A phenolic acid decarboxylase mutant and its application

Abstract

The present invention relates to the field of genetic engineering, and particularly relates to a phenolic acid decarboxylase mutant and its application. The present invention obtained a phenolic acid decarboxylase mutant by mutating the 30th tryptophan of phenolic acid decarboxylase PAD_Cs from Clostridium Clostridium sp. DSM 8431 to leucine and simultaneously mutating the 136th tyrosine to tryptophan. This phenolic acid decarboxylase mutant can catalyze the decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene; it can also use bicarbonate as a carboxyl source to catalyze the carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid. It has excellent enzymatic activity characteristics of temperature stability and acetonitrile stability, retaining more than 80% of the decarboxylase activity after incubation at 55 °C for 2 h, and retaining approximately 36% of the decarboxylase activity after incubation in 50% (v / v) acetonitrile for 2 h. It can significantly increase the yield of p-coumaric acid, reaching 13.05 mM at a substrate concentration of 80 mM, which is 74% higher than that of the original phenolic acid decarboxylase PAD_Cs, and has broad industrial application prospects and CO2 fixation potential.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly relates to a phenolic acid decarboxylase mutant and its application. Background Art

[0002] p -coumaric acid ( p -CA) is a plant - derived secondary metabolite and belongs to phenolic compounds. It is widely distributed in the plant kingdom and mainly exists in fruits, vegetables and grains. p -coumaric acid and its derivatives have various biological activities such as antioxidant, anti - inflammatory, anti - cancer, anti - diabetic and anti - melanogenesis. Due to its strong free radical scavenging activity, it can mitigate the adverse effects of various diseases, including arthritis, neurological diseases and cardiovascular diseases.

[0003] In the prior art, phenolic acid decarboxylase has been widely studied for the biological production of vinylphenol. To further expand the application prospects of phenolic acid decarboxylase in industrial production, it is extremely important to improve its stability. For example, by using the N - terminal engineering of proteins, the N - terminal of the amino acid sequence of phenolic acid decarboxylase from Bacillus amyloliquefaciens ZJH - 01 was extended, improving the alkali resistance and heat resistance of the enzyme; through the combination of ancestral sequence reconstruction and solvent engineering, the thermal stability of the enzyme and the solubility of the substrate p -coumaric acid in the reaction system were improved. The existing protein modification studies on phenolic acid decarboxylase mainly focus on the decarboxylation activity of the enzyme, but the research on the carboxylation activity of phenolic acid decarboxylase and the fixation of CO 2 is still very scarce. Therefore, further research on phenolic acid decarboxylase with carboxylation activity still has extremely high value. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a phenolic acid decarboxylase mutant in view of the deficiencies of the prior art.

[0005] Another technical problem to be solved by the present invention is to provide the application of the above - mentioned phenolic acid decarboxylase mutant.

[0006] To solve the above - mentioned technical problems, the technical scheme adopted by the present invention is as follows:

[0007] A phenolic acid decarboxylase mutant, wherein the phenolic acid decarboxylase mutant is obtained by mutating the 30th tryptophan of phenolic acid decarboxylase PAD_Cs to leucine (W30L), and at the same time mutating the 136th tyrosine to tryptophan (Y136W).

[0008] Among them, the amino acid sequence of the phenolic acid decarboxylase PAD_Cs is shown in SEQ ID NO: 1, and the corresponding codon - optimized nucleotide sequence is shown in SEQ ID NO: 2.

[0009] Among them, the phenolic acid decarboxylase PAD_Cs is derived from Clostridium Clostridiumsp. DSM 8431。

[0010] Among them, the phenolic acid decarboxylase mutant is obtained by first finding two potential amino acid active sites W30 and Y136 based on computer-aided design, then performing saturation mutagenesis on these two sites, preferably obtaining two single-site mutants of phenolic acid decarboxylase, W30L and Y136W, and then performing combined mutagenesis on W30L and Y136W.

[0011] Specifically, the amino acid sequence of the phenolic acid decarboxylase mutant is as shown in SEQ ID NO: 7.

[0012] A DNA molecule encoding the phenolic acid decarboxylase mutant is also within the scope of protection of the present invention.

[0013] Specifically, the coding gene of the DNA molecule is as shown in SEQ ID NO: 8.

[0014] A recombinant expression vector containing the coding gene of the DNA molecule is also within the scope of protection of the present invention.

[0015] A recombinant strain is also within the scope of protection of the present invention.

[0016] Specifically, the recombinant strain is obtained by introducing the coding gene of the DNA molecule into a host cell, or by introducing the coding gene of the DNA molecule into a host cell through the recombinant expression vector.

[0017] Among them, the host cell is Escherichia coli.

[0018] In some embodiments of the present invention, the host cell is E.coli BL21(DE3).

[0019] The application of the described phenolic acid decarboxylase mutant in the catalytic preparation of p-hydroxystyrene is also within the scope of protection of the present invention.

[0020] Among them, in the said catalysis, using p-coumaric acid as a substrate, the phenolic acid decarboxylase mutant is used to catalyze the decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene.

[0021] Specifically, for the said catalysis, the catalytic reaction system is: 10 - 50 mM p-coumaric acid, 0.1 - 1 mg / mL phenolic acid decarboxylase mutant, 50 mM citric acid-sodium citrate buffer at pH 3.0 - 5.5.

[0022] Specifically, for the said catalysis, the catalytic conditions are: reacting at 30 - 80 °C for 5 - 15 min.

[0023] In some embodiments of the present invention, the phenolic acid decarboxylase mutant has excellent enzymatic activity characteristics of temperature stability and acetonitrile stability, and still retains more than 80% of the decarboxylase activity after incubation at 55 °C for 2 h, and still retains about 36% of the decarboxylation activity after incubation in acetonitrile with a volume fraction of 50% for 2 h.

[0024] The application of the phenolic acid decarboxylase mutant in the catalytic preparation of p-coumaric acid is also within the scope protected by the present invention.

[0025] Among them, for the catalysis, using p-hydroxystyrene as the substrate and bicarbonate as the carboxyl source, the phenolic acid decarboxylase mutant is used to catalyze the carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid.

[0026] Specifically, for the catalysis, the catalytic reaction is carried out in a CO 2 environment, and the catalytic reaction system is: freeze-dried whole cells of the phenolic acid decarboxylase mutant at 20 - 40 mg / mL, phosphate buffer at 100 mM with a pH of 5.5 - 7.0, p-hydroxystyrene at 10 - 100 mM, 0 - 20% v / v acetonitrile, 1 - 3 M KHCO 3 .

[0027] Specifically, for the catalysis, the catalytic conditions are: reaction at 30 - 40 °C and 200 - 1000 rpm for 12 - 24 h.

[0028] In some embodiments of the present invention, the phenolic acid decarboxylase mutant can significantly increase the yield of p-coumaric acid. When the substrate concentration is 80 mM, the yield can reach 13.05 mM, which is 74% higher than that of the original phenolic acid decarboxylase PAD_Cs.

[0029] Beneficial effects:

[0030] In the present invention, by using Clostridium ClostridiumThe 30th tryptophan of phenolic acid decarboxylase PAD_Cs from sp. DSM 8431 was mutated to leucine (W30L), and at the same time, the 136th tyrosine was mutated to tryptophan (Y136W), resulting in a mutant of phenolic acid decarboxylase. This mutant of phenolic acid decarboxylase can catalyze the decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene; it can also use bicarbonate as a carboxyl source to catalyze the carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid. It has excellent enzyme activity characteristics of temperature stability and acetonitrile stability, still retains more than 80% of the decarboxylase activity after incubation at 55 °C for 2 h, still retains about 36% of the decarboxylase activity after incubation in 50% (v / v) acetonitrile for 2 h, and can significantly increase the yield of p-coumaric acid. When the substrate concentration is 80 mM, the yield can reach 13.05 mM, which is 74% higher than that of the original phenolic acid decarboxylase PAD_Cs, showing broad industrial application prospects and carbon dioxide fixation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following further specific description of the present invention will be made in conjunction with the drawings, and the above and / or other advantages of the present invention will become clearer.

[0032] Figure 1 It is the SDS-PAGE diagram of phenolic acid decarboxylase PAD_Cs and its single-point and combined mutants. Among them, M: Marker; 1: phenolic acid decarboxylase PAD_Cs; 2: single-point mutant W30L of phenolic acid decarboxylase; 3: single-point mutant Y136W of phenolic acid decarboxylase; 4: combined mutant W30L / Y136W of phenolic acid decarboxylase.

[0033] Figure 2 It is the thermal stability analysis of phenolic acid decarboxylase PAD_Cs and its mutant W30L / Y136W.

[0034] Figure 3 It is the acetonitrile stability analysis of phenolic acid decarboxylase PAD_Cs and its mutant W30L / Y136W.

[0035] Figure 4 It is the effect of different substrate (p-hydroxystyrene) concentrations on the yield of p-coumaric acid synthesized by the combined mutant W30L / Y136W of phenolic acid decarboxylase. DETAILED DESCRIPTION OF THE INVENTION

[0036] The following further specific description of the present invention will be made in conjunction with the specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0037] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0038] In the following examples, the detection method of the high performance liquid chromatography (HPLC) is as follows: all liquid chromatography measurements were carried out using an Agilent HPLC (Model-1200, phase) equipped with C18 (Agilent 5 HC-C18, 250×4.6 mm, 5 μm). The column temperature was 24 °C, the flow rate was 1 mL / min, the program ran for 10 min, and the mobile phase and ultraviolet detection conditions were: using 0.1% H 2 O / TFA and 0.1% MeCN / TFA (40∶60, v / v) as the mobile phase, and spectrophotometric detection was carried out at 280 nm and 320 nm. The concentration of the reaction product in the decarboxylation reaction was calculated based on the detection data at the ultraviolet wavelength of 280 nm, and the concentration of the reaction product in the β-carboxylation reaction was calculated based on the detection data at the ultraviolet wavelength of 320 nm.

[0039] Example 1: Determination of the mutation sites of the phenolic acid decarboxylase mutant

[0040] Download the three-dimensional structure model of the phenolic acid decarboxylase PAD_Cs sequence from the UniProt database, which is from the Clostridium Clostridium sp. DSM 8431 strain, dock the substrate p-hydroxystyrene to the active pocket, select the amino acids within 4 Å of the active site, and combine with the multiple sequence alignment of the reported phenolic acid decarboxylases with carboxylation activity. It was found that the W30 site had a large difference, while the other amino acids were highly conserved. In addition, further according to the sequence conservation, it was found that at the Y136 site, the phenolic acid decarboxylase PAD_Cs was significantly different from other aligned phenolic acid decarboxylases.

[0041] Example 2: Construction, expression and screening of the single-site mutants of the phenolic acid decarboxylase

[0042] 1. Construction of plasmid pET-28a-PAD_Cs

[0043] The sequence of the target phenolic acid decarboxylase (PAD_Cs) gene (WP_090014602.1) after codon optimization (as shown in SEQ ID NO: 2) was handed over to Azenta Life Sciences Company. Using pET-28a(+) as the vector, plasmid pET-28a-PAD_Cs was synthesized and stored in E. coli DH5α.

[0044] 2. Construction and expression of the single-site mutants of the phenolic acid decarboxylase

[0045] (1) Construction of the single-site mutants of the phenolic acid decarboxylase

[0046] Saturation mutagenesis was performed on two sites, namely the W30 site and the Y136 site. The amino acid W at the 30th site was mutated to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y respectively, and the amino acid Y at the 136th site was mutated to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W respectively, resulting in a total of 38 single-site mutants of phenolic acid decarboxylase (W30A, W30C, W30D, W30E, W30F, W30G, W30H, W30I, W30K, W30L, W30M, W30N, W30P, W30Q, W30R, W30S, W30T, W30V, W30Y, Y136A, Y136C, Y136D, Y136E, Y136F, Y136G, Y136H, Y136I, Y136K, Y136L, Y136M, Y136N, Y136P, Y136Q, Y136R, Y136S, Y136T, Y136V, Y136W).

[0047] The specific process is as follows: According to the relevant instructions of the "Mini Plasmid DNA Extraction Kit for yPrp", plasmid pET-28a-PAD_Cs was extracted. Using plasmid pET-28a-PAD_Cs as a template, referring to the Vazyme biological products and operation manuals, the corresponding mutagenic primer pairs were used for whole-plasmid PCR amplification to obtain the site-directed mutagenesis sequence. Among them, for the whole-plasmid PCR, the reaction system included: 12.5 μL of Phanta High-Fidelity Enzyme, 1 μL of template (plasmid pET-28a-PAD_Cs), 1 μL of primer F, 1 μL of primer R, and 9.5 μL of ddH 2 O. The reaction parameters were: 2 min at 95°C, 15 s at 95°C, 15 s at 60°C, and extension at 72°C for 3 min. 30 cycles were set, and 10 min at 72°C. For the above-mentioned single-site mutants of phenolic acid decarboxylase, W30L and Y136W, when using the corresponding mutagenic primer pairs for whole-plasmid PCR amplification to obtain the site-directed mutagenesis sequence, the sequences of the mutagenic primer pairs used are shown in Table 1.

[0048] Table 1 Mutagenic primer pairs for single-site mutants of phenolic acid decarboxylase, W30L and Y136W

[0049]

[0050] The PCR products were digested with Dpn I. After the template digestion was completed, the products were transformed into Escherichia coli competent cells by heat shock method E.coliIn BL21(DE3), it was spread on an LB agar plate containing 100 μg / mL kanamycin sulfate and cultured overnight at 37 °C in an inverted position. The results of the overnight culture mutation were verified by sequence determination by Anhui General Biology Company.

[0051] (2)Expression of single-point mutants of phenolic acid decarboxylase

[0052] The recombinant Escherichia coli of the single-point mutant of phenolic acid decarboxylase with correct sequencing was inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate and cultured overnight at 37 °C and 180 rpm to prepare a seed solution. Then, the seed solution was inoculated into fresh 50 mL of LB liquid medium at an inoculation amount of 2% v / v and cultured at 37 °C and 180 rpm until the OD 600 reached 0.6 - 1.0. It was taken out, cooled in an ice-water bath for 5 min, and an inducer IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.1 mmol / L was added, and induced expression was carried out at 16 °C and 150 rpm for 20 h to obtain a fermentation broth.

[0053] 3. Screening of single-point mutants of phenolic acid decarboxylase

[0054] Due to the long carboxylation reaction time and the presence of the organic co-solvent acetonitrile in the reaction system, the stability of the enzyme is the key to its continuous catalytic action. Therefore, the conversion rate of the single-point mutant in the reaction system with two acetonitrile volume fractions of 12% and 20% was selected as the screening criterion for screening acetonitrile-stable single-point mutants of phenolic acid decarboxylase. The specific process is as follows.

[0055] (1)Preparation of freeze-dried whole cells of single-point mutants of phenolic acid decarboxylase

[0056] The fermentation broth obtained by induced expression of the recombinant Escherichia coli of the single-point mutant of phenolic acid decarboxylase was centrifuged at 4 °C and 12000 rpm for 10 min respectively; the centrifuged supernatant was discarded, the bacterial cells were collected, an appropriate amount of pure water was added to resuspend the precipitated bacterial cells, and the bacterial cells were collected by centrifugation again, repeating 2 - 3 times; the precipitated bacterial cells were placed in a -80 °C ultra-low temperature freezer and pre-frozen for 12 h. The pre-frozen precipitated bacterial cells were quickly placed in a freeze dryer and freeze-dried for 12 - 16 h to obtain freeze-dried bacterial powders of different single-point mutants of phenolic acid decarboxylase, that is, freeze-dried whole cells of single-point mutants of phenolic acid decarboxylase.

[0057] (2)Screening of single-point mutants of phenolic acid decarboxylase

[0058] β-Carboxylation reaction: The 1 mL reaction system is as follows: The freeze-dried whole cells of the combined mutant of phenolic acid decarboxylase at 20 mg / mL are resuspended in 100 mM phosphate buffer at pH 5.5, shaken and hydrated at 600 rpm and 30 °C for 30 min, then 10 mM p-hydroxystyrene (using propylene glycol as the solvent), 12% v / v or 20% v / v acetonitrile, and 3 M KHCO 3 , and react at 30 °C and 600 rpm for 24 h to obtain the reaction solution. The above reaction system is carried out in a covered glass bottle filled with CO 2 (the bottle mouth is wrapped with a sealing film to prevent CO 2 from escaping).

[0059] Centrifuge the reaction solution at 13000 rpm for 15 min, take the supernatant, dilute every 100 μL of the reaction solution with 1 mL of water / acetonitrile mixture (v / v 1:1), and add 3% v / v trifluoroacetic acid (based on the water / acetonitrile mixture, that is, add 30 μL), incubate at room temperature for 5 min, then centrifuge at 13000 rpm for 15 min, and analyze the conversion rate of the β-carboxylation reaction by reverse-phase high-performance liquid chromatography.

[0060] The results are shown in Table 2. It can be seen from the table that at an acetonitrile volume fraction of 12%, most of the single-point mutants of phenolic acid decarboxylase have good conversion rates. When the acetonitrile volume fraction is increased to 20%, the single-point mutants W30L and Y136W of phenolic acid decarboxylase show the best conversion rates, where W30L is 3.71% and Y136W is 2.65%, indicating that these two single-point mutants retain obvious carboxylation activity and have high acetonitrile stability.

[0061] Table 2 Conversion rates of β-carboxylation reactions using different single-point mutants of phenolic acid decarboxylase

[0062]

[0063] Example 3: Construction, expression and purification of the combined mutant W30L / Y136W of phenolic acid decarboxylase

[0064] Using the plasmid of the single-point mutant W30L of phenolic acid decarboxylase as the template, perform PCR amplification with the mutant primer pair Y136W-F and Y136W-R. The PCR amplification process, digestion and transformation of the PCR product, and induction expression of the combined mutant W30L / Y136W bacteria are the same as in Example 2.

[0065] Take the induced fermentation broth, centrifuge at 12,000 rpm for 20 min, discard the supernatant, then resuspend and wash the cells with 20 mM Tris-HCl (pH 7.5) buffer, centrifuge at 12,000 rpm for 20 min, discard the supernatant, and after resuspending again with the buffer, perform ultrasonic disruption. Centrifuge the ultrasonic disruption solution at 12,000 rpm for 20 min, take the supernatant, which is the crude enzyme solution.

[0066] Filter the crude enzyme solution through a 0.22 μm filter membrane. Wash the nickel column with Buffer A (20 mM Tris-HCl, pH 7.5) at a flow rate of 2 mL / min until it is balanced. Inject the protein sample into the injection loop with a syringe, collect the breakthrough peak protein of the sample, and wash the nickel column again with Buffer A (20 mM Tris-HCl, pH 7.5) until no protein is eluted. Use gradient elution method, wash the nickel column with at least 5 times the volume of 15% Buffer B (20 mM Tris-HCl, 500 mM imidazole, pH 7.5) for each gradient, collect the absorbance peak protein during elution until no protein is eluted. The protein solution obtained by nickel column purification is used with a pre-packed desalting column from GE to replace the Buffer containing imidazole with 20 mM Tris-HCl (pH 7.5) buffer to remove the imidazole in the protein solution, and finally obtain the purified enzyme solution.

[0067] Perform SDS-PAGE electrophoresis detection on the purified enzyme solutions of phenolic acid decarboxylase PAD_Cs, single-point mutants W30L and Y136W of phenolic acid decarboxylase, and combined mutant W30L / Y136W of phenolic acid decarboxylase. The results are as Figure 1 shown. The molecular weight of the target protein is 21 kDa, and there are obvious bands at 21 kDa in each lane of the electrophoresis pattern, indicating that the target protein is successfully expressed in each mutant.

[0068] Example 4: Determination of the decarboxylase activity of the combined mutant W30L / Y136W of phenolic acid decarboxylase

[0069] Using p-coumaric acid solution as the substrate to catalyze the formation of p-hydroxystyrene, determine the decarboxylase activity of the combined mutant W30L / Y136W of phenolic acid decarboxylase after incubation at different temperatures and different volume fractions of acetonitrile, so as to evaluate the enzymatic properties of the combined mutant of phenolic acid decarboxylase.

[0070] Among them, the definition of the enzyme activity unit: The amount of enzyme that produces 1 μmol of p-hydroxystyrene per minute is 1 IU, and the decarboxylase activity unit of phenolic acid decarboxylase is IU / mL.

[0071] Among them, the preparation method of the p-coumaric acid solution is as follows: Accurately weigh 8.21 g of p-coumaric acid and place it in a certain amount of pure water. Adjust the pH to 7.0 with NaOH solution. During this period, stir with a magnetic stirrer and monitor the solution pH with a pH meter. After the p-coumaric acid powder is completely dissolved, make up the volume to 1 L. At this time, the final concentration of the p-coumaric acid solution is 50 mM.

[0072] 1. Temperature stability

[0073] After diluting the enzyme solution of the purified phenolic acid decarboxylase combinatorial mutant W30L / Y136W to 0.1 mg / mL, place it in a water bath at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, and 70 °C for incubation for 2 h. In a 1 mL reaction system, add a p-coumaric acid solution with a final concentration of 50 mM, the W30L / Y136W enzyme solution after incubation at different temperatures, and 50 mM citric acid-sodium citrate buffer (pH 5.0), and react at 60 °C for 5 min. Then add 2 mL of acetonitrile to terminate the reaction. After centrifuging the reaction mixture at 4 °C and 12,000 g for 10 min, take the supernatant for high performance liquid chromatography (HPLC) analysis. Use the phenolic acid decarboxylase PAD_Cs (denoted as WT in the figure) as a control.

[0074] Record the initial enzyme activity without temperature incubation as 100%, calculate the relative enzyme activities of the original phenolic acid decarboxylase PAD_Cs and the combinatorial mutant after incubation at each temperature, and draw a curve of the change in residual enzyme activity under different incubation conditions. The results are as Figure 2 shown. The temperature stability of the combinatorial mutant W30L / Y136W is greatly improved compared with the original phenolic acid decarboxylase PAD_Cs. The phenolic acid decarboxylase PAD_Cs can retain 80% of its enzyme activity only when the incubation temperature is less than 35 °C. When incubated at ≥50 °C for 2 h, the phenolic acid decarboxylase PAD_Cs almost loses its activity; while for the combinatorial mutant W30L / Y136W, during the incubation temperature range of 30 - 55 °C, the enzyme activity does not decrease significantly. After incubation at 55 °C for 2 h, it still retains more than 80% of its enzyme activity. In addition, the decarboxylation specific enzyme activity of the combinatorial mutant is increased from 177 IU / mg of the original phenolic acid decarboxylase PAD_Cs to 335 IU / mg, and the decarboxylation specific enzyme activity of the combinatorial mutant is about 1.9 times that of the original phenolic acid decarboxylase PAD_Cs.

[0075] 2. Acetonitrile stability

[0076] After diluting the enzyme solution of the purified phenolic acid decarboxylase combinatorial mutant W30L / Y136W to 0.1 mg / mL, it was incubated on ice at 4 °C for 2 h in acetonitrile with different volume fractions (10% v / v, 20% v / v, 30% v / v, 40% v / v, 50% v / v). In a 1 mL reaction system, a p-coumaric acid solution with a final concentration of 50 mM, the W30L / Y136W enzyme solution incubated with acetonitrile of different volume fractions, and 50 mM citric acid-sodium citrate buffer (pH 5.0) were added, and the reaction was carried out at 60 °C for 5 min. Then 2 mL of acetonitrile was added to terminate the reaction. After centrifuging the resulting mixture at 4 °C and 12,000 g for 10 min, the supernatant was taken for high performance liquid chromatography (HPLC) analysis. The phenolic acid decarboxylase PAD_Cs (denoted as WT in the figure) was used as a control.

[0077] The initial enzyme activity without incubation in acetonitrile was recorded as 100%, and the change curve of the residual enzyme activity under the incubation conditions of acetonitrile with different volume fractions was calculated. The results are as Figure 3 shown. The stability of the mutant W30L / Y136W in acetonitrile was significantly improved compared with the original phenolic acid decarboxylase PAD_Cs. After incubating for 2 h at 10 - 50% acetonitrile volume fractions, the remaining enzyme activity of W30L / Y136W was higher than that of the phenolic acid decarboxylase PAD_Cs. When incubated in 50% volume fraction of acetonitrile, W30L / Y136W still retained about 36% of the activity, while the original enzyme PAD_Cs only had about 16% of the enzyme activity; in addition, the specific enzyme activity of the combinatorial mutant in 50% volume fraction of acetonitrile was still 131 IU / mg, while the original phenolic acid decarboxylase PAD_Cs was only 30 IU / mg, and the specific enzyme activity of the combinatorial mutant was 4.36 times that of the original phenolic acid decarboxylase PAD_Cs.

[0078] Example 5: β-carboxylation reaction of phenolic acid decarboxylase combinatorial mutant W30L / Y136W (catalytic preparation of p-coumaric acid)

[0079] According to the preparation method of the freeze-dried whole cells of the single-site mutant of phenolic acid decarboxylase in Example 1, the freeze-dried whole cells of the phenolic acid decarboxylase combinatorial mutant W30L / Y136W were prepared.

[0080] The 1 mL reaction system for the β-carboxylation reaction was as follows: The freeze-dried whole cells of the 20 mg / mL phenolic acid decarboxylase combinatorial mutant were resuspended in 100 mM phosphate buffer at pH 5.5, and shaken and hydrated at 600 rpm and 30 °C for 30 min. Then different concentrations of p-hydroxystyrene (20 mM, 40 mM, 60 mM, 80 mM, 100 mM, with propylene glycol as the solvent), 0 - 20% v / v acetonitrile, and 3 M KHCO 3, react at 30 °C and 600 rpm for 24 h to obtain a reaction solution. The above reaction system is carried out in a covered glass bottle filled with CO 2 (the bottle mouth is wrapped with a sealing film to prevent CO 2 from escaping). Using phenolic acid decarboxylase PAD_Cs (denoted as WT in the figure) as a control.

[0081] Centrifuge the reaction solution at 13000 rpm for 15 min, take the supernatant, dilute every 100 μL of the reaction solution with 1 mL of water / acetonitrile mixture (v / v 1:1), and add 3% v / v trifluoroacetic acid (based on the water / acetonitrile mixture, that is, add 30 μL). After incubating at room temperature for 5 minutes, centrifuge at 13000 rpm for 15 min, and detect the ultraviolet wavelength data at 320 nm by reversed-phase high-performance liquid chromatography, and calculate the concentration of the reaction product (i.e., p-coumaric acid) at different substrate concentrations.

[0082] The results are as Figure 4 shown. The combined mutant W30L / Y136W can significantly increase the yield of p-coumaric acid. When the substrate concentration is 80 mM, the yield can reach 13.05 mM, which is 74% higher than that of the original phenolic acid decarboxylase PAD_Cs. Even at a substrate concentration of 100 mM, which inhibits the β-carboxylation reaction, the p-coumaric acid yield of the combined mutant W30L / Y136W is still extremely significantly higher than that of the original phenolic acid decarboxylase PAD_Cs, indicating that this combined mutant has broad industrial application prospects and carbon dioxide fixation potential.

[0083] The present invention provides an idea and method for a phenolic acid decarboxylase mutant and its application. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A phenolic acid decarboxylase mutant, characterized in that: The phenolic acid decarboxylase mutant is obtained by mutating the tryptophan at position 30 of the phenolic acid decarboxylase PAD_Cs to leucine and mutating the tyrosine at position 136 to tryptophan; The amino acid sequence of the phenolic acid decarboxylase PAD_Cs is shown in SEQ ID NO:

1.

2. A DNA molecule, characterized in that The DNA molecule encodes the phenolic acid decarboxylase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the coding gene of the DNA molecule according to claim 2.

4. A recombinant strain, characterized in that: The recombinant strain is obtained by introducing the coding gene of the DNA molecule of claim 2 into a host cell, or by introducing the coding gene of the DNA molecule of claim 2 into a host cell via the recombinant expression vector of claim 3.

5. Use of the phenolic acid decarboxylase mutant according to claim 1 in catalyzing the preparation of p-hydroxystyrene.

6. The use according to claim 5, characterized in that: The catalysis uses p-coumaric acid as a substrate and utilizes a phenolic acid decarboxylase mutant to catalyze a decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene; The catalysis reaction system is: 10-50 mM p-coumaric acid, 0.1-1 mg / mL phenolic acid decarboxylase mutant, 50 mM pH 3.0-5.5 citric acid-sodium citrate buffer; the catalysis conditions are: reaction at 30-80°C for 5-15 min.

7. Use of the phenolic acid decarboxylase mutant according to claim 1 in catalyzing the preparation of p-coumaric acid.

8. The use according to claim 7, characterized in that: The catalysis uses p-hydroxystyrene as a substrate and bicarbonate as a carboxyl source, and utilizes a phenolic acid decarboxylase mutant to catalyze a carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid.

9. The use according to claim 8, characterized in that: The catalysis is carried out in a CO2 environment, and the catalytic reaction system is: 20-40 mg / mL freeze-dried whole cells of phenolic acid decarboxylase mutants, 100 mM phosphate buffer with a pH of 5.5-7.0, 10-100 mM p-hydroxystyrene, 0-20% v / v acetonitrile, and 1-3 M KHCO3; the catalysis is carried out under the following catalytic conditions: 30-40° C. and 200-1000 rpm for reaction for 12-24 hours.

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