Phenolic acid decarboxylase mutant for efficiently producing 4-vinyl guaiacol and application of phenolic acid decarboxylase mutant

Abstract

The invention discloses a phenolic acid decarboxylase mutant for efficiently producing 4-vinyl guaiacol and application of the phenolic acid decarboxylase mutant, and belongs to the technical field of gene engineering and enzyme engineering. The 91 amino acid of a wild type sequence as shown in SEQ ID NO.1 has V-I mutation, and the amino acid sequence of the mutated phenolic acid decarboxylase mutant is as shown in SEQ ID NO.2. Compared with wild type phenolic acid decarboxylase, the phenolic acid decarboxylase mutant obtained by the invention has the advantages that the thermal stability and the pH stability are improved, the optimal temperature of the mutant V91I is increased to 50 DEG C, the optimal temperature of the wild type is only 40 DEG C, and the phenolic acid decarboxylase mutant has better tolerance than the wild type at the temperature of 40 DEG C or above. Meanwhile, the pH stability of the mutant is also remarkably improved, so that the phenolic acid decarboxylase mutant can be better applied to the fields of fermented foods and medicines.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and specifically relates to a phenolic acid decarboxylase mutant for efficient production of 4-vinylguaiacol and its uses. Background Technology

[0002] 4-Vinylguaiacol (4-VG) is a volatile phenolic compound with a strong spice and fermented aroma, and is a colorless to pale yellow oily liquid. 4-VG is not only a major aroma component in many foods and beverages, but it can also participate in biopharmaceutical synthesis, possessing health functionalities. Currently, the synthesis of 4-VG on the market is mostly by chemical methods. Chemical methods require high temperature and pressure, and microwave-induced decarboxylation. However, since the main applications of 4-VG are in the food and pharmaceutical fields, there are extremely high requirements for product safety, and substances synthesized by chemical methods have significant safety risks. Researchers have gradually turned to microbial fermentation, plant cell culture, and enzymatic methods. Compared to the other two, enzymatic methods have become the research trend for 4-VG production in recent years due to their renewable, green, and environmentally friendly advantages. Ferulic acid (FA) is a key precursor in the synthesis of 4-vinylguaiacol (4-VG), and its market price is only about 1 / 40th of that of 4-VG, demonstrating extremely high economic conversion potential. Compared to traditional chemical synthesis routes, the enzymatic decarboxylation of FA to 4-VG not only offers mild reaction conditions and a green and environmentally friendly process, but also has significant advantages in raw material cost control and product safety, providing a promising alternative route for the industrial production of 4-VG.

[0003] Phenolic acid decarboxylases (lyases EC4.1.1.102) are bacterial proteases that catalyze the non-oxidative decarboxylation of p-hydroxycinnamic acid to 4-vinyl derivatives without the need for cofactors. Although these enzymes have clear applications in biosynthesis, their practical industrialization faces significant challenges: existing enzyme resources are scarce, and most known phenolic acid decarboxylases exhibit low expression levels, poor stability, and insufficient catalytic efficiency in heterologous expression systems, severely restricting their application in large-scale production. Therefore, it is urgent to expand the microbial sources of phenolic acid decarboxylases and discover more new resources with excellent catalytic properties, high expression potential, and novel properties, providing crucial enzyme support for advancing basic research and application development of this type of enzyme.

[0004] Two-phase biotransformation systems (TPBS) have wide applications in biosynthesis and separation science, consisting of an aqueous phase and a water-insoluble organic phase. Based on the partition coefficients of the substrate and product in the two-phase solution, the product 4-VG dissolves in the organic phase, while the substrate ferulic acid and enzyme remain in the aqueous phase, minimizing the toxicity of the product to the enzyme. There have been reports of producing 4-vinylphenols in two-phase transformation systems using wild-type cells or recombinant E. coli cells equipped with phenolic acid decarboxylase, achieving some success. However, this requires extremely high tolerance to organic solvents and high catalytic efficiency from the enzyme. Therefore, site-specific modification of phenolic acid decarboxylase to obtain a mutant with improved stability and catalytic activity, along with its preparation method and applications, is of great significance. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a phenolic acid decarboxylase mutant for the efficient production of 4-vinylguaiacol and its uses, which can effectively improve the ability of microorganisms to produce tetravinylguaiacol.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a phenolic acid decarboxylase mutant for the efficient production of 4-vinylguaiacol, which has a V→I mutation at amino acid position 91 of the wild-type (bspad) sequence as shown in SEQ ID NO.1, and the amino acid sequence of the mutated phenolic acid decarboxylase mutant is shown in SEQ ID NO.2.

[0007] Wild type: MENFIGSHMIYTYENGWEYEIYIKNDHTIDYRIHSGMVGGRWVRDQEVNIVKLTEGVYKVSWTEPTGTDVSLNFMPNEKRMHGIIFFPKWVHEHPEITVCYQNDYIDVMKESREKYETYPKYVVPEFADITYLNNAGINNETLISEAPYEGMTDDIRAGKLK (SEQ ID NO. 1).

[0008] Mutant: MENFIGSHMIYTYENGWEYEIYIKNDHTIDYRIHSGMVGGRWVRDQEVNIVKLTEGVYKVSWTEPTGTDVSLNFMPNEKRMHGIIFFPKWIHEHPEITVCYQNDYIDVMKESREKYETYPKYVVPEFADITYLNNAGINNETLISEAPYEGMTDDIRAGKLK (SEQ ID NO. 2).

[0009] Another object of the present invention is to provide a method for improving the stability of phenolic acid decarboxylase, wherein the 91st amino acid of the wild-type phenolic acid decarboxylase shown in SEQ ID NO.1 is mutated from V to I to obtain a phenolic acid decarboxylase mutant with higher stability; the amino acid sequence of the phenolic acid decarboxylase mutant is shown in SEQ ID NO.2.

[0010] Furthermore, stability includes pH stability and thermal stability.

[0011] Another object of the present invention is to provide the use of the above-mentioned phenolic acid decarboxylase mutant in food fermentation and / or in the preparation of pharmaceuticals with antioxidant and anti-inflammatory effects.

[0012] Another object of the present invention is to provide the use of the above-mentioned phenolic acid decarboxylase mutant in the preparation of fermentation formulations with better stability.

[0013] Another object of the present invention is to provide a fermentation preparation with better pH and thermal stability, which uses the above-mentioned phenolic acid decarboxylase mutant as the active ingredient.

[0014] The beneficial effects of this invention are: Compared to wild-type phenolic acid decarboxylase, the phenolic acid decarboxylase mutant obtained in this invention exhibits improved thermal and pH stability. The optimal temperature of mutant V91I has increased to 50°C, while the optimal temperature of the wild type is only 40°C, and it shows better tolerance to temperatures above 40°C. Simultaneously, the pH stability of the mutant is also significantly improved, enabling the phenolic acid decarboxylase mutant to be better applied in fermented foods and pharmaceuticals.

[0015] The catalytic efficiency of the phenolic acid decarboxylase mutant obtained in this invention is 1.11 times that of the wild-type enzyme. Using a two-way system of 1-octanol:water phase = 1:3 (v:v) with 400 mm FA (ferulic acid) as substrate, the yield of 4-VG is 12.22 g / DCW. Attached Figure Description

[0016] Figure 1 This is an SDS-PAGE electrophoresis image after nickel ion chelation chromatography; where lane M: protein marker; lane 1: total protein from the IPTG-induced control strain without the pad gene; lane 2: total protein after disruption; lane 3: supernatant after disruption; lane 4: precipitate after disruption; lane 5: flow-through buffer from the nickel ion chelation column; lanes 6-9: imidazole elution buffer at concentrations of 5 mm, 10 mm, 25 mm, and 50 mm, respectively. Figure 2 A comparison of the optimal temperatures of the wild-type enzyme and the mutant enzyme before and after mutation; Figure 3 The effect of temperature before and after mutation on the thermal stability of wild-type and mutant enzymes; Figure 4 A comparison of the optimal pH of the wild-type enzyme and the mutant enzyme before and after mutation; Figure 5 A comparison of the pH stability of the wild-type enzyme and the mutant enzyme before and after mutation; Figure 6 The optimal pH for whole-cell catalysis of the mutant; Figure 7 The optimal temperature for whole-cell catalysis of the mutant; Figure 8 This represents the optimal substrate concentration for whole-cell catalysis. Detailed Implementation

[0017] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0018] Example 1: Site-directed mutagenesis and expression of phenolic acid decarboxylase Physicochemical analysis of the phenolic acid decarboxylase showed that the protein has a molecular weight of 19.10 kDa and is composed of 162 amino acids. Glutamic acid (Glu) is the most abundant amino acid, with 17 amino acids, accounting for 10.5% of the total. The isoelectric point is 5.01, and the instability coefficient is 26.33, less than 40, indicating that the enzyme is stable. Amino acid sequence and spatial structure analysis identified only amino acids at positions 21, 29, 37, 72, and 91 as non-conserved amino acids. I-Mutant 2.0 analysis was used to analyze 19 possible natural amino acid mutations at each of the 21, 29, 37, 72, and 91 amino acid sites. Among all the calculated mutations, only V91I and V91L were stable mutations.

[0019] Primers designed based on V91I and V91L mutations, as shown in Table 1, were used to perform site-directed mutagenesis using the constructed recombinant plasmid pET28a-bspad as a template and whole-plasmid PCR amplification technology. The purified PCR product was inserted into the pET-28a(+) vector using the designed restriction enzyme sites. The resulting plasmid pET-28a(+)-bspad was transformed into *E. coli* DH5α, and positive clones were selected for sequencing verification and preservation.

[0020] The recombinant plasmid was transformed into *E. coli* BL21(DE3). Positive transformants were picked and cultured overnight at 37°C and 180 rpm in 50 ml LB medium (50 μg / ml kanamycin) to prepare seed culture. The seed culture was inoculated at a 2% inoculum into LB medium containing kanamycin resistance. When OD... 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 1 mM, and expression was induced at 18℃ and 180 r / min for 16 h.

[0021] Cells were harvested by centrifugation at 4°C and resuspended in 10 ml of PBS lysis buffer. The cell suspension was sonicated on ice for 15 min, and the cell lysis buffer was centrifuged at 12,000 × g for 20 min at 4°C to obtain the supernatant, which yielded the crude enzyme solution. The crude enzyme solution was then filtered and loaded into a pre-equilibrated nickel ion purification column. After incubation overnight at 4°C with shaking, the flow-through was collected, and the cells were washed three times with different column volumes of imidazole to obtain pure enzyme samples of mutant and wild-type enzymes. All samples were processed and then analyzed by SDS-PAGE gel electrophoresis. The electrophoresis results are shown below. Figure 1 As shown. The purified protein was stored at 4°C, and its enzymatic properties were determined immediately after purification.

[0022] Table 1 Primer sequences for phenolic acid decarboxylase mutation

[0023] Example 2: Determination of the enzymatic properties of wild-type and mutant phenolic acid decarboxylase The enzymatic properties of phenolic acid decarboxylase were determined for both wild-type and mutant strains, as follows: The reaction system consisted of 5 mL of 4 mL Na₂HPO₄-citric acid buffer (pH 6.0), 0.5 mL of 50 mmol / L substrate, and 0.5 mL of phenolic acid decarboxylase solution. The reaction was carried out in a 37°C water bath for 5 min, followed by high-temperature inactivation. The aqueous phase was collected and the amount of ferulic acid consumed was determined using a UV-1200S UV-Vis spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme required to convert 1 μmol of ferulic acid per minute (with the highest enzyme activity being 100%). Based on the study of the effects of pH and temperature on enzyme activity (…),… Figure 2 After substrate-free pre-incubation at the corresponding temperature for a specific time, the thermostability was determined by measuring the residual activity of the enzyme. The results are as follows: Figure 3 As shown. The enzyme was incubated at room temperature for 1 hour in buffer solutions of different pH values. pH stability was assessed by measuring the remaining enzyme activity. The results are shown below. Figure 4 and Figure 5As shown in Table 2, the Michaelis kinetic parameters of phenolic acid decarboxylase and its mutant enzyme were calculated. The protein concentration was determined using Nanodrop. The results are shown in Table 2. The catalytic efficiency of mutant enzyme V91I is 1.11 times that of wild type, while the catalytic efficiency of mutant enzyme V91L is only 0.87 times that of wild type. The catalytic performance is inferior to that of wild type and is not considered.

[0024] Table 2 Kinetic parameters of mutant and wild-type enzymes

[0025] Example 3: Determination of optimal pH and temperature for whole-cell catalysis by mutants The optimal pH and temperature for whole-cell catalysis of the mutant were determined, and the specific procedure is as follows: After induction of expression, the cells were centrifuged at 4°C and 5000 rpm for 20 min to obtain a bacterial pellet. The pellet was washed 2-3 times with PBS buffer, and the cells were resuspended in the buffer solution. The optimal temperature and pH for whole-cell catalysis were determined in a 50 ml Erlenmeyer flask. The reaction conditions and system were the same as in Example 2, except that 0.1 ml of enzyme solution was replaced with 0.1 ml of bacterial culture (OD600 = 0.6-0.8). The optimal pH and temperature are as follows: Figure 6 , Figure 7 As shown.

[0026] Example 4: Determination of optimal pH and temperature for whole-cell catalysis of mutants in a two-phase system The optimal pH and temperature for whole-cell catalysis of the mutant in a two-phase system were determined. Referring to the distribution coefficients of ferulic acid and 4-VG in the aqueous and immiscible organic phases and the activity of phenolic acid decarboxylase in the two-phase system, butanol, 1-octanol, and benzene were found to be relatively suitable solvents. However, considering safety, health, and environmental factors, 1-octanol was chosen as the organic solvent for the two-phase reaction system. The specific process is as follows: (1) Determining the effect of 1-octanol on the whole-cell stability of E. coli BL21 / pET28a-V91I E. coli BL21 / pET28a-V91I whole cells were reacted at 40°C and 180 rpm for 1 h, 12 h, and 40 h in the presence of 1-octanol. The residual enzyme activity was then measured under optimal conditions, with the enzyme activity without organic solvent treatment being taken as 100%. The results are shown in Table 3.

[0027] Table 3. Effects of 1-Octanol on whole-cell stability of E. coli BL21 / pET28a-V91I

[0028] Table 3 shows that 1-octanol can activate the activity of BSPAD. Treatment of BSPAD in different proportions of organic solvents for 12 hours had little effect on its activity, maintaining over 90% of the original activity. However, the activity decreased after 40 hours of treatment. Since the effects of different volume ratios of organic solvents on enzyme activity were relatively small, and considering the extraction and production costs of the product, subsequent experiments were conducted with an organic solvent concentration of 30%.

[0029] (2) Determine the toxicity of the substrate to the enzyme molecule The production of 4-VG was determined at substrate ferulic acid (FA) concentrations of 100, 200, 300, 400, and 500 mM to screen for optimal reaction conditions. In a two-phase system using 1:3 volume ratio of 1-octanol to aqueous buffer, the effect of different substrate concentrations on 4-VG production in whole-cell E. coli BL21 / pET28a-V91I cells was determined by GC-MS. The results are shown below. Figure 8 The specific testing conditions are as follows: GC conditions: The gas chromatography column used was TG-wax (30m×0.25mmx0.25um).

[0030] Temperature program: 40℃ for 2 min, increase to 85℃ at 5℃ / min, hold for 2 min, increase to 110℃ at 8℃ / min, hold for 0 min, increase to 120℃ at 4℃ / min, hold for 1 min, increase to 220℃ at 5℃ / min, hold for 7 min.

[0031] Splitless injection was used, with high-purity helium as the carrier gas at a flow rate of 1.2 mL / min, a solvent delay of 2 min, and an injection port temperature of 250℃.

[0032] MS conditions: electron ion source; electron energy 70 eV; ion source temperature 230℃; interface temperature 230℃; mass scan range 40~400 m / z; scan mode full scan.

[0033] like Figure 8 As shown, the unit yield of FA gradually increased with the FA concentration increasing to 400 mM, reaching a maximum at a concentration of 400 mM, with a unit yield of FA of 12.22 g / DCW. When the pCA concentration exceeded 500 mM, the unit yield of FA began to decrease, demonstrating that the whole cells of E. coli BL21 / pET28a-V91I can tolerate FA at 400 mM.

[0034] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A phenolic acid decarboxylase mutant for efficient production of 4-vinylguaiacol, characterized in that, It has a V→I mutation at amino acid position 91 of the wild-type sequence shown in SEQ ID NO.1, and the amino acid sequence of the phenolic acid decarboxylase mutant is shown in SEQ ID NO.

2.

2. A method for improving the stability of phenolic acid decarboxylase, characterized in that, A point mutation was performed on the 91st amino acid of the wild-type phenolic acid decarboxylase shown in SEQ ID NO.1, changing it from V to I, to obtain a phenolic acid decarboxylase mutant with higher stability; the amino acid sequence of the phenolic acid decarboxylase mutant is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that, Stability includes pH stability and thermal stability.

4. The use of the phenolic acid decarboxylase mutant according to claim 1 in food fermentation and / or in the preparation of drugs with antioxidant and anti-inflammatory effects.

5. Use of the phenolic acid decarboxylase mutant according to claim 1 in the preparation of fermentation preparations with better stability.

6. A fermentation preparation with improved pH and thermal stability, characterized in that, The phenolic acid decarboxylase mutant described in claim 1 is the active ingredient.

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