPEITVCYQNDHIDLMEESREKYETYPKYVVPEFADITYIKNEGINNEKVISEAPYATMADDIRSGKLKFSAAA* (SEQ ID NO:1).

[0063] In this context, "*" represents the stop codon.

[0064] Then, the amino acid sequence of the phenolic acid decarboxylase BcPAD obtained above was imported into the NCBI non-redundant protein database. Based on a sequence identity of 50%-60%, the target phenolic acid decarboxylase gene Mavpad was screened and its complete nucleotide sequence was obtained. The nucleotide sequence information of Mavpad is shown in SEQ ID NO:2.

[0065] GAATTCGCCGCCGCAGGATTTCAGCGGCATCGTTGGCCACCGTTTCATCTACACCTACGCGAACGGCTGGCAGTACGAAATGTACGTTAAAAACGCGACCACCATCGATTACCGTATCCACAGCGGTCACGTTGGCGGCCGTTGGGTTAAAGGCCAGGAAGTTAACCTGGTTCAGCTGGATGATGATAGCTACAAAATCAGCTGGACCGAACCGACCGGCACCTGCGTTGCGGTTAACG TTCTGCCGAGCAAACGTCGTATCCACGGCGTTATCTTCTTCCCGCAGTGGATCCGTCAGCACGGCCAGCGTACCGTTTGCTTCCAGAACGAACACCTGGATGAAATGCGTGCGTACCGT GATCGTGGCCCGACCTACCCGATCTACGAAGTTCCGGAATTTGCGTACATCACCCTGTTCGAATACGTTGGCACCGATGATGAAACCGTTATCGATACCGCGCCGGCGGCCGCTAA (SEQ ID NO:2).

[0066] Example 2: Obtaining the pETDuet-MavPAD recombinant plasmid and constructing the recombinant strain

[0067] 1. Obtain the recombinant plasmid pETDuet-MavPAD

[0068] The obtained sequence was codon-optimized according to the E. coli prokaryotic expression system preference. A 6×His tag and EcoRI were introduced at the front end of the sequence, and then cloned into the vector pETDuet by Sangon Biotech (Shanghai) Co., Ltd. to obtain the recombinant plasmid pETDuet-MavPAD.

[0069] The amino acid sequence information of Mavpad is shown in SEQ ID NO:3:

[0070] MGSSHHHHHHSQDPNSPPQDFSGIVGHRFIYTYANGWQYEMYVKNATTIDYRIHSGHVGGRWVKGQEVNLVQLDDDSYKISWTEPTGTCVAVNVLPSKRRIHGVIFFPQWIRQHGQRTVCFQNEHLDEMRAYRDRGPTYPIYEVPEFAYITLFEYVGTDDETVIDTAPAAA* (SEQ ID NO: 3).

[0071] In this context, "*" represents the stop codon.

[0072] 2. Obtain the pETDuet-MavPAD recombinant strain

[0073] The recombinant plasmid pETDuet-MavPAD obtained above was transformed into *E. coli* TOP10 competent cells using a heat shock method. The heat shock temperature and time were 42℃ and 90s, respectively. After transformation, the strain was plated on LB agar plates (containing 100ug / mL ampicillin), and positive single colonies were picked and inoculated into LB liquid medium. The culture was then incubated at 37℃ and 220 rpm for 12 h with shaking, and the bacterial culture was collected.

[0074] A portion of the bacterial culture was mixed with an equal volume of 50% glycerol, and the other portion of the sample was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The plasmid that was correctly sequenced was recorded as the target plasmid pETDuet-MavPAD, and the corresponding strain was recorded as the pETDuet-MavPAD recombinant strain.

[0075] Example 3: Fermentation expression and purification of pETDuet-MavPAD recombinant strain

[0076] 1. MavPAD fermentation expression

[0077] (1) The recombinant target plasmid culture pETDuet-MavPAD obtained above was inoculated into 100 mL LB medium containing 100 μg / mL ampicillin and cultured in a shake flask at 37℃ and 220 rpm for 10-12 h until OD. 600 = 0.8 - 1.0;

[0078] (2) Inoculate 5% of the bacterial culture obtained in step (1) into 500 mL of ZYM-5052 self-induction medium, and incubate at 37℃ and 200 rpm for 2-3 h until OD. 600 = 0.8 - 1.0, then adjust to 16℃, 200 rpm for 24h induction culture;

[0079] (3) Collect the bacterial cells by centrifuging at 4°C and 6000 rpm for 30 min using a low-temperature high-speed centrifuge in step (2). Wash the precipitate 2-3 times with buffer A to obtain bacterial cells expressing MavPAD protein, which can be used for later use.

[0080] 2. Purification of MavPAD

[0081] First, the collected bacterial cells were resuspended in buffer A at a ratio of 1:10 (w / v). After thoroughly disrupting the cells using an ultrasonic disruptor, the mixture was centrifuged at 4°C and 12,000 rpm for 30 min. The supernatant was the MavPAD crude enzyme solution. The crude enzyme solution was then mixed in a Ni-NTA agarose gel column (purchased from Newprobe) for 1-2 h to ensure sufficient binding of the protein with the Ni packing material. The column was then washed with buffer B1 to remove most of the impurities. The target protein solution was then eluted with buffer B2. The protein concentration was measured to be 3.76 mg / mL using a NanoDrop One microspectrophotometer (purchased from Thermo Fisher Scientific). -1 The total volume was approximately 33 mL, diluted to 2.00 mg / mL using 50 mM phosphate buffer (25 mM Na₂HPO₄·7H₂O, 25 mM NaH₂PO₄, pH 6.0). -1 Aliquot into 2 mL portions using EP tubes and store at 4°C for later use.

[0082] Buffer A contains 6.70 g Na₂HPO₄·7H₂O, 3.00 g NaH₂PO₄, 17.53 g NaCl, and 0.68 g imidazole per liter. Buffer B1 contains 6.70 g Na₂HPO₄·7H₂O, 3.00 g NaH₂PO₄, 17.53 g NaCl, and 3.40 g imidazole per liter. Buffer B2 contains 6.70 g Na₂HPO₄·7H₂O, 3.00 g NaH₂PO₄, 17.53 g NaCl, and 13.62 g imidazole per liter.

[0083] Samples from the above steps were collected and analyzed by SDS-PAGE gel electrophoresis at 120V for 60 minutes. After electrophoresis, Coomassie Brilliant Blue staining was performed for 2 minutes, followed by destaining for 12 hours. The electrophoretic images were observed using a gel imaging analyzer (purchased from Beijing Liuyi Biotechnology Co., Ltd.).

[0084] The results are as follows Figure 1As shown, M represents the protein marker, 1 is the lysate after resuspension and disruption of total bacteria, 2 is the supernatant after centrifugation of total bacteria, 3 is the precipitate after centrifugation of total bacteria, 4 is the supernatant eluted with Ni-NTA buffer, 5 is the supernatant eluted with 50 mM imidazole buffer, and 6 is the supernatant eluted with 200 mM imidazole buffer. This indicates that the molecular weight of the MavPAD protein is approximately 20 kDa, consistent with expectations.

[0085] Example 4: Determination of enzyme activity and stability of MavPAD

[0086] Based on the fact that PAD can independently decarboxylate hydroxycinnamic acid substrates, ferulic acid is one of the common substrates, and its decarboxylation product, 4-vinylguaiacol, can be detected at a UV wavelength of 310 nm. Therefore, this experiment used ferulic acid as a reference substrate to detect the enzyme activity of MavPAD and its optimal temperature and pH.

[0087] 1. Enzyme activity assay

[0088] Product standard curve preparation: Prepare a 25 mM 4-vinylguaiacol-methanol solution as the product stock solution. Then, dilute the product stock solution with methanol to prepare standard solutions with concentrations of 0.125, 0.25, 0.5, 1, 2, 3, and 4 mM, respectively, for HPLC injection detection. Record the peak time and peak area at 310 nm, and plot the product concentration-peak area standard curve.

[0089] Enzyme activity assay: 50 mM phosphate buffer, 10 mM ferulic acid substrate, and 0.2 mg / mL MavPAD pure enzyme solution were added to a 4 mL brown glass reaction flask, with a total reaction volume of 1 mL. The reaction flask was placed on a constant temperature magnetic stirrer and reacted at 300 rpm for 10 min. Immediately after the reaction, 2 mL of methanol was added and mixed thoroughly. The mixture was centrifuged at 12000 rpm for 3 min. 1 mL of the supernatant was taken for HPLC detection, and the peak area of ​​the product at 310 nm was recorded. The enzyme activity was calculated based on the 4-vinylguaiacol standard curve.

[0090] Enzyme activity (1 U) is defined as the amount of enzyme required to convert a substrate into 1 μmol of the corresponding product within 1 minute under the reaction temperature and pH conditions.

[0091] 2. Determination of the optimal temperature

[0092] (1) Optimal temperature

[0093] Prepare a 100 mM ferulic acid-methanol solution as the substrate stock solution. The total reaction volume is 1 mL. Add 100 μL of 2 mg / mL pure enzyme solution and 100 μL of ferulic acid stock solution to 50 mM phosphate buffer (pH 6.0). Incubate at 20, 25, 30, 35, 40, 45, 50, 55, 60, and 70 °C for 10 min at 300 rpm. Sample processing and detection methods are the same as the enzyme activity detection method described above. Each experiment is repeated three times. The enzyme activity of the group with the highest activity is recorded as 100%. Calculate the relative enzyme activity of the remaining groups and plot the temperature-relative enzyme activity curve.

[0094] The results are as follows Figure 2 As shown in A, the optimal temperature for MavPAD is 35℃.

[0095] (2) Temperature stability

[0096] The temperature of the constant temperature water bath was set to 25, 30, 35, 40, 45, and 50℃, respectively. MavPAD was incubated at the set temperature for 0, 0.5, 1, 2, 3, 4, 5, 6, and 12 h, respectively. Samples were then taken to measure enzyme activity. The reaction was performed at 35℃ and 300 rpm for 10 min. Sample processing and detection methods were the same as the enzyme activity detection method described above. Each experiment was repeated three times in parallel. The control group was supplemented with pure enzyme solution that had not undergone any incubation, while all other conditions remained unchanged. The enzyme activity value obtained from the control group was recorded as 100%. The relative enzyme activity of MavPAD after each incubation temperature and time was calculated, and time-relative enzyme activity curves were plotted for different temperatures.

[0097] The results are as follows Figure 2 As shown in B, MavPAD retains approximately 80% of its enzyme activity after incubation at temperatures ranging from 20 to 45°C for 12 hours.

[0098] 3. Determination of optimal pH

[0099] (1) Optimal pH

[0100] Adjust the pH of 50 mM citrate-sodium citrate buffer to 3.0, 4.0, 4.5, and 5.0; adjust the pH of 50 mM Na₂HPO₄·7H₂O-NaH₂PO₄ buffer to 5.0, 6.0, and 7.0; adjust the pH of 50 mM Tris-HCl buffer to 7.0, 8.0, and 9.0; and adjust the pH of 50 mM Na₂CO₃-NaHCO₃ buffer to 9.0, 10.0, and 11.0. Add 100 μL of 2 mg / mL pure enzyme solution and 100 μL of ferulic acid stock solution to each pH buffer, and react at 35℃ and 300 rpm for 10 min. Sample processing and detection methods are the same as the enzyme activity detection methods described above. Each experiment is repeated three times in parallel. The enzyme activity of the group with the highest activity is recorded as 100%, and the relative enzyme activity of the remaining groups is calculated to plot pH-relative enzyme activity curves.

[0101] The results are as follows Figure 3 As shown in A, the optimal pH for MavPAD is 6.0.

[0102] (2) pH stability

[0103] The purified enzyme solution was concentrated to 10 mg / mL using a 3 kDa ultrafiltration tube (purchased from Merck Millipore, USA). 0.6 mL of the concentrated MavPAD enzyme solution was thoroughly mixed with 26.4 mL of each pH buffer and incubated for 0, 0.5, 1, 2, 3, 4, 5, 6, and 12 h, respectively. Then, 900 μL of the incubation mixture was added to the reaction system, and the reaction was carried out at 35°C and 300 rpm for 10 min. Sample processing and detection methods followed the enzyme activity detection method described above. Each experiment was repeated three times. The control group consisted of unincubated pure enzyme solution instead of the pH incubation solution, with all other conditions unchanged. Finally, the enzyme activity value obtained from the control group was recorded as 100%, and the relative enzyme activity of different PADs after incubation at each pH and time was calculated. Time-relative enzyme activity curves were plotted for different pH values.

[0104] The results are as follows Figure 3 As shown in B, MavPAD exhibits residual enzyme activity exceeding 70% after 12 h of incubation at pH 5.0–10.0.

[0105] Example 5: Optimization of the MavPAD decarboxylation reaction system

[0106] 1. Optimization of reaction conditions

[0107] (1) Optimal substrate concentration

[0108] The total reaction volume was 1 mL. 100 μL of 2 mg / mL pure enzyme solution was added to 50 mM phosphate buffer (pH 6.0). The substrate concentration gradient was 1, 5, 10, 15, 20, 30, and 40 mM (different concentrations of substrate stock solution were used to ensure that the methanol content in the system was 10% (v / v)). The reaction was carried out at 35℃ and 300 rpm for 10 min. Sample processing and detection methods were the same as those in Example 4 above for enzyme activity detection. Each experiment was repeated three times in parallel. The enzyme activity of the group with the highest activity was recorded as 100%, and the relative enzyme activity of the remaining groups was calculated to plot the substrate concentration-relative enzyme activity curve.

[0109] The results are as follows Figure 4 As shown, the optimal substrate concentration for the MavPAD-catalyzed decarboxylation reaction is 20 mM.

[0110] (2) Optimal enzyme concentration

[0111] Use 10 mg / mL MavPAD enzyme solution. The total reaction volume is 1 mL. Add 100 μL of 100 mM ferulic acid stock solution to 50 mM phosphate buffer (pH 6.0). The enzyme concentration gradient is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, and 90 μM. React at 35℃ and 300 rpm for 10 min. Sample processing and detection methods are the same as those in Example 4 above for enzyme activity detection. Each experiment is repeated three times. The enzyme activity of the group with the highest activity is recorded as 100%. Calculate the relative enzyme activity of the remaining groups and plot the enzyme concentration-relative enzyme activity curve.

[0112] The results are as follows Figure 5 As shown, the optimal enzyme concentration in the MavPAD-catalyzed decarboxylation reaction system is 40 μM.

[0113] (3) Optimal reaction time

[0114] The total reaction volume was 1 mL. 100 μL of 2 mg / mL pure enzyme solution and 100 μL of 100 mM ferulic acid stock solution were added to 50 mM phosphate buffer (pH 6.0). The reaction was carried out at 35°C and 300 rpm. Samples were taken at reaction times of 10, 20, 30, 60, 120, 180, 240, 300, and 360 min. Sample processing and detection methods were the same as those described in Example 4 above for enzyme activity detection. Each experiment was repeated three times. The enzyme activity of the group with the highest activity was recorded as 100%. The relative enzyme activity of the remaining groups was calculated, and a reaction time-relative enzyme activity curve was plotted.

[0115] The results are as follows Figure 6 As shown, the optimal reaction time for the MavPAD-catalyzed decarboxylation reaction is 60 min.

[0116] Example 6: Decarboxylation Activity Assay of MavPAD

[0117] (1) Product standard curve

[0118] Prepare 25 mM 4-vinylphenol-methanol solution, 25 mM 4-vinylcatechol-methanol solution, and 25 mM 4-vinyleugenol-methanol solution, respectively. The standard curve determination method is the same as in Example 4 above.

[0119] (2) Decarboxylation substrate transformation

[0120] Prepare 200 mM ferulic acid-methanol, 200 mM p-coumaric acid-methanol, 200 mM caffeic acid-methanol, and 200 mM sinapic acid-methanol solutions, respectively. The total reaction volume was 1 mL. Add 100 μL of 8 mg / mL pure enzyme solution and 100 μL of different substrate stock solutions to 50 mM phosphate buffer (pH 6.0). React at 35°C and 300 rpm for 60 min. Dilute the samples 5-fold before loading. Detection was performed according to the enzyme activity detection method in Example 4 above. Each experiment was repeated three times. Calculate the yield of MavPAD for different substrates and plot a substrate-yield bar chart under these reaction conditions.

[0121] See results Figure 7 The results showed that MavPAD catalyzed the formation of coumaric acid, ferulic acid, caffeic acid, and sinapic acid into their corresponding products under the test conditions with yields of 98.12%, 93.37%, 84.26%, and 11.30%, respectively.

[0122] (3) High performance liquid chromatography detection

[0123] High performance liquid chromatography was used to detect the ability of MavPAD to catalyze the synthesis of 4-vinylphenol compounds from hydroxycinnamic acid.

[0124] A Waters e2695 high-performance liquid chromatograph was used, equipped with a SunFire C18 reversed-phase column (4.6 mm × 250 mm) and a 2489 UV-Vis detector. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. The detection temperature was set at 30℃, the flow rate at 1 mL / min, and the UV detection wavelengths were set at 280 nm and 310 nm. The gradient elution program was as follows: 0–8 min: 90% A / 10% B; 8–10 min: 20% A / 80% B; 10–11 min: 80% A / 20% B; 11–13 min: 90% A / 10% B.

[0125] Figure 8 This is a high-performance liquid chromatography (HPLC) chromatogram of the synthesis of 4-vinylphenol compounds from hydroxycinnamic acid catalyzed by MavPAD, as described in this application. A: HPLC chromatogram of the formation of 4-vinylguaiacol from ferulic acid catalyzed by MavPAD; B: HPLC chromatogram of the formation of 4-vinylphenol from p-coumaric acid catalyzed by MavPAD; C: HPLC chromatogram of the formation of 4-vinylcatechol from caffeic acid catalyzed by MavPAD; D: HPLC chromatogram of the formation of 4-vinyleugenol from sinapic acid catalyzed by MavPAD. The detection wavelength was 310 nm (280 nm for 4-vinyleugenol).

[0126] Example 6: MavPAD carboxylation capacity assay

[0127] (1) Product standard curve

[0128] Prepare 25 mM p-coumaric acid-methanol solutions, 25 mM ferulic acid-methanol solutions, 25 mM caffeic acid-methanol solutions, and 25 mM sinapic acid-methanol solutions, respectively. The standard curve determination method is the same as in Example 4 above.

[0129] (2) Decarboxylation substrate test

[0130] Prepare 50 mM 4-vinylphenol-methanol, 50 mM 4-vinylguaiacol-methanol, 50 mM 4-vinylcatechol-methanol, and 50 mM 4-vinyleugenol-methanol solutions, respectively. The total reaction volume was 1 mL. Add 100 μL of 2 mg / mL pure enzyme solution and 100 μL of different substrate stock solutions to 50 mM Tris-HCl buffer (pH 8.5). React at 35°C and 300 rpm for 60 min. The detection method was the same as the enzyme activity detection method in Example 4 above. Each experiment was repeated three times. Calculate the yield of MavPAD for different substrates and plot a substrate-yield bar chart under these reaction conditions.

[0131] Figure 9 Yield analysis of the carboxylation of different 4-vinylphenol substrates catalyzed by MavPAD showed that, under the test conditions, the yields of 4-vinylphenol, 4-vinylguaiacol, 4-vinylcatechol and 4-vinyleugenol to the corresponding products were 20.08%, 15.09%, 12.88% and 12.67%, respectively.

[0132] (3) High performance liquid chromatography detection

[0133] High-performance liquid chromatography (HPLC) was used to detect the effect of MavPAD catalysis on the synthesis of p-hydroxycinnamic acid compounds from 4-vinylphenol. The MavPAD enzyme activity was determined according to previously reported methods. A Waters e2695 HPLC system was used, equipped with a SunFire C18 reversed-phase column (4.6 mm × 250 mm) and a 2489 UV-Vis detector. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. The detection temperature was set at 30℃, the flow rate at 1 mL / min, and the UV detection wavelengths were set at 280 nm and 310 nm. The gradient elution program was as follows: 0–8 min: 90% A / 10% B; 8–10 min: 20% A / 80% B; 10–11 min: 80% A / 20% B; 11–13 min: 90% A / 10% B.

[0134] Figure 10 This document presents high-performance liquid chromatography (HPLC) chromatograms of the synthesis of p-hydroxycinnamic acid compounds from p-vinylphenol catalyzed by MavPAD. A: HPLC chromatogram of the formation of ferulic acid from 4-vinylguaiacol catalyzed by MavPAD; B: HPLC chromatogram of the formation of p-coumaric acid from 4-vinylphenol catalyzed by MavPAD; C: HPLC chromatogram of the formation of caffeic acid from 4-vinylcatechol catalyzed by MavPAD; D: HPLC chromatogram of the formation of sinapic acid from 4-vinyleugenol catalyzed by MavPAD. The detection wavelength was 280 nm.

[0135] In summary, this application provides a novel phenolic acid decarboxylase, MavPAD, its preparation method, and its applications. Specifically, the encoding gene Mavpad was discovered and synthesized from *Mycobacterium avium*, and a recombinant strain was constructed using an *E. coli* heterologous expression system. High-purity recombinant enzyme (yield of 141.5 mg / L) was obtained after induced expression and affinity purification. This enzyme can efficiently catalyze the decarboxylation of four hydroxycinnamic acid substrates—ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid—at 35°C and pH 6.0, generating the corresponding 4-vinylphenol compounds with yields of 93.37%, 98.12%, 85.24%, and 11.21%, respectively. The catalytic activity for sinapic acid is significantly superior to most reported phenolic acid decarboxylases. Meanwhile, under pH 8.5 and CO2 conditions, this enzyme can also reversely catalyze the carboxylation of 4-vinylguaiacol, 4-vinylphenol, 4-vinylcatechol and 4-vinyleugenol to generate the corresponding hydroxycinnamic acid, with a yield between 12% and 20%.

[0136] Furthermore, the novel phenolic acid decarboxylase MavPAD of this application also exhibits good temperature and pH stability (maintaining high activity after 12 h of storage within the range of 25-45℃ and pH 5.0-10.0), providing a new enzyme tool to solve the problems of narrow substrate spectrum, low sinapic acid conversion rate and insufficient stability of existing phenolic acid decarboxylases, and has application prospects in the fields of high-value utilization of lignin, food flavor synthesis and CO2 bioimmobilization.

[0137] The above is a detailed description of the preferred embodiments of this application, but this application is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of this application, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A novel phenolic acid decarboxylase, characterized in that, It can be any of the following: A1) A protein with the amino acid sequence shown in SEQ ID NO: 3; A2) Proteins that share amino acid residue homology greater than 99%, 95%, 90%, 85%, or 80% with the proteins shown in A1) and have the same function; A3) A protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins shown in A1)-A2).

2. The novel phenolic acid decarboxylase according to claim 1, characterized in that, The novel phenolic acid decarboxylase is a protein that has greater than 99% homology with the amino acid residues of the protein shown in A1 and has the same function.

3. The novel phenolic acid decarboxylase according to claim 2, characterized in that, The labels include purification labels and / or solubilization labels.

4. A polynucleotide, characterized in that, Its encoding is a novel phenolic acid decarboxylase as described in any one of claims 1-3.

5. A recombinant expression vector, characterized in that, It contains the polynucleotide as described in claim 4.

6. A recombinant bacterial strain, characterized in that, It comprises the recombinant expression vector as described in claim 5.

7. The method for preparing the novel phenolic acid decarboxylase according to any one of claims 1-3, characterized in that, This includes converting the expression vector encoding the novel phenolic acid decarboxylase into host cells for expression, followed by purification.

8. The use of the novel phenolic acid decarboxylase as described in any one of claims 1-3 in any of the following; B1) Catalyzes the decarboxylation of hydroxycinnamic acid compounds; B2) Reverse catalysis of carboxylation of 4-vinylphenol compounds.

9. The application according to claim 8, characterized in that, The hydroxycinnamic acid compounds include at least one of ferulic acid, p-coumaric acid, caffeic acid, and / or sinapic acid.

10. A method for catalytic decarboxylation of hydroxycinnamic acid compounds, characterized in that, The reaction can be carried out by mixing the novel phenolic acid decarboxylase of any one of claims 1-3 with the hydroxycinnamic acid compound in a reaction system.

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